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

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable anti-tumor 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 trigger 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 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, 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 by knockdown of CRBN or IKZF-3, 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.