Offer, Steven M

Abstract 5-Fluorouracil (5-FU) is a cornerstone drug for the treatment of several aggressive tumors; however, adverse 5-FU toxicity contributes to approximately 1,300 deaths annually in the U.S. While three genetic variations in dihydropyrimidine dehydrogenase (DPD; DPYD gene) are well-established predictors of toxicity (i.e., *2A, I560S, D949V), the contributions of additional reported missense DPYD variants (>130) have not been adequately evaluated in clinical studies. Our lab has previously developed methods to study DPYD variants for potential clinical relevance to 5-FU toxicity using in vitro and cellular approaches (see Offer et al. Cancer Research 2013 & 2014). In the current study, we measured the in vitro enzyme activity of 47 uncharacterized missense variants that have been previously reported in dbSNP and NHLBI ESP databases. Additionally, we assessed the impact of these variants on 5-FU sensitivity in multiple transgenic human cellular models using real-time cellular analysis. Approximately one-third of the studied variants showed significantly reduced enzyme activity compared to wild type. Of the DPD-deficient variants identified in this study, 8 were profoundly deficient (<12.5% of “wildtype” activity), with enzyme activities similar to *2A. Two deficient variants showed modest reductions in enzyme activity (12.5-25% activity), similar to I560S. The remaining 5 deleterious variants exhibited mild deficiency (>25% activity, but significantly lower than “wildtype”), similar to D949V. Cellular sensitivity to 5-FU for cells expressing these variants paralleled the in vitro enzyme activity results, with deleterious variants showing reduced tolerance to 5-FU relative to cells transgenic for wildtype DPD. These trends were observed in multiple cell lines derived from various tissues and tumor types. Overall, our results add to the growing database of deleterious DPYD variations. Many of these variants have been observed primarily in non-Caucasian racial groups, consistent with our previous reports. These data suggest that the deleterious variants classified in this study may be important predictors of 5-FU toxicity, particularly in individuals of non-Caucasian ancestry. Additionally, these data suggest that genetic testing for rare variants may improve the success of predictive tests for 5-FU toxicity. Finally, this study suggests that in vitro and cellular models may be important pharmacogenomic methods to rapidly characterize variants of potential relevance in additional drug-gene pathways. Citation Format: Shikshya Shrestha, Steve Offer, Robert B. Diasio. Functional analysis of rare dihydropyrimidine dehydrogenase variants relevant to 5-fluorouracil toxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5487. doi:10.1158/1538-7445.AM2015-5487

Abstract Purpose: The microRNA miR-27a was recently shown to directly regulate dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. A common polymorphism (rs895819A>G) in the miR-27a genomic region (MIR27A) was associated with reduced DPD activity in healthy volunteers, but the clinical relevance of this effect is still unknown. Here, we assessed the association of MIR27A germline variants with early-onset fluoropyrimidine toxicity. Experimental Design: MIR27A was sequenced in 514 patients with cancer receiving fluoropyrimidine-based chemotherapy. Associations of MIR27A polymorphisms with early-onset (cycles 1–2) fluoropyrimidine toxicity were assessed in the context of known risk variants in the DPD gene (DPYD) and additional covariates associated with toxicity. Results: The association of rs895819A>G with early-onset fluoropyrimidine toxicity was strongly dependent on DPYD risk variant carrier status (Pinteraction = 0.0025). In patients carrying DPYD risk variants, rs895819G was associated with a strongly increased toxicity risk [OR, 7.6; 95% confidence interval (CI), 1.7–34.7; P = 0.0085]. Overall, 71% (12/17) of patients who carried both rs895819G and a DPYD risk variant experienced severe toxicity. In patients without DPYD risk variants, rs895819G was associated with a modest decrease in toxicity risk (OR, 0.62; 95% CI, 0.43–0.9; P = 0.012). Conclusions: These results indicate that miR-27a and rs895819A>G may be clinically relevant for further toxicity risk stratification in carriers of DPYD risk variants. Our data suggest that direct suppression of DPD by miR-27a is primarily relevant in the context of fluoropyrimidine toxicity in patients with reduced DPD activity. However, miR-27a regulation of additional targets may outweigh its effect on DPD in patients without DPYD risk variants. Clin Cancer Res; 21(9); 2038–44. ©2015 AACR.

Abstract Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, DPYD, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify DPYD variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydro-fluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wild-type DPD (120%, P = 0.025; 116%, P = 0.049; 130%, P = 0.0077; 138%, P = 0.048, respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (P = 0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had <25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient DPYD variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent. Cancer Res; 74(9); 2545–54. ©2014 AACR.

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.

Abstract In the 45 years since its development, the pyrimidine analog 5-fluorouracil (5-FU) has become an integral component of many cancer treatments, most notably for the management of colorectal cancer. An appreciable fraction of patients who receive 5-FU suffer severe adverse toxicities, which in extreme cases may result in death. Dihydropyrimidine dehydrogenase (DPD, encoded by DPYD) rapidly degrades 85% of administered 5-FU, and as such, limits the amount of drug available for conversion into active metabolites. Clinical studies have suggested that genetic variations in DPYD increase the risk for 5-FU toxicity, however, there is not a clear consensus about which variations are relevant predictors. In the present study, DPYD variants were expressed in mammalian cells, and the enzymatic activity of expressed protein was determined relative to wild-type (WT). Relative sensitivity to 5-FU for cells expressing DPYD variations was also measured. The DPYD*2A variant (exon 14 deletion caused by IVS14+1G>A) was confirmed to be catalytically inactive. Compared with WT, two variants, S534N and C29R, showed significantly higher enzymatic activity. Cells expressing S534N were more resistant to 5-FU–mediated toxicity compared with cells expressing WT DPYD. These findings support the hypothesis that selected DPYD alleles are protective against severe 5-FU toxicity, and, as a consequence, may decrease the effectiveness of 5-FU an antitumor drug in carriers. In addition, this study shows a method that may be useful for phenotyping other genetic variations in pharmacologically relevant pathways. Cancer Res; 73(6); 1958–68. ©2013 AACR.

Abstract The pyrimidine analog 5-fluorouracil (5-FU) and its prodrug capecitabine are prescribed widely for the management of several common cancers, including colorectal, breast, and pancreatic cancers. 5-FU is activated by conversion to cytotoxic metabolites via the pyrimidine anabolic pathway. In the opposing catabolic pathway, dihydropyrimidine dehydrogenase (DPD, encoded by the DPYD gene) initiates rapid and irreversible conversion of greater than 80% of administered 5-FU to inactive metabolites and limits the pool of 5-FU available for anabolism. Deficiency of DPD has been associated with adverse effects to 5-FU treatment, including mucositis, severe diarrhea, neurotoxicity, and life-threatening hematological toxicity, making DPD activity a potentially important predictor of 5-FU toxicity. Despite considerable study of the DPYD gene and its promoter, little is known about the post-transcriptional regulation of DPD expression at the molecular level. The objective of this study was to determine if DPD expression undergoes microRNA-mediated regulation. Computational analysis of the DPYD 3’ untranslated region (UTR) revealed an evolutionarily conserved seed-binding region for the microRNAs mir27a and mir27b. This finding prompted the hypothesis that both mir27a and mir27b are capable of modulating DPD expression. To determine if mir27a and/or mir27b bind to the DPYD 3’ UTR, HCT116 cells lines were engineered to stably express luciferase reporter constructs wherein the luciferase gene was modified to contain the 3’ UTR sequence from DPYD. As controls, stable cells lines containing mutations in the putative microRNA-binding region were generated concurrently. Two colon cancer cell lines (HT29 and HCT15) were also utilized to determine if mir27a and/or mir27b could modulate endogenous DPD expression. In cell lines expressing the luciferase gene juxtaposed to the DPYD 3’UTR, mimics of mir27a or mir27b resulted in significantly reduced luciferase activity relative to scramble control microRNA. Luciferase levels in mir27a or mir27b transfected lines were also significantly reduced compared to stable luciferase-expressing cells harboring the mutant UTR construct. Over-expression of small RNA inhibitors directed toward mir27a or mir27b led to increased luciferase activity in cells containing the wildtype UTR construct. Finally, over-expression of mir27a or mir27b significantly reduced endogenous DPD expression in both HT29 and HCT15 cells. Based on these findings, we conclude that mir27a and mir27b are capable of post-transcriptional regulation of DPD. To our knowledge this is the first report to show microRNA-mediated modulation of DPD expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2011-4404