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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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.
Supplementary Figures 1–8
2018 NCB LSD1 NMR and HPLC
sgRNA sequences used for LSD1 CRISPR scanning.
log2+1 transformed sgRNA read-count normalized reads for SET-2 treated with GSK-LSD1.
log2+1 transformed sgRNA read-count normalized reads for MV4;11 treated with GSK-LSD1.
log2+1 transformed sgRNA read-count normalized reads for SET-2 CRISPR-suppressor scanning screen.
PCR primers employed for genomic DNA amplification.
Cluster classification for differentially expressed genes in Fig. 6a.
Gene signatures used in Gene Set Enrichment Analysis.