Characterizing and Targeting the Epigenetic Mechanisms Maintaining Neuroblastoma Tumor Initiating Cells

摘要： 癌细胞异质性和存在表型不同的细胞亚群， 肿瘤引发和耐药性能力是癌症治疗中的主要挑战。这些抗药细胞 已知会导致癌症复发，这是治疗失败和高风险癌症死亡的主要原因， 例如小儿神经母细胞瘤（NB）。表观遗传机制导致的基因差异表达失调 功能障碍是癌细胞异质性的主要原因。确定特定表观遗传的作用 在维持肿瘤起始癌症干细胞亚群和开发靶向治疗中的修饰剂， 阻断这些调节剂是有效对抗癌症及其复发的必要条件。 最近，我们发现了一种耐药、高致瘤性、转移性和自我更新的细胞亚群 具有神经母细胞瘤肿瘤起始细胞（TIC）的特征。这个亚群的特征是表面 G-CSF受体（CD 114）的表达，可以逃避初始治疗，并导致难治性和侵袭性 侵袭性复发性疾病。这些TIC可以分化为大量肿瘤细胞（CD 114-）并产生复合物， 在体内具有少至10个细胞的神经母细胞瘤肿瘤。我们最近的初步数据显示，CSF 3R基因 编码CD 114的基因在CD 114 + NB TIC中特异性表达，但在CD 114-本体肿瘤细胞中不表达。我们 假设这种差异基因表达是表观遗传调节的，并支持维持 异构NB TIC。我们还发现，表观遗传调节因子混合谱系白血病-1（MLL 1; H3 K4 me 3甲基转移酶）在CD 114+细胞中过表达，并调节TIC特异性 CSF 3R等基因。MLL 1与WDR 5和Menin形成蛋白复合物，作为活性组蛋白 甲基转移酶表观遗传调节因子。我们的初步数据显示，靶向MLL 1-WDR 5和MLL 1-Menin 使用特异性小分子抑制剂的相互作用抑制NB TIC和体内NB生长和转移。这些 数据支持我们的假设，即表观遗传调节因子维持NB TIC，并指向一种新的治疗方法。 阻断MLL 1活化以抑制NB TIC介导的致瘤性、转移和疾病复发的策略。 在所提出的研究中，我们将确定：a）MLL 1在维持NB TIC亚群中的作用，B）MLL 1在维持NB TIC亚群中的作用， 通过特异性小分子抑制剂阻断表观遗传调节因子对NB肿瘤发生的影响， c）通过组合NB TIC和本体肿瘤细胞同时靶向NB TIC和本体肿瘤细胞的作用， 表观遗传抑制剂与标准化疗药物，和d）进一步开发新的表观遗传抑制剂， 靶向MLL 1表观遗传功能。作为MLL 1、WDR 5和Menin的特异性小分子抑制剂，目前 在实体瘤和白血病的广泛临床前试验中，我们的发现将为NB肿瘤提供见解。 生物学和一种新的基于表观遗传的治疗方法用于高风险NB。

Abstract: Cancer cell heterogeneity and the existence of phenotypically distinct subpopulations of cells with enhanced tumor-initiating and drug-resistant capacities is a major challenge in cancer treatment. These drug-resistant cells are known to drive cancer relapse, which is a major cause of therapy failure and deaths from high-risk cancers, such as pediatric neuroblastoma (NB). Deregulated differential expression of genes due to epigenetic machinery malfunction is the primary cause of cancer cell heterogeneity. Determining the role of specific epigenetic modifiers in maintaining tumor-initiating cancer stem cell sub-populations and developing targeted therapies to block these modifiers is mandatory for effectively battling cancer and its relapse. Recently, we discovered a drug-resistant, highly tumorigenic, metastatic, and self-renewing cell sub-population with features of tumor-initiating cells (TICs) in neuroblastoma. This sub-population, characterized by surface expression of the G-CSF receptor (CD114), can escape initial therapy and cause refractory and aggressively invasive relapsed disease. These TICs can differentiate to bulk tumor cells (CD114-) and produce complex neuroblastoma tumors with as few as 10 cells in vivo. Our recent preliminary data show that the CSF3R gene that codes for CD114 is expressed specifically in CD114+ NB TICs but not in CD114- bulk tumor cells. We hypothesize that this differential gene expression is epigenetically regulated and supports the maintenance of heterogeneous NB TICs. We also found that the epigenetic regulator mixed-lineage leukemia-1 (MLL1; an H3K4me3 methyltransferase) is overexpressed in CD114+ cells and regulates the expression of the TIC specific genes including CSF3R. MLL1 forms a protein complex with WDR5, and Menin to act as an active histone methyltransferase epigenetic regulator. Our preliminary data show that targeting MLL1-WDR5 and MLL1-Menin interactions using specific small molecule inhibitors inhibit NB TICs and in vivo NB growth and metastasis. These data support our hypothesis that epigenetic regulators maintain NB TICs and point towards a novel therapeutic strategy of blocking MLL1 activation to inhibit NB TIC-mediated tumorigenicity, metastasis, and disease relapse. In the proposed research, we will determine: a) the role of MLL1 in maintaining NB TIC subpopulation, b) the effects of blocking epigenetic regulators via specific small molecule inhibitors on NB tumorigenesis and metastasis in vivo, c) the effects of concomitantly targeting both NB TICs and bulk tumor cells by combining epigenetic inhibitors with standard chemotherapy drugs, and d) further developing novel epigenetic inhibitors to target MLL1 epigenetic functions. As specific small molecule inhibitors of MLL1, WDR5, and Menin are currently under extensive pre-clinical trials for solid tumors and leukemias, our findings will provide insights into NB tumor biology and a new epigenetic based therapeutic approach for high-risk NB.