Inhibition of Mcl-1 Synergistically Enhances the Antileukemic Activity of Gilteritinib and MRX-2843 in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia

This study was supported by grants from the National Natural Science Foundation of China (NSGC81800154 to Guan Wang), The Children’s Foundation, LaFontaine Family/U CAN-CER VIVE Foundation, Kids Without Cancer, Decerchio/Guisewite Family, Justin’s Gift, Elana Fund, Ginopolis/Karmanos Endowment, the Ring Screw Textron Endowed Chair for Pediatric Cancer Research. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or the decision to publish the results.

Figure 1. AZD5991 synergistically enhances apoptosis induced by gilteritinib and MRX-2843 in

FLT3

-mutated AML cells. (A)

FLT3

-ITD-positive MOLM-13 and MV4-11 cells treated with varying concentrations of AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination for 24 h were stained with annexin V-FITC/propidium iodide (PI) and analyzed using a flow cytometer. Combination Index (CI) values were calculated using CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. *** indicates

p

< 0.001. (B) MOLM-13 and MV4-11 cells were treated with AZD, gilt, AZD + gilt, MRX, or MRX + AZD for 24 h. Whole cell lysates were subjected to western blot analysis. (C) Normal peripheral blood mononuclear cells (PBMCs) treated with varying concentrations of AZD, gilt, AZD + gilt, MRX, or MRX + AZD for 24 h were stained with annexin V-FITC/PI and analyzed using a flow cytometer. *** indicates

p

< 0.001. (D) Primary AML patient sample cells were treated with varying concentrations of AZD, gilt, or MRX, alone or in combination, for 24 h; then, the cells were stained with annexin V-FITC/propidium iodide (PI) and analyzed using a flow cytometer. CI values were calculated using CompuSyn software. *** indicates

p

< 0.001.

Figure 1. AZD5991 synergistically enhances apoptosis induced by gilteritinib and MRX-2843 in

FLT3

-mutated AML cells. (A)

FLT3

-ITD-positive MOLM-13 and MV4-11 cells treated with varying concentrations of AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination for 24 h were stained with annexin V-FITC/propidium iodide (PI) and analyzed using a flow cytometer. Combination Index (CI) values were calculated using CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. *** indicates

p

< 0.001. (B) MOLM-13 and MV4-11 cells were treated with AZD, gilt, AZD + gilt, MRX, or MRX + AZD for 24 h. Whole cell lysates were subjected to western blot analysis. (C) Normal peripheral blood mononuclear cells (PBMCs) treated with varying concentrations of AZD, gilt, AZD + gilt, MRX, or MRX + AZD for 24 h were stained with annexin V-FITC/PI and analyzed using a flow cytometer. *** indicates

p

< 0.001. (D) Primary AML patient sample cells were treated with varying concentrations of AZD, gilt, or MRX, alone or in combination, for 24 h; then, the cells were stained with annexin V-FITC/propidium iodide (PI) and analyzed using a flow cytometer. CI values were calculated using CompuSyn software. *** indicates

p

< 0.001.

Figure 2. AZD5991 alone and in combination with gilteritinib or MRX-2843 significantly reduces colony-forming capacity of

FLT3

-mutated AML progenitor cells. (A,B) Primary AML patient samples (panel (A)) and normal human umbilical cord blood cells (panel (B)) were treated with AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination, for 24 h, and then plated in methylcellulose. The number of leukemic colonies (AML-CFUs), erythroid, and myeloid colonies were counted 10–14 days later. Data are presented as mean ± SEM. * indicates

p

< 0.05, ** indicates

p

< 0.01, and *** indicates

p

< 0.001 compared to control. ## indicates

p

< 0.01 and ### indicates

p

< 0.001 compared to single-drug treatments. Technical triplicates were performed.

Figure 2. AZD5991 alone and in combination with gilteritinib or MRX-2843 significantly reduces colony-forming capacity of

FLT3

-mutated AML progenitor cells. (A,B) Primary AML patient samples (panel (A)) and normal human umbilical cord blood cells (panel (B)) were treated with AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination, for 24 h, and then plated in methylcellulose. The number of leukemic colonies (AML-CFUs), erythroid, and myeloid colonies were counted 10–14 days later. Data are presented as mean ± SEM. * indicates

p

< 0.05, ** indicates

p

< 0.01, and *** indicates

p

< 0.001 compared to control. ## indicates

p

< 0.01 and ### indicates

p

< 0.001 compared to single-drug treatments. Technical triplicates were performed.

Figure 3. c-Myc plays an important role in the synergistic antileukemic activity of AZD5991 and gilteritinib or MRX-2843 in

FLT3

-mutated AML cells. (A) MOLM-13 cells were treated with AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination, for up to 24 h. Whole cell lysates were subjected to western blotting and probed with the indicated antibodies. Densitometry results (normalized to β-actin and compared to vehicle control at the matching time point) are shown below the corresponding blot. (B) MOLM-13 cells treated with AZD, gilt, or MRX, alone in combination, for up to 24 h, were stained with annexin V-FITC/PI and analyzed using a flow cytometer. *** indicates

p

< 0.001 compared to control and single-drug treatments. (C,D) MV4-11 and primary AML patient sample AML#262 cells were treated with AZD, gilt, MRX, or in combination for 3 h. Whole cell lysates were subjected to western blot analysis. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot in panel (C). After treatment, cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (D)). *** indicates

p

< 0.001 compared to control and single-drug treatments. (E,F) MV4-11 and MOLM-13 cells were treated with vehicle control, AZD, 10058-F4, or AZD + 10058-F4 for 3 h. Whole cell lysates were subjected to western blotting analysis. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot (panel (E)). After treatment, cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (F)). *** indicates

p

< 0.001 compared to control and single-drug treatments. (G) Lentiviral CRISPR/Cas9 knockdown (KD) of c-Myc was performed in MV4-11 cells along with non-target control (NTC). Whole cell lysates were subjected to western blotting (left panel). Cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Annexin V/PI staining and flow cytometry analysis results are shown in the (right panel). ** indicates

p

< 0.01 and ***

p

< 0.001 compared to NTC for the same drug treatment.

Figure 3. c-Myc plays an important role in the synergistic antileukemic activity of AZD5991 and gilteritinib or MRX-2843 in

FLT3

-mutated AML cells. (A) MOLM-13 cells were treated with AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination, for up to 24 h. Whole cell lysates were subjected to western blotting and probed with the indicated antibodies. Densitometry results (normalized to β-actin and compared to vehicle control at the matching time point) are shown below the corresponding blot. (B) MOLM-13 cells treated with AZD, gilt, or MRX, alone in combination, for up to 24 h, were stained with annexin V-FITC/PI and analyzed using a flow cytometer. *** indicates

p

< 0.001 compared to control and single-drug treatments. (C,D) MV4-11 and primary AML patient sample AML#262 cells were treated with AZD, gilt, MRX, or in combination for 3 h. Whole cell lysates were subjected to western blot analysis. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot in panel (C). After treatment, cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (D)). *** indicates

p

< 0.001 compared to control and single-drug treatments. (E,F) MV4-11 and MOLM-13 cells were treated with vehicle control, AZD, 10058-F4, or AZD + 10058-F4 for 3 h. Whole cell lysates were subjected to western blotting analysis. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot (panel (E)). After treatment, cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (F)). *** indicates

p

< 0.001 compared to control and single-drug treatments. (G) Lentiviral CRISPR/Cas9 knockdown (KD) of c-Myc was performed in MV4-11 cells along with non-target control (NTC). Whole cell lysates were subjected to western blotting (left panel). Cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Annexin V/PI staining and flow cytometry analysis results are shown in the (right panel). ** indicates

p

< 0.01 and ***

p

< 0.001 compared to NTC for the same drug treatment.

Figure 4. FLT3 inhibition decreases c-Myc protein via transcriptional regulation through suppression of the MEK/ERK and JAK2/STAT5 pathways. (A) MOLM-13 and MV4-11 cells were treated with AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination, for 3 h. Total RNA was extracted, and real-time RT-PCR was performed. The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. * indicates

p

< 0.05 and *** indicates

p

< 0.001 compared to the vehicle control. (B) MOLM-13 and MV4-11 cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Western blots were generated utilizing whole cell lysates. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (C–E) MOLM-13 and MV4-11 cells were treated with vehicle control, AZD, SCH772984 (SCH), or AZD1480, alone or in combination, for 3 h. Whole cell lysates were subjected to western blotting analysis. Representative western blots are shown in panel (C). Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. After treatment, total RNA was extracted, and real-time RT-PCR was performed (panel (D)). The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. *** indicates

p

< 0.001 compared to vehicle control. Treated cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (E)). *** indicates

p

< 0.001 compared to control and single-drug treatments.

Figure 4. FLT3 inhibition decreases c-Myc protein via transcriptional regulation through suppression of the MEK/ERK and JAK2/STAT5 pathways. (A) MOLM-13 and MV4-11 cells were treated with AZD5991 (AZD), gilteritinib (gilt), or MRX-2843 (MRX), alone or in combination, for 3 h. Total RNA was extracted, and real-time RT-PCR was performed. The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. * indicates

p

< 0.05 and *** indicates

p

< 0.001 compared to the vehicle control. (B) MOLM-13 and MV4-11 cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Western blots were generated utilizing whole cell lysates. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (C–E) MOLM-13 and MV4-11 cells were treated with vehicle control, AZD, SCH772984 (SCH), or AZD1480, alone or in combination, for 3 h. Whole cell lysates were subjected to western blotting analysis. Representative western blots are shown in panel (C). Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. After treatment, total RNA was extracted, and real-time RT-PCR was performed (panel (D)). The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. *** indicates

p

< 0.001 compared to vehicle control. Treated cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (E)). *** indicates

p

< 0.001 compared to control and single-drug treatments.

Figure 5. FLT3 inhibition enhances AZD5991-induced cytochrome

c

release. (A) Proposed mechanism of action of gilteritinib/MXR-2843 in combination with AZD5991. (B,C) MV4-11 cells were treated with vehicle control, AZD5991 (AZD), gilteritinib (gilt), MRX-2843 (MRX), or in combination for 3 h. Cellular fractionation was performed. Mitochondrial and cytosolic fractions were subjected to western blotting analysis. This experiment was performed two independent times in triplicate. One representative image is shown. Relative densitometry measurements were determined, normalized to β-actin or VDAC1, and compared to the vehicle control. Results from one representative experiment are graphed as mean ± SEM in panel (C). * indicates

p

< 0.05, ** indicates

p

< 0.01, and *** indicates

p

< 0.001 compared to vehicle control. # indicates

p

< 0.05 compared to single-drug treatment. (D) MV4-11 cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Western blots were generated utilizing whole cell lysates. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (E,F) MV4-11 cells were treated with vehicle control, AZD, 10058-F4, or in combination for 3 h. Cellular fractionation was performed as described in panel (B). * indicates

p

< 0.05, ** indicates

p

< 0.01 and *** indicates

p

< 0.001 compared to vehicle control. ## indicates

p

< 0.01 compared to single-drug treatment. (G) MV4-11 cells were treated with vehicle control, AZD, 10058-F4, or in combination for 3 h. Western blots were generated utilizing whole cell lysates. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (H,I) shRNA knockdown of Bak and Bax was performed in MV4-11 cells with non-template control (NTC) as the negative control. Whole cell lysates were subjected to western blotting (panel (H)). Cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Annexin V/PI staining and flow cytometry analysis results are shown in panel (I). ***

p

< 0.001 compared to NTC for the same drug treatment.

Figure 5. FLT3 inhibition enhances AZD5991-induced cytochrome

c

release. (A) Proposed mechanism of action of gilteritinib/MXR-2843 in combination with AZD5991. (B,C) MV4-11 cells were treated with vehicle control, AZD5991 (AZD), gilteritinib (gilt), MRX-2843 (MRX), or in combination for 3 h. Cellular fractionation was performed. Mitochondrial and cytosolic fractions were subjected to western blotting analysis. This experiment was performed two independent times in triplicate. One representative image is shown. Relative densitometry measurements were determined, normalized to β-actin or VDAC1, and compared to the vehicle control. Results from one representative experiment are graphed as mean ± SEM in panel (C). * indicates

p

< 0.05, ** indicates

p

< 0.01, and *** indicates

p

< 0.001 compared to vehicle control. # indicates

p

< 0.05 compared to single-drug treatment. (D) MV4-11 cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Western blots were generated utilizing whole cell lysates. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (E,F) MV4-11 cells were treated with vehicle control, AZD, 10058-F4, or in combination for 3 h. Cellular fractionation was performed as described in panel (B). * indicates

p

< 0.05, ** indicates

p

< 0.01 and *** indicates

p

< 0.001 compared to vehicle control. ## indicates

p

< 0.01 compared to single-drug treatment. (G) MV4-11 cells were treated with vehicle control, AZD, 10058-F4, or in combination for 3 h. Western blots were generated utilizing whole cell lysates. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (H,I) shRNA knockdown of Bak and Bax was performed in MV4-11 cells with non-template control (NTC) as the negative control. Whole cell lysates were subjected to western blotting (panel (H)). Cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Annexin V/PI staining and flow cytometry analysis results are shown in panel (I). ***

p

< 0.001 compared to NTC for the same drug treatment.

Figure 6. AZD5991 synergistically enhances apoptosis induced by gilteritinib and MRX-2843 in AraC-resistant AML cells. (A) MOLM-13 and MV4-11 AraC-resistant cells (MOLM-13/AraC-R and MV4-11/AraC-R) were treated with vehicle control, AZD5991 (AZD), gilteritinib (gilt), MRX-2843 (MRX), or in combination for 3 h. Whole cell lysates were subjected to Western blotting. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (B) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Total RNA was extracted, and real-time RT-PCR was performed. The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. ** indicates

p

< 0.01 and *** indicates

p

< 0.001 compared to vehicle control. (C) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Annexin V/PI staining and flow cytometry analysis results are shown. *** indicates

p

< 0.001 compared to vehicle control and single-drug treatment. (D) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 24 h. Annexin V/PI staining and flow cytometry analysis results are shown. *** indicates

p

< 0.001 compared to vehicle control and single-drug treatment. CI values were calculated using CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. (E) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Total RNA was extracted, and real-time RT-PCR was performed. The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. *** indicates

p

< 0.001 compared to control and single-drug treatments. (F,G) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD5991 (AZD), AZD1480, or in combination for 3 h. Whole cell lysates were subjected to western blotting. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot in panel (F). After treatment, cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (G)). *** indicates

p

< 0.001 compared to control and single-drug treatments. (H) Primary AML patient sample AML#246 cells treated with varying concentrations of AZD, gilt, or MRX, alone or in combination, for 24 h were stained with annexin V-FITC/PI and analyzed using a flow cytometer. CI values were calculated using CompuSyn software. *** indicates

p

< 0.001 compared to vehicle and single-drug treatments.

Figure 6. AZD5991 synergistically enhances apoptosis induced by gilteritinib and MRX-2843 in AraC-resistant AML cells. (A) MOLM-13 and MV4-11 AraC-resistant cells (MOLM-13/AraC-R and MV4-11/AraC-R) were treated with vehicle control, AZD5991 (AZD), gilteritinib (gilt), MRX-2843 (MRX), or in combination for 3 h. Whole cell lysates were subjected to Western blotting. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot. (B) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Total RNA was extracted, and real-time RT-PCR was performed. The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. ** indicates

p

< 0.01 and *** indicates

p

< 0.001 compared to vehicle control. (C) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Annexin V/PI staining and flow cytometry analysis results are shown. *** indicates

p

< 0.001 compared to vehicle control and single-drug treatment. (D) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 24 h. Annexin V/PI staining and flow cytometry analysis results are shown. *** indicates

p

< 0.001 compared to vehicle control and single-drug treatment. CI values were calculated using CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. (E) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD, gilt, MRX, or in combination for 3 h. Total RNA was extracted, and real-time RT-PCR was performed. The displayed results represent the mean of three independent experiments, with fold changes calculated via comparative Ct method and normalized to

36B4

transcripts. *** indicates

p

< 0.001 compared to control and single-drug treatments. (F,G) MOLM-13/AraC-R and MV4-11/AraC-R cells were treated with vehicle control, AZD5991 (AZD), AZD1480, or in combination for 3 h. Whole cell lysates were subjected to western blotting. Densitometry results (normalized to β-actin and compared to vehicle control) are shown below the corresponding blot in panel (F). After treatment, cells were stained with annexin V-FITC/PI and analyzed using a flow cytometer (panel (G)). *** indicates

p

< 0.001 compared to control and single-drug treatments. (H) Primary AML patient sample AML#246 cells treated with varying concentrations of AZD, gilt, or MRX, alone or in combination, for 24 h were stained with annexin V-FITC/PI and analyzed using a flow cytometer. CI values were calculated using CompuSyn software. *** indicates

p

< 0.001 compared to vehicle and single-drug treatments.