Citations

Abstract Colorectal cancer (CRC) is the third most common cancer and is expected to cause approximately 50,000 deaths in the US in 2017. The antimetabolite 5-fluorouracil (5-FU) is the cornerstone of first-line adjuvant chemotherapy regimens for CRC. Resistance to 5-FU-based chemotherapies develops over time and is the greatest obstacle to effective management in this disease. However, the resistance mechanisms of 5-FU are largely unknown. Approximately 50% of the CRC cases have activating mutations in KRAS or BRAF. Based on recent clinical data, it is now appreciated that KRAS/BRAF mutations are associated with worse survival in 5-FU-based chemotherapies for CRC. Therefore, there is an urgent unmet need for improved therapeutic options to treat mutant KRAS/BRAF tumors. We analyzed the differential activation status of enhancers in KRAS/BRAF mutant and wildtype CRC cell lines. Enhancers that associated with genes regulating pyrimidine metabolism were significantly enriched in KRAS/BRAF mutant CRC cells. Among them, the differential activation of an enhancer located ~400kb upstream of the DPYD promoter was most significant. By using CRISPR-dCas9 system, specifically targeting of p300 to the enhancer significantly increased DPYD expression, and Ezh2 significantly decreased expression. Knockout (KO) of this enhancer resulted in reductions in DPD expression and increased resistance to 5-FU. In mouse xenograft studies, enhancer-KO cells responded to doses of 5-FU that were otherwise ineffective against control cells. Treating KRAS mutant CRC cells with CBP30, H3K27 acetyl-transferase inhibitor, significantly reduced DPYD expression. Synergistic interactions was identified between CBP30 and 5-FU. Finally, mouse xenograft studies strongly suggest that inhibition of H3K27ac may significantly improve 5-FU effectiveness against KRAS mutant tumors. In summary, the present studies advance our understanding of how mutant KRAS/BRAF epigenetically regulates DPYD expression and results in 5-FU resistance in CRC. We showed that clinical treatment of CRC might be significantly improved by the development of future pharmacologic approaches targeting enhancers that are activated by mutant KRAS/BRAF. Citation Format: Rentian Wu, Qian Nie, Phillip A. Kubica, Zhiquan Wang, Shikshya Shrestha, Colbren S. Trogstad-Isaacson, Calvin R. Jerde, Erin E. Tapper, Kelly J. Bouchonville, Steven M. Offer, Robert B. Diasio. Targeting the mutant KRAS/BRAF-induced enhancer as an approach to overcome resistance to 5-fluorouracil-based chemotherapy in colorectal cancer [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 911.

Abstract 5-Fluorouracil (5-FU) has remained a widely prescribed anti-cancer drug for decades. However, 5-FU–based treatments show inter-individual variability, with up to 34% of 5-FU–treated patients developing severe (grade 3+) adverse toxicity. Dihydropyrimidine dehydrogenase (DPD; DPYD gene) is the rate-limiting enzyme of 5-FU catabolism and converts 80–85% of 5-FU into its inactive metabolites. Three deleterious genetic variations in DPYD are well-established predictors of severe 5-FU toxicity (i.e., *2A, p. I560S, and p. D949V); however, these variants only explain 30–35% of adverse cases. Recently, an intronic variant (rs75017182) has been suggested to contribute to 5-FU–related toxicity by promoting the alternative splicing of DPYD. However, clinical studies are conflicting as to the degree to which rs75017182 affects DPD enzyme function, and the true contribution of the rs75017182 variant towards DPD function and 5-FU toxicity remains unclear and inconclusive. In the present study, we directly examined the effect of the intronic SNP rs75017182 on DPYD mRNA splicing, DPYD expression, and DPD enzyme function. We genotyped 3950 healthy Caucasian volunteers from the Mayo Clinic Biobank to identify the rs75017182, *2A, p.I560S, and p.D949V variants. From the initial cohort, 204 volunteers who were rs75017182 heterozygous carriers were recruited as our study population. Alternative splicing and DPD activity in rs75017182 carriers were measured. The findings were confirmed using a novel mini-gene reporter system in vitro. A moderate, but significant, reduction (30%) was detected in canonically spliced DPYD expression in rs75017182 carriers compared to non-carriers. A correlative reduction (35%) in DPD enzyme activity was observed in those carriers, which was similar to that of D949V carriers (31%). The results demonstrated an association of this deep intronic variant with decreased DPD activity and suggested that rs75017182 may be a predictor of 5-FU toxicity similar to D949V. Citation Format: Qian Nie, Shikshya Shrestha, Erin E. Tapper, Colbren S. Trogstad-Isaacson, Kelly J. Bouchonville, Steven M. Offer, Robert B. Diasio. Quantitative contribution of rs75017182 to dihydropyrimidine dehydrogenase mRNA splicing and enzyme activity [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 3893.

Abstract Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α–HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1α suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

Purpose Overall survival (OS) is the definitive and best-established primary efficacy end point to evaluate diffuse large B-cell lymphoma (DLBCL) therapies, but it requires prolonged follow-up. An earlier end point assessed post-treatment would expedite clinical trial conduct and accelerate patient access to effective new therapies. Our objective was to formally evaluate progression-free survival (PFS) and PFS at 24 months (PFS24) as surrogate end points for OS in first-line DLBCL. Patients and Methods Individual patient data were analyzed from 7,507 patients from 13 multicenter randomized controlled trials of active treatment in previously untreated DLBCL, published after 2002, with sufficient PFS data to predict treatment effects on OS. Trial-level surrogacy examining the correlation of treatment effect estimates of PFS/PFS24 and OS was evaluated using both linear regression ( R2WLS) and Copula bivariable ( R2Copula) models. Prespecified criteria for surrogacy required either R2WLS or R2Copula ≥ 0.80 and neither < 0.7, with lower-bound 95% CI > 0.60. Results Trial-level surrogacy for PFS was strong ( R2WLS = 0.83; R2Copula = 0.85) and met the predefined criteria for surrogacy. At the patient level, PFS strongly correlated with OS. The surrogate threshold effect had a hazard ratio of 0.89. Surrogacy was consistent across comparisons with or without rituximab and with rituximab maintenance trials. Trial-level surrogacy for PFS24 was relatively strong ( R2WLS = 0.77; R2Copula = 0.78) but did not meet prespecified criteria. At the patient level, PFS24 significantly correlated with OS. The surrogate threshold effect had an odds ratio of 1.51. Conclusion This large pooled analysis of individual patient data supports PFS as a surrogate end point for OS in future randomized controlled trials evaluating chemoimmunotherapy in DLBCL. Use of this end point may expedite therapeutic development with the intent of bringing novel therapies to this patient population years before OS results are mature.

Abstract Background: Endometrioid carcinoma (EC) and clear cell carcinoma (CC) histotypes of epithelial ovarian cancer are understudied compared with the more common high-grade serous carcinomas (HGSC). We therefore sought to characterize EC and CC transcriptomes in relation to HGSC. Methods: Following bioinformatics processing and gene abundance normalization, differential expression analysis of RNA sequence data collected on fresh-frozen tumors was completed with nonparametric statistical analysis methods (55 ECs, 19 CCs, 112 HGSCs). Association of gene expression with progression-free survival (PFS) was completed with Cox proportional hazards models. Eight additional multi-histotype expression array datasets (N = 852 patients) were used for replication. Results: In the discovery set, tumors generally clustered together by histotype. Thirty-two protein-coding genes were differentially expressed across histotype (P &lt; 1 × 10−10) and showed similar associations in replication datasets, including MAP2K6, KIAA1324, CDH1, ENTPD5, LAMB1, and DRAM1. Nine genes associated with PFS (P &lt; 0.0001) showed similar associations in replication datasets. In particular, we observed shorter PFS time for CC and EC patients with high gene expression for CCNB2, CORO2A, CSNK1G1, FRMD8, LIN54, LINC00664, PDK1, and PEX6, whereas, the converse was observed for HGSC patients. Conclusions: The results suggest important histotype differences that may aid in the development of treatment options, particularly those for patients with EC or CC. Impact: We present replicated findings on transcriptomic differences and how they relate to clinical outcome for two of the rarer ovarian cancer histotypes of EC and CC, along with comparison with the common histotype of HGSC. Cancer Epidemiol Biomarkers Prev; 27(9); 1101–9. ©2018 AACR.