Immunomodulatory effect of NEDD8-activating enzyme inhibition in Multiple Myeloma: upregulation of NKG2D ligands and sensitization to Natural Killer cell recognition

Sara Petillo,Ricciarda Galandrini,Abdelilah Mekhloufi,Chiara Pighi,Fabrizio Antonangeli,Rossella Paolini,Alessandra Zingoni,Maria Teresa Petrucci,Alessandra Soriani,Angela Santoni,Cinzia Fionda,Cristina Capuano,Rosa Molfetta,Marco Cippitelli

doi:10.1038/s41419-021-04104-w

Abstract

Multiple Myeloma (MM) is an incurable hematologic malignancy of terminally differentiated plasma cells (PCs), where immune interactions play a key role in the control of cancer cell growth and survival. In particular, MM is characterized by a highly immunosuppressive bone marrow microenvironment where the anticancer/cytotoxic activity of Natural Killer (NK) cells is impaired. This study is focused on understanding whether modulation of neddylation can regulate NK cell-activating ligands expression and sensitize MM to NK cell killing. Neddylation is a post-translational modification that adds a ubiquitin-like protein, NEDD8, to selected substrate proteins, affecting their stability, conformation, subcellular localization, and function. We found that pharmacologic inhibition of neddylation using a small-molecule inhibitor, MLN4924/Pevonedistat, increases the expression of the NK cell-activating receptor NKG2D ligands MICA and MICB on the plasma membrane of different MM cell lines and patient-derived PCs, leading to enhanced NK cell degranulation. Mechanistically, MICA expression is upregulated at mRNA level, and this is the result of an increased promoter activity after the inhibition of IRF4 and IKZF3, two transcriptional repressors of this gene. Differently, MLN4924/Pevonedistat induced accumulation of MICB on the plasma membrane with no change of its mRNA levels, indicating a post-translational regulatory mechanism. Moreover, inhibition of neddylation can cooperate with immunomodulatory drugs (IMiDs) in upregulating MICA surface levels in MM cells due to increased expression of CRBN, the cellular target of these drugs. In summary, MLN4924/Pevonedistat sensitizes MM to NK cell recognition, adding novel information on the anticancer activity of neddylation inhibition.

Highlights

Neddylation is a post-translational modification that adds a ubiquitin-like protein, NEDD8, to selected substrate proteins, affecting their stability, conformation/function, and subcellular localization; this in turn can regulate many biological and pathological processes, including cancer progression and immune response [1,2,3,4]. This pathway involves the activity of NAE (NEDD8-activating enzyme, NAE1-UBA3), an enzyme required for the adenylation and activation of the ubiquitin-like molecule NEDD8 [5,6,7], which is transferred to a lysine residue of the target protein by the coordinate action of an E2 NEDD8-conjugating enzyme (UBE2M or UBE2F) [8, 9] and in the final step, NEDD8-E3 ligases catalyze the transfer of NEDD8 from E2 to the target protein [10]
Cullin Ring Ligases (CRLs) are multiprotein complexes composed by a Cullin subunit, to which NEDD8 is covalently attached, which act as a molecular scaffold able to bind to an adaptor protein and a substrate receptor at its N-terminus, and a RING protein at its C-terminus
NKG2DLs [SKO-007(J3), RPMI-8226, U266, ARP-1, JJN-3] [35, 36] experimental setting, and we found IRF4 to be significantly were treated for 72 h with MLN4924/Pevonedistat, a small-molecule inhibitor of NEDD8-activating enzyme (NAE) currently in phase I/II/III clinical trials for patients suffering from solid and haematological malignancies, including MM

Summary

INTRODUCTION

Neddylation is a post-translational modification that adds a ubiquitin-like protein, NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8), to selected substrate proteins, affecting their stability, conformation/function, and subcellular localization; this in turn can regulate many biological and pathological processes, including cancer progression and immune response [1,2,3,4]. We investigated whether MLN4924 could modulate a panel of human MM cell lines already shown to express the expression of these transcription factors (TFs) in our NKG2DLs [SKO-007(J3), RPMI-8226, U266, ARP-1, JJN-3] [35, 36] experimental setting, and we found IRF4 to be significantly were treated for 72 h with MLN4924/Pevonedistat (nanomolar downregulated at mRNA and protein level after neddylation range), a small-molecule inhibitor of NEDD8-activating enzyme (NAE) currently in phase I/II/III clinical trials for patients suffering from solid and haematological malignancies, including MM inhibition (Fig. 5B and C). Analyzing the cell lines showing the higher induction subunit RelA/p65 in SKO-007(J3) cells Overexpression of these ligands, we noticed a significant increase of MICA mRNA of a IkBα dominant negative (S [32A]/S [36A]) resulted in lower levels after 48 h treatment with MLN4924, with no relevant effect IRF4 mRNA levels (Fig. 6C) and upregulated promoter activity, on MICB mRNA levels (Fig. 2) indicating different regulation of mRNA levels and cell surface expression of MICA (Fig. 6D–F), these two ligands by neddylation.

DISCUSSION

MATERIALS AND METHODS

Findings

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