Fig. 6
Patient-derived cells demonstrate clinical utility of combined KL-11743 treatment with Complex I inhibition. A Differential gene expression analysis comparing normal HSPCs, derived from the human embryonic stem cell (hESC) line H1 and two iPSC lines (N-8.2 and N-6.1); KRASWT AML-iPSC-derived LSCs (AML-4.24 and AML-4.16) and KRASG12D AML-iPSC derived LSCs (AML-4.10). Heatmap showing expression of 1447 genes differentially expressed between normal iPSC-HSPCs and AML-iPSC-LSCs using scaled log normalized counts. B–F RNA-seq data from A was re-analyzed to directly compare normal iPSC-HSPCs to AML-iPSC LSCs. Volcano plots represent the log2 fold-change (FC) and p-value of genes enriched in the indicated gene ontology pathways. SLC2A1, a KL-11743 target, is highlighted. The number of genes compared in each pathway as follows: TCA, 28; OXPHOS, 199; Mitochondrial Protein Import: 61; Mitochondrial Protein Translation: 94; Glycolysis: 193. G Normal HSPCs or AML LSCs derived from iPSC lines N-8.2, AML-4.24 (KRASWT) and AML-4.10 (KRASG12D) were treated with DMSO or KL-11743 (500 nM) and OCR was measured following an Agilent XF Cell Mito Stress Test. O: oligomycin (1 μM), F: FCCP (1 μM), R + A: rotenone (0.5 μM) + antimycin A (0.5 μM). H Quantification of basal respiration from G, calculated as the average of the 3 reads preceding injection of oligomycin (O). I Quantification of maximal respiration from G, calculated as the average of the 3 reads immediately following injection of FCCP (F). J Normal HSPCs or AML LSCs derived from iPSC lines N-8.2, AML-4.24 (KRASWT) and AML-4.10 (KRASG12D) were treated with DMSO or KL-11743 (500 nM) for 24 h and ECAR was measured following an Agilent XF Glycolysis Stress Test. G: glucose (10 mM), O: oligomycin (1 μM), 2DG: 2-deoxy-D-glucose (50 mM). K–L Quantification of basal and glucose-stimulated ECAR from H, calculated as the average of the 3 reads immediately before and following injection of glucose (G), respectively. M Normal HSPCs or AML LSCs derived from iPSC lines N-8.2, AML-4.24 (KRASWT) and AML-4.10 (KRASG12D) were treated with DMSO or KL-11743 (500 nM) for 24 h. Apoptosis was measured by AV labeling and flow cytometry. Data are presented as the mean of 3 replicates ± SD. N Cells were treated and analyzed as in M, but with IACS (10 nM) for 24 h. CHX (50 μg/mL) + ABT (1 μM) is a positive control for apoptosis. Data are presented as the mean of 3 replicates ± SD. O Cells were treated and analyzed as in M–N, but with KL-11743 (1 μM) ± IACS (10 nM) for 24 h. Data are presented as the mean of 3 replicates ± SD. P Primary cells from AML patients 1–8 were treated with DMSO (0.1%) or KL-11743 (500 nM) at a density of 100,000 cells/mL, cultured for 24 h, and ECAR was measured following an Agilent XF Glycolysis Stress Test. Data are presented as the glycolysis-specific ECAR, calculated as the difference between basal and glucose-stimulated ECAR using the timepoint immediately before and after the glucose injection. Q Data from P presented as the fold change relative to DMSO. Data are the mean of 3 replicates ± SEM. R Primary cells from AML patients 1–8 were treated with DMSO (0.1%), KL-11743 (500 nM), IACS (10 nM), CHX (50 μg/mL), or indicated combination for 24 h before viability was measured. DMSO and CHX are the negative and positive cell death controls. Data were normalized to the DMSO and CHX treatments as 100 and 0%, respectively