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

Abstract

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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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Shi, J. et al. Discovery of cancer drug targets by CRISPR–Cas9 screening of protein domains. Nat. Biotechnol. 33, 661–667 (2015).

  2. 2.

    Shen, C. et al. NSD3-Short is an adaptor protein that couples BRD4 to the CHD8 chromatin remodeler. Mol. Cell 60, 847–859 (2015).

  3. 3.

    Neggers, J. E. et al. Target identification of small molecules using large-scale CRISPR–Cas mutagenesis scanning of essential genes. Nat. Commun. 9, 502–515 (2018).

  4. 4.

    Ipsaro, J. J. et al. Rapid generation of drug-resistance alleles at endogenous loci using CRISPR–Cas9 indel mutagenesis. PLoS One 12, e0172177 (2017).

  5. 5.

    Donovan, K. F. et al. Creation of novel protein variants with CRISPR/Cas9-mediated mutagenesis: turning a screening by-product into a discovery tool. PLoS One 12, e0170445 (2017).

  6. 6.

    Hakimi, M.-A. et al. A core–BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Proc. Natl Acad. Sci. USA 99, 7420–7425 (2002).

  7. 7.

    Shi, Y. et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941–953 (2004).

  8. 8.

    Tong, J. K., Hassig, C. A., Schnitzler, G. R., Kingston, R. E. & Schreiber, S. L. Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex. Nature 395, 917–921 (1998).

  9. 9.

    Forneris, F., Battaglioli, E., Mattevi, A. & Binda, C. New roles of flavoproteins in molecular cell biology: histone demethylase LSD1 and chromatin. FEBS J. 276, 4304–4312 (2009).

  10. 10.

    Maiques-Diaz, A. & Somervaille, T. C. LSD1: biologic roles and therapeutic targeting. Epigenomics 8, 1103–1116 (2016).

  11. 11.

    Cusan, M. et al. LSD1 inhibition exerts its antileukemic effect by recommissioning PU.1- and C/EBPα-dependent enhancers in AML. Blood 131, 1730–1742 (2018).

  12. 12.

    Harris, WilliamJ. et al. The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells. Cancer Cell 21, 473–487 (2012).

  13. 13.

    Ishikawa, Y. et al. A novel LSD1 inhibitor T-3775440 disrupts GFI1B-containing complex leading to transdifferentiation and impaired growth of AML cells. Mol. Cancer Ther. 16, 273–284 (2017).

  14. 14.

    Maes, T. et al. ORY-1001, a potent and selective covalent KDM1A inhibitor, for the treatment of acute leukemia. Cancer Cell 33, 495–511.e412 (2018).

  15. 15.

    Maiques-Diaz, A. et al. Enhancer activation by pharmacologic displacement of LSD1 from GFI1 induces differentiation in acute myeloid leukemia. Cell Rep. 22, 3641–3659 (2018).

  16. 16.

    McGrath, J. P. et al. Pharmacological inhibition of the histone lysine demethylase KDM1A suppresses the growth of multiple acute myeloid leukemia subtypes. Cancer Res. 76, 1975–1988 (2016).

  17. 17.

    Mohammad, H. P. et al. A DNA hypomethylation signature predicts antitumor activity of LSD1 inhibitors in SCLC. Cancer Cell 28, 57–69 (2015).

  18. 18.

    Schenk, T. et al. Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia. Nat. Med. 18, 605–611 (2012).

  19. 19.

    Takagi, S. et al. LSD1 inhibitor T-3775440 inhibits SCLC cell proliferation by disrupting LSD1 interactions with SNAG domain proteins INSM1 and GFI1B. Cancer Res. 77, 4652–4662 (2017).

  20. 20.

    Yamamoto, R. et al. Selective dissociation between LSD1 and GFI1B by a LSD1 inhibitor NCD38 induces the activation of ERG super-enhancer in erythroleukemia cells. Oncotarget 9, 21007–21021 (2018).

  21. 21.

    Baron, R., Binda, C., Tortorici, M., McCammon, J. A. & Mattevi, A. Molecular mimicry and ligand recognition in binding and catalysis by the histone demethylase LSD1-CoREST complex. Structure 19, 212–220 (2011).

  22. 22.

    Lin, Y. et al. The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. EMBO J. 29, 1803–1816 (2010).

  23. 23.

    Saleque, S., Kim, J., Rooke, H. M. & Orkin, S. H. Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b Is mediated by the cofactors CoREST and LSD1. Mol. Cell 27, 562–572 (2007).

  24. 24.

    Lee, M. G., Wynder, C., Schmidt, D. M., McCafferty, D. G. & Shiekhattar, R. Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem. Biol. 13, 563–567 (2006).

  25. 25.

    Kamburov, A. et al. Comprehensive assessment of cancer missense mutation clustering in protein structures. Proc. Natl Acad. Sci. USA 112, E5486–E5495 (2015).

  26. 26.

    Yang, M. et al. Structural basis of histone demethylation by LSD1 revealed by suicide inactivation. Nature Struct. Mol. Biol. 14, 535–539 (2007).

  27. 27.

    Dhanak, D. Drugging the cancer epigenome. In Proc. 104th Annual Meeting of the American Association for Cancer Research, AACR, Washington DC 6–10 (2013).

  28. 28.

    Du-Cuny, L., Xiao, Q., Xun, G., Zheng, Q. & He, F. Cyano-substituted indole compounds and uses thereof as lsd1 inhibitors. Patent WO/2017/149463 (2017).

  29. 29.

    Pinello, L. et al. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat.Biotechnol. 34, 695–697 (2016).

  30. 30.

    Niwa, H., Sato, S., Hashimoto, T., Matsuno, K. & Umehara, T. Crystal structure of LSD1 in complex with 4-[5-(piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile. Molecules 23, E1538 (2018).

  31. 31.

    Karasulu, B., Patil, M. & Thiel, W. Amine oxidation mediated by lysine-specific demethylase 1: quantum mechanics/molecular mechanics insights into mechanism and role of lysine 661. J. Am. Chem. Soc. 135, 13400–13413 (2013).

  32. 32.

    Forneris, F., Orru, R., Bonivento, D., Chiarelli, L. R. & Mattevi, A. ThermoFAD, a Thermofluor®-adapted flavin ad hoc detection system for protein folding and ligand binding. FEBS J. 276, 2833–2840 (2009).

  33. 33.

    Tortorici, M. et al. Protein recognition by short peptide reversible inhibitors of the chromatin-modifying LSD1/CoREST lysine demethylase. ACS Chem. Biol. 8, 1677–1682 (2013).

  34. 34.

    Clackson, T. et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl Acad. Sci. USA 95, 10437–10442 (1998).

  35. 35.

    Moroy, T., Vassen, L., Wilkes, B. & Khandanpour, C. From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation. Blood 126, 2561–2569 (2015).

  36. 36.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

  37. 37.

    Pilotto, S. et al. Interplay among nucleosomal DNA, histone tails, and corepressor CoREST underlies LSD1-mediated H3 demethylation. Proc. Natl Acad. Sci. USA 112, 2752–2757 (2015).

  38. 38.

    Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

  39. 39.

    Dahl, R., Iyer, S. R., Owens, K. S., Cuylear, D. D. & Simon, M. C. The transcriptional repressor GFI-1 antagonizes PU.1 activity through protein–protein interaction. J. Biol. Chem. 282, 6473–6483 (2007).

  40. 40.

    Cai, S. F., Chen, C. W. & Armstrong, S. A. Drugging chromatin in cancer: recent advances and novel approaches. Mol. Cell 60, 561–570 (2015).

  41. 41.

    Dawson, M. A. The cancer epigenome: concepts, challenges, and therapeutic opportunities. Science 355, 1147–1152 (2017).

  42. 42.

    Dawson, M. A. & Kouzarides, T. Cancer epigenetics: from mechanism to therapy. Cell 150, 12–27 (2012).

  43. 43.

    Helin, K. & Dhanak, D. Chromatin proteins and modifications as drug targets. Nature 502, 480–488 (2013).

  44. 44.

    Kasap, C., Elemento, O. & Kapoor, T. M. DrugTargetSeqR: a genomics- and CRISPR–Cas9-based method to analyze drug targets. Nat. Chem. Biol. 10, 626–628 (2014).

  45. 45.

    Schenone, M., Dančík, V., Wagner, B. K. & Clemons, P. A. Target identification and mechanism of action in chemical biology and drug discovery. Nat. Chem. Biol. 9, 232–240 (2013).

  46. 46.

    Joung, J. et al. Genome-scale CRISPR–Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).

  47. 47.

    Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

  48. 48.

    Schulz-Fincke, J. et al. Structure-activity studies on N-substituted tranylcypromine derivatives lead to selective inhibitors of lysine specific demethylase 1 (LSD1) and potent inducers of leukemic cell differentiation. Eur. J. Med. Chem. 144, 52–67 (2018).

  49. 49.

    Burg, J. M., Gonzalez, J. J., Maksimchuk, K. R. & McCafferty, D. G. Lysine-specific demethylase 1A (KDM1A/LSD1): product recognition and kinetic analysis of full-length histones. Biochemistry 55, 1652–1662 (2016).

  50. 50.

    Forneris, F., Binda, C., Adamo, A., Battaglioli, E. & Mattevi, A. Structural basis of LSD1–CoREST selectivity in histone H3 recognition. J. Biol. Chem. 282, 20070–20074 (2007).

  51. 51.

    Conway, P., Tyka, M. D., DiMaio, F., Konerding, D. E. & Baker, D. Relaxation of backbone bond geometry improves protein energy landscape modeling. Protein Sci. 23, 47–55 (2014).

  52. 52.

    Dral, P. O., Wu, X., Spörkel, L., Koslowski, A. & Thiel, W. Semiempirical quantum-chemical orthogonalization-corrected methods: benchmarks for ground-state properties. J. Chem. Theory Comput. 12, 1097–1120 (2016).

  53. 53.

    Liau, B. B. et al. Adaptive chromatin remodeling drives glioblastoma stem cell plasticity and drug tolerance. Cell Stem Cell 20, 233–246 (2017).

  54. 54.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550–571 (2014).

  55. 55.

    Shen, L., Shao, N., Liu, X. & Nestler, E. ngs.plot: quick mining and visualization of next-generation sequencing data by integrating genomic databases. BMC Genomics 15, 284–298 (2014).

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Acknowledgements

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.