A Novel Small Molecule E-Selectin Inhibitor GMI-1271 Blocks Adhesion of AML Blasts to E-Selectin and Mobilizes Blood Cells in Nodscid IL2Rgc−/− Mice Engrafted with Human AML
Authors: Chien Sylvia , Zhao Xin , Brown Margaret , Saxena Akanksha , Patton John T. , Magnani John L. , Becker Pamela S 
Published: October 2019

Abstract
Abstract 4092
The E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) is expressed by normal hematopoietic stem cells (Merzaban et al Blood 2011) as a functional glycoform of CD44. We observe high level CD44 expression (99%± 1.4%) by blasts from 55 consecutive patients with acute myeloid leukemia (AML) and by putative CD34+CD38−CD123+ leukemia stem cells (LSCs) (99.8% ± 0.6%). The mean fluorescence intensity (MFI) for CD44 expression by AML blasts is one to two logs higher than the MFI for 16 other adhesion receptors. We find that the majority of blasts from patients with AML also express an E-selectin ligand by flow cytometry: >75%% of 22 primary gated blast samples exhibit ≥10% binding of E selectin-IgG chimeric protein with a mean of 22.7% ± 0.17%SD, range 1.8 to 66.2%. We demonstrated that this ligand was HCELL by immunoprecipitation of CD44 from AML cell membranes, followed by staining with HECA 452 antibody that recognizes a functional trisaccharide domain shared by sialyl Lea and sialyl Lex and known to bind to E-selectin. HECA 452 not only detects the functional glycoform of CD44 known as HCELL, a major ligand for E-selectin, but also identifies the human lymphocyte homing receptor CLA (cutaneous lymphocyte antigen). HECA 452 labeled 5 of 6 patient leukemia blast populations, with mean expression 59.0% ± 24.8%. We also observed that HECA 452 antibody labeled CD34+CD38−CD123+ LSCs in addition to leukemic blasts, with a higher percent expression in most cases for the LSCs than the corresponding unfractionated blast population. Moreover, HECA 452 labeled 94% of human AML cells that had been serially engrafted in NODscid IL2Rgc−/− animals, fulfilling the functional definition of LSCs (scid repopulating cells), suggesting that HCELL may be enriched on LSCs. We observe a change in morphology with binding of AML blasts to E-selectin coated plastic, in that they elongate, become more cuboidal and less reflective, in contrast to the non-adherent cells, which remain round and refractile. AML blasts appear to bind to the elongated ends of spindle shaped endothelial cells. The E-selectin-specific inhibitor GMI-1271 (concentration 20 μM) inhibited adhesion of primary human AML cells to E-selectin by an average of 45.0%± 9.1%SD for all patients. For one patient, AML 035, for example, the percent inhibition with GMI-1271 compared to media control was 33.4%± 15.3%SD, p=0.000018. Adhesion to E-selectin did not confer adhesion mediated chemotherapy resistance to daunorubicin or cytarabine observed with adhesion to recombinant fibronectin peptide or immobilized VCAM-1 (Becker et al Blood 2009). We demonstrated that a dual inhibitor of E selectin and CXCR4 (GMI-1215) mobilized human AML engrafted in NODscid IL2Rgc−/− mice (Chien et al Abstract 579, ASH 2011), to a greater degree than we observed with CXCR4 inhibitor plerixafor (Chien et al Abstract 1432, ASH 2011) alone (3–4 fold vs. ∼2 fold). We now report that the E-selectin-specific inhibitor, GMI-1271 (40mg/kg), mobilizes both human and murine cells in immunodeficient xenograft mice engrafted with human AML: there is a 2 fold increase in WBC (p=0.00067) and 2 fold increase in human AML cells (p=0.14) at 3 hrs. An initial experiment with a combination of GMI-1271, daunorubicin, and cytarabine demonstrated greater depletion of human AML from the bone marrow (22% as many AML cells) and spleen (31% as many AML cells) than daunorubicin and cytarabine alone. Additional in vivo studies are in progress. We propose that residence in the bone marrow vascular niche may involve E-selectin and that migration of AML blasts involves critical interactions with the vascular endothelium through E-selectin. This interaction between HCELL expressed by AML and E-selectin might represent a new potential therapeutic target for AML.

Disclosures:
Patton: GlycoMimetics: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership. Becker:GlycoMimetics, Inc: Research Funding.