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Multiple myeloma gammopathies

Immunomodulatory drugs activate NK cells via both Zap-70 and cereblon-dependent pathways

Leukemia volume 35, pages 177–188 (2021)Cite this article

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Abstract

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study, we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly, increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD), independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated, and inhibited or enhanced by KD of CRBN or IKZF3, respectively, independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent, as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity.

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Fig. 1: IMiDs induce phosphorylation of Zap-70 in PBMCs and Jurkat cells.
Fig. 2: IMiDs bind and activate Zap-70.
Fig. 3: CRBN expression does not regulate phosphorylation or protein expression of Zap-70.
Fig. 4: Zap-70 mediates Pom-induced upregulation of NK-cell activity.
Fig. 5: Pom mediates granzyme-B expression via Zap-70.
Fig. 6: Pom upregulates granzyme-B expression via CRBN.
Fig. 7: IKZF3 plays a critical role in Pom-induced GZM-B expression.

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Acknowledgements

We thank Taiho Pharmaceuticals for providing Pom-immobilized beads. This study was supported by the National Institute of Health Grant; SPORE-P50CA100707 (KCA), R01-CA050947 (KCA) and R01-CA178264 (TH and KCA); and the Sheldon and Miriam Medical Research Foundation (KCA). KCA is an American Cancer Society Clinical Research Professor.

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Author notes

  1. These authors contributed equally: Teru Hideshima, Daisuke Ogiya

Authors and Affiliations

  1. Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02215, USA

    Teru Hideshima, Daisuke Ogiya, Jiye Liu, Keiji Kurata, Jooeun Bae & Kenneth C. Anderson

  2. Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, 3-18-15 Kuramoto, Tokushima, 770-8503, Japan

    Takeshi Harada

  3. Department of Chemistry Instrumentation Facility, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Walter Massefski

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Contributions

TH coordinated activities from all authors and performed Western blot, Zap-70 kinase assay, siRNA (Zap-70, CRBN, IKZF1, IKZF3) transfection, NK assay, as well as cell toxicity assay (MTT and cell count), and wrote the paper. DO performed real-time qPCR (GZM-B), NK assay, and purified T cells and NK cells from healthy volunteers. JL performed siRNA (Zap-70, CRBN, IKZF1, and IKZF3) transfection, TH performed Zap-70 pull-down by Pom-beads. KK performed real-time qPCR (GZM-B). JB analyzed the data. WM performed NMR and wrote the paper. KCA managed the project and wrote the paper.

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Correspondence to Kenneth C. Anderson.

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Conflict of interest

KCA serves on advisory boards to Celgene, Millennium, Janssen, Sanofi, Bristol Myers Squibb, Gilead, Precision Biosciences, and Tolero, and is a Scientific Founder of OncoPep and C4 Therapeutics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the paper apart from those disclosed.

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Hideshima, T., Ogiya, D., Liu, J. et al. Immunomodulatory drugs activate NK cells via both Zap-70 and cereblon-dependent pathways. Leukemia 35, 177–188 (2021). https://doi.org/10.1038/s41375-020-0809-x

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