Fig 1: Depletion or degradation of Menin increases sensitivity to treatment with venetoclax or abemaciclib in AML cells.A MOLM13 was transfected with sgRNA against Exon 2 or Exon 6 of Menin or a negative control sgRNA (sgNeg) and incubated for 5 days. Then, total cell lysates were prepared and immunoblot analyses were conducted. The expression levels of GAPDH served as the loading control. B MOLM13 cells were transfected with sgNeg or sg Menin Ex 2 or Ex 6 and incubated for 72 h. Then, cells were treated with the indicated concentrations of venetoclax for 48 h. The % of annexin V-positive, apoptotic cells were determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to sgNeg-transfected MOLM13 cells (determined by a two-tailed, unpaired t test). C MOLM13 cells were transfected with sgNeg or sg Menin Ex 2 or Ex 6 and incubated for 72 h. Following this, cells were treated with the indicated concentrations of abemaciclib for 96 h. The % of TO-PRO-3 iodide-positive, non-viable cells were determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to sgNeg-transfected MOLM13 cells (determined by a two-tailed, unpaired t test). D MOLM13-Menin-FKBP12F36V cells were treated with the indicated concentrations of dTAG-13 for 72 h. At the end of treatment, cell lysates were prepared and immunoblot analyses were conducted for HA-tagged Menin, endogenous Menin, MEIS1, FLT3, CDK6, PBX3, BCL2, and p27. The expression levels of GAPDH served as the loading control. E MOLM13-Menin-FKBP12F36V cells were treated with the indicated concentrations of venetoclax for 48 h alone or co-treated with 500 nM of dTAG-13. The % of TO-PRO-3 iodide-positive, non-viable cells were determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to MOLM13-Menin-FKBP12F36V cells not treated with dTAG-13 (determined by a two-tailed, unpaired t test in GraphPad V8). F MOLM13-Menin-FKBP12F36V cells were treated with the indicated concentrations of abemaciclib for 96 h without or with 500 nM of dTAG-13. The % of TO-PRO-3 iodide-positive cells was determined by flow cytometry. Mean of two independent experiments performed in duplicate ±S.D. **p < 0.01 compared to MOLM13-Menin-FKBP12F36V cells not treated with dTAG-13 (determined by a two-tailed, unpaired t test in GraphPad V8).
Fig 2: 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 ß-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 ß-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.
Fig 3: Menin inhibitor treatment depletes H3K27 acetyl mark on chromatin, depletes mRNA expression of MYC, MEIS1, and FLT3 in cultured and primary AML blasts and depletes Menin, BCL2, and MLL target gene expression levels in phenotypically defined leukemia stem cells.A IGV plots showing signal tag density of H3K27Ac ChIP-Seq at the MEIS1 and FLT3 gene in MOLM13 cells treated with SNDX-50469 for 16 h. Black arrows mark the direction of the coding sequence of each gene. Blue bars beneath the gene indicate significant, log2 fold-changes in signal tag density of H3K27Ac in SNDX-50469-treated cells compared to control cells. B–D MOLM13, OCI-AML3, and PD MLL-AF9, FLT3-TKD AML cells were treated with the indicated concentrations of SNDX-50469 for 16 h. Total RNA was isolated, and reverse transcribed. The resulting cDNAs were used for qPCR as shown. The expression of GAPDH served as the normalization control. E Patient-derived AML cells were treated with 1000 nM of SNDX-50469 for 16 h. Cells were harvested and analyzed by CyTOF analysis utilizing a cocktail of rare metal element-tagged antibodies. Leukemia stem cells were defined by high expression of CLEC12A, CD123, CD244, CD99, and CD33 but low expression of CD11b.
Fig 4: 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 ß-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 ß-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 ß-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.
Fig 5: 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 ß-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 ß-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 ß-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.
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