Targeting epigenetic abnormalities to inhibit cutaneous squamous cell carcinoma

项目总结/摘要 慢性紫外线（UV）辐射不仅诱导基因突变，而且诱导异常的表观遗传 影响转录因子、癌基因和肿瘤抑制因子表达的修饰，导致 皮肤鳞状细胞癌（cSCC）是美国第二大常见癌症， States.更好地理解紫外线相关的表观遗传异常的机制将有助于 提供了抑制皮肤恶性肿瘤的新方法。 值得注意的是，慢性紫外线照射可诱导CDKN 2A基因启动子的DNA超甲基化， 编码肿瘤抑制因子p16 INK 4A和p14 ARF。这种表观遗传改变沉默CDKN 2A，促进肿瘤生长。 生长和转移。然而，目前还不清楚这种表观遗传畸变的逆转是否能 重新激活CDKN 2A并抑制cSCC的恶性表型。在目标1中，我们将使用我们的基于CRISPR-Cas9的 表观基因组编辑工具，以特异性地使CDKN 2A启动子去甲基化，并研究靶向 DNA去甲基化对癌症表型的影响。 UV辐射增加了Myc的表达，Myc是一种致癌转录因子。Myc激活是一种 Myc癌基因家族是癌症的标志，并且Myc癌基因家族在许多人类癌症中失调。当过度表达时， Myc与非典型（低亲和力）基序结合，侵入增强子，并潜在地激活超级增强子 （非编码基因组区域由异常高水平的H3 K27乙酰化定义）。癌症特异性超级 增强子强烈上调致癌基因，从而导致恶性肿瘤。然而，它仍然难以捉摸如何异常 在cSCC中形成超级增强子。我们假设过表达的Myc与低亲和力的Myc结合， 位点，招募组蛋白乙酰转移酶p300，并产生驱动恶性肿瘤的异常超级增强子。 在目标2中，我们将确定过表达的Myc在cSCC中超级增强子形成中的作用。 cSCC的一个子集显示分化差，这与患者生存率差相关。但委员会仍 不清楚是什么赋予低分化cSCC的侵袭性。我们假设小分子 DNA甲基化和/或Myc抑制剂通过重新激活CDKN 2A抑制体内低分化cSCC 和/或抑制Myc相关的超级增强子。在目标3中，我们将使用低分化cSCC患者- 衍生的异种移植物作为临床前模型以评估这些抑制剂对肿瘤生长和转移的作用。 创新技术将用于表观遗传分析：“PIXUL-ChIP”（高通量染色质 剪切以获得稳健的ChIP信号）和“伊娃”（检测特异性DNA的表观遗传可视化测定 单细胞水平的甲基化）。利用我们的表观基因组编辑工具，拟议的研究将阐明 表观遗传异常对cSCC侵袭性的贡献，并揭示了调节cSCC侵袭性的治疗潜力。 表观遗传标记来抑制cSCC。

Project Summary/Abstract Chronic ultraviolet (UV) radiation induces not only genetic mutations but also aberrant epigenetic modifications that affect the expression of transcription factors, oncogenes, and tumor suppressors, leading to the development of cutaneous squamous cell carcinoma (cSCC), the second most common cancer in the United States. A better understanding of the mechanisms underlying UV-associated epigenetic abnormalities will provide novel approaches to inhibit skin malignancies. Strikingly, chronic UV exposure induces DNA hypermethylation at the promoter of CDKN2A gene, which encodes tumor suppressors p16INK4A and p14ARF. This epigenetic alteration silences CDKN2A, promoting tumor growth and metastasis. However, it remains unclear whether the reversal of this epigenetic aberration can reactivate CDKN2A and inhibit malignant phenotypes of cSCC. In Aim 1, we will use our CRISPR-Cas9-based epigenome editing tools to specifically demethylate the CDKN2A promoter and investigate the effect of targeted DNA demethylation on cancer phenotypes. UV radiation increases the expression of Myc, which is an oncogenic transcription factor. Myc activation is a cancer hallmark, and the Myc oncogene family is deregulated in many human cancers. When overexpressed, Myc binds to noncanonical (low-affinity) motifs, invading enhancers, and potentially activates super-enhancers (noncoding genomic regions defined by unusually high levels of H3K27 acetylation). Cancer-specific super- enhancers strongly upregulate oncogenes, driving malignancies. However, it remains elusive how aberrant super-enhancers are formed in cSCC. We hypothesize that overexpressed Myc binds to low-affinity Myc binding sites, recruits histone acetyltransferase p300, and generates aberrant super-enhancers that drive malignancy. In Aim 2, we will determine the role of overexpressed Myc in super-enhancer formation in cSCC. A subset of cSCC displays poor differentiation, which correlates with poor patient survival. However, it remains unclear what confers the aggressiveness of poorly differentiated cSCC. We hypothesize that small-molecule inhibitors of DNA methylation and/or Myc suppress poorly differentiated cSCC in vivo by reactivating CDKN2A and/or inhibiting Myc-associated super-enhancers. In Aim 3, we will use poorly differentiated cSCC patient- derived xenografts as preclinical models to assess the effects of these inhibitors on tumor growth and metastasis. Innovative technologies will be used for epigenetic analyses: “PIXUL-ChIP” (high-throughput chromatin shearing for robust ChIP signals) and “EVA” (epigenetic visualization assay that detects specific DNA methylation at the single-cell level). With our epigenome editing tools, the proposed study will elucidate the contribution of epigenetic abnormalities to cSCC aggressiveness and reveal therapeutic potential of modulating epigenetic marks to suppress cSCC.