Gaël Roué

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.

The Hsp90 inhibitor IPI-504 overcomes bortezomib resistance in mantle cell lymphoma in vitro and in vivo by down-regulation of the prosurvival ER chaperone BiP/Grp78

Despite the promising introduction of the proteasome inhibitor bortezomib in the treatment of mantle cell lymphoma (MCL), not all patients respond, and resistance often appears after initial treatment. By analyzing a set of 18 MCL samples, including cell lines with constitutive or induced resistance to bortezomib, we found a high correlation between loss of sensitivity to the proteasome inhibitor and up-regulation of the prosurvival chaperone BiP/Grp78. BiP/Grp78 stabilization was ensured at a posttranscriptional level by an increase in the chaperoning activity of heat shock protein of 90 kDa (Hsp90). In bortezomib-resistant cells, both BiP/Grp78 knockdown and cell pretreatment with the Hsp90 inhibitor of the ansamycin class, IPI-504, led to synergistic induction of apoptotic cell death when combined with bortezomib. Cell exposure to the IPI-504-bortezomib combination provoked the dissociation of Hsp90/BiP complexes, leading to BiP/Grp78 depletion, inhibition of unfolded protein response, and promotion of NOXA-mediated mitochondrial depolarization. The IPI-504-bortezomib combination also prevented BiP/Grp78 accumulation, thereby promoting apoptosis and inhibiting the growth of bortezomib-resistant tumors in a mouse model of MCL xenotransplantation. These results suggest that targeting unfolded protein response activation by the inhibition of Hsp90 may be an attractive model for the design of a new bortezomib-based combination therapy for MCL.

Selective inhibition of IkappaB kinase sensitizes mantle cell lymphoma B cells to TRAIL by decreasing cellular FLIP level.

In an attempt to circumvent the intrinsic resistance of mantle cell lymphoma (MCL) cells to apoptosis, we have analyzed their sensitivity to the extrinsic apoptotic signal triggered by TRAIL. We show here that TRAIL can trigger apoptosis in a majority of MCL cell lines and primary cultures, irrespective of receptor levels, Bcl-2 family members, or caspase regulator expression. MCL sensitivity to TRAIL was closely linked to the activity of the NF-κB p50 factor and to the consequent expression of cellular FLIP (c-FLIP), which accumulated into the TRAIL-dependent complex in resistant cells. c-FLIP transient knockdown overcame MCL resistance to TRAIL, while NF-κB inhibitors differentially modulated TRAIL cytotoxicity. Indeed, bortezomib increased TRAIL cytotoxic effects in sensitive cells, but led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction. In contrast, the IκB kinase inhibitor BMS-345541 led to decreased c-FLIP expression and allowed all MCL samples to undergo TRAIL-mediated apoptosis. These results present the combination of TRAIL stimulation and IκB kinase inhibition as a new approach to MCL therapy.

BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is a B-cell lymphoid neoplasm with deregulated apoptosis and overexpression of several antiapoptotic BCL-2 proteins. GX15-070/Obatoclax is a small-molecule BH3 mimetic compound that has shown activity against several hematologic malignancies and solid tumors. In the present work, we report that GX15-070 led to the disruption of BCL-2/BIM and MCL-1/BAK complexes in CLL cells, followed by the activation of the mitochondrial apoptotic pathway. CLL cells showed lower sensitivity to GX15-070 than primary mantle cell lymphoma (MCL) ones, in correlation with higher levels of phosphorylated BCL-2 at serine 70 residue (pBCL-2(Ser70)) in CLL cells. Decrease in BCL-2 phosphorylation by extracellular signal-regulated kinase (ERK)1/2 inhibition increased CLL sensitivity to GX15-070, while blocking BCL-2 dephosphorylation using a PP2A antagonist reduced the activity of this BH3 mimetic. GX15-070 activity was increased by cotreatment with the proteasome inhibitor bortezomib. However, as proteasome inhibition led to the accumulation of phosphorylated BCL-2, the degree of interaction between GX15-070 and bortezomib was regulated by basal pBCL-2(Ser70) levels. These results support the role of BCL-2 phosphorylation as a mechanism of resistance to BH3 mimetic compounds, and demonstrate that combination approaches including ERK inhibitors could enhance BH3 mimetics activity both alone or in combination with proteasome inhibitors.

In vitro and In vivo Selective Antitumor Activity of Edelfosine against Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Involving Lipid Rafts

Purpose: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) remain B-cell malignancies with limited therapeutic options. The present study investigates the in vitro and in vivo effect of the phospholipid ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) in MCL and CLL. Experimental Design: Several cell lines, patient-derived tumor cells, and xenografts in severe combined immunodeficient mice were used to examine the anti-MCL and anti-CLL activity of edelfosine. Furthermore, we analyzed the mechanism of action and drug biodistribution of edelfosine in MCL and CLL tumor-bearing severe combined immunodeficient mice. Results: Here, we have found that the phospholipid ether edelfosine was the most potent alkyl-lysophospholipid analogue in killing MCL and CLL cells, including patient-derived primary cells, while sparing normal resting lymphocytes. Alkyl-lysophospholipid analogues ranked edelfosine > perifosine ≫ erucylphosphocholine ≥ miltefosine in their capacity to elicit apoptosis in MCL and CLL cells. Edelfosine induced coclustering of Fas/CD95 death receptor and rafts in MCL and CLL cells. Edelfosine was taken up by malignant cells, whereas normal resting lymphocytes hardly incorporated the drug. Raft disruption by cholesterol depletion inhibited drug uptake, Fas/CD95 clustering, and edelfosine-induced apoptosis. Edelfosine oral administration showed a potent in vivo anticancer activity in MCL and CLL xenograft mouse models, and the drug accumulated dramatically and preferentially in the tumor. Conclusions: Our data indicate that edelfosine accumulates and kills MCL and CLL cells in a rather selective way, and set coclustering of Fas/CD95 and lipid rafts as a new framework in MCL and CLL therapy. Our data support a selective antitumor action of edelfosine. Clin Cancer Res; 16(7); 2046–54. ©2010 AACR.

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.

Bendamustine Is Effective in p53-Deficient B-Cell Neoplasms and Requires Oxidative Stress and Caspase-Independent Signaling

Purpose: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are two incurable B-cell lymphoid neoplasms characterized by distinct clinical presentation and evolution. Bendamustine hydrochloride is a multifunctional, alkylating agent with a purine-like ring system that exhibits activity in multiple cancer models, including CLL and MCL, but whose mechanism is only partially described. Our aim was to analyze the apoptotic pathways activated by bendamustine in CLL and MCL together with the relevance of p53 mutation in determining the response to this drug. Experimental Design: Thirteen CLL/MCL cell lines and primary tumor cells from 8 MCL and 25 CLL patients were cultured for up to 24 h with bendamustine followed by cytotoxic assays, flow cytometry, immunofluorescence, and Western blot analysis of p53 response pathway and apoptosis-related factors. Results: Bendamustine displayed cytotoxic activity on most CLL and MCL primary cells and cell lines irrespective of ZAP-70 expression and p53 status. Bendamustine was found to act synergistically with nucleoside analogues in both CLL and MCL, this combination being effective in p53 mutated cases resistant to standard chemotherapy. Bendamustine cytotoxicity was mediated by the generation of reactive oxygen species and triggering of the intrinsic apoptotic pathway involving up-regulation of PUMA and NOXA, conformational activation of BAX and BAK, and cytosolic release of caspase-related and caspase-unrelated mitochondrial apoptogenic proteins. Conclusions: Our findings support the use of bendamustine as a therapeutic agent, alone or in combination, for CLL and MCL with p53 alterations and describe the molecular basis of its activity in these entities.

Drp1 mediates caspase-independent type III cell death in normal and leukemic cells

ABSTRACT Ligation of CD47 triggers caspase-independent programmed cell death (PCD) in normal and leukemic cells. Here, we characterize the morphological and biochemical features of this type of death and show that it displays the hallmarks of type III PCD. A molecular and biochemical approach has led us to identify a key mediator of this type of death, dynamin-related protein 1 (Drp1). CD47 ligation induces Drp1 translocation from cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 provokes an impairment of the mitochondrial electron transport chain, which results in dissipation of mitochondrial transmembrane potential, reactive oxygen species generation, and a drop in ATP levels. Surprisingly, neither the activation of the most representative proapoptotic members of the Bcl-2 family, such as Bax or Bak, nor the release of apoptogenic proteins AIF (apoptosis-inducing factor), cytochrome c, endonuclease G (EndoG), Omi/HtrA2, or Smac/DIABLO from mitochondria to cytosol is observed. Responsiveness of cells to CD47 ligation increases following Drp1 overexpression, while Drp1 downregulation confers resistance to CD47-mediated death. Importantly, in B-cell chronic lymphocytic leukemia cells, mRNA levels of Drp1 strongly correlate with death sensitivity. Thus, this previously unknown mechanism controlling caspase-independent type III PCD may provide the basis for novel therapeutic approaches to overcome apoptotic avoidance in malignant cells.

Vorinostat-Induced Apoptosis in Mantle Cell Lymphoma Is Mediated by Acetylation of Proapoptotic BH3-Only Gene Promoters

Purpose: Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm with generally poor prognosis, for which current therapies have shown limited efficacy. Vorinostat is a histone deacetylase inhibitor (HDACi) that has been approved for the treatment of cutaneous T-cell lymphoma. Our purpose was to describe the molecular mechanism whereby vorinostat induces apoptosis in MCL with particular emphasis on the role of proapoptotic BH3-only proteins. Experimental Design: The sensitivity to vorinostat was analyzed in eight MCL cell lines and primary cells from 10 MCL patients. Determination of vorinostat mechanism of action was done by flow cytometry, immunoblotting, HDAC activity assay kit, quantitative reverse transcription PCR, chromatin immunoprecipitation, and siRNA-mediated transfection. Results: Vorinostat inhibited total histone deacetylase activity leading to selective toxicity toward tumor cells. Vorinostat-mediated cell death implied the activation of mitochondrial apoptosis, as attested by BAX and BAK conformational changes, mitochondrial depolarization, reactive oxygen species generation, and subsequent caspase-dependent cell death. This phenomenon was linked to H4 hyperacetylation on promoter regions and consequent transcriptional activation of the proapoptotic BH3-only genes BIM, BMF, and NOXA. Selective knockdown of the three corresponding proteins rescued cells from vorinostat-induced apoptosis. Moreover, vorinostat enhanced the activity of the BH3-mimetic ABT-263 in MCL cells, leading to synergistic apoptosis induction. Conclusion: These results indicated that transcriptional upregulation of BH3-only proteins plays an important role in the antitumoral activity of vorinostat in MCL, and that HDACi alone or in combination with BH3-mimetizing agents may represent a promising therapeutic approach for MCL patients. Clin Cancer Res; 17(12); 3956–68. ©2011 AACR.

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.