Author

Xiaofei He

Date of Award

Summer 2020

Document Type

Open Access Dissertation

Department

Pharmacology, Physiology and Neuroscience

First Advisor

Jing Fang

Abstract

G protein-coupled receptor 68 (GPR68), as a proton-sensing receptor, is involved in numerous physiological processes. However, its specific functions in normal hematopoiesis and malignant hematopoietic diseases were unknown. Using a genetic loss of function approach (i.e. whole-body Gpr68-/- mouse model), B lymphocytes were reduced under stressed conditions, such as aging and hematopoietic regeneration. However, hematopoietic stem cells (HSC) from Gpr68-/- mice exhibited comparable function as HSC from wild type mice either under steady-state or upon stress. To determine whether the unaltered HSC function of Gpr68-/- mice was due to compensatory effects of Gpr68 in nonhematopoietic cells, a conditional loss of function model was generated in that Gpr68 was exclusively deleted in hematopoietic tissues (i.e. the Gpr68flox/flox;Vav-cre+ mice). Enhanced competitiveness was exhibited in Gpr68-deficient HSC in older mice, possibly through a Ca2+/caspase pro-apoptotic pathway. However, B lymphocytes did not reduce in Gpr68flox/flox;Vav-cre+ mice. As a reciprocal approach, reverse bone marrow transplantation was performed so that Gpr68 was deleted in non-hematopoietic cells. Intriguingly, B cells were reduced in recipient mice without Gpr68, indicating that loss of Gpr68 in nonhematopoietic cells was responsible for the reduced output of B lymphocytes.

Besides, GPR68 is overexpressed in acute myeloid leukemia (AML) and its overexpression is associated with poor prognosis. The depletion of GPR68 resulted in apoptosis and inhibited in vivo expansion of AML cells, which was associated with a Ca2+/calcineurin pro-survival pathway. To explore the application of GPR68 in AML treatment, the combinational therapies of GPR68 inhibitors with other drugs, i.e., Cytarabine (Ara-C) and Venetoclax (VEN) was studied. Higher levels of GPR68 expression correlated to resistance to chemotherapeutic agents in AML cells. Depletion of GPR68 expression or inhibition of GPR68 activity enhanced the sensitivity to Ara-C and VEN in AML cells. Mechanistic studies revealed that chemotherapeutic agents enhanced GPR68 expression, while glycolysis increased GPR68 activity, and they both contributed to GPR68-mediated chemoresistance. In addition, GPR68 collaborated with BCL-2 leading to an enhanced oxidative phosphorylation that resulted in resistance to VEN due to activating Ca2+ sensitive dehydrogenases.

In summary, GPR68 suppresses normal HSC function with age but enhances chemoresistance in AML cells. It implicates a therapeutic potential of inhibiting GPR68 in damaging leukemia cells while preserving normal hematopoietic cells. Moreover, the excellent performance of GPR68 inhibition in improving the drug sensitivity of AML cells make GPR68 an excellent drug target.

Available for download on Monday, August 15, 2022

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