Les conférences invités du CHO
CHO Invited speakers
One of the fundamental questions in hematopoiesis is how the different blood cell lineage are specified. Elucidating the cellular pathways of blood lineage formation is important not only to understand normal blood cell production, but also to identify the cells-of-origin of hematological malignancies and identify the molecular mechanisms that govern normal hematopoiesis, as well as the perturbations these undergo during leukemogenesis. Single cell biology, which comprises both single cell genomics and single cell-level functional analysis, has in recent years allowed significant advances in these areas. Here, I will describe how single cell transcriptome analysis has allowed us to identify new hematopoietic
progenitor populations and generate more accurate cellular models of murine and human hematopoiesis, and how such improved cellular models can inform the study of the etiology of acute leukemias, using GATA2 and CEBPA mutant bi-lineage acute erythroleukemia (AEL) as an exemplar. In addition, I will discuss the cellular mechanisms by which lineage commitment occurs, and in particular how to distinguish between models involving intermediate oligo-potent progenitors and those predicting direct transition from a multi-potent to an uni-lineage state. Finally, I will address the molecular mechanisms by which the choice between myeloid and megakaryocytic-erythroid lineage occurs at the molecular level, and how this involves the same epigenetic mechanism that allows the generation of bi-lineage leukemia-propagating cells in AEL, underscoring how understanding basic developmental mechanisms informs the study of blood cancers.
Relapse is associated with dismal prognosis in patients with acute leukemias and is typically caused by tumor stem cells which survived treatment, e.g., due to a dormant and resistant phenotype. New therapeutic options are intensively needed to eliminate dormant and resistant leukemia stem cells.
Using patient-derived xenograft (PDX) mouse models and genetic engineering, we showed that acute leukemia cells are able to switch back and forth between a cycling, sensitive state and a dormant, resistant state, both in acute lymphoblastic and acute myeloid leukemia (Ebinger et al., Cancer Cell 2016; Ebinger et al., Haematologica 2020). Resistant cells also evolve upon prolonged treatment of PDX leukemia cells in vivo (Wirth et al.,
Leukemia 2022) and might be restricted to subclones within a single heterogeneic leukemic tumor (Zeller et al., J Hematol Oncol 2022). In search for linchpins to retrieve resistant cells from their protective bone marrow niche, we performed CRISPR/Cas9 dropout screens in PDX models in vivo; we discovered the metalloproteinase ADAM10 as essential molecule and putative novel therapeutic target to reduce the interaction between acute leukemia and the bone marrow niche and to eliminate dormant and resistant acute leukemia stem cells (Bahrami et al., Molecular Cancer 2023).Taken together, dormancy and treatment resistance represent plastic, niche dependent features in acute leukemia stem cells which enables novel treatment options such as targeting cell surface molecules including ADAM10.
Notch1 is a well-established lineage specifier for T cells and amongst the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). Activating mutations in the NOTCH1 gene have also been identified in B cell chronic lymphocytic leukemia and Mantle cell lymphoma, and NOTCH2 mutations are found in splenic marginal zone B cell lymphoma. Thus, the NOTCH pathway has become an attractive therapeutic target for Notch-driven leukemias as well as for Notch driven solid cancers. Current approaches to target Notch signaling are limited to small molecule g-secretase inhibitors (GSIs) and inhibitory antibodies against Notch receptors and ligands. However, due to on-target and off-target
toxicities associated with blocking antibodies and GSIs, these anti-NOTCH agents failed to advance in clinical trials.Here I will describe the discovery of a novel class of small molecules able to target the NOTCH transcription complex. These molecules act as protein-protein interaction inhibitors compromising the assembly of functional NOTCH transcription complex. I will present in vitro and in vivo characterization of the small molecule CB-103 and how this molecule was brought into currently ongoing phase II clinical trials. Moreover, I will discuss the importance to develop novel combination therapies with Notch inhibitors that have not been exploited yet in conventional T-ALL treatment as well as identifying prospective potential resistance mechanism.
Currently there is great interest in targeting mitochondrial oxidative phosphorylation (OXPHOS) in cancer. We have previously reported that chronic myeloid leukaemia (CML) stem cells rewire their metabolism and rely on upregulated OXPHOS for survival. However, currently available OXPHOS inhibitors have drawbacks such as short half-life or limited therapeutic index. Therefore, we performed a drug-repurposing screen to identify FDA-approved drugs that inhibit mitochondrial metabolism in leukaemic cells.
Our metabolism drug screen (of >1,100 FDA-approved drugs screened) highlighted lomerizine dihydrochloride, a L-type Ca2+ channel blocker used as an anti-migraine drug, as a candidate compound effective against
OXPHOS-dependent CML cells. In parallel, transcriptional analysis revealed that Ca2+ channel genes such as TRPC6 and CACNA1D are significantly upregulated in CML stem cells (CD34+CD38-) when compared with normal haematopoietic stem cells (HSCs). Moreover, patient derived CML CD34+ cells were shown to have increased ER mass (the main intracellular Ca2+ store) and subsequent increase in mitochondrial Ca2+ levels compared with normal cells. Therefore, we hypothesised that the observed increase in mitochondrial metabolism in CML stem cells was dependent on TRPC6 and CACNA1D and the increase in mitochondrial Ca2+ levels. Notably, CRISPR-Cas9-mediated TRPC6 or CACNA1D knockout, or lomerizine treatment, significantly reduced cytoplasmic, ER and mitochondrial Ca2+ levels, and decreased mitochondrial oxygen consumption rate, indicative of reduction in OXPHOS. Furthermore, by tracing labelled glucose we observed significant reduction in activity of mitochondria located Ca2+ dependent dehydrogenases and overall reduction in tricarboxylic acid (TCA) cycle activity. Of clinical relevance, lomerizine treatment, alone or in combination with imatinib, targeted CML stem cells in vitro without affecting normal HSCs, and enhanced mouse survival using well-established CML xenograft model.In summary, we reveal for the first time that Ca2+ regulation is disrupted in CML stem cells and influx via TRPC6 and CACNA1D is crucial for maintaining high mitochondrial Ca2+ levels and enhanced mitochondrial OXPHOS. This renders CML stem cells sensitive to lomerizine, and FDA-approved Ca2+ inhibitor identified following metabolism-specific drug-repurposing screening.
Functional attrition of the hematopoietic stem cell (HSC) compartment is thought to be a driver of hematologic aging, leading to the development of a range of age-associated diseases. We have previously identified inflammation and infection as agonists that can promote HSC attrition and aging. In this talk we will explore the impact of intrinsic and extrinsic factors upon the quantitative and qualitative mutation burden of HSCs during aging, as well as the degree of heterogeneity that HSCs demonstrate in lineage bias and self-renewal capacity.
Down’s Syndrome (DS) predisposes individuals to haematological abnormalities, such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood. To understand dysregulated hematopoiesis in DS, we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human foetal liver and bone marrow samples from three disomic and 15 trisomic foetuses. We found that differences in gene expression in DS were both cell type- and environment-dependent. Furthermore, we found multiple lines of evidence that DS haematopoietic stem cells (HSCs) are “primed” to differentiate.
We subsequently established a DS-specific map of enhancer-gene relationships in disomic and trisomic HSCs using 10X Multiome data. By integrating this map with genetic variants associated with blood cell variation, we discovered that trisomy restructured enhancer-gene maps to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Further, as DS mutations display a signature of oxidative stress, we validated both increased mitochondrial mass and oxidative stress in DS, and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Altogether, our single-cell, multi-omic resource provides a high-resolution molecular map of foetal haematopoiesis in Down’s Syndrome and indicates significant enhancer-gene restructuring giving rise to co-occurring
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Adipose depots are frequently found at proximity of invasive human solid tumors such as breast and prostate cancers. In the presence of tumor cells, mature adipocytes exhibit phenotypic changes including dedifferentiation and delipidation and these “Cancer-Associated Adipocytes” contribute to tumor progression through several signaling molecules including lipid metabolites. The nature of this bidirectional crosstalk with hematological malignancies as well as bone metastasis within the bone marrow adipose tissue (BMATs) has only just begun to emerge. Two types of bone marrow adipocytes (BMAds) have been described. The BMAds that are present within the hematopoietic or “red” BMAT and in the “yellow” BMAT where
they are more densely packed. These two different types of BMAds respond differently to external stimuli at least in mouse models where yellow BMAds are named constitutive BM-Ads (cBM-Ads) whereas red BMAds are named regulatory BMAds (rBMAds). Few data have been obtained in human. We will discussed our latest results concerning the specific metabolic phenotype of human BMAds (cBMAds and rBMAds) as compared to “classical” adipose depots. According to this characterization, I will also present our data concerning the signals emanating from rBMAds that modulate tumor behavior with a specific emphasis on their metabolic crosstalk with cancer cells.
References
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Hernandez M, Shin S, Muller C, Attané C. (2022). The role of bone-marrow adipocytes in cancer progression: the impact of obesity. Cancer Metastasis Reviews 41:589-605
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Attané C, Estève D, Chaoui K, Iacovoni J, Corr J, Schiltz O, Moutahir M, Valet P, Reina N, Muller C. (2020). Human bone marrow comprise a new adipocyte subtype with specific lipid metabolism. Cell Reports 30 :949-958
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Attané C and Muller C. Drilling for Oil : Tumor-Surrounding Adipocytes Fueling Cancer.(2020). Trends in Cancer 6 : 563-604.
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Wang YY, Attané C, Milhas D, Dirat D, Dauvillier S, Guerard A, Gilhodes J, Lazar I, Alet N, Laurent V , Le Gonidec S, Biard D, Hervé C, Bost F, Ren GS, Bono F, Escourrou G, Prentki M, Nieto L, Valet P, Muller C (2017). Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells. JCI Insight 2: e87489. doi :10.1172
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B. Dirat, L. Bochet, M. Dabek, D. Daviaud, S. Dauvillier, B. Majed, YY Wang, A. Meulle, B. Salles, S. Le Gonidec, I. Garrido, G. Escourrou , P. Valet, C. Muller (2011), Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion. Cancer Research, 71: 2455-2465
Mutations in the calreticulin (CALR) gene are the most common after JAK2V617F in essential thrombocythemia (ET) and primary myelofibrosis (MF), two non-BCR-ABL myeloproliferative neoplasms (MPNs) characterized by the dysregulation of the megakaryocytic lineage (Klampf T et al, N Engl J Med, 2013; Nangalia J et al, N Engl J Med, 2013). Mutations can be either insertions or deletions in exon 9 of CALR, both of which result in a frameshift that leads to the expression of a novel, positively charged C-terminus as well as the loss of the KDEL endoplasmic reticulum (ER)–retention motif. We and others have shown that the oncogenic activity of CALR mutants (CALRmut) relies on their specific and stable interaction with the thrombopoietin
receptor MPL and the constitutive activation of the JAK2/STAT signaling pathway (Marty C et al, Blood, 2016; Chachoua I et al, Blood, 2016). The interaction between CALRmut and MPL is dependent on the novel C-terminus and originates in the ER but activation of MPL signaling occurs at the membrane. In addition, secreted CALRmut can amplify the oncogenic signaling by acting as a rogue ligand for MPL (Pecquet C et al, Blood, 2023). Thus, the CALRmut stands as a neoantigen at the cell surface paving the way to new targeted therapies. As a first approach to better understand the mechanism of the CALRmut and their roles in the pathophysiology of MPNs, we generated murine retroviral bone marrow transplantation models of the two most frequent CALRmut found in patients, a 52 bp deletion (del52), predominant in MF, and a 5 bp insertion (ins5), more presen t in ET (Marty C et al, Blood, 2016). Second, we developed conditional inducible del52 and ins5 knock-in (KI) mouse models. The del52 KI mice develop a more severe disease than the ins5 animals, including thrombocytosis progressing to MF, MK hyperplasia, hypocellularity of the BM and splenomegaly associated with extramedullary hematopoiesis, amplification of hematopoietic stem cells (HSCs). These phenotypes are amplified by homozygosity. Using competitive engraftments with increasing percentages of homozygous KI cells, we determined that del52 induces a competitive and proliferative advantage of HSCs while ins5 induces a slower competition of wild-type hematopoiesis and little HSC accumulation (Benlabiod C et al, Nat Comm, 2020). This was confirmed by assessing an increased number of HSC divisions in presence of del52 compared to ins5. We have been using these KI mice to understand the mechanisms responsible for the phenotypic differences between the two types of CALRmut, such as the activation of the JAK/STAT pathway and the unfolded protein response, and as pre-clinical models to test potential therapies including interferon alpha and a CALRmut-monoclonal antibody developed in collaboration with Incyte. The clinical and molecular responses of these therapies were assessed in the KI mouse models and show very promising results with the monoclonal antibody in specifically targeting disease-initiating stem cells without compromising wild-type hematopoiesis.
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Cancer is a pure problem of evolutionary biology, from its origin in the history of life to its development in real time in a sick person. I will present various avenues of research currently being explored by evolutionary biologists, including: Why and how did cancer appear with the emergence of multicellularity? Why are some cancers transmissible? Why are some species more vulnerable to cancer than others? What is the evolutionary significance of phenotypic changes in tumour-bearing individuals? Can evolutionary science improve cancer therapies? I will conclude by stating that the traditional separation between medicine and evolutionary ecology remains a fundamental limitation that must be overcome if we are to fully understand complex processes such as cancer.