Abstract

Backgrounds: Precise gene regulation by cell-type-specific enhancers is fundamental to cell fate decision in hematopoietic differentiation. Super-enhancers (SEs) consist of multiple enhancer elements containing a number of transcription factor-binding motifs. Dysregulation of SEs has been recognized to be one of the major causes of leukemogenesis. Recent studies reported that SEs near to oncogenes such as MYC and EVI1 are often aberrantly activated in leukemia cells, while little is known about whether aberrant silencing of SEs near to onco-suppressor genes is involved in leukemia development.

Lysine specific demethylase 1 (LSD1) is a histone modifier that catalyze mono- and di-methylated lysine 4 of histone 3 (H3K4me1/2) directly and acetylated lysine 27 of histone 3 (H3K27ac) via recruiting complex with histone deacetylases 1 (HDAC1) and histone deacetylase 2 (HDAC2). Because both H3K4me1/2 and H3K27ac are activated markers of enhancers, LSD1 is supposed to act as an enhancer repressor. We recently demonstrated that a novel LSD1 inhibitor, NCD38, exerts anti-leukemia effects through inducing myeloid differentiation and activates approximately 500 SEs in leukemia cells. Growth factor independent 1 (GFI1) is an essential regulator for neutrophil development and is strongly upregulated by LSD1 inhibitors. Moreover, the GFI1-SE is included in the list of activated SEs by NCD38. Here we investigate the mechanism and role of the GFI1-SE dysregulation in leukemia cells.

Results: Previously we performed ChIP-seq of histone modifications (H3K4me2/3 and H3K27ac) in a human erythroleukemia cell line (HEL) with or without NCD38, and identified the GFI1-SE regulated by LSD1. To better know if status of the GFI1-SE could influence anti-leukemic effect of LSD1 inhibitors, we compared the H3K27ac level of the GFI1-SE to the GFI1 transcript level, the CD11b level, and cell viability, after treatment with LSD1 inhibitors (NCD38, NCD25 and ORY86) in 4 leukemia cell lines. Activation of the GFI1-SE, which is defined as significant elevation of H3K27ac level, followed by induction of the GFI1 transcript and CD11b was observed in 2 cell lines (HEL and CMK11-5) but not in the remaining 2 cell lines (K562 and UT7-EPO). In conjunction with this response, the anti-leukemic effect was exerted only in the former 2 cell lines. These data suggest that activation of the GFI1-SE seems necessary for anti-leukemic effect of LSD1 inhibitors.

The GFI1-SE has an evolutionarily conserved region which contains several DNA binding motifs of key transcription factors including GATA1 and RUNX1. Thus, to better understand an underlying mechanism of the GFI1-SE regulation, we performed luciferase reporter assay with several deletion mutants. The reporter activity was significantly elevated by LSD1 inhibitors in the vector cloned with the entire region of the GFI1-SE compared with the empty control vector. However, the elevation of the reporter activity was hardly observed with deletion of the conserved region and attenuated with mutation of both GATA1 and RUNX1 motifs. These results suggest that the conserved region is a core regulatory part of the GFI1-SE and is regulated by the coordination of LSD1 and several key transcriptional factors.

Finally, in order to investigate to what extent the GFI1-SE can directly affect leukemia programs by itself alone, we knocked out the GFI1-SE locus using the CRISPR/Cas9-mediated gene editing system in HEL cells. In all 3 GFI1-SE knockout (KO) clones we finally established, GFI1 induction by LSD1 inhibitors was significantly attenuated and consequently CD11b was hardly induced. Next, we compared gene expression profiling after treatment with NCD38 between GFI1-SE KO HEL cells and control HEL cells. Gene set enrichment analysis revealed that gene-sets associated with leukemogenesis and stemness are enriched in GFI1-SE KO cells. These data indicate that deletion of the GFI1-SE suppresses the attenuation of leukemia program by NCD38.

Conclusions: Our study disclosed that the newly identified GFI1-SE is essential for sufficient GFI1 induction and is regulated by the coordination of LSD1 and several key transcriptional factors. Aberrant repression or deletion of the GFI1-SE is closely associated with leukemia programs.

Disclosures

Takaori: Celgene: Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Janssen Pharma: Honoraria; Pfizer​: Honoraria.

Author notes

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Asterisk with author names denotes non-ASH members.