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. 2022 Aug 15;12(8):3829-3842.
eCollection 2022.

BCL-2 inhibitor synergizes with PI3Kδ inhibitor and overcomes FLT3 inhibitor resistance in acute myeloid leukaemia

Ming-Yue Yao  1   2   3   4 , Ya-Fang Wang  3 , Yu Zhao  3   4 , Li-Jun Ling  3   4 , Ye He  2 , Jie Wen  5 , Ming-Yue Zheng  2   6 , Hua-Liang Jiang  1   2   3   4   6 , Cheng-Ying Xie  2   6   7
Affiliations

BCL-2 inhibitor synergizes with PI3Kδ inhibitor and overcomes FLT3 inhibitor resistance in acute myeloid leukaemia

Ming-Yue Yao et al. Am J Cancer Res. .

Abstract

Inhibitors targeting the antiapoptotic molecule BCL-2 have therapeutic potential for the treatment of acute myeloid leukaemia (AML); however, BCL-2 inhibitors such as venetoclax exhibit limited monotherapy efficacy in relapsed or refractory human AML. PI3Kδ/AKT signalling has been shown to be constitutively active in AML patients. Here, we demonstrate that the combination of BCL-2 and PI3Kδ inhibitors exerts synergistic antitumour effects both in vitro and in vivo in AML. Cotreatment with venetoclax and the specific PI3Kδ inhibitor idelalisib significantly enhanced antiproliferative effects and induced caspase-dependent apoptosis in a panel of AML cell lines. The synergistic effects were mechanistically based on the inactivation of AKT/4E-BP-1 signalling and the reduction of MCL-1 expression, which diminished the binding of Bim to MCL-1. Notably, compared with the parental FLT3-ITD-positive MV-4-11, the acquired FLT3 inhibitor quizartinib-resistant xenograft model carrying the F691L mutation, exhibited a markedly higher sensitivity to venetoclax. Furthermore, venetoclax combined with idelalisib led to tumour regression in all animals in this quizartinib-resistant AML model. Thus, these data indicate that combined inhibition of BCL-2 and PI3Kδ may be a promising strategy in AML, especially for patients with FLT3-ITD and/or FLT3-TKD mutations.

Keywords: Acute myeloid leukaemia; BCL-2; FLT3; PI3Kδ; synergistic lethality.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Antiproliferative activity of the co-inhibition of BCL-2 and PI3Kδ in AML cell lines. (A, B) Antiproliferative activity of venetoclax (A) or idelalisib (B) in a panel of AML cell lines. After cells were treated with venetoclax or idelalisib for 72 h, MTT assay was used to assess cell viability. (C) Protein levels of PI3Kδ, BCL-2, BCL-XL, and MCL-1 in a panel of AML cell lines were assessed by Western blotting. (D) Synergistic antiproliferative effects in vitro. After AML cells were treated with venetoclax and/or idelalisib for 72 h, MTT assay was used to assess cell viability. CI was calculated with CalcuSyn DemoVersion 2.0 software. Synergistic efficacy: CI < 1. (E) The differences in survival related to BCL-2 or PI3Kδ mRNA expression were compared in each group (log-rank test). The dotted line indicated the 95% confidence interval. (F) Scatter plot showed the correlation between BCL-2 and PI3Kδ mRNA levels in AML. Statistical analysis was performed using a two-tailed Student’s t-test. R, Spearman’s correlation coefficient.
Figure 2
Figure 2
The combination of BCL-2 and PI3Kδ inhibitors synergistically induces caspase-dependent apoptosis in AML cell lines. (A) AML cell lines were treated with idelalisib and/or venetoclax for 24 h, and then cell apoptosis was assessed by annexin V-FITC/PI staining and FACS analysis. The concentrations used in this study were 10 µM idelalisib, 50 nM, 25 nM, 50 nM, and 10 nM venetoclax for MV-4-11, EOL-1, RS4;11 and MOLM13 cells, respectively. (B) Protein levels of PARP, Caspase 8, Caspase-9, Caspase-3 in MV-4-11 and EOL-1 cells were determined by Western blotting after treatment with idelalisib and/or venetoclax for 24 h. (C, D) MV-4-11 cells were treated with drugs for 24 h and then cell viability and mitochondrial membrane potential were measured by annexin V-FITC/MitoTracker Red CMXRos staining. FITC histograms were obtained by flow cytometry (C), and samples were visualized under a fluorescence microscope (D). The concentrations used in this study were 50 µM Z-VAD-FMK, 5 nM venetoclax, and 10 µM idelalisib. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
The combination of BCL-2 and PI3Kδ inhibitors inhibits the AKT pathway and downregulates MCL-1, resulting in reduced binding of MCL-1 to Bim. (A) Protein levels of BCL-2, MCL-1 and BCL-XL in MV-4-11 and EOL-1 cells were determined via Western blotting after treatment with idelalisib and/or venetoclax for 3 h. (B) MCL-1/Bim interaction in MV-4-11 and EOL-1 cells was measured by co-IP experiment using Bim antibody after 3 h treatment with idelalisib and/or venetoclax, and the corresponding MCL-1 and Bim were analysed by Western blotting. (C) Phosphorylation levels of STAT5, AKT, ERK and 4E-BP-1 in MV-4-11 and EOL-1 cells were analysed by Western blotting after treatment with venetoclax and/or idelalisib for 3 h.
Figure 4
Figure 4
The combination of BCL-2 and PI3Kδ inhibitors overcomes acquired resistance to FLT3 inhibitors in AML cells. (A) After MV-4-11 and MV-4-11/quizartinib cell lines were treated with quizartinib, idelalisib or venetoclax for 72 h, MTT assay was used to assess cell viability. (B) After 3 h treatment with quizartinib in MV-4-11 and MV-4-11/quizartinib cell lines, the phosphorylation status of FLT3 signalling protein and expression of BCL-2 family members were determined by Western blotting. (C) Sequencing of FLT3 in MV-4-11/quizartinib. The “T” to “G” replacement at the codon 691 led to a Phe-to-Leu exchange (F691L). (D) After MV-4-11/quizartinib cells were treated with venetoclax, idelalisib, or a combination for 72 h, MTT assay was used to assess cell viability. (E) Cells were treated with idelalisib (10 µM), venetoclax (50 nM), or their combination for 24 h, and cell apoptosis was measured by annexin V-FITC/PI staining. (F, G) After MV-4-11/quizartinib cells were treated with venetoclax and/or idelalisib for 3 h, the lysates were detected by Western blot with the indicated antibodies. **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
The combination of BCL-2 and FLT3 inhibitors exerts enhanced antitumour activity in mice xenografted with human AML. (A-D) MV-4-11 xenograft mice received vehicle, idelalisib (180 mg/kg), venetoclax (75 mg/kg), their combination or quizartinib (1 mg/kg). Tumour volumes were determined (A), and mouse body weights were measured (B). The mice were euthanized 3 h following the final dose, and then tumours were excised, lysed, and analysed by Western blotting (C) or IHC (D). (E-G) MV-4-11/quizartinib tumour-bearing mice were treated with vehicle or the indicated dosages of idelalisib, venetoclax, their combination or quizartinib. Tumour volumes were measured (E), relative tumour volumes were indicated as percent change from baseline at Day 18 (F), and body weights were shown as the means ± SD (G). **P < 0.01, ***P < 0.001, versus vehicle; ##P < 0.01 versus either single agent alone.
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