From Steroid Resistance to Skeletal Defects: Navigating the Curiosities of Diamond Blackfan Anemia

Transcript

• 00:00Thanks, Diane, I'm so sorry that I
• 00:02don't get to meet you in person's,
• 00:04of course, but it's always fun to to
• 00:07see you and talk science with you.
• 00:09So yeah, today I'm going to
• 00:11talk about 2 two main aspects
• 00:13of what we studying in the lab.
• 00:15The first one is really going to
• 00:17be from my first love and what I've
• 00:20been studying for the past 12 years,
• 00:22which is a ritual poiesis.
• 00:24And in the second part we're going
• 00:26to move towards something that I
• 00:28I was totally a Neo fit for the.
• 00:31And I would say, five years ago,
• 00:34four years ago,
• 00:35which is really looking into
• 00:38skeletal defects in this disease
• 00:40called Diamond Black fan anemia so.
• 00:43Here is my computer of course.
• 00:45OK, here is an overview of the
• 00:47talk in the first spot I'm going
• 00:50to give a general introduction on
• 00:52Diamond Black fan anemia because
• 00:54I'm not sure about the audience,
• 00:56but everyone knows about the disease.
• 00:58Then I'm going to get right into
• 01:00the subject and talk to you about
• 01:02this study that we published last
• 01:04year on the mechanism of action
• 01:07of steroids during normal and
• 01:09disordered humanary choices with a
• 01:10focus on Diamond black fan anemia,
• 01:12and I'll try to.
• 01:14To insist on understanding the
• 01:16developmental differences and
• 01:17the role of this protein P 57.
• 01:19Keep two in the mechanism of
• 01:22action of steroids and then.
• 01:24I will go to modeling the skeletal defects,
• 01:27observing DBA and I'll get back
• 01:29to Erato places at the end.
• 01:31But really the main take home
• 01:33message here is to understand
• 01:35that skeletal defects in Diamond
• 01:37Black fan anemia are mediated.
• 01:39In part, I would say by failure of Leo.
• 01:42Yeah,
• 01:43have you moved past the first slide or no?
• 01:47Yeah, it's not advancing on the screen.
• 01:50I don't know why I think we should.
• 01:55I shouldn't be the cohost Genie
• 01:58because it keeps popping in.
• 02:00You want to be the host?
• 02:02No,
• 02:03I don't want to be the host because I
• 02:06see all the people popping in here.
• 02:08So let me do something.
• 02:10What I was saying is really the
• 02:12take home that the skeletal
• 02:14defects in IVR mediated,
• 02:16in part by the failure of the
• 02:18mesenchymal lineages and how DBA can
• 02:20be a cancer predisposition syndrome
• 02:22and touch a little bit on that idea.
• 02:27Other side moving now. Yes, perfect.
• 02:30So as I mentioned just a little bit
• 02:33of introduction on the clinical
• 02:35features of Diamond Black fan anemia,
• 02:38it Sahara trade hyperplasia an you
• 02:40can see on this bone marrow smear.
• 02:43Although in the US physician don't do
• 02:46a smear anymore on this patients that
• 02:49the bone marrow is pretty normal except
• 02:52for a Pau city of era trade pictures is
• 02:55the patients have congenital anomalies.
• 02:57They have skeletal and growth
• 02:59defects which will be.
• 03:01Focus of the talk today in the second part.
• 03:04Here you can observe, for example,
• 03:06try financial firm in this patient.
• 03:08This was documented by Adriana Blouse
• 03:10and Jeff Lipton several times in
• 03:12the literature and they also have
• 03:14a cancer predisposition and I will
• 03:16insist on that because I think that's
• 03:19one of the most fascinating questions
• 03:21that remained in the field.
• 03:25So just a little bit of history here.
• 03:29The Diamond Black fan anemia,
• 03:31the classic diagnostic criteria was
• 03:33described by Joseph in 1936 and then
• 03:36rebuilt by Diamond Black Fan in 1938.
• 03:38First as pure red cell aplasia.
• 03:41That led to the classic definition of a
• 03:44moderate to severe microcytic anemia.
• 03:46And I'll get back to that at the end
• 03:50because that's what I've been a big
• 03:53problem in the field is to model.
• 03:55Diamond Black fan anemia in the classic
• 03:59mouse model systems that we have
• 04:01because it's very difficult to model A
• 04:04macrocytic anemia with an increase MCV.
• 04:07That is.
• 04:08I complained by reticular cytopenia,
• 04:10a decrease in reticular site due to
• 04:13stress erythropoiesis that happens in the
• 04:15spring in the mouse and as I mentioned,
• 04:18bone marrow is normal cylinder with
• 04:20opacity of red cell precursors.
• 04:22It's diagnosed at an age less than a year.
• 04:27But now we have expanded the definition
• 04:29of DBA with criteria that come with
• 04:32from a more robust Epidemiology as a
• 04:35result of international registries,
• 04:37and I will point out the importance
• 04:39of this clinical registries such as
• 04:42the one we have, a defined Steam,
• 04:45the diamond black fan anemia,
• 04:47registry of North America,
• 04:48but also the ones from Europe in the UK,
• 04:52Germany, Italy, Sweden.
• 04:53Of course France and many others.
• 04:55Gene discovery was very important in helping.
• 04:58Redefined the disease diamond black
• 05:00fan and 23 genes now like categorized
• 05:03as DBA jeans,
• 05:0511 of them discovered through the
• 05:07Diamond Black fan anemia registry
• 05:10and this led to a modern diagnostic
• 05:13criteria because at first Diamond
• 05:15Black fan was discovered as a mutation
• 05:18in a gene encoding ribosomal protein.
• 05:21Whether in the small subunit or
• 05:23in the large subunit.
• 05:25But now we've studies from Vigesaa,
• 05:28Karen and Overs.
• 05:29Getting one mutations and over new
• 05:31mutations found and we're rather call
• 05:34it Diamond black fan anemia syndromes
• 05:36rather than Diamond black fan anemia.
• 05:41OK, so from there where are we going?
• 05:45The damn little bit of wording of the
• 05:48Diamond Black fan anemia registry.
• 05:50As I told you DBA is a rare disease.
• 05:53It's about the incidence is about 7
• 05:56chameleon birth and that computes to
• 05:58about 25 to 30 new patients a year in
• 06:02the United States here at Feinstein
• 06:04in the registry we have about 800
• 06:06patients that are enrolled from Dad.
• 06:09About 788 are coming from North America
• 06:12and the male to female ratio is 1 to one.
• 06:16In the demographics we have 670 patients
• 06:19that are alive which allows us.
• 06:22Kind of a decent access to samples,
• 06:25and that's that's pretty good when
• 06:27you want to do translational research
• 06:29element back to that, 118 are dead.
• 06:32Unfortunately,
• 06:32the median age is 20 three years,
• 06:35So what did they die from?
• 06:37They died from stem cell transplant
• 06:39related complications from iron overload,
• 06:41which is a big issue and I'll get
• 06:43to that because these patients,
• 06:46when they are not steroid responsive,
• 06:48OK they are transfusion dependent
• 06:50and when they get transfusions over,
• 06:52transfusions over transfusions.
• 06:53Of course they accumulate.
• 06:55Iran and Iran is a big issue.
• 06:58They also die from infection,
• 07:00sepsis, often colon cancer,
• 07:02and obviously tumors or from other cancer.
• 07:08OK, the current therapies I touched on the
• 07:11peripheral red blood cells transfusions,
• 07:13but the mainstay of the treatment when a
• 07:15patient is diagnosed with diamond black
• 07:18fan anemia is really corticosteroids.
• 07:20OK, the only cure for the patient
• 07:22is a stem cell transplant.
• 07:25However Anile insist on that
• 07:27the stem cell transplant doesn't
• 07:29protect them from getting cancer.
• 07:31So this is a family that agreed of course
• 07:35to provide this picture and the dad.
• 07:38Is actually.
• 07:41Responsive to steroids and the two
• 07:44daughters have a different phenotype
• 07:46and so that the first daughter.
• 07:49OK here is also having anemia that
• 07:52is responsive to steroid while
• 07:55the second one is transfusion
• 07:57dependent and we really don't know
• 08:00why steroids within the same family
• 08:03can for example lead to a response.
• 08:07Or a resistance,
• 08:08and so that's a problem that one of my MD,
• 08:12PhD student, Ryan Ashley,
• 08:14decided to back off for his PhD
• 08:17is to understand the mechanism of
• 08:19action of glucocorticoids to increase
• 08:21the red cell mass.
• 08:23Because, as I mentioned,
• 08:24it was really unknown.
• 08:26It was really unclear.
• 08:27I should say not unknown but unclear at
• 08:30which stage during era trade differentiation,
• 08:33the glucocorticoids.
• 08:34And here I'm going to just mention
• 08:37dexamethasone in culture was acting.
• 08:39There was some studies that
• 08:41were saying that yeah,
• 08:42glucocorticoid acts early
• 08:44at the BFUE stage OK,
• 08:46especially in the mouse.
• 08:47While over more recent studies by mayor
• 08:50of Sokolowski had shown it was acting
• 08:53later on on the later progenitor,
• 08:55the CFV,
• 08:56while in the human aritro places early
• 08:59studies had showed demonstrated that
• 09:01it was acting on the late progenitor.
• 09:05Indeed,
• 09:05we're not reinventing the wheel right?
• 09:08In 1976 already studies.
• 09:10Beautiful studies had been done showing
• 09:13that demonstrating that the CFU in
• 09:16number was increased as we increase
• 09:20the dexamethasone concentration.
• 09:22So if we go into human arixtra places
• 09:25now everything is happening in the bone
• 09:28marrow from the hematopoietic stem cell,
• 09:30hematopoietic stem and progenitor cell,
• 09:32and here Diane will have to excuse me.
• 09:35But I'm going to bypass all the stages
• 09:38and not argue about which one is which.
• 09:41I'm just going to go directly
• 09:43to the BFG and to the BFUECFUE
• 09:46to the area trade progenitors,
• 09:48which is what is really of
• 09:50our interest today because the
• 09:52terminal differentiation wants.
• 09:54And progenitor is entering the terminal.
• 09:56Differentiation there is not
• 09:58so much that happens, it's.
• 10:00Committed,
• 10:01it's differentiated and you just have
• 10:03four to five cell divisions over 5 days.
• 10:06That leads to the
• 10:09orthochromatic erythrocytes.
• 10:10And the processes of a new creation
• 10:12that we still don't fully understand.
• 10:15Leading to the reticular site that
• 10:18remodels its plasma membrane,
• 10:19degrades all the internal
• 10:21compartments and become the red
• 10:24blood cell that leaves for 120 days.
• 10:27OK.
• 10:29So we started this study by an
• 10:33observation very crude observation.
• 10:35We took CD 34 positive cells the
• 10:38so called hematopoietic stem and
• 10:40progenitor cells that were derived
• 10:42weather from peripheral blood PB
• 10:44for the rest of the talk or from
• 10:46cold blood CB and what we observe
• 10:48this that when we treated these
• 10:50cells with dexamethasone we observed
• 10:53an increase in the cell expansion
• 10:55for the cells that will be derived,
• 10:57derived from an adult source compared
• 10:59to the ones that were derived from
• 11:02the cold blood and actually we
• 11:04saw a decrease in the expansion.
• 11:06And this to us was really intriguing.
• 11:09What was happening here?
• 11:10This was going against not the dogma,
• 11:13but against all the protocols that had
• 11:15been using dexamethasone in their culture.
• 11:17Here we were using a culture systems that
• 11:20was not using any steroids at baseline.
• 11:23So we decided to go deeper into
• 11:26the mechanism.
• 11:27And what we found thanks to the method
• 11:30and that Manada had developed to
• 11:32study the surface markers for Louise
• 11:35and then we validated everything.
• 11:38Of course, with colony forming assays,
• 11:40we observed that it's actually the CFU
• 11:42E from the peripheral blood treated
• 11:45with dexamethasone that we're expanding.
• 11:48You can observe here that none
• 11:50of the BFU is order.
• 11:52CFU is affected except for
• 11:54the peripheral blood treated.
• 12:00When we when we then sorted
• 12:02to cells OK on based on this
• 12:05surface marker expression by fax.
• 12:07By flow cytometry we started the cells.
• 12:10We observed that again it was DCF,
• 12:13UE so-called CFU Ian.
• 12:15Here on code on code because they were
• 12:17not derived from colony forming assays
• 12:20but by surface marker expression.
• 12:22We observed that these were the
• 12:25ones derived from peripheral blood.
• 12:27When we then validated the
• 12:29findings by Colony.
• 12:30Coming essays,
• 12:31we observed that indeed the surface
• 12:34area the colony area for the CFO
• 12:37is formed by peripheral blood were
• 12:39indeed the ones that were responding,
• 12:41and I would not hear that very
• 12:44puzzling and interesting here was
• 12:47that the cold blood at baseline,
• 12:49without dexamethasone,
• 12:50we're having kind of the same size
• 12:52surface area as the peripheral
• 12:55blood treated with dexamethasone,
• 12:56meaning that maybe and here
• 12:58is just speculation,
• 13:00because I have no proof of that, but.
• 13:03Maybe this cells were already.
• 13:06Maximum in their response.
• 13:11The best proof to us would be to
• 13:14go back to the patient, right?
• 13:16Because if we are writing what we are
• 13:19asserting that it's a late progenitor
• 13:21that respond to dexamethasone,
• 13:23we have to prove that in a patient with DB.
• 13:27So we took three patients.
• 13:29OK, that are known to respond to steroids
• 13:31and what we did is that we measure the
• 13:35response to steroid by measuring simply
• 13:37the reticle sight count in the blood.
• 13:40OK, in this patients.
• 13:41After treatment with steroids and
• 13:44so my good friend I knew Nola in her
• 13:46clinic had three patients that she
• 13:49was following and what we did is that
• 13:52she treated them with Prednisone in
• 13:54that case because it was in the clinic,
• 13:57not in vitro and then measure the blood cast.
• 14:00What she observed is that
• 14:02within 7 to 10 days a response.
• 14:04Heretical site response was observed.
• 14:06This if we go back to the basics of
• 14:09very true Poesis tells us that it
• 14:12has to come from a late progenitor.
• 14:15It cannot come for a very early
• 14:17progenitor very early BFUE,
• 14:19because as I showed you before
• 14:21in the diagram in the schematics
• 14:23of human erythropoiesis,
• 14:24it will take way much longer to
• 14:27come from an early progenitor.
• 14:29So having said that,
• 14:32having showed that probably it's.
• 14:34Kind of late for genital that
• 14:36respond to steroids.
• 14:38We decided to go further
• 14:39down into the mechanism,
• 14:41but before going further down
• 14:43into the mechanism we need it.
• 14:45To figure out this,
• 14:47heterogeneity of human error trade
• 14:50progenitors to really try to understand
• 14:53better what is going on here,
• 14:55because this.
• 14:56Hierarchy here, and you're not
• 14:59going to contradict me on that.
• 15:02Is that?
• 15:03Yeah, it's nice,
• 15:04but really it's BFU E2 CFUE,
• 15:06but what else in between?
• 15:08When you look at them under the microscope,
• 15:12the colony forming assays and here
• 15:14provided by young Cheyenne Mohans lab?
• 15:17This is the same plate.
• 15:19Well,
• 15:20it's kind of subjective because
• 15:22all these colonies have different
• 15:24sizes showing probably heterogeneity
• 15:26of the era trade progenitors.
• 15:28An indeed in a previous study we
• 15:32published in 2018 based on these
• 15:34two surface markers CD34 CD 36
• 15:37that would define the BF you
• 15:40here in the lower right quadrant,
• 15:42or the CFU is in the upper left quadrant.
• 15:46We observed again a difference
• 15:49between cornbread and peripheral
• 15:51blood because we observed a double
• 15:54positive population that was
• 15:56present for a long time in culture.
• 15:58In cells derived from peripheral blood,
• 16:01but not much more transient, incorporated.
• 16:05And so we took the bed.
• 16:07Kind of crazy bet that maybe.
• 16:10This double positive population was
• 16:13the one that was responding to steroids.
• 16:16So.
• 16:17We went further and decided to
• 16:20characterize this population.
• 16:21We added surface markers OK,
• 16:24we tested on SA,
• 16:25tested 10s of surface markers.
• 16:27I think an she observed that city
• 16:30105 and CD 71 was giving the best
• 16:35resolution to discriminate between
• 16:37what we call now the immature
• 16:40and mature CFU E.
• 16:42And indeed, when we put them plated them in.
• 16:46People only that give rise to see a few E.
• 16:49This is the definition of a CFU.
• 16:51It responds to Ipoh only.
• 16:53Or incomplete media to generate the beer,
• 16:56and so we there.
• 16:58It's indeed consistent with what she
• 17:00says is that it's it is this image you
• 17:04see FUE that has the potential that I
• 17:07didn't get a chance to answer yet as
• 17:11the potential forming both of them.
• 17:14The BSU and the CFU.
• 17:17OK, when it's placed under any media.
• 17:22So it's not yet a fully committed CFU,
• 17:25E, but it's not a BF UA also, of course.
• 17:28We need single sarony seek for that.
• 17:31To really look at them deeply
• 17:33and characterize them,
• 17:35but it's consistent with what Miraf said,
• 17:37and I'm going to go further down with
• 17:40that because of the mechanism of action.
• 17:43So I'm I'm not going to spend
• 17:45a lot of time here.
• 17:47It's basically this image you see a few.
• 17:50We did respond, but importantly.
• 17:52When we treat.
• 17:54With dexamethasone,
• 17:55we see a reduction of the S
• 17:57phase as may arrive.
• 17:59Did in the mature CFD population.
• 18:03And how is that working so we get
• 18:06into the cell cycle and into the
• 18:08mechanism of Regulation an yeah may
• 18:11have had done everything in the mouse,
• 18:14so that was pretty easy, quote, unquote.
• 18:17To answer,
• 18:17we had P 57 keep to that she had
• 18:21published in cell in Science Advances.
• 18:23OK, that was involved in the
• 18:27regulation of steroids.
• 18:28Indeed,
• 18:29what we observed is that in peripheral blood.
• 18:32OK, there was a downregulation of P57 very
• 18:34early on by the seven of differentiation.
• 18:37Here, we observed that P.
• 18:3957 was totally down,
• 18:41while in the cold blood it
• 18:44was still remaining.
• 18:46P 27 however,
• 18:47was gradually increasing over
• 18:49the 14 days of culture,
• 18:52and here is Alpha Globin's control.
• 18:57Then
• 19:00we looked at the purified CFU E and
• 19:02here really not looking at image
• 19:05services mature because we did not
• 19:07have enough cells to do the Western.
• 19:09So when I say see if you hear is the mix
• 19:13of the two and we observe an increase in
• 19:17the expression levels OK of P57 Kip 2.
• 19:20Under CFIA, derived from peripheral
• 19:22blood but not cold blood.
• 19:24P. 27 wasn't changed.
• 19:26So how do we relate that to DBA now?
• 19:33Well, we went back to our patients
• 19:35with DBA and as I told you the benefit
• 19:38of being part of the Diamond Black
• 19:41fan registries that you have access
• 19:43to samples and patients are really
• 19:45eager to contribute to studies.
• 19:47As I'm sure you know and so we
• 19:48had a transfusion dependent or
• 19:51the steroid responsive.
• 19:52Just a note, it's much easier to get
• 19:54blood from transfusion dependent than
• 19:56the steroid responsive because the
• 19:59steroid responsive don't come to.
• 20:00Clinic they just called to get a
• 20:02refill on their steroids because
• 20:04the treatment works so they don't
• 20:06need to come to clinic.
• 20:08However, the transfusion dependent
• 20:09come over and So what we observe
• 20:12is that the expansion.
• 20:13Was indeed very effective in
• 20:15the series responsive,
• 20:16but not in the transfusion dependent.
• 20:20And here I have to give credit
• 20:22to Ryan because he did a lot of
• 20:25experiments working on about
• 20:2720,000 cells to get Western blots.
• 20:30Because you have a pool city of very
• 20:33trade progenitors as I mentioned.
• 20:35But what we observed is exactly
• 20:38what Diane was asking.
• 20:39OK, and what we what we published is that P.
• 20:4357 OK is actually up regulated.
• 20:47Industry responsive and not into
• 20:50transfusion dependent patients.
• 20:54And so we leave.
• 20:55We left it here on that paper,
• 20:59although we undertook some proteomics
• 21:01and the data are available and in the
• 21:05paper that was published last year,
• 21:07if I can change the slide
• 21:10in JCI and there are over.
• 21:14The targets, notably one that I'm
• 21:16sure is of interest of several people
• 21:19in the audience, such as NL 41,
• 21:22another cell cycle regulator that
• 21:24has been involved in proliferation
• 21:27and differentiation and cancer,
• 21:29and we are actively following up on that.
• 21:33With Pat and Lori Steiner so.
• 21:37In conclusion, for this spot,
• 21:39what I can tell you now is that we start.
• 21:43Understanding not completely,
• 21:44we don't have a complete picture,
• 21:47but still with starting making
• 21:49progress in response to steroid,
• 21:51we think that we can draw a comparison
• 21:54between healthy control patients with TBI,
• 21:57an adult versus neonet.
• 22:00Hematopoietic stem and progenitor
• 22:02cells and probably P 57 Kip, too,
• 22:05is central to the response to
• 22:07steroids on this imagery population.
• 22:10The image you see FUE population that
• 22:13we still have to further characterize
• 22:16an we actively doing that an.
• 22:21Leading to self renewal of this population
• 22:24an increasing the red cell mass.
• 22:26Thinking a lot of people.
• 22:28Of course, the members of the lab.
• 22:31Julian, who's been my partner in
• 22:33crime for the past 10 years with
• 22:36who I probably would be lost.
• 22:38The people in the lab who do an
• 22:41amazing work and the past members.
• 22:44My MD, PhD students that all
• 22:45graduated now and I'll collaborators
• 22:47within the Feinstein, Jeff and and.
• 22:52And of course,
• 22:53our outside collaborators my as
• 22:55I call him my my scientific dad,
• 22:58more Han Bat, who has been following me.
• 23:04I would say listening to me and
• 23:06for the past 10 years also or 12
• 23:09years listening to my complaints
• 23:11and everything very,
• 23:12very patient with me and then
• 23:14this will help us.
• 23:16We've all done older single serving
• 23:18is sick that I didn't have time to
• 23:21present today and you and all our
• 23:24collaborators from the USA and in France.
• 23:27And of course our funding source
• 23:29from NIH hanovers foundations.
• 23:30If you have any questions feel
• 23:33free to send me an email.
• 23:35Thanks for your attention,
• 23:36will be happy to take any questions.
• 23:42Thank you, Leo, for truly excellent talk.