Contributions
Abstract: S121
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 11:45 - 12:00
Location: Room K1
Background
Treatment of patients with MLL-translocated acute myeloid leukemia (AML) with a tranylcypromine-derivative inhibitor of Lysine Specific Demethylase 1 (LSD1/KDM1A) induces differentiation of blast cells in blood and bone marrow (Somervaille et al., Blood 2016). The assumption has been that LSD1 contributes to gene repression by removing monomethyl and dimethyl histone marks from lysine 4 of histone H3 (H3K4me1/me2) and that this is the key activity targeted for potential therapeutic effect. LSD1 also interacts with multiple transcription factors raising the possibility that other mechanisms may be significant.
Aims
We aimed to better characterize and understand LSD1 inhibitors mechanisms of action.
Methods
RNAseq, ATACseq and ChIPseq in human THP1 AML cell line treated with a tranylcypromine-derivative inhibitor (OG86, Oryzon Genomics).
Results
Contrary to what has been assumed, human THP1 AML cells treated with the LSD1 inhibitor OG86 induced rapid transcriptional changes without significant H3K4me1/2 changes in the upregulated LSD1-bound promoters (214/766, 28%) compared with those lacking an LSD1 binding peak (552/766, 72%). Regarding putative enhancer regions, only a modest H3K4me1 reduction in LSD1-bound versus LSD1-unbound enhancers was observed, but a highly significant increase in H3K9ac and H3K27ac, consistently with increase activation of LSD1-bound enhancers. Intriguingly, the transcriptional consequences of LSD1 inhibition mimicked the transcriptional changes of SNAG-domain transcription repressor GFI1 knockdown in THP1 AML cells, and we found that GFI1:LSD1/RCOR1 interaction was disrupted by OG86 treatment in THP1 and MV4,11 AML cells using several unrelated inhibitors.
Interestingly all the strongest GFI1 ChIPseq peaks (based on MACS2 pileup value) exhibited coincident LSD1 and RCOR1 binding (i.e. 98.6% and 88.4% respectively), and a significant GFI1 consensus-binding motif was found on LSD1 and RCOR1 ChIPseq bound regions surrounding the peak centers (MEME-ChIP, p=10-686 and p=10-55 respectively). We observed a loss of LSD1, RCOR1 and GFI1 ChIPseq signal in OG86-treated THP1 AML cells, with the greatest proportional reduction found at the 1,867 sites co-occupied by the three proteins together. The selective loss of LSD1/RCOR1 from GFI1 sites upon inhibition was further supported by the absence of any GFI1 consensus motif found on the residual LSD1 and RCOR1 binding peak sequences in OG86-treated cells. The loss of chromatin binding of the three proteins was also observed using subcellular fractionation analysis. Moreover, LSD1 inhibition in THP1 AML cells promoted differentiation (i.e. increased CD86 expression and reduced clonogenic potential), but cells expressing a doxycycline-regulated GFI1-ZNF DNA binding domain-LSD1 fusion protein completely blocked this differentiation effect, indicating that the myeloid differentiation induced by LSD1 inhibition results from the physical separation of LSD1 and GFI1. Finally, we did not observe any changes on chromatin accessibility (using ATACseq) surrounding sites co-occupied by GFI1, LSD1 and RCOR1 following LSD1 inhibition, but a significant increase of ChIPseq signal for H3K9ac and H3K27ac.
Conclusion
Our data illustrate a paradigm for epigenetic therapy whereby, through disruption of the protein:protein interaction between a transcription repressor and an epigenetic regulator, repression is released and dynamic enhancer acetylation and gene expression ensue.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): acetylation, Gfi-1, Methylation, MLL
Abstract: S121
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 11:45 - 12:00
Location: Room K1
Background
Treatment of patients with MLL-translocated acute myeloid leukemia (AML) with a tranylcypromine-derivative inhibitor of Lysine Specific Demethylase 1 (LSD1/KDM1A) induces differentiation of blast cells in blood and bone marrow (Somervaille et al., Blood 2016). The assumption has been that LSD1 contributes to gene repression by removing monomethyl and dimethyl histone marks from lysine 4 of histone H3 (H3K4me1/me2) and that this is the key activity targeted for potential therapeutic effect. LSD1 also interacts with multiple transcription factors raising the possibility that other mechanisms may be significant.
Aims
We aimed to better characterize and understand LSD1 inhibitors mechanisms of action.
Methods
RNAseq, ATACseq and ChIPseq in human THP1 AML cell line treated with a tranylcypromine-derivative inhibitor (OG86, Oryzon Genomics).
Results
Contrary to what has been assumed, human THP1 AML cells treated with the LSD1 inhibitor OG86 induced rapid transcriptional changes without significant H3K4me1/2 changes in the upregulated LSD1-bound promoters (214/766, 28%) compared with those lacking an LSD1 binding peak (552/766, 72%). Regarding putative enhancer regions, only a modest H3K4me1 reduction in LSD1-bound versus LSD1-unbound enhancers was observed, but a highly significant increase in H3K9ac and H3K27ac, consistently with increase activation of LSD1-bound enhancers. Intriguingly, the transcriptional consequences of LSD1 inhibition mimicked the transcriptional changes of SNAG-domain transcription repressor GFI1 knockdown in THP1 AML cells, and we found that GFI1:LSD1/RCOR1 interaction was disrupted by OG86 treatment in THP1 and MV4,11 AML cells using several unrelated inhibitors.
Interestingly all the strongest GFI1 ChIPseq peaks (based on MACS2 pileup value) exhibited coincident LSD1 and RCOR1 binding (i.e. 98.6% and 88.4% respectively), and a significant GFI1 consensus-binding motif was found on LSD1 and RCOR1 ChIPseq bound regions surrounding the peak centers (MEME-ChIP, p=10-686 and p=10-55 respectively). We observed a loss of LSD1, RCOR1 and GFI1 ChIPseq signal in OG86-treated THP1 AML cells, with the greatest proportional reduction found at the 1,867 sites co-occupied by the three proteins together. The selective loss of LSD1/RCOR1 from GFI1 sites upon inhibition was further supported by the absence of any GFI1 consensus motif found on the residual LSD1 and RCOR1 binding peak sequences in OG86-treated cells. The loss of chromatin binding of the three proteins was also observed using subcellular fractionation analysis. Moreover, LSD1 inhibition in THP1 AML cells promoted differentiation (i.e. increased CD86 expression and reduced clonogenic potential), but cells expressing a doxycycline-regulated GFI1-ZNF DNA binding domain-LSD1 fusion protein completely blocked this differentiation effect, indicating that the myeloid differentiation induced by LSD1 inhibition results from the physical separation of LSD1 and GFI1. Finally, we did not observe any changes on chromatin accessibility (using ATACseq) surrounding sites co-occupied by GFI1, LSD1 and RCOR1 following LSD1 inhibition, but a significant increase of ChIPseq signal for H3K9ac and H3K27ac.
Conclusion
Our data illustrate a paradigm for epigenetic therapy whereby, through disruption of the protein:protein interaction between a transcription repressor and an epigenetic regulator, repression is released and dynamic enhancer acetylation and gene expression ensue.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): acetylation, Gfi-1, Methylation, MLL