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Welcome, everybody,

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wherever you may be.

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Our speaker today is

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Doctor John Turcci.

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John earned a Bachelor of

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Science in Biochemistry from

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Kemson University in

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1985, did his pH.

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D in biochemistry at the

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University of

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Missouri in 1990.

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Did his postdoc training at the

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University of Rochester with Bom

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Bom Bot.

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Robert from Bob Bum Barra

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studying the million Okazaki

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fragment processing.

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John began his independent

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research career at Wright State

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University in 1993,

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studying the recognition and

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repair of cisplatin DNA damage,

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focusing on DNA damage sensors.

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In 2005,

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he joined the Indiana University

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School

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of Medicine,

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where he is now Professor of

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medicine in

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the Division of

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Hematology and Oncology.

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And he's professor of

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biochemistry and molecular

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biology.

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He holds the Tom and Julie Wood.

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Family Foundation endowed

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professorship in lung Cancer

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Research,

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and he serves as the executive

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director of the Tom and Judy

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Wood Center for Lung

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Cancer Research at the Indiana

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University Comprehensive Cancer

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Center.

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His research continues to be

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focused on DNA damage sensors as

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therapeutic targets

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for cancer treatment. Now,

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before I let John start telling

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us about

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some of his labs recent work.

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I just want to remind all of you

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in the audience to put questions

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for him.

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Any questions you might have in

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the chat box?

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And we will ask them on your

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behalf after the seminar is

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finished.

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Without further ado,

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I'm going to hand things over to

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John.

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Alright. Thank you

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Karen for that.

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For that introduction, and to

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everyone, tune will Ken and

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Karen for the

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invitation to present today.

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And you've heard the title.

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You know who I am from that

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intro.

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Quick disclosure and disclaimer

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1 protects the university for

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me.

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The other protects me from the

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university and thinking about

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this presentation,

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I was parking back to when I

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last presented at the NIH and I

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didn't think

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it was that long ago,

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but in fact it was in.

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11 and this is what the group

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looked like back then.

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You can see me in the back there

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a much younger person.

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In fact,

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this was the President at the

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time

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looking pretty good as well.

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Fast forward to where we are

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now. Boy, how things have

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changed.

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So what I'd like to do is is

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take this first slide to

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acknowledge the people who

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have done the work in case we

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don't have time at the end to

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get to them.

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I'm going to tell you about two

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stories today. One.

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Focused on the RPA protein.

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And much of that work was done

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by Matthew Jordan.

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Here a postdoc along with Pam

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van der Beer Caroza, a new

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graduate student.

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Jess and Aditi have joined the

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lab also on that project as

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well.

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The second project I'll tell you

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about is targeting the coup

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protein double strand

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break repair largely done by

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postdoc Pam Mendoza,

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again along with Pam and Joy

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Garrett, who does a lot of our

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ionizing rad.

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Work as well.

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We have great collaborators.

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We'll do.

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A lot of chemistry

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along the way.

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NAV navandi is our chemist.

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He's a Co   and many of our

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grants and and performs all that

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work.

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As I mentioned,

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Joe Dinlock does all the

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radiation with

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us and we have a cadre of

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physician scientists at the IU

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Cancer Center who

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really provide nice perspective

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for us along the way we.

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Also acknowledge Katie Quelzak,

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who is the CEO of Nirex

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Biosciences,

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a startup that.

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Way back in 2009,

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when we thought we were

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targeting the

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nucleotide excision repair,

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we named the company NIREX.

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Reality is we've moved a little

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bit past that and we'll talk

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about damage centers

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in multiple other contexts.

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And so one of the questions we

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ask at the beginning is can we

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target replication,

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stress sensors for

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cancer therapy?

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And just to put this in

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perspective, in a typical

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mammalian S phase,

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we're going to have about 50

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million Okazaki fragments

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synthesized and

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processed.

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And that's just a normal

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unperturbed replication event

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going on.

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And if we work at

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99.9% efficiency.

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Those level of

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efficiency, right?

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We would still expect 50,

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000 potential deleterious events

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in the

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context of trying to replicate

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those chromosomes.

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And so this presents a

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opportunity where in cancer

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cells,

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if you have alterations in how

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these events are occurring,

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you would get even more of these

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replication stress induced

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events that.

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Could potentially be

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used for targeting.

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The reality when you think about

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DNA replication.

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This is from cold spring harvest

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model and everything is

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wonderfully coordinated.

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DNA coming into the heel.

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A case leading strand being

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synthesized

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continuously.

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Discontinuous lagging strand

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incredible orchestration of

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events.

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Most of us in biology know that

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this is often not the case.

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And so it got me thinking a lot

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about coordination of events and

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then these

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things called

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Phantom traffic jams.

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And so if you've seen these,

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if you're on I-5 on the West

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Coast or

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I-65 in the Midwest or I-95 on

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the East Coast?

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Everything seems to be going

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along just fine,

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and then all of a sudden one car

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taps his brakes.

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That causes the car behind and

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the taps their brakes.

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Ultimately what happens is you

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move forward and you say there's

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nothing that

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actually caused this traffic jam

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and you can take this a step

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further and say this

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is a DNA replication

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event and so on.

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Your leading strand you

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encounter an A basic site or you

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encounter a nick in the

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DNA or some type of DNA damage.

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And that results in this traffic

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jam. And if you want to

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incorporate the

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lagging strand model,

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it's just the opposite.

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Side of the highway,

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where they slow down too,

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just because they're watching

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the tragedy on the other side.

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And so normally you know traffic

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stress is not necessarily a bad

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thing.

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You can get through it.

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The real problem is when you

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have traffic catastrophe and so

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these are the major

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events that caused, you know,

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loss of life and and dramatic

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things that

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we'll see in the context of

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replication, very similar

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phenomenon.

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And so when we think about how,

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how to control these events.

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It really gets down to the DNA

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damage and replication stress

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signaling pathways,

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and it's led by a

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series of proteins.

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These ones shown in yellow that

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are the DNA sensors and these

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recognize the

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problems in the DNA.

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And so for this case a stalled

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replication fork.

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If you have RPA binding to the

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single

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stranded DNA that is sensing

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that alteration in the DNA

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structure and can

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signal to.

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Downstream kinases

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that will then.

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Regulate cellular

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pathways and processes.

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If we look at it in the context

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of the coup,

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protein similar binds to DNA

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double strand ends.

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It senses that structure.

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So these are structure specific

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binding proteins.

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It signals to the DNA dependent

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protein kinase which then goes

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in phosphorylates

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all its target proteins to

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elicit the regulation of these

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pathways and

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processes.

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Another sensor is in fact the

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PARP protein.

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It works a little differently in

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that it binds to its DNA sense,

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a DNA Nic,

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for instance,

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but instead of signaling to a

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downstream

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kinase.

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It can auto ADP

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riboscolate itself.

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We'll see a lot of these events

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where you're signaling the

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sensing paramosolate

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itself,

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which then leads to its

300
00:07:22,247 --> 00:07:23,061
association

301
00:07:23,061 --> 00:07:25,089
coupled to activation of DNA PK

302
00:07:25,089 --> 00:07:27,029
which auto phosphorylates and

303
00:07:27,029 --> 00:07:28,033
results in its

304
00:07:28,033 --> 00:07:30,066
removal of the DNA to allow the

305
00:07:30,066 --> 00:07:31,837
repair processes to occur.

306
00:07:31,837 --> 00:07:33,053
And so coordinating

307
00:07:33,053 --> 00:07:34,206
all these events.

308
00:07:34,206 --> 00:07:35,003
Is pretty important

309
00:07:35,003 --> 00:07:35,674
and we thought.

310
00:07:36,808 --> 00:07:38,058
The potential exists that we

311
00:07:38,058 --> 00:07:39,430
could target some of these for

312
00:07:39,430 --> 00:07:39,978
therapeutic

313
00:07:39,978 --> 00:07:40,579
interventions.

314
00:07:40,912 --> 00:07:42,640
So the two I'm gonna tell you

315
00:07:42,640 --> 00:07:44,534
about today are in fact the RPA

316
00:07:44,534 --> 00:07:45,083
protein,

317
00:07:45,083 --> 00:07:46,674
its interaction with single

318
00:07:46,674 --> 00:07:48,539
strand DNA will start there and

319
00:07:48,539 --> 00:07:49,621
then move over to

320
00:07:49,621 --> 00:07:51,225
the coup project that we're

321
00:07:51,225 --> 00:07:52,591
we'll discuss as well.

322
00:07:53,058 --> 00:07:54,632
So RPA is the human single

323
00:07:54,632 --> 00:07:56,328
strand DNA binding protein.

324
00:07:56,328 --> 00:07:58,079
This is the pave structure

325
00:07:58,079 --> 00:07:59,898
published decade plus ago,

326
00:07:59,898 --> 00:08:01,644
that shows that DNA single

327
00:08:01,644 --> 00:08:03,629
strand being wrapped around a

328
00:08:03,629 --> 00:08:05,203
series of OB folds and

329
00:08:05,203 --> 00:08:07,005
we focus on this OB fold B.

330
00:08:07,339 --> 00:08:09,416
It's a major component of high

331
00:08:09,416 --> 00:08:11,009
affinity interactions.

332
00:08:11,009 --> 00:08:13,296
With single strand DNA and when

333
00:08:13,296 --> 00:08:15,567
we performed a high throughput

334
00:08:15,567 --> 00:08:16,248
screen a

335
00:08:16,248 --> 00:08:17,735
number of years ago,

336
00:08:17,735 --> 00:08:20,099
we're able to identify a series

337
00:08:20,099 --> 00:08:20,785
of small

338
00:08:20,785 --> 00:08:22,859
molecules that could bind to

339
00:08:22,859 --> 00:08:24,972
that OB fold B and block the

340
00:08:24,972 --> 00:08:26,858
protein DNA interaction.

341
00:08:26,858 --> 00:08:29,080
So we're showing two compounds

342
00:08:29,080 --> 00:08:30,562
here this tdrl 551,

343
00:08:30,562 --> 00:08:32,332
which is the predecessor

344
00:08:32,332 --> 00:08:33,365
to neurx 329.

345
00:08:33,365 --> 00:08:35,200
It differs only by an addition

346
00:08:35,200 --> 00:08:37,137
of this propyl morpholino group

347
00:08:37,137 --> 00:08:38,136
making an amide

348
00:08:38,136 --> 00:08:39,345
bond instead of this

349
00:08:39,345 --> 00:08:40,372
carboxylic acid.

350
00:08:41,406 --> 00:08:42,140
In vitro.

351
00:08:42,140 --> 00:08:44,402
Either one can inhibit the RPA

352
00:08:44,402 --> 00:08:46,636
single strand DNA interaction

353
00:08:46,636 --> 00:08:47,946
and so just in a

354
00:08:47,946 --> 00:08:50,209
test tube reaction purified 30

355
00:08:50,209 --> 00:08:52,609
base single strand DNA purified

356
00:08:52,609 --> 00:08:53,151
hetero

357
00:08:53,151 --> 00:08:54,889
trimeric human RPA with

358
00:08:54,889 --> 00:08:57,279
titrating increasing amounts of

359
00:08:57,279 --> 00:08:58,590
the inhibitor we

360
00:08:58,590 --> 00:09:00,196
can block that RPADNA

361
00:09:00,196 --> 00:09:02,260
interaction this prep was.

362
00:09:02,260 --> 00:09:04,047
Actually purified out of E coli

363
00:09:04,047 --> 00:09:05,430
in an untagged version,

364
00:09:05,430 --> 00:09:06,756
and so we ran through a

365
00:09:06,756 --> 00:09:08,400
traditional purification and

366
00:09:08,400 --> 00:09:09,868
depending on how good or

367
00:09:09,868 --> 00:09:11,536
bad we are in that purific.

368
00:09:11,536 --> 00:09:13,805
We always get contamination of E

369
00:09:13,805 --> 00:09:16,050
coli SSB which is shown as this

370
00:09:16,050 --> 00:09:17,209
bottom shift in

371
00:09:17,209 --> 00:09:19,341
the DNA and what this shows us

372
00:09:19,341 --> 00:09:21,591
is in fact that these compounds

373
00:09:21,591 --> 00:09:22,681
are relatively

374
00:09:22,681 --> 00:09:24,955
specific for the RPA protein and

375
00:09:24,955 --> 00:09:26,990
not necessarily blocking the

376
00:09:26,990 --> 00:09:28,153
single strand E

377
00:09:28,153 --> 00:09:28,986
coli protein

378
00:09:28,986 --> 00:09:30,722
interaction. So we have.

379
00:09:30,722 --> 00:09:31,670
Pretty good specificity,

380
00:09:31,670 --> 00:09:32,847
pretty good potency in a test

381
00:09:32,847 --> 00:09:33,091
tube.

382
00:09:33,558 --> 00:09:35,241
We can actually do the same

383
00:09:35,241 --> 00:09:37,207
experiment in cells and measure

384
00:09:37,207 --> 00:09:38,730
the RPA DNA interaction

385
00:09:38,730 --> 00:09:39,907
through a proximity

386
00:09:39,907 --> 00:09:40,899
ligation assay.

387
00:09:41,399 --> 00:09:43,370
And either leave these cells

388
00:09:43,370 --> 00:09:45,666
untreated or treat them with the

389
00:09:45,666 --> 00:09:47,172
RK inhibitor for two

390
00:09:47,172 --> 00:09:48,716
hours and we can get a

391
00:09:48,716 --> 00:09:50,504
statistically significant

392
00:09:50,504 --> 00:09:52,577
decrease in the total number

393
00:09:52,577 --> 00:09:54,826
of RPA foci as a function of our

394
00:09:54,826 --> 00:09:55,880
RPA inhibitor.

395
00:09:55,880 --> 00:09:58,072
And if we only select the cells

396
00:09:58,072 --> 00:10:00,051
that are PCNA positive, so.

397
00:10:00,051 --> 00:10:01,788
Those cells that are likely in S

398
00:10:01,788 --> 00:10:03,353
phase, you get more foci per

399
00:10:03,353 --> 00:10:03,688
cell,

400
00:10:03,688 --> 00:10:05,315
but we can still decrease that

401
00:10:05,315 --> 00:10:06,935
dramatically as a function of

402
00:10:06,935 --> 00:10:07,158
our

403
00:10:07,158 --> 00:10:07,692
inhibitor.

404
00:10:07,692 --> 00:10:09,449
So the S phase cells or foci for

405
00:10:09,449 --> 00:10:11,096
our PA, as you might imagine.

406
00:10:11,096 --> 00:10:12,981
Associated with replication and

407
00:10:12,981 --> 00:10:14,969
we can decrease that number. You

408
00:10:14,969 --> 00:10:15,900
might imagine.

409
00:10:15,900 --> 00:10:16,209
Then,

410
00:10:16,209 --> 00:10:18,044
as long as we're blocking RPA

411
00:10:18,044 --> 00:10:18,803
interaction

412
00:10:18,803 --> 00:10:20,272
with DNA in these cells,

413
00:10:20,272 --> 00:10:22,159
this is an H460 non small cell

414
00:10:22,159 --> 00:10:22,474
lung

415
00:10:22,474 --> 00:10:22,901
cancer.

416
00:10:22,901 --> 00:10:24,780
As long as those compounds can

417
00:10:24,780 --> 00:10:25,343
get into

418
00:10:25,343 --> 00:10:27,178
the cells, we can induce cell.

419
00:10:27,178 --> 00:10:27,633
Death.

420
00:10:27,633 --> 00:10:29,496
And so in this metabolic

421
00:10:29,496 --> 00:10:30,815
viability assay,

422
00:10:30,815 --> 00:10:33,032
the 551 compound does not get

423
00:10:33,032 --> 00:10:35,020
into the cells very well.

424
00:10:35,020 --> 00:10:36,553
Largely a function of this

425
00:10:36,553 --> 00:10:37,555
carboxylic acid.

426
00:10:38,323 --> 00:10:40,132
But when we put the morpholino

427
00:10:40,132 --> 00:10:41,459
groups on in the 329.

428
00:10:41,860 --> 00:10:43,239
Or the 2004 derivative,

429
00:10:43,239 --> 00:10:45,088
which is essentially replacing

430
00:10:45,088 --> 00:10:45,397
this

431
00:10:45,397 --> 00:10:46,841
iodine with the bromine.

432
00:10:46,841 --> 00:10:48,758
We get very potent cell killing

433
00:10:48,758 --> 00:10:49,067
in a

434
00:10:49,067 --> 00:10:50,064
pretty tight dose

435
00:10:50,064 --> 00:10:51,002
response curve.

436
00:10:52,570 --> 00:10:54,200
And so we can block the RPA DNA

437
00:10:54,200 --> 00:10:55,716
interaction in test tubes in

438
00:10:55,716 --> 00:10:56,041
cells

439
00:10:56,041 --> 00:10:57,008
induced cell death.

440
00:10:57,542 --> 00:10:59,734
Katie Pozak at Nurex did a whole

441
00:10:59,734 --> 00:11:01,626
series of single agent anti

442
00:11:01,626 --> 00:11:02,747
cancer activity

443
00:11:02,747 --> 00:11:04,816
across a number of cell lines.

444
00:11:04,816 --> 00:11:06,630
And the results demonstrate that

445
00:11:06,630 --> 00:11:08,140
the mechanism of action is

446
00:11:08,140 --> 00:11:08,953
largely tumor

447
00:11:08,953 --> 00:11:09,454
agnostic.

448
00:11:10,021 --> 00:11:11,598
And so we can kill lymphoma

449
00:11:11,598 --> 00:11:13,088
cells, bone cells, breast

450
00:11:13,088 --> 00:11:14,459
cancers, lung cancers,

451
00:11:14,459 --> 00:11:15,974
as well as ovarian cancer.

452
00:11:15,974 --> 00:11:17,586
We see a pretty interesting

453
00:11:17,586 --> 00:11:18,363
distribution

454
00:11:18,363 --> 00:11:20,231
along the different tumor types,

455
00:11:20,231 --> 00:11:22,148
with IC 50s ranging from the low

456
00:11:22,148 --> 00:11:22,567
single

457
00:11:22,567 --> 00:11:24,267
digit micromolar up to the 10

458
00:11:24,267 --> 00:11:26,002
and 15 micromolar, suggesting

459
00:11:26,002 --> 00:11:27,138
that there's some.

460
00:11:27,138 --> 00:11:29,130
Factors that are contributing to

461
00:11:29,130 --> 00:11:31,106
differential sensitivity within

462
00:11:31,106 --> 00:11:31,743
any given

463
00:11:31,743 --> 00:11:32,435
tumor type.

464
00:11:32,435 --> 00:11:34,368
The other thing we noticed was

465
00:11:34,368 --> 00:11:35,013
and a lot

466
00:11:35,013 --> 00:11:35,960
of these cells,

467
00:11:35,960 --> 00:11:37,967
we get an incredibly tight dose

468
00:11:37,967 --> 00:11:38,550
response

469
00:11:38,550 --> 00:11:39,718
curve and this is.

470
00:11:39,718 --> 00:11:41,750
Here in the bottom graph with

471
00:11:41,750 --> 00:11:43,894
the A 2780 ovarian cancer cell

472
00:11:43,894 --> 00:11:45,323
line and the GCT 27

473
00:11:45,323 --> 00:11:47,211
testicular cancer cell line

474
00:11:47,211 --> 00:11:49,284
where we can hit these things

475
00:11:49,284 --> 00:11:50,428
with about four

476
00:11:50,428 --> 00:11:52,253
micromolar drug and elicit

477
00:11:52,253 --> 00:11:54,402
hardly any change in viability

478
00:11:54,402 --> 00:11:55,834
and we go up to six

479
00:11:55,834 --> 00:11:56,234
and a.

480
00:11:56,234 --> 00:11:58,109
Half micromolar drug and we

481
00:11:58,109 --> 00:11:59,637
killed all the cells.

482
00:11:59,637 --> 00:12:01,569
And so this incredibly tight

483
00:12:01,569 --> 00:12:03,614
dose response curve is really

484
00:12:03,614 --> 00:12:04,743
indicative of a

485
00:12:04,743 --> 00:12:05,844
threshold event.

486
00:12:06,511 --> 00:12:07,554
And so as we're thinking about

487
00:12:07,554 --> 00:12:08,622
thresholds and you're thinking

488
00:12:08,622 --> 00:12:09,013
about RPA?

489
00:12:10,048 --> 00:12:12,261
Your mind naturally migrates to

490
00:12:12,261 --> 00:12:13,618
Yuri Lucas's work,

491
00:12:13,618 --> 00:12:15,854
and the idea of RPA exhaustion.

492
00:12:15,854 --> 00:12:18,174
And so we have adopted his model

493
00:12:18,174 --> 00:12:20,468
for our own use and essentially

494
00:12:20,468 --> 00:12:21,726
term it chemical

495
00:12:21,726 --> 00:12:22,820
RPA exhaustion.

496
00:12:22,820 --> 00:12:24,863
And so as the model states,

497
00:12:24,863 --> 00:12:26,955
we've got a certain amount of

498
00:12:26,955 --> 00:12:29,090
RPA in the cell that can hold

499
00:12:29,090 --> 00:12:30,635
the amount of single

500
00:12:30,635 --> 00:12:32,737
strand DNA that you generate.

501
00:12:32,971 --> 00:12:33,938
Let's get that back.

502
00:12:33,938 --> 00:12:34,472
There we go.

503
00:12:35,273 --> 00:12:36,519
For unperturbed replication,

504
00:12:36,519 --> 00:12:37,796
you generate a little single

505
00:12:37,796 --> 00:12:38,343
strand DNA.

506
00:12:38,343 --> 00:12:39,344
You have plenty of RPA.

507
00:12:39,711 --> 00:12:41,185
There for that oncogenic

508
00:12:41,185 --> 00:12:42,414
replication stress.

509
00:12:42,414 --> 00:12:44,048
So now you've got.

510
00:12:45,450 --> 00:12:46,684
The amplification.

511
00:12:46,684 --> 00:12:48,024
Maybe an EGFR mutation,

512
00:12:48,024 --> 00:12:49,937
anything that drives replication

513
00:12:49,937 --> 00:12:50,355
stress

514
00:12:50,355 --> 00:12:51,142
generates more

515
00:12:51,142 --> 00:12:52,323
single stranded DNA.

516
00:12:52,924 --> 00:12:54,630
But you still have enough RPA

517
00:12:54,630 --> 00:12:56,433
protection to handle all those

518
00:12:56,433 --> 00:12:57,395
binding events,

519
00:12:57,395 --> 00:12:58,944
and only when you add adna

520
00:12:58,944 --> 00:13:00,732
damaging agent cisplatin, Dr.

521
00:13:00,732 --> 00:13:02,560
Rubicon Campbellsen whatever is

522
00:13:02,560 --> 00:13:04,311
going to generate more single

523
00:13:04,311 --> 00:13:05,036
strand DNA.

524
00:13:05,036 --> 00:13:06,853
Do you get above this threshold

525
00:13:06,853 --> 00:13:08,594
and end up with a replication

526
00:13:08,594 --> 00:13:09,374
catastrophe?

527
00:13:09,574 --> 00:13:11,130
Or cell death phenomenon.

528
00:13:11,130 --> 00:13:13,171
And So what we can do chemically

529
00:13:13,171 --> 00:13:13,745
is treat

530
00:13:13,745 --> 00:13:14,905
with that low level

531
00:13:14,905 --> 00:13:16,247
of our RPA inhibitor.

532
00:13:16,247 --> 00:13:17,971
We lower the threshold a little,

533
00:13:17,971 --> 00:13:19,465
but there's still enough to

534
00:13:19,465 --> 00:13:20,018
cover the

535
00:13:20,018 --> 00:13:21,553
oncogenic replication stress.

536
00:13:21,553 --> 00:13:23,157
So this would be analogous to

537
00:13:23,157 --> 00:13:24,964
that like 4 micromolar treatment

538
00:13:24,964 --> 00:13:25,924
where we haven't

539
00:13:25,924 --> 00:13:27,348
induced much cell killing.

540
00:13:27,348 --> 00:13:29,032
But if we just put in a little

541
00:13:29,032 --> 00:13:29,594
bit more,

542
00:13:29,594 --> 00:13:31,256
take out two more of the RP as

543
00:13:31,256 --> 00:13:32,697
get below that threshold,

544
00:13:32,697 --> 00:13:34,464
this is where we get replication

545
00:13:34,464 --> 00:13:35,900
catastrophe. So Yuri Luc.

546
00:13:35,900 --> 00:13:37,126
Showed this genetically,

547
00:13:37,126 --> 00:13:38,403
we can do it chemically.

548
00:13:38,403 --> 00:13:40,325
And then the reality is the

549
00:13:40,325 --> 00:13:42,429
difference in the therapeutic

550
00:13:42,429 --> 00:13:44,242
window is defined by how

551
00:13:44,242 --> 00:13:46,310
much RPA we can block to keep

552
00:13:46,310 --> 00:13:48,347
the single strand DNA in the

553
00:13:48,347 --> 00:13:49,948
oncologic replication

554
00:13:49,948 --> 00:13:52,095
stress versus the unperturbed.

555
00:13:52,095 --> 00:13:54,385
And so we can actually hit that

556
00:13:54,385 --> 00:13:56,287
therapeutic window in mice.

557
00:13:56,287 --> 00:13:58,431
And so if we take the non small

558
00:13:58,431 --> 00:14:00,091
cell lung cancer cells,

559
00:14:00,091 --> 00:14:02,186
put them subcutaneously into a

560
00:14:02,186 --> 00:14:03,862
mouse, treat them with.

561
00:14:04,562 --> 00:14:05,616
The inhibitor everyday

562
00:14:05,616 --> 00:14:06,431
for a few weeks.

563
00:14:06,831 --> 00:14:07,999
Or a vehicle.

564
00:14:07,999 --> 00:14:09,918
We can get a statistically

565
00:14:09,918 --> 00:14:12,100
significant decrease in tumor

566
00:14:12,100 --> 00:14:13,905
growth and tumor weight

567
00:14:13,905 --> 00:14:16,045
at the end of the experiment.

568
00:14:16,045 --> 00:14:18,085
So we have a safe margin of

569
00:14:18,085 --> 00:14:19,143
treatment for

570
00:14:19,143 --> 00:14:20,770
these mice with an RPA

571
00:14:20,770 --> 00:14:22,964
inhibitor, indicating that we

572
00:14:22,964 --> 00:14:23,948
can hit this

573
00:14:23,948 --> 00:14:26,008
therapeutic window available

574
00:14:26,008 --> 00:14:28,336
through the chemical exhaustion

575
00:14:28,336 --> 00:14:29,687
model we've also.

576
00:14:29,687 --> 00:14:30,939
Taken this at the patient

577
00:14:30,939 --> 00:14:32,419
derived models, the ones I'll

578
00:14:32,419 --> 00:14:33,491
show you here are an

579
00:14:33,491 --> 00:14:35,003
ovarian cancer spheroid model

580
00:14:35,003 --> 00:14:35,994
that we developed.

581
00:14:35,994 --> 00:14:37,417
From patients that present at

582
00:14:37,417 --> 00:14:38,496
the IU Cancer Center.

583
00:14:40,031 --> 00:14:42,073
We get tissue from them largely

584
00:14:42,073 --> 00:14:44,093
from debulking surgery and can

585
00:14:44,093 --> 00:14:45,169
create a series

586
00:14:45,169 --> 00:14:45,370
of.

587
00:14:48,973 --> 00:14:50,141
Tumor models.

588
00:14:50,141 --> 00:14:51,558
Let's see if this, you know,

589
00:14:51,558 --> 00:14:53,177
these movies don't wanna start.

590
00:14:53,177 --> 00:14:54,379
But that's OK.

591
00:14:55,847 --> 00:14:57,740
So what happens here is if these

592
00:14:57,740 --> 00:14:58,449
were going,

593
00:14:58,449 --> 00:14:59,981
you would see that the RPA

594
00:14:59,981 --> 00:15:01,901
treated tumor spheroids induce a

595
00:15:01,901 --> 00:15:03,221
significant amount of

596
00:15:03,221 --> 00:15:04,748
cell death compared to the

597
00:15:04,748 --> 00:15:06,130
untreated. As these are

598
00:15:06,130 --> 00:15:07,392
monitored over time.

599
00:15:07,392 --> 00:15:09,791
So we have both in vivo activity

600
00:15:09,791 --> 00:15:12,014
in non small cell lung cancer

601
00:15:12,014 --> 00:15:13,164
and in patient

602
00:15:13,164 --> 00:15:15,282
derived human ovarian cancer

603
00:15:15,282 --> 00:15:16,568
spheroid models.

604
00:15:18,303 --> 00:15:19,383
And so then the question is,

605
00:15:19,383 --> 00:15:20,271
how is this happening?

606
00:15:20,271 --> 00:15:21,885
And I'm going to put some work

607
00:15:21,885 --> 00:15:22,907
by Matthew Jordan.

608
00:15:22,907 --> 00:15:23,975
We published this last year.

609
00:15:24,375 --> 00:15:26,423
In scientific reports into one

610
00:15:26,423 --> 00:15:27,378
single slide,

611
00:15:27,378 --> 00:15:29,086
and the idea was if we're

612
00:15:29,086 --> 00:15:31,332
blocking our PA interaction with

613
00:15:31,332 --> 00:15:31,683
DNA,

614
00:15:31,683 --> 00:15:33,724
we would necessarily block the

615
00:15:33,724 --> 00:15:35,602
ATR activation and could we

616
00:15:35,602 --> 00:15:36,854
recapitulate this

617
00:15:36,854 --> 00:15:37,922
in in vitro in a

618
00:15:37,922 --> 00:15:39,324
reconstituted essay.

619
00:15:39,324 --> 00:15:41,509
So Matthew purified the proteins

620
00:15:41,509 --> 00:15:42,260
necessary.

621
00:15:42,260 --> 00:15:43,134
RPA top BP1,

622
00:15:43,134 --> 00:15:45,456
which is an interacting protein

623
00:15:45,456 --> 00:15:45,830
that

624
00:15:45,830 --> 00:15:47,815
works with ATR and ATRIP to

625
00:15:47,815 --> 00:15:49,471
signal to all of these

626
00:15:49,471 --> 00:15:50,902
downstream events.

627
00:15:50,902 --> 00:15:52,049
What we looked at is a

628
00:15:52,049 --> 00:15:53,331
downstream event was the

629
00:15:53,331 --> 00:15:54,238
phosphorylation.

630
00:15:54,439 --> 00:15:55,334
Of P53 here,

631
00:15:55,334 --> 00:15:57,630
as well as the phosphorylation

632
00:15:57,630 --> 00:15:58,242
of RPA.

633
00:15:58,843 --> 00:16:01,079
And so Matthew was able to show

634
00:16:01,079 --> 00:16:03,303
very effectively that with the

635
00:16:03,303 --> 00:16:03,748
right

636
00:16:03,748 --> 00:16:05,483
combination of DNA, RPA,

637
00:16:05,483 --> 00:16:07,491
single strand DNA, top BP1,

638
00:16:07,491 --> 00:16:08,086
aatrip,

639
00:16:08,086 --> 00:16:09,472
we could get robust

640
00:16:09,472 --> 00:16:11,222
phosphorylation of P53,

641
00:16:11,222 --> 00:16:13,527
robust phosphorylation of RPA on

642
00:16:13,527 --> 00:16:15,511
serine 33, as well as three

643
00:16:15,511 --> 00:16:17,128
inning, 21 that were.

644
00:16:17,128 --> 00:16:18,447
All sensitive to the

645
00:16:18,447 --> 00:16:19,897
ATR inhibitor VE 821.

646
00:16:20,531 --> 00:16:22,368
And so we have reconstituted the

647
00:16:22,368 --> 00:16:24,248
ATR signaling pathway, largely a

648
00:16:24,248 --> 00:16:24,836
function,

649
00:16:24,836 --> 00:16:26,267
and this is based on azee

650
00:16:26,267 --> 00:16:28,138
sanjar's work at a University of

651
00:16:28,138 --> 00:16:29,073
North Carolina,

652
00:16:29,073 --> 00:16:30,757
he published this decade plus

653
00:16:30,757 --> 00:16:31,976
ago describing this.

654
00:16:31,976 --> 00:16:34,044
We adapted that and then asked

655
00:16:34,044 --> 00:16:36,087
the question how does our RPA

656
00:16:36,087 --> 00:16:37,215
inhibitors work

657
00:16:37,215 --> 00:16:38,855
on these phosphorylation

658
00:16:38,855 --> 00:16:40,018
events in vitro?

659
00:16:40,018 --> 00:16:41,939
We're able to again effectively

660
00:16:41,939 --> 00:16:43,921
show in a reconstituted system.

661
00:16:43,921 --> 00:16:45,214
We could increase the

662
00:16:45,214 --> 00:16:47,156
concentration of either the 551

663
00:16:47,156 --> 00:16:48,660
or the 329 compound and

664
00:16:48,660 --> 00:16:50,361
potently inhibit P53 phos.

665
00:16:50,361 --> 00:16:51,196
Correlations.

666
00:16:51,196 --> 00:16:53,106
So the inability to find that

667
00:16:53,106 --> 00:16:53,765
DNA leads

668
00:16:53,765 --> 00:16:55,767
to the downstream inhibition of

669
00:16:55,767 --> 00:16:56,801
the ATR kinase.

670
00:16:57,035 --> 00:16:58,173
We published similar

671
00:16:58,173 --> 00:16:58,970
results with.

672
00:17:00,605 --> 00:17:02,020
Showing a decrease in

673
00:17:02,020 --> 00:17:04,108
phosphorylation of RPA itself,

674
00:17:04,108 --> 00:17:06,095
both at serine 33 and three in

675
00:17:06,095 --> 00:17:08,206
21, as well as we monitored the

676
00:17:08,206 --> 00:17:08,546
post

677
00:17:08,546 --> 00:17:10,481
translational modification of

678
00:17:10,481 --> 00:17:12,664
RPA via both phosphorylation and

679
00:17:12,664 --> 00:17:13,551
acetylation.

680
00:17:13,551 --> 00:17:15,606
And so they have impacts on the

681
00:17:15,606 --> 00:17:17,709
ability to block its downstream

682
00:17:17,709 --> 00:17:18,456
signaling.

683
00:17:19,390 --> 00:17:21,262
Probably my most favorite

684
00:17:21,262 --> 00:17:23,396
experiment from this was the

685
00:17:23,396 --> 00:17:25,530
analysis where we looked at

686
00:17:25,530 --> 00:17:27,868
inhibition as a function of ATP

687
00:17:27,868 --> 00:17:29,000
concentration,

688
00:17:29,000 --> 00:17:30,415
and this stems from

689
00:17:30,415 --> 00:17:32,769
conversations where when we use

690
00:17:32,769 --> 00:17:34,439
the V EA21 inhibitor,

691
00:17:34,439 --> 00:17:36,616
we have to use it at 10/20/30

692
00:17:36,616 --> 00:17:38,342
times its published I.

693
00:17:38,342 --> 00:17:40,008
50 values and when Matthew went

694
00:17:40,008 --> 00:17:41,491
back and looked at the very

695
00:17:41,491 --> 00:17:42,480
first papers were

696
00:17:42,480 --> 00:17:43,485
Vertex Therapeutics

697
00:17:43,485 --> 00:17:44,649
was developing these.

698
00:17:45,349 --> 00:17:46,463
He came into my office and said,

699
00:17:46,463 --> 00:17:47,428
did you know that when they

700
00:17:47,428 --> 00:17:47,785
developed

701
00:17:47,785 --> 00:17:48,673
these compounds they were

702
00:17:48,673 --> 00:17:49,454
running their kinase?

703
00:17:49,887 --> 00:17:52,390
Case assays for 24 hours at 25

704
00:17:52,390 --> 00:17:54,785
micro molar ATP and I didn't

705
00:17:54,785 --> 00:17:55,727
know that.

706
00:17:55,727 --> 00:17:57,560
And we would argue that's not

707
00:17:57,560 --> 00:17:59,330
tremendously physiological,

708
00:17:59,330 --> 00:18:01,167
nor is a 24 hour kinase assay

709
00:18:01,167 --> 00:18:03,246
spectacular in in the ensomology

710
00:18:03,246 --> 00:18:03,701
field.

711
00:18:03,701 --> 00:18:04,883
But they got very

712
00:18:04,883 --> 00:18:06,204
potent inhibitors.

713
00:18:06,537 --> 00:18:07,949
But the reality is their

714
00:18:07,949 --> 00:18:09,869
inhibitors are very sensitive to

715
00:18:09,869 --> 00:18:11,008
ATP concentration.

716
00:18:11,008 --> 00:18:12,773
So we can get great inhibition

717
00:18:12,773 --> 00:18:14,693
with DEA 21 at 40 micromolar ATP

718
00:18:14,693 --> 00:18:15,713
in the reaction.

719
00:18:15,713 --> 00:18:17,014
But as we increase it.

720
00:18:17,782 --> 00:18:19,353
The effectiveness of that kinase

721
00:18:19,353 --> 00:18:20,664
and it's a competitive ATP

722
00:18:20,664 --> 00:18:21,219
inhibitor.

723
00:18:21,719 --> 00:18:23,910
Goes down as you might imagine,

724
00:18:23,910 --> 00:18:25,798
whereas the ability of our

725
00:18:25,798 --> 00:18:26,524
compound,

726
00:18:26,524 --> 00:18:27,802
the RPA inhibitor,

727
00:18:27,802 --> 00:18:29,861
is largely unaffected by ATP

728
00:18:29,861 --> 00:18:30,862
concentration.

729
00:18:30,862 --> 00:18:32,263
So we really like this model.

730
00:18:32,263 --> 00:18:34,070
We want to inhibit the kinase,

731
00:18:34,070 --> 00:18:36,045
maybe going after the ATP active

732
00:18:36,045 --> 00:18:36,601
site and

733
00:18:36,601 --> 00:18:38,527
and developing these things with

734
00:18:38,527 --> 00:18:39,370
high potency,

735
00:18:39,370 --> 00:18:40,518
but at very low ATP

736
00:18:40,518 --> 00:18:41,749
concentrations, more

737
00:18:41,749 --> 00:18:43,271
physiological might be a

738
00:18:43,271 --> 00:18:43,841
function

739
00:18:43,841 --> 00:18:44,842
worth looking at.

740
00:18:45,276 --> 00:18:46,383
So we have the ability to

741
00:18:46,383 --> 00:18:47,445
inhibit the ATR kinase.

742
00:18:48,212 --> 00:18:48,937
RPA inhibition of

743
00:18:48,937 --> 00:18:49,747
single strand DNA.

744
00:18:50,114 --> 00:18:52,084
The question is what's happening

745
00:18:52,084 --> 00:18:53,684
at the replication forks,

746
00:18:53,684 --> 00:18:55,587
and so Matthew did these single

747
00:18:55,587 --> 00:18:57,216
molecule molecular combing

748
00:18:57,216 --> 00:18:58,656
experiments ultimately

749
00:18:58,656 --> 00:19:00,515
taking the A549 non small cell

750
00:19:00,515 --> 00:19:01,692
lung cancer cells,

751
00:19:01,692 --> 00:19:03,645
treating them with our inhibitor

752
00:19:03,645 --> 00:19:05,519
or without vehicle control and

753
00:19:05,519 --> 00:19:06,330
then doing a

754
00:19:06,330 --> 00:19:07,363
molecular combing

755
00:19:07,363 --> 00:19:08,699
experiment where you?

756
00:19:08,699 --> 00:19:10,273
Do pulse labeling with iodo

757
00:19:10,273 --> 00:19:11,886
deoxyurating for 30 minutes

758
00:19:11,886 --> 00:19:12,603
followed by

759
00:19:12,603 --> 00:19:13,571
Chloroxyuridine.

760
00:19:13,571 --> 00:19:14,572
Isolate the DNA.

761
00:19:14,572 --> 00:19:15,807
Spread them on slides.

762
00:19:15,807 --> 00:19:17,252
Probe for these with specific

763
00:19:17,252 --> 00:19:18,778
antibodies that give you these

764
00:19:18,778 --> 00:19:19,744
tracks and you can

765
00:19:19,744 --> 00:19:20,211
measure.

766
00:19:20,211 --> 00:19:22,183
Track length and what he showed

767
00:19:22,183 --> 00:19:24,071
very effectively was that the

768
00:19:24,071 --> 00:19:24,982
RPA inhibitor

769
00:19:24,982 --> 00:19:26,173
induced replication

770
00:19:26,173 --> 00:19:27,051
fork slowing.

771
00:19:27,051 --> 00:19:28,566
So we got shorter forks as a

772
00:19:28,566 --> 00:19:30,225
function of treatment with the

773
00:19:30,225 --> 00:19:31,055
RPA inhibitor.

774
00:19:31,055 --> 00:19:32,369
It was statistically

775
00:19:32,369 --> 00:19:34,520
significant. Modest. We go from,

776
00:19:34,520 --> 00:19:35,193
you know,

777
00:19:35,193 --> 00:19:36,782
10 microns to 8 microns,

778
00:19:36,782 --> 00:19:38,410
and so maybe a 10 or 20%

779
00:19:38,410 --> 00:19:39,564
difference where

780
00:19:39,564 --> 00:19:41,408
we saw in much more dramatic

781
00:19:41,408 --> 00:19:42,887
effect was in fact the

782
00:19:42,887 --> 00:19:44,769
replication restart assays.

783
00:19:45,503 --> 00:19:46,942
And so in this case you do the

784
00:19:46,942 --> 00:19:48,508
same type of analysis treat with

785
00:19:48,508 --> 00:19:49,340
IOTA the oxygen.

786
00:19:49,340 --> 00:19:50,842
But now we induce a replication.

787
00:19:51,275 --> 00:19:52,542
Installing event with

788
00:19:52,542 --> 00:19:54,503
hydroxyurea and you put in mirin

789
00:19:54,503 --> 00:19:56,280
which is an inhibitor of the

790
00:19:56,280 --> 00:19:58,028
nuclease MRE 11 that degrades

791
00:19:58,028 --> 00:20:00,017
some of these replication forks.

792
00:20:00,017 --> 00:20:01,947
So we're blocking that event and

793
00:20:01,947 --> 00:20:03,797
then 30 minutes after you wash

794
00:20:03,797 --> 00:20:04,722
it out and add

795
00:20:04,722 --> 00:20:06,546
your chloride in plus or minus

796
00:20:06,546 --> 00:20:07,458
our inhibitor.

797
00:20:07,458 --> 00:20:09,678
So the ability to restart is a

798
00:20:09,678 --> 00:20:11,568
function of this chloride

799
00:20:11,568 --> 00:20:13,231
exothermic deoxyurine

800
00:20:13,231 --> 00:20:13,698
ratio.

801
00:20:13,965 --> 00:20:15,269
And what we see is that

802
00:20:15,269 --> 00:20:17,001
inclusion of the RPA inhibitor

803
00:20:17,001 --> 00:20:18,502
in this second step gives

804
00:20:18,502 --> 00:20:20,221
us a dramatic decrease in the

805
00:20:20,221 --> 00:20:21,405
ability to restart.

806
00:20:21,405 --> 00:20:22,763
Those replication forks,

807
00:20:22,763 --> 00:20:24,509
so we have events occurring at

808
00:20:24,509 --> 00:20:24,742
the

809
00:20:24,742 --> 00:20:26,254
replication fork that could

810
00:20:26,254 --> 00:20:28,045
potentially be impacting these.

811
00:20:29,547 --> 00:20:30,416
Downstream events.

812
00:20:30,416 --> 00:20:31,853
So up till now we can inhibit

813
00:20:31,853 --> 00:20:32,250
the DNA

814
00:20:32,250 --> 00:20:32,817
interaction.

815
00:20:32,817 --> 00:20:34,151
We can block ATR signaling.

816
00:20:34,151 --> 00:20:36,080
We get single agent anti cancer

817
00:20:36,080 --> 00:20:37,920
activity but pretty modest in

818
00:20:37,920 --> 00:20:39,190
vivo from that 8549

819
00:20:39,190 --> 00:20:40,693
experiment I showed you.

820
00:20:40,693 --> 00:20:42,747
And so coupled with that and the

821
00:20:42,747 --> 00:20:43,261
60 cell

822
00:20:43,261 --> 00:20:45,212
line screen that Katie did with

823
00:20:45,212 --> 00:20:46,597
those varying IC 50s,

824
00:20:46,597 --> 00:20:48,169
we thought you know is it

825
00:20:48,169 --> 00:20:49,300
possible that we.

826
00:20:49,300 --> 00:20:50,332
Could identify the

827
00:20:50,332 --> 00:20:51,535
genetic alterations.

828
00:20:51,836 --> 00:20:53,706
That would increase sensitivity

829
00:20:53,706 --> 00:20:54,972
to an RPA inhibitor.

830
00:20:56,507 --> 00:20:57,877
And so we conducted a CRISPR

831
00:20:57,877 --> 00:20:58,709
knockout screen.

832
00:20:58,709 --> 00:21:00,411
And it worked wonderfully with

833
00:21:00,411 --> 00:21:02,147
the 329, because we're able to

834
00:21:02,147 --> 00:21:03,247
treat cells with a

835
00:21:03,247 --> 00:21:04,671
low enough dose that they

836
00:21:04,671 --> 00:21:06,298
maintain their viability and

837
00:21:06,298 --> 00:21:07,518
they're right on the

838
00:21:07,518 --> 00:21:08,093
threshold.

839
00:21:08,093 --> 00:21:09,807
So any little increase in the

840
00:21:09,807 --> 00:21:10,221
single

841
00:21:10,221 --> 00:21:12,054
strand DNA generated is going to

842
00:21:12,054 --> 00:21:13,658
give you cell death. So we,

843
00:21:13,658 --> 00:21:15,662
knocked out a whole series of

844
00:21:15,662 --> 00:21:17,787
DNA damage response genes, 230

845
00:21:17,787 --> 00:21:18,496
in total.

846
00:21:20,097 --> 00:21:21,083
17 of them gave

847
00:21:21,083 --> 00:21:22,266
us positive hits.

848
00:21:22,900 --> 00:21:25,154
That showed up in the ability to

849
00:21:25,154 --> 00:21:27,312
increase the sensitivity to an

850
00:21:27,312 --> 00:21:28,606
RPA inhibitor and

851
00:21:28,606 --> 00:21:29,747
so in this case,

852
00:21:29,747 --> 00:21:32,086
the mutations or the alterations

853
00:21:32,086 --> 00:21:32,743
in these

854
00:21:32,743 --> 00:21:34,073
DNA damage response

855
00:21:34,073 --> 00:21:35,613
and DNA repair genes.

856
00:21:37,148 --> 00:21:38,765
Increase the amount of single

857
00:21:38,765 --> 00:21:40,187
stranded DNA such that it

858
00:21:40,187 --> 00:21:41,552
exceeded that threshold

859
00:21:41,552 --> 00:21:43,263
and gave us the cell death and

860
00:21:43,263 --> 00:21:44,834
so the first one is Okazaki

861
00:21:44,834 --> 00:21:46,057
fragment processing.

862
00:21:46,057 --> 00:21:47,294
This is going to take me back

863
00:21:47,294 --> 00:21:47,892
home to work.

864
00:21:47,892 --> 00:21:49,293
I did 30 years ago.

865
00:21:49,293 --> 00:21:50,710
Next is DNA double

866
00:21:50,710 --> 00:21:52,363
strand brake repair.

867
00:21:52,363 --> 00:21:53,515
And there's also DNA

868
00:21:53,515 --> 00:21:55,402
replication. In fact, #4 this is

869
00:21:55,402 --> 00:21:55,933
in fact,

870
00:21:55,933 --> 00:21:57,234
DNA polymerase epsilon.

871
00:21:57,234 --> 00:21:59,226
Really interesting hit and we're

872
00:21:59,226 --> 00:22:00,471
investigating that,

873
00:22:00,471 --> 00:22:02,146
but I'm going to talk about

874
00:22:02,146 --> 00:22:03,664
these first two where we

875
00:22:03,664 --> 00:22:05,309
validated these by making

876
00:22:05,309 --> 00:22:06,363
CRISPR knockouts.

877
00:22:06,363 --> 00:22:08,080
And so the Okazaki fragment

878
00:22:08,080 --> 00:22:08,779
maturation

879
00:22:08,779 --> 00:22:09,939
function is in fact

880
00:22:09,939 --> 00:22:10,915
the Fen 1 gene.

881
00:22:11,682 --> 00:22:13,126
And this goes back now to my

882
00:22:13,126 --> 00:22:14,758
time in Bob Bambara's lab where

883
00:22:14,758 --> 00:22:15,653
we reconstituted

884
00:22:15,653 --> 00:22:17,054
Okazaki fragment processing.

885
00:22:17,521 --> 00:22:19,862
With fan one and I'm not sure

886
00:22:19,862 --> 00:22:20,992
Bob has ever.

887
00:22:22,660 --> 00:22:24,765
Forgiven me for the contributing

888
00:22:24,765 --> 00:22:26,588
to the incredibly poor name

889
00:22:26,588 --> 00:22:27,331
selection.

890
00:22:27,331 --> 00:22:28,922
When we first found this

891
00:22:28,922 --> 00:22:31,019
protein, we called it the calf.

892
00:22:31,019 --> 00:22:32,169
5 to 3 nuclease,

893
00:22:32,169 --> 00:22:33,871
which didn't really stick.

894
00:22:33,871 --> 00:22:36,007
And then it went by RTH rather.

895
00:22:37,641 --> 00:22:39,697
Only when it was called Fen One

896
00:22:39,697 --> 00:22:41,526
did it really catch on what

897
00:22:41,526 --> 00:22:42,813
we're able to show

898
00:22:42,813 --> 00:22:44,723
here is and Bob and I decades

899
00:22:44,723 --> 00:22:46,607
ago implicated it in Okazaki

900
00:22:46,607 --> 00:22:47,818
fragment process.

901
00:22:48,119 --> 00:22:49,967
A lot of balakrishnan's work a

902
00:22:49,967 --> 00:22:51,791
little later really clarified

903
00:22:51,791 --> 00:22:52,923
the mechanism for

904
00:22:52,923 --> 00:22:54,597
help and one is involved in

905
00:22:54,597 --> 00:22:55,960
removing the RNA DNA,

906
00:22:55,960 --> 00:22:57,834
hybrids and extended fragments

907
00:22:57,834 --> 00:22:58,896
from polymerase,

908
00:22:58,896 --> 00:23:00,053
epsilon and Okazaki

909
00:23:00,053 --> 00:23:01,332
fragment processing.

910
00:23:01,565 --> 00:23:03,505
And So what we did was create a

911
00:23:03,505 --> 00:23:05,557
series of N1 homozygous shown in

912
00:23:05,557 --> 00:23:06,070
red and

913
00:23:06,070 --> 00:23:07,507
heterozygous knockouts.

914
00:23:07,507 --> 00:23:09,507
Each one gives you an increased

915
00:23:09,507 --> 00:23:10,893
sensitivity to the RPA

916
00:23:10,893 --> 00:23:12,910
inhibitor. And so in our model,

917
00:23:12,910 --> 00:23:14,705
we're generating more single

918
00:23:14,705 --> 00:23:16,800
strand DNA as a dysregulation of

919
00:23:16,800 --> 00:23:17,782
these Okazaki.

920
00:23:17,782 --> 00:23:18,883
Fragment processing events.

921
00:23:18,883 --> 00:23:19,853
You can do it through

922
00:23:19,853 --> 00:23:20,684
other mechanisms.

923
00:23:20,684 --> 00:23:21,580
DNA2 and PIF,

924
00:23:21,580 --> 00:23:23,854
one that can ultimately maintain

925
00:23:23,854 --> 00:23:24,535
viability,

926
00:23:24,535 --> 00:23:26,492
but when we block the RPA in

927
00:23:26,492 --> 00:23:27,191
activity,

928
00:23:27,191 --> 00:23:28,764
we get an increase in a

929
00:23:28,764 --> 00:23:30,381
significant increase in

930
00:23:30,381 --> 00:23:31,295
sensitivity.

931
00:23:31,295 --> 00:23:33,269
And so that's consistent with

932
00:23:33,269 --> 00:23:35,496
being at the replication for DNA

933
00:23:35,496 --> 00:23:36,400
replication,

934
00:23:36,400 --> 00:23:38,584
stress increasing sensitivity to

935
00:23:38,584 --> 00:23:40,553
an RPA inhibitor, the second

936
00:23:40,553 --> 00:23:40,905
one.

937
00:23:40,905 --> 00:23:41,772
Double strand, break repair.

938
00:23:41,772 --> 00:23:43,342
We publish this with Sharon

939
00:23:43,342 --> 00:23:45,188
Cantor in her paper a number of

940
00:23:45,188 --> 00:23:45,843
years ago.

941
00:23:46,243 --> 00:23:48,004
Where we demonstrated that in an

942
00:23:48,004 --> 00:23:49,637
ovarian cancer model isogenic

943
00:23:49,637 --> 00:23:50,481
that where the

944
00:23:50,481 --> 00:23:52,108
patient presented with abraca

945
00:23:52,108 --> 00:23:52,950
null mutation,

946
00:23:52,950 --> 00:23:54,667
we get very good sensitivity to

947
00:23:54,667 --> 00:23:55,719
the RPA inhibitor.

948
00:23:55,719 --> 00:23:57,160
When we complemented those

949
00:23:57,160 --> 00:23:58,989
cells, actually we didn't do it.

950
00:23:58,989 --> 00:24:00,692
Someone else complemented those

951
00:24:00,692 --> 00:24:02,318
cells and we showed that that

952
00:24:02,318 --> 00:24:03,327
increased resist.

953
00:24:04,562 --> 00:24:06,401
To the RPA inhibitor and so at

954
00:24:06,401 --> 00:24:08,153
this point, it positions you

955
00:24:08,153 --> 00:24:09,467
right at the lagging

956
00:24:09,467 --> 00:24:11,326
strand positions you in double

957
00:24:11,326 --> 00:24:13,370
strand, break repair and really.

958
00:24:14,105 --> 00:24:14,772
The model of

959
00:24:14,772 --> 00:24:15,940
synthetic lethality.

960
00:24:16,540 --> 00:24:18,685
And gap protection driving the

961
00:24:18,685 --> 00:24:20,804
PARP inhibitor sensitivity in

962
00:24:20,804 --> 00:24:22,046
Bracha deficient

963
00:24:22,046 --> 00:24:22,480
cells.

964
00:24:22,713 --> 00:24:25,009
And so the model that Sharon can

965
00:24:25,009 --> 00:24:26,628
originally espouse and

966
00:24:26,628 --> 00:24:27,952
discovered in his

967
00:24:27,952 --> 00:24:30,030
tremendous data supporting to

968
00:24:30,030 --> 00:24:32,223
date is the idea of the single

969
00:24:32,223 --> 00:24:33,757
strand DNA gaps that

970
00:24:33,757 --> 00:24:34,767
are generated.

971
00:24:34,767 --> 00:24:37,139
If you lose Bracha one or Bracha

972
00:24:37,139 --> 00:24:37,361
2,

973
00:24:37,361 --> 00:24:39,590
or if you inhibit PARP and then

974
00:24:39,590 --> 00:24:41,532
you require other ways to.

975
00:24:41,532 --> 00:24:43,279
Protect those types of gaps,

976
00:24:43,279 --> 00:24:45,261
and RPA is an important feature

977
00:24:45,261 --> 00:24:45,836
in that.

978
00:24:45,836 --> 00:24:47,279
So then we ask the question,

979
00:24:47,279 --> 00:24:48,653
how would an RPA inhibitor

980
00:24:48,653 --> 00:24:49,340
combine with

981
00:24:49,340 --> 00:24:50,747
paap inhibition in a Bracha

982
00:24:50,747 --> 00:24:51,842
deficient cell line?

983
00:24:51,842 --> 00:24:53,747
So we took this directly into

984
00:24:53,747 --> 00:24:55,641
mice having the inhibitor in

985
00:24:55,641 --> 00:24:56,046
hand.

986
00:24:56,680 --> 00:24:58,676
So we did the MBA 436 Bracha 1,

987
00:24:58,676 --> 00:25:00,789
deficient triple negative breast

988
00:25:00,789 --> 00:25:01,252
cancer

989
00:25:01,252 --> 00:25:03,121
implanted these into mice and

990
00:25:03,121 --> 00:25:04,967
then treated them daily with

991
00:25:04,967 --> 00:25:05,956
either the RPA

992
00:25:05,956 --> 00:25:06,992
inhibitor alone,

993
00:25:06,992 --> 00:25:08,926
the PAP inhibitor elap rib or

994
00:25:08,926 --> 00:25:09,193
the

995
00:25:09,193 --> 00:25:09,960
combination.

996
00:25:09,960 --> 00:25:11,641
And we are able to essentially

997
00:25:11,641 --> 00:25:12,930
flatline tumor growth,

998
00:25:12,930 --> 00:25:14,565
as Katie Poilzak likes to say.

999
00:25:14,965 --> 00:25:16,928
With the combination in vivo in

1000
00:25:16,928 --> 00:25:18,998
mice indicating that therapeutic

1001
00:25:18,998 --> 00:25:19,904
window exists

1002
00:25:19,904 --> 00:25:21,682
to exacerbate park inhibitor

1003
00:25:21,682 --> 00:25:23,558
activity and infect stimulate

1004
00:25:23,558 --> 00:25:25,176
the anti cancer activity

1005
00:25:25,176 --> 00:25:26,964
of RPA. In this combination,

1006
00:25:26,964 --> 00:25:28,861
I'll show this picture on the

1007
00:25:28,861 --> 00:25:29,647
right here,

1008
00:25:29,647 --> 00:25:31,549
which is essentially a similar

1009
00:25:31,549 --> 00:25:33,564
model, the same exact cells but

1010
00:25:33,564 --> 00:25:34,084
now in.

1011
00:25:34,084 --> 00:25:36,019
A live cell imaging experiment

1012
00:25:36,019 --> 00:25:38,064
where we can use a single agent

1013
00:25:38,064 --> 00:25:38,856
activity in

1014
00:25:38,856 --> 00:25:40,700
combination where we monitor

1015
00:25:40,700 --> 00:25:42,650
confluence by taking pictures

1016
00:25:42,650 --> 00:25:43,861
every four hours.

1017
00:25:43,861 --> 00:25:45,740
Over A7 day period and you get

1018
00:25:45,740 --> 00:25:47,431
very very similar results.

1019
00:25:47,431 --> 00:25:48,365
Cells grow bot

1020
00:25:48,365 --> 00:25:49,633
fine on their own.

1021
00:25:49,633 --> 00:25:51,042
You hit them with anhibitor.

1022
00:25:51,042 --> 00:25:52,492
They grow fine for about two

1023
00:25:52,492 --> 00:25:52,803
days,

1024
00:25:52,803 --> 00:25:54,318
at which point they then level

1025
00:25:54,318 --> 00:25:55,817
off the RPA inhibitor works a

1026
00:25:55,817 --> 00:25:56,540
little faster

1027
00:25:56,540 --> 00:25:57,048
initially,

1028
00:25:57,048 --> 00:25:58,252
and then actually loses

1029
00:25:58,252 --> 00:25:58,776
activity.

1030
00:25:58,776 --> 00:26:00,381
We know this is a function of

1031
00:26:00,381 --> 00:26:02,129
metabolism, but the combination

1032
00:26:02,129 --> 00:26:03,314
essentially flatline

1033
00:26:03,314 --> 00:26:04,083
cell growth in

1034
00:26:04,083 --> 00:26:05,182
this model as well.

1035
00:26:06,650 --> 00:26:08,160
And so then the real question

1036
00:26:08,160 --> 00:26:09,330
becomes, how are these

1037
00:26:09,330 --> 00:26:10,554
combinations affecting

1038
00:26:10,554 --> 00:26:11,989
replication for dynamics?

1039
00:26:11,989 --> 00:26:13,624
So Matthew went back to the.

1040
00:26:14,158 --> 00:26:15,788
Microscope and combing

1041
00:26:15,788 --> 00:26:17,675
experiments. And did this

1042
00:26:17,675 --> 00:26:19,863
experiment where essentially

1043
00:26:19,863 --> 00:26:21,561
we pretreat with a PARP

1044
00:26:21,561 --> 00:26:23,891
inhibitor or not we include the

1045
00:26:23,891 --> 00:26:25,769
NEURIX 329 RPA inhibitor

1046
00:26:25,769 --> 00:26:27,836
or not or mirin then measure

1047
00:26:27,836 --> 00:26:30,262
chlorideoxyuridine and then do a

1048
00:26:30,262 --> 00:26:30,641
post

1049
00:26:30,641 --> 00:26:33,026
replication S1 nuclease event to

1050
00:26:33,026 --> 00:26:35,112
look for single stranded D.

1051
00:26:35,112 --> 00:26:36,423
That's generated during these

1052
00:26:36,423 --> 00:26:37,579
process, so I'll walk you

1053
00:26:37,579 --> 00:26:38,549
through a few of the

1054
00:26:38,549 --> 00:26:39,528
really important ones.

1055
00:26:39,528 --> 00:26:40,417
We're able to show.

1056
00:26:41,185 --> 00:26:42,315
A PARP inhibitor induced

1057
00:26:42,315 --> 00:26:43,612
increase in replication for

1058
00:26:43,612 --> 00:26:44,622
expeat other people.

1059
00:26:45,055 --> 00:26:46,895
Have observed this as well in

1060
00:26:46,895 --> 00:26:48,835
different cell lines and so in

1061
00:26:48,835 --> 00:26:50,194
this MBA 436 barcode

1062
00:26:50,194 --> 00:26:52,237
1 deficient we see that increase

1063
00:26:52,237 --> 00:26:53,897
in replication for speed.

1064
00:26:53,897 --> 00:26:55,891
So you go from about 5 1/2 up to

1065
00:26:55,891 --> 00:26:56,700
about 6 1/2,

1066
00:26:56,700 --> 00:26:58,647
and that increase is completely

1067
00:26:58,647 --> 00:26:59,903
dependent upon RPA.

1068
00:26:59,903 --> 00:27:01,693
When we put our RPA inhibitor

1069
00:27:01,693 --> 00:27:03,524
in, we drop this back down to

1070
00:27:03,524 --> 00:27:04,408
its baseline.

1071
00:27:04,408 --> 00:27:06,341
So the PARP induced increase in

1072
00:27:06,341 --> 00:27:08,212
fork speed is dependent upon.

1073
00:27:08,412 --> 00:27:08,512
RPA.

1074
00:27:10,147 --> 00:27:10,578
Next,

1075
00:27:10,578 --> 00:27:13,240
RPA inhibition of park induced

1076
00:27:13,240 --> 00:27:13,684
fork

1077
00:27:13,684 --> 00:27:14,785
acceleration.

1078
00:27:15,119 --> 00:27:17,160
Is blocked by Mirren and so

1079
00:27:17,160 --> 00:27:18,822
Mirren is that Emery,

1080
00:27:18,822 --> 00:27:20,930
11 inhibitor that blocks the

1081
00:27:20,930 --> 00:27:23,158
nuclease activity and so that

1082
00:27:23,158 --> 00:27:24,695
shows that the fact

1083
00:27:24,695 --> 00:27:27,022
that we bring this back down in

1084
00:27:27,022 --> 00:27:29,326
the presence of mirin suggests

1085
00:27:29,326 --> 00:27:30,401
that there is

1086
00:27:30,401 --> 00:27:32,531
a nuclease event that RPA is

1087
00:27:32,531 --> 00:27:34,738
inhibiting and so without R.

1088
00:27:34,738 --> 00:27:35,871
You get that degradation

1089
00:27:35,871 --> 00:27:36,674
to look at that.

1090
00:27:36,674 --> 00:27:38,108
We did the S1 dependent

1091
00:27:38,108 --> 00:27:40,149
degradation events of the single

1092
00:27:40,149 --> 00:27:40,978
strand gaps.

1093
00:27:41,645 --> 00:27:43,580
And what you see in untreated

1094
00:27:43,580 --> 00:27:44,581
MBA 436 cells?

1095
00:27:44,581 --> 00:27:45,697
It's just the Bracha 1

1096
00:27:45,697 --> 00:27:46,935
deficiency gives you the

1097
00:27:46,935 --> 00:27:48,585
generation of gaps during these

1098
00:27:48,585 --> 00:27:49,653
replication events.

1099
00:27:49,653 --> 00:27:50,297
We put an RPA

1100
00:27:50,297 --> 00:27:51,288
inhibitor in there.

1101
00:27:51,288 --> 00:27:52,940
It goes down a little bit of

1102
00:27:52,940 --> 00:27:54,864
just the RPA and again you still

1103
00:27:54,864 --> 00:27:56,126
get some degradation

1104
00:27:56,126 --> 00:27:57,494
but not as significant.

1105
00:27:57,961 --> 00:27:59,753
The PARP inhibitor increased in

1106
00:27:59,753 --> 00:28:01,354
replication for speed again

1107
00:28:01,354 --> 00:28:02,066
gives you a

1108
00:28:02,066 --> 00:28:03,784
massive amount of these single

1109
00:28:03,784 --> 00:28:05,600
strand gaps which is even lower

1110
00:28:05,600 --> 00:28:06,303
than these,

1111
00:28:06,303 --> 00:28:08,166
suggesting that those gap events

1112
00:28:08,166 --> 00:28:09,039
are occurring.

1113
00:28:09,039 --> 00:28:11,017
The real important result was

1114
00:28:11,017 --> 00:28:13,184
that the RPA inhibitors renders

1115
00:28:13,184 --> 00:28:13,744
the DNA

1116
00:28:13,744 --> 00:28:14,678
insensitive.

1117
00:28:14,678 --> 00:28:16,938
To S1 and So what that tells us

1118
00:28:16,938 --> 00:28:18,815
is that we've already had

1119
00:28:18,815 --> 00:28:19,416
events.

1120
00:28:20,818 --> 00:28:22,546
Resulting in the degradation of

1121
00:28:22,546 --> 00:28:24,368
this DNA by not having RPA there

1122
00:28:24,368 --> 00:28:25,222
to protect it.

1123
00:28:25,222 --> 00:28:27,422
So RPA protects these gaps from

1124
00:28:27,422 --> 00:28:29,023
degradation and we can

1125
00:28:29,023 --> 00:28:30,260
chemically block

1126
00:28:30,260 --> 00:28:31,362
that protection.

1127
00:28:31,962 --> 00:28:33,715
So then the real question is,

1128
00:28:33,715 --> 00:28:35,575
can we take these local events

1129
00:28:35,575 --> 00:28:36,133
that are

1130
00:28:36,133 --> 00:28:37,512
happening at individual

1131
00:28:37,512 --> 00:28:39,405
replication forks and can these

1132
00:28:39,405 --> 00:28:40,871
lead to global effects?

1133
00:28:42,606 --> 00:28:43,833
And what we did was measure

1134
00:28:43,833 --> 00:28:45,044
micronuclei and chromosome

1135
00:28:45,044 --> 00:28:45,743
pulverization.

1136
00:28:46,076 --> 00:28:47,406
As a measure of genomic

1137
00:28:47,406 --> 00:28:49,235
instability and got some really

1138
00:28:49,235 --> 00:28:50,414
interesting results

1139
00:28:50,414 --> 00:28:52,213
because if you can see and hear

1140
00:28:52,213 --> 00:28:53,083
these NBA 436,

1141
00:28:53,083 --> 00:28:54,929
you get these little micronuclei

1142
00:28:54,929 --> 00:28:56,706
indicated by the red dots in a

1143
00:28:56,706 --> 00:28:57,121
fairly

1144
00:28:57,121 --> 00:28:58,858
high frequency when you put in

1145
00:28:58,858 --> 00:29:00,761
olapy rib, which is a relatively

1146
00:29:00,761 --> 00:29:01,058
non.

1147
00:29:01,058 --> 00:29:02,931
Specific PARP inhibitor inhibits

1148
00:29:02,931 --> 00:29:04,161
part one and Part 2.

1149
00:29:04,161 --> 00:29:05,719
We get a significant increase

1150
00:29:05,719 --> 00:29:07,475
sarooparib, which is a much more

1151
00:29:07,475 --> 00:29:08,298
specific part.

1152
00:29:08,298 --> 00:29:09,656
One inhibitor gives us an

1153
00:29:09,656 --> 00:29:10,634
increase as well.

1154
00:29:11,402 --> 00:29:13,515
The neurix 329 compound doesn't

1155
00:29:13,515 --> 00:29:15,756
affect the the fraction of cells

1156
00:29:15,756 --> 00:29:16,106
with

1157
00:29:16,106 --> 00:29:17,741
micronuclei much at all.

1158
00:29:18,142 --> 00:29:19,946
Compared to the control and in

1159
00:29:19,946 --> 00:29:21,246
fact, when you do the

1160
00:29:21,246 --> 00:29:22,112
combinations,

1161
00:29:22,112 --> 00:29:23,971
there's no significant increase

1162
00:29:23,971 --> 00:29:25,572
by the addition of the 329

1163
00:29:25,572 --> 00:29:26,250
inhibitor.

1164
00:29:26,250 --> 00:29:27,920
So you have the same relative

1165
00:29:27,920 --> 00:29:29,688
number of the micronuclei that

1166
00:29:29,688 --> 00:29:30,454
are forming,

1167
00:29:30,454 --> 00:29:32,010
but we know when we add the

1168
00:29:32,010 --> 00:29:33,892
combination we get a significant

1169
00:29:33,892 --> 00:29:34,892
increase in cell

1170
00:29:34,892 --> 00:29:35,242
death.

1171
00:29:35,242 --> 00:29:36,746
And so as we measure that

1172
00:29:36,746 --> 00:29:37,227
through

1173
00:29:37,227 --> 00:29:38,662
chromosome pulverization.

1174
00:29:38,662 --> 00:29:40,217
So now the idea of massive

1175
00:29:40,217 --> 00:29:41,925
chromosome shattering events

1176
00:29:41,925 --> 00:29:43,267
that we can detect in

1177
00:29:43,267 --> 00:29:44,711
these metaphase spreads,

1178
00:29:44,711 --> 00:29:46,637
if we take a DMSO treated cell,

1179
00:29:46,637 --> 00:29:48,138
you see nice chromosomes.

1180
00:29:48,472 --> 00:29:50,808
329 RPA inhibitor doesn't change

1181
00:29:50,808 --> 00:29:51,975
it much at all.

1182
00:29:53,510 --> 00:29:54,644
Elap rib starts to give you a

1183
00:29:54,644 --> 00:29:55,766
certain degree of chromosome

1184
00:29:55,766 --> 00:29:56,246
shattering.

1185
00:29:56,246 --> 00:29:57,902
The percentage goes up from

1186
00:29:57,902 --> 00:29:59,656
three or four to 10, and the

1187
00:29:59,656 --> 00:30:00,784
degree is pretty.

1188
00:30:02,419 --> 00:30:02,986
Obvious,

1189
00:30:02,986 --> 00:30:05,163
but when the combo of the PARP

1190
00:30:05,163 --> 00:30:05,889
inhibitor

1191
00:30:05,889 --> 00:30:07,865
plus the 329 we get massive

1192
00:30:07,865 --> 00:30:10,060
chromosome shattering events.

1193
00:30:10,060 --> 00:30:11,922
And So what this suggests to us

1194
00:30:11,922 --> 00:30:13,827
is in fact there are activities

1195
00:30:13,827 --> 00:30:14,565
going on in

1196
00:30:14,565 --> 00:30:15,632
these micronuclei.

1197
00:30:15,632 --> 00:30:16,667
They themselves are not

1198
00:30:16,667 --> 00:30:17,701
necessarily giving us.

1199
00:30:18,001 --> 00:30:18,891
The cell death,

1200
00:30:18,891 --> 00:30:20,778
but the dysregulation of events

1201
00:30:20,778 --> 00:30:21,205
within

1202
00:30:21,205 --> 00:30:21,872
micronuclei.

1203
00:30:21,872 --> 00:30:23,789
Maybe it's repair replication,

1204
00:30:23,789 --> 00:30:25,886
RPA protection of those Dna's as

1205
00:30:25,886 --> 00:30:26,410
well as

1206
00:30:26,410 --> 00:30:28,386
nuclear can give us this larger

1207
00:30:28,386 --> 00:30:30,274
effect. And so we think these

1208
00:30:30,274 --> 00:30:31,381
local events can

1209
00:30:31,381 --> 00:30:33,247
really have dramatic impacts.

1210
00:30:33,247 --> 00:30:35,291
Local events at the replication

1211
00:30:35,291 --> 00:30:35,819
fork on

1212
00:30:35,819 --> 00:30:37,120
larger global things.

1213
00:30:38,655 --> 00:30:40,258
So this leads us to the model

1214
00:30:40,258 --> 00:30:41,510
for RPA dependent GAAP

1215
00:30:41,510 --> 00:30:42,192
protection.

1216
00:30:42,826 --> 00:30:45,097
I was at the DNA gap meeting

1217
00:30:45,097 --> 00:30:47,531
Keystone Meeting 2 weeks ago.

1218
00:30:47,798 --> 00:30:49,817
Go in in Seoul, South Korea.

1219
00:30:49,817 --> 00:30:51,963
And I like to say there that,

1220
00:30:51,963 --> 00:30:52,703
you know,

1221
00:30:52,703 --> 00:30:53,921
we're pretty much

1222
00:30:53,921 --> 00:30:55,138
strand agnostic.

1223
00:30:55,138 --> 00:30:56,785
And so whether you get a gap in

1224
00:30:56,785 --> 00:30:58,201
the lagging strand through

1225
00:30:58,201 --> 00:30:58,909
differential

1226
00:30:58,909 --> 00:31:00,528
Okazaki fragment processing or

1227
00:31:00,528 --> 00:31:01,445
leading strands,

1228
00:31:01,445 --> 00:31:02,935
say be running into a damage

1229
00:31:02,935 --> 00:31:04,618
event and then having a primpol

1230
00:31:04,618 --> 00:31:05,649
protein come in to

1231
00:31:05,649 --> 00:31:06,407
generate gaps,

1232
00:31:06,407 --> 00:31:08,085
it really doesn't matter to us

1233
00:31:08,085 --> 00:31:09,419
necessarily anything that

1234
00:31:09,419 --> 00:31:10,754
generates these gaps is.

1235
00:31:10,754 --> 00:31:12,108
Going to rely on a higher

1236
00:31:12,108 --> 00:31:13,835
concentration of RPA to protect

1237
00:31:13,835 --> 00:31:14,057
it,

1238
00:31:14,057 --> 00:31:15,192
and that gives you a

1239
00:31:15,192 --> 00:31:16,326
therapeutic window.

1240
00:31:16,326 --> 00:31:17,534
So in the classic model,

1241
00:31:17,534 --> 00:31:19,186
it's Bracha deficiency gives you

1242
00:31:19,186 --> 00:31:19,496
gaps.

1243
00:31:19,496 --> 00:31:20,693
You had a PARP inhibitor that

1244
00:31:20,693 --> 00:31:21,765
gives you even more gaps.

1245
00:31:21,765 --> 00:31:23,800
You're even more reliant on RPA.

1246
00:31:23,800 --> 00:31:25,695
We think without RPA protection

1247
00:31:25,695 --> 00:31:27,631
then you get degradation either

1248
00:31:27,631 --> 00:31:28,505
of the single

1249
00:31:28,505 --> 00:31:29,973
strands through the MRI,

1250
00:31:29,973 --> 00:31:31,924
11 tip nucleases or potentially

1251
00:31:31,924 --> 00:31:32,175
the

1252
00:31:32,175 --> 00:31:33,243
single strand DNA.

1253
00:31:33,243 --> 00:31:34,533
That's that template and

1254
00:31:34,533 --> 00:31:36,290
ultimately giving you DNA double

1255
00:31:36,290 --> 00:31:37,114
strand breaks.

1256
00:31:37,414 --> 00:31:38,979
Probably a lot of people in the

1257
00:31:38,979 --> 00:31:40,423
audience have contributed to

1258
00:31:40,423 --> 00:31:41,351
this entire table

1259
00:31:41,351 --> 00:31:42,819
that Matthew put together of.

1260
00:31:43,520 --> 00:31:44,696
The different mechanisms by

1261
00:31:44,696 --> 00:31:45,721
which single strand gap

1262
00:31:45,721 --> 00:31:46,657
induction can occur.

1263
00:31:47,124 --> 00:31:48,861
And so top on the list is spend

1264
00:31:48,861 --> 00:31:50,464
one, but April Beck and Paul

1265
00:31:50,464 --> 00:31:51,495
Theta and a whole

1266
00:31:51,495 --> 00:31:52,791
series of other things.

1267
00:31:52,791 --> 00:31:54,300
You go down the bottom and

1268
00:31:54,300 --> 00:31:54,765
there's

1269
00:31:54,765 --> 00:31:56,571
alcalaading agents and platinums

1270
00:31:56,571 --> 00:31:58,184
and any of these things that

1271
00:31:58,184 --> 00:31:59,336
give you DNA damage

1272
00:31:59,336 --> 00:32:01,171
events and a reliance on RPA to.

1273
00:32:01,171 --> 00:32:03,180
Single strand DNA we think is

1274
00:32:03,180 --> 00:32:05,371
worthwhile to try to treat with

1275
00:32:05,371 --> 00:32:06,643
an RPA inhibitor.

1276
00:32:07,244 --> 00:32:08,795
So that's where we stand on the

1277
00:32:08,795 --> 00:32:09,446
RPA project.

1278
00:32:10,414 --> 00:32:12,171
I'm going to switch gears in the

1279
00:32:12,171 --> 00:32:13,798
last half of the talk and and

1280
00:32:13,798 --> 00:32:14,751
jump to our coup

1281
00:32:14,751 --> 00:32:16,463
project where essentially we've

1282
00:32:16,463 --> 00:32:17,955
taken a very similar role.

1283
00:32:17,955 --> 00:32:19,990
We want to understand how the

1284
00:32:19,990 --> 00:32:22,285
pathways work and is it possible

1285
00:32:22,285 --> 00:32:23,360
to target this

1286
00:32:23,360 --> 00:32:24,995
protein DNA interaction?

1287
00:32:24,995 --> 00:32:26,516
So we're showing it here in,

1288
00:32:26,516 --> 00:32:27,331
in the Walker.

1289
00:32:29,132 --> 00:32:29,333
Model.

1290
00:32:29,700 --> 00:32:30,334
Published 20 some years ago.

1291
00:32:30,734 --> 00:32:32,967
Now of how KU interacts with a

1292
00:32:32,967 --> 00:32:35,324
double strand DNA and literally

1293
00:32:35,324 --> 00:32:36,540
the DNA threads

1294
00:32:36,540 --> 00:32:38,937
through this molecule to bind to

1295
00:32:38,937 --> 00:32:40,210
that end of DNA.

1296
00:32:40,210 --> 00:32:42,004
The question we're asking here

1297
00:32:42,004 --> 00:32:43,898
is can we target these specific

1298
00:32:43,898 --> 00:32:44,815
events in lung

1299
00:32:44,815 --> 00:32:46,547
cancer therapy? And so again,

1300
00:32:46,547 --> 00:32:48,518
just to throw out a few numbers,

1301
00:32:48,518 --> 00:32:50,312
there's going to be about 225,

1302
00:32:50,312 --> 00:32:52,210
000 people in the US alone that

1303
00:32:52,210 --> 00:32:52,823
are going

1304
00:32:52,823 --> 00:32:54,191
to be diagnosed with L.

1305
00:32:54,191 --> 00:32:55,021
Cancer this year,

1306
00:32:55,021 --> 00:32:56,576
160 thousand of those are going

1307
00:32:56,576 --> 00:32:56,927
to get

1308
00:32:56,927 --> 00:32:58,395
radiation treatment as part of

1309
00:32:58,395 --> 00:32:59,129
their therapy.

1310
00:32:59,329 --> 00:32:59,602
135,

1311
00:32:59,602 --> 00:33:01,422
000 and get platinum based

1312
00:33:01,422 --> 00:33:02,332
chemotherapy

1313
00:33:02,332 --> 00:33:04,230
and if you put them together

1314
00:33:04,230 --> 00:33:06,040
about 60 will get combined

1315
00:33:06,040 --> 00:33:06,737
platinum.

1316
00:33:06,737 --> 00:33:06,803
IR.

1317
00:33:07,604 --> 00:33:09,461
That combined chemoradiation

1318
00:33:09,461 --> 00:33:11,625
therapy is about the same number

1319
00:33:11,625 --> 00:33:12,843
that are eligible

1320
00:33:12,843 --> 00:33:14,911
for IO therapy based on the 30%

1321
00:33:14,911 --> 00:33:17,029
of patients that can be treated

1322
00:33:17,029 --> 00:33:17,781
with that.

1323
00:33:18,081 --> 00:33:20,152
So the reality in this treatment

1324
00:33:20,152 --> 00:33:22,000
scheme of patients with lung

1325
00:33:22,000 --> 00:33:23,387
cancer is about 200,

1326
00:33:23,387 --> 00:33:24,785
000 patients are going

1327
00:33:24,785 --> 00:33:26,056
to be treated with.

1328
00:33:26,823 --> 00:33:27,532
Therapeutics that

1329
00:33:27,532 --> 00:33:28,325
induce DNA damage.

1330
00:33:28,992 --> 00:33:30,750
Whether it be IR or platinum,

1331
00:33:30,750 --> 00:33:32,489
you're getting adna damaging

1332
00:33:32,489 --> 00:33:33,296
therapeutic.

1333
00:33:33,296 --> 00:33:35,106
And so it becomes important to

1334
00:33:35,106 --> 00:33:37,011
understand how these work and a

1335
00:33:37,011 --> 00:33:38,301
lot of this was done

1336
00:33:38,301 --> 00:33:39,803
in collaboration with our

1337
00:33:39,803 --> 00:33:41,764
clinical colleagues. Shadi Jalal

1338
00:33:41,764 --> 00:33:42,806
and Katie Sears,

1339
00:33:42,806 --> 00:33:44,317
thoracic oncologist and a

1340
00:33:44,317 --> 00:33:46,173
pulmonologist to see and treat

1341
00:33:46,173 --> 00:33:46,977
lung cancer.

1342
00:33:46,977 --> 00:33:48,746
All the time they actually make

1343
00:33:48,746 --> 00:33:50,614
fun of this picture because they

1344
00:33:50,614 --> 00:33:51,314
say none of

1345
00:33:51,314 --> 00:33:52,983
their patients that have lung

1346
00:33:52,983 --> 00:33:54,651
cancer show up. As you know,

1347
00:33:54,651 --> 00:33:56,441
25 year old muscular individual

1348
00:33:56,441 --> 00:33:57,654
with massive tumors.

1349
00:33:58,522 --> 00:34:00,036
Understanding how these double

1350
00:34:00,036 --> 00:34:01,273
strand breaks induced by

1351
00:34:01,273 --> 00:34:02,459
ionizing radiation are

1352
00:34:02,459 --> 00:34:02,966
important,

1353
00:34:02,966 --> 00:34:04,631
and clearly we're going to focus

1354
00:34:04,631 --> 00:34:04,995
on the

1355
00:34:04,995 --> 00:34:05,887
non homologous end

1356
00:34:05,887 --> 00:34:06,730
joining pathway.

1357
00:34:06,730 --> 00:34:08,140
But I put this up and just

1358
00:34:08,140 --> 00:34:09,766
Kersey, the graduate student,

1359
00:34:09,766 --> 00:34:11,481
made this slide for us to really

1360
00:34:11,481 --> 00:34:13,020
highlight the advances we're

1361
00:34:13,020 --> 00:34:13,570
seeing in

1362
00:34:13,570 --> 00:34:14,865
understanding homologous

1363
00:34:14,865 --> 00:34:16,623
recombination, great advances in

1364
00:34:16,623 --> 00:34:17,941
micro homology mediated

1365
00:34:17,941 --> 00:34:19,209
or feta mediated repair.

1366
00:34:19,209 --> 00:34:20,560
These alternate end joining

1367
00:34:20,560 --> 00:34:21,846
pathways in single strand

1368
00:34:21,846 --> 00:34:22,412
annealing.

1369
00:34:23,213 --> 00:34:24,381
There's been really a lot of

1370
00:34:24,381 --> 00:34:25,359
activity and we're very

1371
00:34:25,359 --> 00:34:26,550
interested in understanding

1372
00:34:26,550 --> 00:34:27,617
how all of these proteins.

1373
00:34:28,118 --> 00:34:29,119
Play a role in that.

1374
00:34:29,119 --> 00:34:30,158
Prepare as well,

1375
00:34:30,158 --> 00:34:32,223
but if we're targeting the coup

1376
00:34:32,223 --> 00:34:32,756
protein

1377
00:34:32,756 --> 00:34:34,674
that focuses on non homologous

1378
00:34:34,674 --> 00:34:36,640
end joining pretty closely and

1379
00:34:36,640 --> 00:34:37,360
it got the

1380
00:34:37,360 --> 00:34:39,425
idea of if cou is binding to DNA

1381
00:34:39,425 --> 00:34:41,538
ends, you can absolutely get one

1382
00:34:41,538 --> 00:34:42,199
through a

1383
00:34:42,199 --> 00:34:43,878
ionizing radiation induced

1384
00:34:43,878 --> 00:34:45,235
double strand break.

1385
00:34:45,235 --> 00:34:46,808
But there are a lot of other

1386
00:34:46,808 --> 00:34:48,412
ways to get things that look

1387
00:34:48,412 --> 00:34:49,673
like ends of DNA that

1388
00:34:49,673 --> 00:34:50,688
who could bind to.

1389
00:34:50,688 --> 00:34:52,486
And so one of them is if you're

1390
00:34:52,486 --> 00:34:52,776
on a

1391
00:34:52,776 --> 00:34:53,554
leading strand

1392
00:34:53,554 --> 00:34:54,778
replication, you hit.

1393
00:34:55,445 --> 00:34:55,512
A.

1394
00:34:55,512 --> 00:34:56,567
Some type of damage,

1395
00:34:56,567 --> 00:34:57,781
say a platinum lesion.

1396
00:34:57,781 --> 00:34:58,948
And then you get fork,

1397
00:34:58,948 --> 00:35:00,582
regression and creation of the

1398
00:35:00,582 --> 00:35:01,017
reverse

1399
00:35:01,017 --> 00:35:02,032
chicken foot model.

1400
00:35:02,032 --> 00:35:03,569
This thing here looks like a

1401
00:35:03,569 --> 00:35:03,954
double

1402
00:35:03,954 --> 00:35:04,321
strand.

1403
00:35:04,321 --> 00:35:05,088
End that coupon.

1404
00:35:05,088 --> 00:35:06,718
Bind to if you're replicating in

1405
00:35:06,718 --> 00:35:08,125
the leading strand here and

1406
00:35:08,125 --> 00:35:08,959
going through a

1407
00:35:08,959 --> 00:35:09,212
nick,

1408
00:35:09,212 --> 00:35:10,875
a single break in the phosphorus

1409
00:35:10,875 --> 00:35:11,394
backbone.

1410
00:35:11,394 --> 00:35:13,178
This creates A1 ended or single

1411
00:35:13,178 --> 00:35:14,731
sided double strand break.

1412
00:35:14,731 --> 00:35:16,339
This end essentially creates

1413
00:35:16,339 --> 00:35:18,235
something that KU could bind to,

1414
00:35:18,235 --> 00:35:19,777
and if you consider lagging

1415
00:35:19,777 --> 00:35:21,466
strand gaps in degradation of

1416
00:35:21,466 --> 00:35:22,806
those to create double

1417
00:35:22,806 --> 00:35:23,612
strand breaks.

1418
00:35:23,612 --> 00:35:25,328
Again this thing looks like a

1419
00:35:25,328 --> 00:35:25,742
single

1420
00:35:25,742 --> 00:35:26,176
strand.

1421
00:35:26,176 --> 00:35:27,577
I mean a double strand end.

1422
00:35:27,577 --> 00:35:29,164
That the coup combined to and

1423
00:35:29,164 --> 00:35:30,954
any of these can be processed by

1424
00:35:30,954 --> 00:35:31,681
nucleases to

1425
00:35:31,681 --> 00:35:33,450
generate single strand overhangs

1426
00:35:33,450 --> 00:35:35,044
that then RPA may bind to as

1427
00:35:35,044 --> 00:35:35,385
well.

1428
00:35:35,385 --> 00:35:36,863
And so this idea of keeping

1429
00:35:36,863 --> 00:35:38,595
everything separate from double

1430
00:35:38,595 --> 00:35:39,489
stranded single

1431
00:35:39,489 --> 00:35:40,570
strand binding might

1432
00:35:40,570 --> 00:35:41,758
not actually be true.

1433
00:35:42,559 --> 00:35:43,855
And so while all that's

1434
00:35:43,855 --> 00:35:45,408
interesting, we're going to

1435
00:35:45,408 --> 00:35:46,730
focus on this ionizing

1436
00:35:46,730 --> 00:35:47,291
radiation,

1437
00:35:47,291 --> 00:35:48,847
double strand break coup is

1438
00:35:48,847 --> 00:35:49,366
going to

1439
00:35:49,366 --> 00:35:51,174
slide on to that end and recruit

1440
00:35:51,174 --> 00:35:52,869
the DNA PK catalytic subunit,

1441
00:35:52,869 --> 00:35:54,195
which then catalyzes an

1442
00:35:54,195 --> 00:35:56,039
important auto phosphorylation.

1443
00:35:56,039 --> 00:35:57,552
An event that leads to its

1444
00:35:57,552 --> 00:35:59,391
dissociation to where the other

1445
00:35:59,391 --> 00:36:00,577
proteins like East,

1446
00:36:00,577 --> 00:36:02,258
4X or CC-4 xlf can come in to

1447
00:36:02,258 --> 00:36:04,114
ultimately rejoin that DNA end.

1448
00:36:04,114 --> 00:36:05,959
But this auto phosphorylation is

1449
00:36:05,959 --> 00:36:07,855
something we're going to focus a

1450
00:36:07,855 --> 00:36:08,151
lot.

1451
00:36:09,019 --> 00:36:10,058
And so the question is,

1452
00:36:10,058 --> 00:36:11,539
can we make small molecules that

1453
00:36:11,539 --> 00:36:11,955
do this?

1454
00:36:11,955 --> 00:36:13,223
The answer is yes.

1455
00:36:13,557 --> 00:36:15,458
Again, our chemist.

1456
00:36:15,458 --> 00:36:17,235
We identified an incredibly

1457
00:36:17,235 --> 00:36:19,390
horrible compound, X-80 with low

1458
00:36:19,390 --> 00:36:20,063
affinity,

1459
00:36:20,063 --> 00:36:21,898
low potency, low solubility.

1460
00:36:22,699 --> 00:36:23,886
And so now we made

1461
00:36:23,886 --> 00:36:25,402
about 400 derivatives.

1462
00:36:25,969 --> 00:36:27,466
Largely modifying this

1463
00:36:27,466 --> 00:36:29,685
carboxylic acid here to create a

1464
00:36:29,685 --> 00:36:31,141
compound 68 and 1:49

1465
00:36:31,141 --> 00:36:33,263
that in a very similar assay we

1466
00:36:33,263 --> 00:36:35,431
can show blocks the protein DNA

1467
00:36:35,431 --> 00:36:36,479
interaction of

1468
00:36:36,479 --> 00:36:38,657
coup with a double strand DNA in

1469
00:36:38,657 --> 00:36:40,885
a test tube and that necessarily

1470
00:36:40,885 --> 00:36:41,651
blocks the

1471
00:36:41,651 --> 00:36:43,153
kinase activity so in.

1472
00:36:43,153 --> 00:36:44,974
An in vitro kinase activity we

1473
00:36:44,974 --> 00:36:46,342
can show a very potent

1474
00:36:46,342 --> 00:36:47,524
inhibition of that

1475
00:36:47,524 --> 00:36:49,425
phosphorylation event catalyzed

1476
00:36:49,425 --> 00:36:51,190
by the DNA dependent protein

1477
00:36:51,190 --> 00:36:51,695
kinase.

1478
00:36:52,429 --> 00:36:53,893
So we showed that these

1479
00:36:53,893 --> 00:36:55,866
compounds bind to the protein.

1480
00:36:56,299 --> 00:36:57,234
And not to the DNA.

1481
00:36:57,234 --> 00:36:58,464
We're not looking for DNA

1482
00:36:58,464 --> 00:36:59,782
interaction agents at this

1483
00:36:59,782 --> 00:37:00,136
point.

1484
00:37:00,136 --> 00:37:02,268
And so another derivative 245

1485
00:37:02,268 --> 00:37:04,523
which contains a tetrazol here

1486
00:37:04,523 --> 00:37:05,575
and replacing

1487
00:37:05,575 --> 00:37:07,077
this carboxylic acid.

1488
00:37:08,578 --> 00:37:10,334
Shows very nice increase in

1489
00:37:10,334 --> 00:37:12,457
stability, the thermal stability

1490
00:37:12,457 --> 00:37:13,717
of that protein as

1491
00:37:13,717 --> 00:37:15,827
a function of treating with this

1492
00:37:15,827 --> 00:37:16,553
this drug.

1493
00:37:16,553 --> 00:37:17,921
So the compound binds to the

1494
00:37:17,921 --> 00:37:19,372
protein and makes the protein

1495
00:37:19,372 --> 00:37:20,023
more stable,

1496
00:37:20,023 --> 00:37:21,433
so that when you increase the

1497
00:37:21,433 --> 00:37:22,622
temperature you get less

1498
00:37:22,622 --> 00:37:23,960
denaturation and you get a

1499
00:37:23,960 --> 00:37:25,491
thermal stability shift showing

1500
00:37:25,491 --> 00:37:26,429
that the compound.

1501
00:37:26,763 --> 00:37:28,894
Absolutely interacts with the

1502
00:37:28,894 --> 00:37:31,294
protein. This has been confirmed

1503
00:37:31,294 --> 00:37:32,569
using isothermal

1504
00:37:32,569 --> 00:37:33,734
calorimetry from

1505
00:37:33,734 --> 00:37:34,971
some colleagues.

1506
00:37:34,971 --> 00:37:36,030
Charbonnet in Paris,

1507
00:37:36,030 --> 00:37:37,769
who has done this on some of our

1508
00:37:37,769 --> 00:37:38,041
more

1509
00:37:38,041 --> 00:37:38,975
recent inhibitors.

1510
00:37:39,809 --> 00:37:41,445
Let's add a copy at University

1511
00:37:41,445 --> 00:37:43,233
of Illinois. Chicago showed that

1512
00:37:43,233 --> 00:37:43,680
we can,

1513
00:37:43,680 --> 00:37:44,996
in a reconstituted assay

1514
00:37:44,996 --> 00:37:46,564
measuring non homologous end

1515
00:37:46,564 --> 00:37:47,684
joining so purified

1516
00:37:47,684 --> 00:37:49,172
proteins in a test tube can

1517
00:37:49,172 --> 00:37:50,220
block the joining.

1518
00:37:50,220 --> 00:37:51,865
That results in these multimer

1519
00:37:51,865 --> 00:37:53,382
formations with increase in

1520
00:37:53,382 --> 00:37:54,224
concentration.

1521
00:37:54,557 --> 00:37:56,426
Block not joining and in a

1522
00:37:56,426 --> 00:37:58,771
reconstituted system in vitro we

1523
00:37:58,771 --> 00:38:00,163
can block the auto

1524
00:38:00,163 --> 00:38:02,463
phosphorylation of DNA dependent

1525
00:38:02,463 --> 00:38:04,373
protein kinase and this is

1526
00:38:04,373 --> 00:38:05,769
measured through a

1527
00:38:05,769 --> 00:38:07,938
phospho specific antibody made

1528
00:38:07,938 --> 00:38:10,226
by Susan Lees Miller's group at

1529
00:38:10,226 --> 00:38:11,408
the Serine 2056

1530
00:38:11,408 --> 00:38:12,328
and so as the

1531
00:38:12,328 --> 00:38:13,743
experiment goes in.

1532
00:38:13,743 --> 00:38:15,165
The absence of any DNA you get

1533
00:38:15,165 --> 00:38:16,112
no phosphorylation.

1534
00:38:16,112 --> 00:38:17,506
We put in our positive control,

1535
00:38:17,506 --> 00:38:18,934
which is just adding DNA to the

1536
00:38:18,934 --> 00:38:19,349
reaction

1537
00:38:19,349 --> 00:38:20,328
and the phosphorylates

1538
00:38:20,328 --> 00:38:21,733
wonderfully. We can put any one

1539
00:38:21,733 --> 00:38:22,686
of our inhibitors in

1540
00:38:22,686 --> 00:38:23,537
and they knock them

1541
00:38:23,537 --> 00:38:24,254
out completely.

1542
00:38:25,055 --> 00:38:27,372
Nu 7441 is in fact the catalytic

1543
00:38:27,372 --> 00:38:29,664
subunit inhibitor of DNA PK and

1544
00:38:29,664 --> 00:38:30,994
that blocks it as

1545
00:38:30,994 --> 00:38:31,357
well.

1546
00:38:31,357 --> 00:38:33,587
And just as a negative control

1547
00:38:33,587 --> 00:38:34,331
or neorex

1548
00:38:34,331 --> 00:38:34,622
329,

1549
00:38:34,622 --> 00:38:36,943
the RPA inhibitor has no effect

1550
00:38:36,943 --> 00:38:37,467
on the

1551
00:38:37,467 --> 00:38:39,214
phosphorylation events.

1552
00:38:39,214 --> 00:38:41,324
And so we can block the DNA

1553
00:38:41,324 --> 00:38:41,871
inter.

1554
00:38:41,871 --> 00:38:42,272
We can.

1555
00:38:42,272 --> 00:38:43,682
We know it's binding to the coup

1556
00:38:43,682 --> 00:38:44,607
protein and not DNA.

1557
00:38:44,841 --> 00:38:46,525
We can block nej and and

1558
00:38:46,525 --> 00:38:48,741
reconstitute a damage dependent

1559
00:38:48,741 --> 00:38:50,313
auto phosphorylation.

1560
00:38:50,313 --> 00:38:51,281
Perfectly reasonable.

1561
00:38:51,281 --> 00:38:52,782
And can we do this in cells?

1562
00:38:53,483 --> 00:38:53,750
And.

1563
00:38:54,017 --> 00:38:55,172
The answer is yes.

1564
00:38:55,172 --> 00:38:56,957
In a host cell reactivation

1565
00:38:56,957 --> 00:38:57,354
assay

1566
00:38:57,354 --> 00:38:59,143
measuring non homologous end

1567
00:38:59,143 --> 00:39:01,041
joining. So just transferring

1568
00:39:01,041 --> 00:39:01,891
into cells a

1569
00:39:01,891 --> 00:39:03,308
linearized plasma DNA.

1570
00:39:03,308 --> 00:39:05,429
Then when Circularized gives you

1571
00:39:05,429 --> 00:39:05,562
a

1572
00:39:05,562 --> 00:39:06,776
fluorescent signal,

1573
00:39:06,776 --> 00:39:08,742
we can get good nej efficiency

1574
00:39:08,742 --> 00:39:09,332
and each

1575
00:39:09,332 --> 00:39:11,394
of our compounds with concurrent

1576
00:39:11,394 --> 00:39:13,069
treatment gives us an in.

1577
00:39:13,303 --> 00:39:15,168
Of that rejoining event, again,

1578
00:39:15,168 --> 00:39:16,710
probably my most favorite

1579
00:39:16,710 --> 00:39:17,574
experiment is

1580
00:39:17,574 --> 00:39:18,125
this one,

1581
00:39:18,125 --> 00:39:19,822
where we can demonstrate on

1582
00:39:19,822 --> 00:39:20,577
target coup

1583
00:39:20,577 --> 00:39:22,531
inhibition and sensitization to

1584
00:39:22,531 --> 00:39:23,980
a double strand break.

1585
00:39:23,980 --> 00:39:24,881
Conducing agents.

1586
00:39:24,881 --> 00:39:26,635
So now bleomycin is used as a

1587
00:39:26,635 --> 00:39:27,784
RATHI, or mimetic,

1588
00:39:27,784 --> 00:39:29,401
to induce DNA double strand

1589
00:39:29,401 --> 00:39:31,237
breaks, and in these wild type

1590
00:39:31,237 --> 00:39:32,155
mifs you get a

1591
00:39:32,155 --> 00:39:33,774
pretty decent dose response

1592
00:39:33,774 --> 00:39:35,617
curve. When we put in our coup

1593
00:39:35,617 --> 00:39:36,292
inhibitor,

1594
00:39:36,292 --> 00:39:38,143
we get increased sensitivity to

1595
00:39:38,143 --> 00:39:39,612
that double strand break

1596
00:39:39,612 --> 00:39:40,530
inducing agent

1597
00:39:40,530 --> 00:39:41,498
consistent with.

1598
00:39:41,498 --> 00:39:42,248
Blocking the true

1599
00:39:42,248 --> 00:39:42,999
DNA interaction.

1600
00:39:42,999 --> 00:39:44,117
Blocking DNA PK auto

1601
00:39:44,117 --> 00:39:45,767
phosphorylation, blocking non

1602
00:39:45,767 --> 00:39:47,303
homologous end joining and

1603
00:39:47,303 --> 00:39:48,709
you get a higher level of

1604
00:39:48,709 --> 00:39:50,489
persistent double strand breaks

1605
00:39:50,489 --> 00:39:51,408
that kill those

1606
00:39:51,408 --> 00:39:51,741
cells.

1607
00:39:52,409 --> 00:39:54,387
We do this in a knockout KU 80

1608
00:39:54,387 --> 00:39:55,245
null method.

1609
00:39:55,245 --> 00:39:56,861
So now KU null cell line,

1610
00:39:56,861 --> 00:39:58,851
they're already hypersensitive

1611
00:39:58,851 --> 00:39:59,382
because

1612
00:39:59,382 --> 00:40:01,243
they don't have non homolexen

1613
00:40:01,243 --> 00:40:03,208
joining anymore and we add our

1614
00:40:03,208 --> 00:40:04,387
compound to these

1615
00:40:04,387 --> 00:40:06,252
kunosols and we don't see any

1616
00:40:06,252 --> 00:40:07,759
statistical increase in

1617
00:40:07,759 --> 00:40:09,659
sensitivity as a function of

1618
00:40:09,659 --> 00:40:11,652
inhibiting coup because there's

1619
00:40:11,652 --> 00:40:13,634
no coup in those cells. And so

1620
00:40:13,634 --> 00:40:14,030
this.

1621
00:40:14,030 --> 00:40:15,258
Really tells us that we're

1622
00:40:15,258 --> 00:40:16,463
getting on target coup in

1623
00:40:16,463 --> 00:40:17,667
activation in these wild

1624
00:40:17,667 --> 00:40:18,851
type cells and that's the

1625
00:40:18,851 --> 00:40:20,011
mechanism by which we're

1626
00:40:20,011 --> 00:40:21,171
increasing sensitivity.

1627
00:40:22,372 --> 00:40:24,320
And so Tammy Mendoza here has

1628
00:40:24,320 --> 00:40:26,379
done all this work now so very

1629
00:40:26,379 --> 00:40:27,477
nicely that the

1630
00:40:27,477 --> 00:40:29,491
2:45 I mentioned that tetrazol

1631
00:40:29,491 --> 00:40:31,406
derivative, she could put it

1632
00:40:31,406 --> 00:40:33,116
into cells and doing the

1633
00:40:33,116 --> 00:40:34,339
live cell imaging,

1634
00:40:34,339 --> 00:40:36,224
we get no effect of the 245

1635
00:40:36,224 --> 00:40:36,853
compound

1636
00:40:36,853 --> 00:40:38,655
alone, bleomycin starts to.

1637
00:40:38,655 --> 00:40:39,844
Kill cells on its own,

1638
00:40:39,844 --> 00:40:41,513
and the combination is in fact

1639
00:40:41,513 --> 00:40:41,791
much

1640
00:40:41,791 --> 00:40:42,192
better.

1641
00:40:42,192 --> 00:40:43,732
That works out good for us and

1642
00:40:43,732 --> 00:40:45,211
she showed very convincingly

1643
00:40:45,211 --> 00:40:45,528
that.

1644
00:40:46,329 --> 00:40:47,826
This effect is a function of

1645
00:40:47,826 --> 00:40:49,242
blocking the DNA dependent

1646
00:40:49,242 --> 00:40:50,767
protein kinase by measuring

1647
00:40:50,767 --> 00:40:52,001
the autophosphorylation.

1648
00:40:52,368 --> 00:40:53,209
And now, in an

1649
00:40:53,209 --> 00:40:54,771
immunofluorescence assay.

1650
00:40:54,771 --> 00:40:56,309
And so you get little punctate

1651
00:40:56,309 --> 00:40:57,564
foci when you treat with

1652
00:40:57,564 --> 00:40:58,975
bleomycin you get that DNA

1653
00:40:58,975 --> 00:41:00,610
damage and double strand breaks.

1654
00:41:02,145 --> 00:41:03,969
Jump the gun we put in our 245

1655
00:41:03,969 --> 00:41:05,898
inhibitor and we can completely

1656
00:41:05,898 --> 00:41:06,583
block that

1657
00:41:06,583 --> 00:41:07,851
quantified over here.

1658
00:41:07,851 --> 00:41:09,154
You put in that catalytic

1659
00:41:09,154 --> 00:41:10,866
inhibitor, you can block that as

1660
00:41:10,866 --> 00:41:11,187
well.

1661
00:41:11,187 --> 00:41:13,223
Exactly what you'd expect in

1662
00:41:13,223 --> 00:41:15,462
collaboration with Joy Garrett

1663
00:41:15,462 --> 00:41:16,059
and Joe

1664
00:41:16,059 --> 00:41:17,725
Dinlock did an ionizing

1665
00:41:17,725 --> 00:41:19,946
radiation experiment where she

1666
00:41:19,946 --> 00:41:21,131
showed the same

1667
00:41:21,131 --> 00:41:21,564
event.

1668
00:41:22,031 --> 00:41:25,034
And whoops that one.

1669
00:41:25,335 --> 00:41:27,165
And so I just kind of blow up

1670
00:41:27,165 --> 00:41:28,973
the radiation induced DNA PK

1671
00:41:28,973 --> 00:41:30,006
foci that she's

1672
00:41:30,006 --> 00:41:30,967
able to detect.

1673
00:41:30,967 --> 00:41:32,484
And then when we do the

1674
00:41:32,484 --> 00:41:33,209
concurrent

1675
00:41:33,209 --> 00:41:35,164
treatment of radiation plus our

1676
00:41:35,164 --> 00:41:36,966
inhibitor, we get a decrease

1677
00:41:36,966 --> 00:41:38,381
both in the number of

1678
00:41:38,381 --> 00:41:39,762
foci and the intensity

1679
00:41:39,762 --> 00:41:41,017
of the foci and so.

1680
00:41:41,017 --> 00:41:42,785
This was really nice because now

1681
00:41:42,785 --> 00:41:44,554
we have on target inactivation,

1682
00:41:44,554 --> 00:41:45,981
blocking the pathways that

1683
00:41:45,981 --> 00:41:47,605
increase sensitivity to these

1684
00:41:47,605 --> 00:41:48,725
double strand break

1685
00:41:48,725 --> 00:41:49,592
inducing agents.

1686
00:41:50,026 --> 00:41:52,376
The reality was those compounds

1687
00:41:52,376 --> 00:41:54,865
are very, very difficult to work

1688
00:41:54,865 --> 00:41:55,331
with.

1689
00:41:55,331 --> 00:41:56,389
The solubility

1690
00:41:56,389 --> 00:41:57,901
still wasn't great.

1691
00:41:57,901 --> 00:41:59,540
Cellular uptake was incredibly

1692
00:41:59,540 --> 00:42:00,934
poor and in fact required

1693
00:42:00,934 --> 00:42:02,272
treatment in serum free

1694
00:42:02,272 --> 00:42:02,604
media.

1695
00:42:02,604 --> 00:42:04,193
So every experiment involved

1696
00:42:04,193 --> 00:42:04,874
plating the

1697
00:42:04,874 --> 00:42:06,247
cells, letting them grow,

1698
00:42:06,247 --> 00:42:07,510
washing off the serum,

1699
00:42:07,510 --> 00:42:09,044
then treating with our drug,

1700
00:42:09,044 --> 00:42:10,731
then putting back what we need

1701
00:42:10,731 --> 00:42:11,181
to keep

1702
00:42:11,181 --> 00:42:12,048
the cells alive.

1703
00:42:12,315 --> 00:42:14,703
And so Navi went back to the lab

1704
00:42:14,703 --> 00:42:17,147
again and made a whole series of

1705
00:42:17,147 --> 00:42:17,987
compounds,

1706
00:42:17,987 --> 00:42:20,378
replacing that methylparole with

1707
00:42:20,378 --> 00:42:21,424
an oxen doll.

1708
00:42:21,424 --> 00:42:23,365
And this relatively modest

1709
00:42:23,365 --> 00:42:25,573
modification of this oxendole

1710
00:42:25,573 --> 00:42:27,096
really dramatically

1711
00:42:27,096 --> 00:42:29,129
increased our capacity in a

1712
00:42:29,129 --> 00:42:30,333
number of ways.

1713
00:42:30,333 --> 00:42:30,548
One,

1714
00:42:30,548 --> 00:42:32,252
it docks very nicely using auto

1715
00:42:32,252 --> 00:42:32,802
dog Vena,

1716
00:42:32,802 --> 00:42:34,457
so I'll model and not a crystal

1717
00:42:34,457 --> 00:42:35,738
structure by any means.

1718
00:42:35,738 --> 00:42:37,340
Although we are pursuing that.

1719
00:42:38,107 --> 00:42:40,024
Right in the active site that

1720
00:42:40,024 --> 00:42:41,544
protects that the DNA.

1721
00:42:42,011 --> 00:42:43,179
Has to slide through.

1722
00:42:43,179 --> 00:42:44,721
You can see these two little

1723
00:42:44,721 --> 00:42:45,956
protrubraces here that

1724
00:42:45,956 --> 00:42:47,584
essentially the DNA winds in

1725
00:42:47,584 --> 00:42:48,618
and this blocks it.

1726
00:42:48,618 --> 00:42:50,419
So we block that protein DNA

1727
00:42:50,419 --> 00:42:52,155
interaction, we can block.

1728
00:42:53,656 --> 00:42:54,657
Kinase activity as well.

1729
00:42:54,657 --> 00:42:55,536
Not shown here,

1730
00:42:55,536 --> 00:42:57,285
but the most important result

1731
00:42:57,285 --> 00:42:57,527
was

1732
00:42:57,527 --> 00:42:59,166
cellular uptake in these non

1733
00:42:59,166 --> 00:43:00,630
small cell lung cancers.

1734
00:43:00,630 --> 00:43:02,026
We're getting about 1500

1735
00:43:02,026 --> 00:43:03,631
picamoles per million cells

1736
00:43:03,631 --> 00:43:04,701
across the board,

1737
00:43:04,701 --> 00:43:06,369
even the bad ones are at 500.

1738
00:43:06,603 --> 00:43:08,073
I will tell you the predecessor

1739
00:43:08,073 --> 00:43:09,476
compounds the numbers were in

1740
00:43:09,476 --> 00:43:10,540
the 10 and 20 peak of

1741
00:43:10,540 --> 00:43:10,773
mole.

1742
00:43:10,773 --> 00:43:11,330
So we get a

1743
00:43:11,330 --> 00:43:12,442
significant increase.

1744
00:43:12,775 --> 00:43:13,977
In cellular uptake.

1745
00:43:15,511 --> 00:43:17,132
And Pammy measured in the

1746
00:43:17,132 --> 00:43:18,881
clonagenic survival assay.

1747
00:43:18,881 --> 00:43:20,906
Now either without radiation or

1748
00:43:20,906 --> 00:43:21,951
with radiation,

1749
00:43:21,951 --> 00:43:24,042
the addition of our compound can

1750
00:43:24,042 --> 00:43:25,718
dramatically increase the

1751
00:43:25,718 --> 00:43:26,589
sensitivity.

1752
00:43:26,589 --> 00:43:28,665
So with greater cellular uptake,

1753
00:43:28,665 --> 00:43:30,395
we jumped right into mouse

1754
00:43:30,395 --> 00:43:31,261
experiments.

1755
00:43:31,494 --> 00:43:32,063
In vivo,

1756
00:43:32,063 --> 00:43:34,107
we put the A 549 lung cancer

1757
00:43:34,107 --> 00:43:34,764
cells in

1758
00:43:34,764 --> 00:43:36,600
there, put them into mice,

1759
00:43:36,600 --> 00:43:38,638
treated them with either the

1760
00:43:38,638 --> 00:43:39,002
coup

1761
00:43:39,002 --> 00:43:40,556
inhibitor or IR or the

1762
00:43:40,556 --> 00:43:42,605
combination wait four hours.

1763
00:43:43,172 --> 00:43:44,806
Hit em with ionizing radiation

1764
00:43:44,806 --> 00:43:46,141
and then two hours later

1765
00:43:46,141 --> 00:43:47,310
sacrificed the mice.

1766
00:43:47,310 --> 00:43:48,739
We did direct intratumoral

1767
00:43:48,739 --> 00:43:50,483
injection, so we didn't want to

1768
00:43:50,483 --> 00:43:51,214
wait for PK.

1769
00:43:51,814 --> 00:43:53,403
And then wanted to see if we

1770
00:43:53,403 --> 00:43:55,080
could get this result and she

1771
00:43:55,080 --> 00:43:56,352
showed very nicely in

1772
00:43:56,352 --> 00:43:58,058
an DNA PK auto phosphorylation

1773
00:43:58,058 --> 00:43:59,865
assay measured by Western blood

1774
00:43:59,865 --> 00:44:00,390
from the

1775
00:44:00,390 --> 00:44:01,360
extracted tumors.

1776
00:44:01,360 --> 00:44:02,771
After this two hour post

1777
00:44:02,771 --> 00:44:03,359
radiation

1778
00:44:03,359 --> 00:44:04,829
treatment that the vehicle

1779
00:44:04,829 --> 00:44:06,563
treatment, there's none as you

1780
00:44:06,563 --> 00:44:07,430
might imagine.

1781
00:44:08,498 --> 00:44:09,098
The compound.

1782
00:44:09,098 --> 00:44:10,018
The CU inhibitor doesn't do

1783
00:44:10,018 --> 00:44:10,733
anything on its own.

1784
00:44:10,733 --> 00:44:12,192
Ionizing radiation gives us an

1785
00:44:12,192 --> 00:44:13,603
increase in phosphorylation.

1786
00:44:13,603 --> 00:44:14,937
As you'd imagine.

1787
00:44:14,937 --> 00:44:16,798
And the 2:45. I'm sorry,

1788
00:44:16,798 --> 00:44:19,102
the 3392 inhibitor is able to

1789
00:44:19,102 --> 00:44:19,976
block that

1790
00:44:19,976 --> 00:44:21,044
significantly.

1791
00:44:21,044 --> 00:44:22,744
So we get a significant

1792
00:44:22,744 --> 00:44:24,776
reduction in coup dependent

1793
00:44:24,776 --> 00:44:26,883
DNAPK auto phosphorylation.

1794
00:44:26,883 --> 00:44:29,096
She took it a step further and

1795
00:44:29,096 --> 00:44:30,679
went to the Ki 67 DNA

1796
00:44:30,679 --> 00:44:32,488
proliferation marker in

1797
00:44:32,488 --> 00:44:34,769
tumor slices from the extracted

1798
00:44:34,769 --> 00:44:36,869
tumors of these mice and the

1799
00:44:36,869 --> 00:44:38,594
vehicle controlled the

1800
00:44:38,594 --> 00:44:39,696
brown spots or.

1801
00:44:39,696 --> 00:44:40,930
The Ki 67.

1802
00:44:41,698 --> 00:44:42,281
Or CU inhibitor

1803
00:44:42,281 --> 00:44:43,099
doesn't do anything.

1804
00:44:43,099 --> 00:44:43,900
Thing on it's own.

1805
00:44:45,501 --> 00:44:47,295
The ionizing radiation gives us

1806
00:44:47,295 --> 00:44:48,713
a little decrease in the

1807
00:44:48,713 --> 00:44:50,073
combination is in fact

1808
00:44:50,073 --> 00:44:51,290
better than the mall,

1809
00:44:51,290 --> 00:44:52,955
and so this really set it up

1810
00:44:52,955 --> 00:44:53,609
that we're

1811
00:44:53,609 --> 00:44:55,186
having an in vivo effect of

1812
00:44:55,186 --> 00:44:57,037
these Co inhibitors with direct

1813
00:44:57,037 --> 00:44:57,814
intratumoral

1814
00:44:57,814 --> 00:44:58,381
injection.

1815
00:44:58,881 --> 00:45:00,818
And so we went further and did

1816
00:45:00,818 --> 00:45:02,732
the PK analysis at this point

1817
00:45:02,732 --> 00:45:03,853
and were able to

1818
00:45:03,853 --> 00:45:05,398
with a relatively simple

1819
00:45:05,398 --> 00:45:07,372
formulation, get really decent

1820
00:45:07,372 --> 00:45:08,424
pharmacokinetic

1821
00:45:08,424 --> 00:45:09,125
parameters.

1822
00:45:09,125 --> 00:45:10,815
We get a decent half life of

1823
00:45:10,815 --> 00:45:11,961
about 4 1/2 hours.

1824
00:45:12,528 --> 00:45:14,253
We get a maximum concentration

1825
00:45:14,253 --> 00:45:15,851
that's about twice the IC90

1826
00:45:15,851 --> 00:45:16,265
value,

1827
00:45:16,265 --> 00:45:17,999
so we can maintain that for at

1828
00:45:17,999 --> 00:45:19,766
least two hours as part of the

1829
00:45:19,766 --> 00:45:21,003
treatment and we get

1830
00:45:21,003 --> 00:45:22,565
a really good dose response

1831
00:45:22,565 --> 00:45:24,219
curve in terms of increasing

1832
00:45:24,219 --> 00:45:25,341
doses in the mice.

1833
00:45:25,341 --> 00:45:26,353
We get increase in

1834
00:45:26,353 --> 00:45:27,477
concentrations and.

1835
00:45:27,477 --> 00:45:30,079
So we can hit some really nice.

1836
00:45:31,581 --> 00:45:33,700
In vivo delivery of this drug

1837
00:45:33,700 --> 00:45:36,011
and target coverage in terms of

1838
00:45:36,011 --> 00:45:37,353
blocking the coup

1839
00:45:37,353 --> 00:45:37,920
protein.

1840
00:45:38,221 --> 00:45:39,919
So we did the experiment,

1841
00:45:39,919 --> 00:45:42,024
took the 549 lung cancer cell.

1842
00:45:42,525 --> 00:45:43,926
Put them into the mice.

1843
00:45:43,926 --> 00:45:45,298
Treated them with now

1844
00:45:45,298 --> 00:45:47,165
intraperitoneally. These are

1845
00:45:47,165 --> 00:45:48,564
subcutaneous tumors.

1846
00:45:48,564 --> 00:45:50,006
So they're treated IP.

1847
00:45:50,006 --> 00:45:51,897
And then two hours later hit

1848
00:45:51,897 --> 00:45:52,235
with

1849
00:45:52,235 --> 00:45:53,470
ionizing radiation.

1850
00:45:53,470 --> 00:45:55,472
And we did this regimen once a

1851
00:45:55,472 --> 00:45:56,072
week for

1852
00:45:56,072 --> 00:45:57,373
four weeks in a row.

1853
00:45:57,373 --> 00:45:59,001
So day Zero, day 714 and 21,

1854
00:45:59,001 --> 00:46:00,852
and monitor tumor growth and we

1855
00:46:00,852 --> 00:46:01,210
see a

1856
00:46:01,210 --> 00:46:03,026
little bit of activity from the

1857
00:46:03,026 --> 00:46:03,846
CU inhibitor.

1858
00:46:03,846 --> 00:46:05,656
We see a little bit of activity

1859
00:46:05,656 --> 00:46:07,350
from the ionizing radiation,

1860
00:46:07,350 --> 00:46:08,397
probably more than

1861
00:46:08,397 --> 00:46:09,619
we would have liked.

1862
00:46:09,619 --> 00:46:11,037
We had a titrate that down,

1863
00:46:11,037 --> 00:46:12,455
but the combo in fact was.

1864
00:46:12,455 --> 00:46:14,342
Then all of them put together,

1865
00:46:14,342 --> 00:46:16,085
and so now we have systemic

1866
00:46:16,085 --> 00:46:16,859
delivery of

1867
00:46:16,859 --> 00:46:18,753
the coup inhibitor to increase

1868
00:46:18,753 --> 00:46:20,756
the sensitivity of these tumors

1869
00:46:20,756 --> 00:46:21,531
to ionizing

1870
00:46:21,531 --> 00:46:23,577
radiation with relatively little

1871
00:46:23,577 --> 00:46:25,368
side effects along the way.

1872
00:46:25,368 --> 00:46:27,185
Tumor volume tumor weight goes

1873
00:46:27,185 --> 00:46:28,672
down and so we're pretty

1874
00:46:28,672 --> 00:46:29,972
convinced we have an

1875
00:46:29,972 --> 00:46:31,858
interesting model to understand

1876
00:46:31,858 --> 00:46:33,721
how to block that specific DNA

1877
00:46:33,721 --> 00:46:34,777
PK dependent non

1878
00:46:34,777 --> 00:46:36,298
homologous end joining to

1879
00:46:36,298 --> 00:46:38,105
ultimately increase radiation

1880
00:46:38,105 --> 00:46:38,915
sensitivity.

1881
00:46:39,982 --> 00:46:41,967
And So what I've shown the last

1882
00:46:41,967 --> 00:46:43,119
45 minutes or so,

1883
00:46:43,119 --> 00:46:44,644
we can block protein DNA

1884
00:46:44,644 --> 00:46:46,200
interactions as a viable

1885
00:46:46,200 --> 00:46:47,757
mechanism to target DDR

1886
00:46:47,757 --> 00:46:48,458
signaling.

1887
00:46:48,458 --> 00:46:50,045
So protein DNA interactions are

1888
00:46:50,045 --> 00:46:51,427
often thought undruggable.

1889
00:46:51,427 --> 00:46:53,069
And that's largely from work in

1890
00:46:53,069 --> 00:46:54,764
the transcription factor field,

1891
00:46:54,764 --> 00:46:56,251
where you're relying on, you

1892
00:46:56,251 --> 00:46:57,882
know, single based differences

1893
00:46:57,882 --> 00:46:58,534
to give you

1894
00:46:58,534 --> 00:46:59,167
specificity.

1895
00:46:59,167 --> 00:47:00,683
These proteins are structure

1896
00:47:00,683 --> 00:47:01,170
specific

1897
00:47:01,170 --> 00:47:01,537
events.

1898
00:47:01,537 --> 00:47:02,879
They they bind in a very

1899
00:47:02,879 --> 00:47:04,132
differently and we can

1900
00:47:04,132 --> 00:47:05,842
functionally target them with

1901
00:47:05,842 --> 00:47:07,405
small molecules and blocking

1902
00:47:07,405 --> 00:47:08,411
this can give us.

1903
00:47:09,145 --> 00:47:09,779
Anti cancer activity.

1904
00:47:10,112 --> 00:47:11,919
Single agent in the case of the

1905
00:47:11,919 --> 00:47:13,777
RPA inhibitors combination with

1906
00:47:13,777 --> 00:47:14,016
RPA

1907
00:47:14,016 --> 00:47:15,764
inhibitors in PARP combination

1908
00:47:15,764 --> 00:47:17,381
with coup inhibitors in DNA

1909
00:47:17,381 --> 00:47:17,920
binding,

1910
00:47:17,920 --> 00:47:19,714
so blocking the RPA we think is

1911
00:47:19,714 --> 00:47:21,132
through a gap protection

1912
00:47:21,132 --> 00:47:22,492
mechanism and blocking

1913
00:47:22,492 --> 00:47:24,288
coup in combination with IR is

1914
00:47:24,288 --> 00:47:25,940
through double strand break

1915
00:47:25,940 --> 00:47:27,163
repair and non hom.

1916
00:47:27,163 --> 00:47:27,964
And joining.

1917
00:47:29,432 --> 00:47:30,511
So what I'd like to do is

1918
00:47:30,511 --> 00:47:31,926
acknowledge everybody who's done

1919
00:47:31,926 --> 00:47:32,368
the work.

1920
00:47:32,368 --> 00:47:33,302
I did that previously.

1921
00:47:33,302 --> 00:47:35,264
Want to acknowledge our funding

1922
00:47:35,264 --> 00:47:37,139
sources? Over the years we've

1923
00:47:37,139 --> 00:47:38,174
had significant

1924
00:47:38,174 --> 00:47:40,163
support from the NIH and we are

1925
00:47:40,163 --> 00:47:41,511
very happy for that.

1926
00:47:42,044 --> 00:47:43,728
The reality is it's most of your

1927
00:47:43,728 --> 00:47:45,234
tax dollars going to the NIH

1928
00:47:45,234 --> 00:47:46,148
that decides who

1929
00:47:46,148 --> 00:47:47,149
gets these funding.

1930
00:47:47,149 --> 00:47:48,665
And so we thank you for that

1931
00:47:48,665 --> 00:47:49,151
support.

1932
00:47:49,151 --> 00:47:50,486
And if if we want to keep

1933
00:47:50,486 --> 00:47:51,821
funding through the NIH,

1934
00:47:51,821 --> 00:47:53,518
these are our tax dollars and we

1935
00:47:53,518 --> 00:47:54,790
should make sure we go.

1936
00:47:54,790 --> 00:47:55,191
That way.

1937
00:47:56,993 --> 00:47:58,416
In the absence of that,

1938
00:47:58,416 --> 00:48:00,322
we really do like to thank the

1939
00:48:00,322 --> 00:48:00,830
Tom and

1940
00:48:00,830 --> 00:48:02,639
Julie Wood Family Foundation.

1941
00:48:02,639 --> 00:48:04,238
They have been incredibly

1942
00:48:04,238 --> 00:48:05,134
supportive of

1943
00:48:05,134 --> 00:48:06,323
Indiana University,

1944
00:48:06,323 --> 00:48:08,316
our Simon Cancer Center and our

1945
00:48:08,316 --> 00:48:08,638
lung

1946
00:48:08,638 --> 00:48:09,453
cancer group.

1947
00:48:09,453 --> 00:48:11,387
And when other funding becomes

1948
00:48:11,387 --> 00:48:11,774
more.

1949
00:48:13,609 --> 00:48:14,265
Interesting.

1950
00:48:14,265 --> 00:48:15,728
It's always nice to have a

1951
00:48:15,728 --> 00:48:16,178
backup.

1952
00:48:16,178 --> 00:48:17,848
And our Simon Cancer Center has

1953
00:48:17,848 --> 00:48:19,496
also been very supportive over

1954
00:48:19,496 --> 00:48:20,650
the years as well as

1955
00:48:20,650 --> 00:48:21,724
near ex Biosciences.

1956
00:48:21,724 --> 00:48:23,158
And so the FDA here is for

1957
00:48:23,158 --> 00:48:23,819
funding and

1958
00:48:23,819 --> 00:48:24,529
without that,

1959
00:48:24,529 --> 00:48:26,096
none of this would have been

1960
00:48:26,096 --> 00:48:26,656
possible.

1961
00:48:27,089 --> 00:48:29,101
So I thank them and I thank you

1962
00:48:29,101 --> 00:48:31,229
for the time and I'd be happy to

1963
00:48:31,229 --> 00:48:31,827
take any

1964
00:48:31,827 --> 00:48:32,495
questions.

1965
00:48:36,732 --> 00:48:38,775
Thank you Doctor Turcie for the

1966
00:48:38,775 --> 00:48:40,794
for the wonderful presentation

1967
00:48:40,794 --> 00:48:41,871
for the for the

1968
00:48:41,871 --> 00:48:42,471
audience.

1969
00:48:42,471 --> 00:48:45,134
Please put your questions in the

1970
00:48:45,134 --> 00:48:47,850
chat and we will go through them

1971
00:48:47,850 --> 00:48:49,378
and we have first

1972
00:48:49,378 --> 00:48:51,668
one from it's comments from

1973
00:48:51,668 --> 00:48:53,449
Doctor Robert Sober.

1974
00:48:53,449 --> 00:48:55,017
Sobo is.

1975
00:48:55,017 --> 00:48:57,486
He's a fantastic work and.

1976
00:48:59,055 --> 00:49:00,359
We have a we have a question

1977
00:49:00,359 --> 00:49:01,290
from my colleagues.

1978
00:49:01,624 --> 00:49:03,125
Travis tracker from NCI.

1979
00:49:05,494 --> 00:49:05,728
He said.

1980
00:49:06,062 --> 00:49:06,963
This is a great talk.

1981
00:49:07,496 --> 00:49:09,497
Uh, sorry if I if he misses

1982
00:49:09,497 --> 00:49:11,699
this, is it possible that RPA

1983
00:49:11,699 --> 00:49:12,535
inhibition

1984
00:49:12,535 --> 00:49:14,914
prevents uh Primo uh recruitment

1985
00:49:14,914 --> 00:49:17,268
and re priming that can promote

1986
00:49:17,268 --> 00:49:18,407
gap formation?

1987
00:49:18,674 --> 00:49:20,461
This will potentially prevent

1988
00:49:20,461 --> 00:49:22,473
gaps from being generated rather

1989
00:49:22,473 --> 00:49:23,479
than RPA solely

1990
00:49:23,479 --> 00:49:24,437
being needed for

1991
00:49:24,437 --> 00:49:25,514
their protection.

1992
00:49:26,716 --> 00:49:27,505
Yeah. So I mean that's

1993
00:49:27,505 --> 00:49:28,150
a great question.

1994
00:49:28,150 --> 00:49:29,743
We haven't done that experiment

1995
00:49:29,743 --> 00:49:30,720
and and we agreed.

1996
00:49:30,720 --> 00:49:31,597
So there's two ways

1997
00:49:31,597 --> 00:49:32,521
this can go, right?

1998
00:49:32,521 --> 00:49:34,504
So we know Primpol is stimulated

1999
00:49:34,504 --> 00:49:36,342
by RPA and if we can decrease

2000
00:49:36,342 --> 00:49:37,293
enough of that

2001
00:49:37,293 --> 00:49:38,855
then you wouldn't get the

2002
00:49:38,855 --> 00:49:40,845
primpol re priming on the other

2003
00:49:40,845 --> 00:49:41,230
side.

2004
00:49:41,230 --> 00:49:43,059
So it it necessarily wouldn't

2005
00:49:43,059 --> 00:49:45,116
give you a gap because you don't

2006
00:49:45,116 --> 00:49:46,402
have a piece of DNA

2007
00:49:46,402 --> 00:49:47,503
on the other side.

2008
00:49:47,503 --> 00:49:48,807
What you have is a long single

2009
00:49:48,807 --> 00:49:50,095
strand piece of DNA where the

2010
00:49:50,095 --> 00:49:50,806
prim pole would

2011
00:49:50,806 --> 00:49:51,540
have synthesized.

2012
00:49:51,540 --> 00:49:52,241
And so we think.

2013
00:49:52,942 --> 00:49:53,829
That that would also give you

2014
00:49:53,829 --> 00:49:54,777
the potential for degradation.

2015
00:49:54,977 --> 00:49:56,382
We can't rule out the potential

2016
00:49:56,382 --> 00:49:57,815
of of the Theta mediated events

2017
00:49:57,815 --> 00:49:58,648
on there as well.

2018
00:49:59,081 --> 00:49:59,430
Well,

2019
00:49:59,430 --> 00:50:01,152
so we haven't done those

2020
00:50:01,152 --> 00:50:02,084
experiments,

2021
00:50:02,084 --> 00:50:04,301
but we are actively pursuing how

2022
00:50:04,301 --> 00:50:06,353
primpol re priming events are

2023
00:50:06,353 --> 00:50:07,556
regulated by the

2024
00:50:07,556 --> 00:50:08,691
RPA inhibitor.

2025
00:50:08,691 --> 00:50:10,447
It it's it could go either way

2026
00:50:10,447 --> 00:50:12,028
and and we don't know yet,

2027
00:50:12,028 --> 00:50:13,016
but that's a it's

2028
00:50:13,016 --> 00:50:14,063
a great question.

2029
00:50:17,533 --> 00:50:19,055
Report. Do you want to come out?

2030
00:50:19,055 --> 00:50:19,769
Come off mute.

2031
00:50:19,769 --> 00:50:20,369
Yeah.

2032
00:50:20,736 --> 00:50:21,070
Yeah.

2033
00:50:22,405 --> 00:50:23,741
Thank you for a really

2034
00:50:23,741 --> 00:50:24,774
nice talk, John.

2035
00:50:24,774 --> 00:50:27,172
Elegant work. Beautiful work.

2036
00:50:27,172 --> 00:50:29,462
I was wondering about these

2037
00:50:29,462 --> 00:50:30,479
inhibitors,

2038
00:50:30,479 --> 00:50:32,885
the RPA inhibitor inhibitors.

2039
00:50:32,885 --> 00:50:35,605
You mentioned that there weren't

2040
00:50:35,605 --> 00:50:36,285
so many

2041
00:50:36,285 --> 00:50:38,362
side effects in the mice,

2042
00:50:38,362 --> 00:50:40,523
but in cells and in mice.

2043
00:50:40,523 --> 00:50:41,906
What are the side

2044
00:50:41,906 --> 00:50:43,859
effects of using these?

2045
00:50:43,859 --> 00:50:44,310
I mean,

2046
00:50:44,310 --> 00:50:46,363
you're you're trying to do this

2047
00:50:46,363 --> 00:50:46,562
in

2048
00:50:46,562 --> 00:50:47,296
therapy, so.

2049
00:50:48,330 --> 00:50:48,664
It's relevant.

2050
00:50:48,998 --> 00:50:51,236
And how safe these are and for

2051
00:50:51,236 --> 00:50:53,682
example, do they cost sellers in

2052
00:50:53,682 --> 00:50:54,370
essence?

2053
00:50:55,905 --> 00:50:57,467
Do they cause double strand

2054
00:50:57,467 --> 00:50:58,954
breaks measured by H2X or

2055
00:50:58,954 --> 00:50:59,608
something?

2056
00:51:00,843 --> 00:51:03,057
Yeah. So so, single agent,

2057
00:51:03,057 --> 00:51:05,763
RPA inhibitory activity doesn't

2058
00:51:05,763 --> 00:51:06,549
increase

2059
00:51:06,549 --> 00:51:08,084
our H2 ax signal.

2060
00:51:08,084 --> 00:51:10,366
So we don't see that when we put

2061
00:51:10,366 --> 00:51:12,221
in non cancer cell lines.

2062
00:51:12,221 --> 00:51:14,003
So human peripheral blood

2063
00:51:14,003 --> 00:51:15,357
mononuclear cells,

2064
00:51:15,357 --> 00:51:17,640
you know the IC50 for those come

2065
00:51:17,640 --> 00:51:19,752
out to be about five to seven

2066
00:51:19,752 --> 00:51:21,063
times higher than

2067
00:51:21,063 --> 00:51:23,043
we see for the cancer cells.

2068
00:51:23,043 --> 00:51:25,221
We've also done experiments in

2069
00:51:25,221 --> 00:51:25,801
in non.

2070
00:51:25,801 --> 00:51:26,402
Cancerous.

2071
00:51:27,369 --> 00:51:28,933
Perpetual cell lines like HCC

2072
00:51:28,933 --> 00:51:30,319
293, we see some variable

2073
00:51:30,319 --> 00:51:30,873
activity.

2074
00:51:31,941 --> 00:51:33,623
So we really think that there's

2075
00:51:33,623 --> 00:51:35,234
a fundamental difference even

2076
00:51:35,234 --> 00:51:36,011
with the same

2077
00:51:36,011 --> 00:51:37,698
doubling time, for instance, of

2078
00:51:37,698 --> 00:51:39,212
a, you know, gut epithelial

2079
00:51:39,212 --> 00:51:39,548
cell,

2080
00:51:39,548 --> 00:51:41,237
it does S phase differently and

2081
00:51:41,237 --> 00:51:42,962
generates fewer single stranded

2082
00:51:42,962 --> 00:51:43,853
double stranded

2083
00:51:43,853 --> 00:51:45,001
breaks in DNA damage.

2084
00:51:45,001 --> 00:51:46,797
Hox is lower in them than just a

2085
00:51:46,797 --> 00:51:47,189
cancer

2086
00:51:47,189 --> 00:51:47,456
cell.

2087
00:51:47,456 --> 00:51:48,998
That's dividing at the same

2088
00:51:48,998 --> 00:51:50,799
doubling time, so we think that

2089
00:51:50,799 --> 00:51:52,194
really does give us the

2090
00:51:52,194 --> 00:51:53,496
window we we don't see.

2091
00:51:53,496 --> 00:51:54,312
So there's we

2092
00:51:54,312 --> 00:51:55,631
monitor weight loss.

2093
00:51:55,931 --> 00:51:57,311
We do see some initial with the

2094
00:51:57,311 --> 00:51:57,933
RP inhibitor,

2095
00:51:57,933 --> 00:51:58,581
but they regain

2096
00:51:58,581 --> 00:51:59,401
it pretty quickly.

2097
00:52:00,136 --> 00:52:01,971
There's mild neutropenia.

2098
00:52:02,404 --> 00:52:03,920
That we see in the mouse and

2099
00:52:03,920 --> 00:52:05,274
hematologic differences,

2100
00:52:05,274 --> 00:52:06,008
but we've done

2101
00:52:06,008 --> 00:52:07,476
combinations with platinum.

2102
00:52:07,476 --> 00:52:08,928
We see no increased kidney

2103
00:52:08,928 --> 00:52:10,379
toxicity, liver toxicity,

2104
00:52:10,379 --> 00:52:12,199
so it looks like it's relatively

2105
00:52:12,199 --> 00:52:12,882
safe again.

2106
00:52:12,882 --> 00:52:14,316
You know it's mice and we

2107
00:52:14,316 --> 00:52:15,780
haven't gone on to larger

2108
00:52:15,780 --> 00:52:17,186
rodents and and others.

2109
00:52:17,186 --> 00:52:18,216
But at this point,

2110
00:52:18,216 --> 00:52:20,037
there's no reason to not pursue

2111
00:52:20,037 --> 00:52:20,389
those

2112
00:52:20,389 --> 00:52:20,790
things.

2113
00:52:22,892 --> 00:52:24,426
Very short term experiment.

2114
00:52:24,426 --> 00:52:24,866
Have you done more

2115
00:52:24,866 --> 00:52:25,427
long term experiments?

2116
00:52:25,427 --> 00:52:26,295
They are.

2117
00:52:27,897 --> 00:52:28,239
Yeah,

2118
00:52:28,239 --> 00:52:30,354
we've done some toxicokinetics

2119
00:52:30,354 --> 00:52:30,566
or

2120
00:52:30,566 --> 00:52:32,334
multiple high dosing over.

2121
00:52:33,702 --> 00:52:34,103
I think it's.

2122
00:52:34,470 --> 00:52:35,905
Three month period.

2123
00:52:37,406 --> 00:52:39,255
And we're able to to dose it at

2124
00:52:39,255 --> 00:52:41,203
a level that gives us sufficient

2125
00:52:41,203 --> 00:52:42,178
target coverage

2126
00:52:42,178 --> 00:52:43,917
without significant toxicity,

2127
00:52:43,917 --> 00:52:45,762
but they're really the studies

2128
00:52:45,762 --> 00:52:46,315
that are

2129
00:52:46,315 --> 00:52:46,912
needed at,

2130
00:52:46,912 --> 00:52:48,868
you know for the to getting this

2131
00:52:48,868 --> 00:52:49,418
thing to

2132
00:52:49,418 --> 00:52:50,084
an induced.

2133
00:52:50,084 --> 00:52:52,021
It's GLP tox is the whole game.

2134
00:52:52,021 --> 00:52:52,688
And so we are.

2135
00:52:52,688 --> 00:52:52,855
Yeah.

2136
00:52:52,855 --> 00:52:53,973
Pursuing those those

2137
00:52:53,973 --> 00:52:54,924
experiments now.

2138
00:52:56,058 --> 00:52:57,564
You're making derivatives.

2139
00:52:57,564 --> 00:52:59,343
There may be more safe than or

2140
00:52:59,343 --> 00:52:59,995
something.

2141
00:53:00,796 --> 00:53:01,026
Yeah,

2142
00:53:01,026 --> 00:53:02,482
there's always thoughts of more

2143
00:53:02,482 --> 00:53:02,998
chemistry.

2144
00:53:04,500 --> 00:53:06,720
The the current 329 molecule has

2145
00:53:06,720 --> 00:53:08,778
the requisite characteristics

2146
00:53:08,778 --> 00:53:09,772
that we like,

2147
00:53:09,772 --> 00:53:11,717
but whether that's the final

2148
00:53:11,717 --> 00:53:13,920
clinical candidate or not is is

2149
00:53:13,920 --> 00:53:14,844
still up for

2150
00:53:14,844 --> 00:53:15,311
debate.

2151
00:53:15,644 --> 00:53:17,790
We have other derivatives and

2152
00:53:17,790 --> 00:53:19,048
the question is,

2153
00:53:19,048 --> 00:53:21,023
do you go with a full scale

2154
00:53:21,023 --> 00:53:23,410
resynthesis of new pharmacopores

2155
00:53:23,410 --> 00:53:24,753
or do for a first

2156
00:53:24,753 --> 00:53:26,440
inhuman first in target

2157
00:53:26,440 --> 00:53:28,682
compound. See what we get. And

2158
00:53:28,682 --> 00:53:30,326
that's the challenge.

2159
00:53:31,961 --> 00:53:33,441
The very strong setup you have

2160
00:53:33,441 --> 00:53:34,330
there. Very nice.

2161
00:53:34,330 --> 00:53:34,897
Thank you.

2162
00:53:39,835 --> 00:53:40,236
OK.

2163
00:53:40,236 --> 00:53:42,843
Next question we have Ronit

2164
00:53:42,843 --> 00:53:46,014
Yaden from Nhlbi. She says great

2165
00:53:46,014 --> 00:53:46,609
talk.

2166
00:53:46,609 --> 00:53:48,716
Is there any interest and how do

2167
00:53:48,716 --> 00:53:50,872
you plan to increase the potency

2168
00:53:50,872 --> 00:53:51,614
of various

2169
00:53:51,614 --> 00:53:53,534
compound if there is a global

2170
00:53:53,534 --> 00:53:55,711
development of them for clinical

2171
00:53:55,711 --> 00:53:56,051
use?

2172
00:53:57,586 --> 00:53:59,235
It's a great question and we're

2173
00:53:59,235 --> 00:54:00,990
pursuing structure based design.

2174
00:54:00,990 --> 00:54:02,368
So we're trying to get the

2175
00:54:02,368 --> 00:54:04,048
structures of all of these with

2176
00:54:04,048 --> 00:54:04,860
their targets.

2177
00:54:05,227 --> 00:54:06,966
You know the challenge with a

2178
00:54:06,966 --> 00:54:08,377
protein DNA interaction

2179
00:54:08,377 --> 00:54:09,665
inhibitor is they're

2180
00:54:09,665 --> 00:54:11,388
necessarily hydrophobic and

2181
00:54:11,388 --> 00:54:12,601
solubility is bad.

2182
00:54:12,601 --> 00:54:14,314
And so we've, we've done Co,

2183
00:54:14,314 --> 00:54:15,537
crystal structures,

2184
00:54:15,537 --> 00:54:17,479
cryo am structures and trying to

2185
00:54:17,479 --> 00:54:19,464
get very accurate predictions of

2186
00:54:19,464 --> 00:54:20,209
where these

2187
00:54:20,209 --> 00:54:21,510
things are to give us.

2188
00:54:23,245 --> 00:54:24,894
Structure based design to

2189
00:54:24,894 --> 00:54:26,081
increase potency.

2190
00:54:27,049 --> 00:54:28,490
And so that's really one of the

2191
00:54:28,490 --> 00:54:29,652
the mechanisms to do it.

2192
00:54:29,652 --> 00:54:31,424
I think it the idea of and and

2193
00:54:31,424 --> 00:54:33,255
this is what we did initially,

2194
00:54:33,255 --> 00:54:34,669
which was largely random

2195
00:54:34,669 --> 00:54:36,528
screening and of compounds that

2196
00:54:36,528 --> 00:54:38,027
look similar in creation

2197
00:54:38,027 --> 00:54:39,624
of molecules with different

2198
00:54:39,624 --> 00:54:41,384
pharmacopoeous and ultimately

2199
00:54:41,384 --> 00:54:41,931
ended up

2200
00:54:41,931 --> 00:54:43,640
with ones that give us pretty

2201
00:54:43,640 --> 00:54:45,144
reasonable, you know, low

2202
00:54:45,144 --> 00:54:46,468
micromolar IC 50s in.

2203
00:54:46,468 --> 00:54:47,551
DNA binding assays,

2204
00:54:47,551 --> 00:54:49,304
when we put them in the kinase

2205
00:54:49,304 --> 00:54:49,772
assays,

2206
00:54:49,772 --> 00:54:51,405
we can get them into the, you

2207
00:54:51,405 --> 00:54:53,189
know, 100 nanomolar IC50 ranges

2208
00:54:53,189 --> 00:54:54,109
because they're

2209
00:54:54,109 --> 00:54:55,253
catalytic activities

2210
00:54:55,253 --> 00:54:56,512
versus just a binary.

2211
00:54:56,512 --> 00:54:57,642
Protein interaction.

2212
00:54:57,642 --> 00:54:59,281
And so we think we're close,

2213
00:54:59,281 --> 00:55:00,955
but there's definitely ways to

2214
00:55:00,955 --> 00:55:02,334
go to increase those the

2215
00:55:02,334 --> 00:55:02,851
potency.

2216
00:55:02,851 --> 00:55:04,081
But we think structure

2217
00:55:04,081 --> 00:55:05,087
is the way to go.

2218
00:55:06,622 --> 00:55:07,305
At this point,

2219
00:55:07,305 --> 00:55:08,807
we also are looking at some AI

2220
00:55:08,807 --> 00:55:09,258
versions

2221
00:55:09,258 --> 00:55:10,292
of trying to do this.

2222
00:55:10,292 --> 00:55:12,331
There's a lot of approaches to

2223
00:55:12,331 --> 00:55:14,342
do that, but it would benefit

2224
00:55:14,342 --> 00:55:15,798
from knowing the the

2225
00:55:15,798 --> 00:55:17,362
actual structure of the

2226
00:55:17,362 --> 00:55:19,300
inhibitor with the target as

2227
00:55:19,300 --> 00:55:21,170
opposed to trying to model

2228
00:55:21,170 --> 00:55:22,522
that and then do the

2229
00:55:22,522 --> 00:55:23,806
AI versions of it.

2230
00:55:26,642 --> 00:55:27,176
Great.

2231
00:55:27,176 --> 00:55:29,733
Next we have a question from

2232
00:55:29,733 --> 00:55:30,646
Jimmy Lee

2233
00:55:30,646 --> 00:55:32,314
from NCI. She says.

2234
00:55:32,314 --> 00:55:34,630
Great talk in the MDA MB436B

2235
00:55:34,630 --> 00:55:37,255
BRCA deficient tmbc mouse model

2236
00:55:37,255 --> 00:55:38,187
with an RP

2237
00:55:38,187 --> 00:55:40,671
inhibitor alone tumor start to

2238
00:55:40,671 --> 00:55:43,288
grow again after four weeks and

2239
00:55:43,288 --> 00:55:45,060
the combination with

2240
00:55:45,060 --> 00:55:47,269
PARP inhibitor can cause a

2241
00:55:47,269 --> 00:55:48,797
durable response.

2242
00:55:48,797 --> 00:55:50,733
But have you tried tested?

2243
00:55:52,334 --> 00:55:53,907
RPA inhibitor alone or RPA

2244
00:55:53,907 --> 00:55:55,397
inhibitor plus elaborate

2245
00:55:55,397 --> 00:55:56,205
combination?

2246
00:55:56,839 --> 00:55:58,383
In a pop inhibitor

2247
00:55:58,383 --> 00:55:59,842
resistant model.

2248
00:56:00,709 --> 00:56:02,296
Yeah, that's again another great

2249
00:56:02,296 --> 00:56:03,770
question. And we are actively

2250
00:56:03,770 --> 00:56:04,380
doing that.

2251
00:56:04,380 --> 00:56:06,846
Matthew's generated a series of

2252
00:56:06,846 --> 00:56:09,285
PARP inhibitor resistant lines

2253
00:56:09,285 --> 00:56:10,586
to saroopa rib.

2254
00:56:11,253 --> 00:56:13,101
The the part one specific

2255
00:56:13,101 --> 00:56:15,511
inhibitor and we have very, very

2256
00:56:15,511 --> 00:56:17,092
interesting results.

2257
00:56:17,092 --> 00:56:19,269
So we can get 1000 fold increase

2258
00:56:19,269 --> 00:56:21,009
in resistance to the PAAP

2259
00:56:21,009 --> 00:56:22,331
inhibitor in these

2260
00:56:22,331 --> 00:56:24,371
MDA 436 that are resistant and

2261
00:56:24,371 --> 00:56:26,384
they are equally sensitive to

2262
00:56:26,384 --> 00:56:27,703
the RPA inhibitor.

2263
00:56:27,703 --> 00:56:28,737
So RPA inhibition

2264
00:56:28,737 --> 00:56:29,772
is not affected.

2265
00:56:30,506 --> 00:56:31,351
And so we really

2266
00:56:31,351 --> 00:56:32,408
do like that model.

2267
00:56:33,876 --> 00:56:35,876
Your your question, the RPA,

2268
00:56:35,876 --> 00:56:38,080
resumption of the tumor growth

2269
00:56:38,080 --> 00:56:38,447
that

2270
00:56:38,447 --> 00:56:40,641
resumes after RPA inhibition is

2271
00:56:40,641 --> 00:56:42,163
largely a function of

2272
00:56:42,163 --> 00:56:43,685
pharmacokinetics and

2273
00:56:43,685 --> 00:56:45,054
pharmacodynamics.

2274
00:56:45,054 --> 00:56:47,166
We don't see and I showed you

2275
00:56:47,166 --> 00:56:49,425
the increase in PK parameters,

2276
00:56:49,425 --> 00:56:51,612
the C Max with increasing dose

2277
00:56:51,612 --> 00:56:53,362
of the coup inhibitors.

2278
00:56:53,362 --> 00:56:55,555
We don't see that with the RPA

2279
00:56:55,555 --> 00:56:57,357
inhibitor at its current

2280
00:56:57,357 --> 00:56:58,333
formulation.

2281
00:56:58,333 --> 00:56:59,169
So it really is a

2282
00:56:59,169 --> 00:57:00,102
formulation issue.

2283
00:57:00,102 --> 00:57:01,222
Issue and getting

2284
00:57:01,222 --> 00:57:03,231
bioavailability to be retained

2285
00:57:03,231 --> 00:57:05,441
after multiple dosing events and

2286
00:57:05,441 --> 00:57:07,417
so we think once we crack that

2287
00:57:07,417 --> 00:57:09,436
nut, we'll be able to maintain

2288
00:57:09,436 --> 00:57:10,512
the pressure on

2289
00:57:10,512 --> 00:57:12,551
those tumors in the combination

2290
00:57:12,551 --> 00:57:14,713
that experiment again is a great

2291
00:57:14,713 --> 00:57:14,983
one

2292
00:57:14,983 --> 00:57:16,900
combination PARP RPA and then

2293
00:57:16,900 --> 00:57:18,353
just let them go and.

2294
00:57:18,353 --> 00:57:18,559
See,

2295
00:57:18,559 --> 00:57:20,195
when they come back versus PARP

2296
00:57:20,195 --> 00:57:20,722
inhibitor

2297
00:57:20,722 --> 00:57:21,856
alone and the PARP RPA

2298
00:57:21,856 --> 00:57:23,436
combination, they're long term

2299
00:57:23,436 --> 00:57:24,226
mouse studies.

2300
00:57:24,226 --> 00:57:25,647
And so we're we're trying to

2301
00:57:25,647 --> 00:57:27,307
find somebody to throw us enough

2302
00:57:27,307 --> 00:57:27,930
money to do

2303
00:57:27,930 --> 00:57:28,197
that.

2304
00:57:30,332 --> 00:57:31,084
Hey great.

2305
00:57:31,084 --> 00:57:33,475
I have a actually a a follow up

2306
00:57:33,475 --> 00:57:34,169
question

2307
00:57:34,169 --> 00:57:35,571
with this question.

2308
00:57:35,571 --> 00:57:37,671
So I noticed that many of the

2309
00:57:37,671 --> 00:57:40,033
validation experiment is done in

2310
00:57:40,033 --> 00:57:41,510
a cell line like a.

2311
00:57:41,743 --> 00:57:43,545
8459.

2312
00:57:45,080 --> 00:57:47,262
But have you tried any of these

2313
00:57:47,262 --> 00:57:49,425
inhibitors on the cell line or

2314
00:57:49,425 --> 00:57:50,219
model with

2315
00:57:50,219 --> 00:57:52,419
intrinsic DNA repair deficiency

2316
00:57:52,419 --> 00:57:54,690
like this BRCA deficient model,

2317
00:57:54,690 --> 00:57:56,492
so that the inhibitor may?

2318
00:57:56,758 --> 00:57:58,941
Act as a synthetically

2319
00:57:58,941 --> 00:58:00,529
satity fashion.

2320
00:58:01,530 --> 00:58:01,891
Yeah.

2321
00:58:01,891 --> 00:58:04,199
So we've done that with the RPA

2322
00:58:04,199 --> 00:58:04,999
inhibitors.

2323
00:58:04,999 --> 00:58:07,164
The RPA was really pursued in

2324
00:58:07,164 --> 00:58:07,836
that MDA

2325
00:58:07,836 --> 00:58:09,256
436, which is a BRCA

2326
00:58:09,256 --> 00:58:10,606
1 deficient model.

2327
00:58:11,106 --> 00:58:12,593
So we published and Pami

2328
00:58:12,593 --> 00:58:14,628
published this in the paper last

2329
00:58:14,628 --> 00:58:15,010
year.

2330
00:58:15,277 --> 00:58:17,295
A variety of cell lines with

2331
00:58:17,295 --> 00:58:19,647
differential alterations and and

2332
00:58:19,647 --> 00:58:21,116
we published an ATM

2333
00:58:21,116 --> 00:58:23,195
mutant cell line and the coup

2334
00:58:23,195 --> 00:58:25,248
inhibitor was more potent in

2335
00:58:25,248 --> 00:58:26,421
that cell line.

2336
00:58:27,156 --> 00:58:28,749
The reality is when you get to

2337
00:58:28,749 --> 00:58:30,382
isogenic models, that's really

2338
00:58:30,382 --> 00:58:30,926
the test.

2339
00:58:31,627 --> 00:58:33,353
And so we see less of a

2340
00:58:33,353 --> 00:58:35,591
difference in an isogenic ATM

2341
00:58:35,591 --> 00:58:36,131
model.

2342
00:58:36,131 --> 00:58:37,956
And so we're actually doing the

2343
00:58:37,956 --> 00:58:39,886
CRISPR screens to look for those

2344
00:58:39,886 --> 00:58:40,369
genetic

2345
00:58:40,369 --> 00:58:41,922
alterations that increase

2346
00:58:41,922 --> 00:58:43,972
sensitivity to a coup inhibitor.

2347
00:58:43,972 --> 00:58:44,706
But we agree with you.

2348
00:58:44,706 --> 00:58:45,357
I think those

2349
00:58:45,357 --> 00:58:46,508
experiments are great.

2350
00:58:46,508 --> 00:58:47,747
We just haven't done them yet

2351
00:58:47,747 --> 00:58:48,944
and and they're in process.

2352
00:58:48,944 --> 00:58:50,812
We've got three CRISPR screens

2353
00:58:50,812 --> 00:58:52,851
going currently and are cranking

2354
00:58:52,851 --> 00:58:53,615
through the

2355
00:58:53,615 --> 00:58:55,536
bioinformatics to find out what

2356
00:58:55,536 --> 00:58:57,519
is increasing that sensitivity.

2357
00:58:59,555 --> 00:59:00,042
Great.

2358
00:59:00,042 --> 00:59:02,724
I think we don't have additional

2359
00:59:02,724 --> 00:59:03,528
questions,

2360
00:59:03,528 --> 00:59:05,764
so maybe we'll just wrap up

2361
00:59:05,764 --> 00:59:06,261
here.

2362
00:59:06,261 --> 00:59:08,402
And thank you for Doctor Turcie

2363
00:59:08,402 --> 00:59:10,525
for the insightful and thought

2364
00:59:10,525 --> 00:59:11,233
provoking

2365
00:59:11,233 --> 00:59:13,285
presentation on DNA damage and

2366
00:59:13,285 --> 00:59:15,100
the DNA replication and we

2367
00:59:15,100 --> 00:59:16,705
appreciate everybody's

2368
00:59:16,705 --> 00:59:18,840
participation and we're looking

2369
00:59:18,840 --> 00:59:20,881
forward to continue this this

2370
00:59:20,881 --> 00:59:22,077
session and then

2371
00:59:22,077 --> 00:59:24,208
have a have a great, great day.

2372
00:59:24,208 --> 00:59:25,514
Thank you so much.

2373
00:59:27,049 --> 00:59:27,950
Thank you all.

2374
00:59:28,150 --> 00:59:29,051
Thank you.