Project 2- The ISR effector ATF4 in metabolic reprogramming and survival during Myc-induced tumorigenesis

摘要/总结（项目2） 肿瘤细胞的内源性应激包括癌基因激活，以及外源性应激，如低 氧/营养物质的可用性，引起内质网（ER）的扰动。此外，致癌性 转化的细胞面临着增加的负担，增加的生物合成途径和重新连接的代谢 以满足快速扩散带来的需求。适应随之而来的压力和重建 细胞内稳态的维持是通过激活一个协调的信号转导程序来实现的， 综合应激反应（ISR）。在上一个供资期间，我们证明， 由致癌MYC引起的蛋白质合成，激活ISR的PERK/GCN 2 → eIF 2 β臂，从而 支持MYC诱导的细胞转化。在初步未发表的研究中，我们积累了强大的 证据支持ISR效应子和eIF 2靶向物eIF 2 ATF 4在转化和 肿瘤发生，特别是在具有活化MYC的肿瘤中。然而，ATF 4如何激发差异反应， 在依赖MYC的转化背景下， 无人回应我们将检验ISR激活ATF 4在MYC中起关键作用的假设。 通过促进代谢和翻译适应诱导转化和肿瘤进展 与MYC协调，侧重于三个具体目标。在目标1中，我们将确定关键节点， 细胞代谢和翻译调节，其由ATF 4和c-MYC两者协调调节。 具体地说，我们将描述ATF 4激活Glut-1和eIF 4 E转录的机制， 确定GLUT 1和eIF 4 E在调节糖酵解、翻译和存活过程中的功能需求 体外和体内MYC依赖性转化。在目标2下，我们将描述共同作用的机制。 通过ChIP-seq分析在淋巴瘤、结直肠癌中调节ATF 4和MYC之间的转录靶点 (CRC)和前列腺癌（PCa）细胞表达诱导型MYC，并分析协调调节 基因.然后，我们将确定所鉴定的共调节基因的敲除/敲低的影响， MYC依赖性增殖、凋亡和肿瘤生长的新靶点。最后，根据目标3， 我们将确定ATF 4在PCa和CRC中MYC依赖性转化和肿瘤发生中的作用， 使用与PTENfl/fl：MycTg杂交的条件性敲除ATF 4模型的肿瘤，和小鼠CRC模型 （原位的、同基因的和自发的）以及3D CRC类器官。我们还将与项目1合作， 分析BMAL 1和Clock基因的ATF 4依赖性下调的调节及其在 翻译和淋巴瘤发生。最后，在项目3中，我们将分析ATF 4消融对 I型干扰素途径和肿瘤浸润性细胞毒性T淋巴细胞的活力/效应功能。 这些目标的完成将使我们更好地了解ATF 4在MYC依赖性免疫缺陷中的关键作用。 促肿瘤发生的过程，并可能发现新的目标，治疗干预这些恶性肿瘤。

ABSTRACT/SUMMARY (Project 2) Tumor cell intrinsic stress including oncogene activation, as well as extrinsic stresses, such as low oxygen/nutrient availability, elicit perturbations in the endoplasmic reticulum (ER). Moreover, oncogenically transformed cells face increased burden placed by augmented biosynthetic pathways and rewired metabolism to meet the demands imposed by rapid proliferation. Adaptation to the ensuing stress and re-establishment of cellular homeostasis is achieved via activation of a coordinated signal transduction program termed the Integrated Stress Response (ISR). During the previous funded period, we demonstrated that increased rates of protein synthesis elicited by oncogenic MYC, activate the PERK/GCN2→eIF2 arm of the ISR, thereby supporting MYC-induced cell transformation. In preliminary unpublished studies, we have accumulated strong evidence supporting an essential role for the ISR effector and target of eIF2 ATF4, in transformation and tumorigenesis, particularly in tumors with activated MYC. However, how ATF4 elicits differential responses to various stresses in the context of MYC-dependent transformation is a critical question that remains unanswered. We will test the hypothesis that activation of ATF4 by the ISR plays a critical role in MYC- induced transformation and tumor progression by promoting metabolic and translational adaptation in coordination with MYC by focusing on three specific Aims. In Aim 1 we will identify critical nodes in cellular metabolism and translational regulation which are coordinately regulated by both ATF4 and c-MYC. Specifically, we will delineate the mechanism of Glut-1 and eIF4E transcriptional activation by ATF4 and determine functional requirements of GLUT1 and eIF4E in regulating glycolysis, translation and survival during MYC-dependent transformation in vitro and in vivo. Under Aim 2, we will delineate the mechanism of co- regulation of transcriptional targets between ATF4 and MYC by ChIP-seq analysis in lymphoma, colorectal (CRC) and prostate (PCa) cancer cells expressing inducible forms of MYC and analyze coordinately regulated genes. We will then determine the effects of knockout/knockdown of the identified co-regulated genes and newly identified targets in MYC-dependent proliferation, apoptosis and tumor growth. Finally, under Aim 3, we will determine the role of ATF4 in MYC-dependent transformation and tumorigenesis in PCa and CRC tumors using a conditional knockout ATF4 model crossed with PTENfl/fl:MycTg, and mouse CRC models (orthotopic, syngeneic and spontaneous) as well as 3D CRC organoids. We will also work with Project 1 to analyze the regulation of ATF4-dependent downregulation of BMAL1 and Clock genes and their role in translation and lymphomagenesis. Finally, with Project 3, we will analyze the effects of ATF4 ablation on type I interferon pathway and viability/effector functions of tumor-infiltrating cytotoxic T lymphocytes. Completion of these aims will provide a better understanding of the critical role of ATF4 in MYC-dependent pro-tumorigenic processes and may uncover new targets for therapeutic intervention in these malignancies.