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: 10357003
• 负责人: Catherine Ju-Ying Wu
• 金额: $ 24.96万
• 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/10357003
• 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; Knock-in; Knowledge; Lesion; Letters; Malignant Neoplasms; Mediator of activation protein; 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; Yin; cancer cell; cancer genetics; cancer immunotherapy; 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 肿瘤细胞与微环境的动态相互作用在刺激癌症中起着至关重要的作用 新兴的证据，并可能有助于克隆进化和治疗性抗性的速度。 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），但证据仍然很少。最近，我们生成了几本小说 由B细胞限制驱动的慢性淋巴细胞白血病（CLL）的有条件敲入/输出的小鼠模型 人类CLL病变的表达，提供了模拟人类巨大遗传异质性的机会 免疫能力的微环境中的癌症。当前的项目试图使用单细胞RNA 测序（SCRNA-SEQ）和功能测定，以确定其中的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：（目标1）发现影响 CLL免疫景观上的CLL驾驶员事件。通过SCRNA-SEQ分析，我们将表征 CLL微环境内的免疫景观，检查脾和骨髓（BM） 年龄匹配的对照（CD-19 CRE转基因小鼠）和4个不同遗传定义的CLL的隔室 小鼠模型（CD19-CRE×MDR，MUT-SF3B和ATM，MUT-IKZF3和DNMT3A删除）。建立在我们有趣的基础上 在DNMT3A删除模型中进行的Pilot 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和免疫吞吐量之间的一致或独特的模型依赖性相互作用。 （AIM 2）验证 CLL-TME相互作用的候选者。我们预计我们将确定微环境的变化 在模型中以共同或唯一发现的兴趣，我们将执行流式细胞术，并且 免疫荧光（IF）/免疫组织化学（IHC）在独立的小鼠标本上染色以进行验证。 我们还将进行体外共培养实验和体内移植以研究功能相互作用 在CLL细胞和特定的免疫细胞群之间。确实，我们来自DNMT3A-的试验数据 已删除的CLL小鼠SLEEN已经确定了扩展的树突状细胞的亚群，并且 我们将评估是否还观察到这种变化的其他模型以及此更改的程度 调节CLL的发展。 （AIM 3）确认CLL驾驶员事件在人类CLL的TME中的影响。我们 将跨越鼠标scrna-seq数据与现有的人类cll scrna-seq数据的数据进行比较 在吴实验室。如果/IHC对CLL患者的LN和BM样品的染色/IHC染色以进一步验证 有希望的签名。总共提出的分析将扩大我们有关癌细胞中心如何的知识 功能可以重塑肿瘤免疫微环境，并有可能有助于理性 个性化癌症免疫疗法的设计。