Suppression of GPR56 expression by pyrrole-imidazole polyamide represents a novel therapeutic drug for AML with high EVI1 expression

Abstract

G protein-coupled receptor 56 (GPR56) is highly expressed in acute myeloid leukemia (AML) cells with high EVI1 expression (EVI1high AML). Because GPR56 is a transcriptional target of EVI1 and silencing of GPR56 expression induces apoptosis, we developed a novel drug to suppress GPR56 expression in EVI1high AML cells. For this purpose, we generated pyrrole-imidazole (PI) polyamides specific to GPR56 (PIP/56-1 or PIP/56-2) as nuclease-resistant novel compounds that interfere with the binding of EVI1 to the GPR56 promoter in a sequence-specific manner. Treatment of EVI1high AML cell lines (UCSD/AML1 and Kasumi-3) with PIP/56-1 or PIP/56-2 effectively suppressed GPR56 expression by inhibiting binding of EVI1 to its promoter, leading to suppression of cell growth with increased rates of apoptosis. Moreover, intravenous administration of PIP/56-1 into immunodeficient Balb/c-RJ mice subcutaneously transplanted with UCSD/AML1 cells significantly inhibited tumor growth and extended survival. Furthermore, organ infiltration by leukemia cells in immunodeficient Balb/c-RJ mice, which were intravenously transplanted using UCSD/AML1 cells, was successfully inhibited by PIP/56-1 treatment with no apparent effects on murine hematopoietic cells. In addition, PIP treatment did not inhibit colony formation of human CD34+ progenitor cells. Thus, PI polyamide targeting of GPR56 using our compound is promising, useful, and safe for the treatment of EVI1high AML.

EVI1 dysregulation: impact on biology and therapy of myeloid malignancies

“…EVI1 interactions with the microenvironment are also implicated in apoptosis-resistance. The adhesion molecules ITGA6 (integrin subunit alpha 6) and GPR56 (adhesion G protein-coupled receptor G1) are highly expressed in EVI1-positive AML, and their knockdown leads to increased apoptosis in response to Ara-C treatment and loss of RhoA (ras homolog family member A) signaling, respectively 71 , 72 . In AML, cells high EVI1 expression correlated with high MYC and BCL2 expression, with poorer clinical outcome 73 .…”

“…EVI1-mediated upregulation of the anti-apoptotic Bcl-xL suggests that BH3-mimetic inhibitor targeting this anti-apoptotic protein could also have therapeutic value, alone or in combinations against EVI1-expressing myeloid malignancies. Recently, the small molecule compound pyrrole-imidazole polyamide, which inhibits the DNA-binding activity of N-terminal ZF domain of EVI1, was shown to induce apoptosis of EVI1-expressing AML cells due to downregulation of the EVI1 target GRP56, which was linked to EVI1-mediated resistance to apoptosis 71 . Several therapies targeting anti-apoptotic proteins are in clinical trials, and these agents could potentially be employed in treatment of EVI1-expressing myeloid malignancies.…”

The role of GPR56/ADGRG1 in health and disease

“…EVI1 directly binds to the promoter region of GPR56 and regulates its expression in these EVI1 high AML cells [ 74 ]. Furthermore, therapeutically useful and novel pyrrole-imidazole polyamides (PIPs) have been developed that can specifically bind to the GPR56 promoter and inhibit the binding of EVI1, thereby inhibiting its expression and thus suppressing the cell growth and promoting apoptosis [ 77 , 78 ]. The expression levels of GPR56 and white blood cell count at diagnosis are significantly associated in ALL [ 79 ].…”

Acute Myeloid Leukemia Stem Cells: The Challenges of Phenotypic Heterogeneity

“…Inhibition of GPR56 in leukemic cells decreased BM and tissue infiltration capacity, indicating a functional role in AML LSC. Mechanistically, GPR56 loss was associated with increased leukemic cell apoptosis and impaired ability of LSC to adhere in the BM niche in a RhoA-dependent manner, while colony formation interestingly remained unchanged [ 19 , 100 , 101 ]. Finally, targeting AML cells using a blocking anti-GPR56 antibody demonstrated anti-leukemic activity and prolonged survival in PDX assays [ 101 ].…”