Glycomimetic Antagonist of E-Selectin, GMI-1271, Enhances Therapeutic Activity of the Hypomethylating Agent, 5-Azacitidine, in the KG1 Model of AML

Abstract

Components of the bone marrow stroma afford a permissive environment for the malignant progression of AML. These stromal components include E-selectin, an adhesion molecule expressed in the bone marrow vasculature. Through the recognition and binding of E-selectin by specific ligands on AML blasts, survival pathways are activated within the blasts leading to the development of/increase in chemoresistance. GMI-1271 is a novel E-selectin antagonist that disrupts this process, down-regulating survival pathways and enhancing chemotherapy response with improved survival in mouse xenograft and syngeneic AML tumor models. GMI-1271 is currently in clinical trials for the treatment of AML in combination with intensive chemotherapy; the FDA has granted Breakthrough Therapy Designation to GMI-1271 for the treatment of adults with relapse/refractory AML. Azacitidine (5-AZC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. Vidaza (5-AZC, Celgene) is approved in Europe for the treatment of limited populations with AML. In these studies we evaluated the anti-tumor activity of GMI-1271 in combination with 5-AZC in the KG1 AML tumor model to identify if there was potential for therapeutic benefit of the combination.Seven days post i.v. injection of KG1 cells, female NSG mice (n=10/group) were treated with saline; GMI-1271 (40 mg/kg IP QDx14) alone; 5-AZC (5 mg/kg IP Q3Dx5) alone, or the combination of GMI-1271 and 5-AZC. All treatments were well tolerated. The median survival time (MST) of mice treated with 5-AZC was 88 days and statistically different (P<0.005) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). At study conclusion (Day 110 post tumor injection), 20% of mice treated with 5-AZC remained alive; as expected all mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. Importantly, the therapeutic activity of 5-AZC was significantly enhanced when combined with GMI-1271 (MST>110 days, P=0.0217 compared to 5-AZC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AZC and that GMI-1271 can attenuate this protection.To investigate the nature of the observed in vivo activity of GM-1271 and 5-AZC in the KG1 model, in vitro assays of cytotoxicity and E-selectin ligand expression in KG1 and the KG1a variant were assessed. The IC50 of 5-AZC alone or in combination with a saturating dose of GMI-1271 on KG1 cells was 518 and 528 nM, respectively. The IC50 of 5-AZC alone or in combination with GMI-1271 on KG1a cells was 637 and 711 nM, respectively. These results imply that the enhanced anti-tumor activity observed in vivo was not due to a simple shift in the cytotoxicity profile of 5-AZC. KGI and KG1a cells were subsequently cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AZC and the reactivity of the cells to HECA-452, a surrogate marker of E-selectin ligand, or binding E-selectin was determined by flow cytometry. Treatment with 5-AZC resulted in an increased reactivity of KG1 and KG1a cells to HECA-452 (29 and 27%, respectively compared to nontreated cells) and an increased binding of E-selectin to KG1 cells (32% compared to nontreated cells). Collectively, these results demonstrate that noncytotoxic concentrations of 5-AZC lead to an increased expression of E-selectin ligands on KG1 and KG1a cells.In summary, these studies show that even though the hypomethylating agent 5-AZC improves outcomes with AML, therapeutic potential could be improved. In this regard, 5-AZC at noncytotoxic concentrations promotes the increased expression of E-selectin ligands on AML blasts which may dampen 5-AZC activity. However, attenuation or disruption of E-selectin ligand/E-selectin interactions with GMI-1271 in combination with 5-AZC overcomes this tumor survival pathway with the potential to maximize therapeutic outcome. This work has relevance to patients with AML who do not achieve optimal or long-term response with 5-AZC and other hypomethylating agents. Clinical assessment of GMI-1271 with hypomethylating agents in AML is planned. DisclosuresFogler:GlycoMimetics: Employment, Equity Ownership. Smith:GlycoMimetics: Employment, Equity Ownership. Lee:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.