Vanina Rodriguez

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.

The Human CD38 Monoclonal Antibody Daratumumab Shows Antitumor Activity and Hampers Leukemia-Microenvironment Interactions in Chronic Lymphocytic Leukemia.

Purpose: To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ chronic lymphocytic leukemia (CLL) subtype. Experimental Design: The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP), or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models. Results: Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo whereas exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38+ MEC2 cell line and CLL patient–derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs, and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by downregulation of the matrix metalloproteinase MMP9. Conclusions: These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL. Clin Cancer Res; 23(6); 1493–505. ©2016 AACR.

Disruption of follicular dendritic cells-follicular lymphoma cross-talk by the pan-PI3K inhibitor BKM120 (Buparlisib).

Purpose: To uncover the signaling pathways underlying follicular lymphoma–follicular dendritic cells (FL–FDC) cross-talk and its validation as new targets for therapy. Experimental Design: FL primary cells and cell lines were cocultured in the presence or absence of FDC. After 24 and 48 hours, RNA was isolated from FL cells and subjected to gene expression profiling (GEP) and data meta-analysis using DAVID and GSEA softwares. Blockade of PI3K pathway by the pan-PI3K inhibitor BKM120 (buparlisib; Novartis Pharmaceutical Corporation) and the effect of PI3K inhibition on FL–FDC cross-talk were analyzed by means of ELISA, RT-PCR, human umbilical vein endothelial cell tube formation, adhesion and migration assays, Western blot, and in vivo studies in mouse FL xenografts. Results: GEP of FL–FDC cocultures yields a marked modulation of FL transcriptome by FDC. Pathway assignment by DAVID and GSEA software uncovered an overrepresentation of genes related to angiogenesis, cell adhesion, migration, and serum-response factors. We demonstrate that the addition of the pan-PI3K inhibitor BKM120 to the cocultures was able to downregulate the expression and secretion of proangiogenic factors derived from FL–FDC cocultures, reducing in vitro and in vivo angiogenesis. Moreover, BKM120 efficiently counteracts FDC-mediated cell adhesion and impedes signaling and migration induced by the chemokine CXCL12. BKM120 inhibits both constitutive PI3K/AKT pathway and FDC- or CXCL12-induced PI3K/AKT pathway, hampers FDC survival signaling, and reduces cell proliferation of FL cells in vitro and in mouse xenografts. Conclusions: These data support the use of BKM120 in FL therapy to counteract microenvironment-related survival signaling in FL cells. Clin Cancer Res; 20(13); 3458–71. ©2014 AACR.

The BET bromodomain inhibitor CPI203 overcomes resistance to ABT-199 (venetoclax) by downregulation of BFL-1/A1 in in vitro and in vivo models of MYC +/ BCL2 + double hit lymphoma

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, mostly known as double-hit lymphoma (DHL), is a rare entity characterized by morphologic and molecular features between Burkitt lymphoma and the clinically manageable diffuse large B-cell lymphoma (DLBCL). DHL patients usually undergo a rapidly progressing clinical course associated with resistance to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival inferior to 1 year. ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of a specific subtype of lymphoid neoplasm. In this study, we demonstrate that single-agent ABT-199 efficiently displaces BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+DHL cell lines and primary cultures, as well as in a xenograft mouse model of the disease. We further identify the accumulation of the BCL2-like protein BFL-1 to be a major mechanism involved in acquired resistance to ABT-199. Noteworthy, this phenomenon can be counteracted by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1, the BFL-1 coding gene, as one of the top apoptosis-related gene modulated by this compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 further overcomes resistance to ABT-199 both in vitro and in vivo, engaging synergistic caspase-mediated apoptosis in DHL cultures and tumor xenografts. Together, these findings highlight the relevance of BFL-1 in DH lymphoma-associated drug resistance and support the combined use of a BCL-2 antagonist and a BET inhibitor as a promising therapeutic strategy for patients with aggressive DHL.

The BET bromodomain inhibitor CPI203 improves lenalidomide and dexamethasone activity in in vitro and in vivo models of multiple myeloma by blockade of Ikaros and MYC signaling

Most patients with multiple myeloma treated with current therapies, including immunomodulatory drugs, eventually develop relapsed/refractory disease. Clinical activity of lenalidomide relies on degradation of Ikaros and the consequent reduction in IRF4 expression, both required for myeloma cell survival and involved in the regulation of MYC transcription. Thus, we sought to determine the combinational effect of an MYC-interfering therapy with lenalidomide/dexamethasone. We analyzed the potential therapeutic effect of the combination of the BET bromodomain inhibitor CPI203 with the lenalidomide/dexamethasone regimen in myeloma cell lines. CPI203 exerted a dose-dependent cell growth inhibition in cell lines, indeed in lenalidomide/dexamethasone-resistant cells (median response at 0.5 μM: 65.4%), characterized by G1 cell cycle blockade and a concomitant inhibition of MYC and Ikaros signaling. These effects were potentiated by the addition of lenalidomide/dexamethasone. Results were validated in primary plasma cells from patients with multiple myeloma co-cultured with the mesenchymal stromal cell line stromaNKtert. Consistently, the drug combination evoked a 50% reduction in cell proliferation and correlated with basal Ikaros mRNA expression levels (P=0.04). Finally, in a SCID mouse xenotransplant model of myeloma, addition of CPI203 to lenalidomide/dexamethasone decreased tumor burden, evidenced by a lower glucose uptake and increase in the growth arrest marker GADD45B, with simultaneous downregulation of key transcription factors such as MYC, Ikaros and IRF4. Taken together, our data show that the combination of a BET bromodomain inhibitor with a lenalidomide-based regimen may represent a therapeutic approach to improve the response in relapsed/refractory patients with multiple myeloma, even in cases with suboptimal prior response to immunomodulatory drugs.

The Bruton tyrosine kinase inhibitor CC-292 shows activity in mantle cell lymphoma and synergizes with lenalidomide and NIK inhibitors depending on nuclear factor-κB mutational status

Ibrutinib is a first-in-class covalent inhibitor of Bruton tyrosine kinase (BTK) approved in the European Union and USA for the treatment of patients with relapsed/refractory mantle cell lymphoma (MCL). Despite ibrutinib’s promising activity, recent data suggest that primary and secondary resistance is common in MCL patients treated with this agent. Mutations within the BTK active site (Cys481Ser) have been described in MCL as a secondary mechanism of ibrutinib resistance. For primary resistance unrelated to BTK mutations, recent studies suggest the presence of alternative mechanisms. One investigation indicate that MCL cell lines exhibiting ibrutinib sensitivity depend on chronic activation of the B-cell receptor (BCR) leading to the activation of the classical nuclear factor-kB (NF-kB) pathway, whereas ibrutinib-resistant MCL cell lines rely on the alternative NF-kB pathway and anti-BTK therapy would be unlikely to be of benefit. CC-292 is a highly selective, oral, small molecule inhibitor that shows greater selectivity than ibrutinib against BTK. Both single-agent and combination trials with CC-292 are ongoing in patients with a wide variety of B-cell lymphoproliferative disorders. The aim of this study was to evaluate the antitumor profile of CC-292 in MCL, together with its impact on cellular activation, migration and tumor-stroma crosstalk. We also explored possible combination strategies to enhance CC-292 activity. We first investigated the antitumor effects of CC-292 in five MCL cell lines (REC-1, MINO, UPN-1, MAVER-1 and Z138) after 72 h of treatment. CC-292 (10-1000 nM) had a cytostatic effect in a subset of cell lines, with REC1, MINO and UPN-1 appearing to be the most sensitive, while MAVER-1 and Z138 were the most resistant to

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.

Abstract 1757: The novel BTK inhibitor CC-292 exerts in vitro and in vivo antitumor activity, interferes with adhesion, cell migration, and synergizes with lenalidomide in MCL models

Background B-cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies and has emerged as a new target for therapy with special relevance for tumor cell-microenvironment crosstalk. Recently, blockade of the BCR-related kinase Btk (Bruton tyrosin kinase) with the first-in-class inhibitor Ibrutinib, has shown impressive clinical responses in Mantle cell lymphoma (MCL) and Chronic Lymphocytic Leukemia. However, resistances to treatment have already appeared, opening the door to new BTK inhibitors. In this study, we have evaluated the antitumor profile of a novel and highly selective BTK inhibitor CC-292(Celgene)in MCL. Methods Representative MCL lines (MINO, JEKO-1, REC-1, UPN1, Z138, HBL2, GRANTA, JVM-2) were used for in vitro and in vivo studies. The effects of CC-292 on lymphoma cell growth and apoptosis were analyzed by MTT and Annexin V/PI staining, respectively. BTK phosphorylation at Y223 residue was used as a read-out of BTK activity. Microenvironment-derived BTK activation was mimicked by IgM crosslinking or SDF1α/CXCL12 stimulation. Actin polymerization experiments were performed using Phalloidin-TRITC staining, and adhesion assays were done on EIA/RIA 96-well plates coated with Fibronectin or VCAM-1. In vivo activity was assessed in a subcutaneous mouse xenograft model, where daily oral dosing was started when tumors were palpable and extended for a total of 18 days. Results BTK activation (pBtkY(223)) was detected in all MCL cell lines analyzed. CC-292 (10-1000nM) exerted cytostatic effect in part of the cell lines, where REC-1, MINO, JVM2 and UPN1 were the most sensitive cell lines. No correlation between constitutive Btk activation and response to CC-292 was found. Noteworthy, CC-292 potently inhibited constitutive and IgM/CXCL12-induced Btk activation and interfered with tumor cell-microenvironment interactions, by blocking SDF1α/CXCL12-induced actin polymerization, as well as IgM-induced adhesion to VCAM and FN. In CC-292 sensitive cell lines, the combination with the immunomodulatory drug lenalidomide was synergistic and was accompanied with IRF4 decrease in MINO and REC cell lines. Finally, CC-292 (30mg/kg) showed antitumor activity in vivo in a sc mouse xenograft (UPN-1 cell line), reducing tumor outgrowth by 52% Conclusions In summary, these results warrant further investigation of CC-292 for MCL therapy, both alone and in combination with the immunomodulatory agent lenalidomide. Citation Format: Anna Vidal-Crespo, Vanina Rodriguez, Alba Matas-Cespedes, Elias Campos, Armando Lopez-Guillermo, Gael Roue, Dolors Colomer, Patricia Perez-Galan. The novel BTK inhibitor CC-292 exerts in vitro and in vivo antitumor activity, interferes with adhesion, cell migration, and synergizes with lenalidomide in MCL models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1757. doi:10.1158/1538-7445.AM2014-1757

Loss of the Immune Checkpoint CD85j/LILRB1 on Malignant Plasma Cells Contributes to Immune Escape in Multiple Myeloma

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (LILRB1), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell– and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.