P830: identification of novel target genes correlated with 1q21 amplification in patients with smoldering myeloma and multiple myeloma

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: The gain and/or amplification of the 1q21 (1q21+) region is one of the most common secondary cytogenetic abnormalities present in patients with multiple myeloma (MM). It has been reported that the incidence of the 1q21 copy number increases with disease progression, detected in around 30-40% of smoldering MM (SMM) patients. Several studies indicate that the presence of 1q21+ is associated with a worse clinical outcome and a poor response to MM therapies including proteasome inhibitors (PIs). Recent studies have demonstrated that the number of 1q21 copy (1q21 amp Vs. Gain) in newly diagnosed MM patients (NDMM) has a negative impact in overall survival. Aims: The genes driving amplification of the 1q21 region have not been fully elucidated. This study aims to identify novel targets in the 1q21 region possibly correlated to PIs treatment. Methods: We purified CD138+ primary plasma cells (PCs) from bone marrow (BM) aspirates of a cohort of 29 patients (11 SMM and 18 NDMM). Fluorescent in situ hybridization analysis was performed on all the patients. 48% (14/29) patients carried 1q21+. A score indicating the number of 1q21 copies was calculated based on the FISH hybridization pattern of each patient. The expression profile of all 29 samples was generated using GeneChip ClariomD Arrays (Affymetrix Inc., Santa Clara, CA, USA). The smar package was used to identify differentially expressed genes between 1q21+ and control samples. Results: The expression analysis of our cohort of patients identified proteasome subunits showed PSMB4 and PSMD4 to be significantly upregulated in MM patients 1q21+ compared to control patients (PSMB4 p=0.0006; PSMD4 p=<0.0001 respectively). Furthermore, we found a strong positive correlation between gene expression levels and 1q21 copy number for the proteasome subunits PSMB4 (p=<0.0001, r=0.5631) and PSMD4 (p=<0.0001, r=0.6391). Interestingly, the PSMB4 and PSMD4 expression levels were independent of the disease stage (SMM vs MM) and the expression was driven exclusively by the 1q21 copy number. We evaluated PSMB4 and PSMD4 mRNA and protein expression levels in a 1q21 control MM cell line (OCI-MY5) and a panel of MM cell lines with 1q21 amplification. Our results showed that PSMB4 and PSMD4 expression levels were higher in cell lines with 1q21 amplification, following a 1q21 copy number fashion. Subsequently, we assessed the role of PSMB4 and PSMD4 to the sensibility of PI bortezomib in two myeloma cell lines carrying 1q21+. Our results showed that bortezomib treatment downregulates the protein expression of PSMD4 while PSMB4 was unaffected. Moreover, the MTT viability test correlated with the anti-myeloma effect of bortezomib. Furthermore, we generated a JJN3 PSMB4 and PSMD4-knockdown MM cell line using short hairpin RNA (shRNA) lentivectors. Our functional studies showed that shPSMB4 and shPSMD4 decreased MM cell viability when treated with PI. Moreover, both shPSMB4 and shPSMD4 knockdown cells showed elevated levels of polyubiquitylated proteins, indicating blockade of proteasome-mediated protein degradation. Summary/Conclusion: Our study identified proteasome subunits PSMB4 and PSMD4 to be significantly upregulated in SMM and MM patients with 1q21, correlated with 1q21 copy number but not with disease stage. In addition, knockdown of both, PSMB4 and PSMD4 decreased MM cell proliferation. Therefore, targeting PSMD4 could be a strategy to treat patients with 1q21 amplification. Keywords: Multiple myeloma