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CRISPR-suppressor scanning reveals a nonenzymatic role of LSD1 in AML


Understanding the mechanism of small molecules is a critical challenge in chemical biology and drug discovery. Medicinal chemistry is essential for elucidating drug mechanism, enabling variation of small molecule structure to gain structure–activity relationships (SARs). However, the development of complementary approaches that systematically vary target protein structure could provide equally informative SARs for investigating drug mechanism and protein function. Here we explore the ability of CRISPR–Cas9 mutagenesis to profile the interactions between lysine-specific histone demethylase 1 (LSD1) and chemical inhibitors in the context of acute myeloid leukemia (AML). Through this approach, termed CRISPR-suppressor scanning, we elucidate drug mechanism of action by showing that LSD1 enzyme activity is not required for AML survival and that LSD1 inhibitors instead function by disrupting interactions between LSD1 and the transcription factor GFI1B on chromatin. Our studies clarify how LSD1 inhibitors mechanistically operate in AML and demonstrate how CRISPR-suppressor scanning can uncover novel aspects of target biology.

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Data availability

ChIP–seq and RNA-seq data have been deposited to NCBI GEO (GSE121426). Transformed CRISPR-suppresor scanning reads (log2 + 1) used for Figs. 13 and Supplementary Figs. 13 are supplied in Supplementary Datasets 24.

Code availability

Code employed in Fig. 2 and Supplementary Fig. 2 is available upon reasonable request.

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We thank members of the Liau lab, B. E. Bernstein, M. D. Shair and J. Kim for helpful discussions. We thank Q. Yao for assistance on computational analysis. We thank C. Lee for assistance with figures. We thank S. Miller, K. Zhao, R. Boursiquot, H. Rees and C. Daly for their assistance in high-throughput DNA sequencing. We thank J. Nelson and Z. Niziolek for their assistance with FACS sorting. We thank R. Dahl and M. Simon for providing the M-CSFR-Fluc reporter and MigR1 PU.1 expression plasmid. A.P.S. was supported by the Herchel Smith Graduate Fellowship Program. A.M.F. was supported by award number T32GM007753 from the National Institute of General Medical Sciences. This research was supported by startup funds from Harvard University.

Author information

M.E.V., C.S., A.M.F., A.L.W. and A.P.S. designed, performed and analyzed cell and molecular biology experiments. M.E.V. and A.M.F. designed, performed and analyzed CRISPR–Cas9 screens. A.L.W., P.M.G. and B.D.S. designed, performed and analyzed protein purification and biochemical assays. A.L.W. and E.E.K. performed protein modeling. A.L.W. and Y.P. designed and synthesized molecules. A.P.S. performed computational analysis and edited the manuscript. J.G.D. provided technical advice and oversaw library preparation and sequencing of pooled CRISPR–Cas9 screens. D.E.B. and L.P. provided advice on computational analysis. B.B.L. designed the experimental strategy, performed and analyzed experiments, performed computational analysis, wrote the manuscript and held overall responsibility for the study.

Competing interests

The authors declare no competing interests.

Correspondence to Brian B. Liau.

Supplementary information

Supplementary Information

Supplementary Figures 1–8

Reporting Summary

Supplementary Note 1

Synthetic Procedures

Supplementary Note 2

2018 NCB LSD1 NMR and HPLC

Supplementary Dataset 1

sgRNA sequences used for LSD1 CRISPR scanning.

Supplementary Dataset 2

log2+1 transformed sgRNA read-count normalized reads for SET-2 treated with GSK-LSD1.

Supplementary Dataset 3

log2+1 transformed sgRNA read-count normalized reads for MV4;11 treated with GSK-LSD1.

Supplementary Dataset 4

log2+1 transformed sgRNA read-count normalized reads for SET-2 CRISPR-suppressor scanning screen.

Supplementary Dataset 5

PCR primers employed for genomic DNA amplification.

Supplementary Dataset 6

Cluster classification for differentially expressed genes in Fig. 6a.

Supplementary Dataset 7

Gene signatures used in Gene Set Enrichment Analysis.

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Fig. 1: CRISPR-suppressor scanning identifies regions of LSD1 that mediate its function and susceptibility to pharmacological inhibitors.
Fig. 2: Spatial clustering of CRISPR-suppressor scanning data reveals potential functional hotspots of LSD1 that mediate drug action.
Fig. 3: CRISPR-suppressor scanning enables profiling of LSD1 SARs.
Fig. 4: Identification of enzyme-inactivated LSD1 alleles that maintain AML proliferation and GFI1B binding in the presence of GSK-LSD1.
Fig. 5: An orthogonal drug-complementary GFI1B allele establishes sufficiency of the LSD1–GFI1B interaction for AML survival.
Fig. 6: Drug-resistant AML cells maintain LSD1–GFI1B binding on chromatin and fail to activate GFI1B-bound enhancers in the presence of GSK-LSD1.