Session: 616. Acute Myeloid Leukemia: Novel Therapy, excluding Transplantation: Poster II
Hematology Disease Topics & Pathways:
apoptosis, Biological Processes, pathways
Expression of leukemic oncogenes (i.e. MLL-AF9 and HRASG12D) in mouse hematopoietic stem and progenitor cells, such as Lineage-Sca-1+cKit+ (LSK) cells or granulocyte-monocyte progenitor cells, promote leukemogenesis as evidenced by serial colony formation in vitro and leukemia development in vivo. We found significantly reduced colonies in oncogene-expressing LSK cells from Gpr68 knockout mice compared to wild type mice. Deletion of Gpr68 reduced [Ca2+]c in oncogene-expressing LSK cells. We next examined the function of GPR68 in human AML cell lines. Knockdown of GPR68 with shRNA reduced cell growth and colony formation, and induced apoptosis in AML cells. Knockdown of GPR68 also reduced [Ca2+]c and Mt membrane potential (Δψm) in AML cells, indicating reduced Mt OxPhos. These results suggest that GPR68 regulates the Ca2+/OxPhos pathway in AML cells.
We next examined the cooperative effect of GPR68 and BCL2 by jointly inhibiting their activities with pharmacological agents (i.e. GPR68 antibody and VEN, respectively) in AML cell lines and AML patient-derived xenograft models. Of note, the expression of GPR68 was positively correlated with the sensitivity to VEN in AML cells. For AML cells that were resistant to VEN, GPR68 antibody but not an unrelated antibody increased the sensitivity to VEN by enhancing apoptosis, indicating that GPR68 and BCL2 co-regulate AML cell survival. We next examined the mechanism of this synthetic lethality by measuring cellular respiration. Single treatment with VEN reduced Δψm, ATP production and O2 consumption in AML cells. Cotreatment with VEN and GPR68 antibody further reduced Δψm, ATP production and O2 consumption in AML cells, indicating that GPR68 and BCL2 co-regulate Mt OxPhos. Given that GPR68 releases Ca2+ from ER to cytosol, while BCL2 maintains [Ca2+]m by inhibiting its extrusion from Mt, we hypothesize that the GPR68/BCL2 axis relocates Ca2+ from ER to Mt. As expected, treatment with GPR68 antibody reduced [Ca2+]c. Cotreatment with VEN and GPR68 antibody increased [Ca2+]c, indicating enhanced extrusion of Ca2+ from Mt to cytosol by VEN. Consistently, cotreatment with VEN and GPR68 antibody reduced the activity of isocitrate dehydrogenase, the rate limiting enzyme in the TCA cycle, in AML cells. These results indicate that GPR68 and BCL2 co-regulate the Ca2+/OxPhos pathway in AML cells and that co-inhibition of GPR68 and BCL2 may enhance lethality and overcome VEN resistance.
In summary, our study suggests that the GPR68/BCL2 axis co-regulates AML cell survival by relocating Ca2+ from ER to Mt thus enhancing Mt OxPhos, and that disruption of the GPR68/BCL2 axis provides a novel therapeutic strategy to overcome resistance to VEN.
Disclosures: No relevant conflicts of interest to declare.
See more of: Oral and Poster Abstracts