Diasio, Robert B.

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) is a cornerstone therapy for the treatment of several aggressive tumors. However, nearly 1/3 of patients receiving 5-FU experience severe (grade 3+) adverse 5-FU toxicity. Genetic variations in dihydropyrimidine dehydrogenase (DPD; DPYD gene) are major contributors of 5-FU toxicity. With continued progress in next-generation sequencing technologies, the number of reported genetic variants is substantially increasing; to date, there are more than 450 reported missense variants in the DPYD gene. Only a handful of those variants, including *2A, I560S, D949V, and rs75017182, have been established as predictors of toxicity. Because most of the remaining variants are unstudied, mainly due to their rare occurrence and unequal racial distribution, it is challenging to conduct statistically powered clinical association studies. Therefore, in vitro and computational methods to identify potential toxicity-associated variants have major appeal. The primary goal of the current study was to develop a highly accurate gene-specific in silico classifier to predict deleterious DPYD variants. For this purpose, using a recombinant cellular model, we characterized 69 missense DPYD variants reported in online databases. We then utilized DPD activity data from the 69 variants in combination with data from 87 previously characterized variants (see Offer, SM et al. Cancer Research 2013 & 2014) to develop the DPYD variant classifier model (DPYD-Varifier). DPD protein-specific biochemical and structural features and amino acid conservation scores were integrated into the model's random forest classifier-based algorithm. DPYD-Varifier showed improved accuracy (85% accurate) compared to other in silico tools. Additionally, the model provided mechanistic insights into alterations of DPD function conferred by DPYD variants. In general, deleterious variants were located in closer proximity to important binding/coordinating sites of the DPD protein. Additionally, DPYD-Varifier allowed us to estimate the cumulative allele frequency of all reported deleterious variants in various geographical/racial groups, which can substantially add to our knowledge of DPD deficiency and 5-FU toxicity observed in these populations. In conclusion, the integration of in vitro and accurate computational data from our study permitted classification of relevant DPYD variants, which could be useful to more effectively individualize 5-FU therapy in the future. Citation Format: Shikshya Shrestha, Cheng Zhang, Calvin R. Jerde, Hu Li, Steven M. Offer, Robert B. Diasio. DPYD-Varifier, a computational model to identify 5-FU toxicity-associated DPYD variants [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 3894.

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.

5‐Fluorouracil (5‐FU) is dosed by body surface area, a practice unable to reduce the interindividual variability in exposure. Endorsed by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT), we evaluated clinical evidence and strongly recommend TDM for the management of 5‐FU therapy in patients with colorectal or head‐and‐neck cancer receiving common 5‐FU regimens. Our systematic methodology provides a framework to evaluate published evidence in support of TDM recommendations in oncology.

Deleterious variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) can be highly predictive of clinical toxicity to the widely prescribed chemotherapeutic 5‐fluorouracil (5‐FU). However, there are very limited data pertaining to the functional consequences of the >450 reported no‐synonymous DPYD variants. We developed a DPYD‐specific variant classifier (DPYD‐Varifier) using machine learning and in vitro functional data for 156 missense DPYD variants. The developed model showed 85% accuracy and outperformed other in silico prediction tools. An examination of feature importance within the model provided additional insight into functional aspects of the DPD protein relevant to 5‐FU toxicity. In the absence of clinical data for unstudied variants, prediction tools like DPYD‐Varifier have great potential to individualize medicine and improve the clinical decision‐making process.

The purpose of this guideline is to provide information for the interpretation of clinical dihydropyrimidine dehydrogenase ( DPYD ) genotype tests so that the results can be used to guide dosing of fluoropyrimidines (5‐fluorouracil and capecitabine). Detailed guidelines for the use of fluoropyrimidines, their clinical pharmacology, as well as analyses of cost‐effectiveness are beyond the scope of this document. The Clinical Pharmacogenetics Implementation Consortium (CPIC ® ) guidelines consider the situation of patients for which genotype data are already available (updates available at https://cpicpgx.org/guidelines/guideline‐for‐fluoropyrimidines‐and‐dpyd/ ).

Abstract The antimetabolite 5-fluorouracil (5-FU) is one of the most widely used chemotherapy drugs. Dihydropyrimidine dehydrogenase (DPD) initiates the catabolic degradation of 5-FU to inactive metabolites and is widely accepted as a major determinant of 5-FU response and toxicity. While genetic variants in DPYD have been shown to variants may affect 5-FU metabolism, they are rare events and do not completely explain the reported variability in DPD function or the resultant differences in treatment response. Here, we report that DPYD expression is epigenetically regulated by histone modification at relevant promoter and enhancer regions. Using both chemical inhibitors and genetic approaches, we show that that H3K27 tri-methylation (H3K27me3) at the DPYD promoter is regulated by the epigenetic modifiers Ezh2 and UTX. Promoter H3K27me3 suppresses DPYD expression by inhibiting the binding of the transcription factor PU.1 bindingto the promoter and ,increases sensitivity leading to increased resistance to 5-FU. In a cohort of healthy volunteers, H3K27me3 levels were inversely correlated with An enhancer that regulates DPYD expression was also identified. Modifying the H3K27 status of the DPYD promoter or enhancer modulates DPYD gene expression. Deletions and mutations on the DPYD promoter or enhancer abolish their transcription regulation function. Enrichment of H3K27me3 at the DPYD promoter was negatively correlated with both DPYD expression and DPD enzyme activity in peripheral blood mononuclear cellsspecimens from healthy volunteers. Lastly, tumor expression data suggests that DPYD repression by Ezh2 is a strong predictor ofs poor survival in 5-FU treated cancers. By using a combination of in silico prediction and in vitro reporter assays we also identified a functional enhancer region for DPYD that is also likely to be clinically relevant to 5-FU treatment outcome. Collectively, the presentthese findings suggest that a previously uncharacterized mechanisms regulates DPD expression and may likely contribute to tumor resistance to 5-FU. Citation Format: Rentian Wu, Qian Nie, Erin E. Tapper, Calvin R. Jerde, Steven M. Offer, Robert B. Diasio. Trimethylation and acetylation of Histone H3K27 modulates 5-fluorouracil response by regulating DPYD expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4069. doi:10.1158/1538-7445.AM2017-4069