Oncogenic ASPP2κ: A novel regulator of drug resistance and tumor progression in multiple myeloma

Abstract Treatment options for multiple myeloma (MM) have improved significantly in recent years. Nevertheless, therapy resistance occurs in a proportion of patients, presenting a key mortality factor in advanced tumors. Alterations in the p53pathway are a hallmark of therapy-refractory MM but the underlying mechanisms that lead to inhibition of TP53 tumorsuppressor function are only partially understood. We here show that deregulation of the Apoptosis Stimulating Protein of TP53-2 (ASPP2) is involved in this process. ASPP2 is well known to mediate p53-dependent induction of apoptosis - but also directly interacts with NFκB and BCL-2. This puts ASPP2 into a central position to orchestrate major pathways regulating cellular homeostasis Importantly, we have identified a novel, oncogenic isoform of ASPP2, named ASPP2κ, which is characterized by loss of important p53-, BCL-2 and NFκB-binding sites, thus mediating all classical hallmarks of cancer. We now reveal that ASPP2 is frequently deregulated in MM, promoting drug resistance and tumor progression in vivo, consequently associating with adverse survival rates. A random cohort of newly diagnosed MM patients (n=28) with follow-up data and a healthy bone marrow donor cohort (n=31) were studied to correlate ASSP2κ isoform expression levels with patient outcome data. Notably, the ASPP2 WT isoform is frequently suppressed in CD38+ MM cells, while oncogenic ASPP2κ was found to be highly expressed and functionally active: Isogenic MM isoform-specific shRNA-based cell models (JJN-3, AMO-1 or RPMI-8226 cell lines), stably suppressing ASPP2κ were established, demonstrating significant attenuation of proliferation rates (doubling times decreased by 30% to 40% in all tested models, measured by IncuCyte live cell imager) when compared to EV cell strains. In line, ASPP2κ KD resulted in an increase in induction of apoptosis upon treatment with chemotherapy (melphalan +30% at IC50) in all models. Interestingly, treatment of the parental cell lines with bortezomib led to upregulation of ASPP2κ, supporting the notion that ASPP2κ is a target for proteasomal degradation – and inhibition thereof may fuel a resistance mechanism. Consequently, ASPP2κ_KD cell strains were more responsive to proteasome inhibition (bortezomib or carfilzomib, average +25% at IC50). An ASPP2κ_KD MM Nod/Scid xenotransplant model further demonstrates attenuated tumor engraftment and longer survival of mice bearing ASPP2k-KD cell strains compared to respective EV controls. Consequently,ASPP2κ-interference may be an attractive novel therapeutic approach: We established medicinal siRNAs generated for in vivo use – and identified 2 lead siRNAs that significantly inhibit expression of ASPP2κ (30-80%) along with proliferation rates (> 50%) and sensitize MM cells towards (chemo)therapy (+ av. 25%). Together, our studies suggest that dysfunctional ASPP2 promotes drug resistance and tumor progression. This observation is underlined in a mRNA expression study employing three large MM study cohorts comprising over 1000 MM patients, assessed for ASPP expression patterns (kmplot.com/private edition) - confirming a highly significant correlation of ASPP2 expression levels and event free as well as overall survival (p<0.001). In summary, we demonstrate that ASPP2κ is overexpressed and functionally active in MM. Expression of oncogenic ASPP2κ thereby directly contributes to tumor biology and associates with a more aggressive phenotype as well as drug resistance. Importantly, ASPP2κ-interference sensitizes MM cells for cytotoxic treatment and proteasome inhibition - thus proofing to be a potential novel therapeutic target, which warrants further exploration.