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.
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Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ. 12, 1191–1197 (2005).
Clague, M.J., Coulson, J.M. & Urbé, S. Cellular functions of the DUBs. J. Cell Sci. 125, 277–286 (2012).
Pickart, C.M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. Curr. Opin. Chem. Biol. 8, 610–616 (2004).
Heideker, J. & Wertz, I.E. DUBs, the regulation of cell identity and disease. Biochem. J. 465, 1–26 (2015).
Clague, M.J. et al. Deubiquitylases from genes to organism. Physiol. Rev. 93, 1289–1315 (2013).
Abdul Rehman, S.A. et al. MINDY-1 is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes. Mol. Cell 63, 146–155 (2016).
Sowa, M.E., Bennett, E.J., Gygi, S.P. & Harper, J.W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).
Williams, S.A. et al. USP1 deubiquitinates ID proteins to preserve a mesenchymal stem cell program in osteosarcoma. Cell 146, 918–930 (2011).
Ndubaku, C. & Tsui, V. Inhibiting the deubiquitinating enzymes (DUBs). J. Med. Chem. 58, 1581–1595 (2015).
Atwal, R.S. et al. Kinase inhibitors modulate huntingtin cell localization and toxicity. Nat. Chem. Biol. 7, 453–460 (2011).
Li, Z. & Rana, T.M. A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation. Nat. Commun. 3, 1085 (2012).
Levis, M. FLT3 mutations in acute myeloid leukemia: what is the best approach in 2013? Hematology (Am. Soc. Hematol. Educ. Program) 2013, 220–226 (2013).
Weisberg, E. et al. FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist. Updat. 12, 81–89 (2009).
Stone, R.M. et al. Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation. N. Engl. J. Med. 377, 454–464 (2017).
Oshikawa, G., Nagao, T., Wu, N., Kurosu, T. & Miura, O. c-Cbl and Cbl-b ligases mediate 17-allylaminodemethoxygeldanamycin-induced degradation of autophosphorylated Flt3 kinase with internal tandem duplication through the ubiquitin proteasome pathway. J. Biol. Chem. 286, 30263–30273 (2011).
Sargin, B. et al. Flt3-dependent transformation by inactivating c-Cbl mutations in AML. Blood 110, 1004–1012 (2007).
Ritorto, M.S. et al. Screening of DUB activity and specificity by MALDI-TOF mass spectrometry. Nat. Commun. 5, 4763 (2014).
Reverdy, C. et al. Discovery of specific inhibitors of human USP7/HAUSP deubiquitinating enzyme. Chem. Biol. 19, 467–477 (2012).
Chauhan, D. et al. A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell 22, 345–358 (2012).
Altun, M. et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem. Biol. 18, 1401–1412 (2011).
Liu, J. et al. Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell 147, 223–234 (2011).
Yuan, J., Luo, K., Zhang, L., Cheville, J.C. & Lou, Z. USP10 regulates p53 localization and stability by deubiquitinating p53. Cell 140, 384–396 (2010).
Kessler, B.M. Selective and reversible inhibitors of ubiquitin-specific protease 7: a patent evaluation (WO2013030218). Expert Opin. Ther. Pat. 24, 597–602 (2014).
Weisberg, E. et al. Reversible resistance induced by FLT3 inhibition: a novel resistance mechanism in mutant FLT3-expressing cells. PLoS One 6, e25351 (2011).
Fan, Y.H. et al. USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis. Cell Death Dis. 4, e867 (2013).
De Kouchkovsky, I. & Abdul-Hay, M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J. 6, e441 (2016).
Martelli, M.P., Sportoletti, P., Tiacci, E., Martelli, M.F. & Falini, B. Mutational landscape of AML with normal cytogenetics: biological and clinical implications. Blood Rev. 27, 13–22 (2013).
Weisberg, E. et al. Discovery and characterization of novel mutant FLT3 kinase inhibitors. Mol. Cancer Ther. 9, 2468–2477 (2010).
Warkentin, A.A. et al. Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy. eLife 3, 03445 (2014).
Lavagna-Sévenier, C., Marchetto, S., Birnbaum, D. & Rosnet, O. FLT3 signaling in hematopoietic cells involves CBL, SHC and an unknown P115 as prominent tyrosine-phosphorylated substrates. Leukemia 12, 301–310 (1998).
Dexheimer, T.S. et al. Discovery of ML323 as a novel inhibitor of the USP1/UAF1 deubiquitinase complex. In Probe Reports from the NIH Molecular Libraries Program [Internet] (National Center for Biotechnology Information, 2010). Available at https://www.ncbi.nlm.nih.gov/books/NBK259186/.
Báez-Santos, Y.M., St John, S.E. & Mesecar, A.D. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds. Antiviral Res. 115, 21–38 (2015).
Kelly, L.M. et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood 99, 310–318 (2002).
Matsuo, Y. et al. Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23). Leukemia 11, 1469–1477 (1997).
Armstrong, S.A. et al. Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification. Cancer Cell 3, 173–183 (2003).
Weisberg, E. et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell 1, 433–443 (2002).
Chou, T.C. & Talalay, P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55 (1984).
Weisberg, E. et al. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 7, 129–141 (2005).
Montero, J. et al. Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 160, 977–989 (2015).
Pan, R. et al. Selective BCL-2 inhibition by ABT-199 causes on-target cell death in acute myeloid leukemia. Cancer Discov. 4, 362–375 (2014).
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.).
The authors declare no competing financial interests.
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Weisberg, E., Schauer, N., Yang, J. et al. Inhibition of USP10 induces degradation of oncogenic FLT3. Nat Chem Biol 13, 1207–1215 (2017). https://doi.org/10.1038/nchembio.2486
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