新型抗淋巴瘤小分子化合物DL01靶点的鉴定和作用机制研究

The major challenge for precision therapy of diffuse large B cell lymphoma (DLBCL) lies in its tremendous underlying molecular hetergeneity, and discovery of therapeutic targets and small molecule agents is essential for treatment optimization. DL01, a novel small molecule compound indentified through phenotypic screening, significantly kills lymphoma cells in vitro and in vivo. DL01 induces DNA damage, interestingly, it also represses transcriptions of several critical pro-survival genes such as c-Myc and BCL6, suggesting its multiple mechanisms of action. However, the cellular targets of DL01 remains unknown. In this proposal, integration of DARTS (Drug Affinity Responsive Target Stability）techinique and HuProt proteome microarray will be applied to identify the DL01 targets. With affinity purification, RNA interference, immunoblot, chromatin immunoprecipitation, we aim to elucidate the mechanisms by which DL01 induces apoptosis of lymphoma cells, especially the potential role of DL01 targets in epigenetc transcriptional regulation. Furthermore,we try to determine the physiologic relevance of the targets by animal model and clinic studies. Thus, we would reveal novel therapeutic targets for DLBCL, and provide evidence supporting further development of DL01 as a novel anti-lymphoma agent.

淋巴瘤精确治疗的挑战主要源自分子遗传的高度异质性，发现新的治疗靶点和小分子药物有利于优化治疗策略。DL01是我们通过表型筛选获得的小分子化合物，能在体内外显著杀伤淋巴瘤细胞。前期工作证实，除了诱导DNA损伤之外，DL01明显抑制c-Myc和BCL6等重要生存基因的转录从而促进细胞凋亡，提示DL01具有多重抗淋巴瘤机制，但其作用靶点仍然未知。本项目运用药物亲和反应靶标稳定性（DARTS）技术联合HuProt蛋白质组芯片寻找和鉴定DL01靶点；采用亲和纯化，siRNA，免疫印迹，基因表达谱分析以及染色质免疫共沉淀等方法，初步阐明DL01的分子作用机制，尤其是靶点可能对转录的表观遗传调控；结合动物实验和临床资料分析，明确DL01靶点的生物学意义。通过这一系列研究，不仅为淋巴瘤提供新的治疗靶点，而且为DL01作为抗淋巴瘤新药的进一步研发提供依据。

血液肿瘤精确治疗的挑战主要源自分子遗传的高度异质性，发现新的治疗靶点和小分子药物有利于优化治疗策略。NL101（之前称为DL01）是我们通过表型筛选获得的小分子化合物，前期工作证实，除了诱导DNA损伤之外，NL101明显抑制c-Myc和BCL6等重要生存基因的转录从而促进细胞凋亡，提示NL101具有多重抗肿瘤机制，但具体分子机制未知。本项目先聚焦B细胞淋巴瘤，之后拓展到急性髓系白血病。在B细胞淋巴瘤中，RNA-Seq检测NL101作用的基因表达谱，筛选差异表达基因，其中miR-21表达降低最为显著， 研究证实1) miR-21为NL101 的抗淋巴瘤的重要靶点；2) 初步阐明NL101 通过抑制miR-21抗淋巴瘤的分子机制，首次提出c-Myc/miR-21/MXD1环路是调控B细胞淋巴瘤生长的新机制； 3) 证实miR-21是弥漫大B细胞淋巴瘤独立预后因素，为临床转化提供实验依据。在急性髓系白血病（AML），1）体内外实验表明NL101有显著抗白血病作用；2）采用CRISPR文库筛选和功能实验，证实AKT/mTOR通路在NL-101抗AML机制中发挥重要作用，PTEN是NL-101敏感性的关键基因之一；3）mTOR抑制剂雷帕霉素可以逆转NL-101耐药，提出了NL-101联合雷帕霉素这一新的AML治疗策略。通过本项目研究，我们初步阐明NL101作用分子机制，为其在淋巴瘤和白血病的临床应用奠定了实验基础。