Identification of Pdx1-dependent circuits that reprogram cell identity and promote pancreatic carcinogenesis

细胞特性的丧失是胰腺导管腺癌（PDAC）发生和发展的重要先决条件。恶性细胞转化的特点是遗传改变，但也通过广泛的转录重编程，涉及细胞分化程序的下调和发育信号通路的激活。转录因子Pdx 1最初被鉴定为所有胰腺细胞谱系的主调节因子。我们的实验室最近公布了它作为胰腺腺泡细胞身份的重要监护人的作用。因此，在分化的腺泡细胞中耗尽Pdx 1与致癌基因KrasG12D合作，并诱导快速和广泛的细胞转化，其特征在于腺泡细胞身份的侵蚀和PDAC前体病变和PDAC的形成。然而，这种效应背后的确切机制仍然很难理解。我们的初步数据表明，Pdx 1靶基因涉及Ras信号级联的重要效应子，如Egfr。Ras活性水平是PDAC发展的关键。因此，通过分析Pdx 1和Egfr同时缺失的小鼠模型，结合单细胞分析来定义转录景观，我的目标是确定Pdx 1如何抑制Ras通路活性以抵消KrasG12D驱动的恶性细胞转化。阐明腺泡细胞身份丧失和致癌Kras信号传导之间的背景，将有助于理解逆转恶性细胞转化的方法。由于DNA甲基化介导的表观遗传沉默，肿瘤细胞向更具侵袭性的表型和晚期PDAC转移的重编程通过下调许多胰腺转录因子（包括PDX1）来实现。如我们的数据所示，胰腺癌细胞中Pdx 1的唯一敲低导致肿瘤细胞恶性程度的增强。Pdx 1敲除细胞的特征在于细胞增殖的暂时减少，其通过广泛的转录和代谢重编程绕过，极大地影响谷胱甘肽代谢。谷胱甘肽生物合成中的适应可以通过影响DNA和组蛋白甲基化所需的甲基的可用性对表观遗传景观产生深远的影响。通过应用靶向谷胱甘肽生物合成的药物，我将评估DNA甲基化水平的变化，重点关注在进展过程中沉默的基因。我建议，确定肿瘤细胞补偿Pdx 1损失的机制，将有助于揭示特定疾病的脆弱性，并提供新的治疗策略。

Loss of cell identity is an important prerequisite for the initiation and progression of the pancreatic ductal adenocarcinoma (PDAC). Malignant cell transformation is hallmarked by genetic alterations, but also by a broad transcriptional reprogramming, involving the down-regulation of cell differentiation programs and activation of developmental signaling pathways. The transcription factor Pdx1 was initially identified as the master regulator of all pancreatic cell lineages. Our lab recently unveiled its role as an important guardian of pancreatic acinar cell identity. Consequently, depletion of Pdx1 in differentiated acinar cells cooperated with the oncogene KrasG12D and induced rapid and widespread cell transformation, characterized by an erosion of acinar cell identity and the formation of PDAC precursor lesions and PDAC. However, the exact mechanisms behind the effect remain mostly elusive. Our preliminary data indicate that Pdx1 target genes involve important effectors of the Ras signaling cascade, such as Egfr. Ras activity levels are pivotal for PDAC development. Thus, by analyzing a mouse model with a concomitant loss of Pdx1 and Egfr, combined with single cell analysis to define the transcriptional landscape, I aim to determine how Pdx1 suppresses Ras pathway activity to counteract KrasG12D-driven malignant cell transformation. Elucidating the context between loss of acinar cell identity and oncogenic Kras signaling, will help to comprehend ways to revert malignant cell transformation. Tumor cell reprogramming towards a more aggressive phenotype and metastasis in late stage PDAC is accomplished by the down-regulation of a host of pancreatic transcription factors, including PDX1, due to epigenetic silencing mediated by DNA methylation. As indicated by our data, the sole knockdown of Pdx1 in pancreatic cancer cells leads to the reinforcement of tumor cell malignancy. Pdx1 knockdown cells are characterized by a temporary reduction in cell proliferation that is bypassed through an extensive transcriptional and metabolic reprogramming greatly affecting glutathione metabolism. Adaptations in glutathione biosynthesis can have profound effects on the epigenetic landscape by influencing the availability of methyl groups, needed for DNA and histone methylation. By applying drugs targeting glutathione biosynthesis, I will assess changes in DNA methylation levels, focusing on genes shown to be silenced during progression. I propose that identifying the mechanisms by which tumor cells compensate for Pdx1 loss, will contribute to uncover specific disease vulnerabilities and offer novel therapeutic strategies.