Article | Published:

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

Nature Medicine volume 22, pages 727734 (2016) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    & The application and biology of immunomodulatory drugs (IMiDs) in cancer. Pharmacol. Ther. 136, 56–68 (2012).

  2. 2.

    et al. Myelodysplastic Syndrome-003 Study Investigators. Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N. Engl. J. Med. 355, 1456–1465 (2006).

  3. 3.

    Lenalidomide for del(5q) and non-del(5q) myelodysplastic syndromes. Semin. Hematol. 49, 312–322 (2012).

  4. 4.

    et al. Lenalidomide promotes p53 degradation by inhibiting MDM2 auto-ubiquitination in myelodysplastic syndrome with chromosome 5q deletion. Oncogene 32, 1110–1120 (2013).

  5. 5.

    , & Molecular action of lenalidomide in lymphocytes and hematologic malignancies. Adv. Hematol. 2012, 513702 (2012).

  6. 6.

    et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).

  7. 7.

    et al. Lenalidomide induces ubiquitination and degradation of CK1-α in del(5q) MDS. Nature 523, 183–188 (2015).

  8. 8.

    et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

  9. 9.

    et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014).

  10. 10.

    et al. Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis. Leukemia 24, 748–755 (2010).

  11. 11.

    et al. A novel factor-dependent human myelodysplastic cell line, MDS92, contains hemopoietic cells of several lineages. Br. J. Haematol. 91, 795–799 (1995).

  12. 12.

    et al. Lenalidomide inhibits the malignant clone and upregulates the SPARC gene mapping to the commonly deleted region in 5q– syndrome patients. Proc. Natl. Acad. Sci. USA 104, 11406–11411 (2007).

  13. 13.

    , , & NetWalker: a contextual network analysis tool for functional genomics. BMC Genomics 13, 282 (2012).

  14. 14.

    et al. p53 protein expression independently predicts outcome in patients with lower-risk myelodysplastic syndromes with del(5q). Haematologica 99, 1041–1049 (2014).

  15. 15.

    et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4CRBN. Br. J. Haematol. 164, 811–821 (2014).

  16. 16.

    & Ikaros, Aiolos, and Helios: transcription regulators and lymphoid malignancies. Immunol. Cell Biol. 81, 171–175 (2003).

  17. 17.

    et al. ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids Res. 41, D56–D63 (2013).

  18. 18.

    et al. Proton-sensing G-protein-coupled receptors. Nature 425, 93–98 (2003).

  19. 19.

    , , , & Extracellular acidification elicits spatially and temporally distinct Ca2+ signals. Curr. Biol. 18, 781–785 (2008).

  20. 20.

    et al. An MDS xenograft model utilizing a patient-derived cell line. Leukemia 28, 1142–1145 (2014).

  21. 21.

    et al. Regulated expression of pH-sensing G-protein-coupled receptor 68 identified through chemical biology defines a new drug target for ischemic heart disease. ACS Chem. Biol. 7, 1077–1083 (2012).

  22. 22.

    , , & Metabolic acidosis increases intracellular calcium in bone cells through activation of the proton receptor OGR1. J. Bone Miner. Res. 24, 305–313 (2009).

  23. 23.

    Calcium signaling. Cell 131, 1047–1058 (2007).

  24. 24.

    & The calpains: modular designs and functional diversity. Genome Biol. 8, 218 (2007).

  25. 25.

    , & Impact of genetic insights into calpain biology. J. Biochem. 150, 23–37 (2011).

  26. 26.

    et al. Topography, clinical, and genomic correlates of 5q myeloid malignancies revisited. J. Clin. Oncol. 30, 1343–1349 (2012).

  27. 27.

    et al. Gene expression profiles of CD34+ cells in myelodysplastic syndromes: involvement of interferon-stimulated genes and correlation to FAB subtype and karyotype. Blood 108, 337–345 (2006).

  28. 28.

    et al. Identification of cereblon-binding proteins, and relationship with response and survival after IMiDs in multiple myeloma. Blood 124, 536–545 (2014).

  29. 29.

    et al. Csnk1a1 inhibition has p53-dependent therapeutic efficacy in acute myeloid leukemia. J. Exp. Med. 211, 605–612 (2014).

  30. 30.

    et al. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J. Clin. Oncol. 29, 1971–1979 (2011).

  31. 31.

    , & Inside the tumor: p53 modulates calcium homeostasis. Cell Cycle 14, 933–934 (2015).

  32. 32.

    et al. p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner. Proc. Natl. Acad. Sci. USA 112, 1779–1784 (2015).

  33. 33.

    NFAT proteins: key regulators of T cell development and function. Nat. Rev. Immunol. 5, 472–484 (2005).

  34. 34.

    et al. An erythroid differentiation signature predicts response to lenalidomide in myelodysplastic syndrome. PLoS Med. 5, e35 (2008).

  35. 35.

    et al. Cytotoxic effects of bortezomib in myelodysplastic syndrome–acute myeloid leukemia depend on autophagy-mediated lysosomal degradation of TRAF6 and repression of PSMA1. Blood 120, 858–867 (2012).

  36. 36.

    et al. Ovarian cancer G-protein-coupled receptor 1, a new metastasis suppressor gene in prostate cancer. J. Natl. Cancer Inst. 99, 1313–1327 (2007).

Download references

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.

Author information

Author notes

    • Jing Fang

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

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

Authors

  1. Search for Jing Fang in:

  2. Search for Xiaona Liu in:

  3. Search for Lyndsey Bolanos in:

  4. Search for Brenden Barker in:

  5. Search for Carmela Rigolino in:

  6. Search for Agostino Cortelezzi in:

  7. Search for Esther N Oliva in:

  8. Search for Maria Cuzzola in:

  9. Search for H Leighton Grimes in:

  10. Search for Celia Fontanillo in:

  11. Search for Kakajan Komurov in:

  12. Search for Kyle MacBeth in:

  13. Search for Daniel T Starczynowski in:

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.

Competing interests

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

Corresponding author

Correspondence to Daniel T Starczynowski.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11 and Supplementary Tables 1–2

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nm.4127

Further reading