ALBERT

Description: Key Points SOX11 silencing promotes the shift from a mature B cell into the initial plasmacytic differentiation phenotype in MCL. SOX11 promotes tumor growth of MCL cells in vivo, highlighting its implication in the aggressive behavior of conventional MCL.

Description: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are two different types of mature B-cell non-Hodgkin’s lymphoma (NHL). CLL has an indolent natural history and patients are very responsive to frontline chemotherapy. Unfortunately, multiple relapses are inevitable, and ultimately, no regimen or treatment strategy offers a distinct survival benefit over another. In contrast, patients with MCL generally experience a more aggressive course, with rapid disease progression and also without specific therapeutic options. Bendamustine hydrochloride (Treanda™) is a multifunctional, alkylating agent that exhibits single-agent activity in multiple hematologic and solid tumors. Recently, the combination of bendamustine with rituximab has demonstrated to be a highly active regimen in the treatment of low-grade lymphomas and MCL. However, very little is known about its mode of action. The ability of bendamustine to induce apoptosis in vitro in MCL and CLL cells and the mechanisms implicated in bendamustine-evoked cell death signaling were investigated. Bendamustine exerted cytostatic and cytotoxic effects in 11 MCL cell lines and primary tumor cells from 7 MCL patients and 10 CLL patients independent of their p53 status, and other gene alterations. In vitro treatment of cells with bendamustine induced activation of both p53-dependent and -independent signaling pathways that converged in all cases to the activation of the pro-apoptotic protein Noxa, conformational changes of Bax and Bak, and mitochondrial depolarization. These events led to cytosolic release of the mitochondrial apoptogenic factors cytochrome c, Smac/DIABLO and AIF, and activation of both caspase -dependent and -independent cell death. Genotoxic stress and caspase-independent cell death are often associated with the generation of reactive oxygen species (ROS). We observed that ROS production was a key step in the induction of apoptosis by bendamustine, since pre-incubation of tumor cells with ROS scavengers reverted all the typical hallmarks of apoptosis. Furthermore, bendamustine exerted a cytotoxic effect in p53 deleted CLL cases that were resistant to fludarabine treatment. These findings support the use of bendamustine as a therapeutic agent in MCL and CLL cells and also establish the basis for the use of bendamustine in lymphoid malignancies that show resistance to classic genotoxic agents that depend on cellular p53 status.

Description: Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.

Description: Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Description: 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.

Description: Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm with a mature B-cell phenotype and genetically characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression with the consequent deregulation of cell cycle at the G1-S checkpoint. MCL cells present a constitutive activation of the NF-kappaB pathway which leads to the overexpression of several anti-apoptotic regulators. Recently, MCL cells have been shown to express high levels of the chaperone heat shock protein of 90 kDa (HSP90) and to respond well to the ansamycin derivative 17-AAG, an HSP90 inhibitor. We have analyzed the sensitivity to the novel, highly soluble, 17-AAG derivative IPI-504 (Infinity Pharmaceuticals) on a panel of eleven human MCL cell lines and primary cells from MCL patients, which differ in their p53-dependent pathway status, growth characteristics and sensitivity to cytotoxic drugs. We observed that IPI-504 heterogeneously exerted cytostatic effect among MCL samples, with IC50 ranging from 0.06 to 15.4 μM, irrespective of the mutational status of the client protein p53 and/or expression levels of other client proteins such as cyclin D1 and BCL-2. IPI-504 activity involved the downregulation of the client proteins IKKb and phospho-AKT, and consequent inhibition of NF-kappaB signaling. In the most sensitive samples, these events led to the induction of cell death characterized by loss of mitochondrial membrane potential, activation of caspases and phophatidylserine exposure. IPI-504 cytotoxic activity was increased by cotreatment with pharmacological inhibitors of IKK and AKT. Noteworthy, IPI-504 showed remarkable synergistic interaction with the proteasome inhibitor bortezomib, reaching combination indexes (CIs) between 0.53 and 1.094. Efficiency of this latest combination was found to be correlated with the ratio between HSP90 and the co-chaperone HSP70 (P

Description: Abstract 1647 Mantle cell lymphoma (MCL) is an incurable B-lymphoid neoplasm harboring the t(11;14)(q13;q32) translocation which leads to the overexpression of cyclin D1, with the consequent cell cycle deregulation. Typically, MCL is characterized by bad prognosis and an aggressive course of the disease. Unfortunately, current therapies have shown limited efficacy and relapses occur early, thus our purpose was to evaluate the antitumoral properties of the multikinase inhibitor sorafenib in MCL. Sorafenib is an oral multikinase inhibitor that targets several cancer-specific pathways and directly affects tumor cell proliferation, cell survival and neovascularization. We analyzed the sensitivity to sorafenib in 9 MCL cell lines and 17 primary MCL cells by flow cytometry after annexin V staining. Sorafenib induced apoptosis in MCL cell lines with a mean LD50 of 11.5 ± 5.0 μM at 24 hours, while at 48 hours decreased to 7.1 ± 2.7 μM. In primary MCL cells, the mean LD50 was 13.0 ± 3.6 μM at 24 hours, while at 48 hours it notably decreased to 9.4 ± 3.4 μM. These data indicated that sorafenib exerted a time- and dose-dependent cytotoxic effect in MCL cells. Both in cell lines and primary MCL cells, sorafenib induces rapid dephosphorylation of the BCR (B-Cell Receptor)-associated tyrosine kinases, SYK and LYN, as well as of FAK, a downstream SRC target involved in focal adhesion. In line with this, we demonstrate a strong synergy when combining sorafenib with the SYK inhibitor, R406. In parallel, we show that sorafenib also blocks Mcl-1 and cyclin D1 translation, which promotes an unbalance between pro- and anti-apoptotic proteins and facilitates the release of Bax from cyclin D1. This process leads to the induction of the mitochondrial apoptotic pathway and caspase-dependent and independent mechanisms. Moreover, sorafenib inhibits MCL cell migration as well as actin polymerization in response to CXCL12. FAK siRNA-mediated knockdown partially prevents this inhibitory effect, indicating that FAK is a relevant target for the action of sorafenib in MCL cells. Importantly, this compound resensitizes MCL cells cocultured with bone marrow-derived stromal and follicular dendritic-like cells to bortezomib-induced apoptosis indicating that sorafenib was able to antagonize stroma-mediated resistance in MCL. In conclusion, we provide first evidence on the molecular mechanism of action of the multikinase inhibitor sorafenib in MCL. We propose that this compound inhibits cell migration and stroma-mediated bortezomib resistance by interfering BCR signaling and protein translation. All these results suggest that sorafenib, alone or in combination with bortezomib-based therapies, may represent a promising approach for the treatment of MCL patients. Research funding This work was supported by grants from Ministerio de Ciencia e Innovación (SAF 09/9503) and Redes Temáticas de Investigación Cooperativa de Cáncer from the Instituto de Salud Carlos III RED 2006-20-014 (D.C.). S.X-T. is a recipient of predoctoral fellowship from Ministerio de Ciencia e Innovación (FPU) and M.L-G. holds a contract from Fundación Científica de la Asociación Española contra el Cáncer. Disclosures: No relevant conflicts of interest to declare.

Description: Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm with a mature B-cell phenotype and genetically characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression with the consequent deregulation of cell cycle at the G1-S checkpoint. MCL cells also present a constitutive activation of the NF-kappaB pathway which leads to the overexpression of several anti-apoptotic regulators. We have analyzed sensitivity to the extrinsic apoptotic signal triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on six human MCL cell lines and primary cells from 10 MCL patients, which differ in their p53-dependent pathway status, growth characteristics and sensitivity to cytotoxic drugs. TRAIL has been shown to exert in vivo a selective anti-tumor activity with minimal toxicity on normal cells. We observed that TRAIL was able to trigger apoptosis in a majority of MCL cell lines and primary MCL tumor cells, while sparing normal peripheral B cells. TRAIL-induced cell death was characterized by a time- and dose-dependent loss of membrane potential, Bax and Bak activation, caspase activation and phophatidylserine exposure. MCL sensitivity to TRAIL was irrespective of TRAIL-R1 and TRAIL-R2 receptor levels, Bcl-2 family members or caspase regulators expression, but was closely linked to the activity of the NF-kappaB p50 factor and to the expression of c-FLIP, a NF-kappaB-regulated factor. C-FLIP accumulated into the TRAIL-dependent complex in resistant cells and its transient knockdown overcame MCL resistance to TRAIL. In parallel, NF-kappaB inhibitors differentially modulated TRAIL cytotoxicity. Indeed, sub-toxic doses of bortezomib increased TRAIL cytotoxic effects by up-regulating TRAIL-R2 receptor expression, but also led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction in cells with highest c-FLIP basal level. In contrast, the IkappaB kinase (IKK) inhibitor BMS-354451 allowed to consistent reduction of NF-kappaB activity, decreased total and DISC-associated c-FLIP levels, and sensitized all MCL cells to TRAIL cytotoxic effects. These results indicate that pharmacological enhancement of MCL cells sensitivity to TRAIL does not depend on TRAIL receptors level but is rather regulated by NF-kappaB-regulated c-FLIP expression. Considering that both TRAIL and BMS-345541 have already demonstrated selective cytotoxicity against malignant cells, combining TRAIL, with pharmacological inhibitors of IkappaB kinase signaling may represent an attractive model for the design of a new and rational combination therapy for MCL.

Description: Abstract 287 Mantle cell lymphoma (MCL) is a lymphoproliferative disorder of mature B-cells with an aggressive course and short survival. The proteasome inhibitor bortezomib (BZM) induces clinical responses in up to 50% of patients. Conversely, in half of the cases the lymphoma cells are intrinsically resistant or rapidly develop resistance to BZM. To investigate the mechanisms of BZM resistance, we generated HBL2 and JEKO bortezomib resistant (HBL2-BR, JEKO-BR) derivative lines by continuous culture in sub-lethal concentrations of BZM. After several months, clones of HBL2-BR and JEKO-BR were obtained showing BZM IC50 at 48h of 41.6 and 44.6 nM, compared to 6 and 4.9 nM for the respective parental lines. Acquired resistance to BZM remained stable over months but gradually decreased with extended passages in the absence of BZM, suggesting adaptive changes rather than a single gene mutation as the basis of BZM resistance. BR cells exhibited higher proteasome activity, which was dose-dependently inhibited by higher concentrations of BZM. However, BR cells were able to survive with lower proteasome activity than the parental cells, indicating that BR cells had acquired additional changes. To investigate these changes, we use gene expression profiling (GEP) on Affymetrix U133A plus 2 arrays to compared HBL2-BR (in triplicate) and JEKO-BR (in duplicate) subclones to the corresponding parental lines. Unexpectedly, Gene Set Enrichment Analysis (GSEA) of microarray data revealed reduced expression of the mature B-cell gene signature (including genes for CD19, BLNK, SPIB, SYK) and increased expression of plasma cell differentiation signatures (including genes for CD38, IRF4, BLIMP, CD138) in both HBL-2 BR and JEKO-BR. BR lines also expressed higher protein levels of the master plasma cell regulators BLIMP and IRF4, but did not show enhanced expression of the secretory program controlled by XBP1. Flow cytometry analysis confirmed that BR cells had dramatically reduced expression of B-cell surface markers, including CD19, CD24 and CD52, and expressed plasma cell markers, such as CD38 and CD138. Consistent with a partial plasmacytoid phenotype, BR cells tended to be somewhat larger and more granular than parental cells. Loss of BZM resistance over months of culture in the absence of BZM was paralleled by the recovery of CD19 and CD24 expression and down-regulation of CD38, supporting a mechanistic link between the acquisition of a plasmacytoid phenotype and BZM resistance. We have previously shown that the MCL cell lines Mino and REC-1 are less sensitive to BZM than HBL-2, JEKO and most other MCL cell lines. Here we found that these constitutively resistant cells also showed plasmacytoid features including CD38 and CD138 surface expression, increased granularity and size, and an enlarged endoplasmic reticulum (ER). Combined these changes may enhance the ability of the cells to deal with an increased protein load due to bortezomib inhibition. In addition, we also observed higher expression of IRF4 and its target genes in the constitutively resistant cells, as well as higher IRF4 and CD38 expression in primary tumor cells of patients with poor response to BZM. Given the important role of IRF4 as a survival factor in multiple myeloma, we tested whether BZM treatment could decrease IRF4 expression in MCL cells. Indeed, within 24 hours BZM dose-dependently decreased IRF4 expression and the degree of downregulation of IRF4 correlated with the induction of apoptosis. Knockdown of IRF4 expression by shRNA has been shown to be toxic to myeloma cells (Shaffer et al, Nature 2008). Surprisingly, we found a similar toxic effect of IRF4 knockdown using the same inducible shRNA system in the MCL cell lines HBL2, JEKO and REC, which was more prominent in the latter BZM resistant cell line. These results identify loss of IRF4 expression as an additional mechanism by which BZM may induce cell death. However, overexpression of IRF4 in MCL cells is not sufficient to induce bortezomib resistance, indicating that several components of the plasma cell program cooperate to protect cells from BZM induced apoptosis. Furthermore, we have identified markers of BZM resistance that may be clinically relevant predictors of outcome. Disclosures: No relevant conflicts of interest to declare.