Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.next-generation sequencing | cancer genetics | cancer heterogeneity M antle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13;q32) translocation leading to the overexpression of cyclin D1 (1). CCND1 is a weak oncogene that requires the cooperation of other oncogenic events to transform lymphoid cells (2). Molecular studies have identified alterations in components of the cell-cycle regulation, DNA damage response, and cell survival pathways (3, 4), but the profile of mutated genes contributing to the pathogenesis of MCL and cooperating with CCND1 is not well defined (1). Most MCL cases have a rapid evolution and an aggressive behavior with an unfavorable outcome with current therapies (5). Paradoxically, a subset of patients follows an indolent clinical evolution with stable disease even in the absence of chemotherapy (6, 7). This favorable behavior has been associated with IGHV-mutated (8, 9) and lack of expression of SOX11 (10, 11), a transcription factor highly specific of MCL that contributes to the aggressive behavior of this tumor (12). However, the molecular mechanisms responsible for this clinical heterogeneity are not well understood.To gain insight into the molecular pathogenesis of MCL we performed whole-genome sequencing (WGS) and whole-exome sequencing (WES) of 29 MCL and further investigated mutated genes in an expanded series of patients. We also analyzed the subclonal heterogeneity of the tumors and their modulation during the evolution of the disease. Results Landscape of Mutations in MCL.We performed WGS and WES of 4 and 29 MCL, respectively. These patients were re...

Mantle cell lymphoma (MCL) is one of the most aggressive lymphoid neoplasms whose pathogenesis is not fully understood. The neural transcription factor SOX11 is overexpressed in most MCL but is not detected in other mature B-cell lymphomas or normal lymphoid cells. The specific expression of SOX11 in MCL suggests that it may be an important element in the development of this tumor, but its potential function is not known. Here, we show that SOX11 promotes tumor growth in a MCL-xenotransplant mouse model. Using chromatin immunoprecipitation microarray analysis combined with gene expression profiling upon SOX11 knockdown, we identify target genes and transcriptional programs regulated by SOX11 including the block of mature B-cell differentiation, modulation of cell cycle, apoptosis, and stem cell development. PAX5 emerges as one of the major SOX11 direct targets. SOX11 silencing downregulates PAX5, induces BLIMP1 expression, and promotes the shift from a mature B cell into the initial plasmacytic differentiation phenotype in both primary tumor cells and an in vitro model. Our results suggest that SOX11 contributes to tumor development by altering the terminal B-cell differentiation program of MCL and provide perspectives that may have clinical implications in the diagnosis and design of new therapeutic strategies.

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (b-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by b-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and antiapoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.

Bortezomib therapy has shown promising clinical activity in mantle cell lymphoma (MCL), but the development of resistance to proteasome inhibition may limit its efficacy. To unravel the factors involved in the acquisition of bortezomib resistance in vivo, immunodeficient mice were engrafted with a set of MCL cell lines with different levels of sensitivity to the drug, followed by gene expression profiling of the tumors and functional validation of the identified gene signatures. We observed an increased tumorigenicity of bortezomib-resistant MCL cells in vivo, which was associated with plasmacytic differentiation features, like interferon regulatory factor 4 (IRF4) and Blimp-1 upregulation. Lenalidomide was particularly active in this subgroup of tumors, targeting IRF4 expression and plasmacytic differentiation program, thus overcoming bortezomib resistance. Moreover, repression of the IRF4 target gene MYC in bortezomib-resistant cells by gene knockdown or treatment with CPI203, a BET (bromodomain and extra terminal) bromodomain inhibitor, synergistically induced cell death when combined with lenalidomide. In mice, addition of CPI203 to lenalidomide therapy further decreased tumor burden, involving simultaneous MYC and IRF4 downregulation and apoptosis induction. Together, these results suggest that exacerbated IRF4/MYC signaling is associated to bortezomib resistance in MCL in vivo and warrant clinical evaluation of lenalidomide plus BET inhibitor combination in MCL cases refractory to proteasome inhibition.