Adhesion Of Acute Myeloid Leukemia Blasts To E-Selectin In The Vascular Niche Enhances Their Survival By Mechanisms Such As Wnt Activation

Adhesion within the bone marrow microenvironment enhances leukemia survival and chemoresistance. Both normal hematopoietic stem cells and cancer cells are known to express E-selectin ligands, and adhesion of colon carcinoma cells to E selectin activates survival pathways such as NFκB (Porquet et al., BMC Cancer 11:285, 2011). E-selectin within the bone marrow vascular niche induces proliferation of normal hematopoietic stem cells (HSC), and a selectin inhibitor enhances HSC quiescence and self-renewal (Winkler et al., Nat Med 18:1651, 2012). We therefore initiated a study of E-selectin ligand expression and function by acute myeloid leukemia (AML) blasts to elucidate the potential role of E-selectin in AML biology and chemotherapy resistance.

Primary AML blasts and leukemia stem cells (LSCs) (CD34⁺CD38^-CD123⁺) obtained with informed consent from 40 patients were analyzed for E-selectin ligands by flow cytometry for binding of E-selectin-Fc chimera or by labeling with the HECA452 antibody. Primary AML blasts were engrafted in NODscid IL2Rgc-/- mice for studies of E-selectin inhibitors in combination with chemotherapy. Human Stem Cell Signaling, Leukemia, Apoptosis, and NFκB PCR Arrays (SA Biosciences) were used to analyze gene expression by quantitative RT-PCR after adhesion of primary AML patient blasts to E-selectin coated plates, compared to bovine serum albumin (BSA) coated plates. The activation of the Wnt pathway was studied by luciferase reporter assay.

We find that the majority of primary patient acute myeloid leukemia blasts and leukemia stem cells express an E-selectin ligand, as demonstrated by flow cytometry by binding of E-selectin-Fc chimera and by staining with HECA-452 antibody [that recognizes hematopoietic cell E-/L-selectin ligand (HCELL) and cutaneous lymphocyte antigen (CLA)], as well as by binding to E-selectin coated plates. Flow cytometry analysis reveals that the mean percent binding of E-selectin-Fc chimera is 28% ± 24% (SD) by AML blasts, the mean % staining by the HECA-452 antibody 51% ± 35% (SD). De novo patients tended to have smaller mean fluorescence intensity (MFI) values than relapsed/refractory patients, as follows: for blasts, de novo mean 1441 ± 1127 (SD) vs. relapsed/refractory 4488 ± 4920 (SD) (Wilcoxon p=0.024), and for LSCs, de novo mean 1578 ± 1560 (SD) vs. relapsed/refractory 6601 ± 8498 (SD) (Wilcoxon p=0.061), suggesting upregulation of expression of E-selectin ligand for relapsed as compared to newly diagnosed patients. The specific E-selectin small molecule inhibitor GMI-1271 is able to overcome adhesion mediated chemotherapy resistance of AML in vitro and reduce the leukemia burden of primary AML engrafted NODscid IL2Rgc^-/- mice in combination with chemotherapy agents daunorubicin and cytarabine. Addition of GMI1271 to chemotherapy in this xenograft model reduced the spleen burden at 2 weeks post treatment from 17.1 ± 10.4 X 10⁶ hCD45⁺ cells/spleen to 7.6 ± 5.8 (p=0.04).

To assess the molecular mechanism by which adhesion to E-selectin might protect AML blasts, we first screened with quantitative RT-PCR arrays. We found that adhesion to E-selectin caused upregulation of members of the Wnt and sonic hedgehog pathways for primary AML patient blasts grown on E-selectin vs. BSA coated plates, as well as members of other pathways critical to leukemia such as GM-CSF and IL-3 receptors and Fos. We then confirmed adhesion to E-selectin by AML blasts from 4 different patients enhanced activity of Wnt target genes by Wnt reporter assays. The Wnt reporter assay demonstrated 2-3 fold enhanced activity of Wnt target genes for AML blasts on E-selectin as compared to those on BSA, which increased to 3.3-4.5 fold with addition of Wnt3a. The inhibitor GMI-1271 reduced Wnt activity to 1.4-2.5 fold, similar to XAV939, an inhibitor of the Wnt/β catenin pathway that reduced activity to 1.1-1.8 fold. Similar reduction of Wnt pathway gene expression by GMI-1271 was also observed by the quantitative RT-PCR assay.

These data support a critical role for E-selectin, likely in the vascular bone marrow niche, that promotes survival of AML, that can be targeted with therapeutic intent, and suggests that GMI-1271 should be explored as a treatment for AML in combination with chemotherapy.

Chien:GlycoMimetics, Inc.: Research Funding. Haq:GlycoMimetics, Inc.: Research Funding. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Becker:GlycoMimetics, Inc.: Research Funding.