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International audience; These last 20 years, research on immune tumor microenvironment led to identify some critical recurrent mechanisms used in cancer to escape immune response. Through immune checkpoints, which are cell surface molecules involved in the immune system control, it is now established that tumor cells are able to shutdown the immune response. Due to the complexity and heterogeneity of Non Hodgkin B-cell Lymphomas (NHBLs), it is difficult to understand the precise mechanisms of immune escape and to explain the mitigated effect of immune checkpoints blockade for their treatment. Because genetically engineered mouse models are very reliable tools to improve our understanding of molecular mechanisms involved in B-cell transformation and, at the same time, can be useful preclinical models to predict immune response, we reviewed hereafter some of these models that highlight the immune escape mechanisms of NHBLs and open perspectives on future therapies.

Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.

Escape from immune control must be important in the natural course of B-cell lymphomas, especially for those with activation of NF-κB. The pre-clinical LMP1/CD40-expressing transgenic mouse model is characterized by B-cell specific CD40 signaling responsible for NF-κB continuous activation with a spleen monoclonal B-cell tumor after 1 year in 60% of cases. LMP1/CD40 tumors B-cells expressed high levels of PD-L1. This expression was dependent on activation of either NF-κB, JAK1/JAK2 or BTK pathways since these pathways were activated in tumor B-cells and ex vivo treatment with the inhibitory molecules PHA-408, ruxolitinib and ibrutinib led to decrease of its expression. Treatment of LMP1/CD40-expressing lymphomatous mice with an anti-PD-L1 monoclonal antibody induced tumor regression with decreased spleen content, activation and proliferation rate of B-cells as well as a marked increase in T-cell activation, as assessed by CD62L and CD44 expression. These results highlight the interest of therapies targeting the PD-1/PD-L1 axis in activated lymphomas with PD-L1 expression, with possible synergies with tyrosine kinase inhibitors. Electronic supplementary material The online version of this article (10.1186/s12964-019-0391-x) contains supplementary material, which is available to authorized users.

International audience

Transformation of an indolent B-cell lymphoma is associated with a more aggressive clinical course and poor survival. The role of immune surveillance in the transformation of a B-cell indolent lymphoma towards a more aggressive form is poorly documented. To experimentally address this question, we used the L.CD40 mouse model, which is characterized by B-cell specific continuous CD40 signaling, responsible for spleen indolent clonal or oligoclonal B-cell lymphoma after one year in 60% cases. Immunosuppression was obtained either by T/NK cell depletion or by treatment with the T-cell immunosuppressive drug cyclosporin A. Immunosuppressed L.CD40 mice had larger splenomegaly with increased numbers of B-cells in both spleen and peripheral blood. High-throughput sequencing of immunoglobulin variable segments revealed that clonal expansion was increased in immunosuppressed L.CD40 mice. Tumor B cells of immunosuppressed mice were larger with an immunoblastic aspect, both on blood smears and spleen tissue sections, with increased proliferation rate and increased numbers of activated B-cells. Collectively, these features suggest that immune suppression induced a shift from indolent lymphomas into aggressive ones. Thus, as a preclinical model, immunosuppressed L.CD40 mice reproduce aggressive transformation of an indolent B-cell tumor and highlight the role of the immune surveillance in its clinical course, opening new perspective for immune restoration therapies.

International audience; Bone marrow (BM) produces all blood and immune cells deriving from hematopoietic stem cells (HSCs). The decrease of immune cell production during aging is one of the features of immunosenes-cence. The impact of redox dysregulation in BM aging is still poorly understood. Here we use TP53INP1-deficient (KO) mice endowed with chronic oxidative stress to assess the influence of aging-associated redox alterations in BM homeostasis. We show that TP53INP1 deletion has no impact on aging-related accumulation of HSCs. In contrast, the aging-related contraction of the lymphoid compartment is mitigated in TP53INP1 KO mice. B cells that accumulate in old KO BM are differentiating cells that can mature into functional B cells. Importantly, this phenotype results from B cell-intrinsic events associated with defective redox control. Finally, we show that oxidative stress in aged TP53INP1-deficient mice maintains STAT5 expression and activation in early B cells, driving high Pax5 expression, which provides a molecular mechanism for maintenance of B cell development upon aging.

Somatic hypermutation in variable heavy chain rearranged regions is abrogated in the absence of the 3′ regulatory region enhancer, whereas transcription rate in the Ig heavy chain is only partially reduced., Interactions with cognate antigens recruit activated B cells into germinal centers where they undergo somatic hypermutation (SHM) in V(D)J exons for the generation of high-affinity antibodies. The contribution of IgH transcriptional enhancers in SHM is unclear. The Eμ enhancer upstream of Cμ has a marginal role, whereas the influence of the IgH 3′ regulatory region (3′RR) enhancers (hs3a, hs1,2, hs3b, and hs4) is controversial. To clarify the latter issue, we analyzed mice lacking the whole 30-kb extent of the IgH 3′RR. We show that SHM in VH rearranged regions is almost totally abrogated in 3′RR-deficient mice, whereas the simultaneous Ig heavy chain transcription rate is only partially reduced. In contrast, SHM in κ light chain genes remains unaltered, acquitting for any global SHM defect in our model. Beyond class switch recombination, the IgH 3′RR is a central element that controls heavy chain accessibility to activation-induced deaminase modifications including SHM.

Letter to the Editor., These data were presented in part at the 56th American Society of Hematology meeting, San Francisco, December 2014.

International audience; V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple IgH cis-regulatory elements control B-cell ontogeny. IGCR1 (intergenic control region 1), the DQ52 promoter/enhancer, and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination, whereas there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here, we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B-cell differentiation. In contrast, the earliest antigen-independent steps of B-cell ontogeny would be under the control of only the upstream Cmu elements of the locus.

Although c-myc is classically described as the driving oncogene in Burkitt’s lymphoma (BL), deregulation and mutations of c-myc have been reported in multiple solid tumors and in other mature B cell malignancies such as mantle cell lymphoma (MCL), myeloma, and plasma cell lymphoma (PCL). After translocation into the IgH locus, c-myc is constitutively expressed under the control of active IgH enhancers. Those located in the IgH 3′ regulatory region (3′RR) are master control elements of class switch recombination and of the transcriptional burst associated with plasma cell differentiation. c-myc-3′RR mice are prone to lymphomas with rather homogeneous, most often BL-like, phenotypes with incomplete penetrance (75% tumor incidence) and long latencies (10–12 mo). To reproduce c-myc–induced mature B cell lymphomagenesis in the context of an additional defect often observed in human lymphomas, we intercrossed c-myc-3′RR with p53+/− mice. Double transgenic c-myc-3′RR/p53+/− mice developed lymphoma with short latency (2–4 mo) and full penetrance (100% tumor incidence). The spectrum of B lymphomas occurring in c-myc-3′RR/p53+/− mice was widened, including nonactivated (CD43−) BL, activated (CD43+) BL, MCL-like lymphoma, and PCL, thus showing that 3′RR-mediated deregulation of c-myc can promote various types of B lymphoproliferation in cells that first acquired a p53 defect. c-myc/p53+/− mice closely reproduce many features of BL, MCL, and PCL and provide a novel and efficient model to dissect the molecular events leading to c-myc–induced lymphomagenesis and an important tool to test potential therapeutic agents on malignant B cells featuring various maturation stages.

International audience; The 3' regulatory region (3'RR) located downstream of the IgH gene is the master element that controls class switch recombination and sustains high-level transcription at the plasma-cell stage. This latter role suggests that the 3'RR may be involved in oncogene deregulation during the frequent IgH translocation events associated with B-cell malignancies. A convincing demonstration of the essential contribution of 3'RR in lymphomagenesis has been provided by transgenic animal models. The mouse 3'RR shares a strong structural homology with the regulatory regions located downstream of each human Cα gene. Mouse models exploring the role of the 3'RR in B-cell physiology and in malignancies should provide useful indications about the pathophysiology of human cell lymphocyte proliferation.

The immunoglobulin heavy chain locus (IgH) undergoes multiple changes along B-cell differentiation. In progenitor B cells, V(D)J assembly allows expression of μ heavy chains. In mature B cells, class switch recombination may replace the expressed constant (C)μ gene with a downstream CH gene. Finally, plasma cell differentiation strongly boosts IgH transcription. How the multiple IgH transcriptional enhancers tune these changes is unclear. Here we demonstrate that deletion of the whole IgH 3′ regulatory region (3′RR) allows normal maturation until the stage of IgM/IgD expressing lymphocytes, but nearly abrogates class switch recombination to all CH genes. Although plasma cell numbers are unaffected, we reveal the role of the 3′RR into the transcriptional burst normally associated with plasma cell differentiation. Our study shows that transcriptional changes and recombinations occurring after antigen-encounter appear mainly controlled by the 3′RR working as a single functional unit.

International audience; Cyclin D1 overexpression is associated with mantle cell lymphoma and multiple myeloma. In myeloma, it often results from chromosomal translocations linking the CCND1 gene to the 3' part of the IgH locus constant region. This region includes a single and potent transcriptional regulatory region (RR) 3' of the Calpha gene mostly active in mature B-cells. To check whether this RR alone was sufficient to deregulate CCND1, we generated mice carrying a 3'IgH RR-driven human CCND1 transgene and specifically up-regulating cyclin D1 expression in B-cells. In transgenic B-cells, cyclin D1 enforced cell cycle entry in response to various stimuli (LPS, anti-IgM, anti-CD40) but also increased cell death, so that exaggerated proliferation did not result in peripheral lymphocytosis. Despite exaggerated B-cell entry into G(1) phase, malignant lymphoproliferation did not occur either. Crossing of CCND1-3'IgH RR mice with c-myc-3'IgH RR mice did not reveal accelerated tumorigenesis as compared with c-myc-3'IgH RR mice alone. The data presented here demonstrate that the 3'IgH RR-mediated deregulation of CCND1 in mature B-cells cannot by itself trigger the development of lymphomas and strengthen the concept that cyclin D1 per se is not an armful proto-oncogene. Rather its overexpression in several malignancies might be only a stigma of lymphomagenesis or represent a single hit within a multiple hit process.

International audience

Several studies have reported that regulatory elements located 3′ of the IgH locus (namely hs3a, hs1,2, hs3b, and hs4) might play a role during class switch recombination (CSR) and Ig synthesis. While individual deletion of hs3a or hs1,2 had no effect, pairwise deletion of hs3b (an inverted copy of hs3a) and hs4 markedly affected CSR and Ig expression. Among these two elements, hs4 was tentatively presented with the master role due to its unique status within the 3′ regulatory region: distal position outside repeated regions, early activation in pre-B cells, strong activity throughout B cell ontogeny. To clarify its role, we generated mice with a clean deletion of the hs4 after replacement with a floxed neoR cassette. Surprisingly, and as for previous deletion of hs3a or hs1,2, deletion of hs4 did not affect either in vivo CSR or the secretion level of any Ig isotype. In vitro CSR and Ig secretion in response to LPS and cytokines was not affected either. The only noticeable effects of the hs4 deletion were a decrease in the number of B splenocytes and a decreased membrane IgM expression. In conclusion, while dispensable for CSR and Ig transcription in plasma cells, hs4 mostly appears to contribute to Ig transcription in resting B lymphocytes.

International audience; Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a monoclonal proliferation of lymphocytes with the co-expression of CD5 and CD43, but not of CD23. Typical MCL is associated with overexpression of cyclin D1, and blastoid MCL variants are associated with Myc (alias c-myc) translocations. In this study, we developed a murine model of MCL-like lymphoma by crossing Cdk4(R24C) mice with Myc-3'RR transgenic mice. The Cdk4(R24C) mouse is a knockin strain that expresses a Cdk4 protein that is resistant to inhibition by p16(INK4a) as well as other INK4 family members. Ablation of INK4 control on Cdk4 does not affect lymphomagenesis, B-cell maturation, and functions in Cdk4(R24C) mice. Additionally, B cells were normal in numbers, cell cycle activity, mitogen responsiveness, and Ig synthesis in response to activation. By contrast, breeding Cdk4(R24C) mice with Myc-3'RR transgenic mice prone to develop aggressive Burkitt lymphoma-like lymphoma (CD19(+)IgM(+)IgD(+) cells) leads to the development of clonal blastoid MCL-like lymphoma (CD19(+)IgM(+)CD5(+)CD43(+)CD23(-) cells) in Myc/Cdk4(R24C) mice. Western blot analysis revealed high amounts of Cdk4/cyclin D1 complexes as the main hallmark of these lymphomas. These results indicate that although silent in nonmalignant B cells, a defect in the INK4-Cdk4 checkpoint can participate in lymphomagenesis in conjunction with additional alterations of cell cycle control, a situation that might be reminiscent of the development of human blastoid MCL.

International audience; Mantle cell lymphoma (MCL) is a malignant lymphoproliferative B-cell disorder that does not occur spontaneously in mice but experimental mice model have been developed. Recently two different mice models prone to develop MCL-like lymphomas were generated: c-myc-3'RR/Cdk4(R24C) mice and c-myc-3'RR/p53+/- mice. Comparison of their gene expression profiles does not highlight specific differences other than those in relation with their specific mutational status (i.e., Cdk4(R24C) mutation or p53 mutations). We propose that similarly to typical human MCL and its blastoid or cyclin-D1 variants that correspond to the same genetic entity, MCL-like lymphomas of c-myc-3'RR/ p53+/- mice and c-myc-3'RR/Cdk4(R24C) mice represent a spectrum of the same entity.

Antigen receptor gene loci are among the most complex in mammals. The IgH locus, encoding the immunoglobulin heavy chain (IgH) in B-lineage cells, undergoes major transcription-dependent DNA remodeling events, namely V(D)J recombination, Ig class-switch recombination (CSR), and somatic hypermutation (SHM). Various cis-regulatory elements (encompassing promoters, enhancers, and chromatin insulators) recruit multiple nuclear factors in order to ensure IgH locus regulation by tightly orchestrated physical and/or functional interactions. Among major IgH cis-acting regions, the large 3′ regulatory region (3′RR) located at the 3′ boundary of the locus includes several enhancers and harbors an intriguing quasi-palindromic structure. In this review, we report progress insights made over the past decade in order to describe in more details the structure and functions of IgH 3′RRs in mouse and human. Generation of multiple cellular, transgenic and knock-out models helped out to decipher the function of the IgH 3′ regulatory elements in the context of normal and pathologic B cells. Beside its interest in physiology, the challenge of elucidating the locus-wide cross talk between distant cis-regulatory elements might provide useful insights into the mechanisms that mediate oncogene deregulation after chromosomal translocations onto the IgH locus.

Some of the most potent inflammatory mediators share a lipid origin. They regulate a wide spectrum of cellular processes including cell proliferation and apoptosis. However, the precise roles and ways (if any) in which these compounds impact the growth and apoptosis of leukemic blasts remain incompletely resolved. In spite of this, significant advances have been recently made. Here we briefly review the current knowledge about the production of lipid mediators (prostaglandins, leukotrienes, platelet-activating factor) by leukemic blasts, the enzymatic activities (phospholipaseA2, cyclooxygenases, lipoxygenases) involved in their productions and their effects (through specific membrane bound receptors) on the growth, and apoptosis of leukemic blasts.

International audience; The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear.We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.