Abstract 1591: Integrating protein hydroxylase inhibition into cancer therapeutics: From orbital interactions at hydroxylase active sites to misfolded protein-driven cancer cell death and immunogenic cancer cell apoptosis
Authors: Goering Spencer M. , Singh Sukhwinder , Hanauske Axel-Rainer , Cracchiolo Bernadette M. , Cracchiolo Isabel W. , Sandev Angela A. , Hanauske-Abel Hartmut M. 
Published: June 2023

Abstract
Purpose of study: Residues P/K/D/N/H undergo enzymatic hydroxylations essential for proteins that, like collagens, fibrillins, collectins, and hypoxia-inducible factors, co-determine cancer outcome. Despite disparate protein substrates, these enzymes follow identical catalysis by activating inhaled O2 for hydroxylation - the HAG mechanism, formulated in a 1983 thesis. It details catalysis at subatomic resolution, classifies all possible inhibitors, identifies the first selective inhibitor of collagen and collectin biosynthesis, and defines the numerous 2-oxoacid utilizing dioxygenases (2OUDs). The HAG mechanism is considered ‘seminal’ for drug development (https://pubmed.ncbi.nlm.nih.gov/7857312/). mechanism2OUDs occur in clusters. This predicts HAG inhibitors to act across polygenic hydroxylation-dependent protein families relevant in cancer. Single-drug multi-targeted inhibition is a paradigm for anticancer drug discovery (e.g. Alimta™ [https://ascopubs.org/doi/pdf/10.1200/JCO.1999.17.10.3009]). We tested multi-targeted 2OUD suppression with an FDA-approved medicine, deferiprone (DEF), that per HAG mechanism blocks 2OUD cofactors (2-oxoacid, ascorbate).
Hypothesis: HAG mechanism blockade in multiple 2OUD-reliant pathways triggers a cascade of aftereffects; in cancer cells, this HAG sequence is lethal and immunogenic.
Methods: Culture/metabolic labeling/RNA-seq/flow cytometry of models for high grade serous ovarian cancer (KURAMOCHI), uterine serous cancer (ARK1), and cancer-associated fibroblasts (MRC5).
Results: In a dose-dependent manner at clinical concentrations, DEF inhibited [3H]Pro/[3H]Lys hydroxylation, not incorporation; and caused intracellular accumulation of hydroxylation-deficient collagens (COL1A1, -3A1, -6A1, -11A2, -18A1), fibrillins (FBN1), collectins (C1QA,-B, -C), and HIFs (EPAS1), exceeding controls up to 10-fold. After 96 hrs, accumulation reversed in MRC5, remained elevated in KURAMOCHI, and rose further in ARK1. Activation of the ablative unfolded protein response (UPR), via enhanced DDIT3, TRIB3, BAK1, PMAIP1, HRK expression, coincided with a &gt;80% decline in cancer cell count. In MRC5, DEF downregulated apoptosis markers (cleaved PARP-1, active caspase 3), but upregulated both over controls in KURAMOCHI and ARK1, which also showed enhanced expression of pyroptosis markers (caspase 1, IL-18, gasdermins).
Conclusions: DEF reactivity reveals HAG mechanism inhibition at multiple 2OUDs that hydroxylate P/K/D/N residues in cancer-relevant proteins. The ensuing HAG sequence captures the secondary effects, e.g. HIF2α-driven synthesis of underhydroxylated, therefore UPR-retained COL/FBN, triggering apoptotic cancer cell death that, due to C1q deficiency, must proceed without autoimmunity.
Citation Format: Spencer M. Goering, Sukhwinder Singh, Axel-Rainer Hanauske, Bernadette M. Cracchiolo, Isabel W. Cracchiolo, Angela A. Sandev, Hartmut M. Hanauske-Abel. Integrating protein hydroxylase inhibition into cancer therapeutics: From orbital interactions at hydroxylase active sites to misfolded protein-driven cancer cell death and immunogenic cancer cell apoptosis [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 1591.