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

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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.