Therapeutic Targeting of Epigenetic Mechanisms in Cancer

Transcript

• 00:00Good morning everybody.
• 00:01It's so nice to see everybody here.
• 00:03So let's get started.
• 00:04So this is a special occasion
• 00:06and actually nobody better than
• 00:08Doctor Armstrong to present the
• 00:10Lecturer in honour of Lance Tallman.
• 00:12So this series was established
• 00:15in 2012 by Doctor Marvin Sears.
• 00:17Dr. Sears was a long time chair
• 00:19and founder of the Ophthalmology
• 00:21and Visual Sciences at Yale,
• 00:23and he established this lecture
• 00:24series series in honour of his mother,
• 00:27Lance Tallman,
• 00:27who passed away from leukemia.
• 00:29And it really was the first lecture series
• 00:31dedicated solely to hematologic malignancies.
• 00:33So hematologists are always
• 00:35delighted and it's really intended
• 00:37to bring to Yale pioneers,
• 00:39you know,
• 00:40who study malignant hematologic diseases
• 00:43and then bring treatments to the patient.
• 00:46There's actually nobody better than to give
• 00:48today's lecture than Doctor Scott Armstrong.
• 00:51Doctor Armstrong is the
• 00:52President of Dana Farber,
• 00:54Boston Children's Cancer and Blood
• 00:56Disorder Center and the Chairman of
• 00:59the Department of Pediatric Oncology
• 01:00at Dana Farben Cancer Institute.
• 01:02And since 2016,
• 01:05he serves as the Associate Chief of
• 01:07the Division of Hematology Oncology
• 01:09at Boston Children's Hospital and
• 01:11was previously the Director of the
• 01:13Center for Abidinex Research at
• 01:15Memorial Sloan Kettering Cancer
• 01:17Center and Professor of Pediatrics
• 01:19at Weill Cornell Medical College.
• 01:21I'm not going to go back to
• 01:23medical degrees and all this,
• 01:24so good to have you here.
• 01:25So Roger Armstrong really has you know
• 01:28pioneered research in in epigenetics
• 01:33and studying pediatric cancers,
• 01:34right.
• 01:35And we always learn that studying
• 01:37cancer for example in Pediatrics can
• 01:39then really enlightened mechanism
• 01:41of disease also for adult patients.
• 01:43And I think it's super,
• 01:44super exciting to hear your talk
• 01:46today really bringing basic mechanism
• 01:48all the way from the lab to benefit
• 01:50so many of our patients and we're
• 01:52incredibly excited to have you here.
• 02:01Thank you for the nice introduction
• 02:02and and for the lectureship and
• 02:04the plaque that's very nice.
• 02:06And thank you for coming today.
• 02:08It's not the most beautiful
• 02:09day to be out walking around.
• 02:10So I appreciate you you making it here.
• 02:13And as Stephanie said,
• 02:15I'm going to talk to you today
• 02:17about work we've been doing over
• 02:20the past couple of decades now
• 02:23focused on originally a relatively
• 02:26rare subset of leukemia and then
• 02:28move to more common leukemias
• 02:31and maybe even beyond leukemias.
• 02:33And as many of you probably know
• 02:35that the concept of targeting
• 02:37chromatin or epigenetic based
• 02:39mechanisms been around for quite
• 02:41some time and there have been some
• 02:43therapeutic advances in that regard.
• 02:44But it's been it's stops and
• 02:46starts I would say along the road
• 02:48and hopefully I can convince you
• 02:50that we're maybe finally starting
• 02:52to make some significant go get
• 02:54some significant insights there.
• 02:56So these are my disclosures.
• 02:57I do consult for a number of biotech
• 02:59companies trying to convince them that
• 03:01these mechanisms are relevant that's
• 03:03and sometimes I'm able to do that.
• 03:05And then this patent on amended inhibition,
• 03:08NPM one AML.
• 03:08I'm going to talk about NPM one AML.
• 03:11And more important disclosure is these
• 03:12are the people that do the work.
• 03:14I don't do the work and I'm very lucky
• 03:17to have tremendous fellows in the lab.
• 03:20And actually all of these fellows other
• 03:22than Emily who soon will leave the
• 03:24lab to go start her own have started
• 03:27their own independent lab based careers now.
• 03:30So this is the outline of the
• 03:31talk I'm going to introduce you.
• 03:33Many of you probably know a lot of
• 03:37this to the MLL or CAT or MLL or KMT
• 03:41two ACI can't even remember what the
• 03:44other name is for complex and MLL
• 03:47rearranged leukemias and then move to
• 03:50the therapeutic development of small
• 03:52molecules that target those complexes.
• 03:54Talk a little bit about the clinical
• 03:56translation and resistance mechanisms
• 03:57that we're starting to see to those
• 04:00molecules and then talk about the
• 04:02potential role in other cancers.
• 04:03And as I already mentioned the,
• 04:05the concept of the OR the
• 04:08relevance of epigenetics.
• 04:10And just for those of you that
• 04:11are that are purists in the
• 04:13epigenetic and chromatin space,
• 04:14I will interchangeably use
• 04:16epigenetic and chromatin biology.
• 04:17Actually there there is a group of
• 04:20people that think those two things are
• 04:22not the same thing and the concept
• 04:24that these mechanisms are relevant and
• 04:26cancer has been around for quite some time.
• 04:28This is not a new idea and epigenetics
• 04:31really encompasses many different
• 04:33types of modifications of chromatin,
• 04:36DNA methylation,
• 04:37histone modifications,
• 04:38complexes have proteins that
• 04:41read those histone modifications.
• 04:43The nucleos,
• 04:44there's nucleosome remodeling complexes
• 04:45that you've probably heard about.
• 04:47The bath complex also are
• 04:49frequently mutated in cancer.
• 04:50So we've known that this is relevant,
• 04:52but what to do about it has been
• 04:54a little bit harder to understand
• 04:56and the the kind of simple
• 04:58concept is, is these mechanisms
• 05:00control developmental gene expression
• 05:02and if we were smart enough we'd
• 05:05figure out how to therapeutically
• 05:06target them and reverse those cancer
• 05:09causing gene expression mechanisms and
• 05:11hopefully we're starting to get there.
• 05:13There are some FDA approved drugs that
• 05:15you probably know about H TAC inhibitors
• 05:18and DNA methyl transferase inhibitors.
• 05:19I would say whether or not those molecules
• 05:22are working via epigenetic mechanisms,
• 05:25still a little bit of a question,
• 05:26but indeed those were the first ones
• 05:29that could be working via these some of
• 05:32these mechanisms that were FDA approved.
• 05:34So this is the leukemia that I became
• 05:37most interested in as a fellow
• 05:39back in the late 1990s.
• 05:40Now those are leukemias with
• 05:43rearrangements of the KMT 2A or
• 05:46or MLL gene and in Pediatrics as
• 05:49mentioned I'm a pediatric oncologist.
• 05:52This rearrangement when found in
• 05:53infants with AOL predicts a very poor
• 05:56prognosis and in pediatric leukemia
• 05:58therapy we're actually not used to that.
• 06:00We cure most of our patients with
• 06:02AOL and we find a subset that has a
• 06:05less than 40% long term survival.
• 06:07We that's unusual and this subset
• 06:09is that if an infant comes in that
• 06:12has ALL and has a rearrangement of
• 06:14this gene that it's probably in
• 06:16the 4050% long term survival now.
• 06:23And so back in the late 90s I joined
• 06:26Stan course Meyer's lab to start to
• 06:28learn about that and then obviously
• 06:30ultimately to start my own lab.
• 06:32So this is the the wild
• 06:34type MLL or KMT 2A protein.
• 06:37It's very large, it's in the nucleus,
• 06:39it's about 500K Daltons,
• 06:41makes it has made it difficult to study.
• 06:44It has a number of different
• 06:46domains and is bound to chromatin.
• 06:48We've known that for over 20 years.
• 06:50And when the translocation occurs that
• 06:52in terminus of MLL is fused to the
• 06:54C terminus of what's 100 different
• 06:56fusion proteins also making a little
• 06:59bit complicated to understand.
• 07:00And this is just the history that
• 07:02I won't go through in too much
• 07:04detail other than to say that the
• 07:06wild type MLL protein is was shown
• 07:08in the mid 90s by Stan Course,
• 07:10Mars Group and others to control
• 07:13development of blood system of
• 07:15hematopolysis through presumably
• 07:16control of the homeotic or hox
• 07:18genes that are important in many
• 07:20types of development and but
• 07:22in blood development as well.
• 07:24And that was really actually
• 07:26pointed to that concept by studies
• 07:28done even before that in fly,
• 07:30in fruit flies and Drosophila,
• 07:32showing that the trithorax gene,
• 07:33which is the Drosophila homologue is
• 07:36important for development as well.
• 07:38And then Mike Cleary and Terry,
• 07:40rabbits showed in very nice mouse
• 07:42studies in the late 90s that
• 07:44the MLL fusion proteins indeed
• 07:46do directly induce leukemia.
• 07:48And then David Alice's group showed
• 07:50that in the wild type MLL is a histone
• 07:53modifying enzyme modifies histone H3
• 07:54on lysine 4 through this enzymatic
• 07:56domain here at the C terminus.
• 07:59So this was really the first well
• 08:02characterized chromatin regulator
• 08:03that is known to drive tumor genesis.
• 08:07And so that was really why in the
• 08:10early early 2000s a lot of labs
• 08:12jumped on this to thinking maybe
• 08:14this would give us some insight into
• 08:16chromatin based mechanisms and cancer.
• 08:19So to summarize a lot of work that
• 08:21we did talk thinking about cells
• 08:23of origin of of this type and
• 08:25other types of leukemia.
• 08:26Not going to get into that too
• 08:27much today because I want to
• 08:29get to the therapeutic part.
• 08:30But we were able to show that the
• 08:31MLO fusion when we put it into either
• 08:33stem cells or progenitor cells,
• 08:35either mouse or human,
• 08:36that the MLO fusion can drive
• 08:38the development of leukemia from
• 08:40multiple different cell types
• 08:41in hematopoietic development.
• 08:43And these concepts have now been
• 08:45shown in other types of tumors.
• 08:46But at the time that was a
• 08:48relatively new concept.
• 08:49But what more importantly what it
• 08:50let us do is really characterize
• 08:52the gene expression program that's
• 08:54driven by this MLL fusion protein
• 08:57when we put it into in this case
• 08:59a mouse progenitor cell.
• 09:00And we could look very quickly to
• 09:02see what types of gene expression and
• 09:05chromatin based changes happened when
• 09:07the MLL fusion binds to chromatin.
• 09:09And our work and many,
• 09:11many people's work across the
• 09:13the world with this model
• 09:14originally developed by Mike Cleary's
• 09:16group have I would say that MLO
• 09:19fusion driven leukemia now is about
• 09:20as well characterized mechanistically
• 09:22this as any type of leukemia,
• 09:25probably any type of cancer
• 09:27largely because of this model.
• 09:28So we know where the fusion binds throughout
• 09:31chromatin, which genes it controls.
• 09:33We now have mechanisms of turning the fusion
• 09:35off and we can see what genes get shut off.
• 09:37We understand now quite a bit
• 09:39about what the MLL fusion does.
• 09:42Exactly how it does it is
• 09:44still a little bit unclear,
• 09:46but that is what we and others
• 09:48are really working on now.
• 09:50And of course that is what we
• 09:51need to know and under in order
• 09:53to develop hopefully therapeutics
• 09:55that can target these mechanisms.
• 09:57So this is a very simple actually
• 10:00summary of how the MLL fusion works.
• 10:03So here in Gray is the ML AF9 fusion,
• 10:06the in terminus of MLL bound to
• 10:08some of the proteins normally found
• 10:10in the MLL complex Menon and Ledge
• 10:13F here and those help localize
• 10:15the fusion protein to chromatin.
• 10:17And then the C terminal part of the
• 10:19fusion brings in a number of complexes
• 10:22is histone methyl transferase .1 L,
• 10:23which is a histone H3 lysine
• 10:2679 methyl transferase And this
• 10:28so-called super elongation complex,
• 10:30which is really a fundamental complex
• 10:33for controlling transcription broadly,
• 10:35not just in this setting.
• 10:36Certainly the MLL fusion drags these
• 10:40chromatin regulatory and transcriptional
• 10:42control proteins and complexes to its
• 10:45target genes to drive gene expression.
• 10:48So with that level of understanding,
• 10:51it became easier to go to pharma and
• 10:53biotech and to get them interested
• 10:55in developing small molecules that
• 10:57might target these mechanisms.
• 10:58Even though at the time no one knew
• 11:01if these were mechanisms be relevant
• 11:03beyond this relatively rare disease
• 11:05which is probably A couple thousand
• 11:07patients per year in the United States.
• 11:09But I'll show you,
• 11:10I think we think that indeed and
• 11:12in fact we have now shown in in
• 11:14patients that it it actually is.
• 11:16So each, as I mentioned,
• 11:17each of these labels in red is a small
• 11:20molecule that's been developed to target
• 11:22various components of this large complex.
• 11:25And to summarize broadly on molecules
• 11:28that target the complexes on the right,
• 11:30the kind of general
• 11:32transcriptional complexes,
• 11:33the problem there has primarily been
• 11:35toxicity that actually not too surprising
• 11:38you turn off transcription broadly,
• 11:40we can do that with some of
• 11:41our chemotherapeutic drugs.
• 11:42It's relatively toxic on the
• 11:44left side and to date the problem
• 11:47has actually been efficacy,
• 11:49meaning we can do pre clinical studies
• 11:51and I'll briefly load to this with
• 11:53.1 actually get really impressive
• 11:55changes in gene expression and such.
• 11:57But then we go into patients with
• 11:59the .1 inhibitor get a little bit
• 12:02of clinical signal and but unable
• 12:04to maintain that clinical response.
• 12:07But but toxicity has not primarily
• 12:09been a problem for the molecules
• 12:11on the left side of this figures
• 12:13and that's kind of where we've
• 12:15focused. And I'm going to talk a
• 12:17lot about this protein Menon here,
• 12:19which is really a scaffolding protein
• 12:22that's bound to the MLL fusion
• 12:24and helps keep it on chromatin.
• 12:25I'll show you more about that in a minute.
• 12:28Men and as an important part of the
• 12:30Amylo complex was first demonstrated
• 12:32in 2004 by Michael Cleary's group.
• 12:34And now there are many small molecules.
• 12:36I'll talk about the one we've been
• 12:38working on that disrupt this interaction.
• 12:40I'll show you how in a minute.
• 12:42And that really that concept,
• 12:44the first chemical biology done around
• 12:46this was done by Yolanda Grimbeck is due
• 12:49in Michigan where they developed the
• 12:51first small molecule to bind to minute.
• 12:53So just quickly I'm going to
• 12:55this is kind of jumping ahead,
• 12:56but it's a concept that I think is
• 12:59important in terms when we think
• 13:01about targeting chromatin complexes
• 13:02in leukemia or other diseases.
• 13:04So if this is a even more simplified
• 13:07view of the ML AF9 bound to chromatin
• 13:10through its adapter proteins and
• 13:11bring it in this case the .1 complex.
• 13:14I told you that we worked actually
• 13:16over a decade ago now with a
• 13:19company called Epizyme to make small
• 13:21molecule enzymatic inhibitors of .1.
• 13:23We've shown with genetic studies
• 13:25that that is an important component
• 13:27of this complex and that histone
• 13:29modification that it put places
• 13:31is important for controlling MLO
• 13:33fusion driven gene expression.
• 13:35And that went into clinical trials and
• 13:37we were able to a couple of patients
• 13:39actually went into to remission
• 13:41and many had some response but it
• 13:43they were all pretty transient.
• 13:44And I'm going to show you that
• 13:47that's not the case with the min an
• 13:49inhibitor and this summarizes why
• 13:51we think that is that if you the .1
• 13:54L inhibitor inhibits the enzymatic
• 13:55activity of this protein,
• 13:57but the whole complex remains stuck
• 13:59on chromatin.
• 14:00So it's actually doesn't take a
• 14:02lot for the cell to figure out how
• 14:04to deal with the fact that that
• 14:06enzymatic activity is no longer there.
• 14:08Whereas what I'm going to show
• 14:09you with the MLL Menin inhibitor,
• 14:10it actually disrupts the whole complex
• 14:13and much of the complex lifts and
• 14:15the fusion protein lifts off of
• 14:17chromatin and we think that probably
• 14:19is why the the Menin inhibitor
• 14:22approach is more dramatic.
• 14:24So in 2019 we published the small
• 14:26molecule that we were working on which
• 14:28was at the time called BTP 5O469.
• 14:31The version of it that's in the clinic
• 14:33is called Syndex 5613 or Revu Minib.
• 14:35Get to that in a minute.
• 14:37And this is a very potent small
• 14:39molecule that binds to this pocket on
• 14:41men and this is the men and protein.
• 14:43This is the crystal structure of this
• 14:45protein here in purple and blocks
• 14:48the interaction between men and
• 14:51and the MLL fusion and that leads
• 14:53to as I mentioned the loss of this
• 14:55complex on chromatin,
• 14:56but interestingly enough only
• 14:58does it at very selected sites.
• 15:00This complex actually remains on on
• 15:03chromatin and the wild type complex which
• 15:06would also be potentially disrupted
• 15:08by this molecule remains on chromatin
• 15:11at many sites throughout the genome.
• 15:13But there's a very selected group
• 15:16of genes loci where it's lost.
• 15:18The importance of that is,
• 15:19is that we know genetic inactivation of MLL
• 15:23broadly is toxic that to hematopoietic cells.
• 15:26But there's some reason that this
• 15:28mechanism seems to be only important
• 15:31at certain developmental loci.
• 15:33And we think that may be why we're not
• 15:35seeing tremendous toxicity that you
• 15:37could imagine by lifting this chromatin
• 15:39complexes off the genome broadly.
• 15:41And this is just one example of
• 15:43a preclinical PDX study that we
• 15:46did with an MLL rearranged AML.
• 15:48All of you probably know what
• 15:49PDX studies are,
• 15:50inject the human leukemia into
• 15:52immunodeficient mice and treat the mice here
• 15:55with two cycles if you will of of therapy.
• 15:57And the beauty of this experiment is
• 16:00from a technical standpoint is Syndax
• 16:03actually supplies us with mouse chow
• 16:06that's impregnated with the drugs.
• 16:08So you don't actually have to dose the mice.
• 16:11People in the lab love that fact.
• 16:13So basically you inject the leukemia,
• 16:15change the food and come back in
• 16:16a month and see what happened.
• 16:18And it's obviously not quite that simple,
• 16:19but so we were able to do these experiments
• 16:22with much more speed and much less pain
• 16:25than they than they often can take.
• 16:27And so you can see here that the the
• 16:29mice that were treated with the the MIN,
• 16:31an inhibitor essentially the AML
• 16:33in this PDX model was eradicated,
• 16:36which as most of you know who
• 16:38do these types of experiments.
• 16:40That's not the how these experiments
• 16:42usually go.
• 16:43You might see a prolongation of survival
• 16:46but not this degree of response.
• 16:49Same thing in a model that a lot
• 16:51of people use,
• 16:52this retroviral model of ML AF9
• 16:54that we've used a lot as well,
• 16:55which does lead to a very aggressive
• 16:59mouse AML.
• 17:00And you can see here,
• 17:02we've treated mice that have gotten
• 17:04these cells injected with the,
• 17:06the min,
• 17:06an inhibitor for this period of
• 17:08time and in fact,
• 17:08it eradicated the disease in this model.
• 17:11So that is again very different from
• 17:13what most of the graphs will look like
• 17:16when you do an experiment like this.
• 17:18So really indicating that
• 17:20there's significant potential.
• 17:22So I'm going to switch diseases or
• 17:24switch subtypes of leukemia and tell
• 17:26you a little bit about a different
• 17:28subtype that one is interesting and
• 17:31two actually kind of changed the
• 17:33level of interest from biotech and
• 17:35pharma because it's way more common
• 17:38and that's NPM one mutant AML.
• 17:40And I keep saying that because as
• 17:43you can probably tell in paediatrics
• 17:44and other rare cancers,
• 17:46we're constantly and many of you
• 17:48probably recognize this when we're
• 17:50talking about drug discovery and development,
• 17:52having to convince people to work
• 17:54on the diseases we're interested in
• 17:56because they're indeed quite rare.
• 17:58When you go from 1000 to 2000 patients per
• 18:00year in the United States to 15 into 17,000
• 18:03patients per year in the United States,
• 18:05you actually get a lot more interest.
• 18:07And so that's one of the
• 18:09reasons beyond just the the,
• 18:11the mechanistic interest that
• 18:12I think this is important.
• 18:14So Michael Kuhn, when we were in New York,
• 18:17a fellow at the time asked
• 18:20the question of well, OK,
• 18:21we know that the MLO rearranged leukemias.
• 18:23This is a gene expression data for
• 18:25the HOX genes and MIS ones from Tim
• 18:28LAYS group in a bunch of AM LS,
• 18:30and you probably know how to read these.
• 18:31Each row here is AG in each
• 18:33column with leukemia sample.
• 18:34And here's the MLL rearranged group OX,
• 18:37A cluster we know that's long known.
• 18:39In fact, some of the microarray data
• 18:41that I did as a fellow demonstrated
• 18:43that the Hox A cluster is expressed in
• 18:46MLL rearranged leukemias as is MIS one.
• 18:48Those are two targets of the ML effusion
• 18:51that have been worked on for many decades.
• 18:54It turns out in the NPM one mutant
• 18:56leukemia as you can see here on the right,
• 18:58they have the Hox A cluster as
• 19:01well and B cluster,
• 19:02another Hox cluster and MIS one.
• 19:04So this has also been known for some
• 19:07time that this subset of AML and PM one
• 19:09mutant also tends to express the Hox genes.
• 19:11So Michael using CRISPR which in
• 19:152016 was still relatively new did
• 19:18a what's called a domain scan.
• 19:19I won't get into the details of that,
• 19:21but the point being that the inactivation
• 19:24of MLL and ultimately I mean of men
• 19:27and MLL led to differentiation and
• 19:30ultimately death of NPM one mutant AML cells.
• 19:33And the concept being at the time
• 19:36that the disruption of MLL and
• 19:38Menon in the NPM one state somehow
• 19:41leads to a decrease in the Hox gene
• 19:44expression and therefore ultimately
• 19:46a enemies one expression and therefore
• 19:49ultimately changes in gene expression.
• 19:52Hannah Okleman,
• 19:52who was a a fellow in the lab just
• 19:54recently moved back to Germany
• 19:56to start her own lab,
• 19:57then demonstrated that similar to
• 20:00the MLL fusions that NPM 1 mutations
• 20:04in mouse models could transform
• 20:06multiple hematopoietic cell types,
• 20:09stem cells a little bit more significantly
• 20:11more efficiently than progenitors,
• 20:12but also could transform progenitors
• 20:15as well and.
• 20:17Needless to say,
• 20:18there was still a question as to kind of
• 20:21mechanistically how all this was working.
• 20:23I'm going to summarize some chip seek
• 20:26data here that really shows that at
• 20:29least at this level of understanding,
• 20:32it's quite similar to what we see
• 20:34with the MLL rearranged leukemia.
• 20:37So these are NPM one mutant cell
• 20:39lines where we're doing chromatin
• 20:41immunoprecipitation for men and for
• 20:43example either in a control setting in
• 20:47black PMSO treated cells or the min
• 20:49inhibitor treated cells and you can see
• 20:51that min and comes off of chromatin.
• 20:53We know that when we treat
• 20:54with the min inhibitor
• 20:55min and lifts off of
• 20:57chromatin MLL shown here.
• 20:58Now this is wild type MLL because
• 21:01it's a mutant in PM one setting
• 21:03doesn't come off the Hawks loci.
• 21:04We'd seen that in the MLL fusion setting
• 21:07as well but does come off of MES 1.
• 21:09So this is that concept that I show you
• 21:12that certain loci respond differently to
• 21:14the min and inhibitor and this is showing
• 21:17the RNA SEC or the gene expression.
• 21:20And you can see that when there's
• 21:22a correlation between loss of MLL
• 21:24occupancy and loss of gene expression
• 21:26and this is that looking at that more
• 21:28broadly by what's called a GSDA analysis.
• 21:30So the point being that much like what we saw
• 21:33in the MLL fusions in the NPM one setting,
• 21:36you treat with the men inhibitor
• 21:37men and comes off of chromatin.
• 21:39The MLL protein itself comes off of
• 21:41chromatin at about 100 to 150 loci
• 21:43and those genes lose their expression
• 21:46when the cells differentiate.
• 21:48And then if we go into PDX studies to
• 21:51NPM one mutant flip 3 ITD Co mutant,
• 21:54these are relatively aggressive AM LS.
• 21:57You can see again pretty impressive
• 22:00response in the PDX setting.
• 22:02And for the people who start do these
• 22:04types of experiments in the audience,
• 22:07we actually as you probably know the
• 22:09standard way to do this is put in
• 22:11leukemia into an immunodeficient mouse,
• 22:13the first sign of any leukemia in
• 22:14the peripheral blood of those mice,
• 22:15you start treating them.
• 22:17We actually waited in this experiment
• 22:19on the right until the mice were ill
• 22:21and started treating them and actually
• 22:25we could recover essentially 4 out of
• 22:27five of the mice and eradicate the disease.
• 22:30So we stacked the deck against us and
• 22:32we're still able to to make that or
• 22:35extend the the survival of those mice.
• 22:37So to summarize this at this point
• 22:39we the MIN inhibitor and NPM one
• 22:42mutant AML induces differentiation,
• 22:44reverses leukemia,
• 22:46genic leukemic gene expression,
• 22:48certain genes like MIS one removes
• 22:51MLL from those loci and we get
• 22:54dramatic responses.
• 22:54So that was exciting and that was
• 22:57enough to get Janssen and Ichi
• 22:59and the various other large
• 23:01pharmaceutical companies interested in,
• 23:03in small molecule development
• 23:05of Menon inhibitors.
• 23:06But it's still there was still
• 23:08something here we don't quite or didn't
• 23:12quite understand is why is it that
• 23:14the NPM one mutant AML is depending
• 23:17so much on the MLL Menon complex.
• 23:20So Hannah dug into that as well.
• 23:22You guys probably know this,
• 23:23but this is just a little bit
• 23:25about the mutant NPM one protein.
• 23:27It's shown here.
• 23:29Schematically,
• 23:30it's found mostly in the nucleolus
• 23:33in the wild type setting,
• 23:35but when the mutation occurs,
• 23:37it's a mutation in this nuclear
• 23:39or localization signal that then
• 23:41leads to a nuclear export signal.
• 23:43So the mutant in PM one is largely
• 23:45found in the cytoplasm and that
• 23:47was recognized by the people who
• 23:49initially described this mutation.
• 23:51However,
• 23:52there is some
• 23:54that remains the mutant in PM one
• 23:57in the nucleus and we've taken
• 23:59advantage of a system that many of
• 24:01you probably know about where you
• 24:04can now by CRISPR mediated homologous
• 24:06recombination actually tag if you will,
• 24:10whatever gene or protein of interest with a
• 24:13degradable version of FK PP12 shown here.
• 24:16And actually this cell line was made
• 24:18by Peggy Goodell's group in at Baylor
• 24:21and you have a mutant NPM one protein
• 24:23that has this degradable version
• 24:25of F KBP 12 and you can treat them
• 24:27with a small molecule Protac that
• 24:29will degrade the whole thing.
• 24:30So you can degrade the mutant oncoprotein
• 24:33and look fairly rapidly after degradation
• 24:35as to what's happening and here's
• 24:37how rapidly you get degradation.
• 24:39By 60 minutes you've got about half
• 24:41of the mutant protein gone and by 120
• 24:44minutes essentially all of us gone.
• 24:45So it's relatively rapid and these
• 24:47types of experiments are quite
• 24:49illuminating because you really have
• 24:51very tight control over over the system.
• 24:56And what we see is that we get
• 24:59differentiation when we degrade the
• 25:01mutant onca protein and ultimately
• 25:03apoptosis of the cells as well.
• 25:05And this is just the Western blot looking.
• 25:07We can actually separate the mutant
• 25:09protein from the wild type because
• 25:11it's tagged,
• 25:11it's here and you can see that here.
• 25:14This is the cytoplasmic prep,
• 25:15the nuclear prep and the chromatin
• 25:17prep and here's the mutant in PM one
• 25:19and we can control that by degrading
• 25:21it to show that that signal actually
• 25:23is what we think it is.
• 25:25And in fact there is a fair amount
• 25:27of the mutant in PM one in the
• 25:28nucleus and on chromatin.
• 25:30Then if we do chip seek to say
• 25:32where is it in the nucleus and
• 25:34where are where is it on chromatin.
• 25:36You can see here with with two different
• 25:39antibodies in black that the NPM one mutant,
• 25:42NPM one protein is bound to many
• 25:44of the similar genes that we've
• 25:46learned about with the MLL fusion,
• 25:48the Hox cluster MIS one.
• 25:50And when we degrade it that signal goes away.
• 25:52And I keep saying that because
• 25:54particularly with chip seek experiments,
• 25:55the opportunity for background signal
• 25:57is real and this is makes you feel
• 26:00much better that the signal that
• 26:02you're looking at is indeed the
• 26:04signal that you are interested in.
• 26:05And now you actually can go to primary
• 26:07patient samples with those antibodies
• 26:09and see the NPM one protein mutant
• 26:11NPM one protein bound there as well.
• 26:13And here's the list of the top 50
• 26:15or so genes to which the mutant
• 26:17NPM one protein is bound.
• 26:18And you can see as some of these genes
• 26:20that I've already talked about OX,
• 26:22A&B cluster and a number of other genes
• 26:24that we tend to pay attention to and
• 26:26stare at when we're talking about MLL or
• 26:29looking at MLL rearranged leukemias as well.
• 26:33So,
• 26:34but is it controlling gene expression.
• 26:36So now we have mutant
• 26:38NPM one protein bound to
• 26:40interesting sites in on chromatin
• 26:42and we can degrade it and show
• 26:44that that signal is specific.
• 26:46And now what happens to transcription?
• 26:48So this was 24 hours later,
• 26:50quite a bit later.
• 26:51Most of those genes that I just showed
• 26:53you where the NPM one protein is bound,
• 26:55their expression is down and
• 26:57this is an approach called pro
• 26:59seek which I won't get into the
• 27:00details as to how one does this.
• 27:02So many of you,
• 27:03some of you who work on transcription
• 27:04probably know this technique.
• 27:06But essentially it measures the
• 27:08amount of bound RNA polymerase 2 out
• 27:10throughout the length of the gene as a
• 27:12surrogate for transcriptional activity.
• 27:15And what we can see that as quickly
• 27:17as 30 minutes after treatment of the
• 27:19cells with the NPM one protein degrader,
• 27:22you're already seeing a decrease in
• 27:24transcription at those sites where
• 27:26the NPM one was previously bound.
• 27:28And and if you look at it across
• 27:30the the the all express genes
• 27:32you don't see those changes.
• 27:33So in fact it's the NPM one protein's
• 27:37bound there and somehow controlling
• 27:40transcription of these genes.
• 27:42And one of the ways it's doing
• 27:44it is by keeping RNA Pol two,
• 27:46CK nine that that super elongation
• 27:48complex that I told you is critical for
• 27:50transcription bound to those genes.
• 27:52So when we treat with the degrader
• 27:54NPM one comes off and then much
• 27:56of the transcriptional apparatus
• 27:58comes off of those genes as well.
• 28:00So it's maintaining a state that allows
• 28:03for those critical complexes including
• 28:06pole two to to bind to those low side.
• 28:09I'm going to go through the details of
• 28:12this but just to kind of summarize it
• 28:15that when we degrade mutant in PM one,
• 28:17we lose RNA polymerase two occupancy
• 28:19where the NPM one was previously
• 28:21bound within an hour.
• 28:23So off goes NPM one,
• 28:24off comes pole two and a number
• 28:26of other histone modifications
• 28:28like H3K27 acceleration,
• 28:29some of you know associated with various
• 28:32types of gene expression decreases
• 28:34and then the histone modification,
• 28:36other histone modifications start
• 28:38to decrease somewhat later.
• 28:39For those of you interested in transcription,
• 28:41we can talk more about this in detail later,
• 28:43but it looks like it's like when
• 28:45we degrade the mutant in PM one,
• 28:47the decrease in gene expression
• 28:48is actually biphasic.
• 28:49There's so initially there's a decrease
• 28:52of about 50% probably because pole 2
• 28:55is not quite as there's not as much
• 28:58pull two and other complex occupancy.
• 29:00And then after about 3 days we
• 29:02see a dramatic another dramatic
• 29:04decrease in gene expression.
• 29:06We think that's because now the
• 29:09histone modifications are starting
• 29:12to come in and and work together
• 29:14with whatever the previous mechanism
• 29:15was to fully shut off transcription.
• 29:20So how does this connect to to Menon?
• 29:26So
• 29:31there we go. So we now if you
• 29:33treat with the MIN inhibitor,
• 29:36what happens to this chromatin bound in
• 29:39mutant in PM one and I'll just quickly
• 29:42summarize it by saying you can see here
• 29:45here's the mutant in PM one we treat
• 29:47with the MIN inhibitor doesn't come
• 29:49off the hogs locus but it actually
• 29:50does come off of the mis one locus
• 29:52exactly where we're seeing MLL come off,
• 29:54same thing down here and if you compare
• 29:57that the gene expression again those are
• 29:59the genes that are losing expression.
• 30:02So to summarize what I'm saying here is
• 30:04that when we degrade the mutant NPM one
• 30:07protein with this degrader molecule,
• 30:09we lose RNA pole two CDK 9 and
• 30:12ultimately .1 at those loci.
• 30:14When we treat with the min inhibitor we
• 30:16do the same thing but at a subset of
• 30:19the loci where the NPM one is bound.
• 30:21So very similar to what's happening
• 30:24with the MLL fusion.
• 30:25However, we're now looking to see if
• 30:28indeed the mechanisms are identical.
• 30:30And it turns out that while some
• 30:31of the complexes are overlapping,
• 30:33these are the mechanisms are
• 30:35not perfectly identical.
• 30:37That is,
• 30:37some complexes are important
• 30:39in the MLO fusion,
• 30:41not an NPM one and vice versa.
• 30:42So we're trying to work through those
• 30:45details because as you can imagine,
• 30:47the next step that we want to do
• 30:48is come in and target some of these
• 30:50other complexes with small molecules.
• 30:51So to summarize this part before,
• 30:53now I move to the clinical translation.
• 30:56There are a subset of leukemias that have
• 30:59high level Hox gene expression MIS one,
• 31:01another transcription factor
• 31:02called PBX three.
• 31:04This actually accounts for about
• 31:0640% of human AML and it's ones
• 31:10with these genetic abnormalities,
• 31:13MLO rearrangement,
• 31:14NPM 1 mutation and I'm actually more
• 31:19relatively rare but more common than
• 31:21in adults rearrangement called Newt 98
• 31:24rearrangements in both pediatric and adult.
• 31:26So again accounting for
• 31:28about 40% of patients.
• 31:31So that all while all that was happening,
• 31:35Syndax and another company called cure
• 31:37oncology that many of you know about,
• 31:40we're developing small molecules
• 31:42here Syndax 5613 and here Cure's
• 31:46what's now Zyftominib and these
• 31:47I'll show you some of this,
• 31:49the data from the Cindex trial
• 31:52and some mechanism of resistance.
• 31:54And as I mentioned essentially
• 31:56when the NPM one story came out,
• 31:59we were called by Janssen and actually
• 32:01another couple of other pharmaceutical
• 32:03companies and at least these three
• 32:05now have MIN inhibitors that are
• 32:06right on the tails if you will of
• 32:08the cure of development and Cindex.
• 32:11So this is so now we're into
• 32:15patients with the Syndex 5613.
• 32:17This is just date some of
• 32:19the data from the phase one.
• 32:21This is one of the first patients
• 32:23that was treated at Dana Farber and
• 32:26we were able to get the peripheral
• 32:27blood and here are the blast.
• 32:29And you can see this is with
• 32:31Revumenib that day three,
• 32:32not much has happened.
• 32:33Day seven start to see a decrease
• 32:35in peripheral blast,
• 32:36day 14 further decrease and by day 30
• 32:39at least the peripheral blood blasts
• 32:41are in this case essentially gone.
• 32:44Florian Perner is a postdoc
• 32:45who was doing this.
• 32:46He sorted these cells and looked
• 32:48at gene expression and in fact the
• 32:50gene expression changes that we see
• 32:52here look very similar to what we
• 32:53had seen in the preclinical studies
• 32:56not being a clinical trialist.
• 32:58I'm going to summarize the whole
• 33:00phase one right here with a lot
• 33:02of work from a lot of people and
• 33:04this was published last year.
• 33:07This is the Revue Minib Phase
• 33:09one with Syndex 5613.
• 33:10The other name for it,
• 33:1268 patients with relapsed
• 33:15refractory leukemia.
• 33:16As you probably know,
• 33:17many of these patients have had
• 33:19tremendous numbers of cycles of
• 33:21various types of therapies and the
• 33:24CR rate depending on how you count
• 33:26CRS and for those of you who do
• 33:27clinical trials and A and all we can
• 33:29talk about that is somewhere in the
• 33:3040% range with an overall response
• 33:32rate of about 50% and a median
• 33:35duration of response about nine months.
• 33:37So for in relapse refractory setting,
• 33:39these are actually pretty impressive
• 33:42numbers and the cure oncology
• 33:44small molecule seems to be doing
• 33:47something having similar activity.
• 33:48And in fact some of that data from
• 33:51Janssen was also just printed presented
• 33:53at ASH and it looks like the activity
• 33:55of that molecule is is similar.
• 33:57So indeed there looks like
• 33:59there's significant clinical
• 34:01activity of this approach.
• 34:02This lot slide just reminds me to
• 34:05point out and and then now I'm talking
• 34:08to people who are doing AML clinical
• 34:10trials in the in the audience.
• 34:12An interesting phenomenon that
• 34:13you know better than I do,
• 34:16but that is influencing how these
• 34:17drugs are are able to be developed
• 34:19and that is when you treat patients
• 34:21with them in an inhibitor,
• 34:23many of them will develop this syndrome
• 34:25called differentiation syndrome.
• 34:27But in this setting it looks a little
• 34:30different clinically I'm told,
• 34:31than the differentiation syndrome
• 34:32that you usually see when you
• 34:34treat patients with acute per
• 34:36myelocytic leukemia with ATRA.
• 34:37In fact patients have died from
• 34:40this differentiation syndrome and
• 34:42so that has prompted the FDA to
• 34:44call this a dose limiting toxicity.
• 34:46Think about what that means.
• 34:47It means that your your your dose limiting
• 34:51toxicity is actually occurring as a
• 34:53result of efficacy of your molecules.
• 34:56So we can talk about what So I think
• 34:58the FDA is fighting against the the
• 35:00the some things that they shouldn't be,
• 35:02but that's a that's a whole other soapbox
• 35:05that we can talk about if we want to.
• 35:07That would be like for those of you
• 35:08who treat patients with ALL saying you
• 35:10start to see a little tumor lysis syndrome,
• 35:11we'd better stop treating them
• 35:13because that's bad. No, that's good.
• 35:16So with all this in mind and the
• 35:19clinical activity looking interesting,
• 35:21we figured that it was much like
• 35:23any single targeted agent,
• 35:24there was likely to be some mechanism
• 35:27of resistance to that targeted agent.
• 35:29And right about the time we started
• 35:31thinking about this the Broad Institute
• 35:33developed this screening based on or
• 35:36screening approach based on single
• 35:38nucleotide base editing which in fact
• 35:40what you can do is tile in this case
• 35:44Menon the whole length of the gene
• 35:47with guides that will mutate not
• 35:49every nucleotide because of the way
• 35:51that it's designed but where you can
• 35:53mutate the majority of amino acids
• 35:55across the length of that protein
• 35:59to to basically do an in a
• 36:02cellular mutagenesis screen
• 36:03to see if you can phenotypes.
• 36:06So what Florian decided to do is to
• 36:08get that base editor library made
• 36:10for the minute gene treat cells with
• 36:13the min an inhibitor and see if there
• 36:16were mutations that made the cells
• 36:18resistant to the min an inhibitor.
• 36:21And in fact there were there shown here
• 36:23in two different MLL rearranged lines
• 36:25and interestingly enough we we kind of
• 36:29looking back this was probably silly.
• 36:31We looked at this,
• 36:32the new technique and there's a
• 36:34little bit of noise and we didn't
• 36:36know exactly what to make of
• 36:38it looked kind of interesting.
• 36:39Florian put it in the drawer and kind
• 36:41of didn't do too much more with it
• 36:43until we got a call from Ross Levine
• 36:46and Etan Stein at Memorial Sloan Kettering.
• 36:48Actually,
• 36:49I got a like emergent text from Ross
• 36:51which I thought something really bad
• 36:52had happened and he said we have to talk now.
• 36:55So I called him and he said we
• 36:57found mutations in Menin in samples
• 36:59from patients that have progressed
• 37:01on the Menin inhibitor.
• 37:03And great, you know,
• 37:05what are they?
• 37:06And in fact the first one was this
• 37:08mutation 3 andine 349.
• 37:10So we dug Florian's data out and
• 37:13we're like holy cow,
• 37:14the patients are getting the same
• 37:16mutation that the base header screen
• 37:18had suggested they might get even though
• 37:21we weren't confident in our in our data
• 37:23to go ahead and start studying that.
• 37:25But Needless to say with that information,
• 37:27we started studying this in quite a bit
• 37:30of detail and we went to send X and
• 37:32got samples from a number of patients.
• 37:34And it looks like within two to
• 37:37three months about 40% of the
• 37:40patients had developed.
• 37:41They weren't in fluorid
• 37:42relapse or progression,
• 37:43but had developed a clone with
• 37:46this a min and mutation in them.
• 37:49That's what this is showing here and
• 37:50here is like and those mutations
• 37:52were not present at screening.
• 37:53So this is just a pie chart in
• 37:55red here showing you the size of
• 37:57the clone that has developed the
• 37:58min and mutation.
• 37:59So this is acquired selective
• 38:02mutational resistance to the men,
• 38:04an inhibitor which as most of
• 38:06you probably know is considered
• 38:08a validation of the therapeutic
• 38:10targeting the kinase world when this
• 38:12happens and essentially we think
• 38:14it's saying the same thing here and
• 38:16we found a both in patients with MLO
• 38:18rearranged an NPM one mutant AML.
• 38:20So this is the, if you will,
• 38:22the gold standard for that validation
• 38:24of a therapeutic target in patients
• 38:26that that that you put so much pressure
• 38:29on the target that the cancer mutates.
• 38:31It's such that it's no longer effective.
• 38:33I'll show you why in a minute.
• 38:35And of course as most of you know,
• 38:37we can have to have combinations
• 38:39anyway and we were able to show that
• 38:41we can do the same thing in PDX models.
• 38:43So we take our MLL rearranged
• 38:45or NPM one mutant models,
• 38:46treat them with in an inhibitor and
• 38:49in some cases but not all they will
• 38:52develop the mutations that we see in
• 38:55in the patients and mechanistically
• 38:57we we know how this is working.
• 38:59I won't go into all the details but
• 39:01this is just one chip seek experiment.
• 39:03In the wild type setting you can
• 39:05see Menin comes off of chromatin,
• 39:06this is chip seek increase in
• 39:08concentrations of the Menin inhibitor.
• 39:10But if you have mutated Menin in that
• 39:12cell line, it no longer comes off.
• 39:14And we know now biochemically it's
• 39:17because the binding affinity of the
• 39:19Menin inhibitor has been shifted
• 39:21significantly as a result of those mutations.
• 39:23And in fact,
• 39:24we know this now at the crystal
• 39:26structure level and we know
• 39:28exactly why that's the case.
• 39:29So here's revuminib bound to Menin.
• 39:32You can see over here on the right,
• 39:35these amino acids
• 39:39M327T349-G331, all these are mutations
• 39:40that have been found in patients.
• 39:44Interestingly enough,
• 39:45the wild type MLL protein does not
• 39:49use those amino acids to anchor
• 39:51and that's actually an important
• 39:53concept because if you develop
• 39:55the mutation that where men and
• 39:58the MLL can no longer interact,
• 40:00those cells won't survive that.
• 40:02So that that that's not an
• 40:03option for the cells.
• 40:04They have to mutate something that
• 40:06doesn't affect MLL but does affect
• 40:08the the binding of the inhibitor.
• 40:10And in fact that's exactly what they've done.
• 40:12They've mutated this region of men and
• 40:15that has plays no role in MLL binding.
• 40:18And the way that that happens is
• 40:20essentially right here the yellow
• 40:22is the the min inhibitor bound to
• 40:24wild type min and the purple is the
• 40:27min inhibitor bound to mutant min.
• 40:28And and you can see essentially what's
• 40:31called a steric clash which so the the,
• 40:33the min inhibitor is pushed out a
• 40:35little bit here because of these
• 40:37changes in the amino acid and that
• 40:40leads to a 10 to 100 fold decrease
• 40:42in affinity of this molecule.
• 40:44So this is I find this amazing
• 40:47because it's rare that you get to see
• 40:50molecularly the difference between
• 40:51response and resistance which is really
• 40:54essentially a few angstroms here of
• 40:57this Menon inhibitor binding to to Menon.
• 41:01So this we're we're continuing to work
• 41:04on mechanisms of resistance in the past
• 41:06five or last five or 10 minutes here.
• 41:08I'll tell you about some others,
• 41:11but just getting back to a little
• 41:13bit to that comment I made about
• 41:15dose escalation and stopping your
• 41:18dose escalation before you perhaps
• 41:20get to full potential efficacy.
• 41:22And now we see that we're developing or
• 41:25patients are developing mutations that
• 41:27all they do is shift the curve a little bit.
• 41:31It does make you wonder if you'd had a
• 41:33higher dose and a higher concentration
• 41:35earlier on if you might have prevented
• 41:38the cells from developing those mutations.
• 41:40So we with send X luckily providing
• 41:43us food now with varying amounts
• 41:45of the min an inhibitor.
• 41:47We were able to do a dose response
• 41:49experiment in a PDX model with increasing
• 41:52concentrations of the min an inhibitor.
• 41:54And you can see here that at
• 41:56the lowest concentration point,
• 41:58O3 3%, you see no response.
• 42:01At the minimal the medium concentration
• 42:03here you do see a response and the
• 42:06leukemias progress and the vast majority
• 42:08of them will have developed the mutation.
• 42:10If you then go threefold
• 42:12more of them in an inhibitor,
• 42:14you get a much longer response.
• 42:16In fact,
• 42:16maybe some of them here are cured of the
• 42:19disease and when the resistance occurs,
• 42:21it occurs without the min and mutations.
• 42:23So in fact,
• 42:24a higher concentration does at
• 42:26least in this model prevent the
• 42:28development of those mutations.
• 42:30So you can see if you stop your dose
• 42:32escalation right here because you're
• 42:34getting differentiation syndrome
• 42:35and somebody tells you you have to,
• 42:38you're actually setting up a
• 42:39situation where you're going to
• 42:41get acquired resistance mutations.
• 42:44Having said all that,
• 42:45we're still getting resistance way out
• 42:47here with the single agent at higher doses.
• 42:49So what's that all about and
• 42:52I'll quickly summarize this.
• 42:54Essentially what we're seeing here
• 42:55is that the leukemia cells and
• 42:57this is another phenomenon that's
• 42:59known in other settings now are
• 43:01not mutating the men and they're
• 43:03actually changing their state
• 43:04significantly to lead to a state that
• 43:07we don't understand completely yet,
• 43:09but where they're now no longer
• 43:11dependent on that Hawks niece program.
• 43:14And and in fact interestingly they
• 43:16look much more differentiated.
• 43:17The leukemias themselves almost
• 43:20look like monocytes in terms
• 43:22of their their flow cytometry,
• 43:24but definitely will transplant the
• 43:26disease from 1 bow to the next.
• 43:28So they're not monocytes,
• 43:30but so we're trying to understand
• 43:32this mechanism mode of adaptive
• 43:33resistance a little bit better.
• 43:35Now have developed a model,
• 43:37a cell line model for it and have
• 43:40developed some PDX models as well.
• 43:41But the cell line model actually
• 43:44lets us move to what everyone
• 43:47likes to do now which is a genome
• 43:49wide CRISPR screen to say OK,
• 43:51how did the dependence do the
• 43:53dependencies change when you
• 43:54go from one state to the next?
• 43:56And to summarize,
• 43:57a lot of analysis in fact they do
• 44:00appear to and this is on a subtle
• 44:03transferase CAT6A or MAZ which
• 44:05also rearranged rarely in some
• 44:07leukemias now becomes seems to become
• 44:11relevant in this in this setting.
• 44:13So here is just an experiment
• 44:15showing you that.
• 44:17So we developed resistant leukemia cells
• 44:20that don't have the MIN in mutation.
• 44:21They're resents to the MIN inhibitor.
• 44:23Here it is in blue.
• 44:24But if you treat with the cat
• 44:266A in this case guide,
• 44:28it re sensitizes the the cell
• 44:31line to the MIN inhibitor.
• 44:34But interestingly and interestingly enough,
• 44:36the CAT 6A by itself in the
• 44:38absence of the MIN inhibitor has
• 44:40a little bit of an effect.
• 44:41It's really something about the relationship
• 44:45between CAT6A and Menon that is important.
• 44:48So what's CAT6A?
• 44:49It's a histone has still transferase
• 44:51as well modifies histone H3
• 44:53on various lysine shown here.
• 44:55And if you do now chip seek in either O
• 44:58sensitive leukemia cell lines or resistant,
• 45:01the MLL Menon and CAT6A chip seek
• 45:06data looks very similar and in so here
• 45:08both in the sensitive or the resistant
• 45:10and this is just showing that more
• 45:12broadly so men and Catsix is there,
• 45:14it's on the scene and it becomes seems
• 45:17to become much more important when the
• 45:20cells adapt to the men and inhibitor.
• 45:23Needless to say,
• 45:24we're now doing the experiments to
• 45:26see if this combination in mice will
• 45:29reverse the resistance in patient samples.
• 45:33It won't reverse the resistance
• 45:35to the mutations,
• 45:36but it might reverse the
• 45:38resistance to the adapted form.
• 45:40But the men inhibitor works well enough,
• 45:42it's hard to generate that adaptive form,
• 45:44so it's taking us a little while.
• 45:47I'm going to skip this just for time's sake,
• 45:50but to and just to summarize saying that
• 45:54these complexes here, the .1 complex,
• 45:57the Super elongation complex,
• 45:58I've been talking about MLL and CAT6A.
• 46:02If you look broadly,
• 46:04some of you probably know that Broad
• 46:06Institute's been doing broad CRISPR
• 46:08screens on five 600 cancer cell lines,
• 46:10and they make all this data publicly
• 46:12available in many different ways.
• 46:14You can search that data.
• 46:15If you ask what genes have a
• 46:18similar dependency to Menin
• 46:20throughout all of cancer space,
• 46:23.1 is the next thing on the list.
• 46:26And then and then these other
• 46:28proteins here E&L which is part of
• 46:30the Super elongation complex CAT6.
• 46:33A point being these,
• 46:35it's clear that these complexes
• 46:37are working together somehow
• 46:39throughout many cancer cell lines,
• 46:41Leukemia for sure,
• 46:43but also probably others as well.
• 46:46And in fact that led us to this
• 46:50publication for actually for
• 46:51about a year and a half ago now.
• 46:54Matt Hemming,
• 46:56a paediatric or medical oncology
• 46:58fellow was interested in
• 47:00gastrointestinal stromal tumors
• 47:01and just did a genome wide CRISPR
• 47:04screen because that's how you start
• 47:06every project these days it seems.
• 47:08And in fact cat 6A was one of
• 47:10the top hits in that screen.
• 47:12And then we looked a little bit more in more
• 47:16detail .1 and Menin were in there as well.
• 47:18And the bottom line is,
• 47:19is it appears that this CAT6,
• 47:23A .1 Menin complex cooperation is
• 47:28important in this type of cancer as well.
• 47:32And we don't understand,
• 47:33you might ask why should ask why?
• 47:36We don't completely understand
• 47:37why it's the case.
• 47:39But it does appear that when we
• 47:42inhibit CAT6A and men and in cell
• 47:44lines or in mice or in PDX or
• 47:48xenograft models that a program
• 47:50that Matt had described earlier
• 47:52driven by transcription factors
• 47:54like one called hand one,
• 47:56which is known to be important
• 47:58for controlling lineage associated
• 48:00gene expression in this cancer,
• 48:03certain goes down pretty rapidly.
• 48:05So there's something about the
• 48:07developmental program and this
• 48:08cancer as well that seems to be
• 48:11dependent on these complexes,
• 48:12but you don't really see it as
• 48:15dramatically as in leukemia until you
• 48:17start to combine the the small molecules.
• 48:19Many will do a little bit of it,
• 48:20Mos cats, XA will do a little bit,
• 48:22but when you combine them,
• 48:23you really get a a dramatic response.
• 48:25So the point being that we're
• 48:27looking at this in leukemia,
• 48:28but we're starting to move into
• 48:30some other cancers as well to see if
• 48:34indeed these developmental regulators,
• 48:36if you will, might be relevant there.
• 48:39I just summarized this data and
• 48:42I'll end with this.
• 48:44Pfizer just published a paper
• 48:46about six months ago.
• 48:48They've now developed a clinical
• 48:50grade CAT6A inhibitor and in fact
• 48:52this small molecule is in phase
• 48:54one clinical trials in estrogen
• 48:57receptor positive breast cancer.
• 48:58And it looks like from this paper
• 49:01and we've now done a number of
• 49:03experiments as well that somehow
• 49:05Menon is supporting the ER driven
• 49:08gene expression program.
• 49:09And Needless to say, I mean sorry Cat 6A.
• 49:13Needless to say, we're now looking
• 49:14at this Cat 6A men in combination.
• 49:16And again as much as like that we saw
• 49:18in GIST, it looks like this the ER
• 49:21driven program is highly dependent on
• 49:23those two complexes and we're trying
• 49:25to work through that now as well.
• 49:28So to summarize what I've told you,
• 49:31the MEN in MLO complex is a relevant
• 49:34therapeutic target and snippet subset of
• 49:37AM LS and that acquired somatic mutations
• 49:40in men and are a mechanism of resistance,
• 49:43not the only mechanism of resistance
• 49:45but that clearly validates men and has
• 49:48a therapeutic target in this disease.
• 49:50We're continuing to work on the various
• 49:52types of resistance and we do think
• 49:54that rational combinations like men
• 49:56and CAT6A or men and other things with
• 49:58a lot going on in terms of trying to
• 50:01understand which combinations may
• 50:02prevent development of resistance.
• 50:03And perhaps the most exciting in the
• 50:05longer term is if we can understand
• 50:08where these mechanisms might be
• 50:09important even beyond leukemia.
• 50:11And I think they're going to
• 50:12be opportunities,
• 50:13but we certainly still have work to do to,
• 50:15to prove that.
• 50:16So I've talked much about the people
• 50:18in in the lab that have done the work.
• 50:21These are our collaborators at Dana
• 50:23Farber actually Nathaniel Gray now
• 50:25at Stanford and Ross I mentioned and
• 50:28Chang and Richard who work with Ross
• 50:31at MSK and some of our collaborators
• 50:34throughout HMS community.
• 50:35So thank you,
• 50:37happy to take any questions and
• 50:39thanks for thanks for staying.
• 50:41All
• 50:49right, absolutely spectacular grand
• 50:51rounds really going from basic science to
• 50:54the patient and back and forth and it's
• 50:56absolutely spectacular. Thank you. Yeah
• 50:59and and great talk. So I think
• 51:01the main issue and you know as a
• 51:04clinical investigator in my mind
• 51:05with all epigenetic therapies is,
• 51:07is the therapeutic window as you were
• 51:09saying like how do you actually disrupt
• 51:11translational or transcriptional
• 51:13programs that are relevant to the
• 51:15leukemia but not to the normal tissue.
• 51:18So for example with this index
• 51:20particular drug was this as a result
• 51:23of screening of thousands of molecules
• 51:25and because as you mentioned it,
• 51:27it seems like to disrupt only where
• 51:29it's relevant to the leukemia,
• 51:30but it's not disrupting the MLL
• 51:32interactions that are important for
• 51:34normal hematopoiesis and other functions.
• 51:36So that how did this transition happen?
• 51:38It's just a matter of luck or is
• 51:40it tons of screening of other yeah
• 51:42you know the old saying
• 51:43better lucky than good.
• 51:44I I think that that's what we found here,
• 51:48meaning that for some reason and
• 51:50we're looking into this men and is
• 51:53only critical for MLL wild type.
• 51:55Obviously this mechanism probably wasn't
• 51:58developed during evolution for MLL
• 52:00fusions to localize to certain loci.
• 52:03So MLL as I mentioned is a monstrous protein.
• 52:06It has many domains that combine chromatin.
• 52:09So it's very likely and there's some
• 52:11data to support this that different
• 52:13domains or different binding partners
• 52:16determine localization to different
• 52:18places throughout chromatin.
• 52:19And it just so happens in this it
• 52:21kind of was predicted by some of the
• 52:23early Cleary work that Menon was a
• 52:25unique dependency in these leukemias.
• 52:27Well that's because it's really
• 52:30intersecting exactly with the
• 52:34important MLL fusion driven targets.
• 52:37We don't know the molecular
• 52:39mechanism for that yet,
• 52:40but basically it's a long way of
• 52:42saying we think there's a multi
• 52:43valent interaction between MLL and
• 52:45chromatin and Menon is only important
• 52:47for a subset of that interaction.
• 52:50Scott, fantastic talk.
• 52:51And the the question about
• 52:53the solar cancer part,
• 52:54so you alluded to that you are it's
• 52:56great to see that you and other
• 52:58companies are looking into this aspect.
• 53:00So, so we know that oxygens are
• 53:02often deregulated in solar cancer
• 53:04as well in addition to leukemia.
• 53:06So what happens to men inhibitor,
• 53:09the men and MLL inhibitor monotherapies,
• 53:12do they have any efficacy in
• 53:14solar cancer or you have to
• 53:15really using combinations before
• 53:16you can see something happening?
• 53:18Yeah, it's good question.
• 53:20So actually it's a good point.
• 53:21There are a number of say subtypes of
• 53:24lung cancer that express HOX genes.
• 53:26We actually haven't looked
• 53:27at that probably should,
• 53:29but in the both in the gastrointestinal
• 53:32stromal tumors and the ER
• 53:34positive breast cancer cell lines,
• 53:36the men inhibitor will slow their growth.
• 53:40So they have some effect and that
• 53:42is it looks like through somehow
• 53:44modulating the ER driven program,
• 53:46but it's much more dramatic both
• 53:48the gene expression changes and
• 53:50the inhibition of proliferation if
• 53:52you combine the minute inhibitor
• 53:54and and the cat 6A inhibitor.
• 53:56So how that works, we don't,
• 53:58we don't completely understand yet.
• 54:01It's a way of saying and and predicting
• 54:04and hopefully get the word out
• 54:06before all the trials get shut down,
• 54:08that the single agents might
• 54:10have some activity,
• 54:11but I suspect they won't be home
• 54:13runs and the companies have to
• 54:15have the wherewithal to actually
• 54:17move forward to the combinations.
• 54:19And those of you who've done this
• 54:22before know that can be difficult.
• 54:23So we're going to try to get the
• 54:26word out that you should move
• 54:28the combinations quickly before
• 54:30people lose interest.
• 54:32There's a lot of psychology and
• 54:34sociology that goes into keeping
• 54:36the drug companies interested,
• 54:38so that's a little bit of a soapbox to
• 54:40say the single agents do something.
• 54:42The combination definitely looks better.
• 54:46Manoj, a great talk.
• 54:48My question is about the specificity
• 54:50of both the MLL fusion proteins and
• 54:52the NPM 1C that you alluded to.
• 54:53And Amar was also asking.
• 54:56So I think you probably worked on this
• 54:58on the cancer discovery latest paper,
• 55:00but most of them also seem to be
• 55:02overlapping with like say PRC 2
• 55:04targets or you know are there other
• 55:06mechanisms you think are relevant to
• 55:08why these are so tightly overlapping
• 55:10the fusion proteins and the
• 55:12yeah, so, so it's a good point.
• 55:14So they do overlap significantly with
• 55:16PRC 2 targets and you know as you may
• 55:20remember the the this has been predicted
• 55:23for 3 decades from the Drosophila work.
• 55:25The initial Drosophila work show
• 55:28Polycom and Trithorax actually are
• 55:30known to be genetically genetic
• 55:32antagonists of one another and the the
• 55:35trithorax slash MLL complex controls
• 55:38developmental genes that the PRC two
• 55:40or Polycom complex wants to shut off.
• 55:43So in absolutely this is A these
• 55:46proteins complexes MLL probably
• 55:49Catsix A are inventing.
• 55:50We've shown this in some other settings
• 55:53are preventing the Polycom complexes from
• 55:55coming in and repressing gene expression.
• 55:57So the the way we think that this
• 55:59is working is during hematopoietic
• 56:00development as you go from stem cells to
• 56:03progenitors to fully developed myeloid cells,
• 56:06the Polycom complex at least for a
• 56:08subset of developmental loci are
• 56:10shutting those programs off and the
• 56:12MLL fusion won't let them do that.
• 56:14So they're antagonizing and then
• 56:17Newt 98 fusions and probably NPM
• 56:19one are antagonizing PRC 2 section.
• 56:22Yeah,
• 56:23awesome. I'm going to bring
• 56:24it over to you in a second.
• 56:26We have an online question which I
• 56:27think you probably partially answered
• 56:29and that is what is the mechanism of
• 56:31gene specific targeting of MLL EF9
• 56:33and similarly what you think is the
• 56:35underlying mechanism for the gene target
• 56:37specificity of men and inhibitors.
• 56:39So for the online person, yeah.
• 56:40So it's a good, it's a very good question.
• 56:43We do it. I don't the bottom line is,
• 56:45is we don't know the answer
• 56:46to the second part of that.
• 56:47I mean that's the that's at the moment.
• 56:50Probably the most critical question
• 56:52is why is it that Menon's only
• 56:54important for localization of the
• 56:56MLL compacts to certain loci.
• 56:58So Needless to say,
• 56:59we're looking at various aspects
• 57:00of those loci to try to understand
• 57:03what that's what that's all about.
• 57:04MLL targeting to chromatin broadly has many,
• 57:07probably has many mechanisms,
• 57:09some of its direct there's a domain
• 57:11on MLL that binds to what's called
• 57:13a CPG island which is upstream of
• 57:16many transcriptional start sites.
• 57:17Menon plays a role.
• 57:19There are other accessory proteins
• 57:20that play roles.
• 57:21So I think the cell has just given
• 57:23itself many options to figure out
• 57:25where to put MLL and and each of
• 57:28those mechanisms slightly different.
• 57:31We have a trainee question.
• 57:34So my name is trainee,
• 57:37I just wanted to follow my name used
• 57:38to be that at some point you graduated.
• 57:40I just want to follow up on your
• 57:42comment about combination therapies.
• 57:44So I specifically wanted to ask is there
• 57:46any thought that men and inhibition
• 57:47could convert these resistant like
• 57:49subtypes especially in the pediatric
• 57:50setting to a chemosensitive form if
• 57:52there's thought of combining with
• 57:54chemo to then re sensitize them and
• 57:56potentially cure those patients. Yeah,
• 57:57it's it's a good short answer is
• 58:00we don't know the answer to that.
• 58:02I I think there's a lot to be learned
• 58:05and the beauty of having now multiple
• 58:08small molecule selective small molecules,
• 58:11we can do those types of experiments,
• 58:14but the short answer is we
• 58:16haven't haven't gotten there yet.
• 58:20Yeah, great talk.
• 58:21Have you seen any phenotypic
• 58:23differences in the fusion
• 58:24partners with MML or MLL?
• 58:26You mentioned there's you know,
• 58:27100 different ones.
• 58:27Do they all have the same kind of,
• 58:29you know, break points?
• 58:30Does it change expression?
• 58:31Do you see any variability
• 58:32in the kind of those fusion
• 58:33partners? Yeah, it's a good question
• 58:36that's been asked for many decades
• 58:38and not been answered very well,
• 58:41at least in patient samples because
• 58:42it's hard to get enough patient
• 58:44samples of these subtypes to to ever
• 58:46really do that experiment. Well,
• 58:49at least for the fusion,
• 58:51the different fusion AM LS we've
• 58:53assessed and that have been
• 58:54assessed in patients, it doesn't.
• 58:56It's not clear that the fusion partner
• 58:58is determining men and responsiveness.
• 59:02Is the fusion partner influencing
• 59:04the phenotype of the leukemia?
• 59:06I think that question still still
• 59:10open and there's aren't enough good
• 59:12models to really answer that question.
• 59:18If you delete the at least for AF9,
• 59:21if you delete the C turn much of the AF9,
• 59:23it will no longer be transforming.
• 59:25So that fusion partner is important
• 59:29and and in in the AF9 setting,
• 59:31we think it's important because
• 59:32that's the anchor to drop to pull
• 59:34all those other complexes in.
• 59:35But it's that's a nice simple answer.
• 59:37It's not that simple because
• 59:39some of the fusion proteins
• 59:40don't bind to those complexes.
• 59:42So what they're doing is, is less clear,
• 59:47beautiful talk. Thank you.
• 59:48Obviously there are lots of
• 59:50other chromatin complexes,
• 59:52switch, sniff, polychrome, etcetera.
• 59:54Any any thoughts on those?
• 59:56Are you looking at any exploring
• 59:58any of those other chromatin?
• 01:00:02Yeah, accessibility.
• 01:00:04Epigenetic complexes?
• 01:00:05Sure. So we have over time looked at
• 01:00:11the Polycom complex mostly in leukemia
• 01:00:15and it for whatever reason and this is
• 01:00:18going to be right contradictory to how
• 01:00:19I answered one of my previous questions.
• 01:00:21The Polycom complex does seem
• 01:00:24to be important in the continued
• 01:00:27proliferation of many types of leukemia.
• 01:00:30How that's working and why hard to know,
• 01:00:34but the small molecule PRC 2
• 01:00:37inhibitors or or EZH 2 inhibitors
• 01:00:40don't have tremendous activity.
• 01:00:42So whether or not it's the enzymatic
• 01:00:44activity versus some other part of the
• 01:00:47complex at least in leukemia anyway,
• 01:00:49I don't know.
• 01:00:50And I think that probably it brings
• 01:00:53up a good point that and we've
• 01:00:56done this in the past as well,
• 01:00:57but we have to be careful about
• 01:00:59the thought process that enzymatic
• 01:01:02inhibition of a protein in one of
• 01:01:04these complexes is the same thing as
• 01:01:06complete interactivation of the protein.
• 01:01:08It's not and we now seen that
• 01:01:09many different times.
• 01:01:10So the enzymatic part of the
• 01:01:13proteins is important,
• 01:01:14but there's probably a structural
• 01:01:16component to this that when you
• 01:01:18take the protein completely out and
• 01:01:19this is the same way for EZH 2 the
• 01:01:22the changes are much more dramatic.
• 01:01:23So it gets to the question as to what
• 01:01:25his some modifications are doing and
• 01:01:27that gets to even more deep and complicated.
• 01:01:30But so long answer to yes,
• 01:01:32we've looked at the complexes,
• 01:01:33we haven't looked much at the
• 01:01:36chromatin remodeling complexes.
• 01:01:37Segal Kadosh who some of you may know is
• 01:01:40at Dana Farber and we just let her do that.
• 01:01:42She they're doing a lot in that regard.
• 01:01:46I'm sure they're playing a role here.
• 01:01:48What but how and what we don't know.
• 01:01:51We have one last question and after this
• 01:01:54is actually a session for the trainees,
• 01:01:56very private was Doctor Armstrong.
• 01:01:59Yeah, one last question.
• 01:02:00This is actually related
• 01:02:01to the fusion partners,
• 01:02:03but as you know we often get,
• 01:02:05you know when we do the genetech
• 01:02:07sequencing we get MLL deletions,
• 01:02:08MLL mutations,
• 01:02:09sometimes Trisom 11 or you know PDD.
• 01:02:14And is your sense that beyond
• 01:02:16the fusion MLL Fusion's those
• 01:02:19alterations also have susceptibility
• 01:02:21to an inhibition or yeah,
• 01:02:23as you probably know since you've been
• 01:02:25important in running some of the trials,
• 01:02:27the MLLPTD subtype of AML for
• 01:02:31some of the trials is included.
• 01:02:33There's been an assumption to some extent
• 01:02:36that they should be responsive the PD.
• 01:02:38So we have generated some MLL.
• 01:02:39So MLLPTDS are actually a partial
• 01:02:41tandem duplication of a part of the
• 01:02:44MLL protein and that subset of AML is
• 01:02:46incredibly difficult to treat and it's
• 01:02:48also found in MD's those mutations,
• 01:02:52but that almost tells you immediately
• 01:02:54it's also found in MDSMLL.
• 01:02:56Rearrangements are not found in MDS that
• 01:02:58they're probably something different
• 01:02:59going on in the minute inhibitor
• 01:03:01doesn't have at least in our PDX
• 01:03:03models the same type of activity in
• 01:03:04those PDX as it does MLL rearranged.
• 01:03:07So I think it gets back to this issue
• 01:03:10that we've been discussing that in
• 01:03:12that setting either because of that
• 01:03:15duplication or otherwise the that MLL
• 01:03:18Oncoprotein sticks on chromatin through
• 01:03:20a different mechanism than Menon.
• 01:03:22So we're got a project looking
• 01:03:25at exactly that,
• 01:03:26trying to understand what that is.
• 01:03:28We haven't looked at the deletions.
• 01:03:30I wouldn't predict they would be.