Abstract 3941: Enzymatic nitration in oncology

Abstract

Abstract Background: We have discovered a new class of enzymes, that we name the nitrases, that catalyze the post-translation nitration of specific tyrosine residues of key proteins, several of which regulate important oncogenic pathways and are negatively prognostic in multiple cancer types. Materials And Methods: To identify protein substrates for 31 candidate nitrases, we incubated each nitrase with a 23,000 protein chip in a nitrase assay containing peroxynitrite and using anti-nitrotyrosine antibodies to detect the proteins that were nitrated. We then confirmed this nitration in separate biochemical assays and determined the specific site of nitration by LC-MS. Further, nitrases were evaluated in silico against TCGA gene expression and GISTIC copy number databases for specific cancer associations. Results: We have discovered that several proteins known to regulate tumor cell survival and/or evasion of immune surveillance are specifically nitrated by different nitrases and that their biochemical activities are regulated by this nitration. Pak4, a protein that has been implicated as an oncogenic driver, as well as in immune evasion is specifically nitrated by Nitrase #3 and increases its kinase activity. Interestingly, Nitrase #3 also nitrates Pak5, but not any of the other Pak family members, demonstrating high enzymatic specificity. Targeted inhibition or degradation of nitrated Pak4 or inhibition of Nitrase #3 may have therapeutic potential in oncology. Similarly, RhoA, a protein implicated in the oncogenic state of tumors is nitrated by Nitrase #12. This nitration activates RhoA biochemically and in cells. Bax is a specific substrate for Nitrase #11. Nitration of Bax reduces the cytochrome C release from the mitochondria induced by Bax. For each of these enzymes, we will develop specific inhibitors to determine their therapeutic potential in tumorigenesis. We further performed in silico analysis of nitrases for overall survival association in 17 cancer types. Utilizing TCGA RNA expression data and The Human Protein Atlas, nitrases were screened for association with overall survival in relation to nitrase RNA expression. Nitrases showing higher overall survival with low nitrase RNA expression and p. value <0.001 were identified as negatively prognostic. 10 nitrases showed negative prognostic association, suggesting that they could be favorable therapeutic targets. Three nitrases, N11, N12, N18, were negatively prognostic in renal clear cell carcinoma and nitrases N1 and N13 were negatively prognostic in liver cancer. Conclusions: A new class of enzymes we discovered offers multiple opportunities for development of effective chemotherapeutic agents against a variety of oncogenic targets and in specific cancers that show negative association of high nitrase expression. Citation Format: Arun Kashyap, Sami Hussein, Sahar Mazar, Kate Markham, Anthony Mastracci, Ritu Sharma, Sheerin K. Shahidi-Latham, Irene Griswold-Prenner. Enzymatic nitration in oncology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3941.