Abstract 1324: TRPM2 promotes leukemia proliferation and survival through modulation of ROS and autophagy
Authors: Chen Shu-Jen , Bao Lei , Keefer Kerry , Chen Longgui , Wang JuFang , Zhang Xue-Qian , Shanmughapriya Santhanam , Hirschler-Laszkiewicz Iwona , Madesh Muniswamy , Cheung Joseph Y. , Wang Hong-Gang , Miller Barbara A. 
Published: August 2018

Acute myeloid leukemia (AML) is a heterogeneous group of leukemias that arise in hematopoietic precursors. Understanding the mechanisms involved in AML proliferation and survival is key to development of new therapies. Transient receptor potential melastatin 2 (TRPM2), a calcium permeable ion channel, modulates cell proliferation, survival and oxidative stress-induced cell death and is highly expressed in many cancer cells including AML. To investigate the role of TRPM2 in AML, we depleted the TRPM2 gene in the human AML cell line U937 using clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR) technology. RT-PCR, western blot, and patch clamp analyses confirmed that TRPM2 was depleted in U937 knockout (KO) cells. Cell proliferation and viability measured by XTT and trypan blue exclusion assays were significantly reduced in KO cells after doxorubicin treatment. Expression of wild type TRPM2 (TRPM2-L) restored cell growth and cell survival to doxorubicin-treated TRPM2 KO cells, whereas the TRPM2 channel pore mutant (E960D) or empty vector did not. In a xenograft mouse model, mice were injected with TRPM2 KO or control U937 cells which express luciferase. Mice injected with TRPM2 KO U937 cells showed significantly reduced leukemia growth and enhanced sensitivity to doxorubicin. To examine the mechanisms, mitochondrial ROS were measured with confocal microscopy and MitoSOX Red. ROS were significantly elevated in TRPM2 KO cells compared to scrambled control, and further increased when KO cells were treated with doxorubicin. ATP production, oxygen consumption rate (OCR), and mitochondrial Ca2+ uptake were significantly reduced and mitochondrial membrane potential (ΔΨ) significantly increased in TRPM2 KO cells compared to scrambled control cells treated with doxorubicin, demonstrating dysfunctional mitochondria. Western blot analysis showed that expressions of HIF1/2α, FOXO3a and Nrf2 were downregulated in TRPM2 KO cells, further contributing to the increase in ROS. Expression of autophagy proteins ULK1, Atg7 and Atg5 was significantly reduced and mitochondrial protein Tom20 significantly increased in TRPM2 KO cells, suggesting that depletion of TRPM2 significantly reduced autophagy in AML cells. Reconstitution of wild type TRPM2 but not E960D restored ULK1 expression. In conclusion, depletion of TRPM2 significantly inhibits AML proliferation and increases sensitivity to doxorubicin by reducing mitochondrial function, increasing ROS, and impairing autophagy. TRPM2 contributes significantly to AML survival and may be a novel therapeutic target in leukemia therapy.

Citation Format: Shu-Jen Chen, Lei Bao, Kerry Keefer, Longgui Chen, JuFang Wang, Xue-Qian Zhang, Santhanam Shanmughapriya, Iwona Hirschler-Laszkiewicz, Muniswamy Madesh, Joseph Y. Cheung, Hong-Gang Wang, Barbara A. Miller. TRPM2 promotes leukemia proliferation and survival through modulation of ROS and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1324.