Mechanisms of Serrated Colon Tumor Suppression

缺乏对肿瘤发生起始时刻的了解，使我们在以下方面的能力存在很大差距： 及早发现癌症或制定预防策略。我们这次竞争性续约的主要目标是 在体内定义锯齿状结肠癌期间BRAF致癌突变的直接后果 阵我们将在结肠干细胞中进行这些研究，结肠干细胞是结肠癌的假定起源细胞。 先前资助的工作确定了锯齿状肿瘤发生的新遗传修饰剂。我们发现 致癌BRAFV 600 E突变矛盾地导致干细胞分化而不是形成肿瘤。 然而，当我们灭活肿瘤抑制转录因子CDX 2或SMAD 4时， 肿瘤发生明显恢复。令人兴奋的是，我们发现CDX 2和SMAD 4靶基因可以用于 对人类患者进行锯齿状肿瘤易感性的分层。 这些研究的逻辑延伸是了解发生在茎中的分子机制 细胞对BRAFV 600 E突变的应答。我们将重点关注BRAF的下游转录效应子， pERK，并将利用我们创建的遗传小鼠模型来实现以下目标： 我们以前的工作发现，转录因子CDX 2保留了成熟结肠的特性，而胎儿- 在CDX 2丢失时打开的特定染色质区域。我们预测pERK利用可获得的 CDX 2丢失后的染色质。目的1将比较野生型干细胞中pERK的活性，以及BRAFV 600 E或 CDX 2突变干细胞。最先进的组学方法将允许首次测量pERK 在BRAFV 600 E突变从其内源性基因座表达后数小时内，在干细胞染色质上。我们 将测量pERK结合活性、核定位以及与其蛋白质伴侣的动态相互作用。 目的2将研究BRAFV 600 E突变在干细胞中的下游基因调控后果。 细胞缺乏SMAD 4。我们先前证明SMAD 4缺失使小鼠易患锯齿状肿瘤， SMAD 4在人类肿瘤中经常发生突变。我们预测SMAD 4与pERK一起作用， 促进分化和抑制干细胞更新。表观基因组学方法将映射pERK结合到 ATAC-seq和RNA-seq将分析染色质和转录组的变化。第三，目标 将对结肠损伤/修复周期可以创造一个易感环境的模型进行功能测试。 BRAF-pERK驱动肿瘤发生，预测改变的转录网络允许 在损伤/修复周期中形成锯齿状肿瘤。这些研究将提供重要的临床前 模型，以帮助解释和测试治疗策略，以抑制锯齿状肿瘤在人类的启动。 这些研究具有创新性，具有尖端的组学应用和GEMM， 探索干细胞中致癌pERK的未经测试的机制。成功完成这些研究将 使我们准备测试预防或早期发现锯齿状结肠癌的治疗策略。

A lack of knowledge about the initiating moments of tumorigenesis leaves large gaps in our ability to detect cancer early or develop prevention strategies. Our primary objective of this competitive renewal is to define, in vivo, the immediate consequences of oncogenic mutations in BRAF during Serrated colon cancer formation. We will pursue these studies in colon stem cells, the presumed cell-of-origin of colon cancer. Work from the previous grant identified new genetic modifiers of Serrated tumorigenesis. We found that oncogenic BRAFV600E mutations paradoxically cause stem cells to differentiate rather than form tumors. However, when we inactivated tumor suppressor transcription factors CDX2 or SMAD4, Serrated tumorigenesis was markedly restored. Excitingly, we found that CDX2 and SMAD4 target genes can be used to stratify human patients for their susceptibility to Serrated tumors. The logical extension of these studies is to understand the molecular mechanisms that occur in stem cells in response to BRAFV600E mutations. We will focus on the downstream transcriptional effector of BRAF, pERK, and will leverage the genetic mouse models we created to address the following Aims: Our previous work found that the transcription factor CDX2 preserves mature colon identity, with fetal- specific chromatin regions opening upon CDX2-loss. We predict that pERK takes advantage accessible chromatin upon CDX2 loss. Aim 1 will compare the activity of pERK in wild type stem cells, and BRAFV600E or CDX2-mutant stem cells. State-of-the-art –omics approaches will allow first-of-a-kind measurements of pERK on stem cell chromatin, within hours after the BRAFV600E mutation is expressed from its endogenous locus. We will measure pERK binding activity, nuclear localization, and dynamic interactions with its protein partners. Aim 2 will look at the downstream gene regulatory consequences of the BRAFV600E mutation in stem cells lacking SMAD4. We previously demonstrated that SMAD4 loss predisposes mice to Serrated tumor formation, and SMAD4 is frequently mutated in human tumors. We predict that SMAD4 works with pERK to promote differentiation and suppress stem cell renewal. Epigenomics approaches will map pERK binding to the genome, and ATAC-seq and RNA-seq will profile changes to chromatin and the transcriptome. Finally, Aim 3 will functionally test a model that the injury/repair cycle in the colon can create a susceptible environment for BRAF-pERK to drive tumorigenesis, with the prediction that an altered transcriptional network is permissive to Serrated tumor formation during the injury/repair cycle. These studies would provide important pre-clinical models to help explain and test therapeutic strategies to suppress Serrated tumor initiation in humans. These studies are innovative with cutting edge -omics applications and GEMMs and significant in exploring untested mechanisms of oncogenic pERK in stem cells. Successful completion of these studies will have us poised to test therapeutic strategies for the prevention or early detection of Serrated Colon Cancer.