The Adhesion Molecule “G-Protein Coupled Receptor 56” (Gpr56) Cooperates with the Homeobox Gene Hoxa9 to Induce Acute Myeloid Leukemia in Mice
Authors: Daria Deidre , Muranyi Andrew L , Ihme Susann , Döhner Konstanze , Döhner Hartmut , Feuring-Buske Michaela , Buske Christian 
Published: October 2019

Abstract
Interactions between leukemic cells and the bone marrow microenvironment are essential for the maintenance and progression of myeloid leukemias. G-protein coupled receptor 56 (Gpr56) is an adhesion molecule which collaborates with the extracellular matrix through interaction with collagen III binding and transglutaminase 2, and by this activating the Rho A response pathway. In the hematopoietic hierarchy, GPR56 expression is highest in stem cells and decreasing in expression with differentiation. It is a poor prognostic factor in cytogenetically normal acute myeloid leukemia (AML), and was identified as a member of a signature expressed in functionally validated human AML stem cells (Eppert et al., Nature Medicine 2011). To test its functional relevance for AML, we first analyzed the expression of GPR56 in primary patient samples (n= 74) with different genotypes from the AML dataset available in the TCGA database. Normal karyotype patients showed significantly higher expression of GPR56 (3.1 fold p<0.05), when compared to patient samples with translocations. Analysis within the normal karyotype dataset showed high GPR56 expression independent of NPM1 or FLT3-ITD mutations. Quantification of GPR56 expression by TaqMan PCR confirmed high expression both in the CD34-positive and CD34-negative leukemic subpopulations in normal karyotype AML, indicating high expression of this protein in the compartment enriched for leukemic stem cells. As GPR56 expression was high in normal karyotype AML, known to be characterized by aberrant homeobox expression, we tested the functional relevance of Gpr56 expression in a murine Hoxa9 bone marrow (BM) transplantation model. Aberrant expression of Hoxa9 in 5-FU mobilized stem and progenitor cells increased GPR56 expression 1.3 fold (p<0.05), but failed to induce short latency AML up to an observation time of over 160 days post transplant. Retrovirally engineered aberrant overexpression of Gpr56 alone also did not induce any perturbations with regard to engraftment and cell differentiation over 90 days post transplant in mice. However, overexpression of Hoxa9 together with Gpr56 induced a significant and 1.8 fold increase in colony forming cell activity (CFC) (p<0.05), followed by a 1.6 fold (p<0.05) increase in secondary CFCs when compared to HoxA9 alone. Mice transplanted with BM cells co-transduced with HoxA9 and Gpr56 rapidly developed AML after a median latency of 139 days post transplant (HoxA9 MIEG3 N=8, HoxA9 Gpr56 N=10, p<0.05). The mice displayed splenomegaly (mean 0.47g ± 0.07), and a high expression of myeloid markers on leukemic cells (Mac-1: 31%-57%; Mac-1/Gr-1: 24%-53%; Gr-1: 0.5%-16%). Cells co-transduced with Gpr56 and HoxA9 showed a decrease in Cxcr4 expression, when compared to HoxA9 alone and a 6-fold reduction in CFCs (p<0.05) when cells were introduced to an in-vitro SDF-1 gradient migration assay. However, BM cells co-transduced with Hoxa9 and Gpr56 did not show any significant drop in their homing capacity in vivo. Of note, leukemogenicity of Hoxa9 BM cells co-transduced with Meis1 was dependent on Gpr56 expression, as knockdown of Gpr56 expression via shRNA doubled the latency time in transplanted mice (median latency 55 days compared to 95 days, respectively p<0.05). Knockdown of Gpr56 induced a decrease in cells capable of attaching to the stroma in the adhesion cobble-stone area forming cell assay (CAFC) (6.5 fold reduction). However, cells able to home to the BM in vivoincreased 2.3 fold (p<0.05), suggesting that the delay in disease progression was due to a decrease in attaching to the niche.
In summary, our data characterize Gpr56 as an essential partner in Hoxa9 associated myeloid leukemogenesis, underlining the importance of members of the adhesion-GPCR family of receptors in myeloid leukemogenesis.

Disclosures
No relevant conflicts of interest to declare.