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Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia

Oncogene volume 34, pages 2991–2999 (2015)Cite this article

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Abstract

To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3A572V mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3A572V, as well as the transforming potential of additional JAK3-activating mutations such as JAK3M511I. In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

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Acknowledgements

We thank Sarah Bowden for administrative support. AA is supported by a National Cancer Institute Career Development Award (5 K99 CA151670-02), Collins Foundation, Knight Pilot Project and Friends of Doernbecher grants. JWT is supported by grants from the V-Foundation, Gabrielle’s Angel Foundation and the National Cancer Institute (5R00CA151457-04; 1R01CA183947-01). BJD is a Howard Hughes Medical Institute investigator. This work is also supported by The Leukemia & Lymphoma Society. We thank Dr. Jamie Keck (Dr. Brian Druker’s Laboratory, OHSU) for providing her technical assistance in creating Gateway compatible pMXs-IRES-Puromycin vector.

Author Contributions

AA, RJM, KW-S, RMB and MAD designed the research, performed experiments and wrote the paper. SM-T and BP helped with microarray gene expression analysis. JWT, CET, CAE and BJD provided critical feedback and helped with manuscript preparation.

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Authors and Affiliations

  1. Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA

    A Agarwal, R J MacKenzie, C A Eide, K Watanabe-Smith & B J Druker

  2. Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA

    A Agarwal, R J MacKenzie, C A Eide, K Watanabe-Smith, C E Tognon, S Mongoue-Tchokote, B Park, J W Tyner & B J Druker

  3. Howard Hughes Medical Institute, Portland, OR, USA

    C A Eide, C E Tognon & B J Druker

  4. Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA

    M A Davare

  5. Biostatistics Shared Resource, Oregon Health & Science University, Portland, OR, USA

    S Mongoue-Tchokote & B Park

  6. Department of Pathology, Oregon Health & Science University, Portland, OR, USA

    R M Braziel

  7. Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, OR, USA

    J W Tyner

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Correspondence to A Agarwal.

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Competing interests

OHSU and BJD have a financial interest in Molecular MD (5% equity or less). This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and the Integrity Program Oversight Council. OHSU has clinical trial contracts with Novartis, and Bristol Myers Squibb to pay for patient costs, nurse and data manager salaries and institutional overhead. BJD does not derive salary, nor do their laboratories receive funds, from these contracts. BJD serves as a consultant for Roche and Nodality (Consulting income $10,000 or over).

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Agarwal, A., MacKenzie, R., Eide, C. et al. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia. Oncogene 34, 2991–2999 (2015). https://doi.org/10.1038/onc.2014.243

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