BLR&D Merit Review Research Career Scientist (RCS) Award (IK6)

摘要： 目前，使用免疫检查点抑制剂（ICI），如α-CTLA 4和/或α-PD 1的治疗性治疗， 提高生存率（>50%四年生存），特别是在高度突变的肿瘤。此外，结合ICI 靶向肿瘤癌基因的疗法在某些肿瘤中非常有效。 我们的假设是，免疫学上的“冷”肿瘤对ICI治疗的反应可以通过联合ICI来增强。 靶向致癌驱动因子的治疗也诱导募集CD 8 +T细胞的关键趋化因子的表达 和DC转移到TME，并将肿瘤相关巨噬细胞转变为抗肿瘤表型， 骨髓来源的抑制细胞。为了验证这一假设，我们将利用免疫活性小鼠模型， 携带患者来源的异种移植物（PDX）的人源化小鼠模型，以及人肿瘤共移植物的器官型培养物。 与患者的免疫细胞一起培养，以确定这些特定的精确医学方法是否可以用于靶向治疗 增强对ICI的反应，并提高小鼠的存活率。 我们建议在四个领域推进黑色素瘤和乳腺癌的治疗策略。有两 我们将开发用于黑色素瘤研究的方法：1）使用免疫活性BRAFV 600 E/PTEN-/-， NRASQ 61 K、R/CDKN 2a-/-或三重野生型小鼠模型、人源化PDX模型和人的3D类器官 与从患者分离的免疫细胞共培养的黑色素瘤肿瘤，我们将确定是否用 rigosertib（NRAS通路抑制剂）、CDK 4/6抑制剂+MDM 2拮抗剂联合ICI治疗诱导 肿瘤消退将确定抗肿瘤免疫应答改变的机制。2）。使用CyTOF 和sc-NGS分析，我们将表征来自产生耐药性的黑色素瘤患者的活检组织和PBL， 免疫检查点抑制剂，评估其他检查点蛋白的表达以及新的 驱动突变，并使用这些数据开发新的治疗策略与其他检查点抑制剂， 利用我们的类器官培养模型进行靶向治疗。 对于我们的乳腺癌研究：1）我们将用AKT抑制剂加紫杉醇联合ICI治疗肿瘤 使用免疫活性小鼠模型、人源化PDX模型和器官型模型， 与患者免疫细胞共培养的患者肿瘤的培养物; 2）我们将检查小鼠中的肿瘤生长， 在肿瘤组织或特定免疫细胞群中靶向缺失CXCR 2或CXCR 4， 治疗和免疫治疗。在适当的情况下，CXCR 2和/或CXCR 4拮抗剂可以与药物组合。 靶向治疗，以增强对ICI治疗的反应。 总之，这些研究将为黑色素瘤和乳腺癌的新临床试验设计提供关键信息 与我们的临床合作者，并告知正在进行的治疗的耐药性机制。黑色素瘤和乳腺 癌症可能是我们退伍军人的主要问题， 总体生存率至关重要。

Abstract: Currently, therapeutic treatment with immune checkpoint inhibitors(ICI’s), such as α-CTLA4 and/or α-PD1, are increasing survival rates (>50% four-year survival), particularly in highly mutated tumors. Moreover, combining ICIs with therapies that target tumor oncogenes has been highly effective in some tumors. Our hypothesis is that response to ICI therapy in immunologically “cold” tumors can be enhanced by combining ICIs with therapies targeted to oncogenic drivers that also induce expression of key chemokines that recruit CD8+T cells and DCs to the TME and shift tumor associated macrophages to an anti-tumor phenotype, and reduce the infiltration by myeloid-derived suppressor cells. To test this hypothesis, we will utilize immune competent mouse models, humanized mouse models bearing patient-derived xenograft (PDX), and organotypic cultures of human tumor co- cultured with patient immune cells to determine if these specific precision medicine approaches to targeted therapies enhance response to ICIs, and improve survival in mice. We propose to advance therapeutic strategies for melanoma and breast cancer in four areas. There are two approaches we will develop for melanoma studies: 1) Using immune competent BRAFV600E/PTEN-/-, NRASQ61K,R/CDKN2a-/- or triple wild type mouse models, humanized PDX models and 3D organoids of human melanoma tumors co-cultured with immune cells isolated from the patient, we will determine whether treatment with either rigosertib (NRAS pathway inhibitor), CDK4/6 inhibitor +MDM2 antagonist combined with ICI therapy induces tumor regression. Mechanisms for alterations in anti-tumor immune response will be determined. 2). Using CyTOF and sc-NGS analysis we will characterize biopsy tissue and PBLs from melanoma patients who develop resistance to immune checkpoint inhibitors, evaluate the expression of other checkpoint proteins as well as activation of new driver mutations, and use these data to develop new strategies for treatment with other checkpoint inhibitors and targeted therapies using our organoid culture model. For our breast cancer studies: 1) We will treat tumors with AKT inhibitors plus paclitaxel in combination with ICI therapies or prior to ICI treatment using immune competent mouse models, humanized PDX models, and organotypic cultures of patient tumors co-cultured with patient immune cells; 2) We will examine the tumor growth in mice with targeted deletion of CXCR2 or CXCR4 in tumor tissue or in specific immune cell populations with or without targeted therapies and immune therapy. Where appropriate, CXCR2 and/or CXCR4 antagonists may be combined with the targeted therapies to enhance response to ICI therapy. Altogether these studies will provide key information for the design of new clinical trials I melanoma and breast cancer with our clinical collaborators and inform mechanisms of resistance to ongoing therapies. Melanoma and breast cancer can be major issues for our Veterans and development of improved therapeutic contributions that enhance overall survival is of key importance.