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

Abstract

Purpose: To investigate the molecular mechanism underlying intrinsic resistance to ABT-199.

Experimental design: 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.

Conflict of interest statement

The authors declare no competing financial interests.

©2016 American Association for Cancer Research.

Figures

Figure 1. Mcl-1 binding to released Bim results in increased Mcl-1 protein level and causes resistance to ABT-199 in ABT-199-resistant AML cells

(A) OCI-AML3 cells were treated with ABT-199 (or 200 nM daunorubicin as a positive control) for 24 h and then 5 × 105 cells were subjected to the JC-1 assay. ***indicates p<0.0005. (B) OCI-AML3 cells were treated with ABT-199 for 24 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. (C) OCI-AML3 cells were treated with ABT-199 for 24 h. Bim was immunoprecipitated from whole cell lysates and then subjected to Western blotting. Western blots were probed with the indicated antibody. (D) Mcl-1 was immunoprecipitated from whole cell lysates after 24 h ABT-199 treatment and then subjected to Western blotting and probed with anti-Mcl-1 or -Bim antibody. (E) AML patient sample cells (AML1013) were treated with ABT-199 (or 200 nM daunorubicin as a positive control) for 24 h and then 5 × 105 cells were subjected to the JC-1 assay. ***indicates p<0.0005. (F) AML1013 cells were treated with ABT-199 for 24 h. Whole cell lysates were subjected to Western blotting and probed with anti-Mcl-1, -Bcl-2, -Bcl-xL, or -β-actin antibody. (G) AML1013 cells were treated with ABT-199 for 24 h. Bim was immunoprecipitated from whole cell lysates and then subjected to Western blotting. Western blots were probed with the indicated antibody. *indicates light chain of the Bim antibody. (H) U937, THP-1, CMS, and primary AML patient cells (AML1012) were treated with ABT-199 for 24 h. Whole cell lysates were subjected to Western blotting. Western blots were probed with the indicated antibody. (I) Relative densitometry measurements of Mcl-1 expression were measured using Odyssey Software V3.0. The densitometry measurements are graphed as fold change compared to the no drug control. *indicates p<0.05, **indicates p<0.005, and ***indicates p<0.0005. (J) THP-1 and U937 cells were infected with non-template control (NTC-shRNA) or Bim (Bim-shRNA) shRNA lentivirus. Cells were then treated with or without 2 µM ABT-199 for 24 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the Mcl-1 and Bim densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (K) THP-1 cells were infected with non-template control (NTC, designated THP-1/NTC) or Mcl-1 (designated THP-1/Mcl-1) CRISPR lentivirus. Cells were then treated with or without ABT-199 for 24 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to the no drug treatment control, are indicated. (L) CRISPR knockdown cells were treated with ABT-199 for 24 h and then subjected to annexin V/PI staining and flow cytometry analyses. ***indicates p<0.0005.

Figure 2. ABT-199 treatment results in increased Mcl-1 protein stability in ABT-199-resistant AML cells

(A) OCI-AML3, U937, and CMS cells were treated with ABT-199 for 24 h. Total RNA was isolated and Mcl-1 transcript levels were determined by real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method (comparing samples to the corresponding no drug control). (B) U937 cells were treated with 10 µg/mL cyclohexamide (CHX) alone or in combination with 2 µM ABT-199 for up to 90 min. Whole cell lysates were subjected to Western blotting and probed with anti-Mcl-1 or -β-actin antibody. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (C–E) U937 cells were treated with MG-132 and ABT-199, alone or in combination, for 1 or 24 h, as indicated. Whole cell lysates were subjected to Western blotting and probed with anti-Mcl-1 or -β-actin antibody. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.

Figure 3. Daunorubicin (DNR) synergizes with ABT-199 in U937 AML cell line

(A) U937 cells were treated with ABT-199 and DNR, alone or in combination, for 8 h and 24 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for γH2AX and Mcl-1 densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. Whole cell lysate from the 24 h ABT + DNR treatment was used as the positive control for the 8 h treatment. (B) U937 cells were treated with ABT-199 and DNR, alone or in combination, for 24 h and then subjected to annexin V/PI staining and flow cytometry analyses.***indicates p

Figure 4. Cytarabine synergizes with ABT-199 in AML cell lines

(A) U937 cells were treated with ABT-199 and Ara-C, alone or in combination, for 8 h and 24 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibody. The fold changes for γH2AX and Mcl-1 densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. Whole cell lysate from the 24 h ABT + Ara-C treatment was used as the positive control for the 8 h treatment. (B) U937 cells were treated with ABT-199 and Ara-C, alone or in combination, for 24 h and then subjected to annexin V/PI staining and flow cytometry analyses. **indicates pCt method (comparing samples to the corresponding no drug control). (H) U937 cells were treated as indicated for 24 h. Whole cell lysates were subjected to Western blotting and probed with anti-Mcl-1 or -β-actin antibody. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated.

Figure 5. Daunorubicin and cytarabine synergize with ABT-199 in AML primary patient samples

(A&B) Patient samples, AML1009 (panel A) and AML1010 (panel B), were treated with ABT-199 and DNR, alone or in combination, for 24 h and then subjected to annexin V/PI staining and flow cytometry analyses. CI values were calculated using CompuSyn software. **indicates p50 values were calculated as drug concentrations necessary to inhibit 50% OD590 compared to vehicle control treated cells. The IC50 values are means of duplicates from one experiment due to limited sample. Standard isobologram analysis of anti-leukemic interactions was performed to determine the extent and direction of the antileukemic interaction. The IC50 values of each drug are plotted on the axes; the solid line represents the additive effect, while the points represent the concentrations of each drug resulting in 50% inhibition of proliferation. Points falling below the line indicate synergism whereas those above the line indicate antagonism.

Figure 6. Proposed mechanism of action of ABT-199 alone or in combination with daunorubicin or cytarabine in ABT-199-resistant AML cells

ABT-199 treatment releases Bim from Bcl-2. In sensitive cells, there is an inadequate amount of Mcl-1 to sequester all of the released Bim, resulting in free Bim, which can then activate the canonical apoptosis pathway. In ABT-199-resistant cells, the Bim released from Bcl-2 is sequestered by Mcl-1, stabilizing Mcl-1, and ultimately resulting in survival in the ABT-199-resistant cells. In the combined drug treatment, addition of daunorubicin or cytarabine abolishes Mcl-1 stabilization induced by ABT-199. This results in decreased sequestration of Bim, and allows for activation of Bax/Bak, resulting in apoptosis.