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- [00:02]
A
Welcome to the American Society of Hematology Conversations with Blood Authors. This Blood Podcast episode is hosted by Dr. Laurie Sin. She talks with Dr. Carolina Florian about her article entitled A Notch trans activation to CIS Inhibition Switch Underlies Hematopoietic Stem Cell Aging and she discusses the article off the Shelf Dual CAR Ink T Cell Immunotherapy Eradicates medullary and Leptamin KMT 2 a rearranged leukemia with Dr. Anastasios Caradimitris.
- [00:40]
B
Hi, I'm Laurie Sen, Podcast Editor at Blood and Today I'm joined by Dr. Carolina Florian from Ed Bell in Spain. She's the senior author of a new Blood Journal article entitled A Notch Transactivation to CIS Inhibition Switch Underlies Hematopoietic Stem Cell Aging. Thanks for joining us today.
- [01:04]
C
Thank you Lori for the invitation.
- [01:07]
B
Perhaps you can start by giving us some background on what was previously known about the role of Notch signaling in hematopoiesis and what led you to design this study.
- [01:18]
C
In my lab we are interested in how aging affects hematopoietic stem cells and in particular for this work we decided to focus on the bone marrow microenvironment and on Notch signaling. This is because Notch is one of the most important and developmentally conserved signaling pathway. It was known so far its important role during the early phases of hematopoietic stem cell development and there is wonderful work describing the role of Notch in priming the fate of HSCs during embryogenesis. But in the early days after birth and this is mainly work done with mouse model Notch signaling and Notch activation in hematopoietic stem cells is known to decrease and the rule of Notch signaling later in adult and aged mice was largely unknown. We thought that it was worth investigating it because previously we highlighted some important impact of the Notch signaling during the regeneration of the vasculature and we decided here to deepen our role and deepen our knowledge on the role of Notch signaling for hematopoietic stem cells biology in aging.
- [02:53]
B
Great. Can you briefly summarize your methodological approach and your primary key findings?
- [03:00]
C
One of the major technique that we use in this work is 3D histology. Basically we have very well established in the lab a protocol to image in 3D and provide reconstruction of the position of hematopoietic stem cells within the bone marrow microenvironment and through this technique we were previously able to demonstrate applying deep learning pipeline of image analysis that the localization of HSCs within the bone marrow is not random and is significantly changed with aging and that the position per SE of HSCs within the bone marrow niche has a predictive value to the function of the hscs. Because we are able to understand and determine whether an HSC is young or old just based on its position. We use this technique to investigate how Notch signaling affects activity of hematopoietic stem cells. And we use this technique to see the stem cells and their level with mouse models that have Notch GFP reporter. This model traces Notch activity in the cell with GFP and it's a very sensitive model. And we could show that with aging there is increase in the amount of stem cells that are inactive. And these cells are the one that do expand with aging. We also could measure that they expand in cluster and actually that these cluster of stem cells that expand in the age bone marrow are myeloid bias, accounting for the strong myeloid skewing in the elderly. Moreover, we also use other mouse model, always with this imaging technique, to knock out selectively one of the major Notch ligand in the bone marrow niche. This model is a JAK2 knockout and we prove that this is enough to decrease Notch activity and to induce most of the phenotype that I have just mentioned that we see in age stem cells. Meaning that this effect of expanding HSCs and expanding them in clonal cluster is primarily induced by the decrease of JAK2 ligand expression from the bone marrow vasculature.
- [05:43]
B
You recognize this seemingly adapted switch from Notch transactivation to CIS inhibition that ultimately seems to be maladaptive for hscs. Can you comment on this?
- [05:58]
C
Yes, this is one of the aspects that we discuss in the paper and that we found particularly interesting because we indeed observe this switch where HSCs in aged mice start expressing themselves the JAK2 ligand that is absent from the microenvironment and this induces a cheese inhibition. So crosstalk with the Notch receptor expressed by the hscs and contributes to switch off Notch activity in these cells, leading to the functional impairment that we demonstrate with transplantation assays. And this is actually for us very interest because a similar mechanism was described in the early step of embryogenesis where QIS inhibition was meant to preserve AHSCs in a quiescent state and somehow allow them to move from the most proliferative phase to a more adult phase where they are less proliferative. But here this effect is actually resulting in a kind of, as you said, maladaptive phenotype, because in this context, this leads to the loss of the regenerative potential of these cells. That is actually one of the major problem with aging of hematopoietic stem cells.
- [07:26]
B
In her commentary, Nadia Carleso from City of Hope comments that these findings may actually lead to potential strategies to rejuvenate aged hematopoietic systems. And based on your findings, do you think what types of strategies might be considered?
- [07:42]
C
The commentary is actually a wonderful piece, a wonderful article because it is highlighting some aspect of our work that didn't have enough space to come out in the final manuscript, but are indeed very interesting because we see that this mechanism of notch trans to cheese inhibition switch actually impact on the capacity of HSCs of dividing and their symmetric division increases and this leads to this clonal expansion and loss of regenerative potential. And actually we think that there are possibility of intervention by targeting the surrounding niche and prevent the clonal expansion before it happens. And this is actually what we aim to investigate in the future in the lab, how to target the microenvironment to possibly reduce this clonal myeloid bias expansion.
- [08:44]
B
So moving forward, what are the next questions that you aim to target?
- [08:48]
C
One of the major interests in the lab remains understanding how the microenvironment is changing with aging and whether we can identify targetable pathways and mechanism that can offer us possibility of intervention. And one of the other aspect that we are really working hard on is trying to translate our findings also in human setting. The work that is now published in blood is exclusively on mouse model, mainly because for us having access to a good amount of human biopsies is very complex and still tools are challenging to implement. But we would like really to find a way to translate our finding. And preliminary studies actually show that what we observe in the mice, it's recapitulated also in the human samples. And last but not least, we think that a lot of what we are doing about aging, beside being an important aspect for intervention in the sense of regenerative medicine, can also help our understanding in disease prevention. And so we also look in the direction of clonal hematopoiesis and acute myeloid leukemia that share different features with aging of hematopoietic stem cells and of the hematopoietic system.
- [10:17]
B
Wonderful. Thank you for sharing this with us today. I hope everybody has enjoyed our conversation with Carolina Florian, who's discussed the newly published article A Notch transactivation to CIS inhibition switch underlies Hematopoietic Stem cell aging. This important study offers a new framework for the understanding of hematopoietic stem cell aging which may ultimately inform future regenerative strategies and this article is now available on bloodjournal.org we're also joined today by Dr. Tassos Caradamitras from Imperial College London. He's the co senior author along with Andy Roy of a new blood journal article entitled off the shelf Dual CAR Ink T cell immunotherapy eradicates Medullary and leptomeningeal high risk KMT2 a rearranged leukemia. Thanks for joining us today.
- [11:17]
D
Thank you for the invitation. I'm very excited to be with you.
- [11:20]
B
Perhaps you can start by providing the rationale for why an ink T cell approach was desirable for KMT2 a rearranged leukemia.
- [11:31]
D
Because we study invariant KT cells, the biology and the therapeutic applications and in previous and current work we demonstrated their higher performance when compared with T cells both engineered with the same car. So we demonstrated previously that for B cell lymphoma, currently we have it in multiple myeloma and we thought that would be also the case for high risk acute lymphoblastic leukemia. So that was the motivation of that first and second is because our friends and colleagues in Oxford also work on high risk acute lymphoblastic leukemia. So we teamed up to put our knowledge and expertise together to see if we can develop something that might do better than the established CAR T or tolerable CAR T immunotherapy for this high leukemia, which particularly affects infants, and see if we can improve outcomes at least at the clinical stage.
- [12:27]
B
And what are the key features in the design of this dual targeted CAR ink T cell therapy that really exploits the features of this high risk leukemia?
- [12:37]
D
Well, the obvious of course first target would be CD19, but that is not enough in a clinical setting. We know good number of infants and children actually relapse with CD19 negative disease, so we had to target the second leukemia associated antigen. And so it happened. One of our other friends in Oxford, Tom Milne, is an expert on the transcriptional regulation of CD133, which is almost exclusively expressed in high risk KMT2 a rearranged leukemia. So we agreed that CD19 and CD100 or Chrome 1 would be the two targets that we would go after with a dual car approach and also exploiting the known advantages of INTD over T cells. And that's what we did.
- [13:29]
B
Now it seemed like in doing this study you also uncovered a somewhat novel mechanism that you didn't anticipate that may also be an advantage for this type of car.
- [13:39]
D
Yes, that was one of the exciting aspects of the work. First, we wanted to Explore why CAR NKT were doing better than CAR T in both in vitro and in vivo models. And the mechanism we identified is centered around NKG2D, which is an NK activating receptor, also of course expressed by other innate cells like NKT cells. And we found them to be a steady state be expressed at a higher level of them in T cells. In CAR nkts expression is higher than CAR T counterparts. But that was not unexpected as been described before. The exciting part of that was that when we co culture CAR NKT with leukemia cells and we checked expression of NKG2D the next day it went up from 40% average to 100%. Whereas the upregulation of NKG2D on Car T was very modest. And when we did classical experiments where we had the presence of the CAR in the system, singly or in combination or none of them, we actually established that the upregulation of NKG2D required the car target and the CAR expression. So was CAR target and CAR expression dependent. And when we block this interaction with antibodies against NKG2D, we can actually abrogate the anti leukemia effect of CAR NKT over CAR T. Which meant that if you can model that, you can envisage a scenario where a CAR NKT engages with a CAR target expressing leukemia cell, it kills it, it upregulates NKG2D, and if the leukemia cell next to it doesn't express one or both of the CAR targets, it can still be killed through the NKG2D dependent manner. So that was really a very exciting finding. And this is something that we're planning, we're already actually trying to exploit and enhance. So we can really. And of course NKG2D responds to Myca, MiGB and Eula MPP ligands, which are supposed to be upregulated in stressed tissues, including in malignant tissues.
- [15:58]
B
So another interesting advantage of this type of approach is that it seems to have a better ability to clear cells from the CNS than standard CAR T cell approaches. Why does it have that advantage?
- [16:11]
D
We knew from our previous work that CAR NKT could clear established secondary B cell lymphoma very, very efficiently in preclinical models, of course, whereas CAR T could not do that. So we tested this idea also in the context of leptomeningeal disease, because actually this high risk leukemia has higher incidence of meningeal leukemia than the other types of conventional acute lymphoblastic leukemia. So that was another good model to compare the efficacy of the two. We found that palinget, they can eradicate well established advanced meningeal leukemia as well as bone marrow disease and splenic disease. And that paper of course we didn't explore the mechanisms, but in a subsequent work that we presented Atash a month ago or so, we identify VLA4 as an integrin which is preferential and higher expressed in INKT compared to T cells and that allows CAR NKT to adhere more firmly on endothelial cells, including endothelial cells of the blood brain barrier, so allow them to start the process of transmigration from the systemic circulation into the brain and meningeal spaces. And we demonstrate that also in vivo models, further in vivo models and we think that's one of the main mechanisms that provide this advantage of CAR NKD for treating brain and managerial based blood cancers. But of course we are planning to explore that and exploit it further in other types of brain cancers.
- [17:54]
B
In his commentary, Federico Simonetta from the University of Geneva noted that demonstrating the persistence of these CAR ink T cells in the clinical setting will really be critical to probably prove their value. Do you think that this could be.
- [18:10]
D
A limitation that it has not been tested yet in clinical setting? Well, this is a problem for the whole field of IMQT immunotherapy because we don't have much clinical data at the moment except for data presented clinical data by the Baylor Group. However, it it won't be too long before at least our technology will be tested in the clinical setting. Our technology is licensed to a biotechnology company in Australia and they're expecting to get FDA approval for a phase one clinical trial for BCL lymphoma in Australia and the USA during the course of 2026. And of course that will provide a big boost if things go well for developing also the type of immunotherapy we described in this article, which of course is more rare disease and would require a multi center approach in the uk, Europe. Why not the USA too?
- [19:11]
B
Well, I think this is an exciting time for sure. I hope you enjoyed our conversation today with Tasso's Karim Demetrius who has discussed the newly published article off the Shelf Dual CAR ink T cell immunotherapy eradicates medullary and leptomeningeal high risk KMT2 a rearranged leukemia. This important study demonstrates the promising efficacy of CAR ink T cells targeting CD19 and CD133 for KMT2A rearranged leukemia. This article is now available on bloodjournal.org.
- [19:54]
A
Thank you for listening to this blood podcast of conversations with blood authors to read the articles, visit bloodjournal.org this episode is copyrighted by the American Society of Hematology.