Vascular E-Selectin Mediates Chemo-Resistance in Acute Myeloid Leukemia Initiating Cells Via Canonical Receptors PSGL-1 (CD162) and Hcell (CD44) and AKT Signaling

The vascular adhesion molecule E-selectin is a key component of the bone marrow (BM) vascular niche, awakening otherwise dormant hematopoietic stem cells (HSC) (Winkler et al., Nat Med 2012). Vascular niches also mediate malignant cell survival. We find E-selectin becomes upregulated on BM vasculature during leukemia and that adhesion of BM AML blasts to E-selectin promotes survival signaling. This is unique to E-selectin - not observed with adhesion to P-selectin, PECAM1, or integrin ligands ICAM1, VCAM1. In vivo we find absence (in SELE^-/- mice) or therapeutic blockade of E-selectin by administration of E-selectin antagonist GMI-1271 to mice, induces 9-fold greater chemosensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine therapy in vivo and doubles survival over that achieved by chemotherapy alone. Together these data support the Phase I/II clinical trial of GMI-1271 in combination with intensive chemotherapy to improve efficacy of therapy for AML (NCT02306291), and raise the further questions: 1) what are the AML LIC ligands interacting with E-selectin at the vascular niche and 2) what are the pathways initiated by E-selectin adhesion that mediate chemoresistance.

Using murine models of AML generated by retroviral transduction of the 11q23 fusion oncogene MLL-AF9 into HSC, we find leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. To investigate if this is due to aberrant glycosylation facilitating the generation of de novo AML cell surface receptors, we generated AML from HSC knocked-out for the two canonical E-selectin receptors PSGL1 and CD44. Surprisingly we found although HSC from CD44^-/-PSGL1^-/- mice still bound to E-selectin (indicating HSC express additional E-selectin ligands), AML cells generated from these same HSCs no longer did. Thus the functional E-selectin ligands utilized by murine HSC and AML blasts differ.

When transplanted into recipient mice CD44^-/-PSGL1^-/- AML engrafted with similar disease progression to matching wildtype AML but demonstrated ~100-fold greater sensitivity to high-dose cytarabine chemotherapy. When GMI-1271 was co-administered together with chemotherapy no further boost in chemosensitivity was observed in CD44^-/-PSGL1^-/- AMLs. This complements in vitro data showing E-selectin adhesion-mediated chemoresistance is lost when CD44 and/orPSGL1are absent in AML cells. Together suggesting that CD44 and/or PSGL1 are the key receptors involved in E-selectin-mediated pro-survival signaling.

To understand the pro-survival signaling pathways involved, E-selectin antagonist GMI1271 or saline control were administered 5 days to mice with AML and BM KIT⁺ leukemic blasts sorted. RNA sequencing revealed only a small number (~200) of mRNA transcripts differentially regulated following therapeutic E-selectin blockade. Of note these included down-regulation in several components of the PI3K/AKT/NF-kB signaling pathway frequently involved in cell survival. Indeed AKT Ser⁴⁷³ and NF-kB p65 Ser536 were found to be rapidly phosphorylated within 15 minutes of E-selectin adhesion in AML blasts in vitro. This phosphorylation was specific to E-selectin adhesion and not observed with other vascular adhesion molecules tested suggesting that direct E-selectin-mediated AKT activation in AML cells at the vascular niche may be the potential mechanism driving vascular mediated chemoresistance.

To confirm whether therapeutic blockade of E-selectin alone dampens intracellular AKT signaling in AML blasts, cohorts of mice with wildtype or matching CD44^-/-PSGL1^-/- AML were administered GMI-1271 for 5 days then BM AML blasts collected for signaling studies. Both the administration of E-selectin antagonist or absence of CD44 and/or PSGL-1 dampened AKT^Ser473 phosphorylation in BM AML blasts in vivo, correlating with their heightened sensitivity to chemotherapy .

In summary, these findings suggest E-selectin ligands on specific CD44 and/or PSGL1 glycoforms are the predominant determinants through which the induction/amplification of chemoresistant pathways in AML blasts occur following vascular adhesion and further that these pathways are coupled to an early phosphorylation event of AKT - and finally explain potential mechanism how GMI-1271 administration is able to attenuate vascular-mediated signaling and restore susceptibility of AML to chemotherapy.