Harnessing Hypoxia Biology for Cancer Therapy

Transcript

• 00:00Peter is the chair of
• 00:03therapeutic radiology
• 00:05and the Robert Hunter professor
• 00:07of therapeutic radiology and professor
• 00:09of genetics.
• 00:11Peter did his MD and
• 00:13PhD at Yale.
• 00:16I'm
• 00:17sobered to admit that, in
• 00:19fact, we were here at
• 00:21the same time,
• 00:23in medical school.
• 00:26And, Peter has spent his
• 00:29career at Yale,
• 00:31where
• 00:32he has,
• 00:34focused, of course, on radiation
• 00:36oncology and has led the
• 00:38group,
• 00:39for quite a number of
• 00:40years.
• 00:42And himself is,
• 00:44predominantly
• 00:45a laboratory scientist,
• 00:47who has,
• 00:49worked in a number of
• 00:50areas, but,
• 00:52predominantly in the area of
• 00:53DNA damage repair
• 00:55and is the recipient of
• 00:57an outstanding investigator award that
• 00:59that focuses on that very
• 01:00topic.
• 01:01So without further ado, Peter,
• 01:03it's really a pleasure to
• 01:05have you as our first
• 01:06noon speaker
• 01:08in the current, Eric.
• 01:15Thanks, Eric. I appreciate it.
• 01:16And,
• 01:18nice to be here, everyone.
• 01:20Thank you for coming. And
• 01:21it doesn't hurt that there's
• 01:22a free lunch, so
• 01:24that's great. So I'm I'm
• 01:25gonna tell you about some
• 01:26of the work that we've
• 01:27been doing for a number
• 01:28of years.
• 01:29And so there's gonna be
• 01:30something old and something new,
• 01:33related to hypoxia biology and
• 01:35how you might exploit that
• 01:36for,
• 01:37cancer therapy. So these are,
• 01:39my disclosures.
• 01:42And so,
• 01:43many of you will be
• 01:44familiar with the hallmarks of
• 01:46cancer. It's a series of
• 01:48publications from Hanahan and Weinberg
• 01:50and others that have talked
• 01:52about some of the properties
• 01:53of cancer cells. And what
• 01:55I just wanna call out
• 01:56here is that, tumor hypoxia
• 01:59plays a important role in
• 02:01a number of these. And
• 02:03some of these listed here
• 02:04altered metabolism,
• 02:06inducing invasion, metastasis.
• 02:08There's a literature on that.
• 02:10Of course, hypoxia causes,
• 02:12angiogenesis.
• 02:14But what I'm going to
• 02:15talk about today is our
• 02:16work in which we identified
• 02:17that,
• 02:19one of the impacts of
• 02:20hypoxia is,
• 02:21genetic instability
• 02:23in, cancer.
• 02:24Now this work started,
• 02:26a number of years ago
• 02:28when I was at a
• 02:29Gordon conference
• 02:30on DNA repair mutagenesis,
• 02:33and, one of the speakers
• 02:34was Larry Loeb, who's a
• 02:35very prominent pathologist
• 02:37at University of Washington
• 02:39in the DNA repair field.
• 02:40And he proposed,
• 02:42you know, in the nineties
• 02:43that,
• 02:45there would be a mutator
• 02:46phenotype in cancer cells. And
• 02:48this was based on the
• 02:49idea that
• 02:51the the,
• 02:52frequency of mutations in cancer
• 02:54cells was above and beyond
• 02:56what you would predict from
• 02:57the frequency of mutagenesis
• 02:59and normal cells.
• 03:00So
• 03:01he inferred that there had
• 03:02to be a mutator phenotype.
• 03:04Now at the time, people
• 03:06were focused on, well, there's
• 03:08gonna be mutations in mute
• 03:10in DNA repair mutator genes
• 03:12that's that is going to
• 03:13lead to
• 03:14the increase in mutation frequency.
• 03:17But, as I was sitting
• 03:19in the audience, it occurred
• 03:20to me, well, one of
• 03:21the issues is people do
• 03:22all these studies of,
• 03:24DNA repair mutagenesis
• 03:26in cells and culture,
• 03:27but they're growing in a
• 03:29in a standard,
• 03:30tissue culture dish,
• 03:33in really nice media at
• 03:35twenty percent oxygen.
• 03:37And in fact, most cancer
• 03:39cells do not exist in
• 03:40twenty percent oxygen. They they
• 03:41exist in somewhere between
• 03:43point one to five percent
• 03:45oxygen.
• 03:46And in fact,
• 03:47there is
• 03:49a lot of variety in
• 03:50the level of oxygen tension
• 03:51in tumors that has to
• 03:52do with abnormal and fluctuating
• 03:55perfusion,
• 03:57and then as well distance
• 03:58from the vasculature, which creates
• 04:01a gradient of hypoxia.
• 04:03So
• 04:04my my proposal was,
• 04:06could this,
• 04:07hypoxia that you see in
• 04:09the tumor microenvironment
• 04:10impact genetic instability?
• 04:12Now one of the first
• 04:13grants that I submitted on
• 04:15this was turned down, and
• 04:17one of the reviewers
• 04:18said, well, that can't be
• 04:20possible
• 04:21because the absence of oxygen
• 04:24will actually reduce the amount
• 04:25of oxidation. And, of course,
• 04:26oxygen causes rust
• 04:28and and damage to other,
• 04:31molecules. And, of course, that
• 04:33individual was thinking only chemically,
• 04:35not thinking biologically.
• 04:37So we went on to
• 04:38test this hypothesis,
• 04:40and and this is some
• 04:41older data. And the first
• 04:42dataset here is a a
• 04:44table in which we grew,
• 04:46tumor cells
• 04:48that had a specialized mutation
• 04:50reporter,
• 04:51and we grew them either
• 04:52cells in culture or we
• 04:54use them to form tumors
• 04:55in mice. And And what
• 04:56we consistently saw was the
• 04:58frequency of
• 05:00mutations was higher when the
• 05:01cells were grown as tumors.
• 05:04Then we went on to
• 05:05to study, well, what's the
• 05:06underlying mechanism? First of all,
• 05:08we showed that if you
• 05:09put those cells in hypoxia,
• 05:11you could recapitulate the mutagenesis.
• 05:13So that linked that effect
• 05:15to hypoxia.
• 05:16And we subsequently showed that
• 05:17if you had a damaged
• 05:18plasmid
• 05:20and introduced it into cells
• 05:21that were either normoxic or
• 05:22hypoxic,
• 05:23there was better repair
• 05:25or lesser repair of the
• 05:27damaged plasmid and the hypoxic
• 05:29cells.
• 05:30And this work was, carried
• 05:32out predominantly by Jenling Wan,
• 05:33who's now a faculty member
• 05:35at University
• 05:36of Minnesota in radiation oncology.
• 05:41So then,
• 05:42we went on, to
• 05:45oh, I'm sorry. I skipped
• 05:47one. So thinking about,
• 05:49what we saw in those
• 05:50experiments, we noticed that the
• 05:51pattern of mutations
• 05:53had a fair proportion of
• 05:56changes within repeated sequences, which
• 05:59would be,
• 06:01indicative of a mismatch repair
• 06:02deficiency. So we took a
• 06:04candidate approach and looked at
• 06:05DNA mismatch repair gene expression.
• 06:08And,
• 06:10what you can see here
• 06:11is that in the in
• 06:12the mismatch repair pathway, which
• 06:14involves,
• 06:16proteins, MSH two and MSH
• 06:18six recognizing a mismatch, and
• 06:20then MLH one and PMS
• 06:21two coming on to mediate
• 06:24of downstream effects and steps
• 06:26and repair.
• 06:27The MLH one and PMS
• 06:28two proteins were, decreased in
• 06:31expression levels and hypoxia,
• 06:33and that correlated when we,
• 06:35put in a specific sequence
• 06:37repeated sequence that we could
• 06:38probe for mutation.
• 06:40The frequency mutations was increased
• 06:42in that sequence. So that
• 06:44was the first
• 06:45demonstration on a biological level
• 06:47that there was a repair
• 06:48pathway that was diminished
• 06:50in hypoxic cells.
• 06:52Then we took
• 06:54a a nonbiased approach and
• 06:56did a gene array analysis
• 06:57of,
• 06:59DNA repair expression and hypoxia,
• 07:01and you may see two
• 07:02familiar faces here, Ran, Ranjit,
• 07:05Bindru, who everyone knows, and
• 07:06Susan
• 07:07Gable, who was then Susan
• 07:08Scanlon, both MD PhD students
• 07:10in the lab and now
• 07:11on our faculty here in
• 07:12radiation oncology. And they did
• 07:15some,
• 07:16seminal work in which they
• 07:17looked at the expression
• 07:19of genes that we identified
• 07:20in that screen,
• 07:22including Rad fifty one, BRCA
• 07:24one, and FANC t two
• 07:25in the Fanconi anemia pathway.
• 07:27So Rad fifty one and
• 07:28BRCA one are in the
• 07:29homology dependent repair pathway.
• 07:31And you can see here
• 07:33on, a western blot that
• 07:35Rad fifty one is diminished
• 07:36in the hypoxic component,
• 07:38whereas HIF one as a
• 07:40control is increased.
• 07:42And then here on a
• 07:42northern blot,
• 07:44which people hardly do anymore,
• 07:45BRCA one is decreased in
• 07:47hypoxia, whereas VEGF RNA is
• 07:50increased.
• 07:51And, interestingly, on a western
• 07:53blot, you can see the
• 07:54BRCA one is down. But
• 07:55if you look here closely,
• 07:57you'll see the the protein
• 07:58has actually shifted a little
• 08:00bit up on the, gel
• 08:02mobility, and that has to
• 08:03do with post translational modification
• 08:06in in response to hypoxia.
• 08:08And the same is true
• 08:09with Fanconi anemia.
• 08:10And you see here that,
• 08:11the FANC t two expression
• 08:13is down, and, actually, there's
• 08:15an increase in the second
• 08:16band, which
• 08:17represents ubiquitination.
• 08:21So what is the mechanism?
• 08:22So it turns out that
• 08:23many of these repair genes
• 08:26have common elements in their
• 08:27promoters that respond to the
• 08:29e two f family of
• 08:30transcription factors.
• 08:32And, these factors interact with
• 08:35the pocket proteins that are
• 08:36related to retinoblastoma
• 08:38protein, p one thirty, p
• 08:39one zero seven, and p
• 08:41r b. They form heterodimers
• 08:43that regulate these promoter sites.
• 08:45And what we found is
• 08:46that in response to hypoxia,
• 08:48those pocket proteins become hypophosphorylated
• 08:51due to the activity
• 08:53of a phosphatase called PP
• 08:54two a. And this is
• 08:56a common theme throughout this
• 08:57talk that I'll return to,
• 08:59several times. And, basically, the
• 09:01mechanism is that hypoxia causes
• 09:04p one thirty or the
• 09:05p one zero seven or
• 09:06RB hypophosphorylation
• 09:08formation of e two f
• 09:10heterodimers, and these are repressive
• 09:12and cause the downregulation
• 09:14of DNA repair.
• 09:16We also found that hypoxia
• 09:18causes, changes in expression of
• 09:20microRNAs, and these lead to
• 09:22a cascade of events that
• 09:23regulate,
• 09:24DNA repair as well as
• 09:26adaptation to hypoxia
• 09:28on several levels.
• 09:29But I'm not going to,
• 09:31go into this further in
• 09:32in the talk. Just mention
• 09:34it for completeness.
• 09:36In addition
• 09:38to acute and chronic changes
• 09:40acute and short term changes
• 09:42in gene expression,
• 09:43Yuhang Liu in the lab
• 09:44has shown,
• 09:46that both BRCA one and
• 09:47MLH one become silenced with
• 09:50persistent hypoxia,
• 09:52due to a specific set
• 09:54of epigenetic,
• 09:55changes related to particular,
• 09:58histone,
• 09:59demethylases.
• 10:01And you can see here
• 10:02as an example, persistent downregulation
• 10:05of BRCA1 and prolonged hypoxia.
• 10:08So
• 10:09just
• 10:10an overview,
• 10:12you know, we found,
• 10:13that there are a number
• 10:14of changes in DNA repair
• 10:17pathways related to the hypoxia,
• 10:18but predominantly
• 10:20in the homology dependent repair
• 10:21or homologous recombination
• 10:23pathway
• 10:24and the mismatch repair pathway
• 10:25at the transcriptional,
• 10:27translational, and epigenetic
• 10:29level.
• 10:32And,
• 10:32in addition, I alluded to
• 10:34the fact that there is
• 10:35acute activation of DNA repair
• 10:37and hypoxia,
• 10:39and, this is transient.
• 10:41And you can see that,
• 10:43in response to acute hypoxia,
• 10:45you get activation of check
• 10:47two,
• 10:48which then leads to a
• 10:49phosphorylation
• 10:50of BRCA one. And, actually,
• 10:52as,
• 10:53we accumulated data, there's actually
• 10:54a network of signaling events
• 10:56that happens early in hypoxia
• 10:59followed by, down regulation
• 11:01of the DNA repair expression.
• 11:03So, initially, DNA damage response
• 11:05pathways are activated
• 11:07and then transcriptionally
• 11:09suppressed and then eventually silenced
• 11:11if the hypoxia continues.
• 11:13So, this is basically a
• 11:15summary of a lot of
• 11:16work
• 11:17that shows a scale of
• 11:18time. And in minutes, you
• 11:20get these post translational modifications.
• 11:23But over time, you get
• 11:24suppression of DNA repair expression
• 11:26in specific pathways
• 11:28at the level of
• 11:30translation, transcription, and eventually epigenetic
• 11:33silencing.
• 11:34Now
• 11:35people sometimes
• 11:37ask me when I give
• 11:38these talks, why does this
• 11:40happen?
• 11:41Why would the cell do
• 11:42this? And, you wouldn't think
• 11:44that a a cancer cell
• 11:46that's,
• 11:47you know, looking to survive
• 11:49and and,
• 11:50overcome cancer therapy is interested
• 11:52in down regulating
• 11:54subsets of DNA repair pathways.
• 11:56Well, it turns out that
• 11:58one answer could be related
• 12:00to what people have been
• 12:01doing in the evolutionary biology
• 12:03and microbiology
• 12:04area
• 12:05for a number of years,
• 12:06and this has been, pioneered
• 12:08by Susan Rosenberg at Baylor.
• 12:10And what you see here
• 12:12is, a study where people
• 12:14isolated,
• 12:16e coli in the wild,
• 12:18a lot of different isolates,
• 12:20and then put them under
• 12:21stress. And the stresses could
• 12:23be starvation,
• 12:25in terms of what's in
• 12:26the agar plate as far
• 12:27as, nutrients.
• 12:29Could also be certain antibiotics
• 12:31that are not directly genotoxic.
• 12:34And what they found was
• 12:35an upregulation
• 12:36of a hypermutable
• 12:37state in the E. Coli
• 12:39that increased their ability to
• 12:41evolve and adapt to the
• 12:43stressful environment.
• 12:45And, interestingly,
• 12:46as they started to do
• 12:47the molecular,
• 12:49studies,
• 12:50one of the pathways downregulated
• 12:51in e coli is mismatch
• 12:53repair, and another one is,
• 12:55what they called upregulation of
• 12:57a mutagenic double strand break
• 12:59repair. But what actually happened
• 13:01is they suppressed the normal
• 13:02homologous recombination pathway, and you
• 13:05get an increase in,
• 13:07something similar to nonhomologous end
• 13:09joining. So very similar to
• 13:10what we saw in cancer
• 13:12cells.
• 13:14So
• 13:15if if we wanna look
• 13:16at a parallel, the hypoxia
• 13:18induced genetic instability
• 13:19mirrors what's been seen in
• 13:22e coli and other microorganisms.
• 13:24And and in a way,
• 13:25you could consider cancer
• 13:27as a population of unicellular
• 13:29organisms under stress.
• 13:32Okay. So
• 13:34that's the biology, but now
• 13:35can we exploit it for
• 13:37cancer therapy?
• 13:38So this is work that
• 13:39Denise Heakin in the lab
• 13:40did where she was the
• 13:41first, one to show that
• 13:43hypoxic cells are sensitive
• 13:45to an inhibitor of the,
• 13:47DNA repair enzyme poly ADP
• 13:49ribose polymerase or PARP. Many
• 13:51of you know will know
• 13:52that PARP inhibitors are used
• 13:54in the clinic now for
• 13:55cancers deficient in BRCA one
• 13:56and BRCA
• 13:58two because they are synthetically
• 14:00lethal to,
• 14:01cells deficient hematology dependent repair.
• 14:04And, of course, I I
• 14:06showed you when I show
• 14:07show again here that, hypoxia
• 14:09causes a downregulation
• 14:10of the BRCA pathway,
• 14:12and so those cells are
• 14:13sensitive to PARP inhibitors.
• 14:16So so then,
• 14:18an MD PhD student in
• 14:19the lab, Alana Kaplan, came
• 14:21and we said,
• 14:22can we exploit that biology
• 14:24for cancer therapy in a
• 14:25pharmacologic
• 14:26way?
• 14:27And our thinking was we
• 14:28would use an angiogenesis
• 14:30inhibitor called cediranib,
• 14:33and it's shown here on
• 14:34the upper right, for those
• 14:36of you who are chemically
• 14:37inclined. And it's a small
• 14:39molecule receptor tyrosine kinase inhibitor
• 14:42that was designed to inhibit
• 14:43the VEGF receptor, but, actually,
• 14:45it it inhibits multiple
• 14:47receptor tyrosine kinase,
• 14:50receptor tyrosine kinases,
• 14:54not just the the VEGF
• 14:55receptor.
• 14:56So we we asked, could
• 14:57we use
• 14:58cediranib
• 14:59to cause hypoxia in cancer
• 15:02in tumors and then exploit
• 15:04it? So what Alana did
• 15:06was, treat, we set up
• 15:08tumors in mice, and we
• 15:10treated the mice with cediranib.
• 15:12Then we isolated cells
• 15:14from the tumors, and this
• 15:15says mouse depletion because it's
• 15:17a human tumor. We deplete
• 15:18the mouse cells, and then
• 15:19we isolate the tumor cells.
• 15:22And,
• 15:23you can actually stain them
• 15:24with carbonic anhydra for carbonic
• 15:26anhydrase nine because it's a
• 15:28marker of hypoxia.
• 15:29And in fact, we saw
• 15:30that there was an increase
• 15:32in the hypoxic fraction in
• 15:33this experimental system from twenty
• 15:35to about thirty percent.
• 15:38So we took these cells
• 15:39and we looked at, expression
• 15:41of, rec the DNA repair
• 15:43genes in the HDR pathway,
• 15:45and we actually saw pretty
• 15:46good downregulation.
• 15:47In fact, we saw downregulation
• 15:49that was too good in
• 15:51the sense that it was
• 15:52more than you would expect
• 15:53from that
• 15:55smaller change in the hypoxic
• 15:57fraction.
• 15:58So it was clear that
• 15:59maybe something else was going
• 16:00on.
• 16:02So,
• 16:03what Alana did was we
• 16:04repeated that experiment
• 16:06using that CA nine marker.
• 16:07But instead of just
• 16:09counting those, we actually sorted
• 16:11them, and we collected the
• 16:13normoxic fraction and the hypoxic
• 16:15fraction. And what I'm showing
• 16:16you here is the normoxic
• 16:18fraction. And even in the
• 16:19normoxic fraction,
• 16:22homology dependent repair genes were
• 16:24downregulated, and that was the
• 16:25unexpected result.
• 16:27So what was going on?
• 16:29Well, actually
• 16:31so we then looked at
• 16:32some of the nonmalignant tissues
• 16:34in the mice, and we
• 16:35did not see downregulation. So
• 16:37it was only happening in
• 16:38the tumor.
• 16:39So we looked at bone
• 16:40marrow, liver, lung, and mammary
• 16:42fat pad.
• 16:43And just
• 16:45a little more of a
• 16:46survey, we looked at, some
• 16:48other DNA repair factors, and
• 16:49these are in the nonhomologous
• 16:51end joining pathway,
• 16:52which we already knew from
• 16:53our hypoxia work
• 16:55is that pathway is not
• 16:57affected, and it was also
• 16:59not affected by sedarinib.
• 17:01In,
• 17:02but, also,
• 17:03in this, slide, I'm showing
• 17:05you results of,
• 17:07treatment of cells and culture
• 17:09with sedarinib
• 17:10where you cannot generate hypoxia
• 17:12because there's no vasculature or
• 17:14angiogenesis.
• 17:15This is a direct effect
• 17:16of sedarinib,
• 17:17and you can see that
• 17:18those pathways are downregulated,
• 17:21directly by cediranib. But interestingly,
• 17:24in,
• 17:25bone marrow, CD thirty four
• 17:27positive stem cells, we do
• 17:28not see the same, downregulation.
• 17:32So that is consistent with
• 17:33what we had seen in
• 17:34vivo.
• 17:36Now, functionally, if we look
• 17:38at cells treated with cediranib
• 17:39and then treat them with
• 17:40radiation
• 17:41and and compare them to
• 17:43controls, we see a functional
• 17:44decrease in,
• 17:46DNA repair capacity.
• 17:48And you can see here
• 17:49that,
• 17:50in normal cells, you will
• 17:51see foci of a Rad
• 17:53fifty one coalescing
• 17:55to repair double strand breaks.
• 17:57But in cells treated with
• 17:58cediranib,
• 18:00there is substantially fewer
• 18:02foci indicating that,
• 18:04that pathway has been suppressed.
• 18:07Similarly, if we do an
• 18:09assay looking for,
• 18:11homologous recombination
• 18:12between two fragments of the
• 18:13GFP gene,
• 18:15one of which is cut
• 18:16by an enzyme that can
• 18:17be induced in the cells,
• 18:19we can see that in
• 18:20the presence of cediranib, that
• 18:22recombination
• 18:23event is substantially suppressed and
• 18:25to a similar extent as
• 18:26what you would see if
• 18:27you knock down Radka one
• 18:29or Rad fifty one. But
• 18:31as controls, there's no effect
• 18:33in knocking down the nonhomologous
• 18:35end joining pathway
• 18:36proteins, Kuwaiti and XLF.
• 18:39Now we then return to
• 18:41what Denise had observed in
• 18:43hypoxic cells, and we said,
• 18:44okay.
• 18:45Does siderinib sensitize to a
• 18:47PARP inhibitor? And here's
• 18:49the example that shows,
• 18:51in the in the presence
• 18:52of, cediranib,
• 18:54there is increased sensitivity to
• 18:56elaparib, a commonly used
• 18:58PARP inhibitor in the clinic.
• 19:00So this is a survival
• 19:01current curve on the left,
• 19:03and this is example of
• 19:05molecular markers of, cell death
• 19:07and apoptosis on the right.
• 19:10So what this,
• 19:12data
• 19:13these data show is that
• 19:14cediranib treatment phenocopies the BRCA
• 19:16deficiency
• 19:18and creates a synthetic lethality
• 19:20to PARP inhibitors.
• 19:22Now what's what's the mechanism
• 19:24here? So,
• 19:26I'm not showing you all
• 19:27the data, but one that's
• 19:28particularly striking is we used
• 19:31the papillomavirus
• 19:32e seven protein that doctor
• 19:34DeMeo will be very familiar
• 19:35with. And the e seven
• 19:36protein
• 19:38was known for a long
• 19:39time to interact with the
• 19:40RB family of proteins
• 19:42and cause their degradation and
• 19:44prevent their interaction with the
• 19:46e two f factors.
• 19:47So when we express
• 19:49e seven,
• 19:52we see that there is,
• 19:56abrogation of the sensitivity
• 19:58of sedarinib treated cells to
• 20:01olaparib.
• 20:02So this is one piece
• 20:03of evidence that supports that
• 20:06the downregulation is happening by
• 20:08the same method as happens
• 20:09in hypoxia
• 20:10that is through the,
• 20:12r b e two f
• 20:14family of pro of proteins.
• 20:16And so
• 20:17our conclusion of them, mechanistically,
• 20:19is suderninib has two effects
• 20:21on DNA repair. One is
• 20:23through hypoxia, which was our
• 20:24original
• 20:25hypothesis,
• 20:26and the other one is
• 20:27a direct one through its
• 20:29receptor tyrosine kinase inhibition
• 20:33effects. And and in these
• 20:35particular ovarian cancer cells, we
• 20:37linked it to the platelet
• 20:39derived growth factor receptor.
• 20:42That leads to an upregulation
• 20:44of phosphatase two a
• 20:46and, changes in the
• 20:49phosphorylation of the pocket proteins
• 20:51and that cascade that leads
• 20:53to suppression of repair capacity.
• 20:57So then we took this
• 20:58into an in vivo model
• 21:00in, mice
• 21:02in which we form tumors
• 21:03from those of one of
• 21:04those ovarian cancer cell lines.
• 21:06And,
• 21:07this is the tumor growth
• 21:08suppression curve,
• 21:10and the control or olaparib
• 21:12alone are similar.
• 21:13Sediranib has some effect, but
• 21:15now the combined
• 21:17olaparib and siderinib has the
• 21:19most growth suppression,
• 21:20and that's correlated with an
• 21:22increase in survival.
• 21:24Now when I,
• 21:26first presented this,
• 21:27at a meeting,
• 21:29I I subsequently was contacted
• 21:31by two fairly large pharmaceutical
• 21:34companies.
• 21:35One said, oh, gee. We
• 21:38reproduce that, and we're actually
• 21:39working on,
• 21:41an even better version of
• 21:43cediranib, and we'd like to
• 21:44come talk to you about
• 21:45that.
• 21:47The other one
• 21:48said, oh, well, we can't
• 21:49reproduce what you did, and,
• 21:51actually,
• 21:52we don't like, that in
• 21:54vivo model.
• 21:56We prefer a different one
• 21:57that we don't think that
• 21:58one is,
• 22:00a good one.
• 22:01So, thankfully,
• 22:04Joseph Kim, who I see
• 22:05in the audience here,
• 22:08decided to try a different
• 22:10in vivo model,
• 22:12and that one is called
• 22:13patients in the clinic.
• 22:15And and in his work,
• 22:17reported,
• 22:18in, JCO,
• 22:21he did a trial in,
• 22:22patients with metastatic prostate cancer
• 22:25combining,
• 22:26cidernib and olaparib and saw
• 22:28positive results.
• 22:32So
• 22:33what about other repair pathways?
• 22:35Well, it turns out that,
• 22:37like I said, nonamog is
• 22:39end joining, which you can
• 22:40see here on the left
• 22:41on the right. This is
• 22:42a,
• 22:44heat map of gene expression
• 22:45where blue is down
• 22:47and nonbluer red is up.
• 22:49You can see nonhomologous end
• 22:50joining is not suppressed,
• 22:54but we do see the
• 22:55suppression of,
• 22:56HDR in a different,
• 22:59cancer type, in this case,
• 23:00lung cancer.
• 23:01And we also see some
• 23:03synthetic lethality induced
• 23:05to alaparib in this lung
• 23:07cancer line HCC
• 23:08eight two seven.
• 23:11And then Denise,
• 23:13also looked at,
• 23:15DNA mismatch repair in a
• 23:17series of cancer cell lines
• 23:18as shown here in the
• 23:19middle panel, lung cancer, ovarian,
• 23:22and a glioblastoma.
• 23:23And in this case, DNA
• 23:25mismatch repair as it is
• 23:26in hypoxia is suppressed in
• 23:28response to,
• 23:30sedarinib. And, this is the
• 23:32heat map, and you can
• 23:33see some western blots on
• 23:35the right.
• 23:37Okay.
• 23:38So,
• 23:39at this point,
• 23:41Gary Outwerger, who is a,
• 23:44assistant professor in the GYN
• 23:45oncology,
• 23:47department,
• 23:48here, joined the lab with
• 23:50an interest in uterine serous
• 23:52cancer.
• 23:53And so we knew that
• 23:55siderinib caused a decrease in
• 23:57DNA repair gene expression in
• 23:59specific pathways.
• 24:01But,
• 24:01what impact might it have
• 24:03in uterine cancer?
• 24:05And could there be a
• 24:05role for sudaredim in combination
• 24:08with any DNA repair inhibitors
• 24:09or cell cycle regulators
• 24:11in uterine cancer? And part
• 24:13of the reason we were
• 24:14interested in this is that,
• 24:17there's a fair proportion of
• 24:18uterine cancers that are deficient
• 24:20in DNA mismatch repair.
• 24:22And the ones that are
• 24:23deficient in MMR have a
• 24:25better better natural history and
• 24:27also
• 24:28respond better
• 24:30to immune checkpoint inhibitors.
• 24:33So,
• 24:34Gary went ahead and and
• 24:36surveyed some uterine, cancer,
• 24:38lines that we got from
• 24:39Alessandro
• 24:40Santin here in the GYNOC
• 24:42department. And what he saw
• 24:44was that in the ARC
• 24:45one,
• 24:46on the left and and
• 24:46the HEK one b on
• 24:47the right,
• 24:49the mismatch repair factors
• 24:51were suppressed in response to
• 24:52sudernib at different levels of
• 24:54sensitivity.
• 24:55Interestingly, this ARC four cell
• 24:57line did not show an
• 24:58effect to sudernib alone.
• 25:03We then tested the functional
• 25:04effect on mismatch repair using
• 25:06a reporter system in which
• 25:08a dinucleotide
• 25:09repeat is inserted at the
• 25:11beginning of the reading frame
• 25:13for the green fluorescent protein,
• 25:15and and it's,
• 25:16set up so it's out
• 25:18of frame, so there is
• 25:19no green in the cells.
• 25:20But if there's a mismatch
• 25:22repair deficiency leading to a
• 25:24frame shift, you will see
• 25:25green cells.
• 25:26And so what you see
• 25:27here on the right is
• 25:28this reporter was put into
• 25:30those cell lines. They were
• 25:32treated or not with siderinib,
• 25:33and you can see that
• 25:34there's an increase in the
• 25:36GFP positive fraction after siderinib
• 25:39treatment, notably in the ARC
• 25:41one and the HEK one
• 25:42b
• 25:42and a tiny one in
• 25:44the ARC four.
• 25:47Now
• 25:48having seen this functional decrease,
• 25:50we wondered how could we
• 25:52exploit this?
• 25:54Now we knew that,
• 25:55you know, olaparib was was
• 25:57not known to synergize with
• 26:00mismatch repair deficiencies. So we
• 26:03we,
• 26:04hypothesized that maybe an inhibitor
• 26:07of a cell cycle checkpoint,
• 26:09namely,
• 26:10we one inhibitor might
• 26:12have some combination effect
• 26:15because mismatch repair causes replication
• 26:17stress,
• 26:19on multiple levels.
• 26:20And we we one is
• 26:22a a checkpoint protein that
• 26:24regulates,
• 26:25cell cycle progression at multiple
• 26:27points in the cell cycle
• 26:28as illustrated here, intra s,
• 26:30g two m, and mitotic
• 26:32exit.
• 26:33So we thought there potentially
• 26:35could be an opportunity,
• 26:37for a combined effect.
• 26:39So, Gary went ahead and
• 26:40did a study to look
• 26:42for synergism. And and for
• 26:44those of you not familiar,
• 26:45this is, of cell viability
• 26:47assay where different doses of
• 26:49one agent are on one
• 26:51axis and doses of another
• 26:53on the other. And where
• 26:55you see,
• 26:56hills that are blue
• 26:58are dose combinations where there's
• 27:00statistical evidence for synergism.
• 27:02So in all three cell
• 27:03lines, we saw that these
• 27:04two
• 27:05agents, one cediranib and and
• 27:07a v one inhibitor,
• 27:09which in the slides we
• 27:11mostly call m MK seventeen
• 27:13seventy five or atadavosertib,
• 27:17showed, good synergism.
• 27:20Now,
• 27:21interestingly, the arc four cells
• 27:22had not shown a decrease
• 27:24in mismatch repair
• 27:25gene expression by sediranibalone.
• 27:28But if you can see
• 27:29it here, when we treated
• 27:31them with the combination,
• 27:33we actually saw that there
• 27:34was strong suppression of, DNA
• 27:37mismatch repair expression,
• 27:38which you could measure in,
• 27:40by the increase in, cells,
• 27:43showing GFP expression using that
• 27:45reporter vector that is visualized
• 27:47here and quantified on the
• 27:49right. The same is true
• 27:50on the HEK1 b cells.
• 27:54So what might be going
• 27:55on? Well, it turns out
• 27:57that
• 27:58just like,
• 27:59the BRCA
• 28:00and Rad fifty one promoters,
• 28:02the mismatch repair gene promoters
• 28:04can also respond to e
• 28:05two f and pocket protein,
• 28:08transcription factor complexes.
• 28:10And when we looked at
• 28:12retinoblastoma
• 28:13phosphorylation,
• 28:14we could see that,
• 28:16the we one inhibitor and
• 28:18cediranib
• 28:19synergized to
• 28:21increase the hypophosphorylation
• 28:23or decrease the phosphorylation of
• 28:25r v RB,
• 28:26which creates
• 28:28the repressive
• 28:29complex
• 28:29that then acts on
• 28:33the DNA repair gene promoters.
• 28:35So that was
• 28:37one step in the mechanism.
• 28:39But
• 28:40we wondered, well, why is
• 28:41there synergistic
• 28:43killing?
• 28:45So,
• 28:46we got a clue from
• 28:47the literature.
• 28:49There is a,
• 28:52a group, now at, University
• 28:54of Texas Southwestern led by
• 28:55Guomin Lee who published,
• 28:58this, very interesting paper. Excuse
• 29:00me. I'm just
• 29:02gonna get a little water.
• 29:04Where he looked at
• 29:05MLH one deficiency and its
• 29:07effect on the c gasping
• 29:09pathway.
• 29:13And what he found was
• 29:14when you knock down m
• 29:16l h one,
• 29:17you get an increase in
• 29:19c gas sting signaling in
• 29:21the innate immune,
• 29:23pathway.
• 29:25And his model was that,
• 29:28it's known that MLH one
• 29:31interacts with a exonuclease
• 29:33called XO one. And this
• 29:34is involved in DNA mismatch
• 29:36repair, but also plays a
• 29:37secondary role in end resection
• 29:40during homologous recombination.
• 29:42When MLH one is knocked
• 29:44down,
• 29:45XO one is unleashed
• 29:48and could can
• 29:50mediate,
• 29:52basically dysregulated
• 29:57exonuclease
• 29:58activity on DNA ends,
• 30:00creating,
• 30:01DNA fragments and long single
• 30:03stranded gaps.
• 30:04And what, he's,
• 30:06they showed in this paper
• 30:07is that DNA then gets
• 30:09released into the cytoplasm
• 30:12and can activate the c
• 30:13gas sting pathway, which is
• 30:15the sensor of
• 30:17chromosomal DNA and micronuclei
• 30:19and other DNA, type fragments
• 30:22in the
• 30:23cytoplasm.
• 30:24So the idea
• 30:26is mismatch repair deficiency,
• 30:28too much x o one
• 30:29activity,
• 30:30and then activation of stigast
• 30:32deng. And one way to
• 30:33score that is,
• 30:34the stat one transcription factor
• 30:37factor gets phosphorylated.
• 30:38It's an easy marker of
• 30:39activation of that innate signaling
• 30:41pathway.
• 30:43So we went ahead and
• 30:44looked at that,
• 30:46and we found, in fact,
• 30:48that stat phosphostat
• 30:49one was strongly induced
• 30:52by the combination of cediranib
• 30:54and the wee one inhibitor
• 30:55as shown here,
• 30:57and quantified its its, you
• 30:59know, roughly,
• 31:01ninety fold increase, which is
• 31:03quite striking.
• 31:05And,
• 31:06we were fortunate
• 31:08because,
• 31:10Gary is on a k
• 31:11award with the women's health
• 31:12research
• 31:13program here run by Vicky
• 31:16Abrams and, Yu Taylor.
• 31:18And on his mentoring committee
• 31:19was Akiko Iwasaki
• 31:21who saw this data. And,
• 31:22actually, I had had the
• 31:24privilege of working with Akiko
• 31:25on a paper which reported
• 31:28that ataxia telangiectasia
• 31:31ATM
• 31:32mutated mice,
• 31:34which have,
• 31:36a,
• 31:38our ATM is known to
• 31:40have cerebellar degeneration as one
• 31:42of its phenotypes,
• 31:47linked the ATM deficiency to
• 31:49hyperactivation
• 31:50of line one leading to
• 31:52neurodegeneration.
• 31:53So Akiko said, well, that
• 31:55strong induction of innate immune
• 31:57signaling,
• 31:58that might be related to
• 31:59line one. So we went
• 32:00ahead and looked into that.
• 32:02Now what is line one?
• 32:04It's the long interspersed nuclear
• 32:05elements, which are the, the
• 32:08most common,
• 32:09retro element in the human
• 32:11genome. Most of them are
• 32:12silenced, but some
• 32:14can be expressed.
• 32:16And, here's a diagram of
• 32:18the locus, and it encodes
• 32:19two open reading frames, ORF
• 32:21one and ORF two. ORF
• 32:22two is a reverse transcriptase.
• 32:25And when it when it
• 32:26becomes activated by transcription followed
• 32:28by reverse transcription, it's linked
• 32:30to genomic instability.
• 32:33And and line one cDNA
• 32:35in the cytoplasm is known
• 32:37to activate c gas sting.
• 32:39And, actually, this is sort
• 32:40of a life cycle of
• 32:41the line one elements. And
• 32:43the key thing here is
• 32:45if you can see, you
• 32:46get expression of line one,
• 32:48RNA,
• 32:49and it gets,
• 32:52turned into,
• 32:54cDNA in the,
• 32:57here in the cytoplasm and
• 32:59that activates the c gas
• 33:00sting pathway. No. It can
• 33:02also go back into the
• 33:03nucleus and then reinsert
• 33:05and be another cause of
• 33:06genetic instability.
• 33:09So our hypothesis
• 33:12was that there was
• 33:14suppression of MLH one leading
• 33:16to dysregulated
• 33:17x o one
• 33:19that could lead to line
• 33:20one induction
• 33:21and cDNA production and then
• 33:24activate,
• 33:25c gas sting as line
• 33:27one is cDNA is known
• 33:28to do.
• 33:29So we looked again at,
• 33:31the uterine cancer cell lines,
• 33:33and we saw line one
• 33:35is activated, increased. So this
• 33:37is a western blot for
• 33:38line one open reading frame
• 33:39one, ORF one, is increased.
• 33:42And that correlates with our
• 33:43increase in phosphostat one as
• 33:46seen by western blot in
• 33:47the combination of,
• 33:49cediranib
• 33:50and the w one inhibitor
• 33:52in both ARC four and
• 33:53the HEK one b cell
• 33:54lines.
• 33:55So
• 33:56this is fitting our model
• 33:58that,
• 33:59you get
• 34:00a decreased mismatch of pair
• 34:02leading to line one and
• 34:03c gas sting,
• 34:04and stat phosphostat one.
• 34:07And so
• 34:08the hypothesis still is that
• 34:10exo one is playing a
• 34:11role.
• 34:13Okay.
• 34:14Now
• 34:14for one
• 34:16one part of this, we
• 34:17asked, okay. Is the line
• 34:19one cDNA causing the phosphostat
• 34:22one induction?
• 34:23And one way to test
• 34:24that is use a reverse
• 34:26transcriptase inhibitor called three t
• 34:28c.
• 34:29And this would block
• 34:31the production of the line
• 34:33one cDNA
• 34:34from the line one mRNA.
• 34:37So if you look here,
• 34:38this is,
• 34:39control. This is the combination
• 34:41of cediranib and the wee
• 34:42one inhibitor. MLH one is
• 34:44down.
• 34:45Line one is induced,
• 34:46and fostostat one is induced.
• 34:49Now if you add three
• 34:50t c,
• 34:51you see line one
• 34:54is suppressed back to baseline,
• 34:56and so is fostostat one.
• 34:58Now you can recapitulate
• 35:00that instead of doing the
• 35:01combination of cediranib and v
• 35:03one inhibitor. You acutely knock
• 35:05down MLH one with a
• 35:07s I r n a.
• 35:09You get induction of line
• 35:11one
• 35:12and phosphostat one. Now this
• 35:13part was seen by the
• 35:15Guoam and Li lab. What
• 35:16they hadn't realized was that
• 35:18line one might be playing
• 35:19a role.
• 35:21And this is blocked
• 35:23similarly to the case with
• 35:25the drugs
• 35:26by the reverse transcriptase.
• 35:29So it looked like this
• 35:29is a similar mechanism.
• 35:33Now we went on to
• 35:34test the role for x
• 35:35o one.
• 35:36And in this case, we
• 35:37used
• 35:40siRNA
• 35:40knockdown.
• 35:42So you see here,
• 35:43in this case, we're looking
• 35:44at the impact of acute
• 35:46MLH one knockdown.
• 35:48MLH one knockdown leads to
• 35:51actually a slight increase in
• 35:52levels of x o one,
• 35:54but also line one induction,
• 35:56phosphostat one induction as we
• 35:57saw before.
• 35:59But if you also knock
• 36:00down x o one,
• 36:02you see there's less line
• 36:04one induced and less phosphostat
• 36:06one.
• 36:07Then you you look at
• 36:08that in the setting of
• 36:09the combination of siderinib
• 36:11and the wee one inhibitor.
• 36:14So lipof lipofectamine
• 36:16is just the control for
• 36:17the s I r n
• 36:18a to x o one.
• 36:20And you see here that
• 36:22you get the induction of
• 36:23line one and stat one
• 36:25phosphostat one as we saw
• 36:26before with the combination of
• 36:27the drugs.
• 36:29But when you have knocked
• 36:31down x o one,
• 36:33it abrogates those increases.
• 36:35So you see here there's
• 36:36no induction of line one.
• 36:38In fact, it's below the
• 36:39baseline,
• 36:40and you suppress the induction
• 36:41of phosphostat one.
• 36:45Okay. So now we return
• 36:46to that synergistic killing,
• 36:49and I wanna caution that
• 36:50this is a
• 36:51preliminary result that we are
• 36:53trying to
• 36:54confirm.
• 36:56But it it fit the
• 36:57story so well I couldn't
• 36:58help but show it.
• 37:00And what you see here
• 37:01is that there is killing
• 37:02of the uterine cancer cells
• 37:04by the combination of sildernib
• 37:06and v one inhibitor. But
• 37:08when you add the reverse
• 37:10transcriptase to block the induction
• 37:11of line one, you rescue
• 37:13the cell killing.
• 37:17Now let's go back to
• 37:19an in vivo model.
• 37:22And so,
• 37:24what we did was we
• 37:25tested,
• 37:25whether this applied to a
• 37:28tumor model in mice, and
• 37:29we used the ARC four
• 37:30cells,
• 37:31in
• 37:33in as as a xenograft
• 37:34in nude mice.
• 37:35And you can see here,
• 37:36the blue is the control
• 37:38untreated.
• 37:39These are the two agents,
• 37:41independently,
• 37:43siderinib and, we one inhibitor.
• 37:45And here is the combination,
• 37:47which essentially completely suppressed tumor
• 37:50growth. And you can see
• 37:51an example at
• 37:53at at the time of
• 37:54sacrifice of the animals. There's
• 37:56basically scar tissue left in
• 37:57the combination treatment.
• 37:59And importantly, the mouse weights
• 38:01were stable and so were
• 38:03the blood counts. And this
• 38:04is important because these agents
• 38:06are known to effect,
• 38:08cause myelosuppression.
• 38:11And then we returned to
• 38:12mechanism,
• 38:13and we did immunohistochemistry
• 38:15on those tumors
• 38:17for line one
• 38:18open reading frame one expression.
• 38:20And you can see that
• 38:21in the combination treatment, there's
• 38:23an induction of line one
• 38:25expression
• 38:26consistent with, the model.
• 38:31Now I didn't forget about
• 38:32hypoxia, so we're a little
• 38:34bit back to the future.
• 38:35And it turns out Yuhang,
• 38:37who had done all that
• 38:38work on gene,
• 38:40silencing,
• 38:41has been looking at the
• 38:42innate immune signaling pathway because
• 38:44it turns out that there
• 38:46is some
• 38:47long term silencing of sting
• 38:49in hypoxic cells.
• 38:51But there's initially induction of
• 38:53phosphostat one. So so based
• 38:55on what, Gary had found,
• 38:57we looked at line one
• 38:58in hypoxia, and it turns
• 39:00out line one is induced
• 39:01in hypoxic cells.
• 39:03But that induction
• 39:05can be blocked by the
• 39:06three TC,
• 39:09reverse transcriptase,
• 39:11inhibitors. So more to come
• 39:12on that.
• 39:14So just in summary, what
• 39:16what I've told you is
• 39:17that hypoxia,
• 39:19drives changes in DNA repair
• 39:20and genome instability
• 39:22on multiple levels,
• 39:24that cediranib,
• 39:27phenocopies
• 39:27the effect of hypoxia
• 39:29on homology dependent repair and
• 39:30mismatch repair by engaging a
• 39:32similar signaling meth mechanism.
• 39:36That creates a synthetic lethality
• 39:38with PARP inhibitors that's that
• 39:39has been observed in a
• 39:41clinical trial.
• 39:43And,
• 39:44we have evidence that we
• 39:46won inhibition will synergize
• 39:48with siderinib
• 39:49to both suppress MLH one
• 39:51and activate line one
• 39:53and innate immune signaling,
• 39:55to,
• 39:56kill uterine cancer cells,
• 39:59presumably through immunogenic cell death,
• 40:01although that mechanism needs to
• 40:03be
• 40:04fleshed out.
• 40:06And,
• 40:08we have recent evidence that
• 40:09hypoxia also engages
• 40:12some of the same, pathways
• 40:14possibly through line one.
• 40:16So future directions are to
• 40:17dig a little deeper into
• 40:18this mechanism.
• 40:21As I said, I showed
• 40:22some old,
• 40:23material, but some new material,
• 40:25so this is all still
• 40:26a work in progress.
• 40:27We want to extend to
• 40:28other uterine cancer models and
• 40:30other cancer types,
• 40:32and, we would like to
• 40:33combine with an immune checkpoint
• 40:35inhibitor in a in an
• 40:36immune competent model.
• 40:38We we haven't done that
• 40:39because those were human,
• 40:41cancer lines,
• 40:43not syngeneic with the,
• 40:45immune competent mouse model. And
• 40:47we would like to test
• 40:49this combination in a clinical
• 40:51trial.
• 40:52So,
• 40:53I'll stop there and,
• 40:54happy to, take questions. Thank
• 40:57you.
• 41:08Tommy.
• 41:26Right.
• 41:31Right.
• 41:58So so we we haven't
• 42:00looked at that, but that's
• 42:01very interesting. So Tommy was
• 42:03asking about the fact that
• 42:04three t's I'll go back
• 42:06to the microphone.
• 42:07Three three t c is
• 42:09an antiviral.
• 42:10And,
• 42:12and so what what what
• 42:13is seen clinically
• 42:15that might be related to
• 42:16what we've seen? And, the
• 42:18answer is we don't know,
• 42:19but it's possible that, that
• 42:21could be affecting,
• 42:23response to therapies.
• 42:25The other interesting thing that
• 42:27Gary has proposed is whether
• 42:29three t c, if it
• 42:31in fact suppresses
• 42:33innate immune signaling and inflammation
• 42:35in mismatch repair deficiency,
• 42:38could be used as a
• 42:39chemo preventive
• 42:40for people with Lynch syndrome.
• 42:45And,
• 42:46you know, that remains to
• 42:48be determined, but I think
• 42:49it's something we would like
• 42:51to
• 42:51see if we could model
• 42:52in mice
• 42:53or even in people.
• 42:55Yeah.
• 43:18Right.
• 43:20Yeah. So we haven't looked
• 43:22at small cell lung and,
• 43:23of course, the Schlippet eleven
• 43:25plays a key role in
• 43:26our response,
• 43:28but that would be, you
• 43:29know, very interesting to look
• 43:30at.
• 43:32Yeah.
• 43:33Yeah.
• 43:41Yeah.
• 44:00Yeah. So,
• 44:02just to repeat that, the
• 44:03question has to do with
• 44:04have we you know, can
• 44:06we detect ORF two related
• 44:07to line one?
• 44:10I think as see, as
• 44:11a practical matter, the antibody
• 44:13to ORF one is a
• 44:14really good antibody, and so
• 44:15that's what almost everyone uses
• 44:16in the literature.
• 44:18And so,
• 44:19we haven't looked at ORF
• 44:21two. Or if we did,
• 44:22nobody showed me that data,
• 44:23so I don't think it
• 44:24worked.
• 44:25But, yeah, that's a good
• 44:27point. Now we have the
• 44:28indirect evidence that the reverse
• 44:30transcriptase inhibitor suppresses the,
• 44:33effect, which makes sense because
• 44:35we're looking for line one,
• 44:37but we don't we haven't
• 44:38directly measured or orf two.
• 44:41Yeah. Roy. Peter, I have
• 44:43a question about clinical trials.
• 44:44Actually, two questions.
• 44:46First, the trial of Jo
• 44:47Kim with the Sedernet.
• 44:49Looking at that curve, it
• 44:50looks like there was a
• 44:51positive PFS.
• 44:52So where did that trial
• 44:53go? Has it gone on
• 44:54to a further study? Did
• 44:56any of the molecular correlates
• 44:58confirm some of the, scientific
• 45:00advancements?
• 45:01Yeah. I I I gotta
• 45:02let Joe do it. He's
• 45:03sitting behind you so he
• 45:04can he can answer that.
• 45:11Data before our trials. So
• 45:13I did two trials. One
• 45:14was in prostate cancer and
• 45:15then the other was was
• 45:16in advanced
• 45:23which is shown here is
• 45:24our positive data. So, again,
• 45:25our our new hypothesis was
• 45:27that we'll be able to
• 45:28see the benefit
• 45:30involving, you know, the jawline
• 45:31or condition set up some
• 45:33of the HR g calculations.
• 45:35If you're told surprise, what
• 45:36we saw was actually we
• 45:37saw,
• 45:38most of our benefits in
• 45:39patients with HR proficiency. In
• 45:41other words, the patients with
• 45:42the mutations spread like for
• 45:44those without the mutation, we
• 45:45are able to it's the
• 45:46benefit of the combination. So,
• 45:47actually, our question become actually,
• 45:50whether
• 45:51the,
• 45:52you know, whether this actually
• 45:53prevents or delays the partner
• 45:55need to resist. From that
• 45:56one question to you is
• 45:58that based on what you
• 45:59have seen so far,
• 46:00whether the again, again, it's
• 46:02really a tough thing and
• 46:03losing agent. We do see
• 46:04this in patients
• 46:05like the manifest as, like,
• 46:06a bad the high blood
• 46:08pressure, the fatigue, similar to
• 46:10data, some of the losses
• 46:11there is. If, you know,
• 46:11we do have these adverse
• 46:12strong hypoxia inducing agents. Whether
• 46:14that actually plays a role
• 46:16in getting the vetting or
• 46:17delaying part of the little
• 46:19tests, that's one. And then
• 46:20just to kind of sort
• 46:21of highlight on the,
• 46:23small of the long range
• 46:24data, actually, we have seen
• 46:25we have to have image
• 46:26at half the output of
• 46:27CRM. We had to obtain
• 46:29that part as a scan
• 46:30before and after the same
• 46:31amount of activity directed image
• 46:33that I can see I
• 46:34used by the, CRM. And
• 46:36we actually,
• 46:37saw some early antigen activity
• 46:39in small alarm cancer. We
• 46:41saw about thirty percent response
• 46:43rate and then also in,
• 46:44non small alarm and
• 46:46also in the CNDC as
• 46:47well. In pancreas, we're not
• 46:48able to see,
• 46:50no, you know, benefits at
• 46:51all. So I'll just
• 46:53Yeah. So my question to
• 46:54you is whether
• 46:59Yeah. So that's very interesting,
• 47:01question. And I think what
• 47:03might be going on is
• 47:04that cediranib had has a
• 47:06a little bit of a
• 47:07broad based effect on a
• 47:09number of repair factors in
• 47:11the HDR pathway.
• 47:12So some of the tumors
• 47:14that might have, say, a
• 47:15BRCA deficiency,
• 47:18What sildernib also does is
• 47:20suppress RAD fifty one and
• 47:21some FANC b two and
• 47:23some other
• 47:24factors in the same pathway.
• 47:25So
• 47:26either
• 47:27one is that HDR
• 47:29deficiency occurs at different levels
• 47:31and the sildernib takes it
• 47:32down a little bit more.
• 47:33Or if there's been a
• 47:34reversion,
• 47:36for example, like in BRCA2,
• 47:37there's been some intragenic,
• 47:39deletions that cause a reversion
• 47:41in the phenotype.
• 47:42The siderinib can still suppress
• 47:44expression and cause
• 47:47further
• 47:47HDR deficiency.
• 47:49So it could be that
• 47:51that's the susceptible population where
• 47:53siderinib is gonna have its
• 47:54most effect.
• 47:58And for your other clinical
• 47:59trial with the pembrolizumab, which
• 48:01looks very interesting.
• 48:03I know you're waiting for
• 48:04more preclinical data, but, you
• 48:05know, that's gonna be very
• 48:06hard. The animal models are
• 48:07all good, but not so
• 48:08good. Right. What are the
• 48:09barriers that getting an IIT
• 48:11going? Is there a specific
• 48:12disease that might be best
• 48:14that anybody do in the
• 48:15phase one detector?
• 48:17Where where is that?
• 48:18Well,
• 48:19so I don't know if
• 48:21Gary's in the audience. He's
• 48:22probably in the OR.
• 48:23But he is looking to
• 48:25to do a,
• 48:27clinical trial in uterine.
• 48:29That's his you know, that's
• 48:31where this is leading for
• 48:32him. I think for
• 48:33other,
• 48:34disease,
• 48:35sites, for example,
• 48:37Eric and I were talking
• 48:38about breast.
• 48:39You know, we we need
• 48:40to survey,
• 48:42based on, you know, genetic
• 48:44characteristics of of different, tumor
• 48:46types
• 48:47where there's gonna be susceptibility
• 48:49because we know there's variation.
• 48:50So, for example, in breast,
• 48:52is it triple negative? Is
• 48:53it gonna be,
• 48:54you know, some other subtype?
• 48:56And the same is, you
• 48:57know, there is we have
• 48:59a hint of a difference
• 49:00in lung between the KRAS
• 49:01driven and the EGFR driven.
• 49:04So there's more to learn
• 49:06as far as what tells
• 49:07us
• 49:08that,
• 49:10you know,
• 49:11these strategies could work.
• 49:13And in fact, you know,
• 49:14Eric and I have briefly
• 49:15talked about, you know, sport
• 49:16projects. And I think
• 49:18something in a sport project
• 49:19could be care you know,
• 49:20trying to figure out what's
• 49:21subtype to take this into
• 49:23a trial and then do
• 49:24the trial.
• 49:26Yeah.
• 49:28Yeah.
• 49:42Yes. So we did do
• 49:44that, and that's in Alana's
• 49:46paper. We did a kind
• 49:47of a, I would say,
• 49:48medium sized scale screen.
• 49:50And and a fair number
• 49:52do the same thing, especially
• 49:54the ones that
• 49:55strongly inhibit PDGFR.
• 49:57So that's part of what
• 49:58led to our hypothesis
• 50:00of the pathway.
• 50:02So, you know, I can
• 50:04refer you to there's a
• 50:05table in the supplementary
• 50:06material that has couple hundred
• 50:09agents in it.
• 50:11Joe, you had a question.
• 50:14Add on to that PDGFR
• 50:16question.
• 50:17Just start I mean, it's
• 50:18great work and then about
• 50:20a while. Just start me.
• 50:20Have you looked at cells
• 50:21that over trust PDGFR, or
• 50:23are they did you get
• 50:24a bigger response or mention
• 50:26the p g f r?
• 50:26He he yeah. We we
• 50:27haven't done that, enough to
• 50:29say anything. We I mean,
• 50:29we we thought about doing
• 50:29that, and we did some
• 50:30knockout work and some overexpression
• 50:33work. So
• 50:39there is some correlation, but
• 50:40we haven't done a broad
• 50:41survey.
• 50:42And that, for example, could
• 50:43be something that could, you
• 50:45know, go into a project
• 50:46where we, you know, look
• 50:48at, you know, the cell
• 50:49you know, databases of cell
• 50:51cancer cell lines and things
• 50:52like that.
• 50:55Right. Right.