Mechanisms of Serrated Colon Tumor Suppression

缺乏对肿瘤发生的起始时刻的了解，会在我们的能力上留下巨大的缺口 及早发现癌症或制定预防策略。我们此次竞争性续签的主要目标是 在体内确定锯齿状结肠癌中BRAF癌基因突变的直接后果 队形。我们将继续在结肠干细胞中进行这些研究，这是结肠癌的假定起源细胞。 来自前一项拨款的工作确定了锯齿状肿瘤发生的新遗传修饰物。我们发现 致癌的BRAFV600E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CDX2或Smad4时，锯齿状 肿瘤形成明显恢复。令人兴奋的是，我们发现CDX2和Smad4靶基因可以用来 根据人类患者对锯齿状肿瘤的易感性进行分层。 这些研究的合乎逻辑的延伸是理解茎中发生的分子机制 细胞对BRAFV600E突变的反应。我们将重点关注BRAF的下游转录效应器， 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CDX2保留了成熟的结肠身份，与胎儿- CDX2缺失时打开的特定染色质区域。我们预测，福利利用了可获得的 CDX2丢失时的染色质。目标1将比较PERK在野生型干细胞中的活性，以及BRAFV600E或 CDX2突变干细胞。最先进的组学方法将允许对津贴进行第一次测量 在干细胞染色质上，在BRAFV600E突变从其内源位点表达后的几个小时内。我们 将测量PERK结合活性、核定位以及与其蛋白质伙伴的动态相互作用。 AIM 2将研究BRAFV600E突变在茎中的下游基因调控后果 缺乏Smad4的细胞。我们先前证明Smad4的缺失使小鼠易患锯齿状肿瘤 Smad4在人类肿瘤中经常发生突变。我们预测Smad4与PERK一起工作 促进分化，抑制干细胞更新。表观基因组学方法将把PERK绑定映射到 基因组，atac-seq和rna-seq将描述染色质和转录组的变化。最后，目标3 将从功能上测试结肠中的损伤/修复周期可以为其创造敏感环境的模型 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。这些研究将为临床前提供重要的 帮助解释和测试抑制人类锯齿状肿瘤启动的治疗策略的模型。 这些研究在尖端组学应用和GEMM方面具有创新性，在 探索干细胞中未被测试的致癌作用机制。成功完成这些研究将 让我们做好准备测试预防锯齿状结肠癌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.