Jerde, Calvin R.

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 (5‐FU); however, these studies have focused on European and European‐American populations. Our laboratory 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 understudied 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 nonsynonymous 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.

Abstract 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. Although 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 trimethylation (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 suggest that DPYD repression by Ezh2 predicts poor survival in 5-FU–treated cancers. Collectively, the findings of the present article suggest that a previously uncharacterized mechanism regulates DPD expression and may contribute to tumor resistance to 5-FU. Cancer Res; 76(21); 6362–73. ©2016 AACR.

Abstract Dihydropyrimidine dehydrogenase (DPD) is a major determinant of the efficacy and toxicity of 5-fluorouracil in various cancer therapies. Single nucleotide polymorphisms (SNPs) within DPYD have been studied extensively for years. However, known SNPs do not explain most cases of altered DPD activity and response to 5-FU. Furthermore, variations of DPYD expression in cancer patients have been reported; however, the underlying molecular mechanism is unclear. This suggests that regulation of DPYD expression may be an additional mechanism to control DPD activity. In this study, we focused on epigenetic regulation of DPD and specifically investigated the role of histone methylation on DPYD expression. Inhibition of the H3K27 methyl-transferase Ezh2 by either GSK-126 or a dominant-negative histone H3 mutant significantly increased DPYD expression in various cell lines. The expression of thymidylate synthetase, a major target of 5-fluorouracil toxicity, was not altered. Consistent with elevated DPYD expression, cells that lost H3K27me3 were more resistant to 5-fluorouracil. Furthermore, we demonstrated that Ezh2 and H3K27me3 were enriched on the promoter and the first exon of DPYD in low-DPD expressing cells, but not in cell lines with high DPD levels. Additionally, the enrichment of H3K27me3 on the DPYD promoter was negatively correlated with cellular DPD activity in peripheral mononuclear cells from healthy volunteers. Finally, CRISPR/Cas9-mediated targeting of Ezh2 to the DPYD promoter artificially increased H3K27me3 and suppressed DPYD expression. Collectively, these data suggest that histone methylation can regulate DPYD expression, providing a previously unrecognized mechanism of 5-fluorouracil sensitivity. The tri-methylation of H3K27 is a potential biomarker for guiding individualized 5-FU therapy. Citation Format: Rentian Wu, Steven Offer, Calvin Jerde, Garrett Dunlap, Robert Diasio. Histone H3K27 tri-methylation regulates DPYD expression and cellular sensitivity of 5-fluorouracil. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B49.

Abstract Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) is the rate-limiting enzyme in the uracil catabolic pathway and has a pivotal role in the pharmacokinetics of the commonly prescribed anticancer drug 5-fluorouracil (5-FU). Deficiency of DPD, whether due to inadequate expression or deleterious variants in DPYD, has been linked to severe toxic responses to 5-FU. Little is known about the mechanisms governing DPD expression in the liver. In this report, we show increased accumulation of RNA-induced silencing complex (RISC) proteins on DPYD mRNA in cells overexpressing the highly homologous microRNAs (miRNA) miR-27a and miR-27b. These miRNAs were shown to repress DPD expression through two conserved recognition sites in DPYD. The IC50 of 5-FU for HCT116 cells overexpressing miR-27a or miR-27b was 4.4 μmol/L (both), significantly lower than that for cells expressing a nontargeting (scramble) control miRNA (14.3 μmol/L; P = 3.3 × 10−5 and P = 1.5 × 10−7, respectively). Mouse liver DPD enzyme activity was inversely correlated with expression levels of miR-27a (R2 = 0.49; P = 0.0012) and miR-27b (R2 = 0.29; P = 0.022). A common variant in the hairpin loop region of hsa-mir-27a (rs895819) was also shown to be associated with elevated expression of the miR-27a in a panel of cell lines (P = 0.029) and in a transgenic overexpression model (P = 0.0011). Furthermore, rs895819 was associated with reduced DPD enzyme activity (P = 0.028) in a cohort of 40 healthy volunteers. Taken together, these results suggest that miR-27a and miR-27b expression may be pharmacologically relevant modulators of DPD enzyme function in the liver. Furthermore, our data suggest that rs895819 may be a potential risk allele for 5-FU sensitivity. Mol Cancer Ther; 13(3); 742–51. ©2014 AACR.