Figure 5. Effects of DOT1L-i on gene expression, cell differentiation, and leukemia initiating potential in NPM1^mut AML cells.

(A) (Left panel) Log₂ fold change of HOX genes, MEIS1, and FLT3 between OCI-AML3 cells treated for 7 days with 10μM EPZ4777 or DMSO vehicle control as assessed by RNA-sequencing. Only expressed HOXA- and HOXB-cluster genes are shown with a normalized read count of ≥100 reads within the vehicle control, ≥0.5 log₂ fold change, and a p-value (adjusted for multiple testing) of < 0.05. (Right panel) GSEA of RNA-sequencing data showing enrichment of genes upregulated with EPZ4777 treatment for genes silenced in normal hematopoietic cord blood stem cells.

(B) Gene expression in murine Npm1^CA/+Flt3^ITD/+ and Npm1^CA/+Rosa^SB/+ cells assessed on day 7 of EPZ4777 treatment [10μM] by quantitative PCR.

(C) Dose response curves from cell viability assays after 14 days of EPZ4777 treatment comparing Npm1^CA/+Flt3^ITD/+ cells versus Npm1^CA/+Flt3^ITD/+ cells overexpressing Hoxb4, Meis1, or Hoxa9-Meis1.

(D) Cell differentiation upon DOT1L-i (EPZ4777 [10μM]) as determined by flow cytometry for CD11b expression in OCI-AML3 cells (at day 0, 4, and 7 of treatment).

(E) Morphological changes in Npm1^CA/+Flt3^ITD/+ (upper panel) and Npm1^CA/+Rosa^SB/+ (lower panel) cells consistent with monocytic differentiation in murine cells after 14 days of EPZ4777 [10μM] treatment.

(F) Kaplan-Meier survival curve of mice transplanted with pretreated Npm1^CA/+Rosa^SB/+ leukemia cells (vehicle: n= 8 mice/group; EPZ4777: n=7 mice/group).

(G) White blood cell count and (H) morphology of mice transplanted with pretreated Npm1^CA/+Rosa^SB/+ leukemia cells on day 19 post transplantation.

Data in (A) represent averages of three independently treated replicates per group, data in (B), (C), and (D) represent averages of three independent experiments, each performed in three replicates. Error bars represent the SEM.