Fig. 1

Establishment of acquired resistance to DOT1L inhibition in KMT2A-rearranged ALL cells. A. Graphic overview of acquired resistance induction to DOT1L inhibition in KMT2A::AFF1 + B-cell ALL (SEM) leading to pinometostat-resistant cells (SEM^PINO_RES). B. Viable cell percentage of SEM and SEM^PINO_RES cells in the absence (-) or presence (+) of 50µM pinometostat for 7 days, normalized to cells cultured without pinometostat. Data present the mean +/- the standard deviation (SD) derived from 2 biological replicates. C. IC50 values of viable cells of the indicated cell line models determined using six drug concentrations (0-100 µM) for 14 days. The data illustrates the mean +/- SD from 4 biological replicates, each comprising 3 technical replicates. D. Immunoblot images of H3K79me2 and total histone H3 in SEM and SEM^PINO_RES cells cultured with or without 50 µM pinometostat for 7 days. E. Quantification of H3K79me2 protein expression using densitometry analysis normalized against total histone H3 expression. Data represent fold-changes normalized against untreated SEM cells for 2 biological replicates. F. mRNA expression of DOT1L, KMT2A:AFF1, and HOXA9 determined by qRT-PCR analysis, and G. viable cell percentage in SEM and SEM^PINO_RES at day 2 and day 4 after siRNA-mediated knockdown (KD) relative to non-silencing controls (NSCs). Data of 2 biological replicates ± SD, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. (H) Immunoblot images of DOT1L, H3K79me2, and GAPDH protein expression in SEM and SEM^PINO_RES cells at day 4 following siRNA-mediated KD of DOT1L, and corresponding quantification of (I) DOT1L or (J) H3K79me2 protein expression relative to GAPDH using densitometry analysis.

Differences were statistically evaluated using unpaired t-tests