PROJECT SUMMARY/ABSTRACT Glioma, the most common and aggressive primary brain malignancy, is in dire need of novel therapeutic approaches. Recent findings suggest that neuronal activity promotes tumor proliferation at the tumor-brain interface via excitation of calcium (Ca2+)-permeable glutamate receptors expressed by tumor cells, leading to robust intracellular Ca2+ waves. These exciting observations ascribe a critical role for Ca2+ influx in glioma growth, but they do not explain whether Ca2+ waves are also relevant in the core of tumors, where proliferation occurs in the absence of neuronal activity and synaptic input. Our preliminary data reveal a novel mechanism that allows glioma cells to utilize Ca2+ flux toward their proliferation in the absence of neuron-tumor synapses. Indeed, we found that glioma cells express several types of Ca2+ channels and generate tumor cell-autonomous Ca2+ waves in the absence of neurons or glia. Such waves can be observed with Ca2+ imaging of patient-derived glioblastoma cells cultured without other cell types in vitro, as well as imaging of patient-derived xenografts in the mouse brain in the acute brain slice preparation. These Ca2+ waves are blocked by Co2+ and Cd2+, ionic inhibitors of Ca2+ channels. In search for possible effectors of Ca2+ waves, we performed an unbiased dropout CRISPR screen targeting the “druggable genome” and identified Ca2+/calmodulin-dependent protein kinase 2B (CAMK2b) as a novel essential component of glioma growth. Indeed, pharmacologic inhibition or short hairpin RNA (shRNA) knockdown of CAMK2b impair proliferation of patient-derived glioblastoma cultures in vitro, suggesting CAMK2b essentiality. These preliminary observations lead us to hypothesize that tumor cell-autonomous Ca2+ waves promote glioma growth via activation of CAMK2b and phosphorylation of protein substrates that regulate tumorigenesis. To elucidate this mechanism, we propose the following specific aims: 1) characterize the Ca2+ channels that generate Ca2+ waves in glioma cells and determine their impact on tumor growth in vitro and in vivo; and 2) test the hypothesis that CAMK2b represents a crucial effector of Ca2+ waves and phosphorylates substrates that promote tumor propagation. We have assembled a multidisciplinary team of experts to test this novel hypothesis. The proposed work will lay the foundation for linking glioma cell electroresponsiveness to tumorigenic signaling mechanisms and holds promise for discovery of novel targetable vulnerabilities in this therapy-resistant tumor.