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

January 27, 2021

Information

Lionel Blanc, PhD
Associate Professor, Molecular Medicine and Pediatrics, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell
YCCEH Invited Speaker
January 21, 2021

ID6142

To CiteDCA Citation Guide

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.