Mechanistic Studies of the Synergistic Cytotoxicity of Clofarabine and Gemcitabine in Multiple Myeloma Cell Lines: Relevance of p53-Status

Multiple myeloma (MM) is an abnormal proliferation of plasma cells in the bone marrow. High dose chemotherapy  with autologous stem cell transplantation (ASCT) is an effective treatment for MM. Induction therapies include immunomodulatory drugs, proteasome inhibitor, steroids and DNA alkylating agents in single or tandem ASCT.  Despite the success of ASCT, post-ASCT progression is still a major problem. Based on our previous work, we hypothesized that combination of nucleoside analogs + DNA alkylators could provide synergistic cytotoxicity and may be used as safe and efficacious conditioning therapy in an ASCT-program. We exposed human MM cell lines to various  combinations of clofarabine (Clo) and gemcitabine (Gem) with or without busulfan (Bu) and melphalan (Mel). At less than drug IC₁₀ values, [Bu+Clo], [Bu+Gem], [Mel+Clo], [Mel+Gem] and [Bu+Mel] combinations did not show synergistic cytotoxicity.  However, a combination of [0.25 μM Clo + 3.5 nM Gem] resulted in about 60% inhibition of proliferation of p53-wild type NCI H929 and MM.1R cells;  combination indexes = 0.1 – 0.6 suggested strong synergism. Two p53-mutated MM cell lines, RPMI 8226 and U266B1, were more resistant to these drugs. For example, exposure to [0.4 μM Clo+30 nM Gem] inhibited proliferation of RPMI 8226 only by 25%.   To determine if the cytotoxicity of [Clo+Gem] is mediated by p53, H929 and MM.1R cells were pre-exposed to a p53-inhibitor, pifithrin α. More than 50% of the [Clo+Gem]-mediated cytotoxicity was reversed. The level of p53-regulated proapoptotic PUMA and p21 proteins increased with [Clo+Gem] but the effects were reversed in the presence of pifithrin α. ATM kinase was activated, further supporting involvement of the ATM-p53 pathway in activation of DNA-damage response. Phosphorylation of deoxycytidine kinase (DCK) increased, and proteins involved in DNA-repair and rRNA production were down-regulated. The activation of apoptosis is suggested by the cleavage of PARP1 and caspases 3 and 8. Further, mitochondrial membrane potential (MMP) decreased in cells exposed to [Clo+Gem] consistent with  increased levels of proapoptotic factors cytochrome c, AIF and SMAC/DIABLO in the cytosol, suggesting mitochondrial leaks. These results show that the mechanism of synergistic cytotoxicity of [Clo+Gem] in p53-positive MM cells involves activated DCK, DNA-damage response, decreased MMP, inhibited DNA repair, and nucleolar stress through decreased rRNA. We are determining the effects of [Clo+Gem] in primary cell samples from MM patients; the data will be presented and discussed. Our investigation provides a basis for  introducing nucleoside analog combination(s) in both cytoreductive induction therapy and pre-ASCT therapy in MM, and in individualizing therapy based on p53 status to avoid subjecting patients to ineffective therapy.