Proteasome Activity Not M-Protein Production Influences Multiple Myeloma (MM) Responsiveness to Bortezomib (BZB): The Key Role of PSMB5,

Abstract

Abstract 3491M-protein load may sensitize MM cells to BZB but models thus far have not directly tested that hypothesis, in one case using a subclone of an IgGκ-producing human MM cell line differing 5X from the parent line with respect to intracellular M-protein concentration (Ca Res 2007;67:1783), and in another using MM cell lines differing in proteasome activity (Blood 2009;113:3040). In other models proteasome activity has been increased by exposure to low doses of bortezomib or by over-expression of PSMB5, the gene for the β5c subunit, reducing sensitivity to BZB (Blood 2008;112:2489). To examine more stringently these aspects of the load-capacity hypothesis with respect to responsiveness to BZB, we used ALMC1, MM1S and KMS12-BM MM cells. ALMC1 cells produce an intact IgGλ and excess amyloid-forming λ light chains and provided a target for M-protein IgGλ gene knockdown (KD). All 3 cell lines were used to evaluate the impact of KD of PSMB5 and PSMB8 expression on proteasome-directed lys48-ubiquitinated proteins, proteasome activity, caspase activation and dose-response to BZB.We achieved gene silencing with no apoptosis or loss of viability with optimized use of streptolysin-O permeabilization (J Immun Meth 2008;333:147). To decrease intracellular M-protein, we targeted the ALMC1 IgG heavy chain+λ light chain genes (IgGλ) and to reduce proteasome activity the PSMB5 or PSMB8 (β5i) genes (4 siRNA per gene with scr control; Dharmacon). To test for efficacy of KD we used RT-qPCR for IgGλ, PSMB5 and PSMB8. We optimized flow cytometry assays to test for intracellular M-protein levels with IgG Fc-receptor and λ light chain specific monoclonal antibodies, and to test for proteasome-directed ubiquitinated proteins with anti-ubiquitin lysine48-specific monoclonal antibodies. To test for proteasome activity we used the site-specific luminogenic peptide substrates Suc-LLVY-aminoluciferin (AML) (β5), Z-LRR-AML (β2) and Z-nLPnLD -AML (β1), and for caspase-3/7 activation a similar substrate containing the tetrapeptide DEVD, reagents optimized for cell lysis, protease or caspase and luciferase activity and read-out in relative luminescent units (RLU) (Promega). In 24-hour culture with BZB (0, 1.56, 3.125, 6.25, 12.5 and 25nM), we tested for viability and proliferation by MTT assay (Invitrogen). All KD experiments, and all luciferase and MTT assays (with 104 and 2.5×104 cells/well in triplicate for each situation), were conducted on at least 6 and 3 separate days respectively. Statistical analyses were performed with GraphPad PRISM.Twenty-four hours after KD, IgGλ, PSMB5 and PSMB8 mRNA were decreased by > 90% compared to scr controls. With KD of IgGλ the intracellular M-protein was reduced 10X but with KD of PSMB5 or PSMB8 the M-protein levels were unchanged as was β5 activity. KD of IgGλ, PSMB5 or PSMB8 had no impact on lys48-specific protein levels, or on proteasome or caspase 3/7 activity. Dose-response to BZB after KD of IgGλ or PSMB8 was no different than scr controls but after KD of PSMB5 the BZB dose-response curve was shifted significantly to the left (Figure 1). Among the 3 cell lines there were distinctive differences in baseline proteasome activity (KMS12-BM>ALMC1>MM1S), sensitivity to BZB (MM1S>ALMC1>KMS12-BM), and lys48-specific protein levels (MM1S>ALMC1>KMS12-BM). Results with KD of PSMB5 and PSMB8 in KMS12-BM and MM1S cells showed a similar left-shift of the BZB dose-response curves with KD of PSMB5 but not PSMB8. In addition, β5 and β1 activity was significantly reduced in all 3 cell lines when exposed to BZB after KD of PSMB5 but not PSMB8 (β1 activity is shown in Figure 2). [Display omitted] [Display omitted] These data indicate that proteasome activity, both baseline and post-BZB exposure, influence sensitivity to BZB and that acute reductions in intracellular M-protein levels do not. Results with KD of PSMB5, encoding the β5c subunit, highlight the key role of β5c particularly with respect to control of β1 activity either directly through a role in proteasome assembly and turnover or indirectly as a result of β5c substrates binding non-specifically within the proteasome and blocking the β1 site. Modulation of PSMB5 expression during BZB exposure, perhaps via interference with the TCF11 or Nrf2 feedback loops, may enhance MM cell sensitivity to BZB. Disclosures:No relevant conflicts of interest to declare.