(OA-28) Midnolin Downregulation Promotes Myelomagenesis via Impacting Ubiquitination Independent Pathway of Protein Degradation

Despite advancements in treatment, multiple myeloma (MM) remains incurable, which underscores the need for novel therapeutic targets. Here, we performed a genome-wide CRISPRa screen in 3 MM cell lines to uncover novel tumor suppressor genes that play critical roles in controlling myeloma cell proliferation and survival. 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.

Methods: n/a

Results:

Among the top hits was MIDN, encoding the protein Midnolin, which mediates ubiquitination-independent protein degradation inducing the degradation of many nuclear proteins including stress-induced transcription factors (Fos, EGR1, NR4A1, etc). We confirmed MIDN overexpression (OE) leads to the suppression of proliferation in myeloma, using both stable and inducible ORF systems. Importantly, we observed uniformly lower expression of MIDN in 2 large cohorts of newly diagnosed MM patients (n==507 and n= 319) compared to normal plasma cells (n=21 and n=16). MIDN expression was also significantly lower in premalignant conditions, suggesting an early downregulation in disease progression. Furthermore, MIDN expression was significantly lower in 6 human MM cell lines compared to B cells from 3 healthy donors.

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. 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, proteome and transcriptomic analyses in MIDN OE cells found MIDN overexpression correlated 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. Further mass spectrometry analysis confirmed that Midnolin binds to 19S and 20S proteasomal subunits, as well as to important myeloma nuclear factors including IRF4.

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. It’s been recently shown to inhibit IRF4’s interaction with MIDN at the Catch domain rescuing it from protein degradation and increasing MM cell viability.

Conclusions:

In conclusion, our study shows that downregulation of MIDN, mediating ubiquitination-independent protein degradation, may be a crucial event for MM development and pathogenesis, as its observed universal downregulation increases the key MM cell growth and survival promoting oncoprotein 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.