ALBERT

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: Abstract 3599 CD38 is a cell surface molecule endowed with enzymatic and receptor functions. As an enzyme, CD38 is part of a large family of nucleotide-metabolizing ecto-enzymes (NMEs) involved in the catabolism of extra-cellular nucleotides. Its activity results in the dismantling of NAD and the generation of Ca2+ mobilizing compounds. CD38 also bind CD31, a non substrate ligand, expressed by endothelial and stromal cells. In the neoplastic context, CD38/CD31 interactions lead to increased proliferation, survival and chemotaxis. CD38 is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients and its expression is higher in BM than in peripheral blood cells. Furthermore, the exposure of CLL cells to specific combination of activatory signals (e.g., IL-2, IL-4, CD40L) results in an up-regulation of the molecule, that relies on de novo gene transcription. The human CD38 gene has a genetic single-nucleotide polymorphism (SNP), with a C→G variation in a putative E-box located in the regulatory region. E proteins have the ability to bind to E-box elements and to activate gene transcription. The frequency of the CD38 rare allele (G) has been recently reported to be higher in a subset of CLL patients characterized by clinical and molecular markers of poor prognosis. The aim of this work is to test whether CD38 is a target of E2A at least in CLL cells and if the SNP may affect CD38 gene transcription, influencing the binding affinity of the transcription factor. E2A expression was analyzed in a large cohort of CLL patients (n=72) and in normal B cells. Results indicate that the transcription factor was expressed by the majority of CLL samples, but at higher level in CD38+ ones. Moreover, it was absent in circulating B cells and splenocytes. A positive correlation between the presence of E2A in the nucleus and the surface expression of CD38 in G carrier patients was found. These results suggested that E2A is i) directly associated with CD38 expression and that ii) the binding of the transcription factor is influenced by CD38 genotype. Chromatin immunoprecipitation experiments indicated that E2A directly interacts with the CD38 regulatory region. Furthermore, the binding was stronger in the presence of the G allele. Silencing of E2A resulted in a significant reduction of CD38 surface expression, formally linking these two molecules and confirming the working hypothesis. A direct functional interplay between E2A and CD38 was obtained by mimicking in vitro conditions known to induce CD38 expression through de novo gene transcription. Exposure of CLL cells to TLR-9 ligands and IL-2, both inducers of CD38 expression, resulted in the up-regulation of the molecule. The effect was primarily conditioned by the presence of E2A and then by the G allele. The results obtained in the present work indicate that E2A and CD38 expression are functionally linked in a common pathway and the activity of E-protein is a necessary element for an efficient induction of CD38 transcription. Disclosures: No relevant conflicts of interest to declare.

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.