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1353 Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance in Myeloid Diseases: Poster I
Hematology Disease Topics & Pathways:
Diseases, AML, Myeloid Malignancies
Saturday, December 1, 2018, 6:15 PM-8:15 PM
Hall GH (San Diego Convention Center)

Xiaofei He, B.S.1*, Mark Wunderlich, MS2*, Benjamin Mizukawa, MD2, James C. Mulloy, PhD3, Saran Feng, M.D., Ph.D.1,4*, Lauren Lawley, B.S.1*, Caleb Hawkins, B.S.1*, Wenxin Fan, B.S.1*, Guokun Zhou, Ph.D.5*, Xiang-ming Zha, Ph.D.5*, Jiajia Zhang, PhD6*, Johnie Hodge, M.D., Ph.D.7*, Daping Fan, Ph.D.7*, Stephanie Halene, MD 8 and Jing Fang, MD, PhD1

1Drug Discovery and Biomedical Sciences, University of South Carolina College of Pharmacy, Columbia, SC
2Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
3Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
4Department of Hematology, Shandong Provincial Qianfoshan Hospital, Jinan, China
5Department of Physiology and Cell Biology, University of South Alabama College of Medicine, Mobile, AL
6University of South Carolina Arnold School of Public Health, Columbia
7Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC
8Section of Hematology, Department of Internal Medicine and Yale Cancer Center, Yale Univ. School of Medicine, New Haven, CT

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML.

Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function.

In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway.

In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH