The tumor suppressor gene is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for -mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase , uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines , as well as xenografts . The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage-induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring mutations, indicating that the synthetic lethal relationship between and could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in . SIGNIFICANCE: -mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in -deficient cancers..