Gene expression displayed by K-means clustering. pathway and inhibits the epigenetic regulator LSD1. This medication combination leads to maturation of immature leukemia cells and improves survival in mouse models in a synergistic manner. This therapeutic approach is a promising strategy for the treatment of AML patients with mutations in CEBPA and CSF3R. and members of the and families (Fig. 2and and were among the most highly up-regulated genes identified with LSD1 inhibition, consistent with an autofeedback loop. To confirm that LSD1 inhibition was not altering the expression of either transduced oncogene, we assessed human CEBPA and CSF3R expression in response LSD1 inhibitor treatment and found no significant changes (and = 3/group). (= 2/group). Enhancers were defined as the presence of H3K4me1 PF-AKT400 and H3K27Ac along with the absence of H3K4me3. Differential H3K27Ac signal at enhancers in response to GSK-LSD1 treatment. (and and = 0.0027, Fig. 2and and = 3/dose). (= 3/dose). (= 4 to 5/group). In all cases, values are represented as mean SEM. Survival was assessed by the log-rank test. Significance of other comparisons was assessed by Students test for two group comparisons or ANOVA with Sidaks posttest, as appropriate. Early data demonstrate that inhibition of JAK/STAT signaling with ruxolitinib is effective in patients with CSF3R-mutant chronic neutrophilic leukemia (15). JAK/STAT inhibition has also been suggested as a therapeutic strategy for CEBPA mutant AML irrespective of CSF3R mutational status (12). Our data above suggest that PF-AKT400 LSD1 inhibition might also be an effective therapy in this disease subtype. We therefore assessed disease response to GSK289552 and ruxolitinib in mice harboring CEBPA/CSF3R mutant AML. In mice transplanted with CSF3RT618I alone, marked disease control is achieved with ruxolitinib monotherapy (16). We treated mice harboring CSF3RT618I and CEBPAV314VW with ruxolitinib alone or GSK289552 alone. However, despite in vitro efficacy, neither single agent improved survival, controlled white blood cell (WBC) count, or significantly reduced spleen size (Fig. 3 and = 3/group). (= 3/group). Gene expression displayed by K-means clustering. (= Edn1 5 to 6/group). In all cases, values are represented as mean SEM. ** 0.01. Survival was assessed by log-rank test. Significance of other comparisons was assessed by Students test for two group comparisons or ANOVA with Sidaks posttest, as appropriate. To further characterize the mechanism of synergy, we performed RNA-seq on CEBPA/CSF3R mutant AML cells treated with dimethyl sulfoxide (DMSO), ruxolitinib, GSK-LSD1, or the combination (Fig. 4and and and and and and knockout mice have neutropenia and accumulation of abnormal cells with an intermediate phenotype between monocytes and neutrophils (42). In addition, point mutations in in humans are associated with congenital neutropenia (43). Genetic or pharmacologic inhibition of LSD1 disrupts these repressive complexes, displacing GFI1 from chromatin, leading to enhancer activation (24). Consistent with this, we observed marked up-regulation of gene expression in response to LSD1 inhibitor treatment, consistent with displacement of GFI1 from its own promoter and loss of autoregulatory inhibition. Prior work has demonstrated that LSD1 inhibition reactivates genes with enhancers and promoters PF-AKT400 occupied by known differentiation-promoting transcription factors such as PU.1 and CEBPA (23). Consistent with this, our data for CSF3R/CEBPA mutant AML demonstrated marked reactivation of enhancers in response to LSD1. Additionally, we noted a cluster of genes that show the greatest increase in expression upon combination drug treatment. These genes were associated with a myeloid differentiation signature and showed the strongest enrichment of overlap with PU.1 peaks. Whether LSD1 or JAK/STAT inhibition changes the localization of PU.1 binding is an interesting area for further investigation. Our data also suggest that LSD1 plays an important role in gene activation. We identified a cluster of genes that demonstrated strong down-regulation in response to combined drug treatment. These genes showed enrichment for.