Abstract

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis1. Gain-of-function mutations in the 5′-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL2,3. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5′-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL.

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References

  1. 1.

    & Acute lymphoblastic leukemia in children. N. Engl. J. Med. 373, 1541–1552 (2015)

  2. 2.

    et al. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. Nat. Med. 19, 368–371 (2013)

  3. 3.

    et al. Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia. Nat. Genet. 45, 290–294 (2013)

  4. 4.

    et al. Expression of CD34 and CD7 on human T-cell acute lymphoblastic leukemia discriminates functionally heterogeneous cell populations. Leukemia 25, 1249–1258 (2011)

  5. 5.

    , , , & Expression of CD133 on leukemia-initiating cells in childhood ALL. Blood 113, 3287–3296 (2009)

  6. 6.

    et al. Stromal cells prevent apoptosis of AML cells by up-regulation of anti-apoptotic proteins. Leukemia 16, 1713–1724 (2002)

  7. 7.

    et al. T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments. Nature 538, 518–522 (2016)

  8. 8.

    et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 322, 1377–1380 (2008)

  9. 9.

    et al. Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblastic leukaemia. Nat. Commun. 6, 6604 (2015)

  10. 10.

    et al. Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia. Proc. Natl Acad. Sci. USA 113, 11306–11311 (2016)

  11. 11.

    et al. Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL. Nat. Med. 21, 563–571 (2015)

  12. 12.

    et al. CREBBP mutations in relapsed acute lymphoblastic leukaemia. Nature 471, 235–239 (2011)

  13. 13.

    et al. KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia. Leukemia 29, 1656–1667 (2015)

  14. 14.

    , & High Km soluble 5′-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP. J. Biol. Chem. 263, 18759–18765 (1988)

  15. 15.

    , , , & Cytosolic high Km 5′-nucleotidase and 5′(3′)-deoxyribonucleotidase in substrate cycles involved in nucleotide metabolism. J. Biol. Chem. 276, 6185–6190 (2001)

  16. 16.

    et al. Role of 5′-nucleotidase in thiopurine metabolism: enzyme kinetic profile and association with thio-GMP levels in patients with acute lymphoblastic leukemia during 6-mercaptopurine treatment. Clin. Chim. Acta 361, 95–103 (2005)

  17. 17.

    , & Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382–386 (1998)

  18. 18.

    et al. A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia. Nat. Med. 20, 1130–1137 (2014)

  19. 19.

    & Clonal evolution in leukemia. Nat. Med. 23, 1135–1145 (2017)

  20. 20.

    et al. Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse. J. Exp. Med. 208, 653–661 (2011)

  21. 21.

    et al. Rapid expansion of preexisting nonleukemic hematopoietic clones frequently follows induction therapy for de novo AML. Blood 127, 893–897 (2016)

  22. 22.

    et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 547, 104–108 (2017)

  23. 23.

    & Antibiotic resistance and its cost: is it possible to reverse resistance? Nat. Rev. Microbiol. 8, 260–271 (2010)

  24. 24.

    & Clonal evolution in cancer. Nature 481, 306–313 (2012)

  25. 25.

    et al. Tumor evolutionary directed graphs and the history of chronic lymphocytic leukemia. eLife 3, (2014)

  26. 26.

    et al. The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase. Biochemistry 42, 857–863 (2003)

  27. 27.

    , , , & Pyrimidine homeostasis is accomplished by directed overflow metabolism. Nature 500, 237–241 (2013)

  28. 28.

    et al. Targeting one carbon metabolism with an antimetabolite disrupts pyrimidine homeostasis and induces nucleotide overflow. Cell Reports 15, 2367–2376 (2016)

  29. 29.

    et al. CUTLL1, a novel human T-cell lymphoma cell line with t(7;9) rearrangement, aberrant NOTCH1 activation and high sensitivity to gamma-secretase inhibitors. Leukemia 20, 1279–1287 (2006)

  30. 30.

    . et al. Systematic in vivo structure-function analysis of p300 in hematopoiesis. Blood 114, 4804–4812 (2009)

  31. 31.

    et al. Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities. Endocrinology 148, 3987–3997 (2007)

  32. 32.

    et al. Metabolic reprogramming induces resistance to anti-NOTCH1 therapies in T cell acute lymphoblastic leukemia. Nat. Med. 21, 1182–1189 (2015)

  33. 33.

    et al. Truncating erythropoietin receptor rearrangements in acute lymphoblastic leukemia. Cancer Cell 29, 186–200 (2016)

  34. 34.

    et al. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl Acad. Sci. USA 109, 14508–14513 (2012)

  35. 35.

    et al. Detecting ultralow-frequency mutations by Duplex Sequencing. Nat. Protoc. 9, 2586–2606 (2014)

  36. 36.

    et al. A census of human cancer genes. Nat. Rev. Cancer 4, 177–183 (2004)

  37. 37.

    & The molecular basis of T cell acute lymphoblastic leukemia. J. Clin. Invest. 122, 3398–3406 (2012)

  38. 38.

    et al. Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children’s Oncology Group. Blood 118, 3080–3087 (2011)

  39. 39.

    & ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009)

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Acknowledgements

We are grateful to R. Kopan for the ΔE-NOTCH1 construct and T. Ludwig for the ROSA26Cre-ERT2/+ mouse. This work was supported by the Leukemia & Lymphoma Society Quest for Cures (R0749-14) and Translational Research (6455-15; 6531-18) awards (A.A.F.), an Innovative Research Award from the Alex Lemonade Stand Foundation (A.A.F.), the Chemotherapy Foundation (A.A.F.), National Institutes of Health (NIH) grants R35 CA210065 (A.A.F.), R01 CA206501 (A.A.F.), U54 CA193313 (R.R.), R01 CA185486 (R.R.), U54 CA209997 (R.R.), U10 CA98543 (J.M.G., M.L.L.), P30 CA013696, the Human Specimen Banking Grant U24 CA114766 (J.M.G.), the Stewart Foundation (R.R.) and the American Lebanese Syrian Associated Charities of St Jude Children’s Research Hospital. G.T. was supported by a HHMI International Student Research Fellowship. M.S.M. was supported by a Rally Foundation fellowship. C.L.D. was supported by NIH/NCI T32-CA09503. J.Y. was supported by the China Scholarship Council (CSC 201304910347) and the Ter Meulen Grant of the Royal Netherlands Academy of Arts and Sciences. E.W. was supported by the Dutch Cancer Society (KUN2012-5366).

Author information

Author notes

    • Gannie Tzoneva
    •  & Alberto Ambesi-Impiombato

    Present addresses: Regeneron Pharmaceuticals, Tarrytown, New York, New York 10591, USA (G.T.); PsychoGenics, Paramus, New Jersey 07652, USA (A.A.-I.).

    • Gannie Tzoneva
    •  & Chelsea L. Dieck

    These authors contributed equally to this work.

Affiliations

  1. Institute for Cancer Genetics, Columbia University, New York, New York 10032, USA

    • Gannie Tzoneva
    • , Chelsea L. Dieck
    • , Koichi Oshima
    • , Alberto Ambesi-Impiombato
    • , Marta Sánchez-Martín
    •  & Adolfo A. Ferrando
  2. Department of Systems Biology, Columbia University, New York, New York 10032, USA

    • Chioma J. Madubata
    • , Raul Rabadan
    •  & Adolfo A. Ferrando
  3. Rutgers Cancer Institute, Rutgers University, New Brunswick, New Jersey 08903, USA

    • Hossein Khiabanian
  4. Princess Maxima Center for Pediatric Oncology, Utrecht, 3584 CT, the Netherlands

    • Jiangyan Yu
    •  & Esme Waanders
  5. Department of Human Genetics, Radboud University Medical Center and Radboud Institute for Molecular Life Sciences, Nijmegen, 6525 GA, the Netherlands

    • Jiangyan Yu
  6. Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA

    • Ilaria Iacobucci
    •  & Charles G. Mullighan
  7. Department of Pediatrics, Columbia University Medical Center, New York, New York 10032, USA

    • Maria Luisa Sulis
    •  & Adolfo A. Ferrando
  8. Department of Hematology-Oncology, Saitama Children's Medical Center, Saitama 339-8551, Japan

    • Motohiro Kato
    •  & Katsuyoshi Koh
  9. Onco-Hematology Division, Department, Salute della Donna e del Bambino (SDB), University of Padua, 35128 Padua, Italy

    • Maddalena Paganin
    •  & Giuseppe Basso
  10. Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital, Columbus, Ohio 43205, USA

    • Julie M. Gastier-Foster
  11. Department of Pathology, Ohio State University School of Medicine, Columbus, Ohio 43210, USA

    • Julie M. Gastier-Foster
  12. Department of Pediatrics, Ohio State University School of Medicine, Columbus, Ohio 43210, USA

    • Julie M. Gastier-Foster
  13. Children’s Oncology Group, Arcadia, California 91006, USA

    • Julie M. Gastier-Foster
  14. Department of Pediatrics, University of California, San Francisco, California 94143, USA

    • Mignon L. Loh
  15. Helen Diller Family Comprehensive Cancer Center, San Francisco, California 94115, USA

    • Mignon L. Loh
  16. Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, Berlin, 10117, Germany

    • Renate Kirschner-Schwabe
  17. Department of Biomedical Informatics, Columbia University, New York, New York 10032, USA

    • Raul Rabadan
  18. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York 10032, USA

    • Adolfo A. Ferrando

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Contributions

G.T. and C.L.D. performed biochemical, cellular and animal studies. M.S.-M. and K.O. helped in experimental therapeutic experiments. A.A.-I. and H.K. analysed deep sequencing data. C.J.M. performed ISN analysis. M.L.S., M.K., K.K., M.P., G.B., J.M.G.-F. and M.L.L. provided clinical specimens. J.Y., E.W. and I.I. performed and analysed droplet PCR analyses. R.K.-S. provided clinical samples and correlative analyses of clinical data. C.G.M. supervised droplet PCR analyses; R.R. supervised deep sequencing and ISN analyses. A.A.F. designed the study, supervised the research and wrote the manuscript with G.T. and C.L.D.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Adolfo A. Ferrando.

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Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Metabolomic Analysis of Nt5c2+/R367Q and Nt5c2+/co-R367Q ALL lymphoblasts.

  2. 2.

    Supplementary Table 2

    Metabolomic Analysis of Nt5c2+/R367Q and Nt5c2+/co-R367Q ALL conditioned media.

  3. 3.

    Supplementary Table 3

    Metabolomic Analysis of NT5C2 WT and NT5C2 R367Q expressing CUTLL1 and REH Cells.

  4. 4.

    Supplementary Table 4

    Metabolomic Analysis of Conditioned Media from NT5C2 WT and NT5C2 R367Q expressing CUTLL1 and REH Cells.

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DOI

https://doi.org/10.1038/nature25186

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