Abstract

Oncogenic forms of the kinase FLT3 are important therapeutic targets in acute myeloid leukemia (AML); however, clinical responses to small-molecule kinase inhibitors are short-lived as a result of the rapid emergence of resistance due to point mutations or compensatory increases in FLT3 expression. We sought to develop a complementary pharmacological approach whereby proteasome-mediated FLT3 degradation could be promoted by inhibitors of the deubiquitinating enzymes (DUBs) responsible for cleaving ubiquitin from FLT3. Because the relevant DUBs for FLT3 are not known, we assembled a focused library of most reported small-molecule DUB inhibitors and carried out a cellular phenotypic screen to identify compounds that could induce the degradation of oncogenic FLT3. Subsequent target deconvolution efforts allowed us to identify USP10 as the critical DUB required to stabilize FLT3. Targeting of USP10 showed efficacy in preclinical models of mutant-FLT3 AML, including cell lines, primary patient specimens and mouse models of oncogenic-FLT3-driven leukemia.

  • Compound

    N-(3-(benzyl(methyl)amino)propyl)-9-chloro-5,6,7,8-tetrahydroacridine-2-carboxamide

  • Compound

    1-(5-((2,4-difluorophenyl)thio)-4-nitrothiophen-2-yl)ethan-1-one

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Acknowledgements

We thank D. Ye and S. Walker for their assistance with assessment of luciferase expression indicative of leukemia cell burden in the bone marrow of mice via the Bright-Glo luciferase assay system (Promega, Madison, Wisconsin, USA). Nomo-1, P31-FUJ and NB4 were obtained from G. Gilliland (Fred Hutchinson Cancer Research Center, Seattle, Washington, USA). MV4,11 cells were obtained from A. Letai (Dana-Farber Cancer Institute, Boston, Massachusetts, USA). The human FLT3-ITD-postive AML line MOLM14 was obtained from S. Armstrong (Dana-Farber Cancer Institute, Boston, Massachusetts, USA). PBMCs were generously provided by S. Treon and G. Yang (Dana-Farber Cancer Institute, Boston, Massachusetts, USA). Flag-HA-USP10 was a gift from the Wade Harper lab (Harvard Medical School, Boston, Massachusetts, USA). Work was funded by the Dana-Farber Cancer Institute Accelerator Fund (S.J.B. and E.L.W.), the Leukemia and Lymphoma Society (S.J.B. and E.L.W.), the Chleck Family Foundation (N.J.S.), the National Science Fellowship Graduate Research Fellowship Program (L.D.) and the and Claudia Adams Barr Award (S.J.B. and E.L.W.).

Author information

Author notes

    • Ellen L Weisberg
    • , Nathan J Schauer
    • , Jing Yang
    •  & Ilaria Lamberto

    These authors contributed equally to this work.

Affiliations

  1. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Ellen L Weisberg
    • , Shruti Bhatt
    • , Atsushi Nonami
    • , Chengcheng Meng
    • , Anthony Letai
    • , Renee Wright
    • , Alexandra Christodoulou
    • , Amanda Christie
    • , David M Weinstock
    • , Sophia Adamia
    • , Richard Stone
    • , Dharminder Chauhan
    • , Kenneth C Anderson
    • , Martin Sattler
    •  & James D Griffin
  2. Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Nathan J Schauer
    • , Jing Yang
    • , Ilaria Lamberto
    • , Laura Doherty
    • , Hyuk-Soo Seo
    • , Sirano Dhe-Paganon
    • , Nathanael S Gray
    •  & Sara J Buhrlage
  3. Experimental Therapeutic Core, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

    • Hong Tiv
    •  & Prafulla C Gokhale
  4. MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, Scotland, UK.

    • Maria Stella Ritorto
    • , Virginia De Cesare
    •  & Matthias Trost
  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Nathanael S Gray
    •  & Sara J Buhrlage

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Contributions

E.L.W., S.J.B., N.S.G. and J.D.G. initiated the project, and E.L.W. and S.J.B. oversaw all aspects of the project. E.L.W., N.J.S., J.Y. and I.L. performed biochemical, proliferation, signaling, knockdown, overexpression and immunoprecipitation studies. S.B. and A.L. designed and performed mitochondrial priming experiments. A. Christie, A. Christodoulou and D.M.W. designed and performed primagraft studies. H.T. and P.C.G. designed and performed in vivo bioluminescence studies. M.S.R., V.D.C. and M.T. designed and performed MALDI-TOF DUB assays. S.A. performed flow cytometry experiments. A.N. performed gene-knockdown experiments. S.D.-P. and H.-S.S. were responsible for the generation of USP10 enzyme used in biochemical assays. L.D., C.M. and R.W. performed immunoblotting experiments. R.S. provided AML patient samples. M.S., D.C. and K.C.A. offered valuable scientific feedback and helped with the conception of the research reported in the paper. E.L.W. and S.J.B. wrote the manuscript with input from all other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ellen L Weisberg or Sara J Buhrlage.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 2–7, Supplementary Figures 1–10, Supplementary Note 1.

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    Supplementary Table 1

    List of DUB inhibitors.

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DOI

https://doi.org/10.1038/nchembio.2486