Small molecule probes of MYC stability and function intumorigenesis

MYC稳定性和肿瘤发生功能的小分子探针

• 批准号: 10361512
• 负责人: Sarki A. Abdulkadir
• 金额: $ 55.95万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10361512
• 关键词: 3-Dimensional; ATAC-seq; Affect; Animal Model; Architecture; Binding; Biochemical; Biology; Cancer Model; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cellular Stress Response; Cessation of life; Chemicals; Chromatin; Chromatin Structure; Combined Modality Therapy; Complement; Complex; Computer Assisted; Consensus; Coupled; DNA; DNA Binding; Dependence; Drug Kinetics; Epigenetic Process; Event; Family; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Helix-Turn-Helix Motifs; Human; In Vitro; Knowledge; MYC Family Protein; MYC gene; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Mediating; Modeling; Oncogenes; Oncogenic; Oncoproteins; Output; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Play; Proteasome Inhibitor; Proteins; Publishing; Recurrence; Role; Series; Site; Stress; Testing; Therapeutic; Threonine; Toxicology; Transcriptional Activation; Tumorigenicity; Ubiquitin; Up-Regulation; Work; activating transcription factor; antitumor effect; base; biological adaptation to stress; c-myc Genes; cytokine; efficacy study; genetic manipulation; genome-wide; genomic locus; in silico; in vivo; inhibitor; knock-down; leukemia; member; multicatalytic endopeptidase complex; neoplastic cell; novel; novel strategies; overexpression; programs; recruit; response; screening; small molecule; small molecule inhibitor; transcription factor; transcriptome sequencing; tumor; tumor microenvironment; tumorigenesis

ABSTRACT MYC oncoproteins (including c-MYC, L-MYC and N-MYC) play critical roles in the initiation, progression and recurrence of many human malignancies. Extensive studies indicate that MYC is required to maintain tumor cell survival and proliferation. We have recently used a novel approach that combined computer-aided modeling with a rapid in vivo screen to develop a new series of direct small molecule inhibitors (MYCi’s) that show excellent selectivity, potency and tolerability in multiple MYC-driven cancer models. These compounds demonstrate a dual mechanism of action. First, direct binding of MYCi to MYC in the basic helix-loop-helix (bHLH) region disrupts complex formation with MYC which is required for MYC transcriptional activity. Secondly, binding of MYCi enhances MYC phosphorylation on threonine-58 (pT58) which promotes MYC degradation via the ubiquitin-proteasome pathway. However the key downstream effectors of these events and how they might impact cellular function are unknown. Reduction of MYC protein and enhanced pT58MYC may be expected to have profound effects on MYC family protein interactions with each other and with chromatin. In this regard, we have observed in preliminary studies that MYCi leads to selective loss of MYC at genomic loci enriched for master chromatin regulators (CTCF and FOX), suggesting disruption of the 3D architecture of the MYC-bound genome in response to MYCi. Additionally, unfolded MYC due to MYCi binding and/or enhanced MYC degradation may provoke a cellular stress response. Using unbiased ATAC-seq and RNA-seq approaches, we found that MYCi treatment activates the ATF4/CHOP stress response pathway. Importantly, activation of ATF4/CHOP by MYCi is an on-target, MYC-dependent effect. ATF4 mediates MYCi antitumor activity as ATF4 depletion partially ameliorates the antitumor effects of MYCi. Furthermore, we propose that MYCi-induced ATF4 cytokines modulate the tumor microenvironment. Activation of the ATF4 pathway by MYCi exposes potential therapeutic vulnerabilities for rational combination approaches, such as combination of MYCi with proteasome inhibitors that activates ATF4. Based on the preliminary findings, our central hypotheses is that MYCi inhibits MYC-dependent tumorigenesis by a dual-pronged mode of action. First, MYCi affects MYC family target gene expression by disrupting MYC/MAX interaction and by promoting MYC degradation. Secondly, binding of MYCi to MYC and/or MYC degradation activates an ATF4/CHOP stress response pathway that suppresses tumor cell viability. We propose the following specific aims to test these hypotheses: Aim 1). To investigate the mechanisms by which MYC inhibitor modulates MYC transcriptional activity and the epigenetic landscape. We will investigate the consequences of MYCi treatment on the recruitment of MYC, pT58MYC, and associated factors to chromatin; changes to 3D chromatin architecture; as well as the effects on MYC-driven transcriptional output in tumor cells vitro and in vivo. Aim 2). To define the mechanisms and functional consequences of ATF4/CHOP pathway activation by MYCi. We will determine mechanism of ARF4 upregulation by MYCi; define the role of MYCi- induced ATF4 in regulating target gene expression, cell viability and tumorigenicity; and assess strategies that exploit the consequences of ATF4 activation as a means of enhancing MYCi anti-tumor efficacy. These studies are significant as MYC is implicated in the majority of human cancers. The studies advance the use of MYCi as chemical probes to unmask distinct biology that complements the knowledge derived from genetic manipulations of MYC proteins. The findings will contribute to the efforts aimed at developing small molecule MYCi as potential therapeutics. Specifically, this work indicates that small-molecule MYC inhibitors have an additional anti-tumor effect due to the activation of the ATF4 pathway beyond the antitumor effects of suppressing MYC function. Finally, understanding this on-target ATF4 response provoked by small-molecule MYCi will provide rational strategies for combination therapy to enhance MYCi efficacy.

摘要 MYC癌蛋白(包括c-MYC、L-MYC和N-MYC)在肿瘤的发生、发展和转移中起重要作用。 许多人类恶性肿瘤的复发。广泛的研究表明，MYC是维持肿瘤细胞所必需的 生存和繁殖。我们最近使用了一种新的方法，将计算机辅助建模与 一种快速体内筛选，以开发一系列新的直接小分子抑制剂(MYCI)，显示出良好的 多种MYC驱动的癌症模型的选择性、有效性和耐受性。这些化合物显示出一种 双重作用机制。首先，MYCi与碱性螺旋-环-螺旋(BHLH)区域中的MYC直接结合 破坏MYC转录活动所需的与MYC的复合体的形成。第二，法律的约束力 MYCi增强苏氨酸-58(PT58)上MYC的磷酸化，促进MYC的降解 泛素-蛋白酶体途径。然而，这些事件的关键下游效应者以及它们可能 影响细胞功能的因素尚不清楚。减少MYC蛋白和增强pT58MYC有望 对MYC家族蛋白之间的相互作用以及与染色质的相互作用有深远的影响。在这方面，我们 在初步研究中观察到，MYCi导致在富含MYC的基因组座位上选择性丢失MYC 主要染色质调节剂(CTCF和FOX)，表明MYC结合的3D架构被破坏 基因组对MYCi的反应。此外，由于MYCi结合和/或增强的MYC而展开的MYC 降解可能会引发细胞应激反应。使用不偏不倚的ATAC-SEQ和RNA-SEQ方法，我们 发现MYCi处理激活了ATF4/CHOP应激反应通路。重要的是，激活 MYCi的ATF4/CHOP是一种靶向、MYC依赖的效应。ATF4与ATF4一样介导MYCi抗肿瘤活性 耗竭可部分改善MYCi的抗肿瘤作用。此外，我们认为MYCi诱导的ATF4 细胞因子调节肿瘤微环境。MYCi激活ATF4通路的潜力 合理联合治疗的脆弱性，如MYCi与蛋白酶体联合 激活ATF4的抑制剂。根据初步发现，我们的中心假设是MYCi抑制 MYC依赖的肿瘤发生通过双管齐下的作用模式。首先，MYCi影响MYC家族靶基因 通过干扰MYC/MAX的相互作用和促进MYC的降解来表达。第二，MYCi的约束性 TO MYC和/或MYC降解激活ATF4/CHOP应激反应通路，抑制肿瘤细胞 生存能力。我们提出了以下具体目标来检验这些假设：目标1)。探讨其作用机制 MYC抑制剂通过其调节MYC转录活性和表观遗传格局。我们会调查的 MYCi治疗对MYC、pT58MYC及与染色质相关的因素的影响； 3D染色质结构的变化；以及对MYC驱动的肿瘤细胞转录输出的影响 体外和体内。目标2)。明确ATF4/CHOP通路的机制和功能后果 由MYCi激活。我们将确定MYCi上调ARF4的机制；定义MYCi- 诱导ATF4调节靶基因表达、细胞活性和致瘤性；并评估 利用ATF4激活的结果作为增强MYCi抗肿瘤疗效的一种手段。 这些研究意义重大，因为MYC与大多数人类癌症有关。这些研究推进了 使用MYCi作为化学探针揭开独特的生物学面纱，补充来自 MYC蛋白的遗传操作。这些发现将有助于旨在开发小型 MYCi分子作为潜在的治疗药物。具体地说，这项工作表明，小分子MYC抑制剂 具有额外的抗肿瘤作用，这是由于激活了ATF4途径，而不是 抑制MYC函数。最后，理解小分子引起的这种靶向ATF4反应 MYCi将为提高MYCi疗效提供合理的综合治疗策略。