Fig. 3

Acquired resistance to DOT1L inhibition leads to selective loss of KMT2A-fusion driven PROM1 expression. A.PROM1 mRNA expression in SEM and SEM^PINO_RES cells cultured for 7 days in the absence (-) or presence (+) of 50 µM pinometostat, as determined by RNA-seq. Values indiacte normalized counts with SD derived from four biological replicates for each cell line and condition. ****p < 0.0001. B. Western blot images of PROM1 and GAPDH protein levels in indicated cell line models, and C. quantification of PROM1 expression relative to GAPDH by densitometry analysis. Values indicate mean ± SD PROM1 protein expression as determined in two biological replicates. *p < 0.05. D. Histograms showing the counts of viable cells positive for PROM1/CD133 of indicated cell line models, as determined by flow cytometry (FACS) analysis, and E. Quantification of PROM1/CD133 expression presented as the mean ± SD as determined by two independent FACS experiments. *p < 0.05, **p < 0.005. F. Differences in chomatin accessibility at the PROM1 and TAPT1 gene locus between SEM^PINO_RES and SEM cells as determined by ATAC-sequencing of two biological replicates (on top). Vertical blue lines indicate significant decreases of chromatin accessibility in SEM^PINO_RES cells, whereas grey lines indicate equal chromatin accessibility in both SEM^PINO_RES and SEM. The ATAC-sequencing results are followed by ChIP-sequencing tracks of the same locus showing the distribution of H3K79Me2, H3K27Ac, H3K4Me3, KMT2A in SEM and SEM^PINO_RES cells cultered for 7 days in either the absence (-) or presence (+) of 50 µM pinometostat.

Differences were statistically evaluated using unpaired t-tests