Downregulation of midnolin mediating non-ubiquitin protein degradation and its role in myelomagenesis.

Abstract

Abstract To identify novel tumor suppressor genes that play critical roles in controlling myeloma cell proliferation and survival, we performed a genome-wide CRISPRa screen in 3 MM cell lines. We identified a set of 121 dropout genes that ranked in the top 1% (0.25 FDR) and overlapped in at least 2 multiple myeloma cell lines. From this list by filtering genes expressed in normal plasma cells (NPC) while downregulated in MM, we identified MIDN as one of the top hits. Importantly, we observed uniformly lower expression of MIDN in 2 large cohorts of newly diagnosed MM patients (n=507 and n=319) compared to NPC (n=21 and n=16). MIDN expression was also significantly lower in the myeloma precursor condition suggesting its role earlier on in the disease development. MIDN encodes the protein Midnolin, which was recently described (Gu et al., Science 2023) to mediate ubiquitination-independent protein degradation, inducing the degradation of a number of nuclear proteins including stress-induced transcription factors. We generated both stable and inducible ORF systems for MIDN overexpression and confirmed that MIDN OE leads to the suppression of proliferation in myeloma. Midnolin stably associates with the proteasome and uses a structural domain that incorporates a free β strand to “catch” its substrates for ubiquitin-independent degradation. Mass spectrometry analysis confirmed that Midnolin binds to 19S and 20S proteasomal subunits, as well as to important myeloma nuclear factors, including IRF4. Due to its recently discovered role in degrading nuclear factors, we hypothesized that Midnolin suppresses tumor growth by degrading transcription factors important for MM pathogenesis. Indeed, proteomic and transcriptomic analyses in MIDN OE cells found MIDN overexpression correlating with the downregulation of pathways key to MM cell growth and viability, such as those dependent on nuclear factor IRF4, including plasma cell signature genes. Midnolin contains 3 domains that function in concert to promote proteasomal degradation of bound substrates. It uses a long α Helix to stably bind the proteasome, the Catch domain to interact with substrates, and the ubiquitin-like domain (Ubl) to promote substrate degradation. The Catch domain interacts with IRF4 through formation of an FG-zipper promoting IRF4 degradation and decreased MM cell growth. Inhibiting this interaction of IRF4 with the Catch domain leads to increased MM cell fitness. In conclusion, our study shows that universal downregulation ofMIDN, mediating ubiquitination-independent protein degradation, may be a crucial event for MM development and pathogenesis, by promoting upregulation of key oncoproteins such as IRF4. Moreover, our data provides an initial attempt to evaluate the functional landscape of putative suppressor genes in MM, which may expose new vulnerabilities and provide additional therapeutic opportunities.