G Protein-Coupled Receptor 56 As a Potential Regulator of Normal and Leukemic Stem Cells

Therapy-resistant leukemic stem cells (LSCs) sustain acute myeloid leukemia (AML) and must be eliminated to cure a patient. However, there are no specific anti-LSC therapies, partially due to poor understanding of the unique molecular biology of these cells. Human LSCs share many properties with normal hematopoietic stem cells (HSCs), with similar gene expression profiles and molecular regulators. GPR56 is an adhesion class G-protein coupled receptor (GPCR) that has recently been implicated in the developmental origins of murine HSCs as well as regulating the function of adult murine HSCs, although this is controversial. Previous studies have observed that GPR56 is involved in cell adhesion, migration and differentiation in AML cell lines. Whether GPR56 is part of the molecular regulation of LSCs remains unknown. This study aims to establish whether GPR56 regulates the function of human LSCs and HSCs.

To explore the role of GPR56 in human stem cells, we first investigated the cell-specific expression of GPR56 by qRT-PCR. GPR56 is highly expressed in primary human LSCs, normal blood HSCs, and normal progenitors but not in more mature populations in normal or leukemic blood. Next, to determine if GPR56 is clinically relevant in human disease, we examined GPR56 gene expression in three cohorts of cytogenetically normal AML samples (intermediate risk) and observed a positive correlation with poor outcome across all cohorts (GPR56 P<0.006 in each cohort). Likewise, in a large cohort of AML patients that includes abnormal cytogenetic samples, GPR56 was more highly expressed in poor and intermediate cytogenetic risk patient samples than in good cytogenetic risk patient samples. This pattern was also observed in a cohort of pediatric AML samples, indicating that GPR56 may play a role in both adult and pediatric disease.

We next examined the effect of full length GPR56 overexpression in normal HSC using xenograft assays to determine the functional role of GPR56 in human blood stem cells. Lineage negative cord blood cells transduced with GPR56 overexpression lentiviral vectors were injected into immune-deficient mice and the engraftment of human CD45+ cells was measured by flow cytometry after 12 weeks. In this in vivo analysis, over-expression of GPR56 increased the total engraftment of human cells in immune-deficient recipients compared to cells transduced with a control gene (hRluc, p<0.0001). This advantage was maintained in secondary engraftment (p=0.0375, total 24 weeks). The lineage distribution and percentage of stem and progenitor cells (CD34+, CD90+) was the same in GPR56 overexpressing clones and control cells, indicating that the increased engraftment is due expansion of the stem and progenitor cells and not due to preferential expansion or survival of mature cells.

This data suggests that GPR56 may regulate human blood stem cells, including LSCs, and that GPR56 expression may contribute to poor outcome in high prognostic risk subgroups of human acute myeloid leukemia through effects on leukemic stem cells. We are further exploring a functional role for GPR56 in the regulation of LSCs and HSCs using in vitro and in vivo assays in primary tissue. This will provide insight into the molecular regulation HSCs and LSCs and potentially explain the correlation between high levels of GPR56 and poor outcome in AML patients.