Proteasome Inhibitors and a Fusicoccin Derivative (ISIR-042) Cooperatively Inhibit Proliferation of Myeloma Cells through Induction of C/EBP-Homologous Protein (CHOP/GADD153)

Combinations of bortezomib and novel targeted therapeutic agents may synergistically increase antitumor effect and may overcome specific cellular resistance and/or antiapoptotic processes. However, highly effective combinations of bortezomib with targeted therapeutic agents have not been developed to date. We examined the synergistic effects of various compounds and bortezomib on myeloma cells and identified fusicoccin derivative ISIR-042 as the most potent drug. We developed ISIR-042 that exerts higher cytotoxic effects on hypoxic cells than on normoxic cells and that preferentially inhibits stem/progenitor cells in pancreatic cancer cell lines compared with other chemotherapeutic agents (Kawakami et al, Anticancer Agents Med Chem 2012). In the present study, we determined the synergistic effects of bortezomib and ISIR-042 cotreatment on human multiple myeloma cells. Human multiple myeloma cell lines RPMI8226, KMS-11, and KMS-26 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO₂ in air. Viability of cells cultured with or without the test compounds for indicated time was examined by performing a modified MTT assay.Bone marrow specimens and ascitic fluids were collected from patients at diagnosis or relapse after obtaining written informed consent for sample collection in accordance with institutional policy. The study was approved by the Institutional Review Board of Shimane University. For performing colony-forming assay, the cells (1 × 10⁴/dish) were plated in 1.1 ml semisolid methylcellulose medium supplemented with 0.8% methylcellulose and 20% FBS in triplicates for 10 days. CHOP^-/- and CHOP^+/+ MEF cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Expression of cell surface antigens was analyzed by performing flow cytometry with anti-CD38 or anti-CD138 antibody. Combined effects of myeloma cells in primary culture were analyzed by performing flow cytometry with anti-CD19 and anti-CD138 antibodies. Expression of caspase-10, BCL-2, p62, BCLAF1, LC3, actin, and CHOP was determined by performing western blotting with respective monoclonal antibodies. mRNA expression of genes encoding XBP-1u and XBP-1s was analyzed by performing reverse transcription-PCR. CHOP mRNA levels were measured by performing real-time PCR.

ISIR-042 inhibited the growth of RPMI8226 cells in a concentration-dependent manner and exerted synergistic effects with bortezomib. ISIR-042 also exerted synergistic effects with carfilzomib. Results of flow cytometry showed that the combination of ISIR-042 and bortezomib decreased the number of myeloma cells in the primary culture in a concentration-dependently. Colony-forming assay showed that RPMI8226 cells were more sensitive than normal mouse hematopoietic cells to ISIR-042 and bortezomib cotreatment. ISIR-042 decreased the number of CD38^-CD138^- cells and increased the number of CD38⁺CD138⁺ cells in KMS11 cell population. mRNA expression of genes encoding XBP-1u and XBP-1s was not affected in RPMI8226 cells treated with ISIR-042 or bortezomib alone. However, mRNA expression of the gene encoding XBP-1s significantly increased in RPMI8226 cells cotreated with ISIR-042 and bortezomib. Growth-inhibitory effects of ISIR-042 and bortezomib were not associated with caspase-10 which modulates autophagic response for survival. Expression of autophagic markers such as LC3 and p62 was also examined. ISIR-042 exerted modest effects in the presence or absence of bortezomib. ER stress-mediated CHOP induction promotes the cytotoxic effects of proteasome inhibitors on many cancer cells. Carfilzomib and ISIR-042 cotreatment significantly induced CHOP mRNA expression, suggesting that ISIR-042 and proteasome inhibitor cotreatment induced apoptosis by enhancing ER stress and activating CHOP. This was confirmed using CHOP^-/- MEF cells. Our results showed that carfilzomib and ISIR-042 cotreatment did not exert cytotoxic effects on CHOP^-/- MEF cells. These results suggest that treatment of multiple myelomas with ISIR-042-supplemented proteasome inhibitor-based chemotherapy exerts beneficial anticancer effects.