Binding of Released Bim to Mcl-1 Is Responsible for Resistance to ABT-199 Which Can be Overcome By Combination with Daunorubicin or Cytarabine in Acute Myeloid Leukemia Cells
Authors: Niu Xiaojia , Zhao Jianyun , Ma Jun , Xie Chengzhi , Edwards Holly , Wang Guan , Caldwell J. Timothy , Chu Roland , Wang Zhihong , Lin Hai , Taub Jeffrey W , Ge Yubin 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is a malignant heterogeneous disease characterized by rapid clonal growth of myeloid lineage blood cells. This year there will be an estimated 20,830 new AML cases and an estimated 10,400 deaths from this deadly disease in the United States. Overall survival rates remain low despite advances in treatment with overall survival rates of 25% for adults and 65% for children. Resistance to frontline chemotherapy remains a major cause of treatment failure, highlighting the need for new therapies.
Overexpression of the anti-apoptotic Bcl-2 family members is associated with chemoresistance in leukemic cell line models and with poor clinical outcome. Anti-apoptotic Bcl-2 family members, such as Bcl-2, Bcl-xL, and Mcl-1, sequester pro-apoptotic BH3-only proteins, such as Bim, which activate pro-apoptotic proteins Bax and Bak causing mitochondrial outer membrane permeabilization resulting in cytochrome c release and apoptosis. Thus, inhibition of anti-apoptotic Bcl-2 family members represents a promising approach for the treatment of AML. We previously demonstrated preclinical efficacy of a pan-Bcl-2 inhibitor, GX15-070, in combination with cytarabine in AML cell lines and primary patient samples. Another promising inhibitor, ABT-263, has shown preclinical efficacy, but has been associated with thrombocytopenia due to inhibition of Bcl-xL, thus much attention has been focused on inhibition of Bcl-2. ABT-199, a Bcl-2 selective inhibitor, has demonstrated encouraging results in AML, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, multiple myeloma, and breast cancer. We previously demonstrated that ABT-199 has a wide range of activity in AML cells (Niu X, et al. Leukemia. 2014; 28: 1557-1560.) However, it has limited efficacy in Bcl-xL and Mcl-1 dependent malignancies. Thus, intrinsic drug resistance remains a concern. Understanding the molecular mechanisms of resistance to ABT-199 will allow for rationally designed combination regimens to increase its antileukemic efficacy.
In this study, we investigated the molecular mechanism underlying intrinsic resistance to ABT-199 in AML cells. Immunoprecipitation of Bim from ABT-199 treated cells demonstrated decreased association with Bcl-2, but increased association with Mcl-1, without corresponding change in mitochondrial outer membrane potential. ABT-199 treatment resulted in increased levels of Mcl-1 protein and unchanged or decreased Mcl-1 transcript levels. shRNA knockdown of Bim almost completely abolished ABT-199 treatment-induced increase of Mcl-1 protein levels, suggesting that the association with Bim plays an important role in stabilizing Mcl-1 protein. AML cells treated with ABT-199 in the presence of the protein translation inhibitor cycloheximide resulted in significantly longer Mcl-1 half-life and treatment with the proteasome inhibitor MG-132 resulted in increased Mcl-1 protein level and no further enhancement was detected when treated with combined MG-132 and ABT-199, suggesting that ABT-199 affects Mcl-1 protein stability. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage, decreased Mcl-1 protein levels, decreased association of Mcl-1 with Bim, and synergistic induction of cell death compared to ABT-199 alone, in both AML cell lines and primary patient samples obtained from AML patients at diagnosis independent of their sensitivities to ABT-199, thus providing evidence that screening for ABT-199 resistance is not necessary.
Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance, and this mechanism of resistance can be overcome by combining ABT-199 with daunorubicin or cytarabine in AML cells. Our findings, though in a limited number of primary patient samples, provide new insights into the mechanism of ABT-199 resistance in AML cells and support the clinical development of the combination of daunorubicin or cytarabine and ABT-199 in the treatment of AML.

Disclosures
No relevant conflicts of interest to declare.