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.
Highlights
The ecotropic viral integration site-1 (EVI1) transcription factor is well-known as a marker of poor prognosis for chemotherapy-resistant AML1–6
Since G protein-coupled receptor 56 (GPR56) expression is crucial for cell survival and cell adhesion ability for the bone marrow (BM) niche in EVI1high acute myeloid leukemia (AML) cells[15], we constructed several pyrrole–imidazole polyamide (PIP) compounds that target the EVI1-binding sequence within the GPR56 promoter, which are predicted to inhibit binding of EVI1 to the GPR56 promoter and suppress GPR56 expression
Since GPR56 expression in leukemia stem cells (LSCs) is significantly higher than that in HSCs9,15 and downregulation of GPR56 induces apoptosis of EVI1high AML cells, GPR56 may serve as a potential molecular therapeutic target for EVI1high AML
Summary
The ecotropic viral integration site-1 (EVI1) transcription factor is well-known as a marker of poor prognosis for chemotherapy-resistant AML1–6. To identify novel therapeutic targets in EVI1high AML, we analyzed gene expression profiles of EVI1high AML cells and identified GPR56, CD52 molecule (CD52), integrin α6 (ITGA6), and angiopoietin-1 (ANGPT1) as candidate targets for EVI1high AML12–14. GPR56 has been reported as a novel leukemia stem cell marker for AML16 and is a potential molecular target for refractory AML, including EVI1high AML. To suppress EVI1-dependent GPR56 expression in EVI1high AML cells, in the present study, we developed PIPs, PIP/56-1 and PIP/56-2, that target the EVI1-binding site within the GPR56 promoter[15]. Our results demonstrated that treatment of EVI1high AML cells with PIP/56-1 or PIP/56-2 efficiently inhibits GPR56 expression and suppresses cell growth with concomitant induction of p53-dependent apoptosis. GPR56-PIPs may become a new molecular targeting drug for human EVI1high AML and may possibly benefit other GPR56high AMLs
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