PRECISION METABOLIC THERAPY OF p53 MUTANT TRIPLE NEGATIVE BREAST CANCERS

项目总结 癌症基因组图谱(TCGA)的研究发现，p53是乳腺癌中最常见的突变基因 癌症。特别是，80%的三阴性乳腺癌(TNBCs)含有p53突变。然而，没有 可用于治疗p53突变的TNBC的特异性治疗。相比较而言，癌细胞有不同的代谢需求 对正常细胞的作用，这一观察结果刺激了小分子抑制剂的开发，以靶向新陈代谢 癌细胞生存所特别需要的酶。值得注意的是，尽管有证据支持 代谢途径失控在乳腺癌生长中的作用，以及P53突变的代谢依赖性 TNBC的生长和转移尚未得到全面的鉴定。因此，要明确识别 P53突变型TNBCs的代谢依赖性，我们进行了一项创新的、无偏倚的体内大规模研究 短发夹状RNA(ShRNA)以2000个已知代谢功能的基因为靶点进行筛选。有了这个屏幕， 我们鉴定了N-乙酰鞘氨醇氨基水解酶1(ASAH1)是一种肿瘤形成所必需的酶 P53突变型TNBCs的能力和转移活性。此外，我们还鉴定了两种小分子抑制物 ASAH1具有很强的抗p53突变型TNBC活性，从而表明ASAH1是一种潜在的药物靶点 突变型TNBCs的治疗。这一提议的中心假设是p53突变的TNBC细胞 它们的生存依赖于ASAH1，因此，抑制ASAH1选择性地根除p53突变的TNBCs。 我们的总体目标是确定ASAH1在驱动p53突变的TNBC肿瘤生长和 转移，了解p53突变型TNBCs对ASAH1依赖的机制 评估小分子ASAH1抑制剂治疗p53突变型TNBC的翻译潜力。在目标1中， 我们将通过一系列的研究来确定ASAH1在p53突变的TNBC肿瘤生长和转移中的作用。 补充的，最先进的小鼠模型，概括了TNBC生长和 转移。其中包括一种具有人类免疫系统的高度创新的人性化小鼠模型。在AIM 2，我们将检验我们的假设，即p53通过将ASAH1蛋白隔离在细胞核中来抑制ASAH1活性。 此外，根据我们新的初步结果，我们将确认ASAH1的缺失是否激活了葡萄糖 饥饿反应，通过神经酰胺介导的表达减少导致AMP激酶途径的激活 葡萄糖转运蛋白GLUT1在细胞膜上的结合。在目标3中，我们将评估 免疫抑制和免疫活性人源化小鼠p53突变模型中的ASAH1抑制剂 TNBC，单独使用，或基于我们的初步发现，与BET结构域抑制剂联合使用。总而言之， 我们预测，在本申请中提出的实验结果将确立ASAH1作为一个重要的 P53突变的TNBC细胞固有的脆弱性，阐明了P53依赖的机制- 突变的TNBCs对ASAH1活性的影响，并评估一种治疗P53突变的TNBC的新方法。

PROJECT SUMMARY The Cancer Genome Atlas (TCGA) studies have identified p53 as the most frequently mutated gene in breast cancer. In particular, 80% of triple-negative breast cancers (TNBCs) harbor p53 mutations. However, there is no specific therapy available for treating p53-mutant TNBC. Cancer cells have distinct metabolic needs compared to normal cells, and this observation has spurred the development of small molecule inhibitors to target metabolic enzymes that are specifically needed by cancer cells for survival. Notably, although there is evidence to support a role for metabolic pathway deregulation in breast cancer growth, the metabolic dependencies of p53-mutant TNBC growth and metastasis have not been comprehensively identified. Therefore, to specifically identify the metabolic dependencies of p53-mutant TNBCs, we performed an innovative and unbiased large-scale in vivo short-hairpin RNA (shRNA) screen by targeting 2000 genes with known metabolic functions. With this screen, we identified N-acylsphingosine amidohydrolase 1 (ASAH1) as an enzyme that is necessary for tumor-forming ability and metastatic activity of p53-mutant TNBCs. Additionally, we identified two small molecule inhibitors of ASAH1 with potent anti-p53-mutant TNBC activity, thereby indicating that ASAH1 is a potential drug target for the treatment of p53-mutant TNBCs. The central hypothesis of this proposal is that p53-mutant TNBC cells depend upon ASAH1 for their survival, and thus, ASAH1 inhibition selectively eradicates p53-mutant TNBCs. Our overall objectives are to determine the role of ASAH1 in driving p53-mutant TNBC tumor growth and metastasis, understand the mechanism underlying the dependency of p53-mutant TNBCs on ASAH1, and evaluate the translational potential of small molecule ASAH1 inhibitors for treating p53-mutant TNBC. In Aim 1, we will establish the role of ASAH1 in p53-mutant TNBC tumor growth and metastasis using a series of complementary, state-of-the-art mouse models that recapitulate characteristic features of TNBC growth and metastasis. These include a highly innovative humanized mouse model with a human immune system. In Aim 2, we will test our hypotheses that p53 represses ASAH1 activity by sequestering this protein in the nucleus. Additionally, based on our new preliminary results, we will confirm whether loss of ASAH1 activates the glucose starvation response, leading to AMP kinase pathway activation via a ceramide-mediated reduction in expression of the glucose transporter GLUT1 on the cell membrane. In Aim 3, we will evaluate the translational potential of ASAH1 inhibitors in immunocompromised and immunocompetent humanized mouse models of p53-mutant TNBC, either alone, or based on our preliminary findings, in combination with BET domain inhibitors. Collectively, we predict that the results of the experiments proposed in this application will establish ASAH1 as an important vulnerability inherent to p53-mutant TNBC cells, elucidate the mechanism underlying the dependency of p53- mutant TNBCs on ASAH1 activity, and evaluate a novel therapeutic approach for treatment of p53-mutant TNBC.