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Abstract

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in western world. This disease, with an indolent course and patients responding heterogeneously to recommended therapies, remains incurable. The Eµ-TCL1 mouse model, is a known useful tool for preclinical studies of CLL. In this thesis, I present a detailed in-silico view of CLL specific clonal heterogeneity and T cell tumor microenvironment (TME) as observed in spleen of EµTCL1 mouse and patient lymph nodes during the course of the disease. In the first part, I present clonal evolution orchestrated by dynamics of B cell receptor (BCR) rearrangements and somatic variations, using whole exome sequencing (WES) of serially transplanted Eµ-TCL1 mouse tumors. Low allele frequency mutations that were nonoverlapping between mouse tumors were identified. 10 out of 13 tumors were identified to be oligoclonal. In addition, three distinct patterns of evolving SNV-defined and BCR clonotypes emerged as the disease aggressed from primary to secondary tumor. Interestingly, I identified stereotypic CLL mouse BCRs having Ighv11 and Ighv12 genes that are known to undergo chronic stimulation in response to autoantigens, hence potentially contributing to CLL pathogenesis. These observations signified the importance of clonotype information for accurate interpretation of CLL disease course and drug efficacy, especially during time-series experiments involving adoptively transferred Eµ-TCL1 mouse tumors. Also, trisomy 15 was observed, hypothesizing involvement of Myc overexpression during CLL development in EµTCL1 mouse. It could be stated that, not just the overexpression of Tcl1 gene but other factors also contribute to CLL malignancy in mice. Following this, I investigated genetic (WES) and transcriptomic (RNA-seq) changes in monoclonal Eµ-TCL1 AT (adoptive transfer) mouse tumors, acquired as a result of ibrutinib resistance. Ibrutinib is widely used as a frontline treatment for CLL patients, some of which acquire resistance to the drug after showing an initial response. In mouse tumors, loss of therapeutic efficacy followed by uncontrolled tumor growth was observed at 6 weeks of treatment initiation. Ibrutinib was not able to inflict an observable selection pressure on BCR clonality as well as mutation profile of these tumors in 6 weeks. However, the transcriptional profile of ibrutinib resistant tumors was unique in contrast to untreated ones. From top upregulated genes identified to be putatively involved in ibrutinib resistance, Tbet gene, is currently being followed up for in-vivo studies as a therapeutic target. In the second part of the thesis, I present subpopulations of CD3+ T cell compartment characteristically differentially expressed in the CLL TME as compared to that of controls. This analysis was the first of its kind to have utilized CLL patient lymph nodes (LN) for probing TME at single cell level. Additionally, the patient’s bone marrow (BM) and peripheral blood (PB); as well as the spleens from Eµ-TCL1 AT mice were investigated for CLL infiltrating T cell subpopulations. Single cell (sc) CyTOF (mass cytometry) analysis using a panel of 32 surface protein markers revealed an increased abundance of exhausted phenotype in patient LNs as compared to BM and PB samples from the same patient. This observation raised uncertainty of PB and BM as the tissue of choice for studying CLL linked T cell exhaustion. Intriguingly, Eµ-TCL1 mouse T cell compartments showed presence of IFN-responders, absent from patient CD4+ cell type. 7 out of 12 mouse Cd4+ subpopulations showed expression of Tcytotoxic markers, which could indicate activated subpopulations. The results presented in this thesis provide a detailed view of heterogeneity manifested by 1) Eµ-TCL1 mouse tumors in course of disease progression; 2) the transformed CLL TME in patients and mouse. These findings would prove valuable during mechanistic and drug treatment studies in Eµ-TCL1 mouse and to evaluate their translational potency in CLL clinical setting under the influence of CLL specific tumor niche.