Inhibition of CHK1 Enhances Cell Death Induced By the Bcl-2-Selective Inhibitor ABT-199 in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) remains a challenging disease to treat in both pediatric and adult populations. Resistance to cytarabine (ara-C) and anthracycline [e.g., daunorubicin (DNR)]-based chemotherapy is a major cause of treatment failure in this disease. Therefore, more effective therapies are urgently needed to improve treatment outcome of AML patients.

Anti-apoptotic Bcl-2 family proteins play key roles in the apoptosis pathway. Overexpression of these proteins is associated with chemoresistance and poor clinical outcome. Thus, much attention has been focused on inhibition of the anti-apoptotic Bcl-2 family proteins for the treatment of various malignancies. ABT-199 is a selective Bcl-2 inhibitor that has demonstrated promising results in CLL, as well as other malignancies including AML. We previously demonstrated that ABT-199 has a wide range of activity in AML cells. In addition, we have also identified that the anti-apoptotic protein Mcl-1, in conjunction with the pro-apoptotic protein Bim, is a key player in resistance to ABT-199 in AML cells. Thus, combining ABT-199 with agents that downregulate Mcl-1 could overcome the intrinsic resistance to ABT-199.

Checkpoint kinase 1 (CHK1) is a protein kinase which plays a central role in the DNA damage response (DDR). The DDR represents a complex network of multiple signaling pathways involving cell cycle checkpoints, DNA repair, transcriptional programs, and apoptosis, through which cells maintain genomic integrity following various endogenous or environmental stresses. Inhibition of CHK1 has been demonstrated to induce DNA damage. We and others have also demonstrated that DNA damage results in downregulation of Mcl-1. Thus, it is conceivable that targeting CHK1 may enhance the cytotoxic effects of ABT-199 on AML cells through downregulation of Mcl-1.

In this study, we investigated the combination of LY2603618, a CHK1-selective inhibitor, and the Bcl-2 inhibitor ABT-199 in AML cell lines and primary patient samples. We demonstrated that LY2603618 inhibited proliferation of AML cell lines (n=11) and diagnostic blasts (n=26). Interestingly, all 11 AML cell lines and 23 out of the 26 primary AML patient samples tested showed a LY2603618 IC50 lower than the C_max of LY2603618 (~9 µM) determined in Phase I clinical studies. Annexin V and propidium iodide (PI) staining and flow cytometry analyses revealed that LY2603618 induced Bak-dependent apoptosis in AML cells. As expected, treatment of AML cells with LY2603618 resulted in abolishment of G2 cell cycle check point and DNA double strand breaks (DSBs), which could be, at least partially, blocked by a CDK inhibitor, roscovitine. LY2603618 treatment also resulted in downregulation of Mcl-1, which coincided with the initiation of apoptosis. Overexpression of Mcl-1 in AML cells significantly attenuated apoptosis induced by LY2603618, confirming the critical role of Mcl-1 in apoptosis induced by the agent. Consistent with our hypothesis, simultaneous combination of LY2603618 and ABT-199 resulted in synergistic induction of apoptosis in both AML cell lines and primary patient samples.

Our results demonstrate that LY2603618 synergizes with ABT-199 in AML cells. Our findings provide new insights into overcoming the mechanism of ABT-199 resistance in AML cells and support the clinical development of the combination of ABT-199 and CHK1 inhibitors.