SIRT1去乙酰化修饰GLI2对非经典Hedgehog信号通路和多发性骨髓瘤耐药性调控机制的研究

The Hedgehog signaling plays a very important role in Multiple Myeloma drug resistance, and the post-transcriptional modification of the key transcriptional factor GLI2 is essential for the Hedgehog signaling activation. Recent studies have revealed that GLI2 can be modified by acetylation but the acetylated amino acid sites are still unclear. Moreover, the DNA binding activity of GLI2 after acetylation modification and the details of mechanisms controlling the GLI2 processing as well as the Hh signaling downstream targets also remain obscure. Our preliminary data showed that the protein deacetylase, SIRT1, was highly expressed in MM patients and the SIRT1 expression correlated with advanced MM malignancy; overexpression of SIRT1 stabilized GLI2 protein and activated the hedgehog signaling in consequence; Co-Immunoprecipitation experiment showed that SIRT1 and GLI2 formatted a complex; co-culture with bone marrow stromal cells enhanced the expression of SIRT1 in MM cells. These results indicate that interaction of MM cells with the bone marrow stromal cells may promote the expression of SIRT1, and the latter interacts directly or recruits other molecules to modulate the GLI2 protein in purpose of activating the hedgehog signaling and ultimately results in MM cell drug resistance. In the current study, we will collect clinical MM samples, use in vitro molecular biochemistry experiments and in vivo SCID-hu mice model to mimic the bone marrow microenvironment, and explore the modification mechanisms of SIRT1 on GLI2, as well as the molecular mechanism of MM drug resistance. Our study has important significance in gaining new insights into the reason of MM drug resistance, identifying special targets, and developing new strategy for MM treatment in clinic.

Hh信号通路在多发性骨髓瘤的耐药性中发挥重要作用，其关键转录因子GLI2转录后蛋白水平的修饰是Hh通路活化的关键。最新报道GLI2可以被乙酰化修饰，但是具体的位点、对其DNA结合能力的影响、以及对下游靶基因的调控机制尚不明确。我们发现去乙酰化酶SIRT1在骨髓瘤患者中高表达并且与其恶性程度有相关性；SIRT1通过与GLI2形成复合体阻止蛋白降解并活化Hh通路；骨髓间基质细胞与MM细胞共培养能显著上调SIRT1的表达量。本课题中，我们通过分子、细胞和动物水平的实验验证，结合临床病人标本，进一步深入探讨骨髓微环境上调SIRT1的机制，SIRT1对GLI2蛋白的化学修饰和转录能力的调控，以及促进MM产生耐药性的分子机制。该研究有助于重新认识MM耐药性产生的原因，寻找特异性药物靶点，并指导临床上开发新的治疗方案。

背景：多发性骨髓瘤由于治疗过程中耐药性使得MM最终会复发，这让复发或者难治性MM的治疗面临极大的挑战性。Hedgehog信号通路的活化在肿瘤发生发展中发挥重要作用但是其活化的机制尚不清楚。.主要研究内容： 1、探索SIRT1与硼替佐米诱导的多发性骨髓瘤耐药的相关性；2、研究SIRT1通过hedgehog信号通路促进多发性骨髓瘤耐药性产生的机 .制；3、评估靶向SIRT1对逆转骨髓瘤耐药和骨破坏的效果。.主要结果：1、通过RNA-seq筛选了耐药性MM细胞中286个显著上调的基因和75个明显下调的基因，发现SIRT1作为上调最为显著的基因之一。且在病人标本中增高。以上研究结果表明，SIRT1与蛋白酶体抑制剂相关，蛋白酶体抑制剂能够诱导MM细胞耐药。2、Luciferase实验表明，GLI的转录活性对SIRT1呈现剂量相关性。SIRT1有可能通过介导关键转录因子GLI2一定程度上调控Hh信号通路的激活。3、CO-IP发现SIRT1与GLI2蛋白具有相互作用。通过PLA实验进一步佐证了SIRT1和GLI2之间的交流。4、免疫共沉淀实验表明GLI2被组成的乙酰化修饰。在细胞内过表达GLI2，通过免疫共沉淀，发现GLI2能够与SIRT1蛋白发生相互作用，且SIRT1能够去乙酰化修饰GLI2。5、Luciferase实验表明SIRT1的转录活性被GLI2蛋白逐渐激活。6、使用NOD/SCID小鼠建立xenograft和intrabone模型，结果显示BTZ和EX527联合用药组肿瘤的生长相比较单独用药组和对照组明显得到抑制，且总体生存率显著延长，同时骨破坏程度和面积显著降低。.科学意义: .本项目提出了以下新的科学结论，对认识多发性骨髓瘤耐药性的产生具有重要价值：.1、.SIRT1是一种不依赖于突变但由蛋白酶体抑制剂特异性诱导产生的在多发性骨髓瘤耐药性研究中发挥重要作用的关键调节因子。.2、.SIRT1通过与GLI2相互作用，稳定GLI2蛋白，激活Hh信号通路； .3、.SIRT1是Hh信号通路的直接靶基因，通过形成SIRT1/GLI2/Hedgehog pathway/SIRT1的正性调控通路维持hedgehog的活性；.4、.在体内与体外实验中，靶向SIRT1能够协同硼替佐米提高多发性骨髓瘤细胞对硼替佐米的敏感性。