HERV-K blockade to prevent RAS activation in breast cancer

项目摘要摘要。人类内源性K型逆转录病毒(HERV-K)是一种古老的逆转录病毒，具有 嵌入到人类基因组中，但由于突变的积累，它已经被沉默 在整个进化过程中。我们之前的研究已经证实HERV-K在乳腺癌中过表达 和其他癌症，但不在正常组织中表达，使其成为一个特别独特的治疗靶点。 我们最近还报道了Herv-K调节Ras癌蛋白在乳腺和其他组织中的表达 癌症。尽管激活的RAS突变蛋白是人类癌症中最常见的癌基因，但RAS是 被认为是“无法下药的”，并且还没有在临床上有效地被靶向。然而，这种机制 内源性逆转录病毒通过什么影响RAS的表达和/或激活尚不清楚，该项目是 旨在深入了解这种相互作用的生物学意义。我们的核心假设得到了检验 有观点认为，HERV-K与RAS信号通路的组成部分相互作用，改变RAS的表达。 乳腺癌细胞，主要是三阴性乳腺癌(TNBC)。在目标1的机械层面上，我们将： 1.确定在TNBC细胞中哪些RAS信号蛋白与Herv-K相互作用，是否有 A.HERV-K基因敲除和过度表达 B.三种关键乳腺癌信号通路的抑制剂：RAS/ERK、转化生长因子β和PI3K/AKT 2.评估加速乳腺癌细胞中RAS激活的前馈机制。 第二个目的是评估HERV-K特异性嵌合抗原受体(K-CAR)T细胞的有效性 细胞杀伤Herv-K阳性的TNBC细胞。我们之前的研究表明，K-CAR T细胞是从 乳腺癌患者对乳腺癌细胞有细胞毒作用，抑制细胞生长，但乳房正常 细胞未受影响。移植瘤模型中的肿瘤生长和肿瘤重量也减少了 人乳腺癌细胞，在该模型中肿瘤转移减少。利益的表达 HERV-K在这些乳腺肿瘤中的表达与癌蛋白RAS的表达有关。K-CAR T细胞因此可以 在乳腺癌中有双重作用：溶解肿瘤细胞和阻断RAS的表达。我们建议对这两种情况进行评估 在努力使K-CAR在治疗TNBC患者中发挥最大疗效方面发挥这些作用。我们将评估细胞毒性 从TNBC患者制备的K-CAR T细胞向乳腺癌细胞转化，并将使用TNBC患者- 衍生异种移植(PDX)模型，以确定我们的K-CAR在体内预防肿瘤生长的有效性。在……里面 此外，我们将评估K-CAR联合RAS/ERK、转化生长因子β或PI3K/AK途径抑制剂的联合治疗。 目标2的目标是针对两条主要的乳腺癌途径，其中一条是同时使用K-CAR 溶解肿瘤并靶向RAS信号，第二个使用小分子抑制剂靶向另一个 路径。我们预计联合治疗将显示出相加或甚至协同的效果， 为TNBC患者提供新的免疫治疗机会的目标。

Project Summary Abstract. Human endogenous retrovirus type K (HERV-K) is an ancient retrovirus that has become embedded in the human genome, but it has been silenced as a result of accumulation of mutations throughout evolution. Our previous studies have established that HERV-K is overexpressed in breast cancer and other cancers, but is not expressed in normal tissues, making it a particularly unique therapeutic target. We also recently reported that HERV-K regulates expression of the Ras oncoprotein in breast and other cancers. Although activated Ras mutant proteins are the most prevalent oncogenes in human cancers, Ras is considered to be “undruggable,” and has not been effectively targeted in the clinic. However, the mechanism by which endogenous retroviruses impact expression and/or activation of Ras is unknown, and this project is designed to yield insights into the biological significance of this interaction. The central hypothesis tested in our proposal is that HERV-K interacts with components of Ras signaling pathways to alter expression of Ras in breast cancer cells, primarily triple negative breast cancer (TNBC). At the mechanistic level in Aim 1 we will: 1. Determine which Ras signaling proteins interact with HERV-K in TNBC cells, with and without a. HERV-K knockdown and overexpression b. inhibitors of three key breast cancer signaling pathways: Ras/ERK, TGFβ, and PI3K/AKT 2. Evaluate a feed-forward mechanism that accelerates Ras activation in breast cancer cells. The second Aim will evaluate the effectiveness of HERV-K specific chimeric antigen receptor (K-CAR) T cells in killing HERV-K positive TNBC cells. Our previous studies showed that K-CAR T cells prepared from breast cancer patients were cytotoxic toward breast cancer cells and inhibited cell growth, but normal breast cells were unaffected. Tumor growth and tumor weight was also decreased in xenograft models bearing human breast cancer cells, and tumor metastasis was decreased in this model. Of interest, expression of HERV-K in these breast tumors correlated with expression of the oncoprotein Ras. K-CAR T cells could thus have a dual role in breast cancer: lyse the tumor cells and block Ras expression. We propose to evaluate both of these roles in an effort to maximize K-CAR efficacy in treating TNBC patients. We will evaluate cytotoxicity of K-CAR T cells prepared from TNBC patients toward breast cancer cells, and will use a TNBC patient- derived xenograft (PDX) model to determine efficacy of our K-CAR in tumor growth prevention in vivo. In addition, we will evaluate combined therapies of K-CAR plus Ras/ERK, TGFβ, or PI3K/AK pathway inhibitors. The objective of Aim 2 is to target two major breast cancer pathways, one with K-CAR which simultaneously lyses tumors and targets Ras signaling, and a second with small molecule inhibitors to target an additional pathway. We expect to show an additive or perhaps even a synergistic effect from the combined therapy, with a goal of providing a new immunotherapeutic opportunity for TNBC patients.