Figure 4. DOT1L inhibition reduces STAT5A activation and downregulates STAT5A targets in FLT3-ITD leukemia lines.

(A) MLL-rearranged leukemia lines with genotypes indicated were treated with 100 nM pinometostat (left panel, DOT1L inhibitor) or 30 nM tandutinib (right panel, FLT3 inhibitor MLN518), and relative growth monitored by CellTiter Glo 2.0 assay on the indicated days. Relative viability presented is the mean fraction of luminescence of treated versus side-by-side mock treated cultures (same volume of DMSO) for three independent replicates ± S.E.M. Student’s t-test (** p ≤ 0.01, *** p ≤ 0.001). (B) Western blots of phosphorylated STAT5 (active) or total STAT5A with H3 or HNRNPK as loading controls across the cell lines from panel A treated as indicated; H3K79me2 is monitored in pinometostat-treated lines to confirm inhibition. (C) Time course of gene expression by RT-qPCR, presented as mean fold-change of FLT3, PBX3, PIM1, and MEF2C in MV4;11 cells ± 100 nM pinometostat at each time point indicated ± S.E.M.; n = 3; Student’s t-test (ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, ***** p < 0.00001). (D-E) DOT1L and FLT3 inhibition downregulate STAT5A targets in FLT3-ITD. RT-qPCR expression analysis presented as mean fold-change ± S.E.M. for the indicated transcript in MV4;11 cells treated with indicated inhibitor versus mock-treatment for 7 days. Student’s t-test (** p < 0.01, **** p < 0.0001, ***** p < 0.00001). (F) Proliferation assay as in panel A, with three clonal populations of MV4;11 cells virally transduced, selected, then induced to express shRNA to FLT3 (Green et al., 2015) or a scrambled shRNA (Yuan et al., 2009) control by 1 µg/mL doxycycline. Means of fractional viability relative to uninduced cells ± S.E.M. are shown for three independent experiments; Student’s t-test (** p < 0.01). (G) RT-qPCR analysis of PIM1, PIM2, and ARID3B expression in MV4;11 cells expressing an inducible shRNA targeting FLT3 (Green et al., 2015) for 7 days. Results are depicted as fold-change expression of control cells expressing shRNA to GFP (Scheeren et al., 2005). (H) Proliferation assay of K562, PL-21, and EOL-1 cells treated with 10 µM or 100 nM pinometostat using CellTiter Glo 2.0 to measure viability, showing the luminescence fraction of inhibited over DMSO-treated cells. Means ± SE are shown for three independent experiments. Student’s t-test of day 7 (EOL-1 cells), day 9 (PL-21), or days 9 and 11 (K562 and PL-21) values: ns p > 0.05, * p < 0.05, **** p < 0.0001. (I) Gene expression analysis by RT-qPCR in PL-21 cells treated for 9 days with 10 µM pinometostat. Results are displayed as fold-change over DMSO-treated cells with means ± SE for three independent experiments (ND = not detected). Student’s t-test (**** p < 0.0001). (J) Western blots of (left) cell extract from PL-21 cells treated with 10 µM pinometostat for 9 days and (right) EOL-1 cells treated with 100 nM pinometostat for 7 days and then blotted for H3K79me2 and p-STAT5 with H2B or HNRNPK as a loading controls. (K) Gene expression analysis by RT-qPCR in EOL-1 cells treated for 7 days with 100 nM or 10 µM pinometostat. Results are displayed as fold-change over DMSO-treated cells with means ± SE for three independent experiments. Student’s t-test (ns p > 0.05, **** p < 0.0001).

Figure 4—figure supplement 1. FLT3 knockdown reduces STA5A activation.

(A) RT-qPCR analysis of HOXA9 and MEIS1 expression from three independent experiments of MOLM13 cells treated with 100 nM pinometostat for 7 days. Fold change over DMSO-treated cells is depicted ± S.E.M. Student’s t-test (ns p > 0.05). (B) (left) RT-qPCR analysis of FLT3 and PIM1 expression in MV4;11 cells ± 50 nM SGC0946 for 7 days. Results are displayed as mean fold-change vs. DMSO-treated cells ± S.E.M. of three independent experiments. Student’s t-test (* p < 0.05, *** p ≤ 0.001). (right) Western blots from MV4;11 cells (left) treated with 50 nM SGC0946 for 7 days blotted for phosphorylated STAT5, H3K79me2, or histone H2B as a loading control. (right) MV4;11 cells treated with increasing concentrations of SGC0946 for 7 days blotted for H3K79me2 or H2B as a loading control. (C) Quantitative western blot of whole cell extracts from 3 experiments of MV4;11 cells ± 100 nM pinometostat for 7 days, stained with antibodies for phosphorylated STAT5 or histone H3 as a loading control. The blot includes a dilution series of the extract from the DMSO-treated sample one that encompasses the range of signal observed in each of the replicates and is used to calibrate a relative quantification of phosphorylated STAT5A signal integrated in ImageJ and shown in the bar graph at right as a 65 ± 8% reduction of the DMSO treated samples ± SD. Student’s t-test (** p < 0.01). (D) Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005; Mootha et al., 2003) of the set of downregulated genes in MV4;11 cells ± 100 nM pinometostat compared to genes upregulated by exogenous expression of constitutively active STAT5A in the WIERENGA_STAT5A_TARGETS GROUP1 gene set (Wierenga et al., 2008) from the MSigDB database. (E) Bar graph of Cuffdiff (Trapnell et al., 2012) output for the expression of STAT5A targets ARID3B, PIM1, and PIM2 from RNA-seq in MV4;11 cells ± 100 nM pinometostat. Values are represented as log(10) FPKM + 1 for three independent experiments with standard deviation. Student’s t-test (** p < 0.01, *** p < 0.001). (F) RT-qPCR analysis of PBX3, PIM1, FLT3, and MEF2C expression from three independent experiments of MOLM13 cells treated with 100 nM pinometostat for 7 days. Fold change over DMSO-treated cells is depicted ± S.E.M. Student’s t-test (ns p > 0.05, *** p < 0.001, **** p < 0.0001). (G) Proliferation assay of MV4;11 cells treated with DOT1L or FLT3 inhibitors alone or in combination using CellTiter Glo 2.0 to measure viability, showing the luminescence fraction of inhibited over uninhibited cells. Data are represented as mean ± SE of three independent experiments. Student’s t-test for significance of day 7 values: 100 nM pinometostat vs. combined ** p < 0.01, 30 nM tandutinib vs combined *** p < 0.001. (H) Same as G but cells were treated with DOT1L and PIM1 inhibitors alone or in combination. Student’s t-test of day 7 values: 100 nM pinometostat vs. combined * p < 0.05, 10 µM quercetagenin vs combined ** p < 0.01. Purple asterisks indicate Student’s t-test for significance of day 5 for 100 nM pinometostat vs. combination treatment ** p < 0.01. (I) Western blots of MV4;11 cell extract from clonal cell lines expressing shRNA to FLT3 (clone 3) or GFP blotted for phosphorylated STAT5 or histone H3 as a loading control. (J) Western blots of cell extract from MV4;11 cells treated with 100 nM pinometostat for the indicated number of days and then blotted for H3K79me2 and HNRNPK as a loading control.