MEK-INHIBITORS/ARSENIC TRIOXIDE COMBINATION THERAPY AS A NOVEL TREATMENT OPTION FOR TYROSINE KINASE INHIBITORS-RESISTANT PH+-LEUKEMIA

Despite the unprecedented efficacy observed with Bcr-Abl tyrosine kinase inhibitors (TKIs) in the management of chronic myeloid leukemia (CML), resistance and intolerance towards first-, second- and third-generation Bcr-Abl TKIs are frequently reported. This calls upon the need to identify new therapeutic strategies that may improve the therapeutic outcome of TKI-resistant Ph+-leukemia patients. Based upon previous findings, we have hypothesized that combined treatment with MEK inhibitors and arsenic trioxide (ATO) may provide the wanted cytotoxic effects against TKIs-resistant Bcr-Abl leukemic cells. We therefore first analyzed the pharmacologic interactions between the MEK inhibitor PD0325901 (PD) and ATO on Bcr-Abl-positive leukemia cell lines displaying different levels of resistance to TKIs, using a fixed-ratio treatment paradigm. We found that combination treatment with PD and ATO resulted in a synergistic induction of apoptosis in AR230-R, LAMA-R, Ba/F3 p210Y253F, Ba/F3 p210T315I and K562-R TKI-resistant cell lines. We next determined whether PD and ATO also induced apoptosis of primary cells from patients with BCR-ABL-driven CML and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) who had manifested resistance or intolerance to the TKIs Imatinib, Nilotinib and/or Dasatinib, or carried the T315I BCR-ABL mutation. Mononuclear cells derived from blood or bone marrow of patients with CML or Ph+ ALL harboring native BCR-ABL or BCR-ABLT315I were then treated with PD, ATO or both compounds and cell death was monitored by either sub-G1 or Annexin V/PI analysis. Similarly to cell lines, we found that PD significantly (P<0.02; n=5) increased ATO-induced cell death also in primary TKI-resistant CML or Ph+ ALL cells. We further observed that the combination PD/ATO promoted the accumulation/ activation of the proapoptotic and antiproliferative transcriptionally active (TA)-p73 isoforms and transcription of their proapoptotic targetgenes Bax, Bak, Puma and/or P53AIP1. Consistent with these results, we found that PD greatly enhanced the ATO-induced mitochondrial depolarization, caspase-3 activation in all the analyzed BCR-ABL TKIresistant cell lines, as well as in primary leukemic blast cells derived from patients clinically resistant to TKIs (n=5). Furthermore, in support of the antiapoptotic role of MEK were the findings that experimental overexpression of a constitutively active mutant variant of MEK in TKIresistant leukemia cells caused enhanced phosphorylation of ERK1/2 and protected against ATO-induced cell death. By contrast, the chemical or genetic loss-of-function of MEK consistently and significantly decreased the basal ERK1/2 activity in these engineered leukemia cells and significantly (p<0.001) enhanced their apoptosis in response to ATO. Finally, combination of PD/ATO significantly (p=0.001) prolonged survival of mice carrying BCR-ABL-T315I-induced CML, when compared to either treatment alone. The findings highlight the potential of using a therapeutic approaches combining MEK inhibitors and ATO for the treatment of TKI-resistant Ph+-Leukemia.