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.

Dihydropyrimidine dehydrogenase (DPD; DPYD gene) variants have emerged as reliable predictors of adverse toxicity to the chemotherapy agent 5-fluorouracil (5-FU). The intronic DPYD variant rs75017182 has been recently suggested to promote alternative splicing of DPYD. However, both the extent of alternative splicing and the true contribution of rs75017182 to DPD function remain unclear. In the present study we quantified alternative splicing and DPD enzyme activity in rs75017182 carriers utilizing healthy volunteer specimens from the Mayo Clinic Biobank. Although the alternatively spliced transcript was uniquely detected in rs75017182 carriers, canonically spliced DPYD levels were only reduced by 30% (p=2.8×10−6) relative to controls. Similarly, DPD enzyme function was reduced by 35% (p=0.025). Carriers of the well-studied toxicity-associated variant rs67376798 displayed similar reductions in DPD activity (31% reduction). The modest effects on splicing and function suggest that rs75017182 may have clinical utility as a predictor of 5-FU toxicity similar to rs67376798.

Clinical studies have identified specific genetic variants in dihydropyrimidine dehydrogenase (DPD, DPYD gene) as predictors of severe adverse toxicity to the commonly used chemotherapeutic 5-fluorouracil; however, these studies have focused on European and European-American populations. Our lab recently demonstrated that additional variants in non-European haplotypes are predictive of 5-FU toxicity. The objective of this study was to identify potential risk variants in an under-studied East African population relevant to our institution’s catchment area. The DPYD protein-coding region was sequenced in 588 individuals of Somali or Kenyan ancestry living in central/southeast Minnesota. Twelve novel non-synonymous variants were identified, seven of which significantly decreased DPD activity in vitro. The commonly reported toxicity-associated variants *2A, D949V, and I560S were not detected in any individuals. Overall, this study demonstrates a critical limitation in our knowledge of pharmacogenetic predictors of 5-FU toxicity, which has been based on clinical studies conducted in populations of limited diversity.

The antimetabolite 5-fluorouracil (5-FU) is one of the most widely used chemotherapy drugs. Dihydropyrimidine dehydrogenase (DPD) is a major determinant of 5-FU response and toxicity. While DPYD variants may affect 5-FU metabolism, they do not completely explain the reported variability in DPD function or the resultant differences in treatment response. Here, we report that H3K27 tri-methylation (H3K27me3) at the DPYD promoter regulated by Ezh2 and UTX suppresses DPYD expression by inhibiting transcription factor PU.1 binding, leading to increased resistance to 5-FU. Enrichment of H3K27me3 at the DPYD promoter was negatively correlated with both DPYD expression and DPD enzyme activity in peripheral blood specimens from healthy volunteers. Lastly, tumor expression data suggests that DPYD repression by Ezh2 predicts poor survival in 5-FU-treated cancers. Collectively, the findings of the present manuscript suggest that a previously uncharacterized mechanism regulates DPD expression and may contribute to tumor resistance to 5-FU.

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.

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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