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A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes

Nature Medicine volume 22, pages 727–734 (2016)Cite this article

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Abstract

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

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Figure 1: A genome-wide RNAi screen identifies determinants of LEN sensitivity in MDS.
Figure 2: GPR68 is a critical determinant of LEN sensitivity in MDS.
Figure 3: LEN sensitivity is determined by GPR68-mediated intracellular calcium flux.
Figure 4: Calcium-dependent calpain activity is required for LEN sensitivity.
Figure 5: Loss of CAST expression and increased CAPN1 expression confer sensitivity of MDS cells to LEN.

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Acknowledgements

This work was supported by Cincinnati Children's Hospital Research Foundation (D.T.S.). We thank J. Bailey and V. Summey for assistance with transplantations. The normal bone marrow samples were received through the Normal Donor Repository in the Translational Core Laboratory at Cincinnati Children's Research Foundation, which is supported through the NIDDK-funded Center of Excellence in Molecular Hematology (grant no. P30DK090971; to Y. Zheng (CCHMC)). We thank B. Ebert (Harvard Medical School; Dana-Farber Cancer Institute) for providing the EGI and EGI-IKZF1 vectors, and for his expertise and discussions. In addition, we thank Y. Xu (Indiana University School of Medicine) for providing the MIG-GPR68 vector and K. Tohyama (Kawasaki Medical School) for the MDSL cells.

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Author notes

  1. Jing Fang

    Present address: Present address: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.,

Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, Ohio, USA

    Jing Fang, Xiaona Liu, Lyndsey Bolanos, Brenden Barker, H Leighton Grimes, Kakajan Komurov & Daniel T Starczynowski

  2. Bone Marrow Transplant Unit, Azienda Ospedaliera Bianchi Melacrino Morelli, Reggio Calabria, Italy

    Carmela Rigolino & Maria Cuzzola

  3. Department of Hematology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy

    Agostino Cortelezzi

  4. Hematology Unit, Azienda Ospedaliera Bianchi Melacrino Morelli, Reggio Calabria, Italy

    Esther N Oliva

  5. Celgene Corporation, Seville, Spain

    Celia Fontanillo

  6. Celgene Corporation, San Francisco, California, USA

    Kyle MacBeth

  7. Department of Cancer Biology, University of Cincinnati, Cincinnati, Ohio, USA

    Daniel T Starczynowski

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Contributions

J.F. and D.T.S. designed and interpreted data, and wrote the paper; D.T.S. conceived the study, obtained funding, and coordinated collaborations; J.F., X.L., L.B., B.B., and C.F. performed experiments and analyzed data; C.R., A.C., M.C., and E.N.O. provided and characterized patient samples; K.M. provided important reagents and conceptual input to the design of the study; H.L.G. provided conceptual input to the design of the study; and K.K. performed the bioinformatics analysis of the shRNA screen.

Corresponding author

Correspondence to Daniel T Starczynowski.

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Competing interests

C.F. and K.M. are employees of Celgene.

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Fang, J., Liu, X., Bolanos, L. et al. A calcium- and calpain-dependent pathway determines the response to lenalidomide in myelodysplastic syndromes. Nat Med 22, 727–734 (2016). https://doi.org/10.1038/nm.4127

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