Alexandra Moros

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma

Significance This is a comprehensive whole-genome/whole-exome analysis of mantle cell lymphoma (MCL). We sequenced 29 MCL cases and validated the findings by target sequencing of 172 additional tumors. We identified recurrent mutations in genes regulating chromatin modification and genes such as NOTCH2 that have a major impact on clinical outcome. Additionally, we demonstrated the subclonal heterogeneity of the tumors already at diagnosis and the modulation of the mutational architecture in the progression of the disease. The identification of new molecular mechanisms may open perspectives for the management of MCL patients. 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.

SOX11 regulates PAX5 expression and blocks terminal B-cell differentiation in aggressive mantle cell lymphoma

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.

Synergistic antitumor activity of lenalidomide with the BET bromodomain inhibitor CPI203 in bortezomib-resistant mantle cell 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.

Lipid raft-mediated Akt signaling as a therapeutic target in mantle cell lymphoma

Recent evidence shows that lipid raft membrane domains modulate both cell survival and death. Here, we have found that the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is present in the lipid rafts of mantle cell lymphoma (MCL) cells, and this location seems to be critical for full activation and MCL cell survival. The antitumor lipids (ATLs) edelfosine and perifosine target rafts, and we found that ATLs exerted in vitro and in vivo antitumor activity against MCL cells by displacing Akt as well as key regulatory kinases p-PDK1 (phosphatidylinositol-dependent protein kinase 1), PI3K and mTOR (mammalian TOR) from lipid rafts. This raft reorganization led to Akt dephosphorylation, while proapoptotic Fas/CD95 death receptor was recruited into rafts. Raft integrity was critical for Ser473 Akt phosphorylation. ATL-induced apoptosis appeared to correlate with the basal Akt phosphorylation status in MCL cell lines and primary cultures, and could be potentiated by the PI3K inhibitor wortmannin, or inhibited by the Akt activator pervanadate. Classical Akt inhibitors induced apoptosis in MCL cells. Microenvironmental stimuli, such as CD40 ligation or stromal cell contact, did not prevent ATL-induced apoptosis in MCL cell lines and patient-derived cells. These results highlight the role of raft-mediated PI3K/Akt signaling in MCL cell survival and chemotherapy, thus becoming a new target for MCL treatment.

The EMT activator ZEB1 promotes tumor growth and determines differential response to chemotherapy in mantle cell lymphoma

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 (β-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 β-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 anti-apoptotic (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.

Antitumoral activity of lenalidomide in in vitro and in vivo models of mantle cell lymphoma involves the destabilization of cyclin D1/p27KIP1 complexes.

Purpose: Clinical responses to the immmunomodulatory drug lenalidomide have been observed in patients with relapsed/refractory mantle cell lymphoma (MCL), although its mechanism of action remains partially unknown. We investigated whether the expression and subcellular localization of cyclin D1, a major cell-cycle regulator overexpressed in MCL, and the cyclin-dependent kinase inhibitor p27KIP1, could identify MCL cases sensitive to lenalidomide, and whether the compound could modulate cyclin D1/p27KIP1 complexes in MCL cells. Experimental Design: MCL primary samples and cell lines were analyzed for subcellular levels of cyclin D1/p27KIP1 complexes by Western blot, immunohistochemistry, immunoprecipitation, and flow cytometry. Activity of lenalidomide in vitro and its effect on cyclin D1/p27KIP1 complexes were evaluated by real-time PCR, immunoprecipitation, immunofluorescence, and Western blot. In vivo validation was carried out in a mouse xenograft model of human MCL. Results: We found cyclin D1 and p27KIP1 to be coordinately expressed in all the MCL samples tested. Immunoprecipitation analyses and siRNA assays suggested a direct role of cyclin D1 in the regulation of p27KIP1 levels. The nuclear accumulation of both proteins correlated with MCL cell tumorigenicity in vivo, and sensitivity to lenalidomide activity in vitro and in vivo. Lenalidomide mechanism of action relied on cyclin D1 downregulation and disruption of cyclin D1/p27KIP1 complexes, followed by cytosolic accumulation of p27KIP1, cell proliferation arrest, apoptosis, and angiogenesis inhibition. Conclusions: These results highlight a mechanism of action of lenalidomide in MCL cases with increased tumorigenicity in vivo, which is mediated by the dissociation of cyclin D1/p27KIP1 complexes, and subsequent proliferation blockade and apoptosis induction. Clin Cancer Res; 20(2); 393–403. ©2013 AACR.

Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells

Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.

Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

BackgroundPharmacological inhibition of B cell receptor (BCR) signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton’s kinase (Btk) and spleen tyrosine kinase (Syk) inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents.MethodsWe evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn), in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma.ResultsIQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest.ConclusionsThese results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.

Expression and subcellular localization of cyclin-dependent kinase inhibitor p27 does not correlate with proliferation pattern of mantle lymphoma cells

, September 2014; 55(9): 2204–2206© 2014 Informa UK, Ltd.ISSN: 1042-8194 print / 1029-2403 onlineDOI: 10.3109/10428194.2013.871633 Correspondence: Brigitte Sola, Normandie Univ, UNICAEN, MILPAT (EA 4652), F-14032 Caen, France. Tel: 33-2-3106-8210. Fa x: 33-2-3106-8224. E-mail: brigitte.sola@unicaen.fr

Abstract 1942: Activity of lenalidomide in vitro and in vivo models of bortezomib-resistant mantle cell lymphoma involving the modulation of c-myc/p27 axis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) leading to the overexpression of cyclin D1. As a consequence of its poor responses to conventional chemotherapy and relatively short patient survival, new therapeutic strategies are required. Despite the promising introduction of the proteasome inhibitor bortezomib in the clinical practice, not all the patients respond and relapse frequently occurres after initial response. When comparing the behavior of both bortezomib-resistant and bortezomib-sensitive cell lines in a xenotransplant mouse model, we observed an increased tumorigenecity of bortezomib-resistant cells in vivo, suggesting a major capacity of these tumors to interact with lymphoid microenvironment. As the immunomoduladory drug lenalidomide has been shown to modulate tumor-stroma interaction in several B cell malignancies, we assessed the activity in vitro and in vivo of this agent either alone or combined with the proteasome inhibitor in both bortezomib-resistant and bortezomib-sensitive samples. Lenalidomide single agent was found to exert modest antitumoral activity in 2/10 MCL cell lines, corresponding to those cells with either primary or acquired resistance to the proteasome inhibitor. Conversely, mice bearing bortezomib-resistant tumors and treated for 3 weeks with a 10-50 mg/kg/day regimen of lenalidomide, showed a 30 to 45% reduction in tumor burden when compared to vehicle-treated group (p<0.05). The corresponding biopsies harbored several hallmarks of lenalidomide activity in malignant B cells such as CD80 and CD40L upregulation, together with a remarkable decrease in mitotic index, c-myc down-regulation, p27 cytosolic accumulation and caspase-3 processing. Similarly, bortezomib-resistant MCL cell lines treated for 72h with 1 microM lenalidomide showed lower c-myc levels, as well as p27 accumulation, caspase-3/7 activity and apparition of hypodiploid cells. When combined to bortezomib therapy (0.15 mg/kg, twice a week), lenalidomide induced a 37% and a 66% inhibition of tumor growth when compared to lenalidomide and vehicle groups, respectively (p=0.02). In accordance, lenalidomide showed synergistic effect in vitro with bortezomib in co-culture system associating the MCL cell line Jeko-1 to the dendritic-like cells BDCM, by modifying the secretion pattern of these latest. Altogether, these results suggest that single agent lenalidomide is preferentially effective in MCL cases resistant to bortezomib, by targeting c-myc-driven tumorigenesis. Additionally, lenalidomide may overcome the protection offered by lymphoid tumor microenvironment toward bortezomib treatment, thus warranting a promising clinical activity of lenalidomide-bortezomib combination in MCL cases refractory to bortezomib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1942. doi:1538-7445.AM2012-1942