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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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.


  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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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.

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

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    Life Sciences Reporting Summary

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  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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