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.

项目摘要 肿瘤细胞与其微环境的动态相互作用在刺激癌症中起着重要作用 进展，并可能有助于克隆进化和治疗抗性的步伐。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，我们已经产生了几个新的 慢性淋巴细胞白血病（CLL）的条件性敲入/敲除小鼠模型，由B细胞限制性 人类CLL病变的表达，提供了机会来模拟人类CLL病变的巨大遗传异质性。 在一个有免疫能力的微环境中的癌症。目前的项目试图使用单细胞RNA 测序（scRNA-seq）和功能测定，以确定这些细胞中的CLL-微环境相互作用。 CLL小鼠模型，并验证在人类CLL中的发现。具体而言，我们将：（目标1）发现影响 CLL免疫领域的CLL驱动事件。通过scRNA-seq分析，我们将表征 CLL微环境内的免疫景观，检查脾脏和骨髓（BM） 在年龄匹配的对照（Cd-19 Cre转基因小鼠）和4种不同的遗传定义的CLL中， 小鼠模型（Cd 19-Cre × MDR，mut-Sf 3b &Atm，mut-Ikzf 3和Dnmt 3a-deleted）。基于我们有趣的 在Dnmt 3a缺失模型中进行的scRNA-seq试验研究中，我们现在建议交叉比较 在每个模型中的免疫细胞的组成和转录状态，并确定是否有 CLL和免疫群体之间一致或独特的模型依赖性相互作用。(Aim 2） CLL-TME相互作用的候选介质。我们预计，我们将确定微环境的变化， 兴趣在模型中存在共同或独特之处，我们将进行流式细胞术， 免疫荧光（IF）/免疫组织化学（IHC）染色对独立的小鼠标本进行验证。 我们还将进行体外共培养实验和体内移植来研究功能性相互作用 CLL细胞和特定的免疫细胞群体之间的关系。事实上，我们从Dnmt 3a获得的试验数据- 缺失的CLL小鼠脾脏已经鉴定出了一个扩增的失调树突状细胞亚群， 我们将评估这种变化是否也在其他模型中观察到，以及这种变化的程度 调节CLL的发展。(Aim 3）确认人CLL的TME中CLL驱动事件的影响。我们 将交叉比较小鼠scRNA-seq数据与产生的现有人类CLL scRNA-seq数据 在吴的实验室里我们将对CLL患者的LN和BM样本进行IF/IHC染色，以进一步验证 承诺签名总之，所提出的分析将扩大我们对癌细胞内在 功能可以重塑肿瘤免疫微环境，并有可能有助于合理的治疗。 个性化癌症免疫疗法的设计。