Abstract PR13: p53-related protein kinase is a novel prognostic marker and therapeutic target in multiple myeloma

Abstract

Abstract Background: Multiple myeloma (MM) is a disease characterized by the accumulation of malignant plasma cells in the bone marrow. The introduction of novel drugs, such as immunomodulators (IMIDs), proteasome inhibitors, monoclonal antibodies, and HDAC inhibitors has revolutionized the approach to treat patients. Nevertheless, a better characterization of MM pathogenesis and of the mechanisms of action of current medications is needed to improve patient selection and hence outcomes, by increasing responses to therapies and reducing side effects. To this end, we have developed an approach based on genetic screening and drug characterization that allowed us to identify p53-related protein kinase (TP53RK) as a novel therapeutic target in MM. Methods: Gene expression profiling analysis was performed in primary MM cells and cell lines using Affymetrix U133 Plus 2.0 chips. Knockdown of proteins (TP53RK, c-Myc, p53, RRM1) was carried out using siRNA and/or shRNA. Expression of RNA and protein was assessed by real-time qPCR and immunoblotting, respectively. Cell proliferation was determined by live cell count and MTT assay. Electrophoretic mobility shift assay was used for evaluation of E2F1 activity. Pomalidomide-conjugating beads were generated for pull-down assay. Nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry were used to confirm pomalidomide-Pom binding to TP53RK. Results: We identified a novel protein, called TP53RK, that is expressed at higher levels in malignant plasma cells compared to normal plasma cells. In newly diagnosed MM patients higher TP53RK level confers poor prognosis. Downregulation of TP53RK induces apoptosis and inhibits cellular growth in multiple MM cell lines. TP53RK knockdown inhibits phosphorylation of TP53 (Ser15), promoting downregulation of MYC and E2F1-dependent upregulation of BIM. Importantly, we demonstrated by standard affinity chromatography and nuclear magnetic resonance spectroscopy that lenalidomide and pomalidomide bind to TP53RK affecting its activity, with biologic sequelae similar to TP53RK knockdown. Lenalidomide binds to cereblon (CRBN), a substrate adaptor of the CRL4 E3 ubiquitin ligase, and induces the ubiquitination and degradation of IKZF1 and IKZF3. CRBN knockdown does not alter TP53RK, or p53 axis; conversely, TP53RK downregulation does not modulate CRBN expression, indicating that lenalidomide and pomalidomide independently bind and regulate TP53RK and CRBN. TP53RK knockdown-induced MM cell growth inhibition was observed even in TP53-mutated and TP53-null MM cell lines, suggesting additional molecules targeted by TP53RK. Indeed, we identified RRM1, p18, proteasome inhibitor subunits, and telomerases as potential downstream targets. Hence, our studies demonstrate that either genetic or pharmacologic inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and -independent pathways. Furthermore, TP53RK blockade may be one of the novel mechanisms of action responsible for IMIDs' effects in MM. Conclusions: Our results identify TP53RK as a marker of poor prognosis in MM, validate TP53RK as a novel therapeutic target in this patient subset, and delineate a novel mechanism whereby IMIDs bind TP53RK, inhibit its activity, and trigger apoptotic MM cell death. Ongoing efforts are directed to developing selective TP53RK inhibitors to improve patient outcome in this patient subgroup. This abstract is also being presented as Poster 04. Citation Format: Francesca Cottini, Teru Hideshima, Yoshihisa Nozawa, Hiroto Ohguchi, Teruhiro Utsugi, Kenneth C Anderson. p53-related protein kinase is a novel prognostic marker and therapeutic target in multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR13.