The laminin receptor integrin α7β1 marks a highly migratory cell population and correlates with poor prognosis in Acute Myeloid Leukemia.

Abstract

Abstract Introduction: Interactions with the bone marrow (BM) niche are crucial for self-renewal and survival of acute myeloid leukemia (AML) cells, thereby promoting therapy resistance. Consequently, AML cells express a variety of surface receptors to engage with BM niche cells and extracellular matrix proteins, including laminins. However, despite the association of laminin receptors with stemness in healthy hematopoiesis, their role in AML remains poorly understood. Methods Here we used publicly available datasets (GSE1159, GSE6891 and survival data from the TCGA-AML cohort) next to a panel of seven AML cell lines and 64 primary AML patient samples to comprehensively examine the expression of the laminin receptors integrin α3β1, α6β1, α7β1 and basal cell adhesion molecule (BCAM) in AML stem and non-stem cells using bioinformatic analyses and multiparametric flow cytometry, respectively. The role of integrin α7 was furthermore explored functionally in AML cell lines and primary cells using various in vitro experiments as well as in vivo migration and homing assays in zebrafish and mouse xenografts. Results Analysis of the GSE1159 and GSE6891 datasets indicated that high mRNA expression of all four laminin receptors correlates with poor overall survival. In line, the analysis of primary AML samples (n=40-42) by trend revealed a higher frequency of integrin α6+, integrin α7+ and BCAM+ cells in adverse vs favorable risk AML subtypes (ELN2017). Notably, integrin α6 and α7 displayed the highest cell surface density among the examined laminin receptors with a mean of 27% and 34% of positive cells, respectively, and higher expression on AML cells compared to healthy controls (8% and 7%, respectively). Co-staining of markers used to identify leukemic stem cells (LSC) such as CD34 and NKG2DL indicated that integrin α6, integrin α7 and BCAM expression associates with non-LSC populations, while integrin α3 predominantly marks LSCs. Corroborating these results, colony forming unit (CFU) assays with primary AML samples (n=9) sorted into integrin α7- and integrin α7+ (or CD34+/integrin α7- and CD34+/integrin α7+) populations showed that integrin α7 expression can be used to identify (and refine) the LSC compartment, even in CD34-negative patients. Importantly, re-analyzing survival data from the TCGA-AML cohort, we found that integrin α7 expression can improve the risk prediction in combination with the LSC score, where patients with low LSC and low integrin α7 levels have the best outcome. Additionally, transcriptomic analyses of sorted integrin α7+ vs integrin α7- cells from primary AML cases (n=4) as well as transmigration assays with sorted or unsorted samples (n=4-7) indicated that integrin α7+ cells have enhanced migratory potential, which can be inhibited by treatment with an anti-integrin α7 blocking antibody. In contrast, adhesion to laminin coated wells remained unaffected (n=3). Lastly, xenotransplantation of AML cells pre-treated with the anti-integrin α7 blocking antibody or IgG control into the yolk of zebrafish embryos or the tail vein of NSG mice, respectively, confirmed the requirement of integrin α7 for migration and homing in vivo (11% vs 31% of zebrafish embryos showing active migration; 12 vs 22 cells detected in the bone marrow of NSG mice). Conclusions Our study highlights the association of elevated laminin receptor expression with poor prognosis in AML, and establishes integrin α7 as marker of high migratory leukemic cells. A better understanding of the integrin-mediated interactions of AML cells with the bone marrow niche might be instrumental for the development of future therapies designed to overcome niche-mediated therapy resistance in AML.