Cdc37 suppressing in multiple myeloma blocks proliferation but induces bortezomib resistance

Abstract

PO-302 Amiloride induces potent antitumoral activity in multiple myeloma through the reactivation of mutant p53 E. Rojas, I. Misiewicz-Krzeminska, S.H. Garcia, T. Paino, L.A. Corchete, I. Isidro, M.V. Mateos, E. Ocio, N.C. Gutierrez Hematology Departament, University Hospital, IBSAL IBMCC (USAL-CSIC) Funding: “Instituto de Salud Carlos III” (PI13/00111), “Gerencia Regional de Salud” (BIO/SA57/13), “Asociacion Espanola contra el cancer” (AECC: GCB120981SAN). Background: The antihypertensive agent, amiloride, has been demonstrated to inhibit tumor cell proliferation in different experimental models. Here, we investigate the antimyeloma effect of amiloride in myeloma cell lines and primary samples, and the potential mechanisms involved. Methods: Seven human myeloma cell lines (NCI-H929, JJN3, KMS12-BM, KMS12-PE, U266, MM1S and RPMI-8226) and 6 bone marrow (BM) samples from MM patients were used for the experiments. Cell viability, apoptosis and cell cycle analyses were carried. The measuring of mitochondrial membrane potential (m) was evaluated by flow cytometry analyses. The gene expression was quantified by Taqman assay qRT-PCR. Synergy with other antimyeloma agents was calculated with the “CalcuSyn” software program. Results: To test the antiproliferative effect of amiloride, myeloma cells were treated with increasing concentrations of the compound (0.1mM-1mM) for 24, 48 and 72 hours. Amiloride displayed potent antimyeloma activity in the panel of 7 cell lines. A substantial induction of apoptotic cell death (greater than 40% at 24 h and 60% at 48 h) and cell cycle blockade was observed in all MM cell lines. The apoptosis induced by amiloride was not related to the TP53 mutational status, since amiloride was still able to induce cell death in p53 mutated cell lines, such as U-266, KMS12-BM and JJN3. Moreover, this drug caused a notable decrease of m. Mechanistic studies showed that apoptosis triggered by amiloride was associated with the overexpression of BAX, BAK1, BBC3, TNFRSF10B, FAS, CDKN1B and CDKN1A, even in those MM cell lines in which p53 gene was mutated. The cell death was reduced in MM cell lines under the treatments with p53 inhibitors (pifithrin, PFT and PFT), even in p53 mutated cell lines. These results suggest the reactivation of mutant p53 in amiloride-induced apoptosis in myeloma cells. The double combinations of amiloride with bortezomib, dexamethasone and melphalan were synergistic in NCI-H929, JJN3, RMPI and MM1S cell lines. In vitro studies confirmed the antimyeloma efficacy of amiloride in patient samples and revealed significant lower cytotoxicity in the remaining cell subpopulations compared to tumor plasma cells. Conclusions: Our results demonstrate a potent and selective antimyeloma effect of amiloride independent of p53 status. The present data support the investigation of amiloride as a treatment option for MM patients, either alone or in combination.