TTHX1114 Guards Against Ferroptosis in Primary Rabbit Corneal Endothelial Cells

Posterboard#: B0599

Abstract Number: 4147 - B0599

AuthorBlock: Jessica Weant¹, Anna Stuhlfire¹, Lindsey Myers¹, Sarah Pizzuto¹, Tyler Todd¹, Grace Duffey¹, David Eveleth^1
1Trefoil Therapeutics, San Diego, California, United States;

DisclosureBlock: Jessica Weant, Code E (Employment) Trefoil Therapeutics, Anna Stuhlfire, Code E (Employment) Trefoil Therapeutics, Lindsey Myers, Code E (Employment) Trefoil Therapeutics, Sarah Pizzuto, Code C (Consultant/Contractor) Trefoil Therapeutics, Tyler Todd, Code E (Employment) Trefoil Therapeutics, Grace Duffey, Code E (Employment) Trefoil Therapeutics, David Eveleth, Code O (Owner) Trefoil Therapeutics, Code I (Personal Financial Interest) Trefoil Therapeutics, Code E (Employment) Trefoil Therapeutics

Purpose
Fuchs’ Dystrophy, cataract surgery, and corneal edema are associated with oxidative stress. tert-Butyl hydroperoxide (tBHP) damage affects cell DNA, mitochondria, gene expression profiles, apoptotic signaling, and causes death by ferroptosis. Herein we interrogate a treatment model where our drug, a synthetic analog of FGF1 - TTHX1114 (TTHX), safeguards primary rabbit corneal endothelial cells (pRCEnCs) from ferroptosis.

Methods
pRCEnCs were harvested from Descemet’s Membranes. Cells were subjected to oxidative stress via 50µM tBHP insult for 2 hours and received either 50ng/mL TTHX or serum free media. The MTT assay was used to quantify cell viability. Cells were fixed, permeabilized, and stained with TUNEL, ZO-1, and Hoechst. Cell density and TUNEL counts were performed with ImageJ. RNA extraction and library prep performed with Illumina NEBNext Ultra II RNA Library Prep Kit. Sequencing libraries were clustered on one flow cell and sequenced with Illumina HiSeq. Analysis performed by ROSALIND and DAVID.

Results
MTT absorbance data show loss of pRCEnC viability and loss of cell metabolic function after 50µM tBHP insult. TTHX treatment protects and rescues cell viability and metabolic function. The MTT signal is not significantly different from un-insulted growth media baseline control. Fluorescent stained cell images and cell counts show tBHP insulted TTHX treated cells have clearly defined and regular ZO-1 boarders and no TUNEL stain was observed. TUNEL was observed in other cell protection and rescue treatments. TTHX cell counts: 142.7 ± 54.1. Growth media cell counts: 137.7 ± 17.8. tBHP insult only condition could not be counted for cell density due to cell death, but definite TUNEL staining was present. RNA Seq gene expression shows 50µM tBHP 2-hour insult caused strong upregulation in essential and associated ferroptosis genes: ACSL4, HMOX1, GCL, SLC7A11, SLC3A, and Ferroportin. TTHX treatment blocks upregulation of those genes and promotes enrichment of proliferative pathways and pathways related to oxidative stress. Anti-ferroptotic genes are upregulated in TTHX treated cells, but ferroptosis is not enriched.

Conclusions
By reducing cell loss and DNA breakage; perturbing ferroptotic processes; and upregulating cell survival, proliferation, and anti-ferroptosis genes, a pre/post treatment with 50ng/mL TTHX is ideal for maximizing protection and rescue of pRCEnCs from oxidative stress and cell death.