Co-Evolution of Follicular Lymphoma and its Microenvironment during Disease Formation, Progression and Development of Treatment Resistance

肿瘤微环境对肿瘤的进化具有深远的制约作用。同时，肿瘤细胞诱导自身有利的生长环境，包括对全身治疗的保护。因此，详细了解肿瘤细胞及其微环境之间的动态串扰具有极大的临床意义。滤泡性淋巴瘤（FL）是一种临床和分子高度异质性的B细胞恶性肿瘤，是这种进化模式的一个主要例子。大多数患者表现为晚期疾病，被认为是无法治愈的。我们之前的研究表明，这些患者的治疗结果是由淋巴瘤细胞固有的遗传改变以及免疫细胞衍生的基因表达特征决定的。迄今为止，在疾病形成、进展过程中，FL细胞及其免疫微环境组分之间的共同进化轨迹和功能串扰以及它们对治疗耐药性的影响在很大程度上是未知的。我们建议全面分析FL形成和发展过程中的免疫微环境，以及复发/难治性（r/r）疾病。我们将对不同阶段FL的活检组织进行靶向和全基因组基因表达谱分析。此外，我们将分析免疫化疗（GLSG2000试验）或无化疗方案（替代试验）后r/r FL患者的匹配活检组织。我们的分析将辅以多参数定量成像，提供FL微环境的空间分辨率。这些数据将与基因突变概况和拷贝数改变相结合。同时，我们将在BCL2过表达的转基因小鼠中进行全基因组功能转座子筛选，BCL2是人类FL的分子标志。转座子动员将在B细胞发育的不同阶段启动，以确定恶性转化的特定阶段脆弱性。我们将通过多参数流式细胞术、免疫荧光显微镜和RNA测序分析每个肿瘤的免疫微环境。这将允许遗传扰动与微环境组成和功能状态的关联。此外，我们将进行单细胞RNA和ATAC测序的免疫微环境，在恶性肿瘤前状态和在完全发展的小鼠肿瘤。我们在人类和小鼠研究中的候选致癌改变将使用已建立的体外和体内系统通过crispr - cas9介导的基因编辑进行验证。相应地，我们将从功能性上探究FL免疫微环境，从在已建立的小鼠淋巴瘤中滤泡T辅助细胞的失活开始。破译淋巴瘤细胞及其免疫微环境之间复杂的相互作用的分子机制将为预测、筛选并最终避免和克服疾病进展和治疗耐药性提供信息。

The tumor microenvironment imposes profound constraints upon cancer evolution. At the same time, tumor cells induce their own advantageous growth environment, including protection from systemic therapy. Thus, a detailed understanding of the dynamic crosstalk between tumor cells and their microenvironment is of utmost clinical relevance. Follicular lymphoma (FL), a clinically and molecularly highly heterogeneous B cell malignancy, represents a prime example of this evolving paradigm. Most patients present with advanced stage disease and are considered incurable. We previously showed that treatment outcome of these patients is determined by lymphoma cell-intrinsic genetic alterations as well as immune cell-derived gene expression signatures. To date, the co-evolutionary trajectories and functional crosstalk between FL cells and components of their immune microenvironment during disease formation, progression, and their impact on treatment resistance are largely unknown. We propose to comprehensively profile the immune microenvironment during the formation and progression of FL, and in relapsed/refractory (r/r) disease. We will perform targeted and genome-wide gene expression profiling of biopsies from different stages of FL. In addition, we will analyze matched biopsies from patients with r/r FL after immunochemotherapy (GLSG2000 trial) or chemotherapy-free regimens (Alternative trials). Our analyses will be complemented by multi-parameter quantitative imaging providing spatial resolution of the FL microenvironment. These data will be integrated with gene mutation profiles and copy number alterations. In parallel, we will perform genome-wide functional transposon screens in transgenic mice with BCL2 overexpression, the molecular hallmark of human FL. Transposon mobilization will be initiated at distinct stages of B cell development to identify stage-specific vulnerabilities for malignant transformation. We will profile the immune microenvironment of each tumor by multi-parameter flow cytometry, immunofluorescence microscopy and RNA sequencing. This will allow correlation of genetic perturbations to microenvironmental compositions and functional states. In addition, we will perform single cell RNA and ATAC sequencing of the immune microenvironment, both at premalignant states as well as in fully developed murine tumors. Candidate oncogenic alterations from our studies in man and mice will be validated by CRISPR-Cas9-mediated gene editing using established in vitro and in vivo systems. Correspondingly, we will functionally interrogate the FL immune microenvironment, beginning with the inactivation of follicular T helper cells in established murine lymphomas.Deciphering the molecular mechanisms underlying the complex reciprocal interactions between lymphoma cells and their immune microenvironment will inform approaches to anticipate, screen, and ultimately avoid and overcome disease progression and treatment resistance.