Figure 6.
Jak1 inhibition impairs proliferation of AF10 FP AML cells. (A) Schematic representation of the experiment. BM from Jak1-floxed mice was used to transform lineage-depleted cells with the CALM-AF10 fusion. Transformed cells were transduced with a ER-fused Cre recombinase (ER-Cre) and Jak1 was deleted using 4-hydroxy-tamoxifen (4-OHT) treatment of CFU assays. (B) Number of blast-like (dark blue bars) or differentiated (light blue bars) CFUs per 1000 cells from CALM-AF10–transformed cells upon Jak1 deletion (or Stat3 DN) overexpression is shown for weeks 1, 2, and 3. P values were calculated by comparing each arm to the Jak1+/+ control using the Student t test. *P < .05. No colonies were observed in the Jak1−/− Stat3 DN arm in week 3. (C) A picture of a representative colony in Jak1 wild-type compared with Jak1-deleted CALM-AF10–transformed cells is shown (top) (original magnification ×10) with a Wright-Giemsa–stained cytospins of cells from the CFU assay (bottom) (original magnification ×40). (D) GSEA showing the distribution of CALM-AF10 target genes in RNA-seq data from Jak1 floxed compared with Jak1-deleted cells is shown with the normalized enrichment score (NES) and false discovery rate (FDR) q-value indicated. (E) A heatmap for the top 50 CALM-AF10 target genes with the highest GSEA rank metric score is plotted and shown for 3 Jak1+/+ and 3 Jak1−/− replicates. (F) Total number of colonies from CALM-AF10–transformed cells treated with indicated concentrations of itacitinib are plotted as a percent of DMSO (vehicle)-treated cells. **P < .01. (G) A representative picture of the most common type of colonies in DMSO compared with itacitinib-treated CALM-AF10–transformed cells is shown (original magnification ×10). (H) Cell growth assay showing effect of different concentrations of itacitinib on proliferation of human AML patient cells. P1-P4 are blasts from patients or patient-derived xenograft with MLL-AF10 rearrangements and P5 is from a patient with an MLL-AF9 fusion. Error bars indicate standard deviation (SD) of 3 replicates of each sample. (I) Percent apoptotic cells upon treatment with 10 μM itacitinib are shown as measured by Annexin V staining. Error bars show standard error of mean (SEM) for each sample. *P < .05. ns, nonsignificant.

Jak1 inhibition impairs proliferation of AF10 FP AML cells. (A) Schematic representation of the experiment. BM from Jak1-floxed mice was used to transform lineage-depleted cells with the CALM-AF10 fusion. Transformed cells were transduced with a ER-fused Cre recombinase (ER-Cre) and Jak1 was deleted using 4-hydroxy-tamoxifen (4-OHT) treatment of CFU assays. (B) Number of blast-like (dark blue bars) or differentiated (light blue bars) CFUs per 1000 cells from CALM-AF10–transformed cells upon Jak1 deletion (or Stat3 DN) overexpression is shown for weeks 1, 2, and 3. P values were calculated by comparing each arm to the Jak1+/+ control using the Student t test. *P < .05. No colonies were observed in the Jak1−/− Stat3 DN arm in week 3. (C) A picture of a representative colony in Jak1 wild-type compared with Jak1-deleted CALM-AF10–transformed cells is shown (top) (original magnification ×10) with a Wright-Giemsa–stained cytospins of cells from the CFU assay (bottom) (original magnification ×40). (D) GSEA showing the distribution of CALM-AF10 target genes in RNA-seq data from Jak1 floxed compared with Jak1-deleted cells is shown with the normalized enrichment score (NES) and false discovery rate (FDR) q-value indicated. (E) A heatmap for the top 50 CALM-AF10 target genes with the highest GSEA rank metric score is plotted and shown for 3 Jak1+/+ and 3 Jak1−/− replicates. (F) Total number of colonies from CALM-AF10–transformed cells treated with indicated concentrations of itacitinib are plotted as a percent of DMSO (vehicle)-treated cells. **P < .01. (G) A representative picture of the most common type of colonies in DMSO compared with itacitinib-treated CALM-AF10–transformed cells is shown (original magnification ×10). (H) Cell growth assay showing effect of different concentrations of itacitinib on proliferation of human AML patient cells. P1-P4 are blasts from patients or patient-derived xenograft with MLL-AF10 rearrangements and P5 is from a patient with an MLL-AF9 fusion. Error bars indicate standard deviation (SD) of 3 replicates of each sample. (I) Percent apoptotic cells upon treatment with 10 μM itacitinib are shown as measured by Annexin V staining. Error bars show standard error of mean (SEM) for each sample. *P < .05. ns, nonsignificant.

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