Optimizing Response to Immune Checkpoint Inhibitor Therapy for Breast Cancer: A Role for Inhibitors of the PI3K pathway

转移性乳腺癌（BC）是世界各地妇女的主要健康问题。约40，610名妇女和460名 预计今年美国男性将死于BC。虽然有许多治疗选择激素 受体阳性（HR+）和HER 2 + BC患者，三阴性BC（TNBC）患者的标准治疗主要是 依赖常规化疗和放疗治疗转移性BC的改进选择代表了巨大的 未满足的医疗需求最近，几项临床试验研究了与HR通路中任一种的联合治疗 阻断剂或HER 2拮抗剂和PI 3 K抑制剂，因为PI 3 K途径在细胞内被组成性激活或突变， 超过50%的BC患者。然而，结果显示无进展生存期仅增加2-4个月， 使用的剂量方案存在广泛的3级和4级毒性。免疫检查点的发现 抑制剂（ICI）正在彻底改变癌症治疗，但到目前为止，BC患者对ICI没有表现出强烈的反应 由于低突变负荷和CD 4+和CD 8 + T细胞的最小浸润（“冷”），因此在治疗中， 我们的假设是，免疫“冷”BC对ICI治疗的反应可以通过联合 用紫杉醇（PTX）抑制AKT以诱导免疫原性肿瘤细胞死亡和转移肿瘤相关的免疫原性肿瘤细胞的疗法 将免疫细胞转化为抗肿瘤表型。为了验证这一假设，我们将利用免疫活性小鼠模型， 类器官/免疫细胞共培养物和携带患者来源的异种移植物（PDX）的人源化小鼠模型， 确定PI 3 K通路抑制剂是否增强对ICI的反应并改善小鼠的存活率。有两 具体目标。目标1。开发通过AKT治疗减少TNBC生长的最佳策略 抑制剂ipatasertib联合紫杉醇和免疫检查点抑制剂（ICI）抗CTLA 4+抗PD 1。使用 免疫活性小鼠模型，我们将确定重新编程肿瘤免疫的功能意义， 乳腺肿瘤对AKT抑制的反应与对早期、中期和晚期ICI的反应 治疗期间的时间点。治疗反应的机制将根据对这些结果的分析进行研究。 以下参数：毒性;肿瘤生长;转移;免疫细胞含量（免疫组）;细胞因子/趋化因子 肿瘤、骨髓、肺和血液样品中的表达谱;血管生成;以及应答中的转录组。 和无反应的肿瘤。最先进的技术将包括多重免疫组织化学（IHC）、流式细胞术（FCM）和免疫组织化学（ELISA）。 细胞计数、CyTOF、反相蛋白质分析（RPPA）、RNA测序（RNAseq）、DNA测序（DNAseq） 路径分析。预测小鼠中对治疗的应答的转录组学和免疫组学特征将在本文中描述。 与来自我们现有的正在进行的临床试验和已发表的“响应签名”的RNAseq数据相比26。目标二： 为了确定在两种人TNBC模型中用AKT抑制剂与PTX和ICI组合的治疗的功效： 1)类器官共培养人TNBC加上成纤维细胞、内皮细胞和患者免疫细胞：2）人源化 从TNBC患者建立的患者来源的异种移植物（PDX）小鼠模型。遗传/免疫组签名将 评估并与正在进行的临床试验的数据进行比较。

Metastatic breast cancer (BC) is a major health issue for women across the world. About 40,610 women and 460 men in the US are expected to die this year alone from BC. While there are numerous treatment options for hormone receptor positive (HR+) and HER2+ BC patients, the standard of care for triple negative BC (TNBC) patients largely relies on conventional chemotherapy and radiation. Improved options for treating metastatic BC represent a vast unmet medical need. Recently, several clinical trials investigated combined treatment with either HR pathway blockers or HER2 antagonists and PI3K inhibitors, since the PI3K pathway is constitutively activated or mutated in over 50% of BC patients. However, the results have shown only 2-4 months increase in progression free survival and there is extensive Grade 3 and 4 toxicity with the dosage schedules used. The discovery of immune checkpoint inhibitors (ICIs) is revolutionizing cancer therapy, but thus far BC patients are not showing strong responses to ICI therapy, due to low mutational load and minimal infiltration of CD4+ and CD8+ T cells (“cold”). Our hypothesis is that response to ICI therapy in immunologically “cold” BCs can be enhanced by combining therapies that inhibit AKT with paclitaxel (PTX) to induce immunogenic tumor cell death and shift tumor-associated immune cells to an anti-tumor phenotype. To test this hypothesis, we will utilize immune competent mouse models, organoid/immune cell co-cultures, and humanized mouse models bearing patient-derived xenograft (PDX) to determine if PI3K pathway inhibitors, when enhance response to ICIs, and improve survival in mice. There are two specific aims. Aim 1. To develop the optimal strategy for reducing growth of TNBC through treatment with an AKT inhibitor, ipatasertib, combined with paclitaxel, and immune checkpoint inhibitors (ICIs) anti-CTLA4 + anti-PD1. Using immune competent mouse models, we will determine the functional significance of reprograming the tumor immune environment in mammary tumors in response to AKT inhibition in reference to response to ICIs at early, mid and late time points during therapy. Mechanisms of therapeutic response will be investigated based upon analysis of the following parameters: toxicity; tumor growth; metastasis; immune cell content (immunome); cytokine/chemokine expression profile in tumor, bone marrow, lung and blood samples; angiogenesis; and transcriptome in responding and non- responding tumors. State of the art technology will include multiplex immunohistochemistry (IHC), flow cytometry, CyTOF, reverse phase protein analysis (RPPA), RNA sequencing (RNAseq), DNA sequencing (DNAseq) and pathway analysis. Transcriptomic and immunome signatures predicting response to treatment in mice will be compared to RNAseq data from ongoing clinical trials available to us and published “response signatures” 26. Aim 2: To determine the efficacy of treatment with AKT inhibitors combined with PTX and ICIs in two human TNBC models: 1) organoid co-cultures human TNBC plus fibroblasts, endothelial cells and patient immune cells: 2) humanized patient-derived xenografts (PDX) mouse models established from TNBC patients. Genetic/immunome signatures will be evaluated and compared to data from ongoing clinical trials.