Binding of Released Bim to Mcl-1 is a Mechanism of Intrinsic Resistance to ABT-199 which can be Overcome by Combination with Daunorubicin or Cytarabine in AML Cells.
OBJECTIVE
To investigate the molecular mechanism underlying intrinsic resistance to ABT-199.


METHODS
Western blots and real-time RT-PCR were used to determine levels of Mcl-1 after ABT-199 treatment alone or in combination with cytarabine or daunorubicin. Immunoprecipitation of Bim and Mcl-1 were used to determine the effect of ABT-199 treatment on their interactions with Bcl-2 family members. Lentiviral short hairpin RNA knockdown of Bim and CRISPR knockdown of Mcl-1 were used to confirm their role in resistance to ABT-199. JC-1 assays and flow cytometry were used to determine drug-induced apoptosis.


RESULTS
Immunoprecipitation of Bim from ABT-199-treated cell lines and a primary patient sample 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, unchanged or decreased Mcl-1 transcript levels, and increased Mcl-1 protein half-life, suggesting that the association with Bim plays a role in stabilizing Mcl-1 protein. Combining conventional chemotherapeutic agent cytarabine or daunorubicin with ABT-199 resulted in increased DNA damage along with decreased Mcl-1 protein levels, compared with ABT-199 alone, and synergistic induction of cell death in both AML cell lines and primary patient samples obtained from AML patients at diagnosis.


CONCLUSIONS
Our results demonstrate that sequestration of Bim by Mcl-1 is a mechanism of intrinsic ABT-199 resistance and supports the clinical development of ABT-199 in combination with cytarabine or daunorubicin for the treatment of AML. Clin Cancer Res; 22(17); 4440-51. ©2016 AACR.
National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun, China. Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan.
Xiaojia
X
Niu
National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun, China.
Jianyun
J
Zhao
National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun, China.
Jun
J
Ma
Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan.
Chengzhi
C
Xie
Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan.
Holly
H
Edwards
National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Sciences, Jilin University, Changchun, China.
Guan
G
Wang
MD/PhD Program, Wayne State University School of Medicine, Detroit, Michigan. Cancer Biology Graduate Program, Wayne State University School of Medicine, Detroit, Michigan.
J Timothy
JT
Caldwell
Department of Pathology and Cell Biology, USF Morsani College of Medicine, Tampa, Florida.
Shengyan
S
Xiang
Department of Pathology and Cell Biology, USF Morsani College of Medicine, Tampa, Florida. Cancer Biology and Evolution Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida.
Xiaohong
X
Zhang
Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan. Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan.
Roland
R
Chu
Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan. Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan.
Zhihong J
ZJ
Wang
Department of Hematology and Oncology, The First Hospital of Jilin University, Changchun, China. gey@karmanos.org maillinhai@sina.com jtaub@med.wayne.edu.
Hai
H
Lin
Department of Pediatrics, Wayne State University School of Medicine, Detroit, Michigan. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan. Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Detroit, Michigan. gey@karmanos.org maillinhai@sina.com jtaub@med.wayne.edu.
Jeffrey W
JW
Taub
Department of Oncology, Wayne State University School of Medicine, Detroit, Michigan. Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan. gey@karmanos.org maillinhai@sina.com jtaub@med.wayne.edu.
Yubin
Y
Ge
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000072224:Bcl-2-Like Protein 11 / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003561:Cytarabine / Q000494:pharmacology; D003630:Daunorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology*
D000970:Antineoplastic Agents; D000072224:Bcl-2-Like Protein 11; D019086:Bridged Bicyclo Compounds, Heterocyclic; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D013449:Sulfonamides; D003561:Cytarabine; C579720:venetoclax; D003630:Daunorubicin