Laia Rosich

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.

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.

Counteracting Autophagy Overcomes Resistance to Everolimus in Mantle Cell Lymphoma

Purpose: Mantle cell lymphoma (MCL) is an aggressive B-lymphoid neoplasm with poor response to conventional chemotherapy and short survival. The phosphatidylinositol 3-kinase/Akt/mTOR survival pathway is constitutively activated in MCL cells, thereby making the mTOR inhibition an attractive therapeutic strategy. The first clinical studies of everolimus (RAD001), an mTOR inhibitor, in relapsed MCL patients have reported a significant response. Our aim was to analyze the mechanism related to everolimus resistance/sensitivity in MCL cells. Experimental Design: Sensitivity to everolimus was analyzed in MCL cell lines and primary MCL cells. Everolimus mechanism of action was determined by flow cytometry and Western blot. Particularly, autophagy was studied by LC3BI/II expression, autophagolysosomes detection by flow cytometry and fluorescence microscopy, and siRNA-mediated gene silencing. Results: Everolimus exerted antitumoral effect on MCL cells while sparing normal cells. In MCL cell lines, this phenomenon was associated to G1 cell-cycle arrest, dephosphorylation of the mTOR downstream targets, 4E-BP1 and S6RP, and rephosphorylation of Akt. A synergistic cytotoxic effect was observed between everolimus and an Akt inhibitor, which overcame the compensatory reactivation within the mTOR signaling pathway. Interestingly, MCL cells with low response to this combination showed high levels of autophagy. Accordingly, selective triple knockdown of the autophagy genes ATG7, ATG5 and ATG3, and pretreatment with the autophagy inhibitor hydroxychloroquine, efficiently overcame the resistance to Akt/mTOR inhibitors, leading to the activation of the mitochondrial apoptotic pathway. Conclusions: These results suggest that autophagy induction protects MCL cells from Akt/mTOR targeting and counteracting autophagy may represent an attractive strategy for sensitizing MCL cells to everolimus-based therapy. Clin Cancer Res; 18(19); 5278–89. ©2012 AACR.

The γ-secretase inhibitor PF-03084014 combined with fludarabine antagonizes migration, invasion and angiogenesis in NOTCH1-mutated CLL cells

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), especially for the poor prognostic subgroup of NOTCH1-mutated patients. Here, we report that the γ-secretase inhibitor PF-03084014 inhibits the constitutive Notch activation and induces selective apoptosis in CLL cells carrying NOTCH1 mutations. Combination of PF-03084014 with fludarabine has a synergistic antileukemic effect in primary NOTCH1-mutated CLL cells, even in the presence of the protective stroma. At transcriptional level, PF-03084014 plus fludarabine treatment induces the upregulation of the proapoptotic gene HRK and the downmodulation of MMP9, IL32 and RAC2 genes that are related to invasion and chemotaxis. PF-03084014 also overcomes fludarabine-mediated activation of nuclear factor-κB signaling. Moreover, this combination impairs angiogenesis and CXCL12-induced responses in NOTCH1-mutated CLL cells, in particular those related to tumoral migration and invasion. Importantly, all these collaborative effects are specific for NOTCH1 mutation and do not occur in unmutated cases. In conclusion, we provide evidence that Notch is a therapeutic target in CLL cases with NOTCH1-activating mutations, supporting the use of Notch pathway inhibitors in combination with chemotherapy as a promising approach for the treatment of these high-risk CLL patients.

The phosphatidylinositol-3-kinase inhibitor NVP-BKM120 overcomes resistance signals derived from microenvironment by regulating the Akt/FoxO3a/Bim axis in chronic lymphocytic leukemia cells

Phosphatidylinositol-3-kinase pathway is constitutively activated in chronic lymphocytic leukemia mainly due to microenvironment signals, including stromal cell interaction and CXCR4 and B-cell receptor activation. Because of the importance of phosphatidylinositol-3-kinase signaling in chronic lymphocytic leukemia, we investigated the activity of the NVP-BKM120, an orally available pan class I phosphatidylinositol-3-kinase inhibitor. Sensitivity to NVP-BKM120 was analyzed in chronic lymphocytic leukemia primary samples in the context of B-cell receptor and microenvironment stimulation. NVP-BKM120 promoted mitochondrial apoptosis in most primary cells independently of common prognostic markers. NVP-BKM120 activity induced the blockage of phosphatidylinositol-3-kinase signaling, decreased Akt and FoxO3a phosphorylation leading to concomitant Mcl-1 downregulation and Bim induction. Accordingly, selective knockdown of BIM rescued cells from NVP-BKM120-induced apoptosis, while the kinase inhibitor synergistically enhanced the apoptosis induced by the BH3-mimetic ABT-263. We also found NVP-BKM120 to inhibit B-cell receptor- and stroma-dependent Akt pathway activation, thus sensitizing chronic lymphocytic leukemia cells to bendamustine and fludarabine. Furthermore, NVP-BKM120 down-regulated secretion of chemokines after B-cell receptor stimulation and inhibited cell chemotaxis and actin polymerization upon CXCR4 triggering by CXCL12. Our findings establish that NVP-BKM120 effectively inhibits the phosphatidylinositol-3-kinase signaling pathway and disturbs the protective effect of the tumor microenvironment with the subsequent apoptosis induction through the Akt/FoxO3a/Bim axis. We provide here a strong rationale for undertaking clinical trials of NVP-BKM120 in chronic lymphocytic leukemia patients alone or in combination therapies.

Sorafenib inhibits cell migration and stroma-mediated bortezomib resistance by interfering B-cell receptor signaling and protein translation in mantle cell lymphoma

Purpose: We evaluated the antitumoral properties of the multikinase inhibitor sorafenib in mantle cell lymphoma (MCL), an aggressive B lymphoma for which current therapies have shown limited efficacy. Experimental Design: Sensitivity to sorafenib was analyzed in MCL cell lines and primary samples in the context of BCR and microenvironment simulation. Sorafenib signaling was characterized by quantitative PCR, Western blotting, immunofluorescence, and protein immunoprecipitation. Migration analysis included flow cytometric counting, actin polymerization assays, and siRNA-mediated knockdown of focal adhesion kinase (FAK). In vivo antitumor effect of sorafenib and bortezomib was analyzed in an MCL xenograft mouse model. Results: Sorafenib rapidly dephosphorylates the BCR-associated kinases, Syk and Lyn, as well as FAK, an Src target involved in focal adhesion. In this line, sorafenib displays strong synergy with the Syk inhibitor, R406. Sorafenib also blocks Mcl-1 and cyclin D1 translation, which promotes an imbalance between pro- and antiapoptotic proteins and facilitates Bax release from cyclin D1, leading to the induction of mitochondrial apoptosis and caspase-dependent and -independent mechanisms. Moreover, sorafenib inhibits MCL cell migration and CXCL12-induced actin polymerization. FAK knockdown partially prevents this inhibitory effect, indicating that FAK is a relevant target of sorafenib. Furthermore, sorafenib enhances the antitumoral activity of bortezomib in an MCL xenograft mouse model as well as overcomes stroma-mediated bortezomib resistance in MCL cells. Conclusion: We show for the first time that sorafenib interferes with BCR signaling, protein translation and modulates the microenvironment prosurvival signals in MCL, suggesting that sorafenib, alone or in combination with bortezomib, may represent a promising approach to treat patients with MCL. Clin Cancer Res; 19(3); 586–97. ©2012 AACR.

Autophagy controls everolimus (RAD001) activity in mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive neoplasm, which lacks effective therapy. The mechanistic target of rapamycin (MTOR) kinase inhibitor everolimus (RAD001) has shown activity in preclinical and clinical models of MCL, despite the fact that its mechanism of action has not been fully elucidated. We found that everolimus activity in MCL cells is closely linked to AKT phosphorylation status, and that the prevention of AKT rephosphorylation upon everolimus treatment by means of a selective AKT inhibitor, greatly enhances everolimus activity. Furthermore, our data show that an accumulation of autophagic vacuoles correlates with a lack of efficacy of dual AKT-MTOR targeting and that the complete therapeutic potential of this strategy can be restored by ATG gene selective knockdown or secondary inhibition of autolysosome formation by hydroxychloroquine. We thus demonstrated for the first time that the use of an autophagy inhibitor can overcome resistance to the combination of MTOR and AKT inhibitors in MCL cell lines and primary samples, demonstrating the prosurvival role of autophagy in AKT-MTOR compromised cells, and pointing out some potential opportunities using this triple combinational strategy in hematological malignancies.

Sorafenib targets BCR kinases and blocks migratory and microenvironmental survival signals in CLL cells.

Sorafenib targets BCR kinases and blocks migratory and microenvironmental survival signals in CLL cells

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.

CD69 expression potentially predicts response to bendamustine and its modulation by ibrutinib or idelalisib enhances cytotoxic effect in chronic lymphocytic leukemia

Clinical responses to bendamustine in chronic lymphocytic leukemia (CLL) are highly heterogeneous and no specific markers to predict sensitivity to this drug have been reported. In order to identify biomarkers of response, we analyzed the in vitro activity of bendamustine and the gene expression profile in primary CLL cells. We observed that mRNA expression of CD69 (CD69) and ITGAM (CD11b) constitute the most powerful predictor of response to bendamustine. When we interrogated the predictive value of the corresponding cell surface proteins, the expression of the activation marker CD69 was the most reliable predictor of sensitivity to bendamustine. Importantly, a multivariate analysis revealed that the predictive value of CD69 expression was independent from other clinico-biological CLL features. We also showed that when CLL cells were co-cultured with distinct subtypes of stromal cells, an upregulation of CD69 was accompanied by a reduced sensitivity to bendamustine. In agreement with this, tumor cells derived from lymphoid tumor niches harbored higher CD69 expression and were less sensitive to bendamustine than their peripheral blood counterparts. Furthermore, pretreatment of CD69 high CLL cases with the B-cell receptor (BCR) pathway inhibitors ibrutinib and idelalisib decreased CD69 levels and enhanced bendamustine cytotoxic effect. Collectively, our findings indicate that CD69 could be a predictor of bendamustine response in CLL patients and the combination of clinically-tested BCR signaling inhibitors with bendamustine may represent a promising strategy for bendamustine low responsive CLL cases.