Abstract Transient Receptor Potential Melastatin subfamily member 2 (TRPM2) is highly expressed in many cancers including neuroblastoma. Proline-rich tyrosine kinase 2 (Pyk2) is calcium-sensitive non-receptor tyrosine kinase which enhances cell survival and is activated by calcium influx through TRPM2. Pyk2 also modulates mitochondrial calcium uptake through phosphorylation of the mitochondrial calcium uniporter (MCU), impacting mitochondrial function, and through its effects on CREB, which regulates transcription of MCU. The purpose of this work was to investigate the mechanisms by which TRPM2 regulates cancer cell survival in order to develop new therapeutic strategies based on inhibition of TRPM2. Two models of TRPM2 inhibition were utilized: 1) reducing TRPM2 function in SH-SY5Y cells by expression of the dominant negative construct TRPM2-S, a short isoform of TRPM2, and 2) depletion of TRPM2 expression with CRISPR/Cas9 technology. Treatment with Doxorubicin (Doxo) was used as a model of oxidative stress. Doxorubicin reduced growth of cells in which TRPM2 was inhibited significantly more than control cells. Inhibition of TRPM2 also reduced Pyk2 and CREB expression and activation. Using shRNA technology we showed that Pyk2 mediated reduced expression and activation of CREB and activation of STAT3 in TRPM2 expressing cells. Fractionation experiments showed that depletion of TRPM2 reduced phosphorylation of whole cell and mitochondrial Src and Pyk2 and expression of mitochondrial CREB and MCU. Src has an important role in Pyk2 phosphorylation. In TRPM2 depleted cells, mitochondrial Ca uptake was significantly reduced since both peak mitochondrial calcium uniporter currents and amount of calcium transported (current-time integral) measured in mitoplasts were lower. Wt Pyk2 but not the phospho-deficient mutant Y402F and wt TRPM2 but not the TRPM2 Ca-impermeant mutant E960D, restored viability after Doxo treatment of SH-SY5Y cells in which TRPM2 was inhibited. In conclusion, these results suggest that TRPM2 inhibition leads to reduced viability in neuroblastoma through the Src-Pyk2-CREB-MCU pathway, which plays a key role in regulating mitochondrial function and cellular bioenergetics. Citation Format: Iwona Malgorzata Hirschler-Laszkiewicz, Shu-jen Chen, Lei Bao, JuFang Wang, Xue-Qian Zhang, Santhanam Shanmughapriya, Kerry Keefer, Muniswamy Madesh, Joseph Y. Cheung, Barbara A. Miller. TRPM2 modulates neuroblastoma cell survival through Pyk2, CREB and MCU activation [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 1458.