P424: INVESTIGATING GPR56 FUNCTION IN HUMAN IPSC-DERIVED HEMATOPOIETIC CELLS USING A TET-ON MODEL OF CONSTITUTIVE ACTIVATION

Abstract

Background: GPR56 (ADGRG1) is a widely expressed adhesion G-coupled protein receptor in humans and mice which is implicated to play a role in both haematopoietic development and Acute Myeloid Leukaemia (AML). In AML, there exists a low frequency subpopulation of leukaemic stem cells (LSCs) which, similar to their benign Hematopoietic Stem Cell (HSC) counterparts, exhibit stem cell behaviours. High expression of GPR56 forms part of the molecular signature of these LSCs, and AML cases with high levels of GPR56 at diagnosis have poorer outcomes. Our hypothesis is that GPR56 activity underlies some of the stem cell behaviours seen in both HSCs and LSCs. Aims: To characterise the effects of GPR56 receptor activation so as to better understand its role in AML and hematopoietic development. Methods: We developed a Tet-On human inducible stem cell line (M1) transfected with a construct encoding a truncated GPR56 protein. This constitutively active GPR56 receptor form is overexpressed following doxycycline administration. Flow cytometry-based cell cycle and apoptosis studies were performed on the M1 line and wild type Sci55 (WT) iPSCs +/- doxycycline exposure. IncuCyte® transmigration studies utilising an SDF-α gradient were performed to examine cell migration. Finally, the M1 cells were subjected to a hematopoietic differentiation protocol and assessed for CD34+ CD38- compartment size at day 12 of differentiation. Results: GPR56 activation alters cell cycle progression with significant accumulation of M1 cells in the G0/1 and G2/M phases of the cell cycle compared to WT (p <0.005). GPR56 activation also causes a significant reduction in apoptotic cells in dox-treated M1 cells (p <0.005), despite doxycycline being pro-apoptotic in WT cells (p <0.05). GPR56 activation reduces the number of transmigrating M1 cells in response to an SDF-α gradient (p <0.05). Importantly, GPR56 activation during differentiation increases the CD34+ CD38- compartment size in M1 cells treated with doxycycline at day 12 (p<0.05) as compared to untreated cells Summary/Conclusion: GPR56 activation results in accumulation of undifferentiated M1 iPSCs in the G0/1 and G2/M phases, suggestive of cell cycle checkpoint arrest, and exerts a powerful anti-apoptotic effect. GPR56 activation also reduces cell transmigration in response to SDF-α, suggesting receptor involvement in cell adhesion and migration. Finally, GPR56 activation during hematopoietic differentiation results in an increased yield of CD34+ CD38- cells, suggesting that GPR56 activity may influence the proportion of immature cells arising during haematopoietic differentiation. These results are of interest in relation to normal HSC functions and may indicate the mechanisms through which overexpression of GPR56 (with increased activation) in LSCs contributes to poorer outcomes in AML. Future work will include identification and characterisation of the signalling pathways downstream of the GPR56 receptor to better understand this receptor’s role in the haematopoietic system in health and disease.