Figure 5.
AMKL xenografts depend on prosurvival protein BCL-XL for survival. (A) RNAseq expression values (log2[FPKM]) of selected genes implicated in intrinsic apoptosis pathway in model and patient leukemias from our institutional data set. (B) Crossvalidation of gene expression in a large and published data set of pediatric AMKL.9 (C) Protein levels of BCL-XL (left) and BCL2 (right) in AMKL xenograft cells or normal CB CD34+ cells assessed by intracellular flow cytometry. (D) Apoptosis, assessed by annexin V staining and flow cytometry, (E) relative gene expression detected by quantitative reverse transcription polymerase chain reaction, and (F) prosurvival protein levels quantified by flow cytometry, after short hairpin RNA (shRNA)-mediated KD of prosurvival BCL2 proteins (BCL-XL, BCL2, and BCL-W) in CG2 xenografts (72 hours after infection, 2 selected shRNA per gene). Samples were compared with nontransduced (NT) or cells infected with control shRNA against Renilla (shRenilla). See supplemental Figure 18 for KD studies in mCG2-2 cells. (G) Dose response curves and IC50 values were determined after incubation of AMKL or AML xenografts with DT2216 (BCL-XL proteolysis-targeting chimera) or DMSO for 6 days, followed by viability readout with Cell-Titer Glo. Viability readout was normalized against DMSO for each sample. (H) Amount of apoptosis assessed by annexin V staining and flow cytometry in AMKL xenografts or normal CB CD34+ cells after 72 hours of incubation with DT2216 at 100 nM or 1 μM in comparison with cells treated with DMSO. GATA1s, GATA1 truncation; HOXr, HOX rearrangement; KMT2Ar, KMT2A rearrangement; MFI, mean fluorescence intensity; RBM-MKL, RBM15::MKL1; VHL, von Hippel-Lindau; xAMKL, synthetic model of AMKL; xAML, synthetic model of AML. P values: ∗P < .05; ∗∗P < .005; ∗∗∗∗P < .0001.

AMKL xenografts depend on prosurvival protein BCL-XL for survival. (A) RNAseq expression values (log2[FPKM]) of selected genes implicated in intrinsic apoptosis pathway in model and patient leukemias from our institutional data set. (B) Crossvalidation of gene expression in a large and published data set of pediatric AMKL.9 (C) Protein levels of BCL-XL (left) and BCL2 (right) in AMKL xenograft cells or normal CB CD34+ cells assessed by intracellular flow cytometry. (D) Apoptosis, assessed by annexin V staining and flow cytometry, (E) relative gene expression detected by quantitative reverse transcription polymerase chain reaction, and (F) prosurvival protein levels quantified by flow cytometry, after short hairpin RNA (shRNA)-mediated KD of prosurvival BCL2 proteins (BCL-XL, BCL2, and BCL-W) in CG2 xenografts (72 hours after infection, 2 selected shRNA per gene). Samples were compared with nontransduced (NT) or cells infected with control shRNA against Renilla (shRenilla). See supplemental Figure 18 for KD studies in mCG2-2 cells. (G) Dose response curves and IC50 values were determined after incubation of AMKL or AML xenografts with DT2216 (BCL-XL proteolysis-targeting chimera) or DMSO for 6 days, followed by viability readout with Cell-Titer Glo. Viability readout was normalized against DMSO for each sample. (H) Amount of apoptosis assessed by annexin V staining and flow cytometry in AMKL xenografts or normal CB CD34+ cells after 72 hours of incubation with DT2216 at 100 nM or 1 μM in comparison with cells treated with DMSO. GATA1s, GATA1 truncation; HOXr, HOX rearrangement; KMT2Ar, KMT2A rearrangement; MFI, mean fluorescence intensity; RBM-MKL, RBM15::MKL1; VHL, von Hippel-Lindau; xAMKL, synthetic model of AMKL; xAML, synthetic model of AML. P values: ∗P < .05; ∗∗P < .005; ∗∗∗∗P < .0001.

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