GOOD AFTERNOON FROM BETHESDA,
MARYLAND.
YOU ARE ATTENDING A SESSION OF
THE 21st YEAR OF DEMYSTIFYING
MEDICINE AT THE NATIONAL
INSTITUTES OF HEALTH.
AS WE HAVE POINTED OUT OVER THE
COURSE OF ALL THESE TALKS THAT
THIS IS AN ATTEMPT TO BRIDGE THE
EXCITING DEVELOPMENTS IN
BIOLOGY, ENGINEERING AND
COMPUTER SCIENCES WITH MEDICINE
AND HUMAN HEALTH.
THE LOGO, THE BROOKLYN BRIDGE,
DAVID McCULLOUGH SAID ONCE YOU
BUILD THE BRIDGE LIFE IS NEVER
THE SAME ON EITHER SIDE AND
TODAY'S TALK IS A FURTHER
EXAMPLE OF THAT PRINCIPLE WHICH
WE WILL GET YOU AND THE COURSE
IS TUESDAYS 4:00 TO 6:00 EASTERN
STANDARD TIME APE -- NOW TO ME
MIDDLE OF MAY AND THE CME CODE
IS 52090 AND URGE YOU TO SUBMIT
QUESTIONS VIA THE SEND LIVE
FEEDBACK ON THE VIDEOCAST
DISPLAY.
YOU DO THAT DURING THE TALKS,
THE QUESTION AND ANSWER PERIOD
THAT WILL FOLLOW.
I CALL YOUR ATTENTION TO THE
FACT THAT ALL PREVIOUS SESSIONS
FOR 21 YEARS ARE ARCHIVED AND
THE LINK IS PRESENTED THERE AND
IF YOU HAVE ANY OTHER QUESTIONS
JUST GET IN TOUCH WITH US.
NOW, IN THE LOGO MUCH THE
BROOKLYN BRIDGE, THERE WERE TWO
PEOPLE STANDING.
AND ONE CAN VISUALIZE THAT ONE
OF THEM MAY HAVE SOME DIRECT
RELATIONSHIP TO PATIENTS AND IS
CONCERNED WITH THE TERRIBLE
PROBLEM THAT BOTH INFECTION AND
IN CANCER ONE OF THE MOST
DEVASTATING EFFECTS IS WHEN
NOTHING WORKS BECAUSE OF THE
DEVELOPMENT OF RESISTANCE.
AND THE OTHER THE MOST ADVANCED
TECHNOLOGIES TO TRY TO BEGIN TO
UNDERSTAND HOW THIS HAPPENS.
SO IN CONSIDERING THIS PROPOSE
QUESTIONS WE COULD ALL THINK
ABOUT DURING TODAY'S
PRESENTATIONS.
IS DRUG RESISTANCE PART OF A
LARGER PHENOMENON OF SURVIVAL
AND HOW DOES IT RELATE TO
EVOLUTION, NATURAL SELECTION.
THERE'S A HUGE PRICE THAT'S PAID
FOR THESE EVENTS AS YOU PROBABLY
KNOW VIRUSES AND BACTERIA ARE
KILLED OFF IN THE MILLIONS IF
NOT BILLIONS WHERE AS EUKARYOTIC
CELLS THE PROCESS IS FAR SLOWER.
I THINK THE QUESTION IS NOT WHY
THE BACTERIA MAY BE WHY CANCER
VIRUSES WANT TO SURVIVE BUT THE
QUESTION IS HOW DO THEY DO IT.
AND IF WE UNDERSTOOD HOW WE
THINK ABOUT IT, WE MIGHT
UNDERSTAND HOW WILL ADVANCE
KNOWLEDGE OF THE ORIGIN OF LIFE
AND EVOLUTION.
SO, OUR FIRST SPEAKER IS MICHAEL
GOTTESMAN WHO IS OBVIOUSLY
EXTREMELY WELL KNOWN HERE AND
ABROAD.
MICHAEL GRADUATE FROM HARVARD,
TOOK TRAINING HIS CLINICAL
TRAINING IN BOSTON AND THEN DID
A POST-DOCTORAL FELLOWSHIP IN
MOLECULAR GENETICS AT THE NIH
WITH MARTIN GELARD AND RETURNED
TO HARVARD BRIEFLY AND CAME TO
NIH IN 1976.
IN 1994 UNTIL 2022, MICHAEL WAS
THE DEPUTY DIRECTOR OF THE
INTRAMURAL RESEARCH AT THE NIH
WHICH IS A GIGANTIC CHALLENGING
POSITION.
HE WAS CHIEF OF THE LABORATORY
OF CELL BIOLOGY AT THE NATIONAL
CANCER INSTITUTE AND MICHAEL HAS
WON MANY AWARDS INCLUDING
ELECTION TO THE NATIONAL ACADEMY
OF SCIENCE AND MEDICINE.
THE CAREER PUBLIC HEALTH SERVICE
AWARD OF THE AMERICAN MEDICAL
ASSOCIATION AND SO FORTH.
AND FOR MANY YEARS HE AND HIS
COLLEAGUES HAVE BEEN LEADING
RESEARCH ON DRUG RESISTANCE MAIN
IN CANCER AND APPLICATIONS AND
IN THE COURSE OF THIS HE HAS
TRAINED AN EXTRAORDINARY NUMBER
PEOPLE WHO HAVE BECOME LEADERS
IN THIS FIELD AND THROUGHOUT THE
WORLD.
THE SECOND SPEAKER IS JOHN
DEKKER.
JOHN RECEIVED BOTH AN M.D. AND
Ph.D. FROM HARVARD.
HE DID A PATHOLOGY RESIDENCY
FELLOWSHIP IN MEDICAL
MICROBIOLOGY AT THE MASS GENERAL
HOSPITAL AND CAME TO NIH IN 2013
AS A CLINICAL CENTER SENIOR
STAFF IN THE MICROBIOLOGY PART
OF THE MEDICINE LABORATORY
BRANCH WHERE HE WAS ACTING
CHIEF.
IN 2018 HE WAS AWARDED THE
LASKAR CLINICAL RESEARCH
FELLOWSHIP INVESTIGATOR IN THE
NATIONAL INSTITUTES OF ALLERGY
AND INFECTIOUS DISEASES.
SO JOHN HAS SERVED ON THE
ADVISORY COMMITTEES, BEEN AN
EDITOR OF THE JOURNAL OF
CLINICAL MICROBIOLOGY AND
RECEIVED AWARDS INCLUDING ONE
FOR THE AMERICAN SOCIETY OF
MICROBIOLOGY AND NIH CLINICAL
CENTER AWARD AND IN 2020 A YOUNG
PHYSICIAN SCIENTIST AWARD FROM
THE AMERICAN ASSOCIATION FOR
CLINICAL INVESTIGATION.
JOHN'S FORTE IS USING
NEXT-GENERATION BASED SEQUENCING
METHODS TO STUDY EPIDEMIOLOGY
AND IN INFECTIOUS DISEASE AND
CHIEF OF THE BACTERIAL
PATHOGENESIS ANTIMICROBIAL
RESISTANCE AND STUDIES THE
GENOMIC SESSION OF MICROBIOLOGY
AT NIH.
THE LATEST NEWS JUST RECEIVED
TODAY AND WE CONGRATULATION JOHN
IS THAT TODAY HE RECEIVED TENURE
AT THE NATIONAL INSTITUTES OF
HEALTH.
SO WE WELCOME ALL OF YOU AROUND
THE WORLD AS WELL AS HERE OF
COURSE AT THE NIH WATCHING THIS
ONLINE AND WE LOOK FORWARD TO
THE PRESENTATIONS AND MICHAEL
WOULD YOU --
>> LET ME START BY THANK YOU FOR
THAT INCREDIBLY GENEROUS
INTRODUCTION.
IT'S A DELIGHT TO BE HERE.
THIS IS NOT THE FIRST TIME I'VE
SPOKEN IN THE SESSION AND I
THINK I SPOKE IN THE FIRST YEAR,
21 YEARS AGO.
THAT WAS A LOT EASIER BECAUSE WE
KNEW THE BASIS OF DRUG
RESISTANCE AND NOW, SINCE WE
DON'T THIS IS GOING TO BE A MUCH
MORE INTERESTING TALK THAN THE
OLD ONE.
SO THIS IS CME COURSE AND WE
HAVE OBJECTIVES EXPLAINING HOW
RESISTANCE TO CANCER DRUGS LEADS
TO IMMUNOTHERAPY AND EPNEUM --
ENUMERATE SEVERAL MECHANISMS OF
RESISTANCE WHICH ARE SPECIFIC
FOR CANCER CELL TYPE AND
SELECTING DRUG AND WE WANT TO
BRIDGE THE GAP BETWEEN CLINICAL
AND LABORATORY AND SOMETIMES
THEY'RE ABLE TO BRING PATIENTS
TO ILLUSTRATE COMMENTS THEY'LL
MAKE.
I THOUGHT IT WOULD BE REDUNDANT
TO HEAR ABOUT THE FACT THAT
CANCER BASICALLY IS RESISTANT TO
MANY KINDS OF THERAPY AND THESE
ARE SOME STATISTICS.
YOU CAN SEE FOR BOTH MEN AND
WOMEN THERE'S ABOUT A MILLION
NEW CASE AS A YEAR IN DIFFERENT
SITES AND ABOUT 300,000 PATIENTS
DIE FOR MEN AND WOMEN.
THE PATIENT WHO'S SURVIVE CANCER
ARE FREQUENTLY TREATED
EFFECTIVELY BY SURGERY OR
RADIATION THERAPY IF THE CANCER
IS LOCALIZED.
IF IT SPREAD, ME TTASTASIZES IT
MORE DIFFICULT TO TREAT AND
ALMOST EVERYONE IN THE UNITED
STATES WHEN HAS CANCER THAT HAS
SPREAD GETS CHEMOTHERAPY.
YOU CAN READ IN THE NEWSPAPER
EXCITING RESULTS ABOUT
IMPROVEMENTS IN TREATMENT THAT
EXTEND LIFE AND IMPROVE QUALITY
OF LIFE BUT THE REALITY IS THAT
VIRTUALLY EVERYONE WHO DIES OF
CANCER DIES OF DRUG RESISTANT
CANCER.
SO WE'RE TALKING ABOUT A LOT OF
PATIENTS AND I WOULD IMAGINE
THAT VIRTUALLY EVERYONE IN THE
ROOM KNOWS SOMEBODY WHO DIED OF
DRUG RESISTANT CANCER.
SO THERE'S A KIND OF THEME TO
THE ISSUE OF DRUG RESISTANCE
INCLUDING MICROBIAL AGENTS AND
INFORMATION HOW THIS OCCURS AND
I'LL GO QUICKLY THROUGH THIS AND
THINK IT'S RELEVANT TO WHAT JOHN
HAS TO SAY AS WELL.
WHEN YOU TREAT A PATIENT WITH
CHEMO THERAPY AND USE A DRUG TO
WHICH THEY'RE SENSITIVE AND IT
TURNS OUT THAT SOMETIMES WITHIN
THE POPULATION OF CELLS THAT ARE
BEING TREATED THERE ARE A FEW
CELLS RESISTANT AND WHAT HAPPENS
IS THAT EITHER YOU DON'T GET A
RESPONSE TO THE CHEMOTHERAPY OR
GET A TRANSIENT RESPONSE AND
RESISTANT CELLS GROW OUT.
THE CONCEPT INTRINSIC RESISTANCE
GOES BACK TO RESISTANCE IN
VIRUSES AND BACTERIA AND WE'VE
DONE SIMILAR STUDIES LOOKING AT
RESISTANCE TO VARIOUS DIFFERENT
DRUGS AND CULTURED CELLS AND
WHAT YOU SEE IS THAT IN MANY
CASES THE CELLS THAT EXIST,
PREEXIST IN THE POPULATION.
THEY'RE AT LOW LEVELS BUT
PREEXIST AND NO MATTER WHAT YOU
DO, THEY'LL POP UP.
IT'S NOT TRUE OF ALL RESISTANCE.
IN SOME CASES THE DRUGS
THEMSELVES INDUCE RESISTANCE OR
ENABLE SURVIVAL OF THE CELLS AND
EXAMPLES MAY INCLUDE THE
TRANSPORTERS YOU'LL BE HEARING
LATE ABOUT TODAY, TRANSCRIPTION
FACTORS, METABOLIZING ENZYMES
ALL TO BE INDUCED IN THE SHORT
TERM AND WHEN THEY ARE THE CELLS
CAN SURVIVE FOR A WHILE.
THESE MECHANISMS TEND TO BE
PRETTY INEFFICIENT AND AS SOON
AS THE CELL CAN FIND A WAY TO
FIX A MUTATION FOR EXAMPLE IN A
TARGET UP A SPECIFIC FACTOR
THAT'S ESSENTIAL FOR THE CELLS
TO BE KILLED BY THE DRUG WILL
HAPPEN IN FIXED MUTATIONS AND
WHEN WE SELECT FOR LONG-TERM
RESISTANCE TO DRUG MOST OF WHAT
WE TALK ABOUT IS THE MIXED
MUTATIONS AND MISSING THE
INDUCED RESISTANCE AND I SUSPECT
JOHN WILL HAVE MORE TO SAY ABOUT
THIS PROBLEM.
SO WHAT RAIN SHOWER GOALS I'LL
SPEND ON THE ENERGY DEPENDENT
STORIES AND DETERMINE THE
CLINICAL RELEVANCE OF MECHANISMS
DERIVED FROM IN VITRO STUDIES
AND DEVELOPMENT NEW APPROACHES
TO COMPLOIT OR CIRCUMVENT
CLINICALLY SIGNIFICANT RESISTANT
MECHANISMS AND TALK ABOUT THE
BLOOD BRAIN BARRIER.
SO HOW DO CELLS GET RESISTANT?
IF YOU HAVE A SPECIFICALLY
TARGETED DRUG AIMING FOR EXAMPLE
AT A RECEPTOR ON THE SURFACE AND
SOME TARGET KINASE RECEPTORS AND
BYPASS MUTATIONS ALLOW CANCER
CELLS TO GROW.
A SECOND WAY IS THROUGH WHAT WE
CALL ALTERED CELL PHARMACOLOGY,
FAILURE TO TAKE UP A DRUG.
MECHANISM TO E-FLUX DRUGS AND
SEQUESTER DRUGS AND TO
METABOLIZE DRUGS.
A PANOPLY OF POSSIBILITIES CELLS
CAN USE TO SURVIVE AND THIS IS
AN INTERESTING ISSUE.
I THINK SIMILAR TO ONE RAISED TO
BE A LIFE FORM ON EARTH YOU HAVE
TO BE RESISTANT TO A LOT OF
TOXIC INSULTS.
LIFE EVOLVED IN A TOXIC SEA.
MEMBRANES WERE PUT AROUND LIFE
FORMS AND THERE HAD TO BE A WAY
TO SURVIVE.
SOME TIME AGO WHEN PEOPLE BEGAN
TO KNOCK OUT GENES IN YEAST THEY
DISCOVERED THERE WERE ONLY A FEW
ESSENTIAL GENES UNDER LABORATORY
CONDITIONS BUT HALF THE GENOME
CAN HAVE SENSITIVITY TO
CHEMICALS AND YEAST THAT
SURVIVED BUT WHEN YOU ADD
CHEMICAL Z IT DIDN'T DO AS WELL
AND WILD TYPE AND THE THOUGHT IS
EVERY ONE OF THE GENES IS
SOMETHING THAT GETS EXPRESSED TO
HELP US SURVIVE IN A NASTY
ENVIRONMENT.
ANOTHER WAY IN WHICH RESISTANCE
CAN DEVELOP IS BY MORE DRAMATIC
CHANGES IN DEVELOPMENTAL OR
DIFFERENTIATION PATHWAYS AND THE
CLASSIC WAY FOR CULTURED OR
CANCER CELLS TO GET RESISTANCE
IS A TRANSITION AND IT MEANS THE
CELLS GO FROM AN EPITHELIAL FORM
SAY LIVER CANCER OR COLON
CANCER, THOSE ARE EPITHELIAL
CELLS AND THE CELLS CHANGE THE
DIFFERENTIATION PROFILE IN
BECOMING MORE FIBROBLAST LIKE
AND THE PATTERN TO SENSITIVITY
TO DRUGS CHANGES AND THEY MAY
BECOME QUITE RESISTANCE AND A
WAY OF USING ENTIRE PATHWAYS
EXPRESSED IN CELLS TO CHANGE
EITHER METABOLISM OF DRUGS OR
TRANSPORTIVE DRUGS OR THE
SENSITIVITY THE CELL HAS TO THE
SPECIFIC TARGET IN THE CELL.
YOU CAN CONSIDER FOR EXAMPLE THE
PROBLEM OF PERSISTENCE WHICH IS
DESCRIBED IN BACTERIA AS A
POPULATION OF BACTERIA ABLE TO
SURVIVE IN THE FACE OF
ANTIBIOTIC TREATMENT IN A
VEGETATIVE STATE AND SAME IN
CANCER CELLS THAT STOP DOING
THINGS, THEY BECOME LESS
SENSITIVE BUT THE PRECISE
MECHANISMS BY WHICH THAT
HAPPENED ARE NOT WELL DESCRIBED.
AND WE FINALLY BECAUSE CANCER
CELLS ARE NOT LIVING IN AN OPEN
ENVIRONMENT BUT PART OF AN
ORGANISM THERE ARE LOTS OF WAYS
IN WHICH THE LOCAL ENVIRONMENT
CAN AFFECT THE ABILITY OF THE
CANCER CELLS TO ADVISER AND
BLOOD SUPPLY, OXYGEN
CONCENTRATION, SUCH AS PHYSICAL
PROPERTIES OF THE ENVIRONMENT
AND YOU SEE CHANGES IN DRUG
SENSITIVITY AND RESISTANCE.
IN ADDITION THERE'S THE WHOLE
BUSINESS ABOUT THE MICROBIOME
AND IT AFFECT THE WAY DRUGS ARE
METABOLIZED, HOW THE IMMUNE
SYSTEM RESPONDS TO DRUGS,
ETCETERA.
THIS IS A HUGE AREA.
THERE'S CANCER CELL METRO GEN
EIGHT AND WE THOUGHT THEY WERE
CLONAL AND IF YOU DO SINGLE CELL
SEQUENCES YOU GET AN ARRAY OF
MUTATIONS ALL OF WHICH CAN
CONTRIBUTE TO RESISTANCE.
THE PATHWAY OF CELLS SURVIVAL
ARE QUITE ROBUST IN CANCER
CELLS.
THEY ARE SURVIVORS.
ALL THIS DOESN'T HAPPEN
INSTANTANEOUSLY AND EVOLUTION
OCCURS IN THE TIME ITSELF AND
CANCER CELLS HAVE AN INCREASED
RATE OF GENETIC AND EPIGE GEGEN
AND THE IT'S STACK AGAINST THE
CANCER CELL.
THERE'S TWO WAYS TO STUDY THIS
IN THE LABORATORY FIRST IS AN
APPROPRIATE CULTURED MODEL AND A
CELL DERIVED FROM A SPECIFIC
CANCER AND SELECT FOR RESISTANCE
AND TRY TO IDENTIFY GENES AND
THE ONE WE'RE BEST KNOWN FOR THE
ABC1 AND ABC TRANSPORTER IS
IDENTIFIED BY THE FACT THE GENES
WERE AMPLIFIED.
THE PHENOMENON NOT KNOWN IN
BACTERIA AS WELL.
AND THEN RECENTLY AS THE
TECHNOLOGY HAS CHANGED WE'VE
BEEN USING RNA SEQ INTERROGATING
WHAT THE RNAs ARE EXPRESSED IN
RESISTANT CELLS AND ANY
RESPONSIBLE FOR RESISTANCE?
AND UNTIL RECENTLY TRYING TO
KEEP UP WITH THE TECHNOLOGY
WE'VE USED CRISPR BASED
APPROACHES TO IDENTIFY GENES AND
I SHOULD SAY THE SELECTIONS ARE
SHORT TERM.
WHEN WE SELECT FOR CELLS
RESISTANT WE GROW THEM FOR A
LONG TIME TO BECOME REALLY
RESISTANT.
WHEN WE DO CRISPR THE EXPOSURE
IS TRANSIENT FOR TWO OR THREE
WEEKS.
WE'RE LOOKING FOR SURVIVAL
FACTORS.
GENES WHICH CAN BE INDUCED OR
EXPRESSED THAT WILL ALLOW CELLS
TO SURVIVE UNDER THE SELECTION
CONDITIONS AND I'LL GET INTO
THAT A LITTLE LATER.
OKAY NOW I'LL GO BACK AND TELL
YOU A BIT OF HISTORY BECAUSE
IT'S AN INTERESTING STORY HOW
DID WE HAPPEN TO DISCOVER THE
ABC TRANSPORTER GENES.
WE WERE ASKED BY THE DIRECTOR OF
OUR DIVISION AT THE NATIONAL
CANCER INSTITUTE TO DO SOMETHING
ABOUT THE PROBLEM OF MULTI-DRUG
RESISTANCE.
A PHENOMENON DESCRIBED IN TISSUE
CULTURE AND TRUE CLINICALLY IN
WHICH A PATIENT WHO WAS EXPOSED
TO ONE DRUG BECAME RESISTANT TO
MANY OTHER DRUGS.
HOW CAN THAT BE?
WE BEGAN TO SELECT CELLS AND AS
WE SELECTED TO HIGHER LEVELS OF
RESISTANCE WE MADE SURE THEY
WERE CROSS-RESISTANT.
WE WERE FAVORING THE
AMPLIFICATION OF THE SPECIFIC
MECHANISM THAT LED TO THE
CROSS-RESISTANCE PHENOMENON AND
REPEATED THAT WITH MULTIPLE
DIFFERENT DRUGS AND WE WERE
FORTUNATELY ABLE TO RECRUIT A
FASTIDIOUS TISSUE CULTURE CELL
BIOLOGIST AND THOUGHT AND
THERE'S LITERATURE SHOWING
REDUCTASE INDUCTERS WERE
RESISTANT AND WE STARTED THINK
THE GENE RESPONSIBLE WOULD BE
AMPLIFIED.
IN DEED IT WAS AND I WAS AT A
GORDON CONFERENCE AT A
RELATIVELY YOUNG FACULTY MEMBER
AND A GOOD REASON TO HAVE
CONFERENCES IN PERSON, BY THE
WAY, AND MET THIS TALENTED
SCIENTIST AND WHO WAS RUSSIAN
AND TRYING TO FIND A JOB AND HAD
A POSTER NEXT TO MINE HOW TO
CLONE AMPLIFIED GENES AND HE
RENATURED IT IN A CELL AND THE
GENES THAT WERE HIGH COPY NUMBER
WERE DOUBLE STRANDED AND
HYBRIDIZED MORE QUICKLY AND WERE
ABLE TO PULL PROBES AGAINST GENE
X THAT WAS AMPLIFIED IN THE
CELLS.
YOU CAN SEE ALL THE CELL LINES
WE HAD AND AS THEY BECAME MORE
RESISTANT THE COPY NUMBER
INCREASED THIS IS A CELL BLOT
BASED ON THE PROBE WE WERE ABLE
TO GET THANKS TO IGGOR'S WORK
AND NEXT WAS TO PROVE IT HAD
SOMETHING TO DO WITH DRUG
RESISTANCE AND A POSTDOC WAS
ABLE TOO PULL TOGETHER ALL THE
CDNAs AND WE HAD A LIBRARY OF
THEM AND YOU'RE ALL THINKING WHY
DON'T YOU LOOK UP THE SEQUENCE
AND FIGURE IT OUT.
WE HAD NO SUCH DATABASES AT THE
TIME AND WE PUT TOGETHER AND PUT
IN A VIRUS AND INFECTED CELLS
AND SURE ENOUGH THEY WERE
RESISTANT TO ALL THE DRUGS THEY
WERE RESISTANT TO.
A SINGLE VIRUS WAS ABLE TO
CONFER THE ENTIRE RESISTANCE AND
TURNED OUT COLLEAGUES OF HOURS
DESCRIBED A PROTEIN OF RESISTANT
CELLS CALLED P GLYCOPROTEIN AND
P STOOD FOR PERMEABILITY AND IT
TURNED OUT THE GENE CLONED WAS A
GENE FOR THE PROTEIN AND WE
PUBLISHED A PAPER THAT
DEMONSTRATED THAT.
NOW I'LL GO THROUGH THE STORY OF
ABC TRANSPORTER AND THIS IS AN
EXAMPLE OF A TRANSPORTER THAT
BELONGED TO WHAT BECAME A LARGER
FAMILY OF 48 HUMAN ABA TRANS
PORTERS IN SEVEN CATEGORIES
KNOWN AS ABCDEF AND G.
AND WHAT THEY HAVE IN COMMON IS
THEY HAVE TRANS MEMBRANE REGIONS
AND TWO ATM SITES.
SOME ARE HALF MOLECULES BUT IT'S
LATER SHOWN THEY'RE DIMERIZE TOE
FORM THIS STRUCTURE AND THIS IS
THE STRUCTURE OF P GLYCOPROTEIN
SHOWN HERE.
AND THERE'S MANY EXAMPLES OF
THIS AND IT TURNED OUT THEY'RE
RESPONSIBLE FOR MOVING ALL KINDS
OF THINGS INTO AND OUT OF CELLS
IT AND IN AND OUT OF
COMPARTMENTS OF CELLS AND COUPLE
ENERGY TO THE TRANSPORT PROCESS.
THERE'S A THERE'S TWO EXAMPLES
WHERE THE ENGINE EXISTS THE ATP
BINDING CASSETTE INVOLVED IN
TRANSCRIPTION BUT NO TRANS
MEMBRANE COMPONENT AND THESE ARE
LITTLE ENGINES THAT CAN AND DO
AND PRECISELY WHAT THEY'RE ROLE
IS, IS NOT ENTIRELY CLEAR YET.
WE'RE BACK TO P GLYCOPROTEIN AND
WHY IS THIS GENE THAT PUMPS OUT
OF CELLS AND AGAIN THIS WORK WAS
DONE SHOWING IT WAS EXPRESSED AT
BARRIER FUNCTIONS IN THE HUMAN.
THIS IS AN ULTIMATE HUMAN
TOTALLY ROUND AND YOU CAN SEE
THERE'S PUMPS THAT PUMP THINGS
OUT OF THE PLACENTA.
PUMP THINGS OUD OF OUT OF THE
KIDNEY AND LIVER AND INTESTINE
OUT OF THE BODY.
THERE'S A BLOOD BRAIN BARRIER AS
WELL AS A PLACENTAL BARRIER AND
IF YOU'RE A TUMOR THE BLOOD IS
BUSY PUMPING THINGS IN THE
BLOOD.
IT TURNED OUT THERE WERE DOZENS
AND HUNDREDS AND PROBABLY
THOUSANDS RECOGNIZED BY THIS
SYSTEM AND PUMPED OUT AND SOME
DRUGS ARE LISTED HERE AND
THERE'S INTERESTING STORIES AND
IF I HAD MORE TIME I'D GO INTO
THEM BUT A LOT OF THE DRUGS THAT
ARE IN COMMON USE ARE SUBSTRATES
FOR THE TRANSPORT SYSTEMS.
SO THERE ARE THREE OF THE 48
TRANSPORTERS POLY SPECIFIC AND
CAN RECOGNIZE MANY DIFFERENT
DRUGS.
ONE IS CALLED ABCG2 A DIMER OF
TWO HALF MOLECULES.
ONE IS ABC1 OR PGP SHOWN HERE
AND ONE IS MRP 1 WITH AN
INTERNAL EXTENSION WHICH IS
INTEGRATED INTO THE PLASMA
MEMBRANE AND THE FUNCTION IS NOT
ENTIRELY CLEAR.
YOU CAN CUT IT OFF AND THE
TRANSPORTER STILL WORKS.
SO THESE THINGS LOOK SOMEWHAT
ALIKE BUT NOT IDENTICAL.
THE STRUCTURES HAVE BEEN
DETERMINED BY A VARIETY OF
DIFFERENT LABS IN OUR LABORATORY
AND THERE WERE MAJOR
CONTRIBUTION.
THERE ARE WONDERFUL EXAMPLES BY
STEVE A BUNCH OF PEOPLE
CONTRIBUTED AND WE HAVE SOME
IDEA HOW THE TRANSPORTER
FUNCTIONS.
MOST THE TRANS MEMBRANE REGIONS
STICK IN THE PLASMA MEMBRANE
REGION AND MOST FROM IN THE ATP
AND WE THINK IT ENTERS THROUGH
THE PLASMA MEMBRANE AND ATP
BINDS TO THE SITES AND THE TWO
SITES COME TOGETHER, ATP'S NOT
YET HYDROLYZED BUT CATALYZE THE
ATP BINDING SITE AND THE DRUG IS
EXTRUDED.
THEN THE ATP IS HYDROLYZED,
RELEASED, ATP COMES OFF AND THE
TWO ATP SITES SEPARATE.
THIS IS A VERY GLOBAL
DESCRIPTION.
THE EXACT DETAILS ARE STILL
BEING WORKED OUT.
AND IN PARTICULARLY EXACTLY HOW
IT'S LINKED TO THE TRANSPORT
FUNCTION OF THE TRANSPORTERS.
IF YOU LOOK AT THE SPECIFICITY
OR LACK OF THE SPECIFICITY OF
THE TRANSPORTERS.
THREES A VEN DIAGRAM THAT SHOWS
SOME DRUGS INCLUDING MANY
STANDARD ANTICANCER DRUGS
RECOGNIZED BY ALL THREE
TRANSPORTERS.
SOME SPECIFICALLY BY ONE OR THE
OTHER OR BOTH OR TWO AT A TIME
AND SO UPON SO THIS IS IN THE
HUMAN.
IN THE MOUSE IT'S DIFFERENT AND
IN THE RAD IT'S DIFFERENT AND
WE'VE BEEN WORK ON THE ZEBRAFISH
AND IT'S DIFFERENT AND IT'S A
VERY COMPLICATED AND IT MAKES IT
HARD HOW TO INHIBIT OR FIND
DRUGS WHAT MOST PHARMACEUTICAL
COMPANIES DO IS TRY TO FIND
DRUGS NOT SUBSTRATES FOR ANY OF
THESE TRANSPORTERS AND THAT'S
BEEN TO SOME EXTENT SUCCESSFUL.
SO ONE QUESTION IS HOW ARE THE
DRUGS RECOGNIZED.
IT FLIES IN THE FACE OF WHAT WE
LEARNED ABOUT SPECIFICITY OF
TRANSPORT AND ENZYMATIC
SPECIFICITY AND CAME UP WITH THE
IDEA THE DIFFERENCE IS WAS IN
THE PLASMA MEMBRANE.
THE DRUGS WERE GOING FROM THE
OUTER AND RECOGNIZED IN THE
BINDING SITE WITHIN THE
TRANSPOR
TRANSPORTER -- AND THERE'S NOW
QUITE A BIT OF EVIDENCE
SUGGESTING HOW THEY WORKED AND
THE LACK OF SPECIFICITY NOT
WITHIN THE POLY SPECIFIC DRUG
BINDING SITES BUT THE DELIVERY
IS THROUGH THE HYDROPHOBIC
MEMBRANE.
WITH DISCOVERED THE ORIGINAL
GENE WAS A POLY MORPHIC FORM AND
CLONED OUT OF HELA CELLS AND
ANYBODY NOW KNOWS HELA CAME FROM
A WOMAN CALLED HENRIETTA LACKS
AND THERE'S A NUMBER OF
POLYMORPHISMS FROM PEOPLE WITH
ASI
ASIAN DESCENT AND IT DIDN'T
CHANGE THE AMINO ACID BUT THE
NUCLEOTIDE.
A
AND IT WAS DISCOVERED THIS
POLYMORPHISM EXISTED IN A PLACE
THAT ALLOWED IT TO FORM IN THE
RNA AND WHEN WE DID THE
FOOTPRINTING THERE WAS A LONG
PAUSE THAT OCCURRED WHILE THE
RNA WAS BEING TRANSLATED ON THE
RIBOSOME AND BELIEVE THERE WAS A
SLIGHT ALTERATION IN THE FOLDING
OF THE TRANS MEMBRANE REGION
WHICH IS WHERE THE DRUG BIENGD
SITES RAND THEY MAI HAVE LED TO
THE ALTERATION INCLUDING
INCREASED RESISTANCE.
AND YOU COULD IMAGINE THIS WOULD
HAVE ADVANTAGE DUE TO THE
EXPOSURE OR TOXIC MATERIALS IN
THE PERSON'S DIET.
THEY DON'T CONFER AND THIS IS
TURNED OUT TO BE MORE TRUE THAN
WE THOUGHT ORIGBINDING THOUGHT .
IS AFTER TREATMENT SOME EXPRESS
AND SOME DON'T EXPRESS PGP AND
NOT SURPRISING THEY DON'T
RESPOND TO A LOT OF THE DRUGS WE
USE AND THEY'RE ANIMAL MODELS IN
WHICH CANCER XENO GRAPHS CAN BE
SELECTED IN THE ANIMAL FOR
EXPRESSION AS A REASON FOR
RESISTANCE IN THE ANIMALS.
BASED ON ALL THE DATA WE
CONCLUDED P GLYCO PROTPROTEIN I
ENOUGH FOR RESISTANCE AND MAY
NOT BE NECESSARY, IN OTHER
WORDS, THERE'S OTHER CAUSES OF
DRUG RESISTANCE.
RECENTLY THERE'S BEEN A FAIR
AMOUNT OF ADDITIONAL EVIDENCE
THAT PRETTY MUCH MAKES IT, IT
CAN BE RESPONSIBLE FOR
RESISTANCE TO CANCER AND IN SOME
TUMORS THERE'S HIGH LEVEL OF
EXPRESSION, NOT ALL BUT SOME.
AND SOME CASES THERE'S R
REAARRANGEMENTS THAT TURN ON
EXPRESSION AND THOSE ARE
SELECTED FOR BY THE DRUGS.
-- REARRANGEMENTS THAT TURN ON.
THERE'S SUBSET OF PATIENTS WITH
HIGH LEVELS OF PGP2 AND AS WE
BEGIN TO USE MORE TARGETED DRUGS
WE FIND RESISTANCE DEVELOPING
AGAINST THOSE DRUGS THAT ARE
SUBSTRATES FOR THE TRANSPORTER.
NON-SMALL CELL IS AN EXAMPLE AND
THERE'S AN EXAMPLE A CON GUE --
CONJUGATE AND ONE BASIS OF THE
RESISTANCE OF THE DRUG APPROVED
BY THE FDA TO TREAT HODGKIN'S
LYMPHOMA IS AN EXPRESSION OF THE
PROTEIN AND THOSE THAT ARE
NAYSAYERS MOST OF THE CLINICAL
TRIALS WHERE PATIENTS WERE
TREATED WITH TO THE PGP AND
FAILED TO RESPONSE TO
CHEMOTHERAPY AND THEY SAID PGP
CAN'T BE RESPONSE IN ALMOST ALL
THE TRIALS PGP WASN'T
DEMONSTRATED TO BE PRESENT AND
IF'S ONLY PRESENT IN IF 5% TO
10% THERE'S NO CLINICAL TRIAL TO
DESIGN EXCEPT FOR ONE WHERE
YOU'RE SPECIFICALLY LOOKING AT
PGP EXPRESSING CELLS.
BOTTOM LINE IT'S EXPRESSED IN
SOME DRUG-RESISTANT CANCERS AND
THERE'S THOUGHT IT'S THE CAUSE
BUT THERE'S OTHER MECHANISMS OF
RESISTANCE.
ONE THING THAT BECAME CLEAR TO
US IS THOUGH IT MAY NOT BE
RESPONSIBLE FOR ALL DRUG
RESISTANCE, IT CERTAINLY IN ITS
PHYSIOLOGICAL ROLE OF THE BLOOD
BRAIN BARRIER WAS RESPONSIBLE
FOR KEEPING A LOT OF THE DRUGS
OUT OF THE BRAIN AND TREATING
PRIMARY BRAIN TUMORS IS
ESPECIALLY DIFFICULT BECAUSE
MANY OF THE DRUGS WE'D LIKE TO
USE ARE NOT ABLE TO GET IN THE
BRAIN.
WE BEGAN TO STUDY THE BLOOD
BRAIN BARRIER AND THE TRANS
PORTERS ARE PGP AND ABCG2 AND
THERE'S EXAMPLES HERE.
AND WE LOOK AT OF THE
ACCUMULATION OF THE DRUG IN THE
BRAIN AND RED MEANS A LOT OF
DRUG IN THE BRAIN BUT IF YOU
KNOCK OUT DRUG YOU SEE
REDUNDANCY AND HAVE TO KNOCKOUT
BOTH, INHIBIT BOTH TO GET DRUGS
IN THE BRAIN.
WE WANTED TO STUDY THE
PHENOMENON AND DO A BETTER JOB
OF GET DRUGS IN THE BRAIN.
MATT HALL WHO JUST BECAME A
SCIENTIFIC DIRECTOR OF NCATS A
WEEK AGO SO THERE WAS A MODEL IN
THE MOUSE ARE WE TOOK VOE VOS
IFASE AND IS SUBSTRATE ABCG2 A
TRANSPORTER THAT SITS AT THE
BLOOD BRAIN BARRIER.
THEY COULDN'T GET ACROSS THE
BLOOD BRAIN BARRIER UNLESS YOU
INHIBITED ABCG2 AND YOU LIGHT UP
THE BRAINS OF THESE MICE.
YOU HAVE MORE INHIBITER AND GET
MORE LIGHT.
THE INTERESTING FEATURE OF THE
MODEL IS IT DOESN'T DEPEND ON
THE INHIBITION OF THE
TRANSPORTER.
ANYTHING THAT DISRUPTS THE
JUNCTION BETWEEN THE EPITHELIAL
CELLS ALLOWS THE DRUGS TO GET IN
AND THIS SEEMED LIKE A GOOD
MODEL.
THE THROUGHPUT WAS SOMEWHAT
LIMITED IF WE WANTED TO FOR
DRUGS.
IN FACT WE DECIDED TO START
WORKING WITH ZEBRAFISH.
IT TURNS OUT THE ZEBRAFISH BLOOD
BRAIN BARRIER HAS MANY ATOMIC
SIMILARITIES.
AND WE HAD TO FIGURE OUT WHAT
THE ABC TRANSPORTERS WERE
EXPRESSED IN THE BLOOD BRAIN
BARRIER IN ZEBRAFISH.
TURNS OUT THERE'S A HOMEO --
HOMEOLOGUE AND THIS IS WORK DONE
BY JOE THOMAS IN OUR LAB.
WHAT IS THE SUBSTRATE OF THE
INHIBITOR SPECIFICITY OF THE
ZEBRAFISH MULTI DRUG
TRANSPORTERS WE WERE ABLE TO USE
A MORE SENSITIVE LUCIFERASE AND
THE WORK IS ONGOING AND NEXT
TIME I COME BACK I'LL TELL YOU
ALL ABOUT IT IN DETAIL.
YOU CAN USE CRISPR TO HOOK UP
THE KAS 9 NUCLEASE TO A ZINC
INHIBITER CALLED KRAB OR FOUR
COPIES OF THE VB16 OF THE HERPES
VIRUS AND THEY WORK WELL IF IT
TURNS ON OR TURNS OFF DOWN
STREAM GENES OR KNOCK OUT THE
GENES.
THERE'S LIBRARIES AVAILABLE THAT
HAVE GUARD RNAs FOR THE ENTIRE
GENOME.
I WON'T GO INTO THIS IN DETAIL
BUT YOU CAN INTRODUCE THE CAS9
TOO THE ABOUT WHAT IS OF
INTEREST AND PUT IN THE RNAs.
YOU CAN SELECT FOR THE DRUG YOU
WANT THE CELLS TO BE RESISTANT
TO AND ASK WHAT GENES GET TURNED
ON AND OFF.
THE FIRST EFFORT WAS DONE BY THE
PERSON SHOWN HERE AND SHE DID AN
EXPERIMENT AND ASKED WHAT GENES
MADE CELLS RESISTANT TO TAXOL A
WELL KNOWN ANTI-CANCER DRUG AND
DID A STATISTICAL ANALYSIS OF
THE GUARD RNAs AND FOUND BY TEN
ORDERS OF MAGNITUDE
STATISTICALLY THE GENE LIKELY TO
SURVIVE IN THE CELLS IS OUR
FAVORITE GENE, ABCD1.
THESE EXPERIMENTS TAKE A COUPLE
MONTHS.
IT ORIGINALLY TOOK US TWO TO
THREE YEARS TO CLONE THE
PROTEIN.
THIS IS A VERY EFFICIENT
TECHNOLOGY.
THERE'S OTHER GENES IN HERE AS
WELL BUT SIX LOGS LESS LIKELY TO
BE CRITICAL.
WE REPEATED THE EXPERIMENT WITH
THE SAME RESULTS AND NOW WE'RE
DOING AN EXPERIMENT IN WHICH WE
INHIBIT PGP SO IT CAN'T BE
RESPONSIBLE FOR RESISTANCE AND
ASKED WHETHER ANY OR ALL OF
THESE BECOME THE MAJOR MEDICAL
SYSTEM AND YOU CAN PLAY LOTS OF
GAMES WITH THE SYSTEM AND WE'RE
LOOKING AT RESISTANCE AND EACH
WOULD BE A SEMINAR ON ITS OWN
BUT EVERY TIME WE TAKE A CELL
TYPE OF COLON OR LIVER CANCER,
PANCREATIC CANCER AND A DRUG
USED TO TREAT THAT, WE GET A
WHOLE LUNCH OF GENES THAT CONFER
SENSITIVITY OR RESISTANCE AND
SORTING THROUGH THESE AND
FIGURING OUT WHICH ONES ARE
CLINICALLY IMPORTANT ARE THE
NEXT GENERATION'S JOB.
OKAY.
TWO VERY QUICK STORIES MAYBE
REALLY QUICK.
SO WE HAVE BEEN ALL ALONG
SELECTING CELLS AND LOOKING FOR
RESISTANCE AND HAD A VARYING
CELL LINE RESISTANCE TO AN
IMPORTANT ANTI-CANCER DRUG AND
RESISTANCE IS HARD TO COME BY
WHICH IS WHY IT'S GOOD BUT CAN
SELECT CELLS THAT ARE
RESISTANCE.
IT KNOCKS OUT DNA BY DAMAGING
DNA AND RESISTANCE DEVELOPS BY
IMPROVING DNA REPAIR.
AND WE GOT A GENE THAT MORE
POWERED THAN OTHER CALLED TTPP3.
AND THIS GENE IT TURNS OUT
LITTLE WAS KNOWN AND TURNED OUT
TO BE A GENE WHOSE PRODUCT
STABILIZES MICROTUBE S AND THE
CELLS BECAME SENSITIVE TO THE
DRUG.
AND WE ADDED IT BACK AND CELLS
BECAME RESISTANT AND IT BECAME
CLEAR THEY WERE THE FACTOR AND
WHEN WE DID ADDITIONAL STUDIES
IN THE NEUROLOGY CENTER AND
MICROTUBULES ARE REALLY
IMPORTANT AND THEY WERE CAPABLE
OF DOING IN VITRO MICROTUBULE
ASSAYS AT ACHIEVABLE DOSES AND
LO AND BEHOLD THE MICROTUBULES
WERE NOT HAPPY AND THE DYNAMICS
WERE CHANGE.
WE ADDED TPPP3 AND THEY WERE
STABILIZED AND HAPPY AGAIN AND
IT LOOKS LIKE THERE'S A
MICROTUBULE DAMAGING AGENT AND
IT EXPLAINS SOMETHING ABOUT THE
TOXICITY AND WHY IT'S SUCH A
GOOD DRUG AND HITS THE SENSITIVE
REGIONS OF THE CANCER CELL.
SO THIS IS KIND OF AN
INTERESTING STORY.
AND THEY SELECTED CELLS
RESISTANT TO THE ASSCETYLASE
INHIBITORS AND WE DID A
SELECTION IN THE PRESENCE OF AN
INHIBITER AND DID RNA SEQ
ANALYSIS AND GOT THIS GENE, MCF7
AND TURNS OUT IT'S A METHYL
TRANSFERASE AND OVEREXPRESSION
OF THE GENE WAS ABLE TO
INACTIVATE THE ROMIDEPSIN SO WE
HAVE AN EXAMPLE OF ALMOST
EVERYTHING AND DRUG RESISTANCE
CAN OCCUR IN MANY DIFFERENT WAYS
AND YOU'LL HEAR THE SAME STORY
FROM JOHN IT DEPENDS ON THE CELL
LINE AND DRUG AND THE WAY IN
WHICH YOU CAN MAKE THE
SELECTION.
IT'S MULTI-FACTORIAL AND NEED TO
SEE IF IT'S PREEXISTING OR
OCCURS DURING THE EVOLUTION OF
THE TUMOR.
I'LL GET TO THAT IN A MOMENT.
IF IT'S PREEXISTING IT'S
POSSIBLE THEN TO DO MOLECULAR
SCREENING WITH HIGH SENSITIVITY
AND FIGURE OUT WHAT GENES ARE
LIKELY TO BE RESPONSIBLE AND
MAYBE DEVELOP A THERAPY THAT
TAKES THAT INTO ACCOUNT.
IF IT OCCURS DURING TUMOR WE
NEED SOME WAY OF MONITORING THE
TUMOR.
WE ARE COLLECTING ALL THE DRUG
RESISTANT MECHANISMS AND CRISPR
IS GIVING A NEW SUPPLY OF NEW
ONES AND SO WE'LL GET A CATALOG
AND I'M HOPING THE CATALOG IS
SHORT ENOUGH TO SEE THE
COMPLETED CATALOG BEFORE I'M NOT
WORKING ON THIS ANYMORE.
THE THIRD POINT IS THE CLINICAL
RELEVANCE OF ALL THE MECHANISMS
STILL NEED TO BE DETERMINED.
WE NEED BETTER WAYS TO DO
SCREENING MORE CLINICALLY
RELEVANT AND NEED TO BE ABLE TO
ASSESS THE EXPRESSION OF GENES
AND EVOLUTION OF GENES DURING
THE TUMOR AND ONE WAY IS THROUGH
LIQUID BIOPSIES OR SOME IMAGING
APPROACH AND ON THAT LET ME
THANK A FEW PEOPLE WHO ARE
RESPONSIBLE.
THIS IS MY CURRENT LAB GROUP.
MANY OF WHOM ARE HERE.
MY GOODNESS, EXTRA CREDIT AND
OVER THE YEARS THERE'S BEEN MANY
MANY POST-DOCTORAL FELLOWS AND
MORE SENIOR FELLOW.
ALL THE WORK BEGAN AS A
DESTRUCTION BETWEEN MYSELF AND
OTHER AND I'M GRATEFUL FOR THE
SUPPORT I'VE HAD OVER THE YEARS
FROM THE NIH SO THANK YOU ALL.
>> THANK YOU, VERY MUCH FOR THIS
INSIGHTFUL AND EXCITING
PRESENTATION.
I ENCOURAGE THOSE HERE AND
WATCHING TO PLEASE SUBMIT
QUESTIONS ONLINE.
AS WAS EXPLAINED BEFORE AND WE
WILL HAVE A SESSION AT THE
CONCLUSION OF JOHN DEKKER'S
TALK.
>> OKAY.
SO THANK YOU ALL FOR JOINING.
THANK YOU FOR THE OPPORTUNITY TO
SPEAK AND THANK YOU FOR THAT
VERY GENEROUS INTRODUCTION IN
THE BEGINNING.
IT'S VERY DIFFICULT TO FOLLOW
DR. GOTTESMAN.
I'LL DO MY BEST HERE.
FIRST, I HAVE NO CONFLICTS OF
INTEREST BUT I WILL BE
DISCUSSING A NUMBER OF SPECIFIC
COMMERCIAL PHARMACEUTICAL
PRODUCTS AND THIS DISCUSSION
SHOULD NOT BE CONSTRUED TO
CONSTITUTE ENDORSEMENT BY THE
NIH OR U.S. GOVERNMENT.
THESE WILL OUR LEARNING S BY THE
END OF THE TALK YOU SHOULD BE
ABLE TO DESCRIBE THE GENERAL
CLASSES OF ANTIBIOTIC RESISTANCE
MECHANISMS TO BE USED IN
BACTERIA AND UNDERSTAND THE
OUTCOME OF LACTAMS AND LACK
LACTAMASES AND YOU'LL SEE THE
OUTLINE.
WE'LL START WITH A BROAD
OVERVIEW OF ANTIMICROBIALS AND
LOOK AT RESISTANCE AND LOOK AT
THE EVOLUTION OF BETA LACTAMS
AND THEY'RE THE CLASSIC
ANTIBIOTICS SUCH AS PENICILLIN
AND THERE'S ARGUABLY THE MOST
IMPORTANT CLASS OF ANTIBIOTICS
USED AND THERE'S A LARGE FAMILY
THAT INCLUDE E. COLI AND OTHERS
AND WE'LL TURN TO THE DIFFERENT
BACTERIA AND LOOK WHERE
ANTIBIOTIC RESISTANCE EVOLVES IN
THE AND IN TREATING INFECTIONS
IN OTHERWISE HEALTHY INDIVIDUALS
THEY ENABLED ADVANCES IN SURGERY
AND TRANSPLANTATION THAT WOULD
NOT HAVE BEEN POSSIBLE WITHOUT
THE ABILITY TO CONTROL
INFECTIONS THAT OCCUR IN THE
TISSUE BARRIERS AND IMMUNOLOGIC
DEFENSES.
ANTIBIOTICS ARE THE MOST COMMON
TYPE OF MEDICATION PRESCRIBE AND
ONE OF THE MOST IMPORTANT
MEDICAL INTERVENTIONS MADE.
THE FIRST CONCEPT I WANT TO
START WITH IS IN THINKING ABOUT
THE EVOLUTION OF ANTIBIOTIC
RESISTANCE IT'S ESSENTIAL TO
UNDERSTAND THAT MOST ARE
CHEMICALLY MODIFIED NATURAL
PRODUCTS PRODUCED BY BACTERIA
MOLDS THAT ARE ARMAMENT OF
MICROBIAL WARFARE AND DEFENSE
MECHANISMS HAVE EMERGED TO
PROTECT THE ARMS PRODUCERS AND
THE INTENDED RECIPIENTS AND MANY
LAYERS OF DEFENSIVE AND COUNTER
DEFENSIVE STRATEGY HAVE BEEN
ELABORATED BY THE PARTIES.
THIS IS RELEVANT BECAUSE IT'S
THE ANCIENT DEFENSE MECHANISMS
PLACED UNDER SELECTION BY THE
HUMAN CLINICAL USE OF
DERIVATIVES OF NATURAL
ANTIBIOTICS.
LET'S LOOK AT AN EXAMPLE.
THIS ANTIBODY IS USED TO TREAT
INFECTIONS SUCH AS STAPH.
VANCOMYCIN SUPPRESS THE GROWTH
OF ITS NEIGHBORS COMPETING FOR
LIMITED RESOURCES.
AND ADVANTVANCOMYCIN RESISTANT
EMERGED AND BY THE 1990s THEY
WERE PRESENT IN MOST HOMES
AROUND THE WORLD.
AND VANCOMYCIN RESISTANCE IS
MEDIATED BY A CLUSTER OF GENES.
THERE'S VARIATIONS ON THE GENES
AND THE GENES CAN BE MOBILIZED
BETWEEN BACTERIA AND CONFER
RESISTANCE TO NEW HOST.
WE'LL LOOK IN A LITTLE BIT OF
THE MECHANISMS TO CONFER
MOBILITY TO ANTIMICROBIAL
RESISTANT GENES.
I EMPHASIZE VANCOMYCIN IS A
NATURAL PRODUCT AND INDEED
RESISTANCE TO IT IS NATURAL AND
ALSO ANCIENT IN ORIGIN.
THIS IS DEMONSTRATED BY A STUDY
THAT WAS PERFORMED IN THE YUKON
NEAR ALASKA IN WHICH VANCOMYCIN
RESISTANT GENES WERE DISCOVERED
UNDER 30,000-YEAR-OLD
PERMAFROST.
ON THE LEFT SIDE IS THE
STRATOGRAPHIC DESIGN AND MORE
THAN 6 METERS BELOW THE SURFACE
INCLUDED GENES SIMILAR TO THE
VAN A AND H AND F GENES THAT
MEDIATE VANCOMYCIN RESISTANCE IN
MODERN DAY V.E.R. AND THE
ANCIENT PROTEIN IN BLUE OVERLAYS
WITH THE MODERN DAY PROTEIN
SHOWN IN GREEN.
AND THE NATURAL AND ANCIENT
ORIGINS OF MANY RESISTANT
CATEGORIES HAVE BEEN SHOWN AND
RESISTANCE IS NATURAL AND
ANCIENT IN ITS ORIGINS.
THE SECOND CONCEPT IS RESISTANCE
TO NATURAL ANTIBIOTICS IS
WIDESPREAD IN THE ENVIRONMENT.
THIS IS DEMONSTRATED BY A STUDY
THAT SEQUENCED SPOIL MICROBIOMES
AND RESISTANT GENES IN SOIL
MICROBES.
WHAT WAS FUND THEY WERE LOADED
WITH A LARGE NUMBER OF
ANTMICROBIAL RESISTANT GENES AND
SHOWS THE CLASSES OF RESISTANT
MICRO BIOTICS AND THE GENES ARE
DIVERSELY DISTRIBUTED THROUGHOUT
THE BACTERIAL KINGDOM.
THESE FINDINGS HAVE BEEN
CONFIRMED BY A NUMBER OF
SUBSEQUENT STUDIES AND OTHER
SCENARIOS AND IN ADDITION TO
BEING NATURAL AND ANCIENT
RESISTANCE TO MODERN DAY
ANTIBODIES ARE INCREDIBLY
WIDESPREAD IN THE ENVIRONMENT
AND RANDOMLY SAMPLED SOIL CAN
MEDIATE ANTIBIOTICS AND
ANTIBIOTICS REPRESENT AS A SMALL
SUBSET OF THE WORLD'S NATURAL
ANTIBIOTICS IN PART BECAUSE GOOD
CLINICAL ANTIBIOTICS HAVE BOB
BIO AVAILABLE AND NON-TOXIC AND
HIT TARGETS NOT SHARED BY HUMAN
CELLS AND EUKARYOTICS AND
THERE'S PROCESSES THAN TARGETED,
MOST MODERN ANTIBIOTICS TARGET A
SMALL NUMBER OF THE PROCESSES.
THIS IS RELEVANT AS A
CONSEQUENCE THE ARSENAL OF
MODERN ANTIBODIES GOES TO A
SMALL NUMBER OF RESISTANT
MECHANISMS.
WE'LL LOOK AT SOME OF THE
MECHANISMS THAT CONFER
RESISTANCE IN GROUND NEGATIVE
BACTERIA AND FOCUS ON THEM IN
MORE DETAIL.
THERE'S AN OUTER AND INNER
MEMBRANE.
THE FIRST IS THE FIRST BARRIER
AGAINST ANTIBIOTICS AND MOST ARE
POORLY PERMEABLE AND AROUND THIS
IS ESSENTIAL FOR METABOLISM.
ANTIBIOTICS CAN HIJACK THE
PATHWAYS.
THE FIGURE ON THE LEFT SHOWS THE
STRUCTURE MUCH AN OUT EVER ER --
OUTER MEMBRANE AND CONFER
PRIMERS.
THE FIGURE ON THE RIGHT SHOWS
THE STRUCTURE OF A MON AMER WITH
A MOLECULE OF THE ANTIBODY
MEROPENEM AND IT MAY ALTER THE
STRUCTURE OF THE PERMEATION
PATHWAY AND EXCLUDE ANTIBIOTICS
BUT DOWN REGULATE POINTS THAT
HAVEN'T ESSENTIAL FOR VIABILITY
TO CONFER ANTIBIOTIC RESISTANCE
AND THEY'RE THE FIRST LINE OF
DEFENSE AGAINST ANTIBIOTICS FOR
THE BACTERIA AND THE SECOND
CLASS PUMPS IT OUT BY A NUMBER
OF PUMPS IN TRANSPORTERS AND IN
CONTRAST TO PASSIVE DIFFUSION
THESE ARE MOSTLY ACTIVE OR
SECONDARY ACTIVE TRANSPORTERS
AND THIS ALLOWS THE TRANSPORTERS
TO MOVE ANTIBODIES OUT OF THE
CELL AGAINST CONCENTRATION
GRADIENTS.
THESE ARE FIVE OF THE PRINCIPLE
TRANSPORTERS IN BACTERIAL CELLS
ON THE LEFT AND RIGHT HAND SIDES
ARE THE ABC SUPER FAMILY WHICH
ARE ANCIENT CELLS OF THE
TRANSPORTERS AND EUKARYOTIC
CELLS AND IN BACTERIA THEY'RE
LOCATED IN THE INNER MEMBRANE
AND HYDROLYZE ATP AND CAN MOVE
ANTIBODIES AND OTHER SUBSTRATES
FROM THE CYTOPLASM INTO THE
PERIPLASM.
OR CAN BE CONNECTED WITH
ADDITIONAL MACHINERY THAT ALLOWS
THE MOVEMENT OF SUBSTRATES
THROUGH THE OUTER MEMBRANE INTO
THE CELL.
THE MFS AND SMR FAMILIES OF
TRANSPORTERS CAN MOVE ANTIBODIES
FROM THE CYTOTO CYTOPLASM TO
PERIPLASM AND THE TRANSPORTERS
COUPLE AND CAN MOVE SUBSTRATES
FROM THE CYTOPLASM OR PERIPLASM
ACROSS THE OUTER MEMBRANE OUT OF
THE CELL.
AND SIMILAR TO WHAT WE HEARD IN
THE FIRST TALK THEY CONSTITUTE A
FORMID
FORMIDABLE THE NEXT LINE OF
DEFENSE IS TO HYDROLYZE OR
MODIFY CHEMICALLY AND BACTERIA
HAVE EVOLVED A DIVERSE ARRAY OF
SYSTEMS THAT CAN MODIFY MOST
CLASSES OF ANTIBIOTICS USED IN
MODERN MEDICINE.
THERE'S A MECHANISM GRAM
NEGATIVES USE TO ACTIVATE.
AND THIS IS CARRIED OUT BY AN
ENZYME AND STARTS WITH AN ATP
AND MAKES UP THE STRUCTURE.
THIS AND MODIFY THE SITE OF
ACTION OF THE ANTIBODY AND WE'LL
LOOK AT A CLASS OF ENZYMES IN
MORE DETAIL IN A BIT AND THESE
ARE ENZYMES THAT CROSS-LINK THE
CELL WALLS AND THE MAIN TARGET
FOR BETA LACTAMS AND WHAT
MODIFIES BETWEEN THESE CAN
CONFER RESISTANCE.
AND YOU CAN APPRECIATE THE
INTERACTIONS AND THESE ARE
HIGHLY SPECIFIC CONTRACTIONS AND
SINGLE MUTATIONS CAN ALTER AND
NOW WE'LL LOOK AT A SPECIFIC AND
IMPORTANT AND BETA LACTAMS
INCLUDE PENICILLINS AND THERE'S
A COMMON FAMILY OF BACTERIA THAT
ARE ENTEROGRAM NEGATIVES, E.
COLI AND OTHERS AND IT'S ONE OF
THE MORE SERIOUS PROBLEMS IN
MODERN MEDICINE AND WHY WE'LL
FOCUS ON IT.
AND THIS IS SANDWICHED IN THE
WALL AND THE CROSS LINKS ARE
BETWEEN THE RESIDUES AND SERVE
TO RIGIDIFY THE CELL WALL OF THE
PEPTIDOGLYCAN AND IT CONTAINS A
BOND A STRUCTURAL MIMIC AND THE
NATURAL STRABT AT THE
PEPTIDODASE AND THEY SEAR THE
SITE KNOCKING THEM OUT OF
COMMISSION.
THIS RESULTS IN DECREASED CROSS
LINK OF THE CELL WALL AND
INABILITY TO WITHSTAND STRESSES
AND THIS IS HOW BETA LACTAMS
WORK.
THEY'RE AN ANCIENT CHEMICAL
WEAPON IN RESPONSE THERE'S AN
ANCIENT DEFENSE MECHANISM THESE
ARE THE ENZYMES THAT HYDROLYZE
THE BETA LACTAMS AND A CLASS
THAT'S MORE THAN 100 MILLION
YEARS OLD AND THE CLASSIFICATION
SYSTEM DIVIDE THE BETA
LACTAMASES INTO FOUR GROUPS
BASED ON STRUCTURE AND HOMOLOGY.
THE FOUR GROUPS ARE CLASS A, B,
C AND D.
THE CLASS D ARE ENZYMES AND THE
B AND C ARE HAVE A MECHANISM OF
ACTION IS SIMILAR TO PROTEASES
AND THEY OPEN IT UP AND THE RING
OPENED BETA LACTAM IS NO LONGER
ACTIVE.
AND THESE ARE YOU'VE TO EXTEND
THE LIFE OF THE ACTIVITY OF THE
BETA LACTAMASE AND CLINICALLY
THERE'S TWO CLASSES OF BETA
LACTAMASE INHIBITERS AND THE
CLASS BASED ON THE MOLECULE AND
THERE'S A TAZOBACTAM AND THE
OCTAN
OCTANES.
I TOLD YOU THE CLASSIFICATION
SYSTEM CLASSIFIES BETA LACTA
LACTAMASES INTO A, B, C AND D
AND THE MOST CLASSIC ACTIVATION
RESISTANCE AND THERE'S THREE
PRINCIPLE CLASSES OF BETA
LACTAMASES AND WITH ACTIVITY
HIERARCHICALLY ORGANIZED.
AND WE HAVE ENZYMES THAT
HYDROLYZE FIRST AND SECOND
GENERATIONS AND OCCASIONALLY CAN
HYDROLYZE THIRD AND FOURTH
GENERATIONS.
ABOVE THE EXTENDED SPECTRUM IT'S
EXTENDED BEYOND AND THE THIRD
AND FOURTH GENERATIONS.
AT THE TO THE HIERARCHY WE HAVE
THE CARBAPENEMMASES AND THEY'RE
A CRITICAL CONCERN.
AND THEY'RE RESISTANCE TO MOST
OR ALL AND A SUBSET AREN'T
RESISTANT TO ALL ANTIBIOTICS.
THE SO-CALLED CARBAPENEMASES AND
THESE FALL INTO CLASS A AND D.
ALL ARE IMPORTANT BUT WE'LL
FOCUS ON C -- KPC.
IT WAS FIRST IDENTIFIED IN 199 6
IN NORTH CAROLINA AND ALL RIGHT
-- REPORTED IN 2001 AND IT'S
ASSUMED TO BE DERIVED FROM AN
ENVIRONMENTAL SOURCE.
SOME ARE WIDESPREAD IN THE
ENVIRONMENT AND IT'S NOT KNOWN
EXACTLY HOW THE GENE GOT IN THIS
ICE ISOLATE BUT THIS SPREAD THE
RESISTANCE AROUND THE WORLD
THROUGH HORIZONTAL GENE TRANS
PER.
WE'LL LOOK AT HOW THAT WORKS.
WE HAVE PLASMIDS AND OTHERS AND
THEY'RE THEY CAN CARRY
DIFFERENT GENES INCLUDING
ANTIBIOTIC RESISTANT GENES AND
ENCODE ALL THE MECHANISMS THEY
NEED SO EFFICIENTLY MOVE BETWEEN
BACTERIAL CELLS.
WHEN ONE OF THE GENETIC ELEMENTS
PICKS UP AN ANTIMICROBIAL
RESISTANCE GENE AND INFECTS A
BACTERIUM, IN UNDER SELECTION
PRESSURE WITH TO THE RIGHT
ANTIBIOTIC THE GENE AND HOST CAN
BE CO-SELECTED INTO A
RELATIONSHIP UNLESS IT'S THE
DRIVING FORCE THAT RESULTS IN
HORIZONTAL GENE TRANSFER.
BROAD SPECTRUM RESISTANCE TO
OTHER CLASSES OF ANTIBIOTICS IS
MEDIATED BY TRANSFERRED GENES.
AND THIS IS IMPORTANT BECAUSE
THE HORIZONTAL GENE TRANSFER
REQUIRES A COMMUNITY OF
INTERACTIONS BETWEEN BACTERIA
AND MAY BE FACILITATED BY
ANTIBIOTIC SELECTION.
THIS EXCHANGE HAS BEEN
DEMONSTRATED TO OCCUR IN SOIL,
FARMS, SEWAGE WITHIN THE HOSTILE
ENVIRONMENT INCLUDING WITHIN THE
HOSPITAL SYSTEM OR IN THE
MICROBIOMES OF THE PATIENTS
THEMSELVES AND THERE'S
REMARKABLE DIVERSITY OF GENETIC
ELEMENT IMMEDIATE HORIZONTAL
GENE TRANSFER.
THE KPC GENE EARLY ON ENDED UP
IN A HIGHLY MOBILE TRANSPOSON
AND IT WAS LOCATED WITHIN
PLASMIDS.
THEY'RE EXTRA CHROMOSOMAL DNA
AND IT ENCODED ALL THE MACHINERY
IT NEEDED TO INSERT COPIES OF
ITSELF INTO OTHER PLASMIDS.
INDEED, THIS TRANSPOSON MADE
LOTS OF COPIES AND MOVED AROUND
TO A NUMBER OF PLASMIDS WITH A
VARIETY OF HOST CARRYING THE KPC
GENE WITH IT.
THIS WAS IN COMBINATION AND IT
DISSEMINATED THE DESCENDANTS AND
CAUSED A NUMBER OF OUTBREAKS
AROUND THE WORLD INCLUDING THE
2011 OUTBREAK THAT OCCURRED HERE
IN THE CLINICAL CENTER.
MANY WERE RESISTANT TO CAR C
CARBAPENMENS AND THERE WAS AN
INHIBITER MENTIONED EARLIER WAS
INTRODUCED AND ANTI-BACTAM HAD
STRONG ACTIVITY AGAINST AND THE
COMBINATION WAS EFFECTIVE IN
TRE
TREATING INFECTIONS AND THIS
CHANGED THE NATURE OF THE
OUTBREAK OVERNIGHT IN AREAS
WHERE IT WAS AVAILABLE.
AND THERE WERE REPORTS OF
RESISTANCE TO FOR THE
CEFTAZIDIME LINEAGES WE WERE
BACK TO SQUARE ONE.
WE'LL COME BACK TO CEFTAZIDIME
RESISTANCE AND WE'LL RETURN TO
PSEUDOMONAS AERUGINOSA AND
MULTI-DRUG RESISTANT PSEUDOMONAS
HAS BEEN LABELLED AS BY THE CDC
AND THERE'S OTHER CLASSES OF
ANTIBODIES AND WE LOOKED AT THE
IMPLICATIONS AND IT REQUIRES A
COMMUNITY AND OCCURS IN CERTAIN
CONTEXT AND IT CAN ACT AS A LONE
AGENT AND GENERATE RESISTANCE
DURING THE COURSE OF TREATMENT
AND THIS HAS DIAGNOSTIC,
CLINICAL AND THERAPEUTIC
CONSEQUENCES.
WE'LL LOOK AT THE MECHANISMS
THAT MEDIATE RESISTANCE ENS
ENSUED -- IN PSEUDOMONAS AND THE
GENOME OCCURS MORE THAN 12 AND
THERE'S A VARIETY OF
SPECIFICITIES THAT COVER MOST
THE CLASSES OF ANTIBIOTICS
MODERN PHYSICIANS USE.
THE OPRM EFFLUX PUMP IS
EFFECTIVE AND OVER EXPRESSION OF
THE PUMP IS ONE OF THE MAIN
MECHANISMS CONFIRMED RESISTANCE
AND ALL PSEUDOMONAS AERUGINOSA
HAVE IT INTEGRATED IN THE CHROME
SOME.
IT COMES WITH AN AN ANCIENT
LACTAMASE AND TOGETHER THEY CAN
GENERATE HIGHLY RESISTANT
BACTERIA.
THERE'S NOT MANY WE CAN TURN TO.
TWO OF THE DRUGS ARE BACTAM AND
A COMBINATION OF THE TAZO BACTAM
AND WHEN RESISTANCE EMERGES TO
ONE OR BOTH OF THE DRUGS IT'S A
PRETTY SERIOUS CONCERN.
WE'LL LOOK AT A CLINICAL CASE
FROM THE NIH CLINICAL CENTER IN
WHICH RESISTANCE TO THE TWO
DRUGS, CEFTOLAZIMINE AND THE
OTHER WERE RESISTED BY A PATIENT
WITH LEUKEMIA.
THE PATIENT WAS INITIALLY
ADMITTED TO THE NIH CLINICAL
CENTER UNDER THE PROTOCOL AND
DEVELOPED FEVER AND THE TWO
BLOOD CULTURES BOTH FOR
PSEUDOMONAS AERUGINOSA AND THE
ISOLATES WERE SUSCEPTIBLE AND
THE REGIMENT WAS ADJUSTED TO
CREV CEFTAZIDIME AND THEY DID A
CULTURE OBTAINED FOR PSEUDOMONAS
AERUGINOSA THAT WAS SUSCEPTIBLE
TO CEFTAZIDIME.
THE THERAPY WAS FURTHER ADJUSTED
AND ON DAY EIGHT AMIKACIN WAS
DISCONTINUED AND AFTER DAY 9
HYPOTENSION AND FEVER OCCURRED
AND THIS TIME RESISTANT TO
CEFTAZIDIME AND AMIKACIN AND THE
PATIENT DEVELOPED RESPIRATORY
FAILURE AND EXPIRED AND THE MAIN
POINT I WANT TO MAKE ON THE
SLIDE IS THE INITIAL BLOOD AND
ISOLATES WERE SUSCEPTIBLE TO THE
BACTAM WITHIN MICs OF ONE
MICROGRAM PER MILL BUT OVER A
WEEK A HIGH LEVEL RESISTANCE
EMERGED AND ISOLATES WERE
GREATER THAN 256 WERE ISOLATED.
HOW DID RESISTANCE OCCUR SO
RAPIDLY IN THE CONTEXT UNDER
TREATMENT?
TO ANSWER THE QUESTION WE
SEQUENCED THE GENOME TO THE
ISOLATES IN MY RESEARCH LAB AND
IDENTIFIED THE RESISTANCE
MECHANISM.
AND THIS CONFERRED RESISTANCE TO
AND EXPLAIN THE MECHANISM OF
TREATMENT FAILURE AND TALKS
ABOUT THE RESISTANCE OF
MECHANISMS.
WE'LL LOOP BACK TO THE STORY OF
THE RESIST ANANCE AND SAW MUTATS
CONFERRED RESISTANCE.
AND WE SEE IN PSEUDOMONAS
AERUGINOSA THE CHROMOSOMAL
BACTAMASE CONFERS RESISTANCE AND
ANCIENT ENZYMES CAN CONFER
RESISTANCE TO NEW CLASSES SUCH
AS AVIBACTAM AND BACTERIA HAVE
NOT BEEN ABLE TO EVADE IN SOME
FORM.
THIS SUGGESTS COMPETING WITH
EVOLUTION MAY REQUIRE NOVEL
STRATEGIES.
THERE'S A FEW USED TO COMPLIMENT
ANTIBIOTICS OR WHEN THEY FAIL.
THESE ARE ALL AT EARLY STAGE
DEVELOPMENT AND NOT IN WIDE
SUPPRESS CLINICAL USE.
THE FIRST APPROACH IS VACCINES
TO MONOCLONAL ANTIBODIES AND THE
CONCEPT IS SIMILAR TO OTHER
VACCINES.
IT'S DEVELOPED AGAINST A
MULTI-DRUG RESISTANT STRAIN AND
SIMILAR HOW TO WE VACCINE AGAIN
DIPHTHERIA AND THE GOAL IS NOT
TO BECOME INFECTED WITH THE
BACTERIA IN THE FIRST PLACE.
IN RELATED STRATEGY AMONG
MONOCLONAL ANTIBODIES AND THE
THOUGHT IS THEY'D BE RAISED
AGAINST MULTI-DRUG RESISTANT
STRAINS AND ADMINISTERED TO
TREAT ACUTE INFECTIONS.
A SECOND APPROACH THAT'S
RECEIVED A LOT OF ATTENTION IS
BACTERIA PHAGE THERAPY AND
THEY'RE IDENTIFIED OR RAISED
AGAINST PARTICULAR MULTI-DRUG
RESISTANT STRAINS AND THEY THEN
CONTACT THEIR TARGETS, INFECT
THEM, REPLICATE WITHIN THEM AND
CAUSE LYSIS.
THE STRATEGY HAS BEEN USED IN A
NUMBER OF CASE STUDIES
RELATIVELY EFFECTIVELY BUT ONE
CHALLENGE IS THAT IN MANY OF
THESE CASE STUDIES THE BACTERIA
PHAGES ARE SPECIFIC AND HIGHLY
STALE -- TAIL JORE -- TAILORED
AND THERE'S CLINICAL TRIALS IN
PLANNING STAGE OR UNDERWAY.
AND THE THIRD AND LAST APPROACH
IS CRISPR CAS BASED PROGRAMMABLE
ANTIBIOTICS.
THIGH HAVE BEEN RE-ENGINEERED TO
BE EDITORS AND YOU CAN PROGRAM A
CRISPR CAS SYSTEM IT EDIT OR
DISRUPT A PARTICULAR
ANTIMICROBIAL RESISTANT GENE OR
MDR STRAIN.
SHE'S COULD BE DELIVERED WITH
BACTERIA PHAGES SIMILAR TO THE
SECOND APPROACH WE LOOKED AT.
I HOPE I'VE CONVINCED YOU HUMANS
WILL NOT WIN THE WAR WITH SMALL
MOLECULES AND BY MANY BACTERIA
WE'RE WORSE OFF TODAY THAN A
NUMBER OF TIMES IN THE PAST.
I WILL LEAVE YOU WITH THE
THOUGHT THAT NEW APPROACHES
MAYBE NEEDED FOR CASES OF SEVERE
RESISTANCE OF PAN RESISTANT
ISOLATES AND ENVIRONMENTAL
ANTIBIOTIC RESISTANT RESERVOIRS
AND USAGE IN THE FARMS AND FOOD
CHAIN AND I'D LIKE TO
ACKNOWLEDGE THE NIH CLINICAL
CENTER PATIENTS AND THEIR
FAMILIES WHO PARTNER WITH US IN
THIS IMPORTANT WORK.
I'D LIKE TO THANK THE PEOPLE IN
MY LAB WHO DID THE SEQUENCING IN
THE CLINICAL CASES I SHOWED YOU
AND THANK YOU FOR YOUR
ATTENTION.
THANK YOU.
THIS IS LIKE A BRAVE NEW WORLD I
SUSPECT MOST WHO DON'T WORK IN
EITHER OF THESE AREAS HAVE WE'RE
GRATEFUL FOR OPENING UP OUR
MINDS ANDIZE TO THE COMPLEXITIES
AND CHALLENGE FOR ALL AND WE
HAVE A HUGE NUMBER OF QUESTIONS
AND I'M SURE WE CAN ALL STAY
HERE FOR NEXT THREE HOURS AND
FOR THOSE THEY DON'T GET TO
WE'LL DO OUR BEST TO FORWARD
THEM TO THE SPEAKERS AND MAYBE
MAYBE RESPOND.
ONE QUESTION PEOPLE HAVE ASKED
IS HOW DO CELL LINES DIFFER FROM
PRIMARY CELLS IN TERMS OF
EXPRESSION OF THESE
TRANSPORTERS?
>> HOW MANY WITHOUT SELECTION?
IN OTHER WORDS, IF YOU TAKE A
LIVER CELL AND PUT IT IN CULTURE
AND IT RETAINS SOX THE
DIFFERENTIATED PROPERTIES OF THE
LIVER CELL IT WILL EXPRESS P
GLYCOPROTEIN AND YOU CAN TELL ME
IF THAT'S THE CASE.
>> ALL RIGHT.
>> THE ANSWER IS DEPENDING ON
THE ORIGIN OF THE CELL TYPE.
CELLS WILL GENERALLY RETAIN THE
CHARACTERISTIC OF THE ORIGINAL.
>> SO HAVE ORGANOIDS BECOME PART
OF THE NEW APPROACH?
IN AN EFFORT TO GET GENES MORE
CLINICALLY RELEVANT WE'RE
BEGINNING TO WORK WITH CANCER
ORGANOIDS WHICH ARE PRIMARILY
THE CELLS THAT ARE THE BASIS OF
THE CANCER PLUS PERHAPS
ADDITIONAL CELLS THAT MAY BE
CONTRIBUTING ELEMENTS OF
RESISTANCE AND WE'RE HOPING WITH
THE NEW MODELS THAT WE ARE ABLE
TO GET A SET OF GENES MORE
LIKELY TO BE CLINICALLY RELEVANT
BUT WE DON'T KNOW IF THAT'S THE
CASE.
>> I'M A POSTDOC IN THE V.R.C.
THANK YOU FOR YOUR TALKS THEY
WERE FUN.
MY QUESTION RELATE TO BOTH THEM
AND YOU TOUCHED ON THIS WITH THE
VACCINES AND MONOCLONAL
THERAPIES.
HOW DOES THE IMMUNE SYSTEM
FACTOR IN TO OUR UNDERSTANDING
OF DRUG RESISTANCE OR THERAPY
RESISTANCE IN THE FUTURE?
SPECIFICALLY FIRST FOR THE
CANCER SITUATION.
IF WE HAVE IMMUNOTHERAPY
RESISTANCE WHICH WE'VE SEEN SOME
INSTANCES OF, WHEN THE IMMUNE
SYSTEM GETS INVOLVED, HOW DOES
THAT CHANGE THE FITNESS
LANDSCAPE OF RESISTANCE?
THE CANCER NOW NEEDS TO DEAL
WITH THE IMMUNE SYSTEM USED WITH
RAPIDLY EVOLVING PATHOGENS.
MAYBE ANTIGEN PRESENTATION OR
IMMUNE SIGNALLING IT MAY GET
INTERFERED AND THAT'S ONE I
WANTED TO ASK ABOUT AND FOR
BACTERIA IF YOU'RE NOT USING A
VACCINE DOES THE PROBLEM SHIFT
FROM DRUG RESISTANCE?
AND IS THAT EASIER TO HANDLE
KNOWS WHAT WE KNOW?
>> I HAVE SPECIFICALLY AVOIDED
STUDYING WHY CANCER CELLS ARE
RESISTANT TO IMMUNE THERAPY,
HOWEVER, THERE ARE LOTS OF
INTERACTION OF IMMUNE SYSTEM
WITH THE SURFACE ANTIGENS
RESPONSIBLE FOR DRUG RESISTANCE.
ONE HYPOTHETICAL, WE'RE KIND OF
SURPRISED MORE CANCERS DON'T
EXPRESS ABC TRANSPORTERS.
ONE REASON MAY BE THEIR
ENERGETICALLY NOT VERY FAVORABLE
AND USE A LOT OF ATP AND IF YOU
CAN FIX A MUTATION IT'S A BETTER
WAY AND GLYCOPROTEIN NOT EXPOSED
TO THE BLOOD AND IMMUNE SYSTEM.
IT'S SITTING IN THE KIDNEY,
LIVER, COLON ON EXTERIOR SURFACE
OF CELLS.
IT MAY BE A COMPONENT OF THE
IMMUNE SYSTEM IN REGULATING THE
EXPRESSION OF CERTAIN
TRANSPORTERS.
IT MAY NOT BE DESIRABLE FOR A
CELL TO HAVE A LOT OF THE FUNNY
MOLECULES ON THE SURFACE.
THE OTHER ASPECT WHICH IS REALLY
FASCINATING HAS TO DO WITH THE
MICROBIOME.
THERE'S A FAIR AMOUNT OF
EVIDENCE ONE THING THE
MICROBIOME DOES IS ACTIVATES THE
IMMUNE SYSTEM AND IN ORDER TO
CURE CANCER WITH PLATINUM YOU
NEED THE ACTIVITY OF THE IMMUNE
SYSTEM AND THE ACTIVITY OF THE
DRUG.
IF YOU ANNIHILATE THE MICROBIOME
YOU AFFECT THE ABILITY OF THE
BODY IN THE IMMUNE RESPONSE.
THERE'S DIFFERENT KINDS OF
LEVELS -- I TRY NOT TO GET
INVOLVED BUT IT'S PROBABLY
IMPORTANT IN TREATING CANCER TO
WHY CR THE IMMUNE SYSTEM -- TO
CONSIDER THE IMMUNE SYSTEM.
>> SO, IN TERMS OF VACCINES,
THERE ISN'T JUST ENOUGH
EXPERIENCE BUT THERE'S
EVOLUTIONARY DYNAMICS AND WE CAN
SEE THEM AGAINST PERTUSSIS AND
PNEUMOCAUCUS AND ONE WAY IT'S
USED WITH OTHER VACCINES WITH
EPITOPES AND THAT LIMITS THE
POSSIBILITY OF ESCAPE FROM ONE
AND NOT DOING ANTIBIOTIC
MONOTHERAPY.
SO MY GUESS IS THAT I THINK
VACCINES PERSONALLY THERE'LL BE
A STRATEGY THAT'S PROBABLY GOING
TO BE AN IMPORTANT PIECE OF HOW
WE DEAL WITH MULTI-DRUG
RESISTANT STRAINS IN THE FUTURE.
TO ADDRESS ONE OF THE OTHER
QUESTIONS YOU RAISED AT THE
BEGINNING ANTIBIOTICS IN MANY
CASES DAMAGE BACTERIAL BUT DON'T
KILL THEM AND THE HOST IMMUNE
SYSTEM IS IMPORTANT TO MOP UP
THE DAMAGED CELLS.
WE SEE THIS WHEN WE TREAT
PATIENTS WHO HAVE SERIOUSLY
COMPROMISED IMMUNE SYSTEMS WITH
ANTIBIOTICS THEY TAKE LONGER TO
WORK OR DON'T WORK AT ALL AND
THERE'S A SECOND FEATURE MORE
EVOLUTIONARY FOR EVOLUTION TO
HAPPEN YOU NEED MUTAGENESIS AND
ALL THAT OTHER STUFF.
IF YOU HAVE AN EFFECTIVE IMMUNE
SYSTEM THAT MAINTAINS A SMALL
CRITICAL POPULATION SIZE THERE'S
THE IMMUNE SYSTEM THE ANTIBIOTIC
RESISTANCE IS LESS LIKELY TO
ARISE AND THIS HAS BEEN STUDIED
AND WHERE WE SEE MUTAGENESIS
DRIVEN ANTIBIOTIC RESISTANCE
WHERE WE SEE HYPERMUTATED
BACTERIA IS OFTEN IN THE CONTEXT
OF PATIENTS WITH COMPROMISED
IMMUNITY PARTICULARLY INNATE
IMMUNITY.
>> WE HAVE A QUESTION.
CAN TALK ABOUT RESISTANT GENES
ALTERING AND CREATING
ENVIRONMENTS AND CAN YOU COMMENT
ON THE PERVASIVE PROBLEM THAT
MAY BE IN THE CATEGORY OF
CLIMATE CHANGE.
IS THERE ANYTHING WE CAN DO
ABOUT IT?
>> I KNOW THERE ARE A LOT OF
PEOPLE VERY INTERESTED IN
STUDYING THAT BUT I DON'T THINK
CAN SAY ANYTHING MEANINGFUL
ABOUT IT.
>> WHAT ABOUT THE MICROPLASTIC
ASPECT?
>> THAT'S ANOTHER AREA WHERE I
WOULD SAY I DON'T THINK SHOULD
COMMENT BECAUSE I DON'T THINK I
CAN SAY ANYTHING INTELLIGENT.
>> WE HAVE NOTICED A CHANGE IN
THE PATTERN OF CANCERS IN YOUNG
PEOPLE.
COLON CANCER USED TO BE PRETTY
UNHEARD OF IS NOW BECOMING MORE
COMMON IN YOUNGER PEOPLE.
AND THERE'S A STORY ABOUT
ASBESTOS IN THE ENVIRONMENT THAT
ACT AS INFLAMMATORY FOSI THAT
LEAD TO THELIOMAS AND OTHERS AND
THE WAY THEY COULD INDUCE GENE
EXPRESSION RELATED TO DRUG
RESISTANCE IS NOT UNDERSTOOD BUT
NOT ENTIRELY CRAZY THAT
MICROPLASTICS MAY HAVE A SIMILAR
KIND OF ROLE.
I DON'T WANT TO READ ABOUT THIS
IN THE WASHINGTON POST TOMORROW.
>> PLEASE IDENTIFY YOURSELF.
>> I'M JAMES A MEDICAL STUDENT
DOING RESEARCH IN THE DENNY LAB.
I HAD A QUESTION ABOUT CAREER
DEVELOPMENT.
WHAT MADE YOU SELECT MORE OF THE
RESEARCH FOCUSSED TOWARDS BEING
AN M.D. SERVICE THE MORE
CLINICAL SIDE?
>> SO I WAS ALWAYS INTERESTED IN
RESEARCH.
I GUESS THE QUESTION WOULD BE
WHY DID I GO TO MEDICAL SCHOOL.
THERE'S A LOT OF REASONS.
MOST OF WHICH RELATE THE TO THE
FACT THAT I WAS KIND OF
INTERESTED IN HUMAN MEDICINE AND
HUMAN DISEASE BUT I ALWAYS HAD A
RESEARCH ORIENTATION.
I EVEN IN MEDICAL SCHOOL DIDN'T
THINK I'D ACTUALLY END UP
PRACTICING MEDICINE.
I'M A BOARD CERTIFIED INTERNIST
BUT MY MEDICAL ADVICE IS TO FIND
A GOOD DOCTOR.
I'M AN Ph.D. ORIENTED PHYSICIAN.
I SUSPECT JOHN IS THE SAME.
>> I KNEW FROM EARLY ON I WANTED
TO RESEARCH AND I THINK THAT'S
FOR ANYBODY WHO IS INTERESTED IN
GOING TO MEDICAL SCHOOL IT'S
IMPORTANT TO FIGURE OUT EARLY
ON.
I WENT INTO PATHOLOGY WHICH IS A
FIELD OF MEDICINE WHICH IS
PROBABLY EASIER TO DO RESEARCH
THAN OTHERS.
I WOULD SAY THERE WERE VARIOUS
STAGES IN MY CAREER WHERE I WAS
MORE INTERESTED IN MEDICINE AND
MORE INTERESTED IN RESEARCH BUT
THERE ARE THINGS YOU CAN FIGURE
OUT AS YOU GO ALONG.
A LOT OF IT HAS TO DO BIG --
WITH WHAT YOU'RE SUCCESSFUL AT
TOO.
>> JOHN, WHY DOES PSEUDOMONAS
HAS EXTRA OF EVERYTHING READY TO
GO?
>> I ALWAYS WONDERED MAYBE THE
REVERSE IS WHY DO OTHERS HAVE
THE MICROBIAL RESISTANCE AND WHY
DON'T WE SEE TRANSPORTER
MEDIATED RESISTANCE LIKE WE SEE
IN PSEUDO NOE -- PSEUDOMONAS.
>> IT SEEMS TO WANT TO GET RID
OF THE COMMUNITY.
>> THERE ARE AND THERE'S
NON-FERMENTERS LIKE PSEUDOMONAS
AND WOULD BE INTERESTING TO DO A
STUDY TO FIGURE OUT WHAT THE
ULTIMATE DRIVERS ARE.
IT'S A FASCINATING QUESTION.
>> THERE'S A QUESTION OF THE
BLOOD BRAIN BARRIER.
I REFERRED TO THE PERFORM OF
DEALING WITH SYSTEMS OF THE
BRAIN.
DO YOU WANT TO ELABORATE WHAT
DISEASES OF THE BRAIN YOU'RE
THINK ABOUT AND THE PART B TO
THAT IS WHERE IS PGP EXPRESSED
AND WHERE IS IT IN THE BRAIN?
>> I'LL ANSWER THE SECOND
QUESTION FIRST.
THE P GLYCOPROTEIN IS EXPRESSED
AT HIGH LEVELS IN THE CAPILLARY
ENDOTHELIAL CELLS IN THE BRAIN
IN THE VESSELS THAT FEED THE
PART OF THE BRAIN THAT INTERACTS
WITH THE PER CEREBRAL SPINAL
FLUID PARENCHYMAL PART OF THE
BRAIN.
AND ONE PROBLEM WITH TREATING
DISEASES IS YOU CAN'T GET IN 95%
OF THE DRUGS YOU WANT TO GET IN
BLOCKED BY THE BLOOD BRAIN
BARRIER AND THERE'S EXPRESSIONS
OF TRANSPORTERS OF THE BLOOD
BRAIN BARRIER.
AND CHRONIC DISEASE AND THERE'S
TRANSPORT FUNCTIONS AND SOME
BELIEVE PGP CAN RECOGNIZE AND
PUMP OUT BETA AMYLOID AND THERE
MAY BE A DEFECT THERE.
AND THERE ARE NOW ANTIBODIES
THAT ARE BEING USED BUT THE
EFFICIENCY IS VERY LOW AND YOU
NEED TO BE ABLE TO MANIPULATE
THE BLOOD BRAIN BARRIER TO GET
HIGH LEVELS IN FACT IT THERE WAS
AN ARTICLE IN THE NEW YORK TIMES
AND WASHINGTON JOURNAL USING
ULTRASOUND TO GET HIGHER LEVELS
OF ANTIBODIES IN THE BRAIN.
THE BLOOD BRAIN BARRIER IS A
REAL PROBLEM IN TREATMENT OF
METASTATIC BRAIN TUMORS AND
WE'RE GETTING SO MUCH BETTER AT
TREATING SYSTEMIC METASTASIS
WITH THE SECOND AND THIRD
GENERATION ANTI-CANCER DRUGS YOU
READ ABOUT BUT FOR MANY TUMORS,
LUNG CANCER AND BREAST CANCERS
CAN BE CONTROLLED BUT OCCUR IN
THE BRAIN WHERE THEY'RE MORE
DIFFICULT TO TREAT.
>> THERE'S APPROACHES FOR DRUG
RESISTANCE.
IS THERE SOMETHING COMPARABLE IN
THE MAMMALIAL SYSTEM OF A
SUICIDE DRUG APPROACH?
>> MOST CELLS IF YOU ACTIVATE
THE KILLING MECHANISMS OF WHICH
THERE'S FOUR OR FIVE DIFFERENT
ONES AND YOU CAN DEVELOP DRUGS
THAT WOULD ACTIVATE THE SYSTEMS
AND MANY DO THAT'S THE FINAL
COMMON PATHWAY FOR KILLING.
THAT'S THE ANALOG TO SUICIDE IN
BACTERIA.
>> MY NAME IS JOE ANNA THOMAS
AND I LEARNED A LOT IN YOUR TALK
AND I LEARNED A NEW WORD CALLED
BACTERIAMIA AND MY FIRST
QUESTION WAS IS THAT COMMON OR
SOMETHING YOU CAN USE TO STUDY
THE EVOLUTION WITHIN A PATIENT
AND ARE YOU THEN IDENTIFYING A
PATIENT AND PUTTING THEM ON
LOCKDOWN WORRYING ABOUT THE
PLUMBING OF THE HOSPITAL AND THE
SPREAD OF RESISTANCE?
>> THOSE ARE ALL GREAT
QUESTIONS.
I WOULD SAY FIRST, WITH BACK
TEAR TERRE -- BACTEREMIA NOT
GROWN AND IN A TYPICAL
BACTEREMIA INVOLVES 1 IN 500
COLONY FORMING UNITS MEANING
CELLS PER MILLILITER.
SO THEY'RE NOT DENSE SQUARE IN
THE INTRAVASCULAR SPACE AND SO
NOW THE SECOND QUESTION THOUGH
IN CASES OF PATIENTS WHO AREN'T
IN THAT STAGE CAN WE MONITOR
INFECTIONS AT OTHER SITES BY
SAMPLING BLOOD AND YES WE CAN.
YOU CAN SEE CIRCULATING DNA THAT
CAN BE SEQUENCED ON SHOTGUN
BASED METHODS AND CAN LEARN A
LOT ABOUT INFECTIONS IN OTHER
SITES IN THE BODY AND THERE'S
STUDIES.
TO GET TO THE OTHER QUESTION
WHAT CAN WE LEARN ABOUT
ANTIMICROBIAL RESISTANCE IN THE
EVOLUTION OF ANTIMICROBIAL
RESISTANCE CERTAINLY IN A
PATIENT IN THIS STAGE YOU CAN
GET ISOLATES BUT EVEN WITH AN
PATIENT THAT NOT BACTEREMIA WE
MAY BE ABLE TO DEDUCE RESISTANCE
BY LOOKING AT CIRCULATING DNA
AND WHAT PEOPLE ARE INTERESTED
IN AND THERE'S EXCITING PROGRESS
BEING MADE.
>> THANK YOU.
>> WE HAVE TIME FOR ONE MORE
GENERAL KIND OF QUESTION.
MAYBE BOTH WOULD LIKE TO COMMENT
ABOUT YOUR VIEWS ON DEALING WITH
THE EVOLUTIONARY BIOLOGY OF DRUG
RESISTANCE AND DIFFERENT LIVING
THINGS.
DO WE HAVE ANY KNOWLEDGE ABOUT
THINGS LIKE ARCHIA OR YEAST AND
CAN YOU COMMENT ON ABOUT THIS?
THERE WAS CONCERN MANY YEARS AGO
WHEN THE FIRST ROCKETS WERE
BEING SENT TO THE MOON AND IF
YOU MANAGED TO LAND E. COLI ON
THE MOON IT WOULD COVER THE
SURFACE OR SOMETHING LIKE THAT.
IS THERE THINK IN THE ASTRO
PHYSICAL WORLD ABOUT THE
MICROBIOLOGY OF SPACE AND
POSSIBLE INTERACTION WE SEE ON
EARTH?
TWO SMALL QUESTIONS.
>> I'M A SCIENCE FICTION FAN SO
THESE ARE ALL INTERESTING
QUESTIONS.
GIVEN WE HAVE NOT DEFINITIVELY
DISCOVERED LIFE ANYWHERE ELSE IN
THE LIMITED PART OF THE UNIVERSE
WE'VE EXPLORED I DON'T KNOW HOW
TO ANSWER THAT BUT I THINK THE
UNIVERSE IS LIKELY TEEMING WITH
LIFE AND THERE'S GALAXIES AND
THERE'S A HUNDRED BILLION
GALAXIES WITH A HUN HUN DREAD
BILLION SYSTEMS AND IF WE BRING
BACK AN ALIEN MICROBIAL LIFE
FORM, WHATEVER IT IS BASED ON
SOME POLYMER REPLICATION SYSTEM
THAT FEEDS ON NITROGEN, OXYGEN
AND SULFUR MAYBE IT WOULD
REPLICATE ON EARTH AND IN TERMS
OF ANTIMICROBIAL RESISTANCE I'M
NOT SURE HOW TO MAKE THE
CONNECTION THERE.
>> THERE'S RESISTANCE FITNESS
AND MANY MECHANISMS ARE COSTLY
FOR THE ORGANISM.
AS EVOLUTION GOES ON THE
MECHANISMS WILL BE EVOLVED.
MY WIFE REMINDS ME FREQUENTLY
THAT BACTERIA E. COLI DIVIDE
EVERY 20 MINUTES AND WE DIVIDE
EVERY 20 YEARS.
BACTERIA ARE MORE EVOLVED THAN
HUMANS.
IF THERE'S A WAY TO DO IT, THEY
HAVE FIGURED IT OUT.
THERE'S EXCHANGE OF INFORMATION.
THERE'S SOME EVIDENCE THAT YOU
HEAR ABOUT BACTERIA EXCHANGING
GENES BUT PROBABLY SOME OF THE
GENES WE HAVE IN OUR OWN BODY
WERE DERIVED AT SOME POINT FROM
MICROORGANISMS.
I THINK THIS CONCEPT OF FITNESS
IS REALLY IMPORTANT AND THE
MECHANISMS THAT WORK BEST ARE
THE ONES THAT PICK THE LEAST
AWAY FROM THE NORMAL FUNCTION OF
THE ORGANISM WHATEVER IT IS.
>> WELL, ON BEHALF OF THE SELECT
AUDIENCE HERE TODAY AND THOSE
LISTENING THROUGHOUT THE NIH AND
THROUGHOUT THE WORLD WE WANT TO
THANK BOTH OF YOU FOR A REALLY
EXTRAORDINARILY EXCITING
PRESENTATION.
IT GIVES EVERYBODY A LOT TO
THINK ABOUT AND PARTICULARLY
YOUNG PEOPLE POSTDOCS AND SO
FORTH WHO MAY BE WORKING IN
VASTLY DIFFERENT AREAS OF
SCIENCE AND THERE'S A GREAT
COMMONALITY HERE IN TERMS OF
METHODOLOGY, IDEAS.
THERE'S A LOT OF ROOM FOR BRIDGE
BUILDING.
THANK YOU VERY MUCH FOR A
WONDERFUL PRESENTATION.