Richard Frisbee Memorial Lecture/Mechanisms of B-cell selection as therapeutic targets

January 06, 2021

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Yale Cancer Center Grand Rounds | January 5, 2021

Markus Muschen, MD, PhD

ID6053

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00:00First grand rounds of 2021 and once again
00:03continuing our our mission doing this.
00:06Whether it be in person or by zoom,
00:09we're sticking to the schedule
00:11and making sure that we we advance
00:14the mission of disseminating
00:16and knowledge through this form.
00:19Today is a really special occasion
00:22because it represents something I
00:24think we all look forward to the
00:27annual Frisbee lectureship and
00:28actually to begin the forum I I'm
00:31going to turn it over to Ed Snider,
00:34Doctor Schneider, as you know,
00:36is a professor of laboratory medicine.
00:41Can't send director for.
00:42For membership director of
00:43membership for the Cancer Center,
00:45Leader of blood banking.
00:47Certainly has done a lot of work
00:49over the years in that domain and
00:52has really been the Shepherd for this
00:54lectureship for the past 18 years.
00:56And I want to turn it over to Ed to
00:59share some perspectives and as well
01:02introduce members of the family.
01:05Thank you very much Charlie to pleasure to
01:08introduce the Frisbees for this lectureship.
01:10Rick and Chris Frisby, son Richie,
01:12developed leukemia as a young man that's
01:15a teenager and was the first bone marrow
01:18transplant done in the Connecticut.
01:19It was done by the late Jill Rappaport,
01:22who was running the program at the time.
01:26Richie did not survive first
01:28transplant failed and didn't
01:30survive to have a second transplant,
01:32which was to be his sister.
01:34And in honor of him, in his memory,
01:37the family rich Rick and Christine
01:40set up the Frisby Foundation.
01:42So in 1990 they've given millions of dollars
01:46in Cancer Research and cancer education.
01:49They established the first stem
01:51cell processing lab at Yale,
01:53New Haven long before Smilow had the
01:56first brick lay down the foundation and
01:59that was the precursor of the HCT lab,
02:03which is currently supporting SMILOW in
02:05through Department of Laboratory Medicine.
02:07Went by Diet Doctor Diane Kraus.
02:10This lectureship was established 18
02:12years ago and the current speaker
02:15doctor Marcus Motion is therefore
02:17the 18th speaker and we welcome him.
02:20And we would like to turn it over to
02:22Christine to do or say a few words.
02:24Of I'll be brief,
02:26I just wanted to really thank Yale Dr
02:28Mnuchin, and in particular at Snyder,
02:30who's been very close to us for many
02:33many years and been very supportive
02:35of the foundation and the work we do.
02:37And we love this lectureship. We don't.
02:40Foundations must smaller now.
02:41We don't do that many things,
02:43but this is one thing that we continue doing,
02:46and we're going to continue to fund
02:48this for years to come because we
02:50think it's just very rewarding.
02:52So thank you very much, Ed.
02:54Doctor Fox and Marcus as well at
02:58the Commission.
02:59Thank you Rick and Chris for all
03:01that you've done and for so much
03:03of the work that is being done for
03:06the patients that smile.
03:07Oh well,
03:07we can thank you for for setting
03:09the foundation for this,
03:11so I'll now turn it over to Doctor
03:13Fuchs to introduce our 18th.
03:15Frisbee lecturer Doctor Marcus machine.
03:18It thank you, Ann and Rick and Christine,
03:21thank you for your continued
03:24support of this important
03:25leadership over the past 18 years.
03:27I think this is really been
03:30a wonderful tradition.
03:31Becausw, what this lectureship
03:33has done is brought to Yale.
03:36Really rich cadre of innovators
03:39in developing understanding an
03:41new approaches to human logic,
03:44religion sees which I think is a fitting
03:48legacy and this year's annual Frisbee
03:51lecture is no exception to that.
03:54Impressive in August,
03:56List of lecturers.
03:58Doctor Marcus musician was previously
04:01the chair of the Department of
04:04Systems Biology and the Lee Professor.
04:07Oh, at the City of Hope Cancer Center,
04:11as well as the associate director
04:14of Basic Science an we were very
04:16privileged in the fall of 2020 to bring
04:20and recruit Doctor Mission to Yale.
04:23As our inaugural director for the
04:25Center of Molecular and Cellular
04:27Oncology at the Yale Cancer Center
04:30and Smilow Cancer Hospital as well
04:33as the author and Isabel Bunker,
04:35Professor of Medicine, focused in hematology.
04:38Doctor Mission,
04:39trained in hematology oncology.
04:41His work as his training was both
04:44in the biology of human like you
04:48put in season Immunobiology.
04:50And frankly,
04:51over the past decade or really longer,
04:54he has been a leading innovator
04:57in understanding the evolution of
05:00B cell malignancy's understanding
05:02biology in terms of delivering
05:05new approaches to drug discovery
05:07in these cancers,
05:09Ann and really now advancing that
05:11beyond in terms of the immunobiology
05:14immunotherapy that would be available
05:17for lymphomas and human logic legacies.
05:21Most notably narrow,
05:22even in terms of novel car T therapies,
05:26and I think all of that really
05:28speaks to in many respects what's so
05:31relevant for the Frisby lectureship
05:33Marcus for his many accomplishments
05:35and impressive publication record
05:37has been received countless awards.
05:40I I wouldn't want to take too much
05:43time annunciating all of them,
05:45but they include the NCI outstanding
05:48Investigator award.
05:49Howard, You scholar award, leukemia,
05:51Lymphoma Society scholar award.
05:53Welcome Trust scholar award.
05:55Among many other awards that
05:57recognize his accomplishment,
05:59an innovative and record of innovation and
06:02accomplishment across all of these cancers.
06:05So it's really a pleasure to introduce
06:08Marcus and an in many respects.
06:11Welcome to the faculty of Ovvio
06:15Cancer Center. Orchis, thank you.
06:20Thank you, I'm not sharing my screen.
06:46So first of all, I would like
06:48to thank the Frisby family and
06:51Charlie an ad for me being here.
06:55And since I came to your Cancer
06:58Center last for like I came to
07:01experience that many of us who
07:03are devoted to the cause of.
07:05Now titled can try to leukemia
07:08and leukemia and young tires.
07:10That legacy of Richard D.
07:13Frisbie has inspired many of us
07:15and and today I would like to text
07:19opportunity to present a few new
07:21findings from our lab that over
07:24the past recent years have led to
07:27a new concept that I hope will
07:30help us in the future to treat this
07:34disease is more efficiently than.
07:37We were able to do in the past.
07:39And many of our try to leukemias are
07:43actually derived from lymphocytes
07:44and is a leukemia.
07:46In fact,
07:47represents the most frequent type of
07:50cancer in children and young adults,
07:52and one potential reason for that
07:55is that B cells during the early
07:57development have to go through a
08:00series of genetic modifications
08:02in error combination class,
08:04switching hypermutation with the end
08:07goal for these cells protect us by.
08:10Generating Pi affinity antibodies
08:11and this is a cartoon here from
08:141905 drawn by Powell, Ellie.
08:16She was sitting here in his office
08:19and for those reasons B cells
08:22are an extremely high risk for
08:24malignant transformation.
08:26Actually,
08:27500 times higher than any other somatic cell,
08:30and for this reason and also becausw,
08:33humans can actually live without
08:35the lymphocytes for quite some time.
08:38And we developed a research program
08:41that is centered on specific
08:43vulnerabilities of this very cell type.
08:47So as cancer researchers,
08:48we are always looking for vulnerabilities,
08:51and in this case we're looking
08:54for vulnerabilities.
08:55That are intrinsically encoded in
08:57the nature of the sales making
08:59antibodies and are selected and
09:01ANVISA selection is a scene that
09:04that that I hope I will be able
09:06to present to you on the occasion
09:10of this lecture.
09:11And the reason is that just by the
09:14random nature of recombination,
09:17events of antibody encoding
09:19molecules vast majority,
09:20about 75% of oil newly generated
09:23visas are initially autoreactive,
09:24meaning that their directed against
09:27himself and in these cells have to
09:30be removed from the repertoire,
09:32and this means they have to be
09:35powerful mechanisms in place to
09:38normal development to destroy and
09:40delete yourself from the repertoire.
09:43And although arching theme for
09:45our research in recent years is,
09:47can we actually leverage these
09:49mechanisms that are indeed in the
09:52life and selection and development
09:54of normally lymphocytes for the
09:56treatment of Pisa, leukemia and lymphoma?
10:00So in.
10:00Fact,
10:01the principle of the cell selection
10:03is driven by signals from the B cell
10:06receptor or surface immunolabeling,
10:08and we like to think of this.
10:11Like I said, Goldilocks principle.
10:14Because only if the signal strength
10:17that is elicited from this unit
10:19here is just right,
10:21then this says receive a positive
10:23signal and proliferate and survive,
10:25and this is usually the case
10:28when we have a
10:29balance between activation signals.
10:33Namely, kinase and phosphatase is.
10:36That achieve this balance.
10:37So if the signal is too weak,
10:40for instance,
10:41gives the first parties are just wrong,
10:43or the receptor itself is not
10:46functional and he says die by neglect.
10:48Now we're focusing here on
10:50the other end of the scale,
10:52where the signals overwhelmingly strong,
10:54which is typically the case when this
10:56receptor he is engaged by self antigen,
10:59meaning that these receptors
11:01are out reactive.
11:02And these cells could give rise to autoimmune
11:06disease and have to be eliminated.
11:08And so this principle is not only
11:12relevant to normal B cell evelopment.
11:17In this cartoon here from
11:19a recent publication,
11:20shows that in transformed B cells and
11:23leukemia and lymphoma the signaling
11:25pathway downstream of the beasts
11:27are receptors engaged permutations.
11:29Every step of the way.
11:32And so today I would like to divide
11:34my talk in three areas where we gain
11:38information of how we can leverage
11:41selection for therapeutic benefits.
11:43One comes from inside that we glean
11:46from mutations and deletions and visa
11:49humorists such as leukemia and lymphoma.
11:51Then inside some clinical trials.
11:54So we collaborate with large
11:56clinical trial groups in the United
11:58States and internationally and
12:00look for predictors of clinical.
12:02Outcomes and what we can learn in
12:05terms of therapeutic targeting options.
12:08And then finally how these complicated
12:11oncogenic signaling pathways
12:13intersect and how we can leverage
12:16these interactions again to undermine
12:18oncogenic signaling in these diseases.
12:21So in the first part I'm going to
12:24talk about genetic lesions and what
12:26we have done here based on mutation
12:29data from cosmic and other sources
12:32assembled a set of more than 5 million
12:36somatic mutations in 39 different
12:38types of cancer and look at these
12:41mutations from the angle of whether
12:44the mutation introduce a replacement.
12:47Or effect according capacity of the
12:49gene or whether they are silent,
12:51meaning they are not selected for.
12:53And then in all these diseases we
12:56rank the mutations based on these
12:58replacement over silent ratios.
13:00And we do that in a way that ranks US
13:04based on the cell specific ratios.
13:08We end up with typically mutated genes,
13:10and these are widely known in B cell
13:13tumors like my D8820 and so forth,
13:15But was interesting to us that here
13:18at the top of the list they actually.
13:22Molecules in the PS3 kindness
13:24pass visit our frequently mutated
13:27throughout cancer,
13:28but are unexpectedly spirit were
13:30exempted from in B cell tumors,
13:33and this activating mutations
13:36of the PS3 kinase pathway.
13:39And P-10 and then ship one
13:41night in inventory.
13:42Phosphatases in this pathway.
13:43And this is the catalytic subunit
13:46of peers with kindness itself.
13:48So we studied this in multiple
13:50different directions,
13:51but I would like to focus your
13:54content because it was just such
13:57a subset of striking example and
13:59then as we know for a long time,
14:02pretend deletions and mutations widely
14:04occur throughout many cancer types,
14:06but in 925 cases of B cell image leukemia,
14:09we didn't find any of these mutations.
14:12Again, highlighting the
14:14specificity of this mechanism.
14:16And pretending opposers peers
14:19to kindness signaling by.
14:22I am targeting Pep 3 which is a
14:24central initiator of the PS3 kinase
14:27signaling pathway and I will come
14:29to that back later in my talk.
14:31And to study hyperactivation of the PS3
14:34kindness pathway by dilution of 10,
14:36we develop the mouse model in our lab
14:39based on conditional deletion of the
14:42P 10 gene in our leukemia model and
14:46unlike what we know and solid tumors,
14:49conditional deletion of P.
14:5010 result in Rapid City S of leukemia
14:54cells and if we change the sequence and
14:571st delete return a normal B cells and
15:01then bring in transforming Uncle gene.
15:03Then we basically compromise malignant
15:06transformation and then most importantly,
15:08when we wait for leukemia to
15:10establish and miles at bear these
15:12tumors and then delete speech engine,
15:15this leads to remission and the mice
15:18survive for indefinite periods of time.
15:21We also confirmed that the biochemistry
15:23or the premise here is correct because
15:26we actually do see increased activity,
15:29increased output of the PSC kindness
15:32pathway by increased phosphorylation of AKT.
15:35And most importantly,
15:36we used inhibitors that block the PS3
15:39kinase signaling pathways at multiple levels.
15:42Here,
15:42activation of PS3 kinese bicec using the
15:45sick kinase inhibitor and it's platinum.
15:48BKM 120 is a pan PSD.
15:51Kindness never turn easy.
15:53D 53 E 63 inhibits AKT and all
15:56three of them have in common.
15:59That's actually rescuer protect
16:01leukemia cells from sad as that
16:04would otherwise be in use.
16:06Dapon deletion of the P-10 phosphatase?
16:08So this doesn't mean that these
16:11compounds are counterproductive in
16:12leukemia because actually quite useful.
16:15Our interpretation of this
16:17unexpected result is that.
16:19I was introduction of those inhibitors
16:21restores signaling equilibrium again,
16:23the Goldilocks principle
16:24that these cells need,
16:26whereas deletion of the 10 alone here
16:29introduce a drastic perturbation
16:31which engages negative selection
16:32just as it does for the elimination
16:36of Hartree active users.
16:40We are hopeful that these ideas
16:43these concepts will eventually
16:45make their way into the clinic,
16:47and as an early indication that
16:49that might indeed be the case,
16:52I'm showing you two promising
16:54preclinical results in our lab,
16:56both based on small molecule inhibitors
16:58of these key fast watch cases, namely,
17:01ship one inhibited by 3A amino color stain,
17:04and then pretend which also is.
17:08Target if it was a small molecule
17:11inhibitor and both of them have
17:14desirable on target activity
17:16biochemically and importantly,
17:18their chief Disease Control or disease
17:21burden control for long periods of time,
17:24and she's,
17:25extension or prolongation of overall
17:28survival of mice that bear patient arrives
17:31in a graphs from B cell image leukemia cells.
17:36So we're hoping that this approach
17:39can be developed further in and
17:41that some of these compounds will
17:43make it into the clinical arena.
17:46Now the central premise of this
17:49idea is that this is a mechanism.
17:52Negative selection.
17:53Removal of art reactive says that is
17:56uniquely important in B lymphocytes
17:58and to test this premise we performed
18:01a reprogramming experience.
18:06And in which we should use the VPI flower,
18:09just a transcription factor that can
18:12transform besides into macrophages.
18:13And this is shown here besides expressed in
18:1719 and this marker here is lost overtime
18:20after induction and in favor of Mach one,
18:23which is a macrophage marker.
18:26And indeed, after sometimes you say it,
18:28start to crawl around on the bottom
18:31LCS dishes and like macrophages and
18:33can even phagocytose and importantly.
18:36Coming back to our hypothesis,
18:38if you genetically delete P.
18:4010, Even though genetically identical.
18:43The reprogramming from B to Milo to be 2.
18:47Macrophage fade almost entirely
18:49removes the sensitivity of these cells
18:52to removal of source phosphatases,
18:54which makes sense because fact
18:56macrophages don't make autoantibodies,
18:58there's no need for macrophages to be
19:01negatively selected as beast cells are.
19:03So this gives us confidence that this is
19:07a real mechanism that is reflective of
19:10the nature of the immune system to purge.
19:14Attractive sales and that is possible to
19:17selectively target this vulnerability
19:19in B cell tumors.
19:21Now in this work was done by Gen.
19:24John Shannon, our lab,
19:26and when he worried that this is not
19:28just counterintuitive because you
19:30essentially doing the opposite from what
19:33everyone else is doing in this field,
19:35namely by instead of inhibiting kinases.
19:37VR Pro activating kinases.
19:39But most importantly,
19:40what was worried about what happens
19:42if he hyper activate kinases for
19:44long periods of time,
19:45because that in itself could be dangerous,
19:48so he did an experiment to figure out
19:51what is the shortest period of time.
19:54Home to commit Visa is to say this and he
19:57did this with an engineered hyperactive.
20:00Formosa sick kinase.
20:02Labeled here as GFP.
20:04If you bring in this hyper active
20:07kinase in the presence of stickiness,
20:09inhibitors of GFP labeled cells
20:11remain constant,
20:12'cause there's no hyperactivation
20:13of the pathway.
20:15Now,
20:15if we wash out the inhibitor
20:17cells as expected, rapidly die,
20:19and he found that if there's
20:21a lapse of just three hours,
20:24so removal of simulator for three hours
20:26and then adding it right back that
20:29already is sufficient to commit the sales,
20:32that will be our goal going forward too.
20:35Target is short.
20:37Strong exposure to hyperactivation
20:39probably was click or only dated compounds
20:42that have a short plasma half life.
20:45Now in the second part of my talk,
20:48I'm going to give you 2 examples of how
20:51we can learn from information within
20:54clinical trials and gene expression,
20:57annotation related to outcome.
20:59So what approach is based on
21:02microarray data that we obtain?
21:04Collaboration was a clinical study.
21:06Groups and much of this works
21:10also publicly available.
21:11And for each of these micro area
21:13probe sets that measure expression
21:15of individual transcripts,
21:17we divide the patient courts and
21:19the two groups based on higher than
21:21median versus lower than median
21:23expression in these clinical trials.
21:25And then we asked the question,
21:28is there a difference between those two
21:30groups in terms of clinical outcome?
21:33And if the outcome is more favor
21:35we have here
21:37a blue annotation and its outcome is
21:39more poor, shorter overall survival.
21:41For instance, we have a red annotation
21:44and if the group this heat map based
21:46on the site specific annotations we
21:49come up with a list of genes that
21:52became interesting to us and he at
21:54the very top is 1 molecule that I'm
21:57going to spend the next couple minutes
21:59on the L2 receptor Alpha chain,
22:02also known as C25.
22:06So that was a bit unexpected.
22:09Becausw CD 25 is known as one of
22:12the three chains of the L2 receptor.
22:15Anna typically pairs with the better
22:17chain in the gamma chain to form a
22:20trimeric receptor, and this was step
22:22was active on T cells and in cases.
22:26And it's also important for
22:28formation of regulatory T cells or T.
22:31Rex, and therefore therefore
22:33important to prevent autoimmunity.
22:34Again, important to be self selection.
22:37And here I'm showing you that.
22:41Then if you look at individual visa diseases,
22:45pediatric B cell, leukemia,
22:47CLL, Podiatry, pizza, leukemia,
22:49mantle cell lymphoma,
22:50we see consistent pattern that
22:53the lower half of expression
22:56is related to better outcome.
22:58The top half towards outcome.
23:02The other reason we became interested
23:04is that if you bring in Uncle Gene,
23:07said drive, leukemia,
23:08lymphoma like these are able or LMP 2A.
23:10This leads to upregulation
23:12of C25 on the cell surface.
23:15And also it seems to play a role
23:17in resale developmented save.
23:19So he profile here see 25 M on A levels.
23:24Over the course of the sale
23:26evelopment we find here is striking
23:28peak and the so called Faction D.
23:31And that's interesting,
23:32because if you look at C25 knockout mice,
23:34which we did in our lab.
23:37Compare this to the wild type
23:39animals and look at these fractions
23:41we see here is fraction D.
23:43But distractions entirely missing in the
23:46knockout mice and can also see this here.
23:48This is a defect here.
23:50We still don't know what this actually means,
23:53but we also find that later in
23:56development he says actually are
23:58over represented in fraction F.
24:00So our initial hypothesis was this might
24:05reflect previously unrecognized role
24:08of IL two signaling in B cells and.
24:11I mean,
24:12so we repeated this experiment with
24:15mice that have intact City 25,
24:17but are lacking the L2 cytokine.
24:20But contrary to our hypothesis fraction,
24:22D&F are just fine and be so developmen
24:26is completely unperturbed in these mice.
24:29And I'll see you in this proximity
24:32ligation analysis,
24:32we find that C25 does actually
24:34not bind to any of those other
24:37change of the L2 receptor and does
24:39not respond to iron tools.
24:41So it's not true that C.
24:4325 is in any way related to I
24:46L2 signaling and visas instead.
24:49We found in our proximity ligation assay,
24:52said City 25 associate itself.
24:54With a signal image chain of
24:57the B cell receptor,
24:58which again is responsible for the
25:01Goldilocks principle to keep intact
25:03and equilibrium and intermediate
25:04ram of signaling intensity.
25:06And that's the case in resting B
25:09cells where prices are even more so
25:11the case after the visa receptor was
25:14stimulated with an anti IG M antibody.
25:17This is actually ongoing work in our lab,
25:20by Jay,
25:21wrongly with a research scientist
25:24in my group.
25:25And what he found is actually their CIA.
25:2825 negatively regulates B cell
25:31activation and in the absence of CD 25.
25:34Miles,
25:35develop spontaneous germinal center,
25:37so even without any immunization,
25:39these B cells are autoreactive.
25:42Their escape negative selection
25:44and therefore more attractive,
25:47spontaneous germinal centers
25:49that are antigen independent.
25:51The other observation here was that if
25:55Jerome deleted 325 in human lymphoma sales,
25:58they undergo a particular pattern
26:01of autonomous calcium signaling.
26:03They have autonomous activation.
26:05Do sales are proliferating very fast,
26:08but also for short half life and I quickly,
26:12which is reflected here by
26:14expression of PG restore parenting.
26:17These cells are.
26:18Just easily exhausted and
26:20in that competitive fitness,
26:23so we confirmed this here in a
26:27leukemia model whereby we transformed.
26:3025 mouse cells with the flux see 25
26:34every year and then after activation
26:37of query is illusia CD 25 expression
26:40on the surface and then soon after
26:44this says disappear from culture.
26:46They failed to form any colonies that
26:50cannot initiate leukemia and mice
26:52that bears also leukemias recover and
26:56survive for indefinite periods of time.
26:58Now, Interestingly,
26:59and that's coming back to
27:01signaling feedback control,
27:03we found that upon collisional
27:05City 25 in a similar way like
27:08deletion of P-10 and ship one,
27:11we see that the balance of ether
27:14receptor signaling strength.
27:16It's lost cause we have hyper
27:19activation of kinase substrates
27:21downstream of the visa receptor,
27:23including sick and then loss of phosphatase
27:27activity markers for P-10 and ship one.
27:31So we think those phenomena
27:33might actually be related,
27:34but CD 25 plays a role in maintaining
27:37the Goldilocks principle by
27:40regulating kinases and phosphatases.
27:42Now,
27:42how is this possible?
27:44So the tail of CD 25 is very short
27:47here and it's just 13 amino acids and.
27:51So we looked at what City 25 might bind to.
27:55How does it interact with the
27:58cytoplasmic tail and as a negative
28:00control and using this for a
28:02lot of different experiments,
28:05we introduce a mutation of the
28:07central Seren residue which
28:09destroys the main protein kinase.
28:12He better consensus motive.
28:14And here we are using a bio ID
28:18approach which is based on fusions
28:21between the CD 25 tail and puree,
28:24which is a bacterial biotin ligase
28:27which attaches bio tends to approximate
28:30protein space on the mound and the proximity.
28:34And I,
28:35as expected,
28:36we found that two phosphatases
28:38ship one and PTPN 6 here.
28:41Are in proximity of the tail of
28:43CD 25 and this is not the case.
28:46The tail here is mutated.
28:49And this is also confirmed here in
28:52a more traditional experiment based
28:54on pull down and quiet peace or
28:57ship 1P-10 and PTPN 6 can or bind.
29:00But binding this weekend or entirely
29:03lost when the stay here is mutated.
29:06So in terms of function,
29:08this could be confirmed that indeed see
29:1125 functioned as a powerful negative
29:13regulator of signaling strength.
29:15So if he abusively activate while
29:17types unify, we can block the kite.
29:20Some signal here that would
29:22otherwise be elicited.
29:23So he expresses the 25
29:25and it's wild type form.
29:27The signal is delayed and
29:29almost entirely lost if they
29:31express the mutant.
29:32This depression can still be seen,
29:35but it's much less compared
29:37to the wild type form.
29:40And then in terms of leukemia,
29:42survival and growth be used here,
29:45cameras between the
29:47extracellular part of CD 19,
29:49which is a B cell specific
29:52transmembrane protein and tale of
29:55225 users via type or is mutant
29:57and wild type form can rescue.
30:00Survival of leukemia cells but
30:03seven 268 a mutant cannots again
30:06showing that ability to recruit
30:09phosphatases to negatively
30:10regulate signaling is important for
30:13survival of these leukemia cells.
30:16So we have modeled the interaction
30:19between these molecules and came up
30:21with the structural model for this,
30:24which has rank one PKC better
30:27scaffold at the center.
30:29Wrapped around by PKC better
30:32and this interaction,
30:33he has facilitated by the C25
30:36tear which insert itself here.
30:38So overall we think that he says.
30:42Activate CD 25 downstream of the
30:44visa receptor via sick because
30:46he better phosphorylation of C25,
30:49which then forms a complex with Raekwon
30:52to recruit first parties here to surface.
30:55Which then again provide negative
30:57feedback control so it's like a
31:00circle that goes back to maintain
31:03equilibrium Goldilocks principle
31:05again for the survival of the cells.
31:07So our conclusion is that we think that
31:11negative selection can be leveraged indeed.
31:14For potential therapeutic benefits of.
31:17He said leukemia and lymphoma is
31:20avoided because of phosphatases
31:22hyperactivation of sick or
31:24interference proceeding 25.
31:26Feedback control.
31:27So the goal here would be to.
31:30Push says that I had the upper limit
31:33there or it transformed the after a
31:36powerful activation signal over the
31:38edge by removing feedback control and
31:41balance which will trigger negative
31:43selection of what looks like at the
31:47level of signaling autoreactive B cells.
31:51Um?
31:51Coming back to our.
31:54Database which we find us a very rich
31:58resource for new ideas and concepts.
32:01You found another interesting
32:02outcome predictor that I would
32:04like to introduce to you with the.
32:06It's a recent publication that just
32:09came out a couple weeks ago and that is
32:12focused here on a molecule called IIT M3.
32:15And it's interfering inducible
32:18transmembrane protein.
32:19And as I showed you for C25
32:24to smaller cure is.
32:26An outcome predictor in various.
32:28He said leukemia and lymphoma
32:31subtypes and it's known for long time
32:35initially was found as a specifying
32:38molecules for primordial germ cells.
32:41And then more recently it was
32:43found as an antiviral protein that
32:45can restrict viral replication.
32:47He is shown HIV,
32:48but more recent data shows it also
32:51important for the restriction of
32:53coronavirus and many other viruses,
32:55and what was important to us is that
32:58is actually used as a diagnostic
33:00tool for pediatric leukemia to find
33:03patients that are at high risk.
33:05So it's one probe set on a low density
33:09array to identify patients at high risk.
33:12So we started the function of
33:14items three in a genetic mouse
33:17model and found actually.
33:19Happy says surprisingly,
33:20that are lacking this interferon
33:23inducible transmembrane
33:24protein. I have a defect
33:26in PS3 kind of signaling.
33:28And they are prone to
33:30cell death as shown by P.
33:3153 activation and loss of PCL too.
33:34And importantly, these cells actually
33:37cannot be properly activated to
33:39undergo affinity maturation.
33:41So here is PNA is a German center marker
33:44which is a throwback for affinity maturation.
33:48In in in, in visa here.
33:51So if he smiles are immunized and
33:55nicely form germinal centres but if.
33:58He says that by Adoptively
34:00transferred are lacking item 3.
34:03The amount of general centers
34:04or German centre visa is and
34:07subsequent affinity maturation.
34:08Is this drastically reduced?
34:11And the same is true in leukemia,
34:14so I have items with efficient leukemia.
34:16Says cannot form colonies.
34:18If Transformers disable or N Ross.
34:21There also lacks ability
34:23to initiate leukemia,
34:24and those two models.
34:26And they have a similar phenotype as I
34:29showed you in normally says in terms of
34:32lack of peers with kindness signaling.
34:35Survival and strong expression
34:39of death related or checkpoint
34:43related molecules like P53.
34:46So in terms of structure and mechanism,
34:49we were able to figure out how I've
34:52item three is regulated in Indy.
34:54Lymphocytes is actually very short
34:56protein or 433 amino acids in length
34:59and we found that it can actually
35:02insert itself into the cell membrane.
35:05And this happens when I'm downstream.
35:08Also visa receptor.
35:10Lynn,
35:10another sack family kinases phosphorylate.
35:14I've item three at this tyrosine 20s
35:16that's really a central tiersen,
35:18which leads to recruitment to
35:20the cell membrane,
35:21and then it can easily interact
35:24with the visa receptor or it
35:27becomes internalised again.
35:29So for this reason we studied this
35:31mutation here and actually found that
35:33it can function as an Uncle gene.
35:36So when we introduce us for cinematic
35:39form of white 20 which mimics the
35:42confirmation that is always was formulated.
35:44Into a mouse strain that
35:47carries transgenic BCR ABL,
35:48which has a very long latency to disease.
35:52We actually found that this leads
35:55to increased formation of colonies.
35:57Increased PS3 kinase activity and
36:00also increased activity of the
36:03visa receptor signaling pathway.
36:06And.
36:08And so structurally,
36:10we could show the poisoner interactome
36:12analysis that this form of items
36:15three intersects with multiple central
36:17components of both the PS3 kinase
36:19and visa receptor signaling pathway.
36:21And there's also shown here by these red
36:24dots in this proximity ligation assay,
36:27where if I can three molecules come
36:30in close proximity. And he says.
36:34Now the structural basis for data
36:37set and that was very surprising to
36:39us that I've item 3 can directly
36:42bind to PIP 3 which is initiating
36:45phospholipid and lipid rafts to
36:48initiate PS3 kinase signaling.
36:50And that is unexpected because this
36:53interaction is usually mediated by a
36:55so-called pH domain in larger proteins.
36:57But I've item three is such a short
37:00party in that has no resemblance
37:03of the pH domain,
37:04so we were looking here for and you
37:08structure basis of houses interaction
37:10could happen in the absence of a
37:13pH domain. And we looked at the
37:16conserved intracellular loop of
37:18five items free that that is used
37:21to insert into the cell membrane,
37:24and in doing so we found a cluster of.
37:30Five basic amino acids and of particular
37:33interest is this bracket here would
37:36call it between lysing 83 and license
37:39104 and even though they are 21
37:42amino acids apart from each other,
37:44they come very close here in
37:46the structure analysis,
37:48and they're basically former
37:50clamp to directly interact here.
37:52With this pit three molecule.
37:55So by mutation analysis we were able to
37:58show that the whites are morally cure.
38:01Was this bracket of lice and
38:0483 and license 104 intact?
38:06Is a powerful initiator of PSV kindness
38:09and peace a receptor signaling?
38:12But when these two amino acids
38:14here are mutated through the
38:16brackets along the active,
38:17the entire Lee loses ability.
38:20So that's something that became
38:22really interested in that we hope
38:24to pursue further in collaboration
38:26with our colleagues at.
38:27Yeah,
38:28like a new way of how proteins
38:30can make contact with Pepsi to
38:32initiate PSV kind of signaling
38:34in normal and Uncle Genic Lee.
38:36Transform B says so.
38:38How model is that in the
38:40absence of five items,
38:41we and normal cells also modeling and says.
38:45The molecules that initiate PSU
38:47kind of signaling are scattered
38:50throughout the cell membrane.
38:52Only five items,
38:53three is there acting as a molecular
38:56glues are drawn together and form a
38:58tight complex to initiate signaling.
39:01I'm not coming to the last part of my talk,
39:05which was quite surprising
39:07to some of us and Mr.
39:10Looking for ways to translate that knowledge.
39:13Looking for houses can be
39:15exploited therapeutically,
39:16but it essentially starts from the
39:18question of how do oncogenic pathways,
39:21once activated by mutations?
39:23How do they interact and?
39:27Becoming. Part of an orchestrated move
39:30that is to malignant transformation.
39:33And this idea is based on a concept
39:37that was formulated and long
39:40time ago by fear and Vogelstein.
39:44Here's Arconic concept of Mikey
39:46step malignant transformation by
39:49sequential acquisition of Driver
39:51Uncle Gene set together, then form
39:54the development of colorectal cancer.
39:56So the question here is, is this.
40:00Same in visa is do we.
40:03Is it true that acquisition of addition
40:05mutations lead to more malignant phenotypes?
40:07And how do these oncogenic
40:10pathways interact with each other?
40:12So to answer this question,
40:13we formed a collaboration with Children
40:16psychology Group and Saint Jude.
40:18And studied.
40:20Mutation data from one 1148
40:24cases of ecel image PLA.
40:27And what we did first was what you
40:30would call a mapping analysis of
40:33affinity versus repulsion of pathways.
40:35So basically asking the question.
40:37So activating lesions in one pathway.
40:41Are they?
40:42Do they have affinity to activation of?
40:48Hidden in a different pathway?
40:50Or is there like relationships of mutual
40:52exclusivity and we found a number of
40:54interactions that we are still working on?
40:57But one was really striking to
40:58us and it's an interaction of
41:01repulsion of mutual exclusivity,
41:03as shown here in this cartoon.
41:05And that involves a stat 5 pathway.
41:08The Jack Stat 5 Path pathway and IIRC
41:11home up kinase signaling pathway.
41:15And here I'm showing you the
41:18result based on these 1148 cases.
41:21Many of them have shown here in Green
41:24Spot 5 activating lesions forming
41:26one large cluster up here and then.
41:29Here's another cluster,
41:30but these are activating lesions and only
41:34in 35 cases which is just feed the spend.
41:36We found activation of both pathways,
41:39which which is much lower than than random.
41:43Also, when we look at individual
41:45cases and look at phosphorylation of
41:47Erk or phosphorylation of stat five,
41:50we have a clear cut negative or
41:53inverse relationship between them
41:54and you can also see here I have to
41:57level off Western blot that that
41:59you have either force relation or
42:01step file for false for work and
42:03this leads to different profiles
42:05in terms of correct sensitivity so
42:07traumatic Nip is American emitter.
42:09It was in the herb signaling
42:11pathway that effects on these.
42:13Leukemias hear worse porn atnip effects,
42:18mainly,
42:18the stat 5 signaling pathway which
42:21is affecting those leukemias here.
42:24And I'm.
42:26So we became interested in this small
42:28minority of cases in which we have
42:31indication of activation of both pathways,
42:34even though they seem to
42:36be mutually exclusive.
42:37So wanted to know who they occur in
42:40the same say or how does this work?
42:44And to answer this question,
42:46or we developed in our lap a single
42:48self also protein analysis that allows
42:51us to interrogate her phosphorylation
42:53of STAT 5 and phosphorylation of Erk.
42:56Concurrently in single cells,
42:58and this is here based on the gel
43:02matrix where we can deposit 6400 cells,
43:06single cells and then look at STAT
43:095 and workforce relations events
43:12individually and this year or
43:15four patient arrived.
43:17Cases where we looked at individual
43:19sales and were then actually able
43:22to determine that even though for
43:24all those four cases we get to dual
43:27signal by Western blot, if he.
43:30Use our single cell for supporting analysis.
43:33We see that these are actually two
43:35competing clones, 1 colonial start,
43:37five Zelda clone, here's org,
43:39and that goes for all four cases and we
43:42don't see any double expressing cells.
43:44So our conclusion is at least are
43:46actually rare by colonial diseases,
43:48in which two clones are competing
43:52against each other.
43:53Then we asked what is the reason for that?
43:57So what is the underlying mechanism
43:59that these two pathways just can't go
44:02together and to address this question,
44:04we actually voiced it.
44:06The alternative pathway on the leukemia
44:08said are driven by the other pathway,
44:10meaning that here is a visa able or
44:13start five driven leukemia then was.
44:16And Ross, when Ross driven leukemia was VCR
44:19able and use different models for that.
44:22And here, this colony forming assay
44:25shows if you have one single driver,
44:27either in the rason start.
44:29Five pathways is dramatically
44:31increases number of colonies,
44:33but if we have posed together,
44:35we basically lose or colony
44:37formation capability.
44:38The same holds true for growth,
44:41so single driver nicely lead to outgrows,
44:45but combination of Bosa suppressive.
44:48What was really surprising to us?
44:51This actually,
44:51that if we use genetic ablation of
44:54the diverging or alternative pathway,
44:56even though we basically
44:58remove an Uncle Genic driver,
45:00this actually Slack celebration of
45:02leukemia initiation in this model.
45:04So in this case we have here a
45:07visa able or start five driven
45:10leukemia and we remove perk. This.
45:14Accelerates development of leukemia.
45:16Likewise in a chaos driven leukemia.
45:20Removal of stat 5.
45:23Come initiates faster development
45:26of looking more Genesis.
45:29And biochemically,
45:29we were able to recover too late.
45:32This was small molecule inhibitors
45:35that Rametta Nathan MacKinnon bitter.
45:37Distinguishes her kindness
45:39activity as expected.
45:41But it also induces phosphorylation
45:43of STAT 5, and rocks.
45:45Litten appears opposite effect.
45:47It distinguishes start 5,
45:49but increases per activity.
45:51And so to end here,
45:54this final chapter of my talk,
45:57we came across agonists that
46:00we use for pharmacological
46:02reactivation of diverging pathways.
46:04So in this case BC I hear this,
46:08I 215 is so powerful activator, IIRC agonist.
46:12Strongly activates falsework at the
46:16expense of start 5 and then DPH here.
46:20Is the stat 5 agonist drive start
46:23five phosphorylation but at the
46:25expense of work and this has
46:27interesting activity on the clonal
46:29dynamics of these leukemias in vivo.
46:32So start five leukemia can be converted into,
46:35IIRC, leukemia, and most importantly,
46:37if you combine these pathway
46:39agonist was conventional treatment.
46:41So, for instance,
46:42here on Earth inhibitor with
46:44a Step 5 agonist,
46:46we achieve a dramatic
46:48prolongation of overall survival.
46:50So.
46:52The final conclusion here is that we
46:55propose that diversity of signaling input.
46:58Is actually an important barrier
47:01of malignant transformation and
47:03centralization and convergence
47:04onto one single pathway.
47:06Inactivation of all the other pathways.
47:10Is an early and critical step
47:12of malignant transformation,
47:13and if we achieve.
47:15To reinstate at the very signaling
47:18environment.
47:19That would resemble the interactions
47:22of normal cells were sent by him
47:25and which have multiple receptors.
47:27Multiple cues from the environment.
47:30So we're proposing a strategy
47:32of pharmacological reactivation
47:33that would restore their diverse
47:36signaling environment.
47:37And we hope that this approach can also
47:40be leveraged to overcome convention
47:42mechanisms of black resistance.
47:44So here we have passed their
47:46convergence minimal.
47:47What we called friction and permissive
47:49environment for transformation.
47:50But here if you have divergent pathways,
47:53we actually do have some friction and
47:56create a non permissive environment.
47:59And with that I would like to thank a
48:02number of collaborators at a dinner,
48:04Farber at yeah.
48:06And would like to acknowledge
48:08particular 2 senior members of my lap.
48:10Linda Shannon and Jerome Lee,
48:12who did most of the conceptual
48:14innovation of this work.
48:15Thank you very much.
48:19Marcus that's amazing. Collection
48:22of studies and it is remarkable how
48:27complex and somewhat almost counter
48:30intuitive alot of these pathways are
48:34in B cells and be some legacies.
48:38And it's obviously a delicate balance.
48:39And let me ask you, 'cause you?
48:42You've identified a number of pathways.
48:44That I guess are principally
48:47designed for B cell elimination.
48:49That you could leverage.
48:51How would you potentially
48:53target those pathways?
48:55Or could you target those pathways
48:57in conjunction with the growing
48:59armamentarium of available
49:00therapies for be similar?
49:02Concedes that are now in practice?
49:04Or could you,
49:05could you leverage that combination?
49:09Right in terms of potential for
49:11translation and how we would leverage
49:14hyperactivation of Visa receptor
49:16signaling to engage negative selection,
49:19and I think we have two options, one is.
49:24Already available, but less attractive,
49:27that would be April inhibition or
49:30phosphatases like Ship One and P-10.
49:33I'm attractive cause in the field
49:36phosphatase innovation raises a lot
49:39of questions about specificity.
49:41An alternative approach is one that
49:43Mark Lemon and I have discussed and that
49:47would be to generate a direct agonist
49:50of picky kinase like sick or BDK.
49:53Something fairly upstream in the
49:56visa receptor signaling cascade.
49:58So actually yesterday.
50:01I was at at Mark CBI weekly meeting and
50:03and there was some encouraging feedback
50:05that that might actually be feasable.
50:08So that's an approach that I would
50:10definitely like to pursue in the
50:12future to develop a direct hyper
50:14agonist or one of these key kindness.
50:19Excellent, thank you so and
50:21people should feel free to submit
50:23questions on the on the chat box.
50:26Dying cross as a question.
50:28I don't know if you can see it.
50:32Marcus, I'll just read it.
50:34PLA suggested that CD 25 C
50:3679 eight colocalization is
50:37predominantly intracellular.
50:39What do you think that this indicates?
50:43Great questions so.
50:47We have done an experiment with.
50:50WGA, which is a surface marker and
50:53we we actually find colocalization
50:55of about 60 to 70% of those
50:58interactions with the CD79A and B.
51:01So I don't think it's predominantly,
51:04but the question remains,
51:05there's still a significant amount of
51:08interactions that are was in the sale.
51:10And you thought, indeed,
51:12that is very strange,
51:13because why would the visa receptor
51:16be internalized or somewhat was in
51:19this area associated with CD 25?
51:21And so we can answer this fully.
51:24But in light of the recent study
51:27by whose daughter was published
51:29in nature two years ago,
51:31where he actually shows that in malignant
51:33lymphoma B cell receptor signaling
51:36complexes form of endosomal membranes,
51:38his Tinder sale.
51:39We think that exact same
51:41thing might happen here.
51:43You don't know that,
51:44but that's that would be our explanation,
51:46so I don't think it's predominant.
51:48But I agree with Diane that let
51:50me see intracellular complexes,
51:52and we think they're an endo zones.
51:55Thank you and Diana's second question,
51:58which is CD 25 seems to
52:00prevent autoreactivity.
52:01Do you think this is related
52:04to CD-25 CD 79 interaction?
52:07Does it? Does C25 interact with
52:11surface immuno globulin's? I'm.
52:14So. That's actually a question
52:17that I asked Eric Metra,
52:19who's my collaborator in the
52:21field of autoimmune diseases,
52:22so Eric told us at 3:25,
52:24and that's known by work from
52:27from his group and also others.
52:29It is crucial to maintain
52:32central visa tolerance so that
52:34molecule is not there anymore.
52:37Then central tolerance mechanisms don't work.
52:40I think our signaling studies
52:42just about to clarify how this.
52:44Actually,
52:44you know what this mechanism of that?
52:47I think the link or how C25 interferes
52:49with Visa receptor signaling is not
52:52known in our paper is not published yet,
52:55so we're still working on that.
52:57Honey.
52:58And I I do think,
53:00actually,
53:00that it doesn't act in
53:01service in the global India.
53:05And again, people should
53:07submit their questions online.
53:08Marcus, I mean, could you ever
53:11conceive of you know you identify a?
53:14Be so malignancy that's driven by
53:16jackstadt and you would give them,
53:18IIRC activator.
53:18You know I obviously we focus on how
53:21to inhibit the pathway in cancer,
53:23but is that something you could
53:25conceive as a therapeutic approach?
53:29If I may quote back on my slides
53:31because there's one that I want
53:33to show you. This is study.
53:37Yeah, this study heals. Come by Veronica.
53:40Sex is group so that did you
53:43want to share your slide or? Yes,
53:46I'm going to show this slide again.
53:54OK, so I I hope I'm I
53:57got the question correct.
53:59This is what I I would like to refer.
54:02It's a study by the only car sex is cool.
54:07That it's a trial for patients was NPN,
54:11and they received rocks.
54:12Luton Airport, just Jack.
54:14Start fires inhibitor over long periods of
54:17time is actually going opposite direction,
54:21so they found that these patients
54:23developed in 6% of nine patients out
54:26of 157 was NP ND well developed hybrid
54:30diesel lymphoma that were driven with Keras.
54:34And that's a 15 fold increase risk.
54:38So what they said in this study
54:41is that actually find one reason.
54:43Population of the Start 5 pathway enables.
54:47The transformation of the pre
54:49malignant B cell tumor that carries
54:51the Chaos Legion that essentially
54:52what we did in our genetic experiment.
54:55So I think it can cut both ways so it
54:58can be beneficial if he find ways to
55:00leverage this activity to completely
55:03suppress oncogenic signaling.
55:05But like Veronicas NPN study shows,
55:07it can also go in your opposite
55:09direction if you try to achieve
55:12long-term suppression of 1 pathway,
55:14you might inadvertently activate the other.
55:18So it's really interesting.
55:19'cause obviously if you're
55:20going to use ruxolitinib you,
55:22it's context may be very specific.
55:24I mean, I know it's a small proportion,
55:26but that's a pretty heart risk.
55:35Just waiting to see if any other questions.
55:39Well, I think we're you know,
55:40really, at the top of the hour,
55:42so you know, want to thank Mark is for
55:44it really is an extraordinary talk.
55:47That's creating so much insight
55:49into the biology of AB cells.
55:51Both respect to cancer and autoimmunity.
55:53Wanna thank Doctor, Snyder,
55:55and Snyder for sort of share
55:57continuing to lead this lectureship?
55:59And I want to thank the Frisbees
56:02for their continued support of
56:04our Cancer Center and the mission
56:06and the support of this lecture.
56:11I wish you all a great
56:13day and happy New year.
56:17Thank you. Thank
56:19you, thank you very much.
56:21Thank you Marcus. Take care.