Cell-cycle checkpoint inhibitors as a novel anti-cancer strategy in Glioblastoma multiforme

胶质母细胞瘤（GBM）是成人最常见和最致命的原发性恶性脑肿瘤。确定新疗法的一个挑战是GBM基因型行为的多样性。为了解决这个问题，Beroukhim实验室对78个GBM细胞系进行了全面的分子表征和药物筛选，涵盖了GBM的主要分子类别。肿瘤抑制基因TP53被破坏的细胞系对大多数化合物的反应较差，一般来说，TP53突变的肿瘤也是如此。TP53是癌症中最常见的突变基因，因此在TP53突变细胞中具有活性的化合物可以为大量患有治疗抵抗性癌症的人提供有用的见解。幸运的是，我们发现了一种CHK1/2抑制剂（CHKi），对TP53突变细胞更有效。当肿瘤抑制基因CDKN2A也丢失时，这种抑制剂是最有效的——大约10%的GBM患者的基因丢失。检查点激酶（CHK）是DNA损伤反应（DDR）的重要介质。它们通过在分裂前为细胞提供修复DNA损伤的时间来维持基因组的完整性，或者在损伤不可修复时启动细胞凋亡。目前有几种中国疫苗正在进行临床试验，其中一些已经显示出有希望的活性。因此，我们将在包括GBMs在内的所有癌症中研究CHKi和两种最常见的灭活肿瘤抑制因子之间明显的合成致死关系。我们的具体问题是：1。TP53和CDKN2A的联合破坏是否会产生对CHK1/2抑制剂的易感性？我们将在GBM细胞系中应用CRISPR-CAS9技术，建立TP53和CDKN2A及其组合的等基因模型，以确定TP53和CDKN2A的联合缺失是否会使细胞对CHKi敏感。我们的读数将是细胞活力、增殖、凋亡、细胞周期进展和DDR的变化。这些实验将对决定CHKi的靶点，帮助设计和解释未来的临床试验具有重要意义。评估对CHK1/2抑制的抗性机制。癌症通常对最初有效的靶向治疗产生耐药性。确定这些耐药机制可以提示采取联合治疗方法以延长反应时间。在这里，我们将采用三种方法。首先，我们将测试是否可以通过减缓细胞周期来获得耐药性。其次，我们将对CHKi敏感细胞进行基因组规模的开放阅读框（ORF）筛选，以鉴定表达产生抗性的基因。第三，我们将生成自然产生的CHKi抗性模型，并通过表征其表达变化和新基因改变的发展来评估它们是如何获得抗性的。该项目将评估一种治疗肿瘤的新方法，这些肿瘤基因组中一些最常改变的基因TP53和CDKN2A突变。这些基因改变在癌症中的普遍存在表明，这项工作可能会产生深远的影响。

Glioblastoma (GBM), is the most common and most lethal primary malignant brain tumor in adults. A challenge in identifying new therapies is the diversity in behavior of GBM genotypes. To address this, the Beroukhim lab conducted a comprehensive molecular characterization and drug screen on 78 GBM cell lines spanning the major molecular classes of GBM. Cell lines with disruption of the tumor suppressor gene TP53 showed poor responses to most compounds, as is true of TP53-mutant tumors in general. TP53 is the most commonly mutated gene in cancer, so compounds with activity in TP53 mutant cells could provide helpful insights for a large number of people with treatment-resistant cancers. Fortunately, we identified a CHK1/2 inhibitor (CHKi) that was more effective in TP53 mutant cells. This inhibitor was most effective when the tumor suppressor gene CDKN2A had also been lost—the genetics of about 10% of GBM patients. Checkpoint kinases (CHK) are crucial mediators of the DNA damage response (DDR). They maintain genomic integrity by providing cells time to repair DNA damage before dividing or initiate apoptosis if the damage is irreparable. Several CHKis are currently undergoing clinical trials, with some already showing promising activity. Therefore, we will investigate this apparent synthetic lethal relationship between CHKi and two of the most commonly inactivated tumor suppressors across all cancers including GBMs. Our specific questions are:1. Does joint disruption of TP53 and CDKN2A generate susceptibility to CHK1/2 inhibitors? We will generate isogenic models for TP53 and CDKN2A and the combination by applying CRISPR-CAS9 technology in GBM cell lines, to determine whether joint loss of TP53 and CDKN2A sensitizes the cells to CHKi. Our readouts will be changes in cell viability, proliferation, apoptosis, cell cycle progression and the DDR. These experiments will have implications for deciding whom to target with CHKi, aiding in design and interpretation of future clinical trials.2. Evaluate resistance mechanisms to CHK1/2 inhibition. Cancers often acquire resistance to initially effective targeted therapies. Identifying these resistance mechanisms can indicate combined treatment approaches to extend response times. Here, we will take three approaches. First, we will test if resistance can be gained by slowing down the cell cycle. Second, we will perform a genome-scale open reading frame (ORF) screen on CHKi sensitive cells to identify genes whose expression generates resistance. Third, we will generate naturally arising models of CHKi resistance and evaluate how they gained it by characterization their changes in expression and development of new genetic alterations.This project will evaluate a novel approach to treating tumors with mutations in some of the most frequently altered genes in the cancer genome TP53 and CDKN2A. The prevalence of these genetic alterations across cancers indicates that this work could have a profound impact.