Abstract 4037: The effects of n-acetyltransferase 1 gene knockout on the cytotoxicity of pyrimidine biosynthesis inhibitors in human breast cancer cells

Abstract

Abstract Arylamine N-acetyltransferase 1 (NAT1), a polymorphic drug metabolism enzyme, is often upregulated in breast cancer cells. To develop novel hypotheses on how NAT1 contributes to breast cancer development and progression, NAT1 knockout (KO) cell lines (KO2 and KO5) were created from MDA-MB-231 using CRISPR/Cas9 technology. According to our proteomics and RNA sequencing analyses, NAT1 KO cells have increased cytidine deaminase (CDA), a player in the pyrimidine salvage pathway. Metabolomics data showed NAT1 KO cells had defects in the de novo pyrimidine pathway, which can explain the upregulation of the salvage pathway. Pyrimidine de novo synthesis and salvage pathways are essential for DNA and RNA synthesis and cell growth.We hypothesize that NAT1 plays a key role in maintaining pyrimidine biosynthesis in breast cancer cells, and hence, exhibit increased sensitivity to some of chemotherapeutic drugs that depends on pyrimidine metabolic pathways for their cytotoxicity. Parental, KO2, and KO5 MDA-MB-231 breast cancer cell lines were treated with several different classes of drugs: CDA inhibitors: Tetrahydrouridine and zebularine; pyrimidine biosynthesis inhibitors teriflunomide and leflunomide; DNA damaging agent doxorubicin; and cytidine analogues 5-hydroxymethyl-2’-deoxycytidine, 5’-formyl-2’-deoxycytidine, and 5’-deoxy-5-fluoropyridine at varying concentrations for 4 days. Cell viability was assessed by Alamarblue assay. Differences between cell lines were assessed for statistical significance by two-way ANOVA. The results were as follows Tetrahydrouridine: No statistically significant difference in cell viability between parental and NAT1 KOs except at 200 µM p= 0.0002Zebularine: Statistically significant difference between parental and NAT1 KOs at concentrations above 60 µM at p < 0.0001Teriflunomide: Statistically significant difference between parental and NAT1 KOs between 20 and 60 µM at p < 0.0001Leflunomide: No statistically significant difference between parental and NAT1 KOs except at 200 µM at p = 0.00065’- Formyl- 2'- Deoxycytidine: Statistically significant difference between parental and NAT1 KOs above 12 µM at p = 0.0042Doxorubicin: No statistically significant difference between parental and NAT1 KOs 5-Hydroxymethyl-2’-deoxycytidine: No statistically significant difference between parental and NAT15’-Deoxy-5-fluorocytidine: No statistically significant difference between parental and NAT1 KOs Whereas the results with many of the drugs were inconsistent with our initial hypothesis, it is possible that the NAT1 KO cells are more resistant to CDA or pyrimidine biosynthesis inhibitors because of the upregulation of CDA. Understanding the pharmacogenomic similarities in Tetrahydrouridine, zebularine and teriflunomide’s may help develop a new hypothesis. Citation Format: Afi Henriette Tagnedji, Kyung U. Hong, David W. Hein. The effects of n-acetyltransferase 1 gene knockout on the cytotoxicity of pyrimidine biosynthesis inhibitors in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4037.