Identification of a GABP Feedback Loop and its Role in Tumor Cell Immortality

项目摘要 人类癌症的一个基本特征是它们有能力克服复制衰老和 实现细胞永生。在体细胞分化过程中通常是沉默的，90%的人类肿瘤 在肿瘤发生的早期重新激活端粒酶逆转录酶(TERT)的表达以实现 细胞永生。端粒酶的催化亚单位TERT与RNA模板分子形成络合物 TERC和其他蛋白质结合并延伸染色体末端的重复序列，称为 端粒。最近，TERT启动子(TERTp)中的非编码突变在许多研究中被描述 癌症。这些TERTp突变是所有癌症中最常见的非编码突变，是 许多癌症中最常见的突变，如83%的IDH野生型胶质母细胞瘤(GBM)，最常见的 常见且致命的成人脑瘤。这些突变导致了典型的E26的形成 转换特定(ETS)结合基序，与TERTp原生ETS站点协同工作，以招募 特定的ETS转录因子，GA结合蛋白(GABP)复合体。我们的实验室证明了这一点 GABP的募集对于TERT的重新激活和维持肿瘤细胞的永生是必要的。 此外，我们的实验室还表明，通过CRISPR-Cas9介导的四聚体形成亚单位的突变， GABPB1L(B1L)，认为GABP的四聚体形式是维持肿瘤细胞永生所必需的。 有趣的是，虽然CRISPR-Cas9介导的B1L消融并没有显示出TERT的减少 表达后，它确实显示GABP二聚体特异性亚基GABPB1S(B1S)大幅增加。重要的是，它 这种B1s表达的增加可能是由于GABP四聚体介导的负反馈环。 此外，初步证据表明，B1s的这种增加可能允许含有二聚体的B1s结合 突变的TERTp并维持TERT的表达。从广义上讲，这项研究旨在阐明 在B1L消除状态下维持TERT表达的机制。在这样做的过程中， 研究将确定B1L减少过程中B1S表达上调的机制，并将 确定含有B1S的GABP二聚体是否为突变体TERTp的有效调节因子。端粒酶有 长期以来一直是逆转肿瘤细胞永生的有吸引力的治疗靶点；然而，尝试靶向 由于端粒酶表达干细胞，端粒酶大多不成功。先前的研究表明， 在TERTp突变的癌症中靶向B1L将允许肿瘤特异性抑制端粒酶，然而，我们的 数据显示，这种方法比之前认为的要复杂得多。重要的是， 这些调查的结果将指导设计和开发有效和持久的治疗方法 靶向GABP-TERT轴逆转TERTp突变癌的肿瘤细胞永生化 胶质母细胞瘤、黑色素瘤、膀胱癌和许多其他疾病。

Project Summary A fundamental hallmark of human cancers is their ability to overcome replicative senescence and achieve cellular immortality. Normally silenced during differentiation in somatic cells, 90% of human tumors reactivate Telomerase Reverse Transcriptase (TERT) expression early during tumorigenesis to achieve cellular immortality. TERT, the catalytic subunit of telomerase, complexes with the RNA template molecule TERC and other proteins and binds to and extends the repetitive sequences at chromosomal ends, known as telomeres. Recently, non-coding mutations in the TERT promoter (TERTp) have been described in many cancers. These TERTp mutations are the most common non-coding mutation across all cancers and are the most frequent mutations in many cancers, such as 83% of IDH wildtype glioblastomas (GBM), the most common and deadly form of adult brain tumors. These mutations result in the formation of canonical E26 Transformation Specific (ETS) binding motifs that work in tandem with TERTp native ETS sites to recruit a specific ETS transcription factor, the GA-binding protein (GABP) complex. Our lab has demonstrated that this recruitment of GABP is necessary for TERT reactivation and maintenance of tumor cell immortality. Furthermore, our lab has shown, through CRISPR-cas9 mediated mutation of the tetramer forming subunit, GABPB1L (B1L), that the tetrameric form of GABP is necessary for this maintenance of tumor cell immortality. Interestingly, while CRISPR-Cas9 mediated ablation of B1L does not show a reduction in TERT expression, it does show a large increase in the GABP dimer specific subunit, GABPB1S (B1S). Importantly, it seems that this increase in B1S expression may be due to a GABP tetramer mediated negative feedback loop. Furthermore, preliminary evidence suggests that this increase in B1S may allow B1S containing dimers to bind to the mutant TERTp and maintain TERT expression. Broadly, this study aims to elucidate the molecular mechanisms underlying the maintenance of TERT expression during states of B1L elimination. In doing so, this study will determine the mechanisms underlying B1S expression upregulation during B1L reduction and will determine if B1S-containing GABP dimers are competent regulators of the mutant TERTp. Telomerase has long been an attractive therapeutic target for the reversal of tumor cell immortality; however, attempts to target telomerase have been mostly unsuccessful due to telomerase expressing stem cells. Prior studies suggest that targeting B1L in TERTp mutant cancers would allow for tumor specific inhibition of telomerase, however, our data have revealed that this approach is more complicated than previously appreciated. Importantly, the findings from these investigations will guide the design and development of effective and durable therapies targeting the GABP-TERT axis for the reversal of tumor cell immortality in TERTp mutant cancers such as glioblastoma, melanoma, bladder cancer, and many others.