Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Inhibition of neddylation enhances Multiple Myeloma recognition and killing mediated by Natural Killer cells.

Multiple Myeloma (MM) is a cancer of terminally differentiated plasma cells that arises in the bone marrow. The importance of Natural Killer (NK) cells in the control of MM has been well described as well as the impairment of NK cell effector functions during disease progression. MLN4924 is a small molecule inhibitor of neddylation, currently involved in many clinical trials, also for the treatment of MM. Neddylation is a post-translational modification that adds a ubiquitin-like molecule, NEDD8, to the substrate protein, affecting protein stability, conformation, and subcellular localization. The best characterized neddylation substrates are the Cullin Ring Ligases (CRLs), the largest family of E3 ubiquitin ligases in eukaryotes, that require the binding of NEDD8 for their activation. Neddylation inhibition thus results in the stabilization and accumulation of CRL substrates and this triggers multiple cellular responses that account for the anti-cancer activity of MLN4924. We aimed to investigate the immunomodulatory properties of MLN4924 in the context of MM and to dissect how neddylation is involved in MM cell recognition by NK cells. We observed that treatment of MM cells with MLN4924 increased NK cell recognition and killing through the upregulation of the stress-induced ligand MICA and MICB on the plasma membrane, which are recognized by the NK cell activating receptor NKG2D. After MLN4924 treatment, we observed a higher MICA promoter activity, result of the downregulation of the transcriptional repressors IRF4 and IKZF3, transcription factors (TFs) essential for MM growth and survival. IRF4 downmodulation is a consequence of impaired NF-κB activation after neddylation inhibition because the NF-κB inhibitor and a CRL1βTRCP substrate IκBα is no longer degraded. With a different mechanism, we observed a redistribution of MICB on the plasma membrane after MLN4924 treatment, without any change in the total protein amount. The inhibition of CRL3-SPOPL complex, regulator of the endolysosomal pathway, could explain the accumulation observed for MICB at the cell surface, since MICB expression is regulated by an active recycling through clathrin-dependent endocytosis. Moreover, MLN4924 by blocking the degradation of CRBN, substrate of CRL1/SCF-Fbxo7 complex, enhanced sensitivity of these cells to IMiDs and further upregulated MICA expression in combination to Lenalidomide. In this context, MICB is not modulated by Lenalidomide in MM cells. We also observed that MLN4924, alone and even more in combination with the monoclonal antibodies Daratumumab or Elotuzumab, currently used in therapy, directly enhanced NK cell degranulation and killing of MM cells. This was a result of the stabilization of the CRL substrate Rac1 and RhoA, Rho GTPases involved in cytoskeletal rearrangements and cytotoxic granule release after target recognition, as confirmed by the increased levels of F-actin in MLN4924-treated NK cells contacting target cells. Furthermore, neddylation inhibition partially counteracted the TGFβ-mediated impairment of NK cell effector functions by preventing the activation of the CRL4-AMBRA1 complex that mediate non-proteolytic polyubiquitylation of Smad4 to enhance its transcriptional activity. Our results suggest that neddylation inhibition could be a strategy to enhance NK cell-mediated immunotherapy in Multiple Myeloma.