Blockade of Ubiquitin Receptor Rpn13 in Plasmacytoid Dendritic Cells Enhances Anti-Myeloma Immunity

Introduction Although proteasome inhibitor (PI) based combination therapies achieve remarkable responses multiple myeloma (MM), emergence of PI resistance is common. The mechanism(s) of PI-resistance include tumor-intrinsic factors such as mutations of the 20S proteasomal subunits, and/or tumor-extrinsic cellular components in the BM microenvironment. Interactions of BM accessory cells, immune effector cells, and tumor cells confer both drug-resistance and immune suppression in MM. For example, we showed that interactions of MM plasmacytoid dendritic cells (pDCs) with MM cells and with T/NK cells both confer immune suppression via immune checkpoints, as well as trigger MM cell growth by inducing secretion of MM cell growth factors. We recently reported that targeting proteasome-associated ubiquitin receptor Rpn13 triggers cytotoxicity and overcomes tumor-intrinsic PI-resistance in MM (Song et al, Leukemia 2016;30:1877). Here we utilized our co-culture models of patient pDCs, T cells, NK cells, and autologous MM cells to characterize the immune sequelae of Rpn13 inhibition.

Methods Analysis of pDCs activation Purified patient-pDCs (n =7) were treated with Rpn13 inhibitor RA190 (0.05 µM) for 24h, followed by multicolor staining using fluorophore-conjugated Abs against pDC activation/maturation markers CD80, CD83, and CD86. Transient transfections Purified MM patient pDCs were transfected with Rpn13-siRNA using TransIT-X2 transfection Kit,and analyzed for alterations in maturation markers. CTL/NK activity assays Purified MM-BM CD8⁺ T- or NK-cells (n = 8) were co-cultured with autologous BM-pDCs (pDC:T/NK; 1:10 ratio) for 3 days, in the presence or absence of Rpn13 inhibitor RA190 (100 nM). After washing, cells were cultured for 24h with autologous MM cells pre-stained with CellTracker/CellTrace Violet (10 T/NK:1 MM), followed by 7-AAD staining and quantification of CTL-or NK cell-mediated MM cell lysis by FACS.

Results 1) RA190 triggers significant upregulation of maturation markers CD80, CD83, and CD86 on MM-pDCs (fold change vs untreated: CD80: 1.2; p = 0.007; CD83: 2.15; p = 0.006; CD86: 1.4; p = 0.003). In contrast, bortezomib-treated pDCs showed no significant upregulation of these markers. 2) Similar to pharmacological inhibition of Rpn13 with RA190, Rpn13-siRNA increased CD80 (1.76-fold), CD83 (3.12-fold), and CD86 (2.28-fold) expression on MM pDCs (p<0.01). Of note, both RA190 and bortezomib block protein degradation via proteasome, but only RA190 activates pDCs. 3) RA190 treatment increases pDC-induced MM-specific CD8⁺ CTL activity, as well as NK cell-mediated cytolytic activity against autologous tumor cells, evidenced by decreased viable patient MM cells. 4) Treatment of MM-pDCs with RA190 increases expression of calnexin, a molecular chaperone protein of endoplasmic reticulum which regulates immune co-stimulatory molecules, immune-regulatory signaling, and restores the ability of pDCs to induce proliferation of MM-specific CTLs or NK cells. These findings were also confirmed using pDC cell line CAL-1.

Conclusions Our prior findings showed that inhibition of UbR Rpn13 overcomes intrinsic PI-resistance in MM cells. Here we show that targeting Rpn13 also triggers anti-MM immune responses. Rpn13 blockade therefore represents a novel therapeutic approach to overcome both PI-resistance and immune suppression in MM.