Abstract 2030: Oncological relevance of protein hydroxylase inhibitors (PHI): results of testing an emerging concept with an orally active pioneer medicine that blocks the hydroxylations of proline and lysine residues
Authors: Hanauske-Abel Hartmut M. , Cracchiolo Bernadette M. , Singh Sukhwinder , Hanauske Axel-Rainer 
Published: July 2021

Abstract
Background & Objectives: 40 years ago, the HAG mechanism specified the sequence of orbital interactions that underlie the synthesis of peptide-bound hydroxyproline and hydroxylysine by ketoglutarate-utilizing dioxygenases. Instantly reducing this insight to practice, the HAG mechanism revealed the pyridine-dicarboxylate class of PHIs as part of its general, now repeatedly validated classification of PHIs. The HAG mechanism provided the physicochemical basis for breakthroughs in matrix formation and oxygen sensing, yet its clinical impact is less evident. We employed the HAG mechanism to discover pioneer PHIs among approved medicines. We hypothesize that such drugs trap cancer cells between the hydroxylation-requiring stability of matrix proteins and the hydroxylation-requiring instability of oxygen sensing proteins – between hammer and anvil.
Methods & Results: To test our hypothesis, we relied on the PHI activity of deferiprone (DEF) at clinically relevant concentrations in cultures of normal fibroblasts (MRC5) and of an extensively annotated cancer cell line (ARK1), representative for highly lethal uterine malignancies. In MRC5, labeling with radioactive proline/lysine revealed DEF suppresses extracellular and total hydroxyproline/hydroxylysine and increases residual intracellular hydroxyproline/hydroxylysine, indicating secretory blockade of underhydroxylated thus unstable collagenous material. DEF had no effect on proline/lysine incorporation into MRC5 protein and did not reduce MRC5 survival. In ARK1, DEF likewise caused intracellular retention of collagens, established by flow cytometry using antibodies monospecific for the mainly expressed collagens type IV, VI, and XVIII. In repeated RNA-seq, expression of EGLN3, the protein hydroxylase that limits stability especially of hypoxia-inducible factor 2α (HIF-2α), at 48 hr increased in controls (+4.8 fold, FDR p &lt;10-6), but by DEF decreased (-2.9 fold, FDR p &lt;10-6) despite DEF-enhanced HIF-2α expression (+3 fold, FDR p &lt;10-6). DEF thus paradoxically secured HIF-sustained synthesis of collagen despite DEF-blocked hydroxylation of collagen. Persistent collagen misfolding in the endoplasmic reticulum (ER) triggered expression of ER stress response and cell cycle arrest genes, e.g. DDIT3 (+2.3 fold, FDR p &lt;10-6) as well as expression of apoptosis-mediating genes, e.g. HRK (+8.5 fold, FDR p &lt;10-6). Apoptosis occurred in over 90% ARK1 at 96 hr.
Conclusions: Our results confirm the concept that joint inhibition of HIF hydroxylation and of collagen hydroxylation in cancer cells renders matrix formation and oxygen sensing irreconcilably conflicting, resolved by apoptosis induction. Definition of the clinical implications, if any, may use several medicines that, like DEF, are identified by the HAG mechanism as PHIs.
Citation Format: Hartmut M. Hanauske-Abel, Bernadette M. Cracchiolo, Sukhwinder Singh, Axel-Rainer Hanauske. Oncological relevance of protein hydroxylase inhibitors (PHI): results of testing an emerging concept with an orally active pioneer medicine that blocks the hydroxylations of proline and lysine residues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2030.