Defining the impact of mutational drivers on the immune microenvironment of CLL

定义突变驱动因素对 CLL 免疫微环境的影响

• 批准号: 10558675
• 负责人: Catherine Ju-Ying Wu
• 金额: $ 20.39万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10558675
• 关键词: Address; Affect; Age; B lymphoid malignancy; B-Lymphocytes; Biological Assay; Biological Models; Bone Marrow; Cells; Chromosome abnormality; Chronic Lymphocytic Leukemia; Clonal Evolution; Coculture Techniques; Collaborations; DNMT3a; Data; Dendritic Cells; Dependence; Development; Disease; Disease Progression; Ensure; Event; Flow Cytometry; Genetic; Genetic Heterogeneity; Genetic Transcription; Genetically Engineered Mouse; Heterogeneity; Human; Immune; Immunocompetent; Immunofluorescence Immunologic; Immunotherapy; In Vitro; Institution; Knock-in; Knowledge; Lesion; Letters; Malignant Neoplasms; Maps; Mediator; Modeling; Mus; Mutation; Oncogenic; Pathway interactions; Patients; Pilot Projects; Play; Population; Principal Investigator; Resolution; Role; Sampling; Series; Signal Transduction; Specimen; Spleen; Stains; Transgenic Mice; Transplantation; Validation; Writing; Yin; cancer cell; cancer genetics; cancer immunotherapy; candidate validation; chromosome 13q loss; chronic lymphocytic leukemia cell; comparative; disease heterogeneity; driver mutation; experimental study; genetic makeup; human disease; immune function; in vivo; individual patient; insight; interest; lymph node microenvironment; lymph nodes; mouse model; neoplastic cell; notch protein; novel; rational design; response; single-cell RNA sequencing; therapy resistant; trafficking; transcriptome sequencing; treatment response; tumor; tumor microenvironment; tumor progression; tumor-immune system interactions

Project Summary The dynamic interactions of tumor cells with their microenvironment play an essential role in stimulating cancer progression, and likely contribute to the pace of clonal evolution and therapeutic resistance. Emerging evidence suggests that genetic features of cancer cells may impact composition and function of immune cells within the tumor microenvironment (TME), yet evidence for this remains scant. Recently, we have generated several novel conditional knock-in/out mouse models of chronic lymphocytic leukemia (CLL) driven by B-cell restricted expression of human CLL lesions, providing opportunities to model the vast genetic heterogeneity of human cancers within an immune-competent microenvironment. This current projects seeks to use single cell RNA sequencing (scRNA-seq) and functional assays to determine the CLL-microenvironment interactions in these CLL mouse models, and validate the findings in human CLLs. In particular, we will: (Aim 1) Discover the impact of CLL driver events on the CLL immune landscape. Through scRNA-seq analyses, we will characterize the immune landscape within the CLL microenvironment, examining both the spleen and bone marrow (BM) compartments in age-matched controls (Cd-19 Cre transgenic mice) and 4 different genetically-defined CLL mouse models (Cd19-Cre × MDR, mut-Sf3b&Atm, mut-Ikzf3 and Dnmt3a-deleted). Building on our intriguing pilot scRNA-seq study conducted in the Dnmt3a-deleted model, we now propose to cross compare the composition and transcriptional states of immune cells in each model, and to determine whether there are consistent or unique model-dependent interactions between CLL and immune populations. (Aim 2) Validate candidate mediators of CLL-TME interactions. We anticipate that we will identify microenvironment changes of interest either found in common or unique amongst the models, and we will perform flow cytometry and immunofluorescence (IF)/immunohistochemical (IHC) staining on independent mouse specimens for validation. We will also perform in vitro co-culture experiments and in vivo transplantation to study the functional interaction between CLL cells and particular immune cell populations of interest. Indeed, our pilot data from the Dnmt3a- deleted CLL mouse spleen has already identified an expanded subpopulation of dysregulated dendritic cells, and we will evaluate if this change is also observed across the other models and the extent to which this change regulates CLL development. (Aim 3) Confirm the effects of CLL driver events in the TMEs of human CLL. We will cross compare the mouse scRNA-seq data with that of an existing human CLL scRNA-seq data generated in the Wu lab. We will perform IF/IHC staining of LN and BM samples from CLL patients to further validate promising signatures. Altogether, the proposed analyses will expand our knowledge on how cancer cell-intrinsic features can remodel the tumor immune microenvironment, and has the potential to contribute to the rational design of personalized cancer immunotherapy.

项目概要 肿瘤细胞与其微环境的动态相互作用在刺激癌症中发挥着重要作用 进展，并可能有助于克隆进化和治疗耐药的速度。新出现的证据 表明癌细胞的遗传特征可能会影响免疫细胞的组成和功能 肿瘤微环境（TME），但这方面的证据仍然很少。最近，我们创作了几部小说 B细胞限制性慢性淋巴细胞白血病（CLL）条件性敲入/敲除小鼠模型 人类 CLL 病变的表达，为模拟人类巨大的遗传异质性提供了机会 免疫功能强大的微环境中的癌症。当前的项目寻求使用单细胞 RNA 测序 (scRNA-seq) 和功能测定以确定这些疾病中的 CLL-微环境相互作用 CLL 小鼠模型，并验证人类 CLL 中的发现。特别是，我们将：（目标 1）发现影响 CLL 免疫景观中的 CLL 驱动事件。通过 scRNA-seq 分析，我们将表征 CLL 微环境中的免疫景观，检查脾脏和骨髓 (BM) 年龄匹配对照（Cd-19 Cre 转基因小鼠）和 4 种不同基因定义的 CLL 中的区室 小鼠模型（Cd19-Cre × MDR、mut-Sf3b&Atm、mut-Ikzf3 和 Dnmt3a 删除）。建立在我们有趣的 在 Dnmt3a 删除模型中进行的试点 scRNA-seq 研究，我们现在建议交叉比较 每个模型中免疫细胞的组成和转录状态，并确定是否存在 CLL 和免疫群体之间一致或独特的模型依赖性相互作用。 （目标 2）验证 CLL-TME 相互作用的候选中介者。我们预计我们将识别微环境的变化 在模型中发现共同或独特的兴趣，我们将进行流式细胞术和 对独立小鼠样本进行免疫荧光 (IF)/免疫组织化学 (IHC) 染色以进行验证。 我们还将进行体外共培养实验和体内移植来研究功能相互作用 CLL 细胞和感兴趣的特定免疫细胞群之间的关系。事实上，我们来自 Dnmt3a 的试点数据- 删除的 CLL 小鼠脾脏已经鉴定出一个扩大的失调树突状细胞亚群，并且 我们将评估其他模型是否也观察到这种变化以及这种变化的程度 调节 CLL 的发展。 （目标 3）确认 CLL 驱动事件对人类 CLL 的 TME 的影响。我们 将交叉比较小鼠 scRNA-seq 数据与生成的现有人类 CLL scRNA-seq 数据 在吴实验室。我们将对 CLL 患者的 LN 和 BM 样本进行 IF/IHC 染色，以进一步验证 有希望的签名。总而言之，所提出的分析将扩展我们对癌细胞内在如何 特征可以重塑肿瘤免疫微环境，并有可能有助于合理的治疗 个性化癌症免疫治疗的设计。