Abstract LB-275: Targeting poor-prognosis subtypes of prostate cancer by inhibition of DNA repair gene ribonucleotide reductase small subunit M2
Authors: Mazzu Ying Z. , Armenia Joshua , Chakraborty Goutam , Yoshikawa Yuki , Gerke Travis A. , Coggins Si Ana A. , Qiu Xintao , Atiq Mohammad , Stopsack Konrad H. , Lee Gwo-Shu Mary , Long Henry W. , Kim Baek , Freedman Matthew L. , Pomerantz Mark M. , Mucci Lorelei A. , Kantoff Philip W. 
Published: August 2018

Abstract
Studies reveal defective DNA repair contribute to prostate cancer (PC) progression. We hypothesize that overexpression of DNA repair genes could also contribute to poorer outcomes in PC. The nucleotide metabolism enzyme ribonucleotide reductase (RNR) is essential for DNA synthesis and DNA repair by producing dNTPs. The small subunit M2 (RRM2), as the rate-limiting RNR subunit, is frequently up-regulated in cancers. Clinically, targeting of RRM2 with small molecules is being tested in multiple cancers, but there is little knowledge of RRM2 function in PC. The analysis of multiple PC clinical cohorts (total include 1602 cases) revealed that high RRM2 level was associated with poor clinical outcomes, including a higher likelihood of metastasis (p&lt;0.001), biochemical recurrence (p&lt;0.001), and lethality (p&lt;0.0001). In PC cells, knockdown of RRM2 inhibited dNTP production and induced DNA damage, which led to significant cell growth inhibition, major S phase arrest, and apoptosis. Overexpression of RRM2 promoted epithelial-mesenchymal transition (EMT) by increasing the expression of multiple EMT markers. Furthermore, the small molecule RRM2 inhibitor (COH29) induced a similar phenotype as knocking down RRM2 in PC cells. RNA-Seq analysis in siRRM2 or COH29 treated PC cells provided a global assessment of RRM2-regulated transcriptome changes. GSEA analysis revealed that inhibition of RRM2 could activate biological processes including cell cycle checkpoint, DNA damage response, and apoptotic signaling. COH29 treatment could target genes highly enriched in PC. We further applied an RRM2-regulated gene signature (from RNA-Seq datasets) to TCGA and Taylor cohorts. Intriguingly, the RRM2 signature was highly correlated with metastasis and disease free survival (p&lt;0.001). Furthermore, inhibition of RRM2 specifically targets poor prognostic luminal subtypes (PCS1 subtype; Lum B in PAM50 classifier) recently reported. Besides transcriptome changes, protein kinase arrays showed that AKT/mTOR and SFK-STAT signaling were repressed by inhibition of RRM2. These oncogenic signaling pathways are crucial for EMT program. Amplification of RRM2 is rare in PC and transcriptional activation of RRM2 may play a major role in overexpression of RRM2. H3K27ac ChIP-Seq from tissues revealed more activated RRM2 promoter in PC than in normal prostate. 13 potential RRM2-targeting transcription factors (TFs) were identified by integrating clinical cohorts and a TF database. They showed a positive correlation with RRM2 expression in PC cohorts. Among these TFs, FOXM1 was reported to be the master driver of the aggressive luminal subtype of PC. We revealed that FOXM1 expression was associated with clinical outcomes. The ChIP-PCR and luciferase reporter assays provided evidence of physical binding of FOXM1 to the RRM2 promoter in PC cells. Knockdown of FOXM1 significantly repressed RRM2 mRNA and protein levels. Altogether, FOXM1-regulated transcriptional activation contributes to overexpression of RRM2. Intriguingly, COH29 can also repress FOXM1 expression, which leads to transcription repression of RRM2. Altogether, our study elucidated the molecular mechanisms underlying RRM2 oncogenic functions and the transcriptional regulation of RRM2 in PC cells. We suggest that RRM2 can be a novel therapeutic target for PC treatment.
Citation Format: Ying Z. Mazzu, Joshua Armenia, Goutam Chakraborty, Yuki Yoshikawa, Travis A. Gerke, Si Ana A. Coggins, Xintao Qiu, Mohammad Atiq, Konrad H. Stopsack, Gwo-Shu Mary Lee, Henry W. Long, Baek Kim, Matthew L. Freedman, Mark M. Pomerantz, Lorelei A. Mucci, Philip W. Kantoff. Targeting poor-prognosis subtypes of prostate cancer by inhibition of DNA repair gene ribonucleotide reductase small subunit M2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-275.