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WELL, WHY DON'T WE GO AHEAD AND

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GET STARTED.

3
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THANK YOU ALL FOR COMING DOWN.

4
00:00:12,775 --> 00:00:15,178
THOSE OF YOU WHO DON'T KNOW ME

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00:00:15,178 --> 00:00:17,247
I'M DAN Mc VICAR FROM UP IN

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00:00:17,247 --> 00:00:18,515
FREDERICK AND IT'S A REAL

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PLEASURE FOR ME THIS AFTERNOON

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TO INTRODUCE OUR SPEAKER DEBBIE

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00:00:23,186 --> 00:00:23,453
MORRISON.

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I THINK MANY OF YOU KNOW DEBBIE

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AND KNOW AND KNOW HER HISTORY

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VERY WELL BUT I'M GOING TO

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00:00:29,058 --> 00:00:32,128
REPEAT IT FOR YOU ANYWAY.

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SO SHE'S INITIALLY DID HER

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UNDERGRADUATE WORK IN NASHVILLE,

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00:00:35,798 --> 00:00:37,867
AND THEN COMPLETED HER Ph.D.

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AT VANTER BUILT WITH RICHARD

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MOYER AND PUBLISHED A CELL PAPER

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THERE, NOT BAD FOR A GRADUATE

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STUDENT LOOKING AT THE USE OF

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00:00:46,843 --> 00:00:51,347
CELLULAR RNA POLYMERASE BY P. PX

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00:00:51,347 --> 00:00:51,614
VIRUSES.

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00:00:51,614 --> 00:00:54,350
BUT THEN SHE MOVED TO THE

24
00:00:54,350 --> 00:00:57,854
DANA-FARBER AS A POST DOC AND

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00:00:57,854 --> 00:00:59,355
JOINED TOM LAMBERT'S LAB THERE

26
00:00:59,355 --> 00:01:00,723
AND MADE HER STUDY ON THE REST

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OF HER CAREER ON WHICH IS

28
00:01:05,461 --> 00:01:06,863
TYROSINE PHOSPHOR LEAN AND

29
00:01:06,863 --> 00:01:07,130
SIGNALS.

30
00:01:07,130 --> 00:01:10,900
SHE STARTED WORKING ON R,A ,--RF

31
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AND YOU WILL HEAR A LOT ABOUT

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THAT TODAY, PUBLISHED A SECOND

33
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CELL PAPER, TALKING ABOUT

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TYROSINE PHOSPHORYLATION BY PDGF

35
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RECEPTOR AND ITS ACTIVATION OF

36
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--RAF, TO GIVE YOU AN IDEA OF

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THE FIELD IT WAS TYROSINE AND

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PHOSPHORYLATION SIGNALING WAS

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STARTING TO BE ACCEPTED AND IN

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FACT, I THOUGHT IT WAS REALLY

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FUN ACTUALLY THAT ON HER CV, SHE

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HAS A TITLE TYROSINE

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PHOSPHORYLATION IS SIGNAL

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TRANSDUCTION, AT THE TIME YOU

45
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HAD TO MAKE THAT ARGUMENT BUT

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YOU DON'T HAVE TO DO THAT

47
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ANYMORE.

48
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BUT THAT WAS A LONG LINE OF

49
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SEMINOLE WORK REALLY ON RAF 1

50
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FROM DEBBIE'S LAB.

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SHE JOINED THE LAB UP IN

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FREDERICK WHICH GEORGE WAS THE

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DIRECTOR, RIGHT?

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GEORGE VANNEDDER WOOD WHO MANY

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OF YOU PROBABLY KNOW, SHE WAS

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00:02:01,084 --> 00:02:02,952
THEN PROMOTED UP TO SENIOR

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00:02:02,952 --> 00:02:04,220
INVESTIGATOR AND TENURED WITHIN

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00:02:04,220 --> 00:02:08,458
THE NCI UP THERE.

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DEBBIE'S WORK AND REALLY

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REACHING AND FANTASTIC.

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SHE'S BEEN FEATURED MULTIPLE

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TIMES BY THE CCR LEADERSHIP AND

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00:02:15,131 --> 00:02:16,199
INNER MILESTONES AND THINGS LIKE

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00:02:16,199 --> 00:02:18,968
THAT AND SHE HAS MULTIPLE NIH

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DIRECTOR AWARDS FOR HER

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DISSECTION OF THE SIGNALING AND

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RAS AND R,A F AND, WOOING OUT

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DETAILS OF THAT PATHWAY, SHE'S

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00:02:26,676 --> 00:02:28,211
ALSO BEEN RECOGNIZED IMPORTANTLY

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WELL OUTSIDE OF THE CCR, SHE WON

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THE PFIZER RESEARCH AWARD FROM

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THE UNIVERSITY OF MONITOR TRIAL,

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A SIGNALING, LRI TRANSDUCTION

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AWARD FROM THE ROYAL COLLEGE OF

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SURGEONS IN LONDON AND THE KARL

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BIER AWARD FROM THE UNIVERSITY

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OF WISCONSIN MADISON AND OF

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COURSE MANY OF YOU KNOW THAT JUF

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00:02:50,166 --> 00:02:54,504
IN 2002 SHE WAS ELECTED TO THE

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00:02:54,504 --> 00:02:56,806
NAGGAL ACADEMY OF SCIENCES.

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SORRY?

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WHAT DID I SAY?

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TWO.

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TWENTY-TWO.

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YOU SHOULD HAVE BEEN ELECTED IN

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2002--WE THAT WOULD SHOW YOUR

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00:03:05,748 --> 00:03:07,083
AGE, WE DON'T WANT TO DO THAT.

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00:03:07,083 --> 00:03:08,818
THANK YOU FOR THE CORRECTION.

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>> DEBBIE'S LAB CONTINUES
TO BE

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A WORLD LEADER IN THE FUNCTION

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00:03:14,023 --> 00:03:16,526
OF RAF AND THEY HAVE FOCUSED ON

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00:03:16,526 --> 00:03:18,761
THE KEY SIGNAL TRANSDUCTION OF

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00:03:18,761 --> 00:03:21,998
LOCATION, LOCATION, LOCATION, SO

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SHE'S BEEN CRITICAL IN

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00:03:22,865 --> 00:03:24,133
DISSECTING THE ROLE OF SCAFFOLD

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PROTEINS AND HOW THEY COORDINATE

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00:03:26,135 --> 00:03:27,070
THESE ACTIVATION COMPLEXES AND

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CONTINUES TO INCREASE OUR

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00:03:29,138 --> 00:03:31,674
FUNDAMENTAL UNDERSTANDING OF RAS

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00:03:31,674 --> 00:03:35,578
AND RAF SIGNALING AND IN MANY

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00:03:35,578 --> 00:03:37,447
CASES NOW THROUGH CLOSE RAS

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00:03:37,447 --> 00:03:40,783
INITIATIVE AND THE RAS OPERATIVE

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00:03:40,783 --> 00:03:41,851
HERES HERE AT NIH AND TODAY SHE

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00:03:41,851 --> 00:03:43,252
WILL TELL US ABOUT HER ONGOING

105
00:03:43,252 --> 00:03:45,722
WORK AND THE TITLE OF HER TALK

106
00:03:45,722 --> 00:03:49,092
IS UNDERSTANDING RAF KINASE

107
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DISEASE REGULATION IN HUMAN

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00:03:50,460 --> 00:03:51,494
DISEASE STATES EMPLOY.

109
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THE DETAILS DO MATTER!

110
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SO YOU WILL HEAR DETAILS.

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00:03:53,896 --> 00:03:57,066
THANKS SO MUCH DEBBIE.

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00:03:57,066 --> 00:03:58,501
>> WELL, I WANT TO SAY
FIRST OF

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ALL, IT'S A REAL HONOR TO BE

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00:04:00,036 --> 00:04:05,608
HERE TO GIVE THE GRAND ROUND AND

115
00:04:05,608 --> 00:04:07,276
IT'S GOD TO SEE THE FACES THAT I

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DON'T SEE ALL THE TIME UP IN

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FREDERICK.

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SO I HOPE WHEN NIGH TALK'S OVER

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00:04:11,347 --> 00:04:13,416
AND YOU WALK ARK WAY, YOU WILL

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REALIZE HOW IMPORTANT THE

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DETAILS FOR FOR UNDERSTANDING

122
00:04:19,455 --> 00:04:20,323
RAF REGULATION.

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00:04:20,323 --> 00:04:22,759
SO MY LAB HAS REALLY HAD A LONG

124
00:04:22,759 --> 00:04:24,827
STANDING INTEREST IN STUDYING

125
00:04:24,827 --> 00:04:29,232
THE SIGNAL TRANSDUCTION PATHWAY

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THAT'S REGULATED BY THE RAF

127
00:04:32,668 --> 00:04:34,070
GTPACES, OFTEN IT'S REFERRED TO

128
00:04:34,070 --> 00:04:35,638
AS A MOLECULAR SWITCH TO TURN ON

129
00:04:35,638 --> 00:04:38,241
SIGNALING IN THE CELL AND MANY

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TIMES THAT ACTIVATING SWITCH IS

131
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WHEN THE CELL RECEIVES SIGNALS

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FROM RECEIPTOR TYROSEEN KINASES

133
00:04:44,380 --> 00:04:48,684
ON THE CELL SURFACE AND RAS ONCE

134
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ITS ACT VAILTED WITH INTERACT

135
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WITH A NUMBER OF EFFECTIVE 

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00:04:51,788 --> 00:04:54,357
CASCADINGS BUT 1 OF ESSENTIAL

137
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CAS CADE IS THE ERK CASCADE

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WHICH IS COMPRISED OF THE RAF,

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MEK AND ERK PROTEIN KINASES.

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NOT ONLY DOES THIS PLAY A

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PATHWAY ROLE IN NORMAL GROWTH

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AND DEVELOPMENTAL SIGNALS, A

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NUMBER OF THE COMPONENTS OF THE

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PATHWAY HAD FOUND TO BE MUTATE

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INDEED HUMAN KANSASER AND IN

146
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PARTICULAR KRAS IS MUTATED AT

147
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90% OF THE PANCREATIC CANCER AND

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BRAF IS MUTATED AT ABOUT 60% OF

149
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THE MALIGNANT MELANOMAS.

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BUT IN ADDITION TO BEING MUTATE

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INDEED CANCER, THESE PROTEINS

152
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CAN CONTAIN GERM LINE MUTATIONS

153
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THAT WILL CAUSE A GROUP OF

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RELATED DEVELOPMENTAL DISORDERS

155
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THAT ARE KNOWN COLLECTIVELY NOW

156
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AS THE RAS-OP A THIES.

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AND BECAUSE OF THE FUNCTION OF

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THIS PATHWAY IN YOU HAD NANOG

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DISEASE STATES THERE'S BEEN A

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LOT OF INTEREST TO FIGURE OUT

161
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HOW CAN WE TARGET THIS PATHWAY?

162
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AND SO, FOR THERAPEUTIC AND

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INTERVENTION, BOTH IN CANCER AND

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IN THESE DEVELOPMENTAL

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DISORDERS.

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BUT I THINK THE 1 THING THAT

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WE'VE REALLY LEARNED IS

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RESEARCHERS OVER THE YEARS, IS

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THAT IN ORDER TO TARGET THE

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PATHWAY, IT'S REALLY IMPORTANT

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THAT YOU UNDERSTAND HOW IT

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NORMALLY FUNCTIONS AND

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UNFORTUNATELY, OR MAYBE

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FORTUNATELY FOR ME BECAUSE IT'S

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GIIVE ME JOB SECURITY IS THE

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RAFS ARE REALLY VERY COMPLEX

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WHEN IT COMES TO THEIR

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REGULATION.

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AND SO JUST TO SHOW YOU A

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SCHEMATIC FOR THOSE OF YOU WHO

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DON'T AND THINK ABOUT THE RAFS

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24/7, THEY'RE A GROUP OF PROTEIN

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KINASES AND THEY CONTAIN A

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CATALYTIC DOMAIN AT THE

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C-TERMINAL REGION AND A

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REGULATORY DOMAIN AT THE END

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TERMINUS AND THIS HAS A RAS

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BINDING DOMAIN, ASSISTING RICH

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DOMAIN AND A C3 ARHINE, AND IF

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WE THINK ABOUT THEIR INTRINSIC

191
00:07:07,990 --> 00:07:10,927
CATALYTIC ACTIVITY, BRAF HAS THE

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HIGHEST INTRINSIC ACTIVITY

193
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FOLLOWED BY CRAF AND THEN ARAF.

194
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AND WHAT WE'VE LEARNED MAINLY

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HISTORICALLY A LOT OF OUR

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INFORMATION THROUGH THE YEARS

197
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ABOUT THE RAFS CAME FROM STUDIES

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OF BIOCHECKICAL STUDIES WITH

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00:07:30,146 --> 00:07:34,350
MUTE ANTS OR WITH THE STUDY OF

200
00:07:34,350 --> 00:07:35,117
INDIVIDUAL PROTEIN DOMAINS.

201
00:07:35,117 --> 00:07:38,287
AND SO WHAT WE LEARNED WAS THAT

202
00:07:38,287 --> 00:07:40,723
AND I THINK THIS WAS PROBABLY

203
00:07:40,723 --> 00:07:42,858
THE FIRST REAL BREAK THROUGH IN

204
00:07:42,858 --> 00:07:45,061
UNDERSTANDING RAF WAS THAT IT

205
00:07:45,061 --> 00:07:47,063
CAN DIRECTLY INTERACT WITH RAS

206
00:07:47,063 --> 00:07:49,465
AND THAT'S MEDIATED THROUGH THE

207
00:07:49,465 --> 00:07:52,468
RAS BINDING DOMAIN BUT ONCE RAS

208
00:07:52,468 --> 00:07:53,970
INTERACTS, HAS A HIGH AFFINITY

209
00:07:53,970 --> 00:07:56,639
BINDING TO THE RBD, IT CAN ALSO

210
00:07:56,639 --> 00:07:59,875
MAKE CONTACTS WITH THE CYSTINE

211
00:07:59,875 --> 00:08:00,977
RICH DOMAIN.

212
00:08:00,977 --> 00:08:04,046
THE CYSTINE RICH DOMAIN WAS

213
00:08:04,046 --> 00:08:05,047
PROBABLY FIRST CHARACTERIZED

214
00:08:05,047 --> 00:08:08,351
BASED UPON ITS ABILITY TO

215
00:08:08,351 --> 00:08:08,918
INTERACT WITH FOSTER NURSED

216
00:08:08,918 --> 00:08:10,486
FOCUSED ON TILE SERIES POINTSINE

217
00:08:10,486 --> 00:08:16,325
IN THE MEMBRANE, AND THEN THERE

218
00:08:16,325 --> 00:08:26,902
ARE ALSO 1433 BINDING SITES AND

219
00:08:26,902 --> 00:08:29,405
1433 OF-THEY'RE DIMERS,

220
00:08:29,405 --> 00:08:31,107
CONSTITTIVE BINDERS THAT

221
00:08:31,107 --> 00:08:33,376
INTERACT WITH THE PROTEINS AND

222
00:08:33,376 --> 00:08:36,912
THE RAFS CAN ALSO DIMERIZE AND

223
00:08:36,912 --> 00:08:38,848
THEY CAN INTERACT WITH THEIR

224
00:08:38,848 --> 00:08:40,149
DOWN STREAM SUBSTRATE WHICH IS

225
00:08:40,149 --> 00:08:40,383
MEK.

226
00:08:40,383 --> 00:08:42,818
SO TO KIND OF PUT THIS

227
00:08:42,818 --> 00:08:44,854
ALTOGETHER IN CONTEXT, WHAT WE

228
00:08:44,854 --> 00:08:48,924
KNOW IS THAT IN A QUIE ESTIMATE

229
00:08:48,924 --> 00:08:51,193
THADENT CELL, BOTH BRAF AND CRAF

230
00:08:51,193 --> 00:08:54,397
ARE FOUND IN THE CYTOSOL AS

231
00:08:54,397 --> 00:08:55,998
INACTIVE MONITOR MERS AND I WILL

232
00:08:55,998 --> 00:08:58,834
JUST POINT OUT HERE THAT BRAF

233
00:08:58,834 --> 00:09:02,405
AND CRAF ARE THE PRIMARY DISEASE

234
00:09:02,405 --> 00:09:03,172
DRIVERS.

235
00:09:03,172 --> 00:09:05,941
BRAF IS PROMINENT DRIVER IN

236
00:09:05,941 --> 00:09:07,843
HUMAN CANCER, CRAF HAS NOT BEEN

237
00:09:07,843 --> 00:09:10,346
CHARACTERIZED SO MUCH AS BEING A

238
00:09:10,346 --> 00:09:12,548
CANCER DRIVER BUT BOTH OF THEM

239
00:09:12,548 --> 00:09:16,619
ARE DRIVERS OF THE DEVELOPMENTAL

240
00:09:16,619 --> 00:09:18,354
DISORDERS AND ARAF HAS NOT

241
00:09:18,354 --> 00:09:19,655
REALLY BEEN PICKED UP AS A

242
00:09:19,655 --> 00:09:23,059
DRIVER OF THE RAS-OP A THIES AND

243
00:09:23,059 --> 00:09:25,027
ISN'T REALLY KNOWN TO BE A

244
00:09:25,027 --> 00:09:26,162
PRIMARY DRIVER IN CANCER EITHER.

245
00:09:26,162 --> 00:09:29,365
SO LESS IS KNOWN ABOUT ARAF.

246
00:09:29,365 --> 00:09:32,268
BUT BRAF AND CRAF ARE ACTIVATED

247
00:09:32,268 --> 00:09:35,204
IN RESPONSE TO RAF'S ACTIVATION

248
00:09:35,204 --> 00:09:36,872
AND RTK SIGNALING AND THEY EXIST

249
00:09:36,872 --> 00:09:39,408
AS MONOMERS IN A QUIESCENT CELL.

250
00:09:39,408 --> 00:09:42,478
BUT THEN WHEN SIGNALS ARE

251
00:09:42,478 --> 00:09:45,748
RECEIVED BY THE CELLS AND RAS

252
00:09:45,748 --> 00:09:48,150
BECOMES ACTIVATED IN ITS ON

253
00:09:48,150 --> 00:09:49,852
STATE, IT CAN DIRECTLY INTERACT

254
00:09:49,852 --> 00:09:52,621
WITH THE RAF RBD AND THEN THAT

255
00:09:52,621 --> 00:09:54,223
ALLOWS THE CRD TO INTERACT WITH

256
00:09:54,223 --> 00:09:58,294
BOTH THE MEMBRANE AND TO CONTAKE

257
00:09:58,294 --> 00:09:58,861
RAS AS WELL.

258
00:09:58,861 --> 00:10:02,598
WE ALSO KNOW THAT 1433 COMES OFF

259
00:10:02,598 --> 00:10:06,202
OF THIS END TERMINAL SITE WHICH

260
00:10:06,202 --> 00:10:08,938
EXPOSES IT TO BE

261
00:10:08,938 --> 00:10:10,172
DEPHOSPHORYLATED BY A COMPLEX OF

262
00:10:10,172 --> 00:10:13,142
FOSTER 

263
00:10:13,142 --> 00:10:14,510
PHOSPHATASE COMPLEX THAT'S KNOWN

264
00:10:14,510 --> 00:10:16,312
AS SMP AND THERE'S BEEN A LOT OF

265
00:10:16,312 --> 00:10:20,149
STUDIES ON THE SMP COMPLEX AND

266
00:10:20,149 --> 00:10:22,885
ITS ROLE IN KRAS DRIVEN CANCERS

267
00:10:22,885 --> 00:10:23,185
AS WELL.

268
00:10:23,185 --> 00:10:25,821
BUT WE ALSO KNOW THAT THE RAF

269
00:10:25,821 --> 00:10:30,025
KINASE DOMAINS CAN DIMERIZE AND

270
00:10:30,025 --> 00:10:31,961
THEIR DIMERIZATION RESULTS IN

271
00:10:31,961 --> 00:10:32,261
ACTIVATION.

272
00:10:32,261 --> 00:10:34,296
AND I JUST WANT TO SHOW YOU HERE

273
00:10:34,296 --> 00:10:37,133
SOME OF THE PRIMARY DATA THAT

274
00:10:37,133 --> 00:10:40,402
KIND OF DEPICTS THAT, AND HERE

275
00:10:40,402 --> 00:10:42,371
WE'RE LOOKING AT SERUM STAR

276
00:10:42,371 --> 00:10:44,774
CELLS THAT HAVE EITHER BEEN

277
00:10:44,774 --> 00:10:46,008
SERUM STARVED OR TREATED WITH

278
00:10:46,008 --> 00:10:49,178
EGF AND WE'RE LOOKING AT EITHER

279
00:10:49,178 --> 00:10:50,913
THE WILD-TYPE ERAF PROTEIN OR

280
00:10:50,913 --> 00:10:53,649
CRAF OR A MUTATION IN THE

281
00:10:53,649 --> 00:10:54,383
DIMERIZATION MOTIF.

282
00:10:54,383 --> 00:10:59,889
SO THERE'S A CRITICAL RTKR MOTIF

283
00:10:59,889 --> 00:11:02,224
THAT IS ESSENTIAL FOR THE RAF TO

284
00:11:02,224 --> 00:11:08,497
DIMERIZE AND IF YOU MUTATE THIS

285
00:11:08,497 --> 00:11:10,065
RGENINE, TO HISTIDINE, WHAT YOU

286
00:11:10,065 --> 00:11:11,867
FIND IS YOU DISRUPT ALL

287
00:11:11,867 --> 00:11:13,169
DIMERIZATION AND IN RESPONSE TO

288
00:11:13,169 --> 00:11:14,203
GROWTH FACTOR SIGNALING YOU SEE

289
00:11:14,203 --> 00:11:18,240
NO IPT GREATER CREASE

290
00:11:18,240 --> 00:11:19,041
IN--INCREASE IN ACTIVITY WHERE

291
00:11:19,041 --> 00:11:21,343
NORMALLY YOU SEE AN INCREASE IN

292
00:11:21,343 --> 00:11:23,179
ACTIVITY AND RESPONSE TO BRAF

293
00:11:23,179 --> 00:11:25,114
AND CRAF AND YOU CAN SEE THAT IT

294
00:11:25,114 --> 00:11:28,417
CORRELATES WITH INCREASED

295
00:11:28,417 --> 00:11:28,751
DIMERIZATION.

296
00:11:28,751 --> 00:11:30,553
BRAF HAS A LITTLE BIT OF ABILITY

297
00:11:30,553 --> 00:11:33,455
WE TEND TO SEE SOME JEIMER

298
00:11:33,455 --> 00:11:35,825
DIMERIZATION, IT DOES GO UP, THE

299
00:11:35,825 --> 00:11:41,564
SAME IS TRUE BUT IT'S LESS FOR

300
00:11:41,564 --> 00:11:41,764
CRAF.

301
00:11:41,764 --> 00:11:43,399
BUT THE 1 THING I WILL SAY THAT

302
00:11:43,399 --> 00:11:44,166
WE DON'T COMPLETELY UNDERSTAND

303
00:11:44,166 --> 00:11:45,835
RIGHT NOW IS THAT THERE IS

304
00:11:45,835 --> 00:11:47,469
REALLY A PREFERENCE FOR BC

305
00:11:47,469 --> 00:11:49,338
DIMERS IN RESPONSE TO RAS

306
00:11:49,338 --> 00:11:50,606
ACTIVATION AND THAT'S SOMETHING

307
00:11:50,606 --> 00:11:52,741
THAT'S GOING TO CONTINUE TO KEEP

308
00:11:52,741 --> 00:11:56,111
US BUSY AND TRYING TO UNDERSTAND

309
00:11:56,111 --> 00:11:59,114
HOW THERE IS SELECTIVITY FOR THE

310
00:11:59,114 --> 00:12:01,383
DIFFERENT RAF FAMILY MEMBERS AND

311
00:12:01,383 --> 00:12:02,017
PROMOTING DIMERS.

312
00:12:02,017 --> 00:12:04,353
BUT FOR THIS, IN GROWTH FACTORS

313
00:12:04,353 --> 00:12:08,057
SIGNALING, IT'S THE BC DIMERS.

314
00:12:08,057 --> 00:12:10,926
SO, WHAT WE KNOW IS THAT

315
00:12:10,926 --> 00:12:13,162
DIMERIZATION IS ABSOLUTELY

316
00:12:13,162 --> 00:12:16,866
CRITICAL FOR ACTIVATION AND DOWN

317
00:12:16,866 --> 00:12:18,767
STREAM SIGNAL TRANSDITION TO MEK

318
00:12:18,767 --> 00:12:21,403
AND ERK, THE THING GOES ON AND

319
00:12:21,403 --> 00:12:22,571
TELLS THE CELL TRANSMITS SIGNALS

320
00:12:22,571 --> 00:12:25,674
TO TELL THE CELL TO DO A VARIETY

321
00:12:25,674 --> 00:12:26,508
OF DIFFERENT THINGS.

322
00:12:26,508 --> 00:12:29,778
BUT NOT ONLY IN GROWTH FACTOR

323
00:12:29,778 --> 00:12:31,013
SIGNALING IS DIMERIZATION

324
00:12:31,013 --> 00:12:34,350
CRITICAL IT ALSO PLAYS A ROLE IN

325
00:12:34,350 --> 00:12:36,952
HUMAN DISEASE STATES AS WELL.

326
00:12:36,952 --> 00:12:39,255
SO YOU CAN IMAGINE THAT

327
00:12:39,255 --> 00:12:41,790
DIMERIZATION IS CRITICAL IN

328
00:12:41,790 --> 00:12:45,995
ORDER FOR RECEPTOR TYROSEEN

329
00:12:45,995 --> 00:12:49,098
KINASES OR RAS PROTEINS THAT ARE

330
00:12:49,098 --> 00:12:51,400
MOOITATED SO IF ANY

331
00:12:51,400 --> 00:12:52,835
CONSTITTATIVELY RAS PROTEIN OR

332
00:12:52,835 --> 00:12:55,371
RECEPTOR IS GOING TO RECRUIT THE

333
00:12:55,371 --> 00:12:56,839
RAFs AND THEY ARE NEEDED FOR

334
00:12:56,839 --> 00:13:00,442
THE DOWN STREAM SIGNALING TO MEK

335
00:13:00,442 --> 00:13:01,510
AND ERK.

336
00:13:01,510 --> 00:13:04,647
AND ALSO IT'S CRITICAL FOR CLASS

337
00:13:04,647 --> 00:13:08,984
2 AND CLASS 3 DISEASE ASSOCIATED

338
00:13:08,984 --> 00:13:10,419
RAF MIEWITANTS AND NEAL ROSEN

339
00:13:10,419 --> 00:13:11,820
SEVERAL YEARS AGO, I THINK

340
00:13:11,820 --> 00:13:13,455
REALLY HAD A TOUR DEFORCE EFFORT

341
00:13:13,455 --> 00:13:19,361
TO TRY TO CHARACTERIZE ONCA

342
00:13:19,361 --> 00:13:20,162
GENIC RAF MUTANTS.

343
00:13:20,162 --> 00:13:22,398
AND THE BRAF THE 1 THAT PROBABLY

344
00:13:22,398 --> 00:13:24,433
EVERYONE IS MOST FAMILIAR WITH

345
00:13:24,433 --> 00:13:27,202
IS V600 E, IT'S THE PRIMARY

346
00:13:27,202 --> 00:13:28,871
DRIVER IN MALIGNANT ELANNOMA AND

347
00:13:28,871 --> 00:13:34,009
WHAT IS REALLY UNIQUE ABOUT THIS

348
00:13:34,009 --> 00:13:35,377
MUTATION, THE VAILING TO GLUE

349
00:13:35,377 --> 00:13:36,078
TISSUE AND MACROPHAGESSIC ACID

350
00:13:36,078 --> 00:13:38,580
IS THAT SINGLE MUTATION HAS

351
00:13:38,580 --> 00:13:41,116
ALLOWED A SALT BRIDGE TO FORM

352
00:13:41,116 --> 00:13:42,651
THAT ALLOWS THE CATALYTIC DOMAIN

353
00:13:42,651 --> 00:13:44,687
TO ASSUME THE ACTIVE

354
00:13:44,687 --> 00:13:47,589
CONFIRMATION IN THE ABSENCE OF

355
00:13:47,589 --> 00:13:47,923
DIMERIZATION.

356
00:13:47,923 --> 00:13:51,126
AND I WILL SHOW THAT A LITTLE

357
00:13:51,126 --> 00:13:52,861
BIT LATER BUT FOR THE MAIORITY

358
00:13:52,861 --> 00:13:55,064
OF THE OTHER MUTANTS THAT ARE

359
00:13:55,064 --> 00:13:59,802
CALLED CLASS 2, THEY HAVE THIS

360
00:13:59,802 --> 00:14:02,338
ABILITY TO SELF-DIMERIZE AND IN

361
00:14:02,338 --> 00:14:03,572
THAT CASE, DIMERIZATION IS

362
00:14:03,572 --> 00:14:07,209
REQUIRED FOR THEM TO TRANSMIT A

363
00:14:07,209 --> 00:14:08,444
SIGNAL.

364
00:14:08,444 --> 00:14:12,481
AND I THINK 1 OF THE VERY FIRST

365
00:14:12,481 --> 00:14:14,249
BIG SURPRISES IN THE RAF FIELD

366
00:14:14,249 --> 00:14:15,851
CAME WITH THE DEVELOPMENT OF

367
00:14:15,851 --> 00:14:26,261
SOME OF THE TARGETED INHIBITORS,

368
00:14:26,261 --> 00:14:28,731
BRAF NIB, AND THEY WERE GREAT,

369
00:14:28,731 --> 00:14:32,034
THEY WERE SUPPRESSING BSE

370
00:14:32,034 --> 00:14:33,569
ACTIVITY AND BLOCKING SIGNAL

371
00:14:33,569 --> 00:14:34,803
TRANSMISSION TO MEK.

372
00:14:34,803 --> 00:14:36,005
BUT WHAT WAS COMPLETELY

373
00:14:36,005 --> 00:14:37,973
UNEXPECTED AT THE TIME WAS THAT

374
00:14:37,973 --> 00:14:42,077
HE'S INHIBITORS HAD THIS

375
00:14:42,077 --> 00:14:42,711
PARADOXICAL ACTIVATING EFFECT

376
00:14:42,711 --> 00:14:45,581
AND WHAT I MEAN BY THAT WAS THAT

377
00:14:45,581 --> 00:14:49,151
WHEN THEY BOUND TO EITHER A

378
00:14:49,151 --> 00:14:50,986
HETERODIMER OR THE SELF-DIMERS,

379
00:14:50,986 --> 00:14:53,689
WHAT THEY ACTUALLY DID WAS THEY

380
00:14:53,689 --> 00:14:55,791
STABILIZED THE DIMERS, AND THEY

381
00:14:55,791 --> 00:14:59,828
HAD THIS PROPERTY OF ONCE THEY

382
00:14:59,828 --> 00:15:03,165
WERE BOUND IT CAUSED NEGATIVE

383
00:15:03,165 --> 00:15:05,467
COOPERATIVITY AND ALLOWING THE

384
00:15:05,467 --> 00:15:08,871
INHIBITOR TO BIND TO THE OTHER

385
00:15:08,871 --> 00:15:11,040
PROTOMER, SO THE INHIBITOR COULD

386
00:15:11,040 --> 00:15:13,142
BIND HERE BUT THEN YOU HAD

387
00:15:13,142 --> 00:15:14,543
REALLY REDUCED ABILITY FOR THE

388
00:15:14,543 --> 00:15:18,747
INHIBITOR TO GET INTO THE PRO

389
00:15:18,747 --> 00:15:20,182
TOMMER, AND SO SIGNALING STILL

390
00:15:20,182 --> 00:15:22,084
WENT TO MEK AND ERK.

391
00:15:22,084 --> 00:15:24,219
IN FACT, IT WAS AUGMENTED AND

392
00:15:24,219 --> 00:15:25,454
THIS IS 1 OF THE PRIMARY

393
00:15:25,454 --> 00:15:33,829
CONTRIBUTORS TO THE DRUG

394
00:15:33,829 --> 00:15:34,763
RESISTANCE TO VINMIRATANIB IN

395
00:15:34,763 --> 00:15:35,831
THE LAB AS WELL.

396
00:15:35,831 --> 00:15:37,933
SO IT RAISES THE QUESTION, THAT

397
00:15:37,933 --> 00:15:39,334
DIMERIZATION IS CRITICAL FOR

398
00:15:39,334 --> 00:15:42,071
NORMAL SIGNALING THROUGH RAF BUT

399
00:15:42,071 --> 00:15:45,441
IT CAN ALSO AFFECT THE EFFICACY

400
00:15:45,441 --> 00:15:49,812
OF RAF INHIBITORS AND CO PROMOTE

401
00:15:49,812 --> 00:15:50,446
DRUG RESISTANCE.

402
00:15:50,446 --> 00:15:52,714
SO THERE ARE BETTER WAYS TO

403
00:15:52,714 --> 00:15:53,515
TARGET THE RAFS?

404
00:15:53,515 --> 00:15:55,350
AND WE THOUGHT IT WOULD BE

405
00:15:55,350 --> 00:15:57,319
IMPORTANT AND I GUESS--OUR LAB

406
00:15:57,319 --> 00:15:59,154
WASN'T THE ONLY 1, EVERYONE

407
00:15:59,154 --> 00:16:00,255
WORKING ON RAF WANTED TO KNOW

408
00:16:00,255 --> 00:16:04,426
WHAT IS THE STRUCTURAL CHANGES

409
00:16:04,426 --> 00:16:06,528
THAT OCCUR WHEN BRAF TRANSITIONS

410
00:16:06,528 --> 00:16:09,731
FROM AN INACTIVE MONITOR MERTO

411
00:16:09,731 --> 00:16:10,399
AN ACTIVE DIMER.

412
00:16:10,399 --> 00:16:14,703
AND SO WE HAD A PHENOMENAL

413
00:16:14,703 --> 00:16:18,774
COLLABORATION WITH JULIANA

414
00:16:18,774 --> 00:16:22,678
MARTINEZ FIESCO, AND PING CHANG

415
00:16:22,678 --> 00:16:23,879
ON STRUCTURAL BIOLOGY UP IN

416
00:16:23,879 --> 00:16:24,146
FREDERICK.

417
00:16:24,146 --> 00:16:30,219
AND WHAT WOE DID IS WE TOOK OUR

418
00:16:30,219 --> 00:16:31,286
BRAF CONSTRUCT AND WE GENERATED

419
00:16:31,286 --> 00:16:33,755
HUMAN CELL LINES THAT WERE

420
00:16:33,755 --> 00:16:37,493
STABLY EXPRESSING THIS TAGGED

421
00:16:37,493 --> 00:16:37,826
PROTEIN.

422
00:16:37,826 --> 00:16:40,896
AND IT TURNS OUT THAT ACTUALLY

423
00:16:40,896 --> 00:16:42,197
THIS WAS VERY IMPORTANT TO HAVE

424
00:16:42,197 --> 00:16:44,533
IT IN HUMAN CELLS WHERE IT BE

425
00:16:44,533 --> 00:16:45,701
PROPERLY MODIFIED IN THE CELL

426
00:16:45,701 --> 00:16:48,437
AND WE COULD SERUM STARVE IT AND

427
00:16:48,437 --> 00:16:50,172
WE COULD ALSO GROWTH FACTOR

428
00:16:50,172 --> 00:16:50,606
TREAT IT.

429
00:16:50,606 --> 00:16:55,911
AND WE WERE ABLE TO SOLVE THE

430
00:16:55,911 --> 00:16:56,912
STRUCTURE, [INDISCERNIBLE]

431
00:16:56,912 --> 00:17:02,885
STRUCTURE OF 3 RAF COMPLEXES.

432
00:17:02,885 --> 00:17:04,586
SO THIS IS SHOWING THE DIMER

433
00:17:04,586 --> 00:17:05,554
COMPLEX AND THERE HAD BEEN A

434
00:17:05,554 --> 00:17:06,955
NUMBER OF THE DIMER COMPLEXES

435
00:17:06,955 --> 00:17:09,558
THAT HAVE BEEN REPORTED BUT THIS

436
00:17:09,558 --> 00:17:10,859
IS ACTUALLY THE FULL LENGTH

437
00:17:10,859 --> 00:17:13,695
PROTEIN BUT THE ONLY THING THAT

438
00:17:13,695 --> 00:17:17,466
WAS RESOLVED WAS THE KINASE

439
00:17:17,466 --> 00:17:20,869
DOMAIN AND 1433 DIMER POUND TO

440
00:17:20,869 --> 00:17:23,238
EACH PROTOMER OF THE RAF DIMER.

441
00:17:23,238 --> 00:17:25,874
THE ENTIRE REGULATORY DOMAIN WAS

442
00:17:25,874 --> 00:17:28,343
NOT RESOLVED AND WE AREN'T THE

443
00:17:28,343 --> 00:17:32,748
ONLY 1S WHO SOLVE THIS

444
00:17:32,748 --> 00:17:35,784
STRUCTURE, JOHN KIRRIAN AND MIKE

445
00:17:35,784 --> 00:17:38,754
[INDISCERNIBLE] ALSO, AND FOR

446
00:17:38,754 --> 00:17:40,489
ALL OF THESE COMPOWBDS THIS WAS

447
00:17:40,489 --> 00:17:41,456
NOT RESOLVED LIKELY DUE TO

448
00:17:41,456 --> 00:17:43,859
FLEXIBILITY IN THE ABSENCE OF

449
00:17:43,859 --> 00:17:46,295
ITS NORMAL MEMBRANE BINDING

450
00:17:46,295 --> 00:17:46,562
PARTNERS.

451
00:17:46,562 --> 00:17:53,001
BUT WHAT WAS REALLY EXCITING FOR

452
00:17:53,001 --> 00:17:57,139
US IS THAT WE WERE ABLE TO

453
00:17:57,139 --> 00:17:58,640
IDENTIFY 2 AUTOINHIBITTED

454
00:17:58,640 --> 00:17:59,808
COMPLEXES.

455
00:17:59,808 --> 00:18:00,976
ONE THAT CONTAINED--I HAVE TO

456
00:18:00,976 --> 00:18:02,678
FIND IT, 1 THAT CONTAINED MEK,

457
00:18:02,678 --> 00:18:05,414
AND 1 THAT DID NOT, BUT THE

458
00:18:05,414 --> 00:18:07,616
OTHER REGIONS EXCEPT FOR THE MEK

459
00:18:07,616 --> 00:18:12,254
BINDING WERE REALLY VIRTUALLY

460
00:18:12,254 --> 00:18:12,688
IDENTICAL.

461
00:18:12,688 --> 00:18:18,694
AND WHAT WAS UNIQUE, MIKE YEK

462
00:18:18,694 --> 00:18:23,899
HAS ALSO PUBLISHED A

463
00:18:23,899 --> 00:18:25,033
[INDISCERNIBLE] MONOMERBUT OURS

464
00:18:25,033 --> 00:18:26,868
ENDED UP BEING HIGHER RESOLUTION

465
00:18:26,868 --> 00:18:29,204
AND WE WERE ABLE TO UNIQUELY

466
00:18:29,204 --> 00:18:31,740
RESOLVE THE RBD WHICH IS SO

467
00:18:31,740 --> 00:18:33,108
CRITICAL FOR RAF ACTIVATION AND

468
00:18:33,108 --> 00:18:36,545
WE WERE ABLE TO DETERMINE ITS

469
00:18:36,545 --> 00:18:37,112
POSITION AND ORIENTATION.

470
00:18:37,112 --> 00:18:39,548
SO NOW THAT WE'VE GOT THE

471
00:18:39,548 --> 00:18:41,917
COMPLEX, WHAT CAN IT TELL US

472
00:18:41,917 --> 00:18:45,120
ABOUT THE ACTIVATION MECHANISMS.

473
00:18:45,120 --> 00:18:47,689
WE WERE ABLE TO DECIDE FOR THAT,

474
00:18:47,689 --> 00:18:49,458
THAT THE KINASE DOMAIN IS IN THE

475
00:18:49,458 --> 00:18:53,562
ACTIVE STATE, AND SO FOR KINASE

476
00:18:53,562 --> 00:18:56,732
OFFICKIAN ADOS, THAT MEANS THIS

477
00:18:56,732 --> 00:19:01,236
CRITICAL ALPHA C HELIX IS IN THE

478
00:19:01,236 --> 00:19:11,913
OUTPOSITION AND ITION AND WHAT -

479
00:19:29,698 --> 00:19:31,033
 BUT IN,A DITION TO THAT IT'S

480
00:19:31,033 --> 00:19:32,801
NOT JUST THE KINASE DOMAIN, YOU

481
00:19:32,801 --> 00:19:35,304
ALSO HAVE THIS DIMER OF 1433

482
00:19:35,304 --> 00:19:40,642
HERE AND ACTUALLY THE 1433 DIMER

483
00:19:40,642 --> 00:19:43,512
OBSCURES THAT CRITICAL RTKR

484
00:19:43,512 --> 00:19:45,347
MOTIF THAT'S REQUIRED FOR

485
00:19:45,347 --> 00:19:46,381
DIMERIZATION AND ANOTHER THING

486
00:19:46,381 --> 00:19:48,016
THAT I THINK I WANT TO POINT OUT

487
00:19:48,016 --> 00:19:52,754
THAT I THINK PEOPLE DON'T FULLY

488
00:19:52,754 --> 00:19:54,589
APPRECIATE SOMETIMES IS THIS

489
00:19:54,589 --> 00:19:56,425
ALPHA C HELIX IS IMMEDIATELY

490
00:19:56,425 --> 00:19:59,594
ADJACENT TO THIS RTKR MOTIF,

491
00:19:59,594 --> 00:20:01,530
THEY ARE SIDE BY SIDE.

492
00:20:01,530 --> 00:20:05,901
AND SO MOVEMENT OF THIS ALPHA C

493
00:20:05,901 --> 00:20:09,671
HELIX OR INTERACTIONS HERE CAN

494
00:20:09,671 --> 00:20:14,042
TRANSFER BACK AND FORTH.

495
00:20:14,042 --> 00:20:18,246
AND SO, WE KNOW THE CYSTINE RICH

496
00:20:18,246 --> 00:20:19,648
DOMAIN IS IN THE CENTER OF THE

497
00:20:19,648 --> 00:20:22,751
COMPLEX AND YOU CAN SEE HERE

498
00:20:22,751 --> 00:20:26,955
THAT IT CONTACTS ACTUALLY MAKES

499
00:20:26,955 --> 00:20:27,923
CONTACTS WITH BOTH 1433 POLYMERS

500
00:20:27,923 --> 00:20:31,593
AND THE KINASE DOMAIN AND THE

501
00:20:31,593 --> 00:20:35,097
RBD MAKES CONTACTS WITH THIS

502
00:20:35,097 --> 00:20:36,765
PROTOMEROF 1433 THAT'S BOUND TO

503
00:20:36,765 --> 00:20:39,101
THE C-TERMINAL SITE.

504
00:20:39,101 --> 00:20:43,372
SO, WHEN YOU HAVE ACTIVATION, WE

505
00:20:43,372 --> 00:20:45,640
KNOW THAT THE RBDs ARE

506
00:20:45,640 --> 00:20:46,575
RELEASED AND 1433 IS RELEASED

507
00:20:46,575 --> 00:20:50,679
FROM THE END TERMINAL AND IS

508
00:20:50,679 --> 00:20:53,548
THAT SITE'S DEPHOSPHORYLATED

509
00:20:53,548 --> 00:20:54,983
THERE'S A REARRANGEMENT.

510
00:20:54,983 --> 00:20:56,618
THERE'S THE DIMER INTERFACE IS

511
00:20:56,618 --> 00:21:00,489
EXPOSED AND ONCE YOU HAVE THESE

512
00:21:00,489 --> 00:21:01,656
DIMERIZATION, IT'S THOUGHT TO

513
00:21:01,656 --> 00:21:05,127
THEN CAUSE THIS ALPHA C HELIX TO

514
00:21:05,127 --> 00:21:09,431
DROP DOWN TO MELT THIS

515
00:21:09,431 --> 00:21:11,299
INHIBITORY TURN.

516
00:21:11,299 --> 00:21:13,902
AND YOU HAVE 1433 REARRANGING

517
00:21:13,902 --> 00:21:17,239
AND BINDING TO BOTH PROTOMERS OF

518
00:21:17,239 --> 00:21:20,509
THE RAF DIMER AND THE WORK FROM

519
00:21:20,509 --> 00:21:21,810
GENENTECH GROUP HAS REALLY SHOWN

520
00:21:21,810 --> 00:21:24,546
THIS IS ESSENTIAL FOR KEEPING

521
00:21:24,546 --> 00:21:28,183
THIS KINASE DOMAINS DIMERIZED

522
00:21:28,183 --> 00:21:30,318
DURING CATALIAISONS, AS IT BINDS

523
00:21:30,318 --> 00:21:31,520
MEK, PHOSPHORYLATES IT AND

524
00:21:31,520 --> 00:21:33,488
RELEASED, IN EK THAT YOU NEED

525
00:21:33,488 --> 00:21:35,957
THE 1433 THERE TO KIND OF HOLD

526
00:21:35,957 --> 00:21:36,892
THE 2 TOGETHER EMPLOY KIND OF

527
00:21:36,892 --> 00:21:41,062
THINK OF IT LIKE A SEE SAW GOING

528
00:21:41,062 --> 00:21:45,500
BACK AND FORTH FROM 1433 HOLDS

529
00:21:45,500 --> 00:21:46,902
THEM TOGETHER, BUT YOU CAN ALSO

530
00:21:46,902 --> 00:21:51,773
SEE, I MEAN, THERE'S A LOT THAT

531
00:21:51,773 --> 00:21:52,441
WE DON'T UNDERSTAND.

532
00:21:52,441 --> 00:21:55,744
AND SO WE WERE STARTING TO

533
00:21:55,744 --> 00:21:57,045
WONDER, IS THERE WAYS THAT WE

534
00:21:57,045 --> 00:21:58,914
CAN FILL IN SOME OF THESE

535
00:21:58,914 --> 00:22:02,884
MISSING STRUCTURES, SO WHEN THE

536
00:22:02,884 --> 00:22:07,322
RAFS BEGIN TO YOU KNOW INFOLD OR

537
00:22:07,322 --> 00:22:09,157
THE AUTOINHIBITTED COMPLEX KIND

538
00:22:09,157 --> 00:22:15,797
OF UNRAVELS, WHAT HAPPENS FIRST 

539
00:22:15,797 --> 00:22:17,399
YOU KNOW DOES 1433 COME OFF,

540
00:22:17,399 --> 00:22:18,266
DOES KINASE COME OUT?

541
00:22:18,266 --> 00:22:20,068
AND WE THINK IT MADE BE

542
00:22:20,068 --> 00:22:22,237
IMPORTANT AGAIN BECAUSE IT MAY

543
00:22:22,237 --> 00:22:23,772
IDENTIFY NOW TARGETS AND

544
00:22:23,772 --> 00:22:24,973
APPROACHES FOR CANCER THERAPY.

545
00:22:24,973 --> 00:22:25,674
AND IN PARTICULAR WE WANT TO

546
00:22:25,674 --> 00:22:29,211
KNOW WHAT IS THE EXACT FUNCTION

547
00:22:29,211 --> 00:22:31,680
OF THE RAS RAF INTERACTION

548
00:22:31,680 --> 00:22:32,280
BECAUSE IT'S KIND OF

549
00:22:32,280 --> 00:22:33,782
CONTROVERSIAL IN THE FIELD RIGHT

550
00:22:33,782 --> 00:22:37,319
NOW IS ALL RAS DOES IT LOCALIZE

551
00:22:37,319 --> 00:22:39,454
RAF TO THE MEMBRANE OR DOES IT

552
00:22:39,454 --> 00:22:44,493
PLAY A MORE ACTIVE ROLE IN

553
00:22:44,493 --> 00:22:45,193
DISASSEMBLING THE AUTOINHIBITTED

554
00:22:45,193 --> 00:22:45,994
COMPLEX IS WHAT IS THE

555
00:22:45,994 --> 00:22:48,697
CONTRIBUTION OF THE PLASMA

556
00:22:48,697 --> 00:22:52,067
MEMBRANE, AND HOW IMPORTANT IS

557
00:22:52,067 --> 00:22:53,034
THIS PHOSPHATASE COMPLEX.

558
00:22:53,034 --> 00:22:54,469
WELL, WE'VE BEEN REALLY

559
00:22:54,469 --> 00:22:57,439
FORTUNATE NOW THAT WE'RE PART OF

560
00:22:57,439 --> 00:22:59,074
THIS ADMIRAL PROJECT AND IT'S AN

561
00:22:59,074 --> 00:23:02,077
AI DRIVEN, IT STANDS FOR AI

562
00:23:02,077 --> 00:23:03,912
DRIVEN MULTISCALE INVESTIGATION

563
00:23:03,912 --> 00:23:09,284
OF RAS, RAF ACTIVATION LIFE

564
00:23:09,284 --> 00:23:10,952
CYCLE AND IT IS COLLABORATION

565
00:23:10,952 --> 00:23:13,655
BETWEEN THE NATIONAL CANCER

566
00:23:13,655 --> 00:23:15,524
INSTITUTE, THE NCI, RAS

567
00:23:15,524 --> 00:23:19,160
INITIATIVE AND ALSO TAKES

568
00:23:19,160 --> 00:23:23,098
ADVANTAGE OF THE COMPUTER

569
00:23:23,098 --> 00:23:24,299
CAPABILITIES PRESENT AT LAWRENCE

570
00:23:24,299 --> 00:23:25,767
LIVER MOORE NATIONAL LABS WHICH

571
00:23:25,767 --> 00:23:31,806
ARE AMONGST THE MOST POWERFUL

572
00:23:31,806 --> 00:23:33,174
AND ACTUALLY GLOBALLY SO WE'RE

573
00:23:33,174 --> 00:23:35,210
GOING FROM STRUCTURES IN KNOWN

574
00:23:35,210 --> 00:23:40,949
BIOLOGY, WE'RE DOING LARGE SCALE

575
00:23:40,949 --> 00:23:41,917
AND LONG COMPUTATIONAL MODELS

576
00:23:41,917 --> 00:23:44,319
AND THEN COMING BACK AND TESTING

577
00:23:44,319 --> 00:23:46,855
AND VALIDATING THE MODELS.

578
00:23:46,855 --> 00:23:49,090
AND SO WHEN WE BEGAN THIS, WE

579
00:23:49,090 --> 00:23:52,060
KIND OF START WIDE OUR STRUCTURE

580
00:23:52,060 --> 00:23:53,962
AND SO, THIS IS KIND OF A SPACE

581
00:23:53,962 --> 00:23:56,131
FILLING MODEL OF THAT AND WHAT

582
00:23:56,131 --> 00:23:57,899
YOU CAN SEE IS THE RBD IS

583
00:23:57,899 --> 00:24:00,435
SITTING ON TOP OF THIS 1433

584
00:24:00,435 --> 00:24:03,572
PROTOMER, AND IF WE LOOK AT THE

585
00:24:03,572 --> 00:24:04,139
RESIDUES INVOLVED IN THIS

586
00:24:04,139 --> 00:24:07,108
INTERACTION AND IT WAS QUITE A

587
00:24:07,108 --> 00:24:08,176
LARGE CONTEXT SURFACE AREA, YOU

588
00:24:08,176 --> 00:24:14,282
CAN SEE IT ALSO HAS A DEGREE

589
00:24:14,282 --> 00:24:17,986
CONSIDERABLE ELECTROSTATIC

590
00:24:17,986 --> 00:24:18,787
CHARGE COMPLIMENTARY.

591
00:24:18,787 --> 00:24:21,489
AND THE DYNAMIC SIMULATIONS THAT

592
00:24:21,489 --> 00:24:23,825
WERE   PERFORMED AT THE

593
00:24:23,825 --> 00:24:25,026
LIVERMOORE LAB WERE ABLE TO

594
00:24:25,026 --> 00:24:29,097
IDENTIFY A NUMBER OF POTENTIAL

595
00:24:29,097 --> 00:24:30,365
HYDROGEN BONDS THAT CAN FORM

596
00:24:30,365 --> 00:24:36,471
HERE BETWEEN THIS IS THE ALPHA

597
00:24:36,471 --> 00:24:40,609
HELIXES OF THE RBD LOOP 2 AND

598
00:24:40,609 --> 00:24:44,412
ALPHA HELIXES 8 AND 9 IN THE

599
00:24:44,412 --> 00:24:46,348
1433 PROTOMER.

600
00:24:46,348 --> 00:24:48,183
BUT, IN THIS PARTICULAR

601
00:24:48,183 --> 00:24:52,854
ORIENTATION, WHAT YOU CAN ALSO

602
00:24:52,854 --> 00:24:57,058
SEE IS THAT THE 4 CRITICAL BASIC

603
00:24:57,058 --> 00:25:00,462
RESIDUES THAT MAKE IONIC

604
00:25:00,462 --> 00:25:02,364
INTERACTIONS WITH RAS ARE

605
00:25:02,364 --> 00:25:02,964
LARGELY EXPOSED.

606
00:25:02,964 --> 00:25:06,635
AND WE CAN SEE IN OUR STRUCTURE

607
00:25:06,635 --> 00:25:08,937
THAT  THERE WAS A POCKET FOR RAS

608
00:25:08,937 --> 00:25:10,905
TO FIT IN HERE SO WHEN WE WEPT

609
00:25:10,905 --> 00:25:13,908
ON TO DO THE MODELING AS RAS

610
00:25:13,908 --> 00:25:16,845
WOULD ENGAGE WITH THE BASIC

611
00:25:16,845 --> 00:25:21,249
RESDUES, WHAT WE SAW IS THAT IT

612
00:25:21,249 --> 00:25:22,450
WOULD GENERATE ASTERIC CLASS AT

613
00:25:22,450 --> 00:25:26,721
THIS INTERFACE AND IN ADDITION

614
00:25:26,721 --> 00:25:29,124
WHILE THESE ACIDIC RESIDUES ARE

615
00:25:29,124 --> 00:25:30,058
VERY COMPLIMENTARY WITH THE

616
00:25:30,058 --> 00:25:34,029
BASIC CHARGES AND THE RBD WHAT

617
00:25:34,029 --> 00:25:37,132
YOU CAN SEE IS THE 2 RESIDUES

618
00:25:37,132 --> 00:25:40,235
DOWN HERE TO THE GLUTEAMILLIOIC

619
00:25:40,235 --> 00:25:42,504
ACID RESIDUES WOULD CAUSE A

620
00:25:42,504 --> 00:25:44,072
REPULINGS HERE AGAIN AT THE

621
00:25:44,072 --> 00:25:47,909
INTERFACE SO BOTH A CLASS AND A

622
00:25:47,909 --> 00:25:48,643
ELECTROSTATIC REPULINGS.

623
00:25:48,643 --> 00:25:52,447
SO OUR QUESTION WAS, DOES THE

624
00:25:52,447 --> 00:25:55,350
RAS RBD CONTACT ACTUALLY

625
00:25:55,350 --> 00:25:57,686
INITIATE THE DISASSEMBLY OF THE

626
00:25:57,686 --> 00:25:58,787
AUTOINHIBITTED COMPLEX.

627
00:25:58,787 --> 00:26:00,955
AND SO, WE WONDERED, YOU KNOW

628
00:26:00,955 --> 00:26:04,392
CAN WE DEVELOP AN INVITRO RAF

629
00:26:04,392 --> 00:26:06,361
ACTIVATION ASSAY TO CONFIRM THE

630
00:26:06,361 --> 00:26:09,431
IMPORTANCE OF VARIOUS COMPONENTS

631
00:26:09,431 --> 00:26:11,966
THAT FACILITATE THE MONITOR

632
00:26:11,966 --> 00:26:13,334
MERTO DIMER TRANSITION AND CAN

633
00:26:13,334 --> 00:26:15,236
THIS HELP ORDER THE STEPS THAT

634
00:26:15,236 --> 00:26:19,708
ARE REQUIRED FOR THIS

635
00:26:19,708 --> 00:26:20,008
TRANSITION.

636
00:26:20,008 --> 00:26:23,812
SO BASICALLY WHAT WE DID WAS WE

637
00:26:23,812 --> 00:26:25,880
TOOK OUR PURIFIED BRAF MONOMERS

638
00:26:25,880 --> 00:26:29,884
FROM THE HUMAN CELLS THAT WE HAD

639
00:26:29,884 --> 00:26:36,524
AND WE INCUBATED THEM IN TEST

640
00:26:36,524 --> 00:26:38,793
TUBES, EITHER KRAS THAT WAS

641
00:26:38,793 --> 00:26:40,528
FULLY PROCESSED AND GTP LOADED,

642
00:26:40,528 --> 00:26:42,464
LIPO STUDIES OF MULTIPLE

643
00:26:42,464 --> 00:26:43,732
ENDOCRINES THAT CRATE 30 PRGT

644
00:26:43,732 --> 00:26:45,066
FOSTER NURSED FETILE SERIES

645
00:26:45,066 --> 00:26:48,503
POINTSINE OR IS THE SMP COMPLEX

646
00:26:48,503 --> 00:26:51,940
WE HOPED WOULD BE ABLE TO

647
00:26:51,940 --> 00:26:52,907
PHOSPHORYLATE THAT PROTEIN 1433

648
00:26:52,907 --> 00:26:54,576
BINDING SITE OR A COMBINATION.

649
00:26:54,576 --> 00:26:58,913
AND THEN WE ADDED KINASE BUFFER

650
00:26:58,913 --> 00:27:02,117
AND DID OLD SCHOOL P32 ATP.

651
00:27:02,117 --> 00:27:04,819
AND THEN RAN A GEL AND ANALYZED

652
00:27:04,819 --> 00:27:07,455
IT FOR THE PHOSPHORYLATION OF

653
00:27:07,455 --> 00:27:11,693
MEK THAT WE THEN ADDED TO THE

654
00:27:11,693 --> 00:27:11,960
SAMPLES.

655
00:27:11,960 --> 00:27:14,028
AND ACTUALLY, I THINK THE DESIGN

656
00:27:14,028 --> 00:27:16,431
OF THIS ASSAY WAS REALLY

657
00:27:16,431 --> 00:27:18,099
CRITICAL IN THAT IT'S ONLY

658
00:27:18,099 --> 00:27:20,268
MONITORING WHAT IS HAPPENING IN

659
00:27:20,268 --> 00:27:23,705
THE TEST TUBE BECAUSE WE'RE ONLY

660
00:27:23,705 --> 00:27:25,406
MONITORING WHETHER THE BRAF

661
00:27:25,406 --> 00:27:29,310
ACTIVITY ALL BY ITSELF OR DOES

662
00:27:29,310 --> 00:27:35,116
IT CHANGE IN THE PRESENCE OF ANY

663
00:27:35,116 --> 00:27:35,617
OF THESE ADDITIONS.

664
00:27:35,617 --> 00:27:37,085
AND SO THESE ARE THE RESULTS

665
00:27:37,085 --> 00:27:39,120
THAT WE FOUND THAT BASICALLY

666
00:27:39,120 --> 00:27:42,590
THERE'S A LOW BASAL ACTIVITY OF

667
00:27:42,590 --> 00:27:43,525
THE AUTOINHIBITTOR MONOMERAND WE

668
00:27:43,525 --> 00:27:46,327
SEE ABOUT A 2 OF THIS 3 FOLD

669
00:27:46,327 --> 00:27:52,200
INCREASE WHEN WE ADD IN KRAS, WE

670
00:27:52,200 --> 00:27:53,701
CONSISTENTLY SEE A FALLER EFFECT

671
00:27:53,701 --> 00:27:54,369
WITH THE LIPO STUDIES OF

672
00:27:54,369 --> 00:27:55,970
MULTIPLE ENDOCRINES AND VERY

673
00:27:55,970 --> 00:27:58,039
LITTLE EECT WITH THE SMP COMPLEX

674
00:27:58,039 --> 00:27:59,574
AND WHAT I THINK'S REALLY NICE

675
00:27:59,574 --> 00:28:01,676
ABOUT THIS, YOU CAN ACTUALLY, WE

676
00:28:01,676 --> 00:28:04,112
CUT OUT AND COUNT THE MEK THAT'S

677
00:28:04,112 --> 00:28:05,280
PHOSPHORYLATED BUT BEFORE WE DO

678
00:28:05,280 --> 00:28:06,815
THAT WE JUST ECPOSE IT AND SO

679
00:28:06,815 --> 00:28:09,851
VISUALLY YOU CAN JUST SEE, YES,

680
00:28:09,851 --> 00:28:12,153
THERE'S A MAJOR INCREASE IN RAF

681
00:28:12,153 --> 00:28:16,257
ACTIVITY IN THE PRESENCE OF

682
00:28:16,257 --> 00:28:17,559
ADDING FULLY PROCESSED KRAS.

683
00:28:17,559 --> 00:28:21,196
AND WE COULD ALSO IN THE SAMPLES

684
00:28:21,196 --> 00:28:22,363
THAT WE'RE ADDING, BECAUSE WE

685
00:28:22,363 --> 00:28:23,798
RAN EVERYTHING ON A GEL, WE CAN

686
00:28:23,798 --> 00:28:28,369
PROBE TO SEE, OKAY, DID SEE SMP

687
00:28:28,369 --> 00:28:28,970
ACTUALLY DEPHOSPHORYLATE THE

688
00:28:28,970 --> 00:28:29,938
SITE THAT WAS IT SUPPOSED TO AND

689
00:28:29,938 --> 00:28:31,973
YOU CAN SEE THAT WE ARE SEEING A

690
00:28:31,973 --> 00:28:34,442
REDUCTION IN THE PHOSPHORYLATION

691
00:28:34,442 --> 00:28:34,776
THERE.

692
00:28:34,776 --> 00:28:37,045
AND EVEN THIS EXPERIMENT GAVE US

693
00:28:37,045 --> 00:28:39,881
2 IMPORTANT PIECES OF

694
00:28:39,881 --> 00:28:40,815
INFORMATION THAT BRAF AT LEAST

695
00:28:40,815 --> 00:28:45,353
IN A TEST TUBE, CAN BE ACTIVATED

696
00:28:45,353 --> 00:28:46,688
WITHOUT DEPHOSPHORYLATION OF

697
00:28:46,688 --> 00:28:48,923
THIS 1433 BINDING SITE AND THAT

698
00:28:48,923 --> 00:28:49,958
DEPHOSPHORYLATION OF THIS

699
00:28:49,958 --> 00:28:54,796
BINDING SITE ON ITS OWN ISN'T

700
00:28:54,796 --> 00:28:57,866
ENOUGH TO ACTIVATE BRAF.

701
00:28:57,866 --> 00:29:00,902
SO WE WANTED TO DO THE PROPER

702
00:29:00,902 --> 00:29:02,103
CONTROLS, WE WANTED TO MAKE SURE

703
00:29:02,103 --> 00:29:06,608
THE ACTIVATION WE'RE SEEING WITH

704
00:29:06,608 --> 00:29:08,509
KRAS IS ACTUALLY DEPENDENT ON

705
00:29:08,509 --> 00:29:10,845
THE ACTIVATION STATE OF KRAS.

706
00:29:10,845 --> 00:29:15,283
SO WHEN IT'S INACTIVE WHEN IT'S

707
00:29:15,283 --> 00:29:20,154
GDP-BOUND BUT HERE IS AN THE

708
00:29:20,154 --> 00:29:21,122
HYDROLYZABLE ANALOGUE OF GTP AND

709
00:29:21,122 --> 00:29:28,296
YOU CAN SEE AGAIN, WE'RE SEEING

710
00:29:28,296 --> 00:29:30,598
ACTIVATION BUT WITH GDP, IT'S

711
00:29:30,598 --> 00:29:34,569
NOT, YOU AND SEE IT OVER HERE AS

712
00:29:34,569 --> 00:29:34,769
WELL.

713
00:29:34,769 --> 00:29:36,537
ANOTHER THING THAT WAS I THINK

714
00:29:36,537 --> 00:29:40,141
REALLY IMPORTANT TO US IS OKAY

715
00:29:40,141 --> 00:29:43,478
SO RAS IS ACTIVATING BUT IS

716
00:29:43,478 --> 00:29:44,312
DIMERIZATION REQUIRED.

717
00:29:44,312 --> 00:29:46,180
IS RAS PROMOTING THE DIMERS TO

718
00:29:46,180 --> 00:29:49,751
FORM, AND SO WE ADDED IN THE

719
00:29:49,751 --> 00:29:53,354
CONTROL OF MUTATING THE CRITICAL

720
00:29:53,354 --> 00:29:56,891
DIMER INTERFACE WITH THIS

721
00:29:56,891 --> 00:29:57,725
RGENINE TO HISTIDINE MUTATION

722
00:29:57,725 --> 00:29:59,594
EMPLOY AND WHAT WE FOUND WAS

723
00:29:59,594 --> 00:30:01,896
THAT FOR BRAF TO BE ACTIVATED BY

724
00:30:01,896 --> 00:30:05,767
RAF IT HAD TO BE DIMERIZATION

725
00:30:05,767 --> 00:30:06,167
COMPETENT.

726
00:30:06,167 --> 00:30:09,203
AND 1 OTHER THING WE DID WAS THE

727
00:30:09,203 --> 00:30:11,940
TITRATION CURVE WE'RE ADDING

728
00:30:11,940 --> 00:30:13,107
INCREASING AMOUNTS OF KRAS AND

729
00:30:13,107 --> 00:30:16,210
AS YOU CAN SEE, THAT THERE IS A

730
00:30:16,210 --> 00:30:17,478
DOSE RESPONSE EFFECT HERE AND

731
00:30:17,478 --> 00:30:21,749
CAN YOU  VISUALLY SEE IT HERE IN

732
00:30:21,749 --> 00:30:23,284
THE GEL THAT WE'RE GETTING

733
00:30:23,284 --> 00:30:24,419
INCREASING LEVELS OF ACTIVATION

734
00:30:24,419 --> 00:30:26,654
WITH THE MORE RAS THAT WE ADD.

735
00:30:26,654 --> 00:30:29,190
SO WE WANTED TO GO BACK AND SAY

736
00:30:29,190 --> 00:30:33,661
TO LOOK AT WHAT ABOUT THE PFs

737
00:30:33,661 --> 00:30:34,329
CONTAINING LIPO STUDIES OF

738
00:30:34,329 --> 00:30:35,096
MULTIPLE ENDOCRINES, MAYBE WE

739
00:30:35,096 --> 00:30:36,397
WERE JUST AT THE WRONG

740
00:30:36,397 --> 00:30:39,901
CONCENTRATION, SO WE ALSO DID A

741
00:30:39,901 --> 00:30:42,203
DOSE RESPONSE AND INCREASING

742
00:30:42,203 --> 00:30:43,638
LEVELS OF THE LIPOSOMES, AND

743
00:30:43,638 --> 00:30:46,841
WHAT WE FOUND WAS THAT WE KIND

744
00:30:46,841 --> 00:30:49,310
OF PLATEAUED, BUT WE NEVER SAW

745
00:30:49,310 --> 00:30:53,748
MORE THAN A 2 FOLD INCREASE IN

746
00:30:53,748 --> 00:30:54,282
ACTIVATION.

747
00:30:54,282 --> 00:30:56,718
BUT THEN, WHEN WE MIXED THE LIPO

748
00:30:56,718 --> 00:30:59,554
STUDIES S

749
00:30:59,554 --> 00:31:02,123
OMES WITH THE RAS WHAT WE

750
00:31:02,123 --> 00:31:04,292
SAW WAS AN AUGMENTING EFFECT

751
00:31:04,292 --> 00:31:05,493
HERE OF THE ACTIVATION LEVEL

752
00:31:05,493 --> 00:31:08,496
THAT WE'RE ACHIEVING IF WE HAVE

753
00:31:08,496 --> 00:31:12,834
FULLY PROCESSED KRAS PLUS THE

754
00:31:12,834 --> 00:31:13,101
LIPOSOMES.

755
00:31:13,101 --> 00:31:14,736
BUT REMEMBER, THESE ASSAYS ARE

756
00:31:14,736 --> 00:31:17,171
DONE IN THE TEST TUBES SO

757
00:31:17,171 --> 00:31:18,773
THEY'RE IN SOLUTION, SO WE'RE

758
00:31:18,773 --> 00:31:20,742
KIND OF DEPENDING ON THEM MIXING

759
00:31:20,742 --> 00:31:22,810
AROUND IN THE TEST TUBE, SO WE

760
00:31:22,810 --> 00:31:28,282
THOUGHT IT WOULD BE IMPORTANT TO

761
00:31:28,282 --> 00:31:29,550
TRY TO RECAPITULATE A MORE

762
00:31:29,550 --> 00:31:30,885
AUTHENTIC ENVIRONMENT.

763
00:31:30,885 --> 00:31:33,855
SO IN COLLABORATION WITH SUZANNE

764
00:31:33,855 --> 00:31:37,125
SANDIN IN THE RAS INITIATIVE,

765
00:31:37,125 --> 00:31:39,127
SHE WAS ABLE TO GENERATE FOR US

766
00:31:39,127 --> 00:31:41,796
SOME LIPID NANO DISKS THAT

767
00:31:41,796 --> 00:31:43,765
CONTAINED KRAS, WITH KRAS

768
00:31:43,765 --> 00:31:46,501
TETHERED ON TO THE NANO DISK.

769
00:31:46,501 --> 00:31:49,003
AND SO, THESE ARE THE RESULTS

770
00:31:49,003 --> 00:31:50,171
FROM THAT EXPERIMENT THERE.

771
00:31:50,171 --> 00:31:53,374
AS YOU CAN SEE, IF THE NEW NANO

772
00:31:53,374 --> 00:31:57,345
DISK DID NOT HAVE KRAS PRESENT

773
00:31:57,345 --> 00:31:58,813
WE SAW VERY LITTLE ACTIVATION

774
00:31:58,813 --> 00:32:01,916
BUT THEN FOR THOSE THAT HAD 3

775
00:32:01,916 --> 00:32:04,018
MOLECULES OF KRAS, IT WAS UP TO

776
00:32:04,018 --> 00:32:07,155
REALLY ALMOST 7 FOLD INCREASE IN

777
00:32:07,155 --> 00:32:11,893
ACTIVITY AND IF SHE ADDED MORE

778
00:32:11,893 --> 00:32:13,728
KRAS MOLECULES, PROTEINS, SHE

779
00:32:13,728 --> 00:32:16,030
DOUBLED IT TO 6 KRAS THERE AND

780
00:32:16,030 --> 00:32:19,233
THEN WE SAW AN EVEN FURTHER

781
00:32:19,233 --> 00:32:21,969
INCREASE AND AGAIN, THIS IS A

782
00:32:21,969 --> 00:32:23,805
RAS DEPENDENT EFFECT WE'RE

783
00:32:23,805 --> 00:32:28,776
SEEING IN THE CONTEXT OF THE

784
00:32:28,776 --> 00:32:32,246
LIPID THAT IS PRESENT ON THE

785
00:32:32,246 --> 00:32:32,980
NANO DISK.

786
00:32:32,980 --> 00:32:34,282
SO PUTTING ALL THIS TOGETHER, I

787
00:32:34,282 --> 00:32:36,818
THINK WE HAVE LEARNED A LOT

788
00:32:36,818 --> 00:32:39,554
ABOUT IN OF THE MOLECULAR

789
00:32:39,554 --> 00:32:42,824
MECHANISMS AND SO, IF YOU LOOK

790
00:32:42,824 --> 00:32:44,926
WE ARE OUR AUTOINHIBITTOR

791
00:32:44,926 --> 00:32:47,228
MONOMERAND THEN WHEN YOU HAVE

792
00:32:47,228 --> 00:32:48,629
RAS, BECOMES GTP LOADED AND

793
00:32:48,629 --> 00:32:52,066
ACTIVE IN THE RBD CAN INTERACT,

794
00:32:52,066 --> 00:32:55,636
IT CAUSES THIS CLASH WITH THIS

795
00:32:55,636 --> 00:33:01,075
PROTOMEROF 1433 THAT GENERATES A

796
00:33:01,075 --> 00:33:03,277
CLASH AND CAUSES THE REPULINGS

797
00:33:03,277 --> 00:33:07,849
OF 1433 THAT STARTS TO UNRAVEL

798
00:33:07,849 --> 00:33:09,083
THIS AUTOINHIBITTED COMPLEX.

799
00:33:09,083 --> 00:33:11,953
AND THAT THEN ALLOWS SMP TO COME

800
00:33:11,953 --> 00:33:13,788
IN AND DEPHOSPHORYLATE THAT.

801
00:33:13,788 --> 00:33:16,424
AND I THINK EVEN THOUGH SMP

802
00:33:16,424 --> 00:33:17,492
WASN'T CRITICAL IN OUR TEST

803
00:33:17,492 --> 00:33:19,760
TUBE, I DO FULLY BELIEVE THAT IT

804
00:33:19,760 --> 00:33:21,829
IS IMPORTANT IN THE CELL BECAUSE

805
00:33:21,829 --> 00:33:24,265
REMEMBER, IN THE CELL WHEN YOU

806
00:33:24,265 --> 00:33:26,200
HAVE GROWTH FACTOR SIGNALS

807
00:33:26,200 --> 00:33:27,368
COMING IN, THERE'S A LOT GOING

808
00:33:27,368 --> 00:33:30,171
ON AT THE MEMBRANE, IT'S NOT

809
00:33:30,171 --> 00:33:31,539
JUST RAS SIGNALING, YOU HAVE

810
00:33:31,539 --> 00:33:33,541
ACTIVATION OF OTHER KINASES AND

811
00:33:33,541 --> 00:33:36,644
PHOSPHATASES BUT WHAT THIS DOES,

812
00:33:36,644 --> 00:33:39,046
BY DEPHOSPHORYLATING THIS SITE,

813
00:33:39,046 --> 00:33:40,915
THIS ACTUALLY PROVIDES THE

814
00:33:40,915 --> 00:33:45,019
DIRECTIONALITY OF  SIGNALING.

815
00:33:45,019 --> 00:33:46,888
IT DOESN'T ALLOW THIS 1433 TO

816
00:33:46,888 --> 00:33:49,857
COME BACK AND REBIND AND IT

817
00:33:49,857 --> 00:33:52,126
CAUSES THE DIRECTIONALITY TO BE

818
00:33:52,126 --> 00:34:00,034
FORCED INTO THE DIMER FORMATION.

819
00:34:00,034 --> 00:34:03,371
SO I'M GOING TO SWITCH GEARS

820
00:34:03,371 --> 00:34:04,071
COMPLETELY NOW.

821
00:34:04,071 --> 00:34:07,909
SO WE'VE TAKEN A LOT WE LEARNED

822
00:34:07,909 --> 00:34:08,809
ABOUT THE MOLECULAR MECHANISM

823
00:34:08,809 --> 00:34:10,645
BUT I WANT TO TALK ABOUT ITS

824
00:34:10,645 --> 00:34:13,981
ROLE IN HUMAN CANCER AND THE

825
00:34:13,981 --> 00:34:16,651
OTHER RAS-OP A THIES AS WELL.

826
00:34:16,651 --> 00:34:18,953
AND BRAF IS A DRIVER IN BOTH.

827
00:34:18,953 --> 00:34:21,455
SO FOR BRAF MOST OF THE

828
00:34:21,455 --> 00:34:24,392
ONCOGENIC DRIVERS OCCUR WITHIN

829
00:34:24,392 --> 00:34:25,693
THE CATALYTIC DOMAIN AND THESE

830
00:34:25,693 --> 00:34:27,495
ARE MUTATIONS THAT ARE OFTEN

831
00:34:27,495 --> 00:34:28,596
STRONG GAIN OF FUNCTION AND

832
00:34:28,596 --> 00:34:31,499
THEY'VE BEEN SHOWN TO BE

833
00:34:31,499 --> 00:34:32,366
EMBRYONIC LETHAL.

834
00:34:32,366 --> 00:34:36,504
BUT FOR THE

835
00:34:36,504 --> 00:34:38,739
RAS-OPOTHAY MUTATIONS THEY TEND

836
00:34:38,739 --> 00:34:40,541
TO BE WEAKER AND THE REAL HOT

837
00:34:40,541 --> 00:34:42,376
SPOT IS THIS PSYCHIATRISTIN RICH

838
00:34:42,376 --> 00:34:43,744
DOMAIN.

839
00:34:43,744 --> 00:34:45,713
BUT I WANT TO MAKE A POINT HERE

840
00:34:45,713 --> 00:34:46,814
TOO, I THINK IT'S IMPORTANT TO

841
00:34:46,814 --> 00:34:48,282
THIS AUDIENCE THAT SOME OF THE

842
00:34:48,282 --> 00:34:56,457
MUTANTS THAT ARE OBSERVED IN THE

843
00:34:56,457 --> 00:34:58,693
RAF'S OP A THIESS HAVE BEEN

844
00:34:58,693 --> 00:35:00,995
FOUND TO OCCUR WITH OTHER

845
00:35:00,995 --> 00:35:02,296
MUTATIONS WITH OTHER UPSTREAM

846
00:35:02,296 --> 00:35:04,966
COMPONENTS OF THE RAS PATHWAYS.

847
00:35:04,966 --> 00:35:07,368
SUCH AS RTKs, RAFs AND

848
00:35:07,368 --> 00:35:07,969
NSWOBDERFUL--WONDERFUL KNOW 1

849
00:35:07,969 --> 00:35:09,971
EMPLOY SEE WE CAN SEE SOME OF

850
00:35:09,971 --> 00:35:12,707
THESE WEAKER GAIN OF FUNCTION

851
00:35:12,707 --> 00:35:13,541
RAF MUTATIONS, CO DEARING WITH

852
00:35:13,541 --> 00:35:16,210
SOME OF THE OTHER UPSTREAM

853
00:35:16,210 --> 00:35:16,978
COMPONENTS AS WELL.

854
00:35:16,978 --> 00:35:19,280
AND I THINK WHAT WE'RE--WHAT I

855
00:35:19,280 --> 00:35:20,581
HOPE TO SHOW YOU ABOUT WHAT

856
00:35:20,581 --> 00:35:25,820
WE'VE LEARNED ABOUT THE RAS OP A

857
00:35:25,820 --> 00:35:26,520
THIES, WILL BE IMPORTANT

858
00:35:26,520 --> 00:35:28,055
INFORMATION WHEN WE'RE IN THIS

859
00:35:28,055 --> 00:35:28,823
ERA OF PERSONALIZED MEDICINE AND

860
00:35:28,823 --> 00:35:31,058
YOU LOOK AT THE MUTATIONS THAT A

861
00:35:31,058 --> 00:35:35,463
TUMOR HAS AS YOU START TO DECIDE

862
00:35:35,463 --> 00:35:36,931
WHAT THERAPIES OR WHAT TARGETED

863
00:35:36,931 --> 00:35:41,669
THERAPIES YOU WANT TO USE. 

864
00:35:41,669 --> 00:35:45,873
SO THE RAS-OP A THIES, THEY'RE

865
00:35:45,873 --> 00:35:48,075
DEVELOPMENTAL, THEY'RE OCCURRING

866
00:35:48,075 --> 00:35:48,576
IN 1-2000 LIVE BIGGERS.

867
00:35:48,576 --> 00:35:52,346
THERE ARE A NUMBER OF DIFFERENT

868
00:35:52,346 --> 00:35:53,247
SYNDROMES.

869
00:35:53,247 --> 00:35:55,316
CRAF HAS BEEN FOUND MOST OFFIN

870
00:35:55,316 --> 00:35:57,151
IN [INDISCERNIBLE] SYNDROME AND

871
00:35:57,151 --> 00:36:01,489
BRAF IS A PRIMARY DRIVER IN

872
00:36:01,489 --> 00:36:03,057
CARDIO FACIALIAL SYNDROME, AND

873
00:36:03,057 --> 00:36:04,091
ALTHOUGH THEY'RE DISTINCT THEY

874
00:36:04,091 --> 00:36:08,429
CAN BE BROADLY CHARACTERIZED BY

875
00:36:08,429 --> 00:36:11,766
DISTINCT CARDIO FACIAL FEATURES,

876
00:36:11,766 --> 00:36:14,201
OFTEN CONGENITAL HEART DEFECS,

877
00:36:14,201 --> 00:36:14,902
SHORT STATURE, AUTISM TRAITS AND

878
00:36:14,902 --> 00:36:16,871
FOR SOME OF THEM AN INCREASED

879
00:36:16,871 --> 00:36:18,639
RISK OF CERTAIN CHILDHOOD

880
00:36:18,639 --> 00:36:19,006
CANCERS.

881
00:36:19,006 --> 00:36:22,543
SO WE WONDERED WHY IS BRAF CRD A

882
00:36:22,543 --> 00:36:28,115
HOT SPOT FOR THE RASOP A

883
00:36:28,115 --> 00:36:28,449
THY MUTATIONS.

884
00:36:28,449 --> 00:36:30,117
SO NOW YOU NEED TO KNOW WHAT IS

885
00:36:30,117 --> 00:36:33,487
THE CRD IN IT'S CALLED THE

886
00:36:33,487 --> 00:36:34,522
CYSTINE RICH DOMAIN.

887
00:36:34,522 --> 00:36:35,489
THERE ARE 2 TYPES.

888
00:36:35,489 --> 00:36:40,895
THERE'S A TYPICAL AND A AN

889
00:36:40,895 --> 00:36:41,362
ATYPICAL.

890
00:36:41,362 --> 00:36:42,396
EVERYONE PROBABLY KNOWS THEM

891
00:36:42,396 --> 00:36:43,831
MOST BECAUSE THEY'RE FOUND IN

892
00:36:43,831 --> 00:36:45,700
PROTIEN KINASE C, AND IN PROTIEN

893
00:36:45,700 --> 00:36:52,406
KINASE C, THEY'RE TYPICAL

894
00:36:52,406 --> 00:36:58,245
DOMAINS AND THEY BIND DIAZAL

895
00:36:58,245 --> 00:37:00,414
GLYCEROL BUT THE ATYPICAL 1S DO

896
00:37:00,414 --> 00:37:03,984
NOT, THEY'RE FOUND IN NUMEROUS

897
00:37:03,984 --> 00:37:04,719
SIGNALING MOLECULES, THEY CAN

898
00:37:04,719 --> 00:37:07,355
IRPT ACT WITH LIPIDS AND

899
00:37:07,355 --> 00:37:09,023
PROTEINS AND THEY'RE OFTEN

900
00:37:09,023 --> 00:37:12,326
INVOLVED IN REGULATING CATALYTIC

901
00:37:12,326 --> 00:37:12,993
ACTIVITY AND LOCALIZATION.

902
00:37:12,993 --> 00:37:16,030
SO WHAT DO WE KNOW ABOUT THE RAF

903
00:37:16,030 --> 00:37:24,638
CRD, WELL IT DOESN'T BIND DAZAL

904
00:37:24,638 --> 00:37:26,273
GLYCEROL IT WAS FIRST

905
00:37:26,273 --> 00:37:28,175
CHARACTERIZED AS BEING A FOSTER

906
00:37:28,175 --> 00:37:29,577
NURSED FOCUSED ON TERILE CYSTINE

907
00:37:29,577 --> 00:37:31,412
BINDING PARTNER AND IT WAS

908
00:37:31,412 --> 00:37:33,948
THOUGHT TO HELP FACILITATE THE

909
00:37:33,948 --> 00:37:35,015
INTERACTION WITH THE MEMBRANE

910
00:37:35,015 --> 00:37:36,917
AND ALLOWING IT ALSO TO CONTACT

911
00:37:36,917 --> 00:37:38,352
RAS.

912
00:37:38,352 --> 00:37:41,589
BUT WHEN WE SAW THE

913
00:37:41,589 --> 00:37:42,523
AUTOINHIBITTED CONFIRMATION AND

914
00:37:42,523 --> 00:37:45,359
WE SAW THE CENTRAL POSITION OF

915
00:37:45,359 --> 00:37:47,795
THE CRD, YOU KNOW WE WONDERED IF

916
00:37:47,795 --> 00:37:52,700
IT ALSO PLAYS A ROLE IN RAF

917
00:37:52,700 --> 00:37:53,067
AUTOINHIBITION.

918
00:37:53,067 --> 00:37:55,836
AND I THINK WHAT WE'RE LEARNING

919
00:37:55,836 --> 00:37:57,638
1 THING THAT'S--I THINK IS

920
00:37:57,638 --> 00:38:01,175
BECOMING A TREND IS SOME OF

921
00:38:01,175 --> 00:38:03,344
THESE REGULATORY DOMAINS IN RAF

922
00:38:03,344 --> 00:38:04,645
ACTUALLY HAVE DUAL FUNCTIONS.

923
00:38:04,645 --> 00:38:06,847
THEY FUNCTION TO HELP MAINTAIN

924
00:38:06,847 --> 00:38:07,548
THE AUTOINHIBITTED STATE BUT

925
00:38:07,548 --> 00:38:10,418
THEY ALSO HAVE AN OPPOSING

926
00:38:10,418 --> 00:38:12,253
FUNCTION AND PROMOTING

927
00:38:12,253 --> 00:38:12,820
ACTIVATION.

928
00:38:12,820 --> 00:38:17,191
AND WE WANTED TO KNOW DOES THE

929
00:38:17,191 --> 00:38:22,329
RAS OP A THY MUTATIONS  MUTATIOR

930
00:38:22,329 --> 00:38:22,730
THESE FUNCTIONS.

931
00:38:22,730 --> 00:38:25,032
SO WHEN YOU LOOK AT THE CYSTINE

932
00:38:25,032 --> 00:38:26,634
RICH DOMAIN, YOU LOOK AT 2

933
00:38:26,634 --> 00:38:27,435
GROUPS.

934
00:38:27,435 --> 00:38:28,936
ONE IS THIS LOOP 1 WHICH

935
00:38:28,936 --> 00:38:33,107
INTERACTING WITH THE MEMBRANE

936
00:38:33,107 --> 00:38:35,342
AND CONTACT FOSTER NURSED FET

937
00:38:35,342 --> 00:38:36,177
ILLEGALS SIRRINE.

938
00:38:36,177 --> 00:38:38,579
ELEVEN% ARE FOUND IN THIS REGION

939
00:38:38,579 --> 00:38:39,947
BUT THE REMAINDER ARE FOUND

940
00:38:39,947 --> 00:38:44,752
ELSEWHERE IN THE DOMAIN AND THEY

941
00:38:44,752 --> 00:38:46,754
TEND TO OR SUBSTITUTION THAT

942
00:38:46,754 --> 00:38:49,089
INTRODUCE A BASIC RESIDUE WITH

943
00:38:49,089 --> 00:38:53,527
THIS Q257 R ACCOUNTING FOR 67%

944
00:38:53,527 --> 00:38:59,433
OF THE CRD MUTATIONS AND SO, HOW

945
00:38:59,433 --> 00:39:00,234
DO THEY FUNCTION?

946
00:39:00,234 --> 00:39:02,470
SO WE HAD 3 ASSAYS THAT WE

947
00:39:02,470 --> 00:39:04,705
WANTED TO MONITOR, THE FIRST

948
00:39:04,705 --> 00:39:06,540
BEING THE ABILITY TO BIND FOSTER

949
00:39:06,540 --> 00:39:07,708
NURSED FET ILLEGALS SERIES

950
00:39:07,708 --> 00:39:09,410
POINTSINE SINCE THAT WAS KIND OF

951
00:39:09,410 --> 00:39:12,847
THE MOST WELL KNOWN SO WE WORKED

952
00:39:12,847 --> 00:39:15,082
WITH ANDY STEPHENS GROUP IN THE

953
00:39:15,082 --> 00:39:16,984
RAS MINISHIAATIVE TO DO

954
00:39:16,984 --> 00:39:18,586
PLACEMENT RESIDENCE, ANALYSIS OF

955
00:39:18,586 --> 00:39:19,753
LIPID BINDING SO THIS WE'RE

956
00:39:19,753 --> 00:39:22,289
USING JUST OUR ICE ISOLATED

957
00:39:22,289 --> 00:39:24,658
CYSTINE RICH DOMAIN OF BRAF AND

958
00:39:24,658 --> 00:39:26,160
LOOKING FOR ITS ABILITY TO BIND

959
00:39:26,160 --> 00:39:29,930
NANO DISKS THAT CONTAINED 30%

960
00:39:29,930 --> 00:39:30,564
FOSTER NURSED FOCUSED ON TILE

961
00:39:30,564 --> 00:39:33,000
SERIES POINTSINE AND WE CAN

962
00:39:33,000 --> 00:39:33,601
MONITORING THE BINDING THERE.

963
00:39:33,601 --> 00:39:38,339
OF BUT IF WE FLOW THE SAME

964
00:39:38,339 --> 00:39:41,842
PURIFIED CRD OVER A NANO DISK

965
00:39:41,842 --> 00:39:46,881
THAT HAS 0 FOSTER NURSED FET

966
00:39:46,881 --> 00:39:48,282
ILLEGALS SERINE, AND HAVE

967
00:39:48,282 --> 00:39:49,750
CLONING WE SEE LITTLE BINDING SO

968
00:39:49,750 --> 00:40:00,261
IT IS SPECIFIC FOR THE PHOSPHO

969
00:40:00,861 --> 00:40:01,228
FETAL SERINE.

970
00:40:01,228 --> 00:40:03,531
BUT WE DIDN'T SEE ANY MUTATIONS

971
00:40:03,531 --> 00:40:04,899
IN THE LOOP 1 BUT WE FOUND THAT

972
00:40:04,899 --> 00:40:06,667
THE 1S THAT INTRODUCE THIS

973
00:40:06,667 --> 00:40:09,003
POSITIVE CHARGE WE DID SEE A

974
00:40:09,003 --> 00:40:12,473
SIGNIFICANT INCREASE IN THE

975
00:40:12,473 --> 00:40:13,040
INTERACTION WITH THE FOSTER

976
00:40:13,040 --> 00:40:15,242
NURSED FOCUSED ON TITLE SERINE,

977
00:40:15,242 --> 00:40:17,044
SO IF WE GO BACK AND LOOK AT THE

978
00:40:17,044 --> 00:40:19,547
STRUCTURE AND THIS IS A SURFACE

979
00:40:19,547 --> 00:40:21,215
CHARGE DISTRIBUTION OF THE

980
00:40:21,215 --> 00:40:22,316
CYSTINE RICH DOMAIN AND WE'RE

981
00:40:22,316 --> 00:40:24,385
LOOKINGA THE IT NOW AS THE

982
00:40:24,385 --> 00:40:25,553
MEMBRANE BINDING VIEW, AND SO

983
00:40:25,553 --> 00:40:29,089
YOU CAN SEE HERE'S THE

984
00:40:29,089 --> 00:40:35,262
HYDROPHOBIC POCKET AND IT'S

985
00:40:35,262 --> 00:40:37,464
ACTUALLY VERY HYDROPHOBIC--BUT

986
00:40:37,464 --> 00:40:43,103
THE INTRODUCTION OF RASOP A

987
00:40:43,103 --> 00:40:45,239
THY MUTATIONS HERE ACTUALLY MAKE

988
00:40:45,239 --> 00:40:47,575
THE SURFACE OF THIS DOMAIN MORE

989
00:40:47,575 --> 00:40:49,176
POSITIVE 3 CHARGED WHICH WE

990
00:40:49,176 --> 00:40:54,248
THINK IS CAUSING A MORE

991
00:40:54,248 --> 00:40:55,416
POSITIVELY CHARGED SURFACE WOULD

992
00:40:55,416 --> 00:40:56,483
BE CONDUCIVE FOR THE

993
00:40:56,483 --> 00:40:59,119
INTERACTIONS WITH THE NEGATIVELY

994
00:40:59,119 --> 00:40:59,887
CHARGED PLASMA MEMBRANE.

995
00:40:59,887 --> 00:41:01,755
SO THAT KIND OF, I THINK, I'VE

996
00:41:01,755 --> 00:41:04,625
REALLY ENJOYED SO MUCH ABOUT

997
00:41:04,625 --> 00:41:07,695
THIS WORK IS THAT YOU CAN PUT

998
00:41:07,695 --> 00:41:08,662
STRUCTURE AND FUNCTION TOGETHER

999
00:41:08,662 --> 00:41:10,431
AND YOU CAN KIND OF GET, OKAY,

1000
00:41:10,431 --> 00:41:11,465
THIS MAKES SENSE, THIS IS THE

1001
00:41:11,465 --> 00:41:14,768
REASON WHY THESE MUTATIONS ARE

1002
00:41:14,768 --> 00:41:15,936
HAVING THIS EFFECT AND

1003
00:41:15,936 --> 00:41:18,639
ESPECIALLY IF YOU THINK THIS

1004
00:41:18,639 --> 00:41:21,375
Q257 R WHICH IS THE PREDOMINANT

1005
00:41:21,375 --> 00:41:24,945
MUTATION AND HOW CLOSE IT IS TO

1006
00:41:24,945 --> 00:41:25,679
THE HYDROPHOBIC POCKET.

1007
00:41:25,679 --> 00:41:28,582
SO NEXT WE LOOK AT

1008
00:41:28,582 --> 00:41:29,650
AUTOINHIBITION, AND FOR THIS WE

1009
00:41:29,650 --> 00:41:31,051
KNEW, IT WOULD GOING TO BE

1010
00:41:31,051 --> 00:41:35,856
REALLY CRITICAL TO COME UP WITH

1011
00:41:35,856 --> 00:41:39,827
A LIFESTYLE ASSAY, SO WE

1012
00:41:39,827 --> 00:41:40,561
DEVELOPED LIFESTYLE PROXIMITY

1013
00:41:40,561 --> 00:41:42,096
BASED ASSAY TO MONITOR OUR

1014
00:41:42,096 --> 00:41:44,031
INHIBITION AND IT WAS BASED ON

1015
00:41:44,031 --> 00:41:45,599
THESE PREVIOUS FINDINGS THAT IF

1016
00:41:45,599 --> 00:41:49,136
YOU JUST EXPRESS THE ISOLATED

1017
00:41:49,136 --> 00:41:52,473
KINASE DOMAIN OF BRAF IN A CELL,

1018
00:41:52,473 --> 00:41:56,110
IT CAN DIMERIZE ON ITS OWN AND

1019
00:41:56,110 --> 00:42:01,081
TRANSMIT A SIGNAL TO PMEK, BUT

1020
00:42:01,081 --> 00:42:05,052
IF YOU EXPRESS ANISEOLATED REG

1021
00:42:05,052 --> 00:42:07,454
DOMAIN WITH THE KINASE DOMAIN,

1022
00:42:07,454 --> 00:42:11,125
THEY CAN ACTUALLY FORM AN

1023
00:42:11,125 --> 00:42:14,495
AUTOINHIBITTED MONOMERKIND OF

1024
00:42:14,495 --> 00:42:17,297
ACTING IN TRANS, AND BLOCK

1025
00:42:17,297 --> 00:42:17,765
SIGNALING TO PMEK.

1026
00:42:17,765 --> 00:42:20,067
SO WE WANTED TO KNOW IF WE

1027
00:42:20,067 --> 00:42:23,504
TAGGED 1 WITH AN ENERGY DONOR

1028
00:42:23,504 --> 00:42:27,174
AND AN ENERGY ACCEPTOR COULD WE

1029
00:42:27,174 --> 00:42:28,609
THEN DETECT AUTOINHIBITION?

1030
00:42:28,609 --> 00:42:30,744
AND SO THAT'S SHOWN HERE, SO WE

1031
00:42:30,744 --> 00:42:32,346
HAD CONSTANT LEVELS OF THE

1032
00:42:32,346 --> 00:42:35,015
KINASE DOMAIN, AND AS YOU CAN

1033
00:42:35,015 --> 00:42:36,617
SEE, INCREASING AMOUNTS OF THE

1034
00:42:36,617 --> 00:42:39,486
REG DOMAIN, AND AS WE ADDED THE

1035
00:42:39,486 --> 00:42:46,060
REG DOMAIN, WE ARE DETEBTING A

1036
00:42:46,060 --> 00:42:47,761
BRETT SIGNAL IN A DOSE DEPENDENT

1037
00:42:47,761 --> 00:42:48,762
MANNER AND WHAT I THINK IS

1038
00:42:48,762 --> 00:42:51,598
REALLY IMPORTANT IS THAT YOU CAN

1039
00:42:51,598 --> 00:42:53,467
SEE A DOSE DEPENDENT

1040
00:42:53,467 --> 00:42:54,535
TRANSMISSION AND THE SIGNAL TO

1041
00:42:54,535 --> 00:42:54,735
MEK.

1042
00:42:54,735 --> 00:42:56,437
SO YOU CAN SEE THE KINASE

1043
00:42:56,437 --> 00:42:57,971
SIGNALING TO MEK WITHOUT THE REG

1044
00:42:57,971 --> 00:43:01,775
DOMAIN BUT IT GOES AWAY WHEN THE

1045
00:43:01,775 --> 00:43:02,609
REG DOMAIN COMBINE.

1046
00:43:02,609 --> 00:43:04,878
SO THAT'S GREAT.

1047
00:43:04,878 --> 00:43:05,913
WE CAN DETECT AUTOINHIBITION BUT

1048
00:43:05,913 --> 00:43:09,516
WHAT'S CRITICAL CAN WE DETECT

1049
00:43:09,516 --> 00:43:10,417
DISRUPTION OF AUTOINHIBITION?

1050
00:43:10,417 --> 00:43:12,286
SO WE ADDED A FEW EXTRA

1051
00:43:12,286 --> 00:43:12,553
CONTROLS.

1052
00:43:12,553 --> 00:43:14,488
SO WE KNOW THAT THE REG DOMAIN

1053
00:43:14,488 --> 00:43:17,624
CAN INTERACT WITH RAS, IF THERE

1054
00:43:17,624 --> 00:43:18,759
THEY'RE SEPARATED THEN THAT

1055
00:43:18,759 --> 00:43:21,061
SHOULD FREE THE REG DOMAIN AND

1056
00:43:21,061 --> 00:43:22,663
WOULD SEPARATE THE 2 AND WE

1057
00:43:22,663 --> 00:43:24,164
WOULDN'T SEE THE SIGNAL AND

1058
00:43:24,164 --> 00:43:26,467
THAT'S ACTUALLY WHAT WE SAW WAS

1059
00:43:26,467 --> 00:43:30,871
A REDUCTION IN THE BRETT SIGNAL.

1060
00:43:30,871 --> 00:43:37,678
WE ALSO NEEDED V600 DISRUPTED

1061
00:43:37,678 --> 00:43:39,146
THE MUTATION STATE AND WE SAW

1062
00:43:39,146 --> 00:43:41,348
THAT AGAIN AND WE AWE A

1063
00:43:41,348 --> 00:43:42,750
DISRUPTION IN AUTOINHIBITION

1064
00:43:42,750 --> 00:43:45,486
EMPLOY SO WE INCORPORATED ALL OF

1065
00:43:45,486 --> 00:43:48,188
OUR CRD MUTATIONS AND WITH ALL

1066
00:43:48,188 --> 00:43:53,961
OF THE EXCEPTION OF THIS E275-K

1067
00:43:53,961 --> 00:43:57,231
MUTATION THEY ALL DISRUPTED

1068
00:43:57,231 --> 00:43:57,831
AUTOINHIKING HIB THORSITION

1069
00:43:57,831 --> 00:43:59,566
INDICATING THAT THE CRD DOES

1070
00:43:59,566 --> 00:44:02,436
INDEED PLAY A CRITICAL ROLE IN

1071
00:44:02,436 --> 00:44:03,270
AUTOINHIBITION AND AGAIN IF WE

1072
00:44:03,270 --> 00:44:04,805
LOOK AT THE STRUCTURE, WE CAN

1073
00:44:04,805 --> 00:44:07,641
SEE THAT THESE RESIDUES ARE IN

1074
00:44:07,641 --> 00:44:11,411
CONTACT WITH BOTH THE 1433

1075
00:44:11,411 --> 00:44:13,680
PROTOMERAND WITH BRAF BUT THE

1076
00:44:13,680 --> 00:44:15,783
275 ALONE IS KIND OF OUT THERE,

1077
00:44:15,783 --> 00:44:18,318
IT'S NOT MAKING INTERACTIONS SO

1078
00:44:18,318 --> 00:44:20,621
IT SHOULD BE AUTOINHIBITTED.

1079
00:44:20,621 --> 00:44:23,056
AND SO THIS KIND OF DIVIDED THE

1080
00:44:23,056 --> 00:44:25,359
CATEGORIES OF THE MUTANTS INTO

1081
00:44:25,359 --> 00:44:26,393
THOSE THAT RELIEVE

1082
00:44:26,393 --> 00:44:27,427
AUTOINHIBITION WHICH ARE ALL 4

1083
00:44:27,427 --> 00:44:29,463
OF THESE AND THIS 1 BEING THE

1084
00:44:29,463 --> 00:44:33,767
ONLY 1 THAT JUST HAS THE EFFECT

1085
00:44:33,767 --> 00:44:34,334
OF INCREASING FOSTER NURSED

1086
00:44:34,334 --> 00:44:35,602
FOCUSED ON AT THE TIME ILLEGALS

1087
00:44:35,602 --> 00:44:37,571
SIRRINE BINDING SO WE'RE

1088
00:44:37,571 --> 00:44:39,072
ANYTHING TO BIOLOGICAL FUNCTION

1089
00:44:39,072 --> 00:44:43,944
NOW, AND GO THROUGH THIS PRETTY

1090
00:44:43,944 --> 00:44:46,046
QUICKLY, WE CAN FOCUS ON ASSAYS

1091
00:44:46,046 --> 00:44:46,880
LOOKING AT PROLIFERATION AND ALL

1092
00:44:46,880 --> 00:44:51,018
OF 1S THAT RELIEVED

1093
00:44:51,018 --> 00:44:51,819
AUTOINHIBITION, ACTUALLY INDUCED

1094
00:44:51,819 --> 00:44:52,920
FOCUS FORMATION AND IT WAS

1095
00:44:52,920 --> 00:44:54,788
SEVERELY REDUCED FOR THIS MUTANT

1096
00:44:54,788 --> 00:44:57,925
WHICH ONLY HAS INCREASED BINDING

1097
00:44:57,925 --> 00:45:00,294
AND IF WE LOOK AT CELLS AT THE

1098
00:45:00,294 --> 00:45:03,163
PMEK LEVELS AGAIN IT CORRELATED

1099
00:45:03,163 --> 00:45:03,630
WELL.

1100
00:45:03,630 --> 00:45:04,698
BUT WE WERE REALLY FORTUNATE TO

1101
00:45:04,698 --> 00:45:08,135
BE ABLE TO COLLABORATE WITH THE

1102
00:45:08,135 --> 00:45:10,437
ZEBRAFISH FACILITY BECAUSE WE

1103
00:45:10,437 --> 00:45:12,739
WANTED A MORE BIOLOGICAL ASSAY.

1104
00:45:12,739 --> 00:45:14,107
SO WE'RE USING ZEB RAW DATA FISH

1105
00:45:14,107 --> 00:45:16,376
AND WE'RE INYECTING MUTANTS INTO

1106
00:45:16,376 --> 00:45:18,045
THE EMBRYOS AND WE HAVE 3 ASSAYS

1107
00:45:18,045 --> 00:45:19,413
THAT WE'RE LOOKING AT.

1108
00:45:19,413 --> 00:45:23,517
ONE IS THE ELONGATION OR THE

1109
00:45:23,517 --> 00:45:26,486
MEASUREMENT OF THE EMBRYOAXIS

1110
00:45:26,486 --> 00:45:29,857
AND IT IS, IF YOU HAVE ENHANCED

1111
00:45:29,857 --> 00:45:32,559
SIGNALING OF THE PATHWAY WHAT

1112
00:45:32,559 --> 00:45:34,761
YOU GET IS ELONGATED EMBRYOS AND

1113
00:45:34,761 --> 00:45:39,233
THIS IS A SIGN OF DEFECT ANDS

1114
00:45:39,233 --> 00:45:40,000
CONVERGENT EXTENSION MOVEMENTS.

1115
00:45:40,000 --> 00:45:43,904
WE ALSO LOOKED AT 3 DAYS AT HARD

1116
00:45:43,904 --> 00:45:49,109
EDEMA AND WITH THAT SHOWN HERE

1117
00:45:49,109 --> 00:45:50,677
AS--HEART EDEMA AND WITH THAT

1118
00:45:50,677 --> 00:45:51,778
SHOWN HERE MEASURING HEART

1119
00:45:51,778 --> 00:45:54,748
DEFEBTS AND FAILURE, AND WE ALSO

1120
00:45:54,748 --> 00:45:56,116
LOOKED AT THE [INDISCERNIBLE]

1121
00:45:56,116 --> 00:45:58,151
ANGLE AND THIS IS KNOWN TO BE A

1122
00:45:58,151 --> 00:45:59,920
MEASURE OF HEAD WIDTH, EYE

1123
00:45:59,920 --> 00:46:03,690
SPACES WHICH IS A CHARACTERISTIC

1124
00:46:03,690 --> 00:46:07,394
OF THE RASOP A THIES.

1125
00:46:07,394 --> 00:46:08,061
SO WE TOOK REPRESENTATIVES FROM

1126
00:46:08,061 --> 00:46:09,496
EACH OF OUR CLASSES, HERE YOU

1127
00:46:09,496 --> 00:46:11,031
CAN SEE THE EXPRESSION IN THE

1128
00:46:11,031 --> 00:46:11,932
EMBRYOS AND WAWE FOUND THAT WAS

1129
00:46:11,932 --> 00:46:15,302
THAT THE 1S THAT RELIEVED

1130
00:46:15,302 --> 00:46:20,774
AUTOINHIBITION ACTUALLY W

1131
00:46:20,774 --> 00:46:21,675
ERE--HAD SIGNIFICANT DEFECT

1132
00:46:21,675 --> 00:46:24,845
ANDS THERE WAS DEFECTS FOR THE

1133
00:46:24,845 --> 00:46:28,582
EE275 K AS WELL BUT IT WAS MUCH

1134
00:46:28,582 --> 00:46:29,082
LESS SIGNIFICANT.

1135
00:46:29,082 --> 00:46:31,285
IF WE LOOK AT HEART EDEMA,

1136
00:46:31,285 --> 00:46:35,989
AGAIN, THE 2 THAT MUTANTS THAT

1137
00:46:35,989 --> 00:46:38,191
DISRUPT AUTOINHIBITION, WE SAW

1138
00:46:38,191 --> 00:46:39,393
SIGNIFICANT DEFECTS AND AGAIN,

1139
00:46:39,393 --> 00:46:41,795
IT WAS REDUCED FOR THE MUTATION

1140
00:46:41,795 --> 00:46:45,465
THAT ONLY HAD INCREASED BINDING,

1141
00:46:45,465 --> 00:46:48,669
PS-BINDING AND I THINK FINALLY,

1142
00:46:48,669 --> 00:46:50,404
WE LOOKED AT [INDISCERNIBLE]

1143
00:46:50,404 --> 00:46:53,240
ANGLE AND AGAIN, HERE WE

1144
00:46:53,240 --> 00:46:55,943
ACTUALLY SAW NO INCREASE AT ALL

1145
00:46:55,943 --> 00:46:58,478
WHERE WITH BOTH OF THE MUTANTS

1146
00:46:58,478 --> 00:47:00,113
THAT HAVE AUTOINHIBITION

1147
00:47:00,113 --> 00:47:02,382
RELIEVED, YOU DO SEE THE

1148
00:47:02,382 --> 00:47:04,284
INCREASE.

1149
00:47:04,284 --> 00:47:06,053
AND WE'RE FORTUNATE THAT THESE

1150
00:47:06,053 --> 00:47:08,188
MUTATIONS HAVE BEEN REPORTED IN

1151
00:47:08,188 --> 00:47:09,256
CHARACTERIZED AND I KNOW THAT

1152
00:47:09,256 --> 00:47:11,558
THEY'RE NOT A LOT OF THE

1153
00:47:11,558 --> 00:47:15,262
NUMBERS, BUT WHAT YOU FIND IS

1154
00:47:15,262 --> 00:47:20,867
THAT THIS GROUP OF ASSAYS DO

1155
00:47:20,867 --> 00:47:22,369
CORRELATE WELL WITH WHAT HAS

1156
00:47:22,369 --> 00:47:24,104
BEEN OBSERVED WITH THE PATIENTS

1157
00:47:24,104 --> 00:47:27,607
AND IN PARTICULAR, THIS WIDE SET

1158
00:47:27,607 --> 00:47:28,875
EYES IS OBSERVED IN ALL THE

1159
00:47:28,875 --> 00:47:31,979
PATIENTS THAT HAVE THE MUTANTS

1160
00:47:31,979 --> 00:47:33,113
THAT DISRUPT AUTOINHIBITION IS

1161
00:47:33,113 --> 00:47:35,048
IT'S NOT OBSERVED IN THE

1162
00:47:35,048 --> 00:47:39,353
PATIENTS THAT HAVE THE E275 K

1163
00:47:39,353 --> 00:47:42,990
MUTATION AND OVERALL, THESE

1164
00:47:42,990 --> 00:47:45,092
PATIENTS, THE SEVERITY OF THE

1165
00:47:45,092 --> 00:47:48,762
DEFECTS OR THE PHENOTYPE IS

1166
00:47:48,762 --> 00:47:49,830
GREATLY REDUCED IN COMPARISON TO

1167
00:47:49,830 --> 00:47:50,030
THESE.

1168
00:47:50,030 --> 00:47:52,366
SO I THINK THIS IS REALLY

1169
00:47:52,366 --> 00:47:55,002
TELLING US THAT RELIEF OF

1170
00:47:55,002 --> 00:47:57,504
AUTOINHIBITION IS REALLY THE

1171
00:47:57,504 --> 00:48:00,941
MAJOR DETERMINANT FOR THESE

1172
00:48:00,941 --> 00:48:03,510
RASOP A THY CRD MUTATIONS AND

1173
00:48:03,510 --> 00:48:05,979
HOW SEVERE THE DEFECTS ARE GOING

1174
00:48:05,979 --> 00:48:06,346
TO BE.

1175
00:48:06,346 --> 00:48:10,317
SO I THINK WHAT I WOULD LIKE--I

1176
00:48:10,317 --> 00:48:12,285
HOPE THIS HAS CONVINCED YOU THAT

1177
00:48:12,285 --> 00:48:15,022
THE STUDY OF THIS DISEASE

1178
00:48:15,022 --> 00:48:17,524
ASSOCIATED MUTATIONS CAN REVEAL

1179
00:48:17,524 --> 00:48:18,925
IMPORTANT REGULATORY INFORMATION

1180
00:48:18,925 --> 00:48:21,061
AND IT DEMONSTRATES THE CRITICAL

1181
00:48:21,061 --> 00:48:25,032
ROLE OF THE CRD AND HOW TAKING A

1182
00:48:25,032 --> 00:48:27,601
MULTIFACETED APPROACH OF LOOKING

1183
00:48:27,601 --> 00:48:30,804
AT THE STRUCTURE, DOING

1184
00:48:30,804 --> 00:48:31,738
BIOCHEMISTRY, HAVING LIVE CELL

1185
00:48:31,738 --> 00:48:34,841
ASSAYS AND THEN GOING TO

1186
00:48:34,841 --> 00:48:37,244
AN ANIMAL MODEL SYSTEM THAT DOES

1187
00:48:37,244 --> 00:48:39,212
FULLY KIND OF REPRESENT OR IS A

1188
00:48:39,212 --> 00:48:41,815
GOOD MODEL FOR THE HUMAN DISEASE

1189
00:48:41,815 --> 00:48:44,885
CAN BE VERY BENEFICIAL SO WITH

1190
00:48:44,885 --> 00:48:48,955
OUR ASSAYS IN PLACE, WE HAVE

1191
00:48:48,955 --> 00:48:53,527
BEEN INTERACTING WITH THE NCI

1192
00:48:53,527 --> 00:48:55,195
ADVANCING RASOP A THIES, WHICH

1193
00:48:55,195 --> 00:48:57,197
IS A COLLABORATION BETWEEN THE

1194
00:48:57,197 --> 00:48:59,533
CLINICAL GENETICS BRANCH, THE

1195
00:48:59,533 --> 00:49:01,568
PEDIATRIC ONCOLOGY BRANCH AND

1196
00:49:01,568 --> 00:49:02,869
THE NCI RAS INITIATIVE AS WELL

1197
00:49:02,869 --> 00:49:06,873
AND IT'S TAKING A GENETICS FIRST

1198
00:49:06,873 --> 00:49:08,141
APPROACH TO IDENTIFY NEW MUTANTS

1199
00:49:08,141 --> 00:49:11,912
AND WE HAVE THESE ASSAYS, THE

1200
00:49:11,912 --> 00:49:14,214
ZEBBIA FISH ASSAYS IN PLACE AND

1201
00:49:14,214 --> 00:49:16,883
IN FACT, MARIO IS IN OUR

1202
00:49:16,883 --> 00:49:20,220
LABORATORY IN THE LCDS AND

1203
00:49:20,220 --> 00:49:22,055
UTILIZING THESE ASSAYS NOW IN

1204
00:49:22,055 --> 00:49:27,861
ANALYZING SOME NEW RAS MUTANTS,

1205
00:49:27,861 --> 00:49:28,361
HRAS MUTANTS.

1206
00:49:28,361 --> 00:49:30,163
AND WE ALSO WANT TO USE IT TO

1207
00:49:30,163 --> 00:49:31,898
DEVELOP NEW THERAPIES SO I WILL

1208
00:49:31,898 --> 00:49:35,235
GIVE A SHOUT OUT TO ALSO THE

1209
00:49:35,235 --> 00:49:36,536
MOLECULAR TARGETS, PROTEIN WHICH

1210
00:49:36,536 --> 00:49:38,171
IS LOCATED IN FREDERICK, AND IT

1211
00:49:38,171 --> 00:49:40,340
HAS THE LARGEST AND MOST DIVERSE

1212
00:49:40,340 --> 00:49:42,709
COLLECTION OF THE NATURAL

1213
00:49:42,709 --> 00:49:46,179
PRODUCTS AND THERE ARE OVER

1214
00:49:46,179 --> 00:49:48,014
200,000 PARTIALLY PURIFIED

1215
00:49:48,014 --> 00:49:52,085
FRACTIONS FROM OVER 80,000

1216
00:49:52,085 --> 00:50:01,128
ORGANISMS BOTH AQUATIC, MARINE,

1217
00:50:01,128 --> 00:50:02,062
AND OTHERS.

1218
00:50:02,062 --> 00:50:03,029
AND WE IDENTIFIED A NATURAL

1219
00:50:03,029 --> 00:50:04,898
PRODUCT THAT IS A COMPOUND

1220
00:50:04,898 --> 00:50:08,935
THAT'S ISOLATED FROM THE EDIBLE

1221
00:50:08,935 --> 00:50:11,037
PLANTS YOU SEE CANNED WHAT,

1222
00:50:11,037 --> 00:50:12,873
WE'RE CALLING IT A DIMER

1223
00:50:12,873 --> 00:50:15,008
DISRUPTER AND IT'S JUST SHOWING

1224
00:50:15,008 --> 00:50:17,744
IT CAN DISRUPT BRAF, CRAF DIMERS

1225
00:50:17,744 --> 00:50:19,346
AND SUPPRESSES SIGNALING TO

1226
00:50:19,346 --> 00:50:23,817
PMEK, AND WHEN WE INCUBATE OUR

1227
00:50:23,817 --> 00:50:25,452
EMBRYOS WITH THE DIMER

1228
00:50:25,452 --> 00:50:28,321
DISRUPTER, HERE YOU CAN SEE THE

1229
00:50:28,321 --> 00:50:29,956
ELONGATIONS THAT'S INDUCED BY

1230
00:50:29,956 --> 00:50:31,424
THIS RAS OPERATING GLOBALLYATHY

1231
00:50:31,424 --> 00:50:32,993
MUTANT AND THAT IT'S SUPPRESSED

1232
00:50:32,993 --> 00:50:37,397
HERE IF IT'S IN THE PRESENCE OF

1233
00:50:37,397 --> 00:50:37,964
THE DIMER DISRUPTER.

1234
00:50:37,964 --> 00:50:40,167
AND SOMETHING THAT WE'RE REALLY,

1235
00:50:40,167 --> 00:50:41,134
REALLY EXCITED ABOUT, WE

1236
00:50:41,134 --> 00:50:44,404
THOUGHT, WELL IF IT'S

1237
00:50:44,404 --> 00:50:45,038
DISRUPTING, DIMERIZATION HERE,

1238
00:50:45,038 --> 00:50:50,210
MAYBE LET'S ANY BACK AND RETHINK

1239
00:50:50,210 --> 00:50:52,279
ABOUT THE RESISTANCE TO

1240
00:50:52,279 --> 00:50:54,014
[INDISCERNIBLE] RAF AND 

1241
00:50:54,014 --> 00:50:54,281
MELANOMA.

1242
00:50:54,281 --> 00:50:55,215
BECAUSE WHAT WE LEARNED IN MEL

1243
00:50:55,215 --> 00:51:00,020
AN OHM IS THAT THE REASON

1244
00:51:00,020 --> 00:51:01,087
RESISTANCE IS DEVELOPED IS OFTEN

1245
00:51:01,087 --> 00:51:05,458
DUE TO MUTATIONS AND UPTREME

1246
00:51:05,458 --> 00:51:07,093
COMPONENTS, LIKE THE ACQUISITION

1247
00:51:07,093 --> 00:51:08,261
OF RAS MUTATIONS OR THE

1248
00:51:08,261 --> 00:51:10,697
OUTGROWTH OF A MUTATION ACTUALLY

1249
00:51:10,697 --> 00:51:13,667
IN A V600 E WHICH IS A

1250
00:51:13,667 --> 00:51:15,335
TRUNCATION MUTANT THAT

1251
00:51:15,335 --> 00:51:17,571
CONSTITTATIVELY DIMERIZES AND SO

1252
00:51:17,571 --> 00:51:21,107
AGAIN, WHEN YOU HAVE RESISTANCE,

1253
00:51:21,107 --> 00:51:23,310
WHAT HAPPENS IS YOU GET RESTORE

1254
00:51:23,310 --> 00:51:31,451
STIGINALLING AND ALL OF THESE

1255
00:51:31,451 --> 00:51:32,852
MECHANISMS REQUIRE RAF

1256
00:51:32,852 --> 00:51:33,520
DIMERIZATION.

1257
00:51:33,520 --> 00:51:37,190
SO CAN WE CO TREAT WITH THE RAF

1258
00:51:37,190 --> 00:51:42,095
DISRUPTERROR INHIBIT THE

1259
00:51:42,095 --> 00:51:43,029
ACQUISITION OF RESISTANCE TO

1260
00:51:43,029 --> 00:51:44,197
[INDISCERNIBLE] AND THIS IS

1261
00:51:44,197 --> 00:51:45,298
SHOWING THE EXPERIMENTS ARE

1262
00:51:45,298 --> 00:51:48,435
DONE, YOU CULTURE, THIS IS SKML5

1263
00:51:48,435 --> 00:51:52,439
HERE AND WE'RE GROWING THEM

1264
00:51:52,439 --> 00:51:53,106
CONSTITTATIVELY IN TREATMENT, I

1265
00:51:53,106 --> 00:51:54,608
THINK HAD IS A 3-4 WEEK

1266
00:51:54,608 --> 00:51:56,977
TREATMENT AND YOU GET THE

1267
00:51:56,977 --> 00:52:00,146
EMERGES ENSEL OF RESISTANT CELLS

1268
00:52:00,146 --> 00:52:03,550
BUT AS YOU CAN HERE IN THE

1269
00:52:03,550 --> 00:52:05,552
COMBINATION OF THE DIMER

1270
00:52:05,552 --> 00:52:07,254
DISRUPTER, WE'RE ACTUALLY

1271
00:52:07,254 --> 00:52:08,655
PREVENTING THE RESISTANCE CELLS

1272
00:52:08,655 --> 00:52:10,590
FROM COMING OUT WHERE BY ITSELF,

1273
00:52:10,590 --> 00:52:12,192
IT REALLY HAS VERY LITTLE

1274
00:52:12,192 --> 00:52:14,928
EFFECT, AND WE FOUND THIS TO BE

1275
00:52:14,928 --> 00:52:16,730
THE CASE IN A NUMBER OF THE

1276
00:52:16,730 --> 00:52:18,565
MELANOMA LINES THAT WE LOOKED AT

1277
00:52:18,565 --> 00:52:22,002
AND WE'RE WORKING NOW TO TRY TO

1278
00:52:22,002 --> 00:52:24,537
OPTIMIZE, YOU KNOW THE STRUCTURE

1279
00:52:24,537 --> 00:52:27,340
FUNCTION ACTIVITY OF THESE

1280
00:52:27,340 --> 00:52:27,641
MOLECULES.

1281
00:52:27,641 --> 00:52:28,975
SO I'LL STOP THERE AND THANK THE

1282
00:52:28,975 --> 00:52:32,479
PEOPLE THAT DID THE WORK AND SAY

1283
00:52:32,479 --> 00:52:34,714
THAT DOING SCIENCE TAKES A

1284
00:52:34,714 --> 00:52:36,316
VILLAGE AND I'M REALLY FORTUNATE

1285
00:52:36,316 --> 00:52:38,518
AND THANKFUL THAT MY VILLAGE HAS

1286
00:52:38,518 --> 00:52:40,253
BEEN THE NCI BECAUSE AS YOU CAN

1287
00:52:40,253 --> 00:52:42,355
SEE HERE I'VE REALLY BENEFITED

1288
00:52:42,355 --> 00:52:44,557
FROM OUR COLLABORATORS AND THE

1289
00:52:44,557 --> 00:52:47,327
STRUCTURAL BIOLOGY LAB, THE

1290
00:52:47,327 --> 00:52:50,297
MOLECULAR TARGETS, THE R,A S

1291
00:52:50,297 --> 00:52:51,798
INITIATIVE AND OUR NOW

1292
00:52:51,798 --> 00:52:53,900
COLLABBATION WITH THE LAWRENCE

1293
00:52:53,900 --> 00:52:55,268
LIVERMOORE, AND I WANT TO SHOUT

1294
00:52:55,268 --> 00:52:56,936
OUT THAT DAVE DURAN IN MY LAB

1295
00:52:56,936 --> 00:53:01,174
THE 1 WHO REALLY WORKED WITH AND

1296
00:53:01,174 --> 00:53:02,175
SOLVED THE STRUCTURES.

1297
00:53:02,175 --> 00:53:04,511
DAN'S BEEN DOING KEEN ACE

1298
00:53:04,511 --> 00:53:09,549
ASSAYS, RUSSELL DID ALL OF THE

1299
00:53:09,549 --> 00:53:12,018
RASOP A THY ANALYSIS TOGETHER

1300
00:53:12,018 --> 00:53:13,219
WITH OUR ZEBRAFISH CORE

1301
00:53:13,219 --> 00:53:14,621
FACILITY, I WILL STOP THERE AND

1302
00:53:14,621 --> 00:53:21,294
ANSWER ANY QUESTIONS AND THANK

1303
00:53:21,294 --> 00:53:21,461
YOU.

1304
00:53:21,461 --> 00:53:21,795
[ APPLAUSE ]

1305
00:53:21,795 --> 00:53:24,664
>> REALLY NICE. 

1306
00:53:24,664 --> 00:53:27,100
SO I WILL TAKE INTRODUCERS

1307
00:53:27,100 --> 00:53:27,400
PREROGATIVE.

1308
00:53:27,400 --> 00:53:28,768
SO IF I'M FOLLOWING THE MODEL

1309
00:53:28,768 --> 00:53:31,438
YOU SHOULD PERHAPS SEE, YOU GUYS

1310
00:53:31,438 --> 00:53:32,739
FIGURED THIS MODEL OUT SO THE

1311
00:53:32,739 --> 00:53:33,707
TUMOR SHOULD HAVE MAYBE AND

1312
00:53:33,707 --> 00:53:35,241
WOULD HAVE MUTATIONS ON THE

1313
00:53:35,241 --> 00:53:41,014
1433, THAT WOULD PREVENT IT FROM

1314
00:53:41,014 --> 00:53:42,882
THAT AUTOINHIBITTORY COMPLEX,

1315
00:53:42,882 --> 00:53:43,083
RIGHT?

1316
00:53:43,083 --> 00:53:44,684
SORE IS DUS RUPGZ OF THAT AND

1317
00:53:44,684 --> 00:53:48,521
INTERACTION NOT ENOUGH TO EXPOSE

1318
00:53:48,521 --> 00:53:50,824
THE LOOP FOR DEPHOSPHORYLATION?

1319
00:53:50,824 --> 00:53:53,827
>> SO THERE'S GOING TO BE
THE

1320
00:53:53,827 --> 00:53:56,596
SELECTIVE PRESSURE, I GUESS FOR,

1321
00:53:56,596 --> 00:53:59,532
OKAY, SO YOU'RE TALKING ABOUT ON

1322
00:53:59,532 --> 00:54:04,070
A MUTATION IN 1433.

1323
00:54:04,070 --> 00:54:04,371
>> RIGHT.

1324
00:54:04,371 --> 00:54:05,338
>> WELL 1433 IS KNOW WOOF
THOSE

1325
00:54:05,338 --> 00:54:07,307
IT HAS A DUAL FUNCTION.

1326
00:54:07,307 --> 00:54:11,745
IT'S REQUIRED TO STABILIZE THE

1327
00:54:11,745 --> 00:54:13,580
AUTOINHIBIT AND MONOMERBUT IT IS

1328
00:54:13,580 --> 00:54:14,514
REQUIRED FOR DIMER STABILITY

1329
00:54:14,514 --> 00:54:19,319
BECAUSE IF YOU MUTATE THOSE

1330
00:54:19,319 --> 00:54:19,986
C-TERMINAL SITES--

1331
00:54:19,986 --> 00:54:22,355
>> IT MIGHT KICK AWAY BUT
IT

1332
00:54:22,355 --> 00:54:23,323
WON'T HOLD--

1333
00:54:23,323 --> 00:54:24,391
>> NO, IT WON'T HOLD.

1334
00:54:24,391 --> 00:54:25,458
IN FACT MUTATION OF THAT SITE

1335
00:54:25,458 --> 00:54:27,227
CAN BE A DOMINANT NEGATIVE IF

1336
00:54:27,227 --> 00:54:31,064
YOU OVEREXPRESS IT IN CELLS.

1337
00:54:31,064 --> 00:54:34,601
BUT, I WILL SAY, WHICH IS A

1338
00:54:34,601 --> 00:54:38,338
LITTLE--IT'S NOT YOUR QUESTION,

1339
00:54:38,338 --> 00:54:39,773
BUT FOR CRAF THAT 1433 BINDING

1340
00:54:39,773 --> 00:54:43,309
SITE THAT'S INVOLVED IN THE

1341
00:54:43,309 --> 00:54:45,111
AUTOINHIBITION, AND INTERNAL 1

1342
00:54:45,111 --> 00:54:50,917
THAT'S THE CRAF HOT SPOT IN THE

1343
00:54:50,917 --> 00:54:53,353
RASOP A THIES, AND THAT'S A

1344
00:54:53,353 --> 00:54:54,988
COMMON MUTATION PICKED UP IN

1345
00:54:54,988 --> 00:54:57,490
DRUG RESISTANCE AS WELL, IN SO

1346
00:54:57,490 --> 00:54:59,793
THEN WHY DO WE HAVE RAF A

1347
00:54:59,793 --> 00:55:00,059
ANYWAY?

1348
00:55:00,059 --> 00:55:01,928
IT DOESN'T SEEM TO DO ANYTHING?

1349
00:55:01,928 --> 00:55:03,763
>> WELL WE FOUND OUT--

1350
00:55:03,763 --> 00:55:06,733
>> WELL YOU SHOW TODAY
WASN'T

1351
00:55:06,733 --> 00:55:09,536
INTERACTIVE, IS IT DOMINANT

1352
00:55:09,536 --> 00:55:10,103
NEGATIVE?

1353
00:55:10,103 --> 00:55:11,438
>> NO, IT'S NOT A DOMINANT

1354
00:55:11,438 --> 00:55:12,472
NEGATIVE, SO THERE'S A QUESTION

1355
00:55:12,472 --> 00:55:13,706
OF ARE WE NOT LOOKING IN THE

1356
00:55:13,706 --> 00:55:15,742
RIGHT PLACE OR IN THE RIGHT

1357
00:55:15,742 --> 00:55:17,277
CONDITION IN. 

1358
00:55:17,277 --> 00:55:17,744
>> RIGHT, RIGHT.

1359
00:55:17,744 --> 00:55:23,082
ALL RIGHT, OTHER QUESTIONS?

1360
00:55:23,082 --> 00:55:24,884
>>  DEBBIE BEAUTIFUL,
BEAUTIFUL

1361
00:55:24,884 --> 00:55:25,285
TALK AND RESEARCH.

1362
00:55:25,285 --> 00:55:27,787
QUESTION WITH OUR DIMERIZATION

1363
00:55:27,787 --> 00:55:31,391
RISK ASSESSMENTS DID YOU WANTER

1364
00:55:31,391 --> 00:55:31,624
 DID YOU

1365
00:55:31,624 --> 00:55:33,326
WANT--DISRUPTER, 1 OF THE

1366
00:55:33,326 --> 00:55:34,260
QUESTIONS IS [INDISCERNIBLE]

1367
00:55:34,260 --> 00:55:36,930
GROUP HAS SHOWN BEAUTIFULLY THAT

1368
00:55:36,930 --> 00:55:37,964
THE MEK INHIBITORS WORK WELL

1369
00:55:37,964 --> 00:55:43,203
WITH A CERTAIN GROUP OF NF1

1370
00:55:43,203 --> 00:55:44,537
MUTANT PATIENTS BUT YOU GD LOOK

1371
00:55:44,537 --> 00:55:45,705
AND SEE THE PHENOTYPE WHEN YOU

1372
00:55:45,705 --> 00:55:49,309
INTERFERE WITH THE DIMERIZATION

1373
00:55:49,309 --> 00:55:52,946
OF THE RAF TO WHAT DEGREE DOES

1374
00:55:52,946 --> 00:55:55,748
IT MIMIC OR IS IT DISTINCT FROM

1375
00:55:55,748 --> 00:55:59,619
PI3--I'M SORRY NOT PI3 KINASE

1376
00:55:59,619 --> 00:56:01,221
BUT MEK INHIBITION OR THOSE

1377
00:56:01,221 --> 00:56:03,423
KINDS OF EXPERIMENTS TO TRY TO

1378
00:56:03,423 --> 00:56:07,494
HAVE IMPACT WITH OTHER RASOP A

1379
00:56:07,494 --> 00:56:07,961
THIES.

1380
00:56:07,961 --> 00:56:08,795
>> YOU KNOW RIGHT NOW
WE'RE

1381
00:56:08,795 --> 00:56:12,298
TRYING TO FIND THE EXACT--IT'S

1382
00:56:12,298 --> 00:56:13,466
DISRUPTING DIMERIZATION, BUT HOW

1383
00:56:13,466 --> 00:56:15,201
IS IT DISRUPTING DIMERIZATION IN

1384
00:56:15,201 --> 00:56:18,037
AND WE'RE WORKING AND DOING SOME

1385
00:56:18,037 --> 00:56:19,572
OF THESE CANNED WHAT EXPERIMENTS

1386
00:56:19,572 --> 00:56:20,940
RIGHT NOW LOOKING FOR IF IT

1387
00:56:20,940 --> 00:56:24,377
FINDS DOES IT TABLIZE THE--IS IT

1388
00:56:24,377 --> 00:56:28,481
THERMAL STABILITY AND DOING MASS

1389
00:56:28,481 --> 00:56:29,015
SPEC. ON IT?

1390
00:56:29,015 --> 00:56:31,618
IT DOESN'T SEEM TO BE A DIRECT

1391
00:56:31,618 --> 00:56:34,554
BINDER OF THE RAF OR OF THE MEK

1392
00:56:34,554 --> 00:56:36,723
PATHWAY, BUT IT DOES SEEM TO

1393
00:56:36,723 --> 00:56:40,460
HAVE EFFECTS MODULATING THE 1433

1394
00:56:40,460 --> 00:56:44,030
INTERACTION WHICH IS CRITICAL

1395
00:56:44,030 --> 00:56:45,331
FOR DIMERIZATION, TABLE

1396
00:56:45,331 --> 00:56:46,666
DIMERIZATION, BUT AGAIN THIS IS

1397
00:56:46,666 --> 00:56:50,703
IN THE EARLY STAGES OF SORTING

1398
00:56:50,703 --> 00:56:51,137
THAT OUT.

1399
00:56:51,137 --> 00:56:53,206
BUT MY QUESTION WAS THAT, TOO.

1400
00:56:53,206 --> 00:56:57,410
IS IT, YOU KNOW IS IT BINDING TO

1401
00:56:57,410 --> 00:56:59,245
RAF AND DISRUPTING OR IS IT

1402
00:56:59,245 --> 00:57:00,880
HAVING EFFECTS SIMILAR TO THE

1403
00:57:00,880 --> 00:57:05,552
MEK INHIBITOR BUT IT SEEMS TO BE

1404
00:57:05,552 --> 00:57:05,818
DIFFERENT.

1405
00:57:05,818 --> 00:57:07,220
>> BEAUTIFUL STORY,
BEAUTIFUL

1406
00:57:07,220 --> 00:57:09,923
TALK, FANTASTIC OVER THE YEARS.

1407
00:57:09,923 --> 00:57:10,990
QUICK QUESTION, MAYBE YOU

1408
00:57:10,990 --> 00:57:12,859
MENTIONED THIS AND I MISSED IT,

1409
00:57:12,859 --> 00:57:18,264
DOES THE DIMER DISRUPTOR

1410
00:57:18,264 --> 00:57:19,699
INTERFERE WITH KRAS CELL

1411
00:57:19,699 --> 00:57:20,633
PROLIFERATION OR GROWING IN.

1412
00:57:20,633 --> 00:57:21,734
>> THAT'S A GOOD QUESTION.

1413
00:57:21,734 --> 00:57:25,471
SO YES AND NO.

1414
00:57:25,471 --> 00:57:30,343
SO IT CAN HAVE AN EFFECT IF IT'S

1415
00:57:30,343 --> 00:57:33,513
IN MELANOMA WHERE THERE'S NOT A

1416
00:57:33,513 --> 00:57:34,314
LOT OF UPSTREAM SIGNAL FREE

1417
00:57:34,314 --> 00:57:36,649
RADICALS GENERATED OTHER RTKs,

1418
00:57:36,649 --> 00:57:38,818
BUT WE'VE--AND THAT'S WHY WE'VE

1419
00:57:38,818 --> 00:57:43,690
KIND OF WANTED TO FURTHER

1420
00:57:43,690 --> 00:57:44,257
INCREASE ITS EFFICACY.

1421
00:57:44,257 --> 00:57:46,993
IF YOU HAVE LIKE IN A COLORECTAL

1422
00:57:46,993 --> 00:57:48,328
CANCER WHERE YOU'VE GOT AND YOU

1423
00:57:48,328 --> 00:57:50,096
HAVE A RAS MUTATION BUT THERE'S

1424
00:57:50,096 --> 00:57:52,332
A LOT OF SIGNALING COMING FROM

1425
00:57:52,332 --> 00:57:54,434
THE EGF RECEPTOR, IT'S NOT VERY

1426
00:57:54,434 --> 00:57:57,236
EFFECTIVE, SO, IT SEEMS, YOU

1427
00:57:57,236 --> 00:58:00,173
KNOW, IT'S IN THE MELANOMAS

1428
00:58:00,173 --> 00:58:02,008
WHERE REALLY, YOU DON'T HAVE A

1429
00:58:02,008 --> 00:58:04,177
LOT OF SIGNALING UPSTREAM OF RAF

1430
00:58:04,177 --> 00:58:07,146
SO IT'S NOT THAT POTENT.

1431
00:58:07,146 --> 00:58:08,681
>> SO BY EXTENSION WOULD
YOU

1432
00:58:08,681 --> 00:58:11,951
ENVISION THE DIMER DISRUPTER TO

1433
00:58:11,951 --> 00:58:16,255
BE AN OBLIGATE COLD DRUG AS

1434
00:58:16,255 --> 00:58:17,523
[INDISCERNIBLE] INHIBITOR

1435
00:58:17,523 --> 00:58:19,626
TREATMENTS, HER 2 INHIBITORS, IS

1436
00:58:19,626 --> 00:58:21,127
THAT WHAT YOU'RE THINKING?

1437
00:58:21,127 --> 00:58:21,861
>> WELL, YOU KNOW THAT'S

1438
00:58:21,861 --> 00:58:25,031
SOMETHING WE HAVEN'T DONE. 

1439
00:58:25,031 --> 00:58:27,967
THOSE ARE EXPERIMENTS ACTUALLY

1440
00:58:27,967 --> 00:58:30,403
THAT'S ON WELL, EXPERIMENTS WE

1441
00:58:30,403 --> 00:58:32,338
WANT TO DO.

1442
00:58:32,338 --> 00:58:32,538
YES.

1443
00:58:32,538 --> 00:58:36,242
WE'RE LOOKING AT IT.

1444
00:58:36,242 --> 00:58:38,811
WE STARTED IT WITH THE RAF

1445
00:58:38,811 --> 00:58:41,547
INHIBITOR, THE CO TREATMENT OF

1446
00:58:41,547 --> 00:58:42,615
THE RAF INHIBITOR, WE NEED TO

1447
00:58:42,615 --> 00:58:48,121
GO, WE NEED TO DO THE EGF

1448
00:58:48,121 --> 00:58:48,888
RECEPTOR, SOMETHING UPSTREAM. 

1449
00:58:48,888 --> 00:58:54,627
>> OTHER QUESTIONS IN
QUESTIONS?

1450
00:58:54,627 --> 00:58:55,662
ALL RIGHT.

1451
00:58:55,662 --> 00:58:56,129
LET'S THANK DEBBIE.

1452
00:58:56,129 --> 00:59:06,472
THANK YOU SO MUCH.