Bcl-2 phosphorylation confers resistance on chronic lymphocytic leukaemia cells to the BH3 mimetics ABT-737, ABT-263 and ABT-199 by impeding direct binding.
OBJECTIVE
Although the ongoing clinical trials of ABT-263 and ABT-199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge.


METHODS
The LD50 values of ABT-737, ABT-263 and ABT-199 in a number of primary CLL cells from 40 patients, were determined. The levels of Bcl-2 family proteins, including phosphorylated Bcl-2 (pBcl-2) and their interactions were measured by immunoblotting and co-immunoprecipitation. In vitro binding assays were performed by isothermal titration calorimetry and ELISA. BH3 profiling in isolated mitochondria was analysed.


RESULTS
The ratio of (Mcl-1 + pBcl-2) to Bcl-2 expression provided the most significant predictive marker for the cytotoxic potential of ABT-737, ABT-263 and ABT-199 in the panel of CLL samples. Mechanistically, pBcl-2 inhibited the effects of the ABT compounds on the displacement of Bax and Bim from Bcl-2, thereby suppressing mitochondrial apoptosis. The ABT compounds exhibited 100-300-fold lower binding affinity to the glutamic acid, phosphomimetic, mutant of Bcl-2 (T69E, S70E and S87E; EEE-Bcl-2). BH3 peptides exhibited different rank orders of binding affinities to full-length WT-Bcl-2 and full-length EEE-Bcl-2.


CONCLUSIONS
Our study suggested that a structural alteration in the BH3-binding groove was induced by phosphorylation of Bcl-2. Our data also provided a framework to overcome resistance of CLL cells to the ABT compounds by combining pBcl-2 kinase inhibitors with the ABT compounds.
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, China.
Ting
T
Song
School of Life Science and Technology, Dalian University of Technology, Dalian, China.
Gaobo
G
Chai
School of Life Science and Technology, Dalian University of Technology, Dalian, China.
Yubo
Y
Liu
School of Life Science and Technology, Dalian University of Technology, Dalian, China.
Xiaoyan
X
Yu
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, China.
Ziqian
Z
Wang
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, China.
Zhichao
Z
Zhang
D000814:Aniline Compounds / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001713:Biphenyl Compounds / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009596:Nitrophenols / Q000494:pharmacology*; D010446:Peptide Fragments; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology*
C501332:ABT-737; D000814:Aniline Compounds; D000970:Antineoplastic Agents; C474007:Bax protein (53-86); D001713:Biphenyl Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D009596:Nitrophenols; D010446:Peptide Fragments; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; C579720:venetoclax; C528561:navitoclax