Signaling Pathways in the Pathogenesis and Treatment of Diamond Blackfan Anemia

Transcript

• 00:17Alright guys, why don't we go ahead
• 00:19and get started so we're having a
• 00:21zoom webinar format for this talk so
• 00:23Doctor Sakamoto will give her talk and
• 00:24then at the end we'll have questions.
• 00:26I think you're going to have to
• 00:28put your questions either in the
• 00:30Q&A or in the chat and then I can
• 00:32read them to her just given
• 00:34the format of this talk.
• 00:36So I'm Jeannie Hendrickson.
• 00:37I'm representing the
• 00:38Yellow Cooperative Center
• 00:40of Excellence in hematology
• 00:41for the enrichment program,
• 00:42and we're extremely happy to have
• 00:45Doctor Sakamoto who's giving a talk,
• 00:47a talk to us all the way from Stanford.
• 00:50Virtually, of course,
• 00:51she's a professor in pediatric
• 00:53hematology oncology at Stanford,
• 00:54and her research involves signaling
• 00:56pathways and gene regulation in
• 00:58normal and aberrant masterpieces,
• 01:00including bone marrow failure.
• 01:01And today she's going to focus her talk on
• 01:04this as it relates to Diamond Black fan.
• 01:07Nina, so I will turn it over
• 01:09to her and again thank you so
• 01:11much for joining us today.
• 01:14Thank you so much Gene.
• 01:15I really appreciate the invitation.
• 01:17I also want to thank Pat and Diane
• 01:19especially for this kind of rotation
• 01:21and what I'm going to do today for the
• 01:24next 45 minutes or so is to talk about
• 01:26some of the recent work that we've
• 01:28been doing on signaling pathways in
• 01:30the pathogenesis and treatment of TBI.
• 01:33So just to say I have no good disclosure,
• 01:35I have no disclosures that
• 01:36are related to this work.
• 01:38So Diamond life and anemia is a
• 01:40very rare boomer failure syndrome,
• 01:42and I'll discuss some of the background
• 01:45and clinical features of this disease.
• 01:48Also, the role of an email like
• 01:50high knees in DBA models and then
• 01:53targeting and OK for the treatment
• 01:56of DBA and the last part of the talk.
• 01:59I will talk about a new project on 71
• 02:02in erythropoiesis and EPA pathogenesis.
• 02:06So DPA is a very rare congenital
• 02:08bone marrow failure syndrome that's
• 02:10associated with macrocytic anemia,
• 02:13congenital defects, Anna risk of cancer.
• 02:15This disease is generally diagnosed in
• 02:18early childhood, less than a year of age.
• 02:21The incidence is seven in a million
• 02:24and approximately 20 to 40 new cases
• 02:27are diagnosed per year in the US,
• 02:30in Canada.
• 02:34Download iPad anemia.
• 02:36Clinical features are quite diverse and
• 02:39there are many patients who present
• 02:42with short stature shown here and
• 02:46these Twins upper limb abnormalities
• 02:48including thumb and facial and pallet
• 02:52malformations which can occur with certain
• 02:55ribosomal protein subunit mutations.
• 02:58Patients can present with small eyes,
• 03:00kidney defects, but only for 30% of
• 03:03all patients have physical findings,
• 03:05which makes it very challenging
• 03:08when you have a patient who may not
• 03:11manifest significant anemia and may
• 03:13be identified by their adult intern,
• 03:16internist, physician,
• 03:17who notices that they have microcytosis
• 03:19but no other physical features,
• 03:21and we've had a few of these patients
• 03:25come to clinic in our at Stanford.
• 03:29The treatment for Diamondback anemia
• 03:31is typically steroids, chronic ritzel,
• 03:33transfusions and stem cell transplantation.
• 03:36For patients who are steroid refractory
• 03:38or chronically transfusion dependent,
• 03:40and these are all associated
• 03:43with significant morbidities,
• 03:44including immunosuppression,
• 03:45iron overload, graph versus host disease,
• 03:48there are newer therapies that have
• 03:50come been proposed.
• 03:52For example, L, leucine, the amino acid.
• 03:55Many of you are familiar
• 03:57with the clinical trial.
• 03:59It's been directed by Jeff Lipton,
• 04:02an Adreno blocos.
• 04:03They showed a modest
• 04:05improvement with leucine,
• 04:06although the doses were
• 04:08much lower and for safety,
• 04:10but there were some patients who
• 04:13did experience improvement in
• 04:14the transfusion requirements.
• 04:16So Tatter septis Eligant wrapped that
• 04:19inhibits the TGF beta signaling pathways
• 04:21that also has been shown recently
• 04:24to improve with police in patients,
• 04:27for example with Milo dysplastic syndrome.
• 04:30But most of them are being
• 04:33cronian investigated.
• 04:37Why does summer deficiency in bone
• 04:39marrow failure syndrome is become
• 04:41more and more common in diseases
• 04:43such as Shockman Diamond syndrome
• 04:45which is another congenital bone
• 04:48marrow failure syndrome with other
• 04:50physical findings, deletion 5 Q.
• 04:52Milo dysplastic syndromes which
• 04:54is associated with our PS14
• 04:57haploinsufficiency that's been described
• 04:58by Ben Ebras group several years ago.
• 05:01These are all considered diseases
• 05:03that are ribosome opathy's with
• 05:05defective ribosome Biogenesis.
• 05:07And function.
• 05:08Over 80% of DBA patients have
• 05:11mutations in ribosomal protein.
• 05:13Some units resulting in ribosome dysfunction,
• 05:16an impaired protein translation.
• 05:19This is just a pie chart that shows
• 05:22the various percentages of patients
• 05:24who have ribosomal mutations.
• 05:26You can see that among the most
• 05:29common is RPS 1925% RP 11.
• 05:31About 5% in RP 11 and five have been
• 05:34associated more with craniofacial defects,
• 05:37and there are several other
• 05:39mutations have been identified but
• 05:4130% have non ribosomal mutations.
• 05:43In an example is what Vijay Sankar
• 05:46had reported that God would.
• 05:48Mutations can also contribute
• 05:49to this disease.
• 05:51But there are several more mutations
• 05:52that have yet to be identified.
• 05:56This is a schematic that basically
• 05:58summarizes some of the defects that
• 06:00have been identified in the ribosomes
• 06:03are made synthesis pathway primarily
• 06:05the Treacher Collins syndrome, which,
• 06:08Interestingly does not usually
• 06:10manifest with bone marrow findings.
• 06:12We have just keratosis congenita
• 06:14which most people are familiar
• 06:17with that can cause fibrosis as
• 06:19well as bone marrow failure.
• 06:21Cartilage hair hypoplasia has been
• 06:23associated with macrocytic anemia.
• 06:25And then of course, TBA,
• 06:27which can involve the small subunit
• 06:29or large subunit 60 S or 40th,
• 06:31and then shockman diamond syndrome.
• 06:36It's still very interesting that
• 06:38this disease, which is germline
• 06:40mutation of these Robertson,
• 06:42will proteins in other mutations
• 06:44involved primarily the erythroid
• 06:47lineages and what I thought is in
• 06:49this very simplified version of Rip
• 06:52Oasis is that the cells increase
• 06:54in number as they become Earth,
• 06:56will glass and somehow this results in
• 06:59increased requirement for ribosomes,
• 07:01most likely due to increase.
• 07:03Protein translation is required for
• 07:05cell proliferation and differentiation.
• 07:09The normal hematopoietic treated Mary.
• 07:11Are you familiar with already from?
• 07:13He might have put stem cells to
• 07:15come in my local gender Mega
• 07:17Carey Service Rd Ripper genders,
• 07:18and then the birth funding
• 07:20isn't calling from Eunice.
• 07:21Very earlier it'll bus stage all lead to
• 07:24eventually the maturing of red blood cells.
• 07:27In DBA, these very subunit mutations
• 07:30that have been described seemed
• 07:32to be a result in a block in
• 07:35early committed with blaster be.
• 07:37If you be a few istage,
• 07:40mostly resulting from haploinsufficiency
• 07:42inducing mutation and we know that
• 07:45homozygous mutations result in
• 07:47mouse models as well as in humans.
• 07:50Embryonic lethality so helpful
• 07:52insufficiency or heterozygous
• 07:53mutations are typically what results.
• 07:55What results in this disease.
• 07:58Ultimately leading to anemia.
• 08:01So we started out looking at
• 08:03variety of signaling molecules,
• 08:05one that was of interest to us initially
• 08:08was a transcription factor MYB and
• 08:10we know that this is a very important
• 08:14protein that regulates erythropoiesis.
• 08:16It's also associated with my little keemia.
• 08:19When Aberrantly expressed and it's been
• 08:22reported that one of the kinases that
• 08:25activates or phosphorylates Mibiz,
• 08:27NIMAL, Iconis,
• 08:28and this kinese is Assyrian
• 08:30threatening kinese.
• 08:31That is a revolutionary and evolutionary
• 08:34conserved margin activated protein
• 08:36kinase in the map kinase family member.
• 08:39Originally described when mutated
• 08:41result results in I development
• 08:44defects in just saffola is highly
• 08:46expressed in neural tissues and
• 08:49plays a critical role in a number of
• 08:52important cellular functions through
• 08:54the regulation of various transcription
• 08:57transcription factors such as nib.
• 09:00An OK also regulate signaling
• 09:02pathways involving wind beta catenin
• 09:04active in aisle 6 an notch,
• 09:05which are all very critical
• 09:08for normal hematopoiesis.
• 09:10So this is work done by Mark Walsh,
• 09:13who is a former postdoctoral
• 09:14fellow in my lab,
• 09:15and now is an instructor in Pediatrics.
• 09:19We used a model that had been used
• 09:21prior in my lab where we would
• 09:24transduced human CD 34 positive cells,
• 09:27hematopoietic stem progenitor
• 09:28cells with small hairpin RNAs,
• 09:30then knocked down either RPS 19 or RPO 11.
• 09:34Making lentiviral constructs with
• 09:36transduced and then show that we
• 09:39could get help low or half the
• 09:42expression at the protein level by
• 09:44this Western blot or M RNA levels.
• 09:47And this is the model that we used.
• 09:49And when Mark looked at the expression
• 09:52of an OK throughout, he matter.
• 09:54Police is we didn't see any
• 09:56significant differences in our bar.
• 09:58RPS 19 RP 11 knock down now.
• 10:01In contrast,
• 10:02looking at phosphorylation or activation
• 10:04of an OK when Mark noticed was that
• 10:08taking three different substrates,
• 10:09either an OK itself this is auto
• 10:13phosphorylates, nib or wrapped,
• 10:14or that there is significant increase
• 10:17in the activation of this kinese
• 10:20in the 1st 10 days of hematopoiesis
• 10:23and so this we decided to focus
• 10:26on an OK and looking at mid target
• 10:29genes with RPS 19 lockdown.
• 10:32Again, HSBC is,
• 10:33we see that their expression
• 10:34of Alamo two in California were
• 10:37significantly decreased.
• 10:38However,
• 10:38with the expression or knockdown of an OK,
• 10:41we see that that we could partially
• 10:43rescue the expression of these two genes.
• 10:46Obviously there other Trump,
• 10:47you know the regulators of nib,
• 10:49so that makes a lot of sense to us.
• 10:52I'm sorry, Kathy,
• 10:53can you go back one slide?
• 10:57What is one more? I'm sorry.
• 10:59What are you showing on the PC MIB?
• 11:03One forward PC MIB an?
• 11:05What's the PC MIB? There is
• 11:07middling. Phosphorylated
• 11:08yeah phosphorylated. MIB is
• 11:10that active? Is phosphorylated
• 11:11MIB active? Yes yes
• 11:13so. So that is a key phosphorylation
• 11:16site that's recognized by in OK. OK,
• 11:19so any OK is phosphorylating
• 11:21in activating them? Yes OK
• 11:24just clarifying yes and I'll talk
• 11:26more about that in a few slides.
• 11:29Yeah and so so now.
• 11:32How does an OK regulate nib actually what
• 11:35happens is that activated and OK then
• 11:38phosphorylates mid to ubiquitinated nib,
• 11:41an result in 26 S proteasome degradation.
• 11:44So if we have an OK and we knock it down.
• 11:49RPS 19 meter put extent progenitor cells.
• 11:52What we actually see is stabilization of MIB.
• 11:55So you see that the protein
• 11:57levels are higher and Konan OK.
• 11:59It's knocked down and this
• 12:01again this is in RPS.
• 12:0319 knockdown cells so you can see that
• 12:06if we again look at phosphorylated nip,
• 12:09that compared to when we knock down
• 12:11in OK we see higher levels of mid
• 12:14phosphorylation as we would expect
• 12:16because MIB results in degradation
• 12:18and OK phosphorylation results in.
• 12:20Amid degradation and also here only
• 12:23milk is ubiquitinated without an OK.
• 12:26So you see higher levels,
• 12:29so without an OK it's not degraded.
• 12:34So we also looked at C71 expression in
• 12:38this schematic schema that you see to
• 12:41indicate Orthop Oasis will Mark did was to
• 12:44isolate the C 71 positive cells and then
• 12:47to perform a Western blot with antibody
• 12:50specific for three nine, 298 on in.
• 12:53OK so here we see that only in C 71
• 12:57positive cells do we see an OK activation
• 13:00phosphorylation at this site but not in C 71.
• 13:05Get of cells and that's whether you have
• 13:07herpes 19 knocked down or control. Again,
• 13:10if we immunoprecipitated and OK in cells,
• 13:14either control or RPS 19 knockdown cells,
• 13:17we see that the phosphorylation in CD
• 13:2071 cells of these three substrates
• 13:23is is upregulated,
• 13:24and that's what we would expect because
• 13:28we get hyper activation of an OK.
• 13:31So this is only again in C 171671
• 13:34positive cells but not in other lineages.
• 13:40Furthermore, we looked at the
• 13:42activation of MLK in mouse models,
• 13:45so we collaborated with new on
• 13:48Flickr at Lund University in Sweden,
• 13:51who provided to us stem cells from RPS.
• 13:5519 knockdown mice.
• 13:56It's catcher cycling inducible.
• 13:58Similarly, we examine the stem cells from RP.
• 14:0211 flox mice, which is provided to us by
• 14:05Manuel Serrano from Barcelona, Spain.
• 14:08This is also.
• 14:10Tamoxifen induced to lose one allele
• 14:13of RP 11 gene and what we see is in
• 14:16these mice with which have anemia that
• 14:19the activity of in OK is much higher
• 14:23than our control mice or minus docs.
• 14:26In both the RPS 19 and RPL 11
• 14:29knockdown mice mouse cells.
• 14:31We also examined patient samples and
• 14:33this is a collaboration with Hannah
• 14:35Gosda from Boston Children's Hospital.
• 14:38She provided to us three
• 14:40different patient samples.
• 14:41With these mutations in our PS 19
• 14:44Mark then examined the relative in OK
• 14:48kindness activity compared to healthy
• 14:50control cells and show that that the
• 14:54activity was significantly increased.
• 14:56And finally,
• 14:57in collaboration with here Mitsunaga,
• 14:59which he had Stanford,
• 15:00his postdoctoral fellow,
• 15:02to she developed,
• 15:03I PS cells from two different patients
• 15:05who are diagnosed at Stanford,
• 15:07who had the typical clinical
• 15:09and physical features of DVA.
• 15:11And one of them had an RPS 26 mutation.
• 15:14The other one had an unknown mutation,
• 15:17but in all three clones that
• 15:19we analyzed from I PS cells,
• 15:21we see hyperactivation of an OK using
• 15:24the substrates that I mentioned.
• 15:26And OK leban rector.
• 15:31So given this in all of the
• 15:33models that we examined for DBA,
• 15:35regardless of the mutation,
• 15:36we were able to see an OK activation.
• 15:40And so we wanted to test the hypothesis
• 15:42could in OK be a possible target for therapy.
• 15:46The way that we looked at this was to
• 15:49study the number of two thirty 5235 cells,
• 15:52which is a reflection
• 15:53of birthright expansion.
• 15:54We see that in our PS 19 knockdown cells,
• 15:58if we treat with sin LK.
• 16:00To knock down MLK,
• 16:01we see increase in the numbers of these
• 16:04are three projector cells or three cells,
• 16:07in contrast to absence of in OK or.
• 16:10Design OK or a control escape in
• 16:12OK and the point here is that
• 16:15in the patients with DPA,
• 16:18generally speaking they are treated
• 16:20with steroids or transfused in
• 16:22when the hemoglobin is below 8.
• 16:24So our goal is not to necessarily improve
• 16:28the hemoglobin up to normal levels,
• 16:30but rather to increase or decreases
• 16:32sufficiently so that they will no longer
• 16:35need story therapy or wrestle transfusions.
• 16:38So that's really our goal.
• 16:42You have a hand raise. Pat Gallagher
• 16:45is raising his hand. OK, yes?
• 16:50Let me think we have to figure
• 16:52out how to let him talk.
• 16:54Hold on one second so I can do it.
• 16:56I can do it. Go ahead.
• 16:58I'm good guys are the question I got OK?
• 17:02Do you put my hand down there?
• 17:05I don't know if I can do know how to do that.
• 17:09I had a question though on the
• 17:11previous slide you normalized
• 17:12the healthy control cells.
• 17:14Yes to 100% on the previous slide.
• 17:16Yeah, and had to go back and if you
• 17:19knock down you had the sin LK and you
• 17:21gave the absolute number of cells.
• 17:24Is that affected in the healthy controls?
• 17:26Well certainly with DPA
• 17:27patient samples there are lower numbers
• 17:29of cells, but in spite of this we see
• 17:32hyperactivation of energy. So I'm talking
• 17:34bout in the controls on the left. In
• 17:37the controls, if we knock if we if
• 17:39we look at the numbers of cells.
• 17:42I don't know if we actually have done that.
• 17:45I mean normally we would try to
• 17:48normalize the cell's cell number.
• 17:51To make it equal on both sides,
• 17:53but yet it's still we see hyperactivation of.
• 17:57I mean we see lower levels of an OK
• 18:00activation. Yeah thanks yeah.
• 18:03So in collaboration with Johan,
• 18:05we were able to look at.
• 18:07He gave us 8 compounds that he had screened.
• 18:12Using a 14,000 compound library in his DBA
• 18:15RPS 19 knockdown mice trying to go back,
• 18:19but I can't. And what we saw was
• 18:23these eight compounds and compounds.
• 18:25Six and eight were the most effective
• 18:28as far as their third expansion.
• 18:30And then if we looked at the Enoch activity,
• 18:34the typical substrates that
• 18:35we've examined in the past,
• 18:37we see that in these two compounds we were
• 18:40able to see significantly decrease activity.
• 18:43Now here we have our Pierce
• 18:4519 knockout cells again,
• 18:46and we see with this is with sin LK.
• 18:50So when we add this compound is
• 18:52Scituate which is a TGF beta inhibitor.
• 18:55Which has an OK as an off target.
• 18:59We see that there's improvement
• 19:01in other places.
• 19:02However, if we knock down in OK,
• 19:04we still see an improvement.
• 19:06So just by knocking down and OK,
• 19:08we see an improvement as we would expect,
• 19:11and then the treatment also in combination
• 19:14with an saw an OK gave about equal
• 19:16amount of improvement of earth places.
• 19:19So what the suggested to us
• 19:21was that the effect of this SD,
• 19:23two ages most most likely
• 19:25due primarily to an OK.
• 19:27So pushing up in OK activity.
• 19:29There's no effect of Miley
• 19:30sales as we previously seen.
• 19:34This presentation also within OK
• 19:37Nebbish and improves with pre sis of
• 19:40of our mouse models again which we had
• 19:44shown but turn 19119 increased with
• 19:46treatment of our this TGF beta inhibitor.
• 19:50Same with RPL 11 in the fold was
• 19:53different a little bit but two to three
• 19:57fold two year 424 fold stimulation.
• 20:00A production of rich white cells.
• 20:05We we also looked at human.
• 20:07Our cell models, including our PSAT,
• 20:09knocked down and 11 knock down.
• 20:12We see again Approvement at our DBA cells.
• 20:15No ST208 with SD too late,
• 20:17so we see an improvement anywhere
• 20:20from four and a half to about 7
• 20:23fold and then finally a patient
• 20:25sample we because he 235 levels.
• 20:28And found where would CSD 208 that
• 20:30we see increase in our ourselves
• 20:33with red cells and above 2 fold
• 20:35increase by quantitating.
• 20:37So how does this occur?
• 20:39Well one of the reasons why we think
• 20:41MLK may be important for treatment such
• 20:44as losing is that Lucy and may require
• 20:48which is amino acid as I mentioned
• 20:50before in clinical trials that it
• 20:53may require active mtor complex and
• 20:55pathways to be activated or induced.
• 20:58And Raptor me and this whole complex.
• 21:01We believe this is attached to lysosome.
• 21:05In the case of an OK phosphorylating Raptor,
• 21:08we hypothesize that this released
• 21:10this complex from the license zone,
• 21:13thereby enabling translation to occur.
• 21:15So Mark perform evening up
• 21:17fluorescent experiments where he
• 21:19labeled Raptor with green life,
• 21:21some in red and then looked at
• 21:24colocalization in the case of
• 21:26RPS Anki knockdown cells where he
• 21:29saw was that the Raptor was cold.
• 21:31Localising with their lices own.
• 21:34And as attached seem to be attached
• 21:37or interacting with each other,
• 21:40whereas with an OK knock down we
• 21:42don't see wrapped or any longer
• 21:45colocalizes with their license home,
• 21:48so this is 1 possible role model that
• 21:51we propose for which YNLK inhibitors
• 21:54might actually work together with lysine
• 21:57to enhance the erythropoietic effect.
• 22:00And this is another experiment where
• 22:03we basically took our cell model RPS
• 22:0619 and looked at the number of two
• 22:09CD 235 cells and you can see here.
• 22:12So control losing increasing
• 22:14orbital Boyces St 208, again RTF,
• 22:16beta inhibitor and the combination of
• 22:19the two seemed to increase even more.
• 22:22The effects through synergistic and
• 22:24synergism and then the we looked at.
• 22:27Also for EBT one which is a downstream
• 22:30target of optimizing implore.
• 22:32And showed that the phosphorylation
• 22:34was also enhanced when we combine the
• 22:37leucine with SD in our PS90 knockdown models.
• 22:42So in summary of this,
• 22:44part of the talk we showed that in
• 22:47OK is activated in every projectors
• 22:49from DBA patient samples as well as
• 22:53our other human and mouse models.
• 22:55Pharmacological genetic inhibition of
• 22:57NLC increases with regenerx expansion
• 23:00in our models and then OK appears to
• 23:03exert influence on putting translation
• 23:04in DBA through the mtor pathway.
• 23:07So our focus was next to begin
• 23:10to identify potential therapies.
• 23:12That would target in OK.
• 23:14Unfortunately, the SD 208 compound is
• 23:17not ready for clinical application.
• 23:19It doesn't have the appropriate
• 23:22physical chemical properties,
• 23:23including solubility,
• 23:24to be able to be converted to the clinic.
• 23:28And So what we show is that we
• 23:31worked with a number of mid chemistry
• 23:34consultants from our Spark program,
• 23:37which is a program at Stanford to
• 23:40convert projects and lab to the clinics.
• 23:43And were able to examine a number
• 23:46of these compounds,
• 23:47all of which have different primary targets.
• 23:50But we tested a number of them.
• 23:52This is just a representative
• 23:54experiment showing in OK in
• 23:56vitro kinase activity descend,
• 23:58everyone is familiar with which
• 24:00is a tyrosine kinase inhibitor
• 24:02used to treat CML and carafe,
• 24:04and if it would be rough inhibitor OTS.
• 24:06167 is a milk inhibitor suppen
• 24:09assertive is an important hitter in
• 24:12St 208 is our TGF beta inhibitor.
• 24:14So just to show an example,
• 24:16we use low and high concentrations.
• 24:18We able to show significant decrease in OK
• 24:22activity with this particular drug OTS 167.
• 24:25We examined some of these compounds for their
• 24:28ability to increased risk of Oasis in RPS,
• 24:3119 lockdown models and here's
• 24:33our TGF beta inhibitor.
• 24:35We see various levels of
• 24:37increase in Rip Oasis,
• 24:39but mostly due to RTS 167.
• 24:42We also looked for phosphorylation
• 24:44of Raptor again,
• 24:45which is the target of which
• 24:47is in the Mentor complex and we
• 24:50see that there was an increase
• 24:52in phosphorylation of Raptor.
• 24:55But decrease with OTS 167 within OK activity.
• 25:02So our lead compound we decided
• 25:04to focus on was OTS 167.
• 25:07This is a milk inhibitor.
• 25:09It's currently under being studied
• 25:11in Phase 1/2 clinical trials and
• 25:13particularly in advance to acute
• 25:15leukemia as well as lung cancer.
• 25:17The drug was developed by uncle with
• 25:20therapy scientists and their bins.
• 25:22Number of studies that show that drug is
• 25:25still effective when milk is knocked out,
• 25:28so there are clearly other targets involved.
• 25:32This compound was shown in RP slinky
• 25:35knockdown cells again to increase
• 25:37every thread expansion and when
• 25:39we combine our knockdown oven OK
• 25:42with this inhibitor we did not see
• 25:44significant increase in irithyll.
• 25:46Police is suggesting to us again that
• 25:50the primary target of OTS in this
• 25:52system to improve it or places in OK
• 25:55and this drug was dosed at 200 animal
• 25:58or every three days for one cycle.
• 26:04To Sir to to understand whether
• 26:06there there there was toxicity
• 26:08to normal or through blast.
• 26:11We looked at every expansion
• 26:13as well as DBA cells.
• 26:15Knockdown cells with artist 19 we see
• 26:18that the maximum effect was at 300 animal,
• 26:21but we begin to see an effect in
• 26:24North places as early as 30 nanomolar,
• 26:27which shows greater than
• 26:29tenfold therapeutic window.
• 26:30Since the IC50 and normal.
• 26:32Or healthy with Glass was
• 26:34about 480 animal are.
• 26:36With my lead cells we saw slightly
• 26:39more sensitivity of this compound.
• 26:41Again, we can see 30 an animal or an
• 26:44increase in it with the police is,
• 26:47but then the myeloid cell we
• 26:49began to see decreased more
• 26:51significantly around 300 nanomolar,
• 26:53so Even so we believe the
• 26:55therapeutic window is about 10 fold.
• 26:57And again, this is an in vitro assay system.
• 27:02So in conclusion,
• 27:03Altius 167 appears to improve our
• 27:06ethical thesis in our DBA models in
• 27:09vitro with very little toxicity.
• 27:11There have been previous reports
• 27:13in Nora Blastoma and breast cancer
• 27:16Xenografted mouse models treated
• 27:17with OTS 167 twice a week for three
• 27:21weeks over the course of a month or so,
• 27:24and none of those mice developed
• 27:26bone marrow toxicity.
• 27:28There are also other inhibitors to
• 27:30indicate that potentially they.
• 27:32May be effective,
• 27:33including John Conyers inhibitors
• 27:35and we're currently testing those
• 27:38and finally experiments to really
• 27:40test this is necessary in vivo
• 27:42in order for us to proceed to
• 27:44clinical trial stage.
• 27:48Another interesting observation
• 27:49that Mark made as far as Enoch
• 27:52expression is the fact that metformin,
• 27:54the commonly used medication
• 27:56for type 2 diabetes,
• 27:58inhibits an OK expression in small
• 28:00cell lung cancer cells,
• 28:02and this drug also improves the
• 28:04effect of hematopoiesis and delays
• 28:07tumors in Fanconi mice.
• 28:08This has been reported to be and metformin
• 28:11to be protective against aldehydes,
• 28:14which is one of the toxins
• 28:17thought to affect inhibit.
• 28:18He met up with stem cells and
• 28:21he's in this disease.
• 28:22There is currently a phase two
• 28:24trial with metformin in Fanconi
• 28:26patients that's being directed by
• 28:29Akiko Shimamura Boston Children's.
• 28:31So what is the mechanism by which in Oak
• 28:33expression is inhibited by metformin?
• 28:35Well, one of the things we
• 28:38looked at was first of all,
• 28:40just metformin improve the cell
• 28:42numbers of C235 cells and we
• 28:44show that both in RPS 19 RP.
• 28:4611 Knocked down models that it
• 28:48does increase the production
• 28:49of these research senators.
• 28:51In contrast,
• 28:51there's no effect of moderate cells.
• 28:53We don't see any phenotype,
• 28:55and then it would be a few economy.
• 28:58Or if you eat colonies,
• 29:00we see an increased number.
• 29:02With metformin. In contrast to controls.
• 29:06CD 235 arthritis sales.
• 29:10Metformin increases by five or
• 29:12six fold two to six fold and
• 29:14then be a few mirrors for it's
• 29:16also about two or three fold.
• 29:18We've seen increasing without no
• 29:20effect on the mileage ourselves.
• 29:24The metformin also improves erythropoiesis
• 29:26by inhibiting anoche activity.
• 29:28Here we have again in OK
• 29:31phosphorylation of in OK Mabel Rector.
• 29:34In all cases we see that the metformin
• 29:38as well as SD 208 TGF beta inhibitor
• 29:43decreases the activity of a van.
• 29:46OK in our PS90 knockdown model.
• 29:49The RNA expression also decreases
• 29:51which is interesting which is.
• 29:54Primary mechanism by which we
• 29:57believe metformin inhibits an OK.
• 30:00Anorith Rd expansion we see
• 30:02again with knockdown of RPS 19.
• 30:04Any cells and knocked down again OK at
• 30:07the same time we see that just knock down
• 30:10of in OK and improves mini expansion.
• 30:13There is replaces but also just say it not
• 30:16kind of in OK here as well as metformin.
• 30:19So metformin not done OK in the
• 30:21form it again no significant
• 30:23change which again suggested some
• 30:25informants working through an OK.
• 30:30Sorry.
• 30:33This is a slide which shows our
• 30:36treatment of zebrafish models in
• 30:38collaboration with Scholin at UCLA.
• 30:41He created a DBA model
• 30:43using RPS 19 morpholinos.
• 30:45This is a phenotype of the fish.
• 30:48After approximately 5 days you can see
• 30:51that these embryos show significant
• 30:53anemia compared to controls with
• 30:55the treatment with metformin.
• 30:57We see that there is again increase
• 31:00in risk reduction as indicated
• 31:02by staining with Odeon sitting.
• 31:05Which binds to hemoglobin.
• 31:09So how does metformin
• 31:11regulate in OK expression?
• 31:13One of the ideas is through micro RNAs,
• 31:17and so Mark created a number of
• 31:20truncation mutants that would include
• 31:22include a variety of micro ironies
• 31:25for which we can then try to identify
• 31:29the specific mechanism by which
• 31:31metformin effects in OK expression.
• 31:35So this is showing the levels in
• 31:38humans that mirror 30 a mere 26
• 31:42increases with metformin treatment.
• 31:44So this is again in human primary
• 31:47cells versus mice that do not show
• 31:51this increase in May 26 expression.
• 31:54So the idea here is metformin could be
• 31:57inducing near 26 which binds to the
• 32:01three prime UTR event OK resulting
• 32:04in inhibition of by of expression.
• 32:07So this is a luciferase constant
• 32:10Reporter construct showing activity as
• 32:12a reflection of transcription of in OK.
• 32:14You can see that with the R in micro
• 32:17RNA 26 mimetic that there is we see
• 32:21decreased transcription also with
• 32:23181 which is in the same region
• 32:26the truncation mutant as mere 26
• 32:28we see a decrease in me.
• 32:31Yeah can you
• 32:32clarify what you mean by a mere 26
• 32:35mimetic? It's it's a similar.
• 32:37The structure, as a mere 26,
• 32:39so it's acting as if it were a mere
• 32:4226 and binding to that site, so I hope
• 32:45it's done with like with the
• 32:47retrovirus like with a hairpin or.
• 32:48Yes, I believe so, yeah.
• 32:51And the same thing with an OK if
• 32:54we we see that in OK expression
• 32:57at the protein level is decreased.
• 33:00But a sponge which basically is
• 33:02exactly what it described it in
• 33:05no longer allows me or 26 to bind
• 33:07to the three point Muti UTR.
• 33:10We see that there is again
• 33:12increase in OK at the Mr. NY.
• 33:14Also prefer at the Reporter assay
• 33:17and also the protein level.
• 33:20And also the Mirror 181 on the top
• 33:23didn't affect, but on the bottom did
• 33:25what's going on with that?
• 33:27Let me see for here.
• 33:29Yeah, I mean that's a good question whether
• 33:32the mere 181 it may not be a specific
• 33:36so that it doesn't sound consistent,
• 33:38because you would expect me
• 33:40or 181 to increase there too.
• 33:42So you know, not all the mirror when
• 33:45he I guess they could be inhibiting
• 33:48or blocking other mere sites.
• 33:50So, but that's something we
• 33:52need to look at more carefully,
• 33:53but thanks for noticing, yeah.
• 33:56An Emmy this metformin also
• 33:58mediates everything voices,
• 33:59so again we see the C235 increase
• 34:02with the mimetic that Foreman or both.
• 34:06Finding interrupted right
• 34:07'cause they're happening.
• 34:08So there's a question from the audience.
• 34:11Is the difference of metformin
• 34:12effect on human versus mouse
• 34:14near 26 due to changes in mere
• 34:1726 transcription or processing?
• 34:18Yeah, that's a really good question.
• 34:21I don't think we really know that.
• 34:23We haven't really focused on the
• 34:25mouse system, but we do know that it's
• 34:28different and you know that there are
• 34:31clearly differences at the genomic level
• 34:33and that the exact reason for that.
• 34:36We're not sure. So it's a good.
• 34:39It's a great question, yeah?
• 34:42So both in the mouses are
• 34:45in the mouse system.
• 34:47We see an increase turn 119 and also 235.
• 34:51We see increase in CD 235 R 3119
• 34:55the expression as you would expect
• 34:58goes down with metformin treatment
• 35:00here in our PS90 knockdown cells.
• 35:04So, to summarize,
• 35:05metformin improves with crisis
• 35:06in our models of DPA.
• 35:08It decreases in OK expression through
• 35:10mere 26 A and targeting mirrors is
• 35:13a possible approach to DBA therapy.
• 35:15There now companies that are
• 35:17trying to make medics or sponges
• 35:19for clinical application.
• 35:20Although it's very in early stages.
• 35:25OK, for the last part of the talk I'm
• 35:28going to focus on a new project that I
• 35:32appreciate Diane and Vanessa's input.
• 35:35This is a protein set B1 which is
• 35:38special 80 rich binding protein one
• 35:40and we initially did a many years ago.
• 35:44Actually did a RNA seek experiment
• 35:47with fetal liver human CD 34 positive
• 35:50cells that were transduced with RPS 19.
• 35:54Like if I were constructs and found 560
• 35:56or so genes that are differentially
• 35:58expressed and then we cross reference that
• 36:01with the list of genes identifying earlier.
• 36:04It'll poesis in a paper published by
• 36:07Mohan Orla and also Pat Gallagher and
• 36:09found about 1700 genes over lapping were
• 36:12about 42 genes that we then analyze
• 36:15in a variety of our cell model systems
• 36:18and hematopoietic stem cells both in
• 36:20control an RPS 19 knockdown cells
• 36:22and found that among those that were.
• 36:25Mostly regulated,
• 36:26the Sepy one was very interesting to
• 36:29us since not much had been described
• 36:31at all on the role of sappy one during
• 36:35Aritha Poesis So sappy one is a
• 36:38protein that basically forms chromatin
• 36:41loops and regulates transcription.
• 36:44And there's a number of their number
• 36:46of papers that have very nicely
• 36:49described its expression and rolling
• 36:51hematopoietic differentiation.
• 36:52It's moderately expressed in HS
• 36:54season is required for self renewal.
• 36:57It's induced in lymphopoiesis and
• 36:59required for T cell expansion.
• 37:01Knockout mouse have defects in
• 37:03lymphopoiesis and then downregulation
• 37:05in Milo.
• 37:06Police has been demonstrated in its
• 37:09requirement for PU .1 regulation in
• 37:12in common Milo progenitor cells.
• 37:14So Mark looked at sappi 1M RNA
• 37:17expression anarchist.
• 37:1819 knockdown cells,
• 37:19day OD five.
• 37:21He showed that the expression
• 37:23decrease more rapidly in our PS 19
• 37:26knockdown cells compared to controls
• 37:28and then also at the protein level.
• 37:31We see that there is much more
• 37:33decrease in the protein levels.
• 37:36Is that being one day 5?
• 37:40The colony essays and liquid culture
• 37:42essays that we perform showed
• 37:45that in colony essays in RPS.
• 37:4719 knockdown.
• 37:48HSBC's that we did not see significant
• 37:51increase in colony numbers per
• 37:53plate with three expressions.
• 37:55We expressed that being one about
• 37:57to fold in these knockdown cells,
• 38:00we can see any much increase in
• 38:03liquid culture. However, we sell a CD.
• 38:06235 sales that there was increase in.
• 38:09With happy one overexpression.
• 38:12But although there was no increase
• 38:15in the colony numbers,
• 38:17we could see by visualization that on
• 38:19the colonies were huge for much larger,
• 38:22suggesting that there was a
• 38:24proliferation of these cells,
• 38:26and we did see also a normal controls,
• 38:29but not as dramatically as in our case,
• 38:3219 knockdown cells.
• 38:34There's also a hand up from. Yeah, it's
• 38:37Vince. OK, I've is.
• 38:42Sorry, that was sorry that
• 38:44was accidentally did that.
• 38:46Oh alright, never mind.
• 38:47So colony assays we see that in
• 38:50knockdown of sappy when in normal
• 38:53cells or healthy progenitors we see a
• 38:56slight decrease in an doxycycline or 71
• 38:59knockdown was 235 in liquid culture.
• 39:01So we do see a mild decrease in
• 39:05erythroid colonies and or through glass.
• 39:08So sad he went on.
• 39:10Regulation does not dramatically
• 39:11impact anemia phenotype in DBA.
• 39:13So what is the function of 71?
• 39:16So healthy uncommitted Mylar
• 39:18projectors we see that.
• 39:19So again,
• 39:20here's our colony assay results
• 39:23showing mild decrease in be a few.
• 39:25Ease the culture.
• 39:27The same 235 cells but in megakaryocytes
• 39:30regenerators we see at least a 30%
• 39:33decrease in with that be one knockdown.
• 39:36So our hypothesis at this point
• 39:38was that although in DPA we see
• 39:41a block here early erythroblast
• 39:44stage or seek to the 235 stage.
• 39:47That perhaps happy one is
• 39:49acting upstream of this,
• 39:51affecting the megakaryocyte
• 39:53recite progenitor stage.
• 39:55And so here we have again
• 39:57our as our PS90 knockdown.
• 39:59Showing calling or calling numbers.
• 40:01Interesting Lee with CFU GM.
• 40:03We see an increase in colonies.
• 40:07C235 expansion was noted with RPS 19.
• 40:10Knock down an 71.
• 40:14We did see also 11B expansion
• 40:17and overexpression.
• 40:18We also see CD 41 increase
• 40:21more even more significantly.
• 40:25How does this happen then?
• 40:27What is the mechanism downstream of that?
• 40:30Be? One well, one of the things one of
• 40:33the genes that has been very important
• 40:36recently has been a heat shock.
• 40:3970th proteins that's encoded by
• 40:41the gene zedex, HSP, 1A1B, and 1A,
• 40:43and there are three different papers.
• 40:46Sorry, there are three different
• 40:48papers that have been describing
• 40:50by Hermione and Leah Dacosta.
• 40:51The role of HSP 70 in regulating gotta one.
• 40:56An agency 70 is thought to interact
• 40:58with God and one to prevent the caspase
• 41:01dependent cleavage of God in one,
• 41:03so basically is stabilizing that
• 41:05complex or God or one to allow
• 41:08it to to to activate genes,
• 41:10and this is just a profile of chromosomes
• 41:13looking at various genes so they are not
• 41:16affected by sappy one or influence to
• 41:18rescued by setting one in the orange.
• 41:21So here are the two genes and if we
• 41:24knock down step one, we see that.
• 41:27This HP A1A expression goes down.
• 41:30Similarly,
• 41:30if we knock down at SABI one
• 41:33we see HSP one being Arnie,
• 41:36decreasing ansim with the protein levels.
• 41:41If we then look examine our
• 41:43peacemaking knockdown cells and
• 41:45then overexpress sappy one,
• 41:47we see that there's a rescue and
• 41:49the expression of these two genes
• 41:52and also at the protein level here.
• 41:58To understand what sappy one might be doing,
• 42:02and regulating house regulating it,
• 42:05just P1A1A and the HPA 1B.
• 42:08Mark performed chromatin
• 42:09immunoprecipitation assays with antibodies
• 42:12that are specific to these H3K914,
• 42:14desolation HK4 trimethylation
• 42:16HCC 27 five metalation,
• 42:18and these are the sites that set the
• 42:23one binds and what we see is that.
• 42:27There's also commented peaks which that
• 42:29with the H3K29 acetylation and HGK
• 42:324 trimethylation but not with H3K27
• 42:35Trimethylation. So here's our model.
• 42:37Here is the HSP A1A gene. Here's.
• 42:41Here are the seven one binding sites,
• 42:44and here are the predicted enhancer and
• 42:47promoter sites that we would expect,
• 42:50and so sappy one promotes the looping
• 42:53so that all of these will be enclosed.
• 42:57Doximity to facilitate transcription
• 42:59of these two genes,
• 43:00another approach that Mark Hughes
• 43:03was as chromatin confirmation capture
• 43:05essay where you basically treat
• 43:07the cells with formalin,
• 43:09then digest using restriction enzymes
• 43:11and then perform PCR and what he
• 43:15showed was that with our with our
• 43:18control we see two nice peaks at the
• 43:21sites of the Enhancement Promoter
• 43:23regions with RPS 19 knock down,
• 43:25this is blunted.
• 43:26With knock down as happy when we
• 43:28basically don't see these peaks and
• 43:31then with re expression of happy
• 43:33when we see rescue and again the
• 43:35peaks appear again.
• 43:37Another way to validate these data
• 43:40and look at it functionally is to
• 43:43perform a new technology known as Cloud Nine,
• 43:46and this is a approach that
• 43:49was developed by Kevin Wang at
• 43:51Stanford where you use Casper,
• 43:53Chris crisper CAS 9 to basically
• 43:56target certain sequences in the genome,
• 43:58treat with abscisic acid,
• 44:00which then forms and reversibly
• 44:02loop that's induced,
• 44:04and then you can see the effects downstream.
• 44:08So this is what Mark did and this
• 44:10is again showing our sappy one,
• 44:12and the various promoters
• 44:13and enhancer region,
• 44:14and again using G Arnie dimer pairs.
• 44:17If you knock down S isep or sappy
• 44:19one treat with this, I said the one,
• 44:22you see that there's no expression
• 44:24of these two.
• 44:25He drop protein genes.
• 44:27If we then induce looping using
• 44:30Cloud nine at at the EP1,
• 44:32which is here to transducer to
• 44:35facilitate expression of his HSP.
• 44:37A1A we see that there's expression
• 44:40but not with the other JPA 1D.
• 44:43If you then loop at EP2,
• 44:46which is here so that the enhancer
• 44:49is next to the P2 for HSV one a B1B,
• 44:53you see that there's expression of
• 44:56this gene and also slightly of HPA 1A.
• 45:00And then finally this is just a
• 45:03control showing there's no induction Eugene.
• 45:06So correlating with that is also
• 45:09me P expansion.
• 45:10We we knocked down SFB one,
• 45:13but then we expressed it.
• 45:15We see increase.
• 45:16We also see it by looping of P1
• 45:20and P2 increase in comparison
• 45:22to controls and we don't see any
• 45:25effect on HCS or the CMP population.
• 45:29So can
• 45:29I just clarify what's the experimental
• 45:32design here for this expansion
• 45:34and the CMP right? Starting with
• 45:37this is looking at 34 positive CD,
• 45:4071 low population of cells,
• 45:42but they've been cultured in vitro
• 45:44plus and minus for how yes, all of these
• 45:47yeah and then treated with the cloud 9,
• 45:50so you're then treating with the
• 45:53dimer peers and then with abscisic
• 45:55acid to induce the looping. But
• 45:57what you're starting with me P.
• 46:01We're starting at the beginning and
• 46:03then waiting until the stage where
• 46:05we would see these cells appear.
• 46:07OK, yeah. Yeah, and then we also look.
• 46:12We also examine the Seppi 1S I or any
• 46:16induced Rizzo Blastic Buster Apolysis
• 46:18showed that with the looping at P1
• 46:22and P2 we see increasing with robust
• 46:24increase in CD41A positive cells
• 46:27but not in Salem be my light cells.
• 46:32The similar findings were also
• 46:34observed in cells that had SHRPS 19.
• 46:36So in our PS90 knockdown cells
• 46:38the same thing happened.
• 46:40If we loop it P1 we see
• 46:43the expression of HSP 1A.
• 46:45If we loop at P2P CHSP A1B
• 46:47expression and then controls,
• 46:49we don't see and then this is just
• 46:52Western blot showing the expression
• 46:54a day five for both of these.
• 46:58Experiments.
• 47:01So looping appears.
• 47:02He also rescues Happy one,
• 47:04defects and healthy megakaryocytes.
• 47:07Here's colony essays showing
• 47:09our looping experiments and
• 47:11knockdown of 71 so by looping
• 47:14itself we can see increasing BFUE.
• 47:17Nothing with the CFU GM.
• 47:19Also, in liquid culture we see
• 47:21just with looping alone we can see
• 47:24improvement and C 235 and 41 a,
• 47:26but not in CD11B.
• 47:27So again, that tells us that
• 47:30the looping is critical for the
• 47:32transcription of these genes to
• 47:34take place and also for the effects
• 47:38on CD235 and C41 proliferation.
• 47:40Finally, to link this all back to God.
• 47:43Oh one. As I mentioned earlier,
• 47:45the God one is regulated HSP 70 being
• 47:48available to prevent the degradation of God.
• 47:51Oh one.
• 47:52So here we see 235 positive
• 47:54cells E 41 positive cells,
• 47:56gotta one expression.
• 47:57And if we then take our knockdown
• 48:00of essay of sappy one,
• 48:01we see that the looping can restore
• 48:04the expression of God in one because
• 48:07now you're getting HP 70 expression.
• 48:09So it's able to prevent
• 48:11degradation of God 01.
• 48:13Godwin expression is just shown
• 48:15here showing the increase in
• 48:17expression correlating with with
• 48:19the data I just talked about in
• 48:22an MVP expansion also correlated
• 48:24with with the God one expression.
• 48:26So if you did a western on
• 48:29the top right for the HSP 70,
• 48:32it would parallel the gotta one.
• 48:34That's what we believe. Yes, yes.
• 48:36And so that goes back to our model where we
• 48:40are in normal conditions that 71 gradually
• 48:43decreases as cells become more committed.
• 48:47But we do see HP 70 increasing, which
• 48:50prevents gotten one from being degraded.
• 48:53There's some city state level there,
• 48:56but obviously once committed
• 48:57to pull through blast stage,
• 49:00we see significant increase in God,
• 49:02one in order to transduced jeans that are
• 49:05required for earthway differentiation,
• 49:08terminal differentiation.
• 49:09We knocked down sappy one prematurely,
• 49:12or, in the case of RPS,
• 49:1419, insufficiency, seven.
• 49:15That would be 1 levels are much lower,
• 49:18which does not allow HSP 70 P.
• 49:21It just be 70 should be expressed,
• 49:24which then blunts the expression of God.
• 49:26Oh, one so that we don't get the
• 49:29normal increase and got a wine.
• 49:32And we believe this contributes
• 49:34to the anemia phenotype.
• 49:36It's just a schematic of what we
• 49:39believe is happening in CMP, ME P.
• 49:42The ratio relative ratios that
• 49:43occur at this stage normally,
• 49:46and then at the risible stage
• 49:48would receive an abundance of God.
• 49:50One protein that basically
• 49:52overwhelms the HSP 70,
• 49:53but that we have enough to be able
• 49:56to induce Raceway specific genes,
• 49:58so I'd like to end there.
• 50:01And then just to acknowledge
• 50:02people who have done the work.
• 50:03I think I've been knowledge Mark
• 50:05throughout the talk as well
• 50:06as the other Members I lab who
• 50:08contributed to this project.
• 50:09The collaborators at Stanford
• 50:10and also the other collaborators
• 50:12that not at Stanford in,
• 50:14particularly Diana Vanessa,
• 50:15for their help in doing the experiments
• 50:17with happy One and Megakaryocytic with
• 50:19recite progenitors and the funding
• 50:21sources that supported this work.
• 50:23So I'm happy to answer questions.