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

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.