Novel non saccharide glycosaminoglycan mimetic inhibit colorectal cancer stem cells by selectively targeting key tyrosine kinase receptors.

Introduction: Unique sulfated non-saccharide glycosaminoglycan mimetic (NSGM) of heparin hexasaccharide (HS06), G2.2, selectively inhibited CSCs via inhibition of specific tyrosine kinase receptors, resulting in selective activation of p38 mitogen-activated protein kinases (MAPK). To further improve the potency and pharmacology of G2.2, novel analogs MQD1 and MQD1-8C (cholesterol-modified to improve oral activity and potency) were rationally designed using computational molecular modeling and molecular dynamics. The goal of the present studies was to examine the efficacy, toxicities, and mechanisms of novel NSGMs. Method: HT-29 and HCT-116 colorectal cancer (CRC) cells were grown as 3D spheroids or monolayers and treated with NSGMs (0→1mM) to assess 1◦→3◦ spheroid formation and viability (MTT), respectively. NCr nude mice were inoculated with 105 CD133 hi/CXCR4 hi (Dual hi) HT-29 cells s.c. and were treated with FUOX (5-FU 25mg/kg and oxaliplatin 2mg/kg weekly x3) to further enrich CSCs in vivo, followed by the second randomization to the vehicle, FUOX, NSGM (100 mg/kg 3 times a week IP or 5 times a week oral gavage), and/or a combination of NSGM + FUOX x 3 weeks. Xenografts were analyzed ex vivo for CSC expression and function. Additionally, toxicity was assessed in normal intestinal stem cells ex vivo using an organoid formation assay. Standard methods such as western blotting and flow cytometry were performed for protein analysis, and Q-PCR was used for RNA analysis. Human Receptor Tyrosine Kinase Proteome array (R&D) and PamGene Kinase profiling were performed to decipher the mechanism of action. Results: All tested NSGMs inhibited 1◦→3◦ spheroid formation and CSC markers (CD133, LGR5, OCT4, NANOG) expression. All NSGMs inhibited xenograft growth when given i.p. In addition, MQD1-8C also inhibited xenograft growth when given orally despite harboring several sulfates in its backbone. Furthermore, post-treatment xenograft cells demonstrated sustained ex vivo growth inhibition for up to 4 passages in the absence of further treatment, strongly suggesting inhibition of self-renewal. More importantly, MQD1-8C displayed minimal toxicity, as observed through biochemical assessments of vital organ function, cell counts, and ex vivo analyses of normal intestinal stem/progenitor cell (NSC) function. Interestingly, the parent molecule G2.2. Conclusion: Hence, MQD1-8C is a potent and highly selective inhibitor of IGF-1R representing a first-in-class orally active anti-CSC agent to treat CRC.