BACKGROUND The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

中文翻译：

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组蛋白去甲基化酶KDM5B的抑制作用直接诱导肿瘤抑制蛋白HEXIM1在癌细胞中的重新表达。

背景技术我们小组等已报道了六亚甲基双乙酰胺（HMBA）诱导蛋白1（HEXIM1）在乳腺癌，前列腺癌，黑色素瘤和AML中的抑癌作用。增加的HEXIM1表达引起癌细胞分化并抑制其增殖和转移。从历史上看，HEXIM1是用杂化极性化合物HMBA在实验上诱导的，但由于缺乏已知靶标，不良的药理学特性和不利的ADMETox特性，HMBA在临床上较差。因此，HEXIM1诱导是一种引人入胜的癌症治疗方法，但需要比HMBA更好的化学工具。方法我们使用化学蛋白质组学方法，生物素-NeutrAvidin下拉测定法，表面等离振子共振，和分子对接。使用染色质免疫沉淀测定，RT-PCR，western印迹和shRNA消耗KDM5B，评估了KDM5B和KDM5B抑制剂对HEXIM1的调节。使用蛋白质印迹，分化测定，增殖测定和乳腺癌转移的小鼠模型评估了KDM5B抑制剂对乳腺癌细胞表型的调节。通过使用shRNA消耗HEXIM1，然后进行蛋白印迹，分化测定和增殖测定，确定HEXIM1在KDM5B抑制剂作用中的相对作用。结果我们确定了高度可药物治疗的靶标KDM5B，该靶标被HEXIM1的小分子诱导剂抑制。RNAi抑制KDM5B诱导HEXIM1表达，从而验证了H3K4me3 / 2脱甲基酶KDM5B对肿瘤抑制因子HEXIM1的特异性负调控。已知的KDM5B抑制剂还能够诱导HEXIM1表达，抑制细胞增殖，诱导分化，增强对癌症化学疗法的敏感性以及抑制乳腺肿瘤转移。结论HMBA和4a1通过抑制KDM5B诱导HEXIM1表达。使用KDM5B抑制剂，HEXIM1表达水平的上调在抑制乳腺癌细胞的增殖中起关键作用。基于我们确定的新型分子支架，该分子支架更有效地诱导了HEXIM1的表达和数据，以证明KDM5B是这些化合物的靶标，我们开辟了新的先导发现和优化方向。并抑制乳腺肿瘤转移。结论HMBA和4a1通过抑制KDM5B诱导HEXIM1表达。使用KDM5B抑制剂，HEXIM1表达水平的上调在抑制乳腺癌细胞的增殖中起关键作用。基于我们确定的新型分子支架，该分子支架更有效地诱导了HEXIM1的表达和数据，以证明KDM5B是这些化合物的靶标，我们开辟了新的先导发现和优化方向。并抑制乳腺肿瘤转移。结论HMBA和4a1通过抑制KDM5B诱导HEXIM1表达。使用KDM5B抑制剂，HEXIM1表达水平的上调在抑制乳腺癌细胞的增殖中起关键作用。基于我们确定的新型分子支架，该分子支架更有效地诱导了HEXIM1的表达和数据，以证明KDM5B是这些化合物的靶标，我们开辟了新的线索发现和优化方向。