LSD1调控非组蛋白底物CDK4稳定性的分子机制以及联合应用抑制剂对骨肉瘤增殖影响的机制研究

骨肉瘤作为最常见的骨组织产生的恶性肿瘤，临床治疗的主要问题为化疗药物靶向性不强且选择有限。表观遗传调控研究的快速发展为解决上述问题提供了有利条件。本课题组前期研究发现，去甲基化酶LSD1抑制剂处理骨肉瘤细胞导致周期调控激酶CDK4蛋白质稳定性降低，并与其泛素化水平相关，联合应用LSD1与CDK4抑制剂有效提高了抑瘤效果。因此，提出科学假说，CDK4是LSD1的非组蛋白底物，CDK4的甲基化与泛素化修饰相互调控影响蛋白质稳定性。本项目拟主要运用质谱鉴定、体外甲基化、体外泛素化、RNA-seq、CHIP-seq等方法，明确LSD1介导CDK4去甲基化、CDK4甲基化与泛素化相互调控、联合应用二者抑制剂对骨肉瘤细胞转录组的调控以及抑制骨肉瘤生长等分子机制。本课题的完成将揭示表观调控分子LSD1的多重生理功能，重要药物标靶CDK4的翻译后修饰调控机制，也将为临床应用新的骨肉瘤治疗药物提供理论依据。

Osteosarcoma, as the most common malignant tumor arising from bone tissue faces the problem of weak drug targeting and limited choice in clinical treatment. In recent years enormous progress has been made in epigenetic related research and has provided favorable conditions for solving the problems above mentioned. We found that LSD1 inhibition resulted in a dramatic decrease of the stability of CDK4 protein in osteosarcoma cells, which was related to its ubiquitination. Further data demonstrated that the treatment of CDK 4/6 inhibitor in combination with LSD1 inhibitor significantly suppressed the proliferation of osteosarcoma cells. We hypothesize that CDK4 is a novel non-histone substrate of LSD1 and LSD1 regulates CDK4 ubiquitination through demethylation. Our approaches include mass spectrometry identification, in vitro methylation assay, in vitro ubiquitination assay, RNA-seq,CHIP-seq. Our objective is to clarify the molecular mechanism of LSD1 demethylasing CDK4 and the crosstalk between ubiquitination and methylation of CDK4. To identify the differentially expressed genes associated with combined inhibition, the transcriptome analysis will be made. The discoveries made from the proposed study will provide a new mechanism to understand the crosstalk between ubiquitination and methylation of CDK4 and the multifunction of LSD1 for regulating a novel non-histone substrate. And this will also offer a solid mechanistic basis for the development of new druggable molecules for osteosarcoma treatment.