Genetically Engineered Mouse Models Support a Major Role of Immune Checkpoint-Dependent Immunosurveillance Escape in B-Cell Lymphomas.

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.

Continuous MYD88 Activation Is Associated With Expansion and Then Transformation of IgM Differentiating Plasma Cells.

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.

Pre-clinical blocking of PD-L1 molecule, which expression is down regulated by NF-κB, JAK1/JAK2 and BTK inhibitors, induces regression of activated B-cell lymphoma.

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.

TP53INP1 deficiency maintains murine B lymphopoiesis in aged bone marrow through redox-controlled IL-7R/STAT5 signaling.

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 immunosenescence. 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.

The IgH 3' regulatory region and c-myc-induced B-cell lymphomagenesis.

Deregulation and mutations of c-myc have been reported in multiple mature B-cell malignancies such as Burkitt lymphoma, myeloma and plasma cell lymphoma. After translocation into the immunoglobulin heavy chain (IgH) locus, c-myc is constitutively expressed under the control of active IgH cis-regulatory enhancers. Those located in the IgH 3' regulatory region (3'RR) are master control elements of transcription. Over the past decade numerous convincing demonstrations of 3'RR's contribution to mature c-myc-induced lymphomagenesis have been made using transgenic models with various types of IgH-c-myc translocations and transgenes. This review highlights how IgH 3'RR physiological functions play a critical role in c-myc deregulation during lymphomagenesis.

PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression.

Hematopoietic stem cells (HSCs) give rise to all blood populations due to their long-term self-renewal and multipotent differentiation capacities. Because they have to persist throughout an organism's life span, HSCs tightly regulate the balance between proliferation and quiescence. Here, we investigated the role of the transcription factor promyelocytic leukemia zinc finger (plzf) in HSC fate using the Zbtb16(lu/lu)mouse model, which harbors a natural spontaneous mutation that inactivates plzf. Regenerative stress revealed that Zbtb16(lu/lu)HSCs had a lineage-skewing potential from lymphopoiesis toward myelopoiesis, an increase in the long-term-HSC pool, and a decreased repopulation potential. Furthermore, oldplzf-mutant HSCs present an amplified aging phenotype, suggesting that plzf controls age-related pathway. We found that Zbtb16(lu/lu)HSCs harbor a transcriptional signature associated with a loss of stemness and cell cycle deregulation. Lastly, cell cycle analyses revealed an important role for plzf in the regulation of the G1-S transition of HSCs. Our study reveals a new role for plzf in regulating HSC function that is linked to cell cycle regulation, and positions plzf as a key player in controlling HSC homeostasis.

Deciphering the importance of the palindromic architecture of the immunoglobulin heavy-chain 3' regulatory region.

The IgH 3' regulatory region (3'RR) controls class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The mouse 3'RR contains four enhancer elements with hs1,2 flanked by inverted repeated sequences and the centre of a 25-kb palindrome bounded by two hs3 enhancer inverted copies (hs3a and hs3b). hs4 lies downstream of the palindrome. In mammals, evolution maintained this unique palindromic arrangement, suggesting that it is functionally significant. Here we report that deconstructing the palindromic IgH 3'RR strongly affects its function even when enhancers are preserved. CSR and IgH transcription appear to be poorly dependent on the 3'RR architecture and it is more or less preserved, provided 3'RR enhancers are present. By contrast, a 'palindromic effect' significantly lowers VH germline transcription, AID recruitment and SHM. In conclusion, this work indicates that the IgH 3'RR does not simply pile up enhancer units but also optimally exposes them into a functional architecture of crucial importance.

The IgH 3' regulatory region controls somatic hypermutation in germinal center B cells.

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.

Mantle cell lymphoma-like lymphomas in c-myc-3'RR/p53+/- mice and c-myc-3'RR/Cdk4R24C mice: differential oncogenic mechanisms but similar cellular origin.

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.

A defect of the INK4-Cdk4 checkpoint and Myc collaborate in blastoid mantle cell lymphoma-like lymphoma formation in mice.

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.

Enhancers located in heavy chain regulatory region (hs3a, hs1,2, hs3b, and hs4) are dispensable for diversity of VDJ recombination.

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.

A p53 defect sensitizes various stages of B cell development to lymphomagenesis in mice carrying an IgH 3' regulatory region-driven c-myc transgene.

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.

Characterisation of genome-wide PLZF/RARA target genes.

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.

The IgH locus 3' regulatory region: pulling the strings from behind.

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.

Lipid mediators and human leukemic blasts.

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 (phospholipase A(2), cyclooxygenases, lipoxygenases) involved in their productions and their effects (through specific membrane bound receptors) on the growth, and apoptosis of leukemic blasts.

The IgH 3' regulatory region and its implication in lymphomagenesis.

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.

Genomic deletion of the whole IgH 3' regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes.

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 C(H) 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 C(H) 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.