Person:

Du, Ting

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib-resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death. Genetic knockdown and biochemical blockade of SOD1 with LCS-1 sensitizes bortezomib-resistant MM cells to bortezomib. SOD1 inhibitor LCS-1 decreases viability in MM cell lines and patient cells. LCS-1-induced cell death is associated with: (i) increase in superoxide and ROS levels; (ii) activation of caspases, and p53/p21 signaling; (iii) decrease in MCL-1, BCL2, CDC2, cyclin B1 and c-Myc; (iv) ER stress response; and (v) inhibition of proteasome function. In animal model studies, LCS-1 inhibits xenografted bortezomib-resistant human MM cell growth and prolongs host survival. Our studies therefore show that targeting Rpn13 overcomes bortezomib-resistance by decreasing cellular SOD1 levels, and provide the rationale for novel therapeutics targeting SOD1 to improve patient outcome in MM.

Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin–proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.

Interaction of plasmacytoid dendritic cells (pDCs) with multiple myeloma (MM) cells, T- or NK-effector cells in the bone marrow (BM) microenvironment induces tumor cell growth, as well as inhibits innate and adaptive immune responses. Defining pDC-MM interaction-triggered immunosuppressive mechanism(s) will enable design of interventional therapies to augment anti-MM immunity. In the present study, we show that pDC-MM interactions induce metabolic enzyme Ecto-5’ Nucleotidase/CD73 in both pDCs and MM cells. Gene expression database from MM patients showed that CD73 levels inversely correlates with overall survival. Using our pDC-MM coculture models, we found that blockade of CD73 with anti-CD73 Abs: decreases adenosine levels; activates MM patient pDCs; and triggers cytotoxic T lymphocytes (CTL) activity against autologous patient MM cells. Combination of anti-CD73 Abs and an immune-stimulating agent TLR-7 agonist enhances autologous MM-specific CD8+ CTL activity. Taken together, our preclinical data suggest that the therapeutic targeting of CD73, alone or in combination with TLR-7 agonist, represents a promising novel strategy to restore host anti-MM immunity.