performed most of other experiments. cell renal cell carcinoma, medulloblastoma, chronic myelomonocytic leukemia, acute lymphoblastic Syk leukemia, transitional cell carcinoma of the bladder, and high-grade muscle-invasive urothelial bladder carcinoma and others12,13. UTX and the other major H3K27 demethylase JMJD3 (KDM6B) have been reported to play major functions in cell proliferation and malignancy in several different experimental systems. UTX was reported to regulate expression of RB and RB-binding proteins by directly binding to their promoters and cause cell cycle arrest in main human fibroblasts14. Pralidoxime Iodide UTX negatively regulates proliferation, clonogenicity, adhesion and xenograft tumorigenicity of multiple myeloma15. Jmjd3, but not Utx, is usually induced by the Ras-Raf signaling and activates p16Ink4a and p19Arf Pralidoxime Iodide to suppress cell proliferation in mouse embryonic fibroblasts16,17. In the Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) mouse model, Notch1 increases Jmjd3 expression likely via NFkB, which in turn enhances the expression of Notch1 target genes in cooperation with Notch1, while Utx suppresses the T-ALL formation18. Somewhat paradoxically, however, knockdown of UTX suppresses proliferation of breast malignancy cell lines in vitro and the invasiveness of their xenografts in mice19, and UTX promotes the proliferation and migration of estrogen receptor-positive breast malignancy cells20. Thus the contribution of UTX and JMJD3 to malignancy formation and progression appears to be highly context-dependent, which remains to be mechanistically and comprehensively explained. GSK-J1 is usually a potent KDM6 (both JMJD3 and UTX) inhibitor in in vitro binding and enzyme assays21. GSK-J4 is the cell-permeable form of, and is converted to, GSK-J1 and inhibits TNF production in human main macrophages21. GSK-J4 has been considered as a therapeutic tool against numerous malignancies22 such as T-ALL, pediatric brainstem glioma, diffuse intrinsic pontine glioma, Pralidoxime Iodide non-small cell lung malignancy, breast malignancy stem cells, castration-resistant prostate malignancy, and high-risk neuroblastoma. However, these studies mainly focused on inhibitory effects on cell proliferation, and the molecular mechanisms involved in these therapeutic effects remain poorly comprehended. In a subsequent study, also using an in vitro inhibition and cell-based assays, GSK-J4 significantly inhibited other KDMs such as KDM5B and KDM5C23. While this discrepancy in target specificity could be due to difference in intracellular conversion rates from GSK-J4 to GSK-J124, further studies around the GSK-J4 specificity are required. To gain insights into UTX functions for malignancy formation and maintenance, we utilized the KDM6 inhibitor, GSK-J4, and UTX-deficient cells created using the CRISPR-Cas9 editing system in conjunction with complementation with exogenous UTX. We found that ATF4 and its downstream target genes are highly and rapidly induced by GSK-J4 independently of the canonical ER stress response and that one of the integrated stress Pralidoxime Iodide response (ISR) kinases, HRI, is mainly responsible for the ATF4 induction. Expression of the ATF4 target genes was specifically suppressed by UTX complementation. It is unlikely that activation of the ATF4-CHOP cascade by GSK-J4 is usually a single or major determinant of apoptosis induction. These results revealed a unique physiological effect of GSK-J4 and provide insights into therapeutic use of KDM6 inhibitors. Results GSK-J4 induces expression of ATF4 and its target genes independently of the unfolded protein response (UPR) We have found that GSK-J4 treatment up-regulated the mRNA expression levels of genes targeted by ATF4 (expression, in epithelial cells: obvious cell renal cell carcinoma (ccRCC) 786-O cells, main renal proximal tubule epithelial cells (PRETEC) and colorectal malignancy HCT116 cells (Fig.?1A). The induction of the ATF4 target genes by GSK-J4 was high especially in HCT116..