Lymphomas are malignant diseases involving the lymphatic system. Arsenic trioxide (As2O3) is a current therapeutic agent for acute promyelocytic leukaemia (APL).APL cells are sensitive to As2O3, with As2O3directly targeting the PML-RARA protein that plays an important role in the oncogenesis of APL. In order to discover the potential of As2O3as a treatment of lymphoma, understanding of the molecular mechanism of As2O3in human lymphoma cells is essential. In this thesis, we showed that the MYC gene is a therapeutic target for As2O3in B-cell lymphomas and the CCND1 (cyclin D1) gene is another therapeutic target for As2O3in mantle cell lymphoma (MCL), a subtype of non-Hodgkin lymphoma (NHL). Both real-time RT-PCR and immunoblotting analysis showed that the expression levels of MYC in all B-cell lymphoma cell lines were down-regulated at both mRNA and protein level after As2O3treatment. The expression levels of MYC were also found to positively correlate with the arsenic sensitivity as measured by MTT assay. Hence, the higher the level of MYC expression, the higher the arsenic sensitivity of human B-cell lymphoma cell lines. Besides, the change of downstream genes after modulation of MYC expression level by As2O3 treatment was investigated. The expression level of CDKN1A and CDKN1B was increased after As2O3 treatment. Interestingly, the growth rate of MYC over-expressing lymphoma cell lines decreased significantly after As2O3treatment, while there was no significant decrease in colony formation assay in lymphoma cells without MYC over-expression. Immunoblotting analysis showed that As2O3could degrade the cyclin D1 protein in mantle cell lymphoma cell lines in a dose-dependent manner. Real-time RT-PCR analysis also showed that the mRNA level of CCND1gene was decreased after As2O3treatment. We also demonstrated that As2O3-induced cyclin D1 protein degradation was related to the proteasome pathway. The growth rate of MCL cell line decreased significantly after As2O3treatmentby using colony formation assay. Human water channel protein, aquaporin 9 (AQP9) has been demonstrated to facilitate the arsenic uptake in human leukaemia cells. In this thesis, we showed that the expression levels of AQP9were found to positively correlate with the arsenic sensitivity as measured by MTT assay in B-cell lymphoma cells. We also demonstrated that dexamethasone could up-regulate AQP9expressions at both mRNA and protein levels in human B-cell lymphoma cell lines. These results not only suggest that As2O3is a potential therapy for B-cell lymphomas, especially for those MYC-over-expressed B-cell lymphomas and MCL, but also indicate that MYC may act as a biomarker for predicting the clinical behaviour of B-cell lymphoma patients to the As2O3treatment.Moreover, dexamethasone pre-treatment may enhance the therapeutic effect of As2O3by up-regulating AQP9expression in B-cell lymphomas. / published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Medical Sciences

T-cell lymphomas (TCLs) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas derived from post-thymic mature T- and NK-cells broadly classified peripheral (PTCL) or cutaneous (CTCL), indicating either a nodal or skin-homing disease, respectively. One of the main clinical challenges contributing to dismal outcomes for TCL patients is both the lack of curative treatment strategies and the high rate of relapse for currently approved therapies, underscoring the need for identification of novel targeted therapies for the treatment of TCL. While combination therapeutic strategies have been proposed and show great promise in pre-clinical and clinical trials for PTCL and CTCL, none are yet approved. Additional contributing factors toward the difficulty in studying TCL and the high rate of therapeutic failure is the highly heterogeneous genetic and molecular mechanisms driving TCL as well as the poorly understood role of non-tumor microenvironment cells in the pathogenesis of TCL. Indeed, while several studies have suggested that tumor associated macrophages play both a significant functional role in supporting tumor maintenance and are therapeutically targetable, less is known about potential tumor supporting roles of other cell microenvironment populations. Here, I used an unbiased and high-throughput approach to discover novel drug combinations in CTCL and to characterize at the single-cell level relevant molecular mechanisms driving T-cell lymphomagenesis. First, I demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an inhibitor of the epidermal growth factor receptor (EGFR) family, produces strongly synergistic antitumor effects in CTCL models, both in vitro and in vivo, using mechanisms of action that involve down-regulation of the JAK-STAT signaling pathway. This result suggests a potential therapeutic role for the combination of HDAC inhibitors with afatinib in the treatment of CTCL that had not been previously recognized. Second, we developed single-nuclei analysis on a cohort of 30 TCL (PTCL-NOS, AITL, epstein barr virus positive PTCL) and 6 normal patient samples to identify and deconvolute genomic and functional mechanisms contributing to T-cell lymphomagenesis. Here, I implemented a comprehensive bioinformatics pipeline for the analysis of sparse single-nuclei transcriptomic data and characterized heterogeneous molecular mechanisms driving T-cell lymphomagenesis, such as the upregulation of the PI3K-AKT-mTOR and WNT signaling pathways in a subset of 5/9 PTCL-NOS and 7/19 AITL samples, respectively. Additionally, I identified the enrichment of both the macrophage compartment in PTCL-NOS and AITL, and the specific enrichment of CD8+ T cells in AITL. These results suggest a correlation between patient-specific characteristics, such as mutational status, and possibly therapeutically targetable molecular mechanisms driving neoplastic cell growth that warrants further investigation.

Indiana University-Purdue University Indianapolis (IUPUI) / Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We previously reported that chemotherapy-treated lymphoma patients acquire a deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNy production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer cells (NKCs). Peripheral blood mononucleated cells (PBMCs) from healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNy production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines have higher tumoricidal activity than those stimulated with cytokines alone using in vitro tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation at target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.