LYMPHOCYTE DEVELOPMENT AND LYMPHOID DISORDERS
The team “Lymphocyte development and lymphoid disorders” (U976; Team 5), is the continuation of the Inserm unit UMR 1126, which was created in 2014. Our research addresses fundamental questions related to the molecular mechanisms controlling the differentiation of hematopoietic stem cells towards lymphocytes and driving B cell oncogenesis. Based on our results, we seek to develop and evaluate novel treatments against B cell malignancies.
The main goals of our research are:
- To identify the molecular mechanisms which regulate the emergence of early lymphoid cells during human and mouse development and those leading to a decline in lymphoid production later in life.
- To investigate the molecular basis of MM progression and develop novel treatments with a special focus on Chimeric Antigen Receptor (CAR) -T cell based therapy.
Since the creation of our team in January 2014, our studies have:
- revealed a new architecture of fetal human lymphopoiesis,
- identified a new pathway involving the regulation of heterochromatin structure, which is impaired during aging and impacts on B cell commitment,
- shed new light on the activity of the Ets1 transcription factor on lymphoid development,
- provided insights to the role of the histone methyltransferase MMSET and long noncoding RNAs in the progression of Multiple Myeloma (MM).
Molecular mechanisms regulating lymphopoiesis during development and aging
A new road map of human lymphoid development
Project manager: B. Canque
Over the last twelve years, the group lead by B Canque has been working in the field of human lymphopoiesis which led to identification of a fetal population of T-lineage-primed thymus colonizers (Haddad et al. Immunity, 2006) and more recently to the development of a humanized mouse model to investigate the mechanisms regulating the emergence and dynamics of human lymphocytes. This led to the finding that human lymphoid development stems from functionally specialized subsets of CD127- and CD127+ early lymphoid progenitors (ELPs) which originate from multipotent intermediates referred to as LMDPs (Lympho-Mono-Dendritic Progenitors) (Alhaj Hussen et al. Immunity, 2017). This asymmetrical two-family model is further supported by the observation that the T cell potential segregates with the CD127- ELPs, and that CD127- and CD127+ NKIPs or pro-B cells differ as to both growth factor requirements and differentiation potentials. Over the last three years, the Canque group has focused on: (i) the mechanisms governing acquisition of lymphoid competence and subsequent restriction during progression from multipotent HSC to the stage of lymphoid-restricted progenitor; (ii) on the cell-intrinsic and extrinsic regulation of genetic networks controlling differential emergence of the CD127- and CD127+ ELPs.
Identification of new pathways linking growth stimulation to transcription factor activity and normal or oncogenic B cell differentiation
Project manager: JC. Bories
Cooperation between the B Cell Receptor (BCR) and cytokine receptors is critical for the efficiency and the specificity of B cell responses to pathogens. Upon interaction with their cognate ligand, these receptors induce activation of intracellular transduction cascades, which trigger the changes in gene expression that are required for cell proliferation, survival or differentiation. Proteins acting with or downstream of receptors have been extensively studied and were shown to be involved not only in the normal development of the immune system, but also in malignant transformation of T and B cells. However, the nuclear factors that translate signals into changes in gene expression remain incompletely characterized. Our work has focused on Ets-1, the founding member of a family of winged helix-turn-helix transcription factors (Leprince et al., 1983; Nunn et al., 1983) that plays a critical role in the control of lymphoid cell development and has been implicated in multiple biological processes including haematopoiesis, angiogenesis, and tumor progression (Dittmer, 2003).
Over the last 20 years, the group headed by JC Bories has provided major insights into the role of Ets-1 in T and B cell development (Bories et al., 1995; Eyquem et al., 2004a; Eyquem et al., 2004b; Mouly et al., 2010; Nguyen et al., 2012; Cauchy et al. Nucleic Acids Res. 2016). However, despite extensive international efforts, the molecular networks with which Ets-1 cooperates to regulate the development and the function of the immune system is still unclear.
To address this we are using genetic tools and Ets-1 deficient mice to investigate the function of this transcription factor in the response of early B cells to signaling from the pre-BCR and/or the IL-7R, and the role of its deregulation in lymphomatogenesis.
The role of heterochromatin in hematopoietic stem cell aging
Project managers: David Garrick & Michele Goodhardt
The focus of this project is to understand how epigenetic changes contribute to the aging of the hematopoietic system and to age-associated haematological malignancy. Hematopoietic stem cells (HSC) are responsible for the life-long maintenance of all lymphoid and myeloid blood cells. However, the function of HSC changes as we age, contributing to a decline in immune function and an elevated prevalence of haematological malignancies in the elderly (Goodhardt et al 2020). One of the most prominent changes occurring with age is a decline in the ability of HSC to generate antibody-producing B-lymphocytes. Recent studies have also shown that chronic low-level inflammation, which is a feature of aging, can directly affect HSC and cause aging-like phenotypic changes in the hematopoietic compartment. However, the molecular mechanisms underlying HSC aging remain poorly understood.
A characteristic feature of aging in various cell types and model organisms is a decline in the integrity of heterochromatin domains, which are highly condensed and transcriptionally repressed regions of the genome that are essential for genome stability and the regulation of gene expression. Our recent studies of HSC chromatin have shown a reduction with age in global levels of histone H3 lysine 9 trimethylation (H3K9me3), a modification critical for heterochromatin formation (Djeghloul et al., 2016). This age-related loss of heterochromatin integrity is associated with decreased expression of the histone methyltransferase SUV39H1 in HSC and leads to transcriptional derepression of genomic repeat elements and to decreased B lymphoid output. We are currently investigating the impact of advancing age on the different heterochromatin compartments of human HSC, their contribution to functional changes in these aging stem cells and whether reinforcing this epigenetic axis can improve HSC function in elderly individuals. We are also carrying out transcriptomic and epigenomic profiling in mouse models to investigate the effects of chronic inflammation on HSC heterochromatin and to characterise the molecular pathways downstream of these epigenetic changes.
Pathophysiology of Multiple Myeloma and development of novel therapies
Multiple Myeloma (MM) is a genetically complex and incurable disease which is characterized by an abnormal clonal plasma cell infiltration in the bone marrow. Chromosomal translocations in which different oncogenes, such as the multiple myeloma SET domain (MMSET; also known as WHSC1) gene in t(4;14), are placed under the control of the enhancers of the Ig loci, are observed in around half of cases. These translocations, which result from aberrant IgH class switch recombination (CSR), are associated with adverse prognoses. During the last years, we have constituted a very large cohort of MM patients, including more than 200 cases carrying a t(4,14) translocation, in which genetic, biological and clinical features were extensively investigated (Chandesris et al., 2007; Hutchison et al., 2012; Jaccard et al., 2007; Moreau et al., 2011; Szalat et al., 2011). Using this series of patients, we have confirmed that the t(4,14) translocation indicates a very poor prognosis (Karlin et al., 2011), and that even the recently developed treatments have little effect on the course of t(4,14)-positive disease. We also revealed that the breakpoint within the MMSET locus may explain, in part, the prognostic heterogeneity of t(4;14) gammopathies (Lazareth A. et al. Haematologica. 2015). However, the molecular bases of this poor outcome remain poorly understood and effective treatment is still lacking. Our aims are to characterize the molecular mechanisms cooperating in plasma cell tumor progression and to develop novel treatments against MM.
The role of MMSET in terminal B cell differentiation and MM progression.
Project managers: Bertrand Arnulf, Jean-Paul Fermand and Jean-Christophe Bories
Terminal B cell differentiation relies on major transcriptional and epigenetic changes that allow immunoglobulin class switch recombination (CSR) and expression of a plasma cell- specific genetic program. MMSET (NSD2) is a histone methyl transferase which catalyzes H3K36 di and tri-methylation and which is deregulated by the t(4;14) translocation in around 15% of patients with Multiple Myeloma (MM). In order to gain insights into the function of MMSET in B cells, we have generated mice with B and T cells bearing deletion of the Whsc1 gene and therefore devoid of MMSET HMT activity. We found that deletion of MMSET inhibits CSR without affecting either IgH germline transcription or joining of DSBs within S regions by classical nonhomologous end joining (C-NHEJ). Instead, we find that MMSET inactivation leads to decreased AID recruitment and DSBs at the upstream donor Sμ region. Our findings suggest a role for the HMT MMSET in promoting AID-mediated DNA breaks during CSR (Nguyen et al. Proc Natl Acad Sci U S A. 2017).
Based on these results, we are now investigating potential cooperation between MMSET and AID (or other APOBEC family members) in genomic instability during terminal B cell differentiation.
The contribution of long non-coding RNAs to the pathophysiology of MM
Project managers: David Garrick
Over the last decade, long non-coding RNAs (lncRNA) have emerged as important modifiers of epigenetic state and chromatin structure. LncRNAs play critical regulatory roles during normal developmental and differentiation processes, and deregulation of lncRNA expression is increasingly implicated in the onset and etiology of malignant disease. In our group, we are investigating the role of lncRNAs in MM, using genome engineering (CRISPR and CRISPRi), transcriptomic and epigenetic approaches. Our recent studies have shown that one lncRNA, called CRNDE, is increased in tumour plasma cells of MM patients where it contributes to disease progression by sensitizing the cells to pro-tumorigenic IL6 signalling (David et al., 2020). In our ongoing studies we are carrying out high-throughput screens to further characterise the function of CRNDE by identifying its interacting partner proteins, as well as to identify other lncRNAs that could contribute to the etiology of MM.
Development of novel T cell based therapies for MM
Project managers: Bertrand Arnulf, Jean-Paul Fermand and Jean-Christophe Bories
Novel immune based therapies have shown promising results in the treatment of MM. Monoclonal antibodies (mAb) targeting CD38, such as daratumumab, have shown good therapeutic efficacy, both alone or in combination with normal standard of care regiments. However, many patients eventually relapse because of resistance mechanisms. Bi-specific T-cell engaging (BiTE) antibodies belong to a new class of immunotherapeutic agents that can recognize, on the one hand, a specific antigen on the surface of the target cells (i.e. tumor antigen) and, on the other hand, the CD3 on T lymphocytes. By activating T cells through the CD3 complex and recruiting them in the proximity of the target cells, BiTEs efficiently induce T-cell mediated cytotoxicity. We recently, developed and evaluated Bi38-3, a new anti-CD38/CD3 Bispecific T cell-engager antibody, which triggers specific T cell-mediated lysis of CD38-positive MM cells in vitro, ex vivo and in vivo (Fayon et al. 2020). Bi38-3 shows no toxicities on B, T and NK cells in vitro and showed efficacy even in patients that relapse after daratumumab therapy. Altogether, the data presented in this study identifies Bi38-3 as a selective and efficient compound for the treatment of MM, that could be used both as a front-line agent or at relapse (alone or in combination with additional drugs), and which should be evaluated further in MM patients. Based on these results, we have generated novel Chimeric Antigen Receptors (CAR) targeting CD38 at the surface of tumor plasma cells. In collaboration with the clinical departments of the St Louis Hospital (Paris) and the Intergroupe Francophone du Myélome (IFM), our goal is now to modify the CAR-T therapeutic strategy in order to ameliorate both its specificity and safety in MM patients.