Abstract 6353: Cholesterol modification enhances potency and pharmacokinetic properties of a selective cancer stem cell targeting agent

Increasing evidence supports the concept that tumor-initiating cancer stem cells (CSCs) are responsible for the relapse and reconstitution of formerly treated tumors. Previously, our lab identified a potent and selective inhibitor of colorectal CSCs, named G2.2, from a library of non-saccharide glycosaminoglycan mimetics (NSGMs). To further improve upon the potency, pharmacokinetics, and drug-like properties of G2.2, cholesterol-modified analogs were rationally designed using computational molecular modeling and molecular dynamics studies. A small group of designed NSGMs, labeled as G2C, G5C and G8C, were synthesized for biological evaluation. These agents and the parent NSGM G2.2 were studied for their ability to inhibit the growth of primary spheroids from a panel of 15 human colorectal cancer (CRC) cells representative of the consensus molecular subtypes of human CRC. Spheroid formation frequencies (SFFs) of cell lines were measured using limiting dilution assays (LDAs), and IC50 of inhibition was evaluated from dose-response curves for monolayer growth and primary spheroid growth in the presence of NSGMs. Selectivity of CSC targeting was assessed from the ratio of IC50,monolayer to IC50,spheroid. Pharmacokinetics (PK) and bioavailability in rodents were deduced following IV and PO administrations. In-vivo therapeutic potential of the NSGMs was studied in CSC-induced xenograft models aided by ex-vivo CSC phenotype characterization. Each NSGM preferentially targeted 3D spheroidal growth in comparison to monolayer cellular growth by 12- to 450-fold, suggesting excellent CSC selectivity. Cholesterol modification of G2.2 enhanced in-vitro spheroid inhibition potency across the entire panel of cell lines on average by 3- to 21-fold. G8C showed the highest inhibition potency with an IC50 of 1 to 10 μM across cell lines. Cells with higher SFF were several-fold more sensitive to NSGMs, suggesting that basal expression of CSCs could predict NSGM response. Robust reductions in tumor volumes were observed in mice treated with G5C (100 mg/kg i.p. 3x a week) following an intial-treatment with oxaliplatin and 5-fluorouracil (weekly x3) compared to vehicle controls. This reduction was concomitant with a 3.9-fold reduction in colonic crypt stemness marker LGR5+ cells and 2.6-fold decrease in xenograft-derived tertiary spheroids. No gross or organ specific toxicity was found in animals treated with G5C. Finally, cholesterol modification greatly improved PK and oral bioavailability of parent G2.2. Overall, cholesterol modification of CSC-selective G2.2 was found to enhance in vitro potency against spheroidal growth without compromising selectivity toward tumor initiating CSCs. The G2.2 analogs offered robust tumor reductions in vivo with essentially no off target toxicity, and serve as promising and novel potential cancer therapeutics. Citation Format: Connor P. O9Hara, Rio S. Boothello, Shravan Morla, Alberto Vera, Daniel K. Afosah, Nehru V. Sankaranarayanan, Balaji Nagarajan, Chetna Sharon, Bhaumik B. Patel, Umesh R. Desai. Cholesterol modification enhances potency and pharmacokinetic properties of a selective cancer stem cell targeting agent [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6353.