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The microenvironment strongly influences mantle cell lymphoma (MCL) survival, proliferation, and chemoresistance. However, little is known regarding the molecular characterization of lymphoma niches. Here, we focused on the interplay between MCL cells and the associated monocytes/macrophages. Using circulating MCL cells (n = 58), we showed that, through the secretion of CSF1 and, to a lesser extent, IL-10, MCL polarized monocytes into specific CD163+ M2-like macrophages (MϕMCL). In turn, MϕMCL favored lymphoma survival and proliferation ex vivo. We next demonstrated that BTK inhibition abrogated CSF1 and IL-10 production in MCL cells, leading to the inhibition of macrophage polarization and consequently resulting in the suppression of microenvironment-dependent MCL expansion. In vivo, we showed that CSF1 and IL-10 plasma concentrations were higher in MCL patients than in healthy donors, and that monocytes from MCL patients overexpressed CD163. Further analyses of serial samples from ibrutinib-treated patients (n = 8) highlighted a rapid decrease of CSF1, IL-10, and CD163 in responsive patients. Finally, we showed that targeting the CSF1R abrogated MϕMCL-dependent MCL survival, irrespective of their sensitivity to ibrutinib. These data reinforced the role of the microenvironment in lymphoma and suggested that macrophages are a potential target for developing novel therapeutic strategies in MCL.

In this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53wt cell lines (P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53wt cell lines, but not the TP53abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency.

Multiple myeloma is a plasma cell malignancy that escapes from apoptosis by heterogeneously over-expressing anti-apoptotic BCL2 proteins. Myeloma cells with a t(11;14) translocation present a particular vulnerability to BCL2 inhibition while a majority of myeloma cells relies on MCL1 for survival. The present study aimed to determine whether the combination of BCL2 and MCL1 inhibitors at low doses could be of benefit for myeloma cells beyond the single selective inhibition of BCL2 or MCL1. We identified that half of patients were not efficiently targeted neither by BCL2 inhibitor nor MCL1 inhibitor. Seventy percent of these myeloma samples, either from patients at diagnosis or relapse, presented a marked increase of apoptosis upon low dose combination of both inhibitors. Interestingly, primary cells from a patient in progression under venetoclax treatment were not sensitive ex vivo to neither venetoclax nor to MCL1 inhibitor, whereas the combination of both efficiently induced cell death. This finding suggests that the combination could overcome venetoclax resistance. The efficacy of the combination was also confirmed in U266 xenograft model resistant to BCL2 and MCL1 inhibitors. Mechanistically, we demonstrated that the combination of both inhibitors favors apoptosis in a BAX/BAK dependent manner. We showed that activated BAX was readily increased upon the inhibitor combination leading to the formation of BAK/BAX hetero-complexes. We found that BCLXL remains a major resistant factor of cell death induced by this combination. The present study supports a rational for the clinical use of venetoclax/S63845 combination in myeloma patients with the potential to elicit significant clinical activity when both single inhibitors would not be effective but also to overcome developed in vivo venetoclax resistance.

International audience; B-cell receptor (BCR) signaling pathways and interactions with the tumor microenvironment account for mantle cell lymphoma (MCL) cells survival in lymphoid organs. In several MCL cases, the WNT/β-catenin canonical pathway is activated and β-catenin accumulates into the nucleus. As both BCR and β-catenin are important mediators of cell survival and interaction with the microenvironment, we investigated the crosstalk between BCR and WNT/β-catenin signaling and analyzed their impact on cellular homeostasis as well as their targeting by specific inhibitors. β-catenin was detected in all leukemic MCL samples and its level of expression rapidly increased upon BCR stimulation. This stabilization was hampered by the BCR-pathway inhibitor Ibrutinib, supporting β-catenin as an effector of the BCR signaling. In parallel, MCL cells as compared with normal B cells expressed elevated levels of WNT16, a NF-κB target gene. Its expression increased further upon BCR stimulation to participate to the stabilization of β-catenin. Upon BCR stimulation, β-catenin translocated into the nucleus but did not induce a Wnt-like transcriptional response, i.e., TCF/LEF dependent. β-catenin rather participated to the regulation of NF-κB transcriptional targets, such as IL6, IL8, and IL1. Oligo pull down and chromatin immunoprecipitation experiments demonstrated that β-catenin is part of a protein complex that binds the NF-κB DNA consensus sequence, strengthening the idea of an association between the two proteins. An inhibitor targeting β-catenin transcriptional interactions hindered both NF-κB DNA recruitment and induced primary MCL cells apoptosis. Thus, β-catenin likely represents another player through which BCR signaling impacts on MCL cell survival.

Background Multiple myeloma (MM) is a malignancy of plasma cells that largely remains incurable. The search for new therapeutic targets is therefore essential. In addition to a wide panel of genetic mutations, epigenetic alterations also appear as important players in the development of this cancer, thereby offering the possibility to reveal novel approaches and targets for effective therapeutic intervention. Results Here, we show that a higher expression of the lysine methyltransferase SETD8, which is responsible for the mono-methylation of histone H4 at lysine 20, is an adverse prognosis factor associated with a poor outcome in two cohorts of newly diagnosed patients. Primary malignant plasma cells are particularly addicted to the activity of this epigenetic enzyme. Indeed, the inhibition of SETD8 by the chemical compound UNC-0379 and the subsequent decrease in histone H4 methylation at lysine 20 are highly toxic in MM cells compared to normal cells from the bone marrow microenvironment. At the molecular level, RNA sequencing and functional studies revealed that SETD8 inhibition induces a mature non-proliferating plasma cell signature and, as observed in other cancers, triggers an activation of the tumor suppressor p53, which together cause an impairment of myeloma cell proliferation and survival. However, a deadly level of replicative stress was also observed in p53-deficient myeloma cells treated with UNC-0379, indicating that the cytotoxicity associated with SETD8 inhibition is not necessarily dependent on p53 activation. Consistent with this, UNC-0379 triggers a p53-independent nucleolar stress characterized by nucleolin delocalization and reduction of nucleolar RNA synthesis. Finally, we showed that SETD8 inhibition is strongly synergistic with melphalan and may overcome resistance to this alkylating agent widely used in MM treatment. Conclusions Altogether, our data indicate that the up-regulation of the epigenetic enzyme SETD8 is associated with a poor outcome and the deregulation of major signaling pathways in MM. Moreover, we provide evidences that myeloma cells are dependent on SETD8 activity and its pharmacological inhibition synergizes with melphalan, which could be beneficial to improve MM treatment in high-risk patients whatever their status for p53. Supplementary Information The online version contains supplementary material available at 10.1186/s13148-021-01160-z.

BCL2-family proteins have a central role in the mitochondrial apoptosis machinery and their expression is known to be deregulated in many cancer types. Effort in the development of small molecules that selectively target anti-apoptotic members of this family i.e., Bcl-2, Bcl-xL, Mcl-1 recently opened novel therapeutic opportunities. Among these apoptosis-inducing agents, BH3-mimetics (i.e., venetoclax) led to promising preclinical and clinical activity in B cell malignancies. However, several mechanisms of intrinsic or acquired resistance have been described ex vivo therefore predictive markers of response as well as mechanism-based combinations have to be designed. In the present study, we analyzed the expression of the BCL2-family genes across 10 mature B cell malignancies through computational normalization of 21 publicly available Affimetrix datasets gathering 1,219 patient samples. To better understand the deregulation of anti- and pro-apoptotic members of the BCL2-family in hematological disorders, we first compared gene expression profiles of malignant B cells to their relative normal control (naïve B cell to plasma cells, n = 37). We further assessed BCL2-family expression according to tissue localization i.e., peripheral blood, bone marrow, and lymph node, molecular subgroups or disease status i.e., indolent to aggressive. Across all cancer types, we showed that anti-apoptotic genes are upregulated while pro-apoptotic genes are downregulated when compared to normal counterpart cells. Of interest, our analysis highlighted that, independently of the nature of malignant B cells, the pro-apoptotic BH3-only BCL2L11 and PMAIP1 are deeply repressed in tumor niches, suggesting a central role of the microenvironment in their regulation. In addition, we showed selective modulations across molecular subgroups and showed that the BCL2-family expression profile was related to tumor aggressiveness. Finally, by integrating recent data on venetoclax-monotherapy clinical activity with the expression of BCL2-family members involved in the venetoclax response, we determined that the ratio (BCL2+BCL2L11+BAX)/BCL2L1 was the strongest predictor of venetoclax response for mature B cell malignancies in vivo.

Mantle cell lymphoma (MCL) accumulates in lymphoid organs, but disseminates early on in extranodal tissues. Although proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem, few studies have assessed MCL microenvironment. We therefore cocultured primary circulating MCL cells from 21 patients several weeks ex vivo with stromal or lymphoid-like (CD40L) cells to determine which interactions could support their proliferation. We showed that coculture with lymphoid-like cells, but not stromal cells, induced cell-cycle progression, which was amplified by MCL-specific cytokines (insulin-like growth factor-1, B-cell activating factor, interleukin-6, interleukin-10). Of interest, we showed that our model recapitulated the MCL in situ molecular signatures (ie, proliferation, NF-κB, and survival signatures). We further demonstrated that proliferating MCL harbored an imbalance in Bcl-2 family expression, leading to a consequent loss of mitochondrial priming. Of interest, this loss of priming was overcome by the type II anti-CD20 antibody obinutuzumab, which counteracted Bcl-xL induction through NF-κB inhibition. Finally, we showed that the mitochondrial priming directly correlated with the sensitivity toward venetoclax and alkylating drugs. By identifying the microenvironment as the major support for proliferation and drug resistance in MCL, our results highlight a selective approach to target the lymphoma niche.

The aggressive clinical behavior of mantle cell lymphoma (MCL) and its short-term response to treatments highlight a need for novel options. The microenvironment strongly controls MCL cell survival, proliferation and chemoresistance, nevertheless little is known regarding the interactions that occur in the tumor niches. Here, we studied the interplay between primary MCL cells (n=55) and macrophages and identified mechanism-based targeted strategies to disrupt this dialogue. Using ex vivo co-cultures of peripheral blood MCL cells and monocytes (healthy donors), we showed that monocytes greatly improved MCL cell survival, whereas MCL cells poorly survived when cultured alone (co-culture, 85.9%; alone, 6.5%; D7; n=17; p During co-culture, primary MCL cells polarized monocytes into MCL-associated-macrophages (MϕMCL). To define the nature of MϕMCL, we studied their phenotype and function with 12 markers differentially expressed in M1 and M2 macrophages (CD14, CD68, CD163, CD11b, CD86, HLA-DR, CD16, CD23, IL10, IL6, IGF1, BAFF). The analysis showed that even though MϕMCL distinctly clustered from both M1 and M2, they shared more similarities with M2 (CD163high, CD11blow, IL10+, IGF1+). To determine how MCL cells polarized monocytes into CD163high MϕMCL, we analyzed the expression of known M2-polarizing factors and observed that CSF1 and IL10 were overexpressed in MCL cells compared to normal B cells (GEP, p In vivo, we showed higher levels of CSF1 and IL-10 in the plasma of MCL patients (n=28) compared to match-aged HD (p In conclusion, through secretion of IL-10 and CSF1, MCL cells polarized monocytes into M2-like macrophages (MϕMCL), which in turn support tumor survival and proliferation. Ibrutinib counteracts the MCL/MϕMCL dialogue through inhibition of CSF1 production and, consequently, impairs the MϕMCL-dependent expansion of ibrutinib-sensitive MCL cells. Of note, our in vivo retrospective analysis highlights that CSF1, IL-10 and CD163 modulations might be early markers of ibrutinib response. A larger cohort of MCL patients treated with ibrutinib is now necessary to confirm the strength of this soluble and cellular signature. Lastly, targeting the CSF1R is an alternative to disrupt the MCL/MϕMCL pro-tumoral dialogue, especially for ibrutinib-refractory patients for which no therapeutic alternative is available. Disclosures Moreau: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Le Gouill:Roche: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria.

Mantle cell lymphoma (MCL) accumulates in lymphoid organs but disseminates early on in extranodal tissues. However, there is relatively little information regarding the nature of surrounding cells and soluble factors and the resulting molecular regulations induced in MCL. Further investigations that integrate the key role of the microenvironment are now needed to overcome tumor drug resistance in protective niches. The benefits of such an approach have been recently reinforced by studies reporting that selective inhibition of BTK or PI3Kδ, critical kinases of the BCR and CXCR4 pathways, prevents the homing of MCL, leading to peripheral lymphocytosis. However, the consequences of cell egress in the peripheral blood on proliferation and survival are still unclear and need further investigations. In spite of the significant level of the proliferation index Ki67 in secondary lymphoid organs and the elevated percentage of circulating malignant cells, we did not detect any proliferating peripheral blood MCL cells, which were arrested in the G1-phase of the cell cycle. The proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem. In situ, lymphoma B-cells are in close contact with cells of mesenchymal or immune origin. To determine whether stromal (hMSC) or lymphoid-like (CD40L) interactions could support survival and proliferation of MCL, primary circulating MCL cells from 21 patients were cocultured several weeks ex vivo. In all samples tested we showed that CD40L signal, but not hMSC, induced cell-cycle progression, which was amplified by a MCL-specific cytokine cocktail, defined by assessment of cytokine receptor expression in situ. Using this system, we were able to maintain proliferation of primary MCL cells for several weeks, and most MCL samples remained dependent on the extrinsic signal over time. Of major interest, using RNA-seq, we showed that 70% of genes induced ex vivo are expressed in lymph nodes, confirming the relevance of the coculture. We further observed that our model recapitulated in situ MCL molecular signatures such as proliferation, BCR/NFkB and survival signatures. Among the genes related to survival, coculture resulted in anti- and pro-apoptotic Bcl-2 family member modulation. We demonstrated that an anti-apoptotic Bcl-xL protein increase was associated with a striking downregulation of pro-apoptotic Bim, Bax and Bak. Of note, this unbalanced regulation seemed to be specific to MCL cells as it did not occur in naïve CD5+ B cells or memory B cells. We provided evidences that Bcl-xL upregulation was responsible for loss of mitochondrial priming and drug resistance. Using BH3-profiling, we showed a dynamic sequestration of the BH3-only activator Bim by Bcl-xL proteins at the mitochondrial level. In addition, we showed that the mitochondrial priming directly correlated with the sensitivity toward BH3-mimetics (venetoclax) and alkylating drugs (bendamustine). Our results thus highlight molecular basis for microenvironment-dependent modulation of the mitochondrial priming and consequent drug resistance in primary MCL cells. Thus, whereas venetoclax is highly efficient in inducing apoptosis in peripheral blood MCL cells, tumor cells protected into their niches could be resistant and in fine induce relapse. Using our ex vivo model, we further demonstrated that, in rational combinations, anti-CD20 antibody (obinutuzumab) can suppress survival and overcomes drug resistance to venetoclax exploiting NFkB/Bcl-xL-specific dependence of primary MCL cells. Moreover, ibrutinib, which mediates indirect Bcl-xL down-modulation upon BTK-dependent lymphocytosis, could also increase in vivo venetoclax efficacy. Our ongoing OAsIs Trial for MCL patients (obinutuzumab, ibrutinib and venetoclax, NTC#02558816) will rapidly determine in vivoefficacy. In summary, we reported here the development of a reproducible ex vivo coculture model for primary MCL cells. This model has provided new insights into microenvironment-dependent proliferation and Bcl-2 family regulation, which are central components of survival and drug resistance. Our increased understanding of intrinsic abnormalities, the development of highly selective inhibitors and integration of the microenvironment offer new opportunities to design mechanism-based strategies that should overcome drug resistance in MCL and potentially other B cell malignancies Disclosures Moreau: Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria.