At Clinically Achievable Concentrations Obatoclax (GX15-070) Potentiates The Cytotoxic Effect Of Cytarabine On Acute Myeloid Leukemia Cells By Enhancing DNA Damage

AML accounts for one-fourth of acute leukemias in children, but is responsible for more than half of the pediatric acute leukemia deaths. Most adult AML patients treated with chemotherapeutic regimens relapse and die of their disease or associated complications. One major cause of treatment failure in this disease is resistance to anthracycline [e.g. daunorubicin (DNR)] and cytarabine (ara-C)-based chemotherapy. Therefore more effective therapies are urgently needed. One common cancer-causing defect arises from the overexpression of the anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-xL, and Mcl-1, which inactivate the intrinsic apoptotic pathway in many types of cancer cells. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in leukemia cell lines and with a poor clinical outcome in studies of adult patients with leukemia, including AML. Therefore, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents for the treatment of leukemia.

In this study, we investigated the effects of clinically achievable doses (175 nM and lower) of obatoclax (a hydrophobic small molecule pan-Bcl-2 inhibitor that binds to the BH3-binding site of Bcl-2, Bcl-xL and Mcl-1) on cytarabine sensitivity in AML cell lines (n=5) and diagnostic AML blasts (n=6). The majority of the AML cell lines and diagnostic AML blast samples expressed Bcl-2, Bcl-xL, and/or Mcl-1 proteins as determined by Western blots, though the levels were variable and did not correlate with sensitivities to cytarabine or obatoclax. Simultaneous combination of obatoclax and cytarabine resulted in synergistic inhibition of proliferation of 4 AML cell lines and 4 diagnostic AML blast samples. Cytarabine IC₅₀s were reduced by 3.4-26.6 fold in the presence of 175 nM obatoclax. The two drugs also synergistically induced apoptosis in THP-1 and OCI-AML3 cells (combination index values ranged from 0.23 to 0.55). Bax activation was detected in the combined drug treatments but not in the individual drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential (MMP). Interestingly, co-treatment of THP-1 and OCI-AML3 cells with cytarabine and obatoclax resulted in decreased levels of Mcl-1 and increased nuclear accumulation of Bcl-2 and Bcl-xL that can occur as early as 24h after combined treatment with cytarabine and obatoclax. Apoptosis and MMP loss induced by the combination of cytarabine and obatoclax could be completely attenuated by ectopic overexpression of Bcl-xL. Emerging evidence suggests that Bcl-2, Bcl-xL, and Mcl-1 also possess nuclear functions and can influence the repair of DNA double-strand breaks (DSBs) and/or the regulation of cell cycle checkpoints. It is conceivable that obatoclax may target the nuclear functions of these anti-apoptotic Bcl-2 family proteins to enhance ara-C-induced apoptosis in AML cells. Interestingly, combined drug treatment showed an increase in DNA DSBs, as indicated by increased levels of phosphorylated H2AX (γH2AX) and comet assay, in both THP-1 and OCI-AML3 cells. Further, γH2AX foci coincided with increased nuclear localization of Bcl-2 and Bcl-xL. Furthermore, a time course experiment revealed that γH2AX levels in AML cells treated with combined cytarabine and obatoclax positively correlated with cleavage of caspase-3 and PARP and inversely correlated with Mcl-1 levels. Additional experiments suggest that induction of a decrease of Mcl-1 and nuclear accumulation of Bcl-2 and Bcl-xL may represent a common mechanism underlying apoptosis induced by DNA DSBs.

Our study provides convincing evidence that obatoclax enhances cytarabine-induced DNA DSBs which may trigger a decrease of Mcl-1 and nuclear accumulation of Bcl-2 and Bcl-xL, tipping the balance toward apoptosis. Our new findings strongly support the clinical use of obatoclax in combination with DNA damaging agents for treating AML and possibly a broader range of malignancies.