Nicolas Robert

Characterization of a Transitional Preplasmablast Population in the Process of Human B Cell to Plasma Cell Differentiation

The early steps of differentiation of human B cells into plasma cells are poorly known. We report a transitional population of CD20low/−CD38− preplasmablasts along differentiation of human memory B cells into plasma cells in vitro. Preplasmablasts lack documented B cell or plasma cell (CD20, CD38, and CD138) markers, express CD30 and IL-6R, and secrete Igs at a weaker level than do plasmablasts or plasma cells. These preplasmablasts further differentiate into CD20−CD38highCD138− plasmablasts and then CD20−CD38highCD138+ plasma cells. Preplasmablasts were fully characterized in terms of whole genome transcriptome profiling and phenotype. Preplasmablasts coexpress B and plasma cell transcription factors, but at a reduced level compared with B cells, plasmablasts, or plasma cells. They express the unspliced form of XBP1 mRNA mainly, whereas plasmablasts and plasma cells express essentially the spliced form. An in vivo counterpart (CD19+CD20low/−CD38−IL-6R+ cells) of in vitro-generated preplasmablasts could be detected in human lymph nodes (0.06% of CD19+ cells) and tonsils (0.05% of CD19+ cells). An open access “B to Plasma Cell Atlas,” which makes it possible to interrogate gene expression in the process of B cell to plasma cell differentiation, is provided. Taken together, our findings show the existence of a transitional preplasmablast population using an in vitro model of plasma cell generation and of its in vivo counterpart in various lymphoid tissues.

APRIL and TACI interact with syndecan‐1 on the surface of multiple myeloma cells to form an essential survival loop

BLyS and APRIL share two receptors – transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B‐cell maturation antigen (BCMA) – and BLyS binds to a third receptor, BAFF‐R. We previously reported that TACI gene expression is a good indicator of a BLyS‐binding receptor in human multiple myeloma cell lines (HMCLs), unlike BCMA, which is expressed by all HMCLs or BAFF‐R which is typically not expressed by late‐stage B cells. We hypothesised a link between APRIL and TACI through syndecan‐1, similar to the situation reported for FGF and FGFR. We observed very strong binding of APRIL, but not BLyS, at the surface of all syndecan‐1+ HMCLs and primary multiple myeloma cells (MMC). All syndecan‐1+ HMCLs and MMC could also bind TACI‐Fc, but not BCMA‐Fc or BAFF‐R‐Fc molecules. Binding of APRIL or TACI‐Fc was abrogated by heparin or cell pretreatment with heparitinase, which cleaves heparan sulfate chains. The growth factor activity of APRIL on MMC was also inhibited by heparin. Our data identify syndecan‐1 as a co‐receptor for APRIL and TACI at the cell surface of MMC, promoting the activation of an APRIL/TACI pathway that induces survival and proliferation in MMC.

Targeting NF-kappaB pathway with an IKK2 inhibitor induces inhibition of multiple myeloma cell growth

The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IκB kinase/nuclear factor‐κB (IKK/NF‐κB). The present study aimed at investigating the effect of a pharmaceutical IKK2 inhibitor, the anilinopyrimidine derivative AS602868, on the in vitro growth of 14 human MM cell lines (HMCL) and primary cells from 13 patients. AS602868 induced a clear dose‐dependent inhibition of MM cell growth on both HMCL and primary MM cells, which was the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Combination of AS602868 with suboptimal doses of melphalan or Velcade showed an additive effect in growth inhibition of HMCL. AS602868 also induced apoptosis of primary myeloma cells. Importantly, AS602868 did not alter the survival of other bone marrow mononuclear cells (CD138−) co‐cultured with primary MM (CD138+) cells, except for CD34+ haematopoietic stem cells. The results demonstrate the important role of NF‐κB in maintaining the survival of MM cells and suggest that a pharmacological inhibition of the NF‐κB pathway by the IKK2 inhibitor AS602868 can efficiently kill HMCL and primary myeloma cells and therefore might represent an innovative approach for treating MM patients.

IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors

The recent understanding of plasma cell (PC) biology has been obtained mainly from murine models. The current concept is that plasmablasts home to the BM and further differentiate into long-lived PCs (LLPCs). These LLPCs survive for months in contact with a complex niche comprising stromal cells (SCs) and hematopoietic cells, both producing recruitment and survival factors. Using a multi-step culture system, we show here the possibility to differentiate human memory B cells into LLPCs surviving for at least 4 months in vitro and producing immunoglobulins continuously. A remarkable feature is that IL-6 is mandatory to generate LLPCs in vitro together with either APRIL or soluble factors produced by SCs, unrelated to APRIL/BAFF, SDF-1, or IGF-1. These LLPCs are out of the cell cycle, express highly PC transcription factors and surface markers. This model shows a remarkable robustness of human LLPCs, which can survive and produce highly immunoglobulins for months in vitro without the contact with niche cells, providing the presence of a minimal cocktail of growth factors and nutrients. This model should be useful to understand further normal PC biology and its deregulation in premalignant or malignant PC disorders.

Differential effects of lenalidomide during plasma cell differentiation.

Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide.

Characterization of human FCRL4-positive B cells

FCRL4 is an immunoregulatory receptor that belongs to the Fc receptor-like (FCRL) family. In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) localized in sub-epithelial regions of lymphoid tissues. Expansion of FCRL4+ B cells has been observed in blood and other tissues in various infectious and autoimmune disorders. Currently, the mechanisms involved in pathological FCRL4+ B cell generation are actively studied, but they remain elusive. As in vivo FCRL4+ cells are difficult to access and to isolate, here we developed a culture system to generate in vitro FCRL4+ B cells from purified MBCs upon stimulation with soluble CD40 ligand and/or CpG DNA to mimic T-cell dependent and/or T-cell independent activation, respectively. After 4 days of stimulation, FCRL4+ B cells represented 17% of all generated cells. Transcriptomic and phenotypic analyses of in vitro generated FCRL4+ cells demonstrated that they were closely related to FCRL4+ tonsillar MBCs. They strongly expressed inhibitory receptor genes, as observed in exhausted FCRL4+ MBCs from blood samples of HIV-infected individuals with high viremia. In agreement, cell cycle genes were significantly downregulated and the number of cell divisions was two-fold lower in in vitro generated FCRL4+ than FCRL4- cells. Finally, due to their reduced proliferation and differentiation potential, FCRL4+ cells were less prone to differentiate into plasma cells, differently from FCRL4- cells. Our in vitro model could be of major interest for studying the biology of normal and pathological FCRL4+ cells.

DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy.MethodsSince HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment.ResultsWe report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines.ConclusionIn conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

Global miRNA expression analysis identifies novel key regulators of plasma cell differentiation and malignant plasma cell

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that attenuate expression of their mRNA targets. Here, we developed a new method and an R package, to easily infer candidate miRNA–mRNA target interactions that could be functional during a given biological process. Using this method, we described, for the first time, a comprehensive integrated analysis of miRNAs and mRNAs during human normal plasma cell differentiation (PCD). Our results reveal 63 miRNAs with significant temporal changes in their expression during normal PCD. We derived a high-confidence network of 295 target relationships comprising 47 miRNAs and 141 targets. These relationships include new examples of miRNAs that appear to coordinately regulate multiple members of critical pathways associated with PCD. Consistent with this, we have experimentally validated a role for the miRNA-30b/c/d-mediated regulation of key PCD factors (IRF4, PRDM1, ELL2 and ARID3A). Furthermore, we found that 24 PCD stage-specific miRNAs are aberrantly overexpressed in multiple myeloma (MM) tumor plasma cells compared to their normal counterpart, suggesting that MM cells frequently acquired expression changes in miRNAs already undergoing dynamic expression modulation during normal PCD. Altogether, our analysis identifies candidate novel key miRNAs regulating networks of significance for normal PCD and malignant plasma cell biology.

Loss of RASSF4 Expression in Multiple Myeloma Promotes RAS-Driven Malignant Progression

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155-68. ©2017 AACR.

PRC2 targeting is a therapeutic strategy for EZ score defined high-risk multiple myeloma patients and overcome resistance to IMiDs

BackgroundMultiple myeloma (MM) is a malignant plasma cell disease with a poor survival, characterized by the accumulation of myeloma cells (MMCs) within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with drug resistance.MethodsWe identified a significant upregulation of the polycomb repressive complex 2 (PRC2) core genes in MM cells in association with proliferation. We used EPZ-6438, a specific small molecule inhibitor of EZH2 methyltransferase activity, to evaluate its effects on MM cells phenotype and gene expression prolile.ResultsPRC2 targeting results in growth inhibition due to cell cycle arrest and apoptosis together with polycomb, DNA methylation, TP53, and RB1 target genes induction. Resistance to EZH2 inhibitor is mediated by DNA methylation of PRC2 target genes. We also demonstrate a synergistic effect of EPZ-6438 and lenalidomide, a conventional drug used for MM treatment, activating B cell transcription factors and tumor suppressor gene expression in concert with MYC repression. We establish a gene expression-based EZ score allowing to identify poor prognosis patients that could benefit from EZH2 inhibitor treatment.ConclusionsThese data suggest that PRC2 targeting in association with IMiDs could have a therapeutic interest in MM patients characterized by high EZ score values, reactivating B cell transcription factors, and tumor suppressor genes.