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S. Hultmark et al.
dates were histone deacetylase inhibitors as well as CPI- 203 (Figure 5B and Online Supplementary Figure S6), a well- known BET inhibitor of BRD4.31 Notably, while displaying little to no differentiating effect on its own, CPI-203 enhanced the H4-driven increase of CD11b expression, but more strikingly led to a 22-fold increase of the CD11b pro- tein intensity compared to 6-fold with H4 alone (Figure 5C, Online Supplementary Figure S7, Online Supplementary Table S3). In additional primary samples, the combination enhanced the differentiating effect of H4 on two out of two FLT3 wild-type AML-M5 samples and in one out of three FLT3-mutated AML-M5 primary samples (Figure 5D, Online Supplementary Figure S7 and Online Supplementary Table S3). The AML-M5 cell lines followed the same pat- tern, with THP1 (FLT3 wild-type) showing a clear response while MM6 and MOLM-13 (FLT3 mutated) did not respond to the addition of CPI-203 (Online Supplementary
Figures S7 and S8, Online Supplementary Table S3). Finally, the combination of H4 and the BET inhibitor had no addi- tional effect on the other primary samples regardless of FAB-type or of FLT3 mutation status, except the cell line HL60 (FLT3 wild-type) which responded to the combina- tion (Figure 5D, Online Supplementary Figure S8 and Online Supplementary Table S3). The healthy control also respond- ed to the combination with an increase of CD11b+ cells. However, the increase was still below the 20% threshold, and no morphological changes were detected (Figure 5D, E, Online Supplementary Figure S7 and Online Supplementary Table S3). Taken together, our findings indicate that among the H4-sensitive primary samples, the combination with the BET inhibitor predominantly affects monocytic (M5) AML.
GSEA of upregulated genes in H4-treated AML-3 cells revealed strong enrichment of MYC target genes (Figure
ABC
DEF
Figure 5. Combinatorial screen identified a BET inhibitor as an enhancer of H4-induced differentiation. (A) Cells from primary AML-3 were treated with 176 com- pounds (5 mM, 0.5 mM, and 0.05 mM) with defined targets alone and in combination with compound H4 (10 mM). After 4 days, the combinatorial differentiation response was assayed by increased expression of CD11b using flow cytometry. (B) The most potent compounds in combination with H4 are shown in the concentra- tion that did not reduce cell number more than 75% relative to treatment with H4 alone (the data are shown as mean ± standard deviation [SD] of technical repli- cates). (C) Representative histogram plots showing the combinatorial differentiation effect of H4 (10 mM) and CPI-203 (0.05 mM) in AML-3 cells. Lower graphs show fold-change of mean fluorescent intensity (MFI) of CD11b relative to dimethylsulfoxide (DMSO). The data are shown as mean ± standard error of mean (SEM) from independent experiments designated with individual symbols. (*P<0.05, **P<0.01, ns not significant, using one-way analysis of variance [ANOVA]). (D) Combining H4 (10 mM) and the BET inhibitor CPI-203 (0.05 mM) enhanced differentiation in two out of two FLT3 wild-type and one out of three FLT3-ITD/mutated monocytic AML-M5 samples, while none of 15 samples of other M-types responded to the combination. The healthy control was CD34+ umbilical cord blood. Responders (green dashed line) to the combination had to exceed the 20% cutoff (dotted line) in increased CD11b expression to be categorized as responding. (E) As in panel (C). Healthy control (CD34+ umbilical cord blood) n=3. (**P<0.01, ns not significant, using one-way ANOVA). (F) MYC was dispensable for the differentiation response in primary-monocytic AML-3 cells and the monocytic THP1 cell line. Cells were treated with H4 10 mM and CPI-203 0.05 mM alone and in combination. MYC levels are displayed as fold-change compared to DMSO for each time-point. The data are shown as mean ± SD of technical replicates.DMSO: dimethylsulfoxide; ITD: internal tandem duplication; mut: mutated; WT: wild-type: MFI: mean fluorescent intensity.
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