Background and Rationale 20S proteasome-based therapies are the mainstay of treatment of patients with multiple myeloma (MM); however, resistance to proteasome inhibitor (PI) therapies commonly develops underlying relapse of disease. We showed that inhibition of 19S-associated ubiquitin receptor (UbR) ADRM1/Rpn13, upstream of 20S proteasome, inhibits MM cell growth and overcomes PI-resistance in MM. hRpn13 recognizes polyubiquitinated proteins and facilitates their disassembly via 19S-associated deubiquitinating enzyme UCHL5, allowing for protein degradation via 20S proteasomal catalytic activities. To date, however, hRpn13-modulated protein substrates and downstream signaling remains unclear. Here, we utilized multiplexed proteomics with tandem mass spectrometry; GeneOntology (GO) enrichment; as well as pathway database KEGG and Reactome to identify hRpn13-associated signaling molecules and delineate functionally significant biological pathways.

Materials and Methods CRISPR-Cas9 genome editing was performed to generate hRpn13-knockout (hRpn13-KO) HCT116 cells which were subjected to multiplexed proteomics. Functionally relevant proteins were categorized using GO, as well as KEGG- and Reactome-pathway analysis. The prognostic relevance of identified proteins was derived using Gene Expression Profiling (GEP) databases on uniformly treated MM patients. Protein expression level was determined by western blotting. CRISPR-Cas9-mediated hRpn13 deletion was confirmed using PCR and sequencing, as well as immunoblot analysis. Statistical significance was assessed with Student's t test

Results Among 8766 proteins analyzed, 206 proteins were down-regulated, whereas 65 proteins were significantly up-regulated in hRpn13-KO cells compared to hRpn13-WT cells. Targeted hRpn13-deletion upregulated physiological pathways related to amino acid biosynthesis and transport, metabolism, proliferation, and apoptosis. We validated 5 such upregulated proteins (SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A) using immunoblotting. As for hRpn13 deletion, treatment of MM.1S cells with Rpn13 inhibitor RA190 triggered a similar increase in SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A levels. These data indicate that SLC1A3, THBS1, GLYR1, GCLM, and TIMM8A proteins are direct substrates of UbR hRpn13. On the other hand, hRpn13-deletion decreased levels of proteins involved in cell adhesion, biological regulation, antigen recognition, and extracellular matrix interactions. Clinical relevance in MM: We next screened 271 proteins against GEP datasets on MM patients (GSE6477; GSE13591; GSE6691). Among the 65 molecules upregulated in hRpn13-KO cells, 18 were expressed at significantly lower levels in MM patients vs normal plasma cells; and importantly, 2 (SLC1A3, THBS1) of these 18 proteins correlated with poor survival. In a functional validation study, blockade of hRpn13 with RA190 induced increased SLC1A3 and THBS1 levels, suggesting that targeting SLC1A3/THBS1 may restore abnormal amino acid metabolism in MM. We next screened 206 of 271 proteins that were downregulated in Rpn13-KO cells in GEP database of MM patients. Results show that 32 of these 206 molecules were highly expressed in MM patient samples vsnormal plasma cells, and that 2 (SLC16A7 and DBF4) of 32 correlated with poor survival. SLC16A7 encodes for monocarboxylate transporter 2 (MCT2), a key component of glycolysis, and DBF4 regulates DNA replication/cell proliferation. Importantly, RA190 decreased SLC16A and DB4 levels. Together, these data suggest that: 1) SLC16A, DBF4, SLC1A3, and THBS1 are downstream signaling targets of hRpn13; and 2) hRpn13-inhibition triggered MM cell death involves blockade of elevated glycolysis and DNA replication/cell growth via SLC16A and DBF4, respectively, as well as restoration of normal amino acid synthesis via SLC1A3.

Conclusion Collectively, our study utilized CRISPR gene-editing, biochemical, and molecular strategies to identify UbR hRpn13-mediated proteomic alterations. We identified novel targets including SLC16A7, DBF4, SLC1A3, and Thrombospondin-1 which may serve as prognostic biomarkers in MM. Importantly, our findings further support hRpn13-directed therapeutics, as well as preclinical evaluation of novel strategies targeting SLC16A, DBF4, SLC1A3, and Thrombospondin-1, to enhance cytotoxicity and improve patient outcome in MM.

Disclosures

Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

Author notes

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Asterisk with author names denotes non-ASH members.