Mechanisms by which of GPCR Signaling Inhibits Acute Myeloid Leukemia

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Project Summary: Despite recent advances in cancer therapy the 5-year overall survival rate of Acute Myeloid Leukemia (AML) remains around 30%. The first line standard of care therapy for AML is chemotherapy, which is ineffective in relapsed patients and often too toxic to be administered to older patients. Targeted therapy such as venetoclax and decitabine have only shown modest effects in the clinic, calling for different approaches in targeting AML. Our lab has shown that one such approach is through the activation of the Gs-coupled G-protein coupled receptor, G-protein Coupled Estrogen Receptor (GPER). Work from our lab has shown that pharmacological activation of GPER with LNS8801 induces differentiation, promotes growth inhibition, and drives immunogenicity in melanoma without observable toxicity in animal models. I have shown that primary AML cells and AML cell lines that are exposed to LNS8801 induce cell death, which is likely through apoptosis based on positive Annexin V and PI staining, in addition to growth arrest. LNS8801 induced cell death is accompanied by the depletion of Mcl-1, which is an anti-apoptotic protein that is often overexpressed in cancer cells. Additionally, I found that death response to GPER activation highly correlates with the mutational status of FLT3, specifically FLT3-ITD. FLT3-ITD mutants maintain survival through constitutive activation of AKT, which consequently maintains Mcl-1 expression. Although FLT3 inhibitors are used in clinic, patients inevitably succumb to disease due to development of resistance. FLT3-ITD vulnerability to GPER activation indicates that LNS8801 may be able to be used as a novel therapeutic approach for FLT3-ITD mutant patients. Seeing that activation of GPER may lower the apoptotic threshold in AML, I tested whether LNS8801 can enhance the efficacy of cytarabine in vitro and showed that combination therapy is more effective than either drug alone. Based on these preliminary data, I will perform experiments to 1) Define the mechanism by which Gs-coupled GPCRs induce apoptosis in AML cells and to 2) test efficacy of LNS8801 alone and combination with cytarabine in vivo AML models. In aim 1, I will first determine whether the depletion of Mcl-1 is responsible for cell death by genetically manipulating the expression of Mcl-1 in the presence or absence of drug. Next, I will determine whether Mcl-1 depletion is mediated through AKT depletion. I will then test whether FLT3-ITD mutation renders cells more vulnerable to GPCR activation by measuring the viability of a panel of FLT3-ITD and FLT3wt primary samples that are exposed to GPCR agonists. I will also overexpress FLT3-ITD in FLT3wt and FLT3 null cells to test whether they become more sensitive to GPCR activation. Next, in aim 2, I will test whether LNS8801 alone can effectively inhibit AML in vivo. I will also determine whether LNS8801 enhances the efficacy of cytarabine in vivo with limited toxicity. Together, these aims will define the mechanism of Gs- coupled GPCR induced apoptosis in AML cells and test the efficacy of LNS8801 in vivo, which may further help development of novel therapeutics for AML, especially for the more vulnerable and older patients.