Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

Tumors have evolved elaborate mechanisms for evading immune detection, such as production of immunoinhibitory cytokines and down-regulation of major histocompatibility complex (MHC) expression. We have studied PAX3-FKHR as an example of an oncogenic fusion protein associated with an aggressive metastatic cancer. We show that PAX3-FKHR alters expression of genes that are normally regulated by Janus kinase/signal transducer and activator of transcription (STAT) signaling pathways. This occurs as a result of a specific interaction between PAX3-FKHR and the STAT3 transcription factor, which results in a dramatic reduction in tumor MHC expression, and an alteration in local cytokine concentrations to inhibit surrounding inflammatory cells and immune detection. Collectively, these data show that an oncogenic transcription factor can promote tumor growth and tissue invasion while inhibiting local inflammatory and immune responses. This is the first time that an immunomodulatory role has been described for an oncogenic fusion protein

The development of pulsed dendritic cell therapeutic vaccine strategies for the treatment of paediatric solid tumours and leukaemias

Anti-tumour immune responses are a balance between host immunosurveillance and immune escape mechanisms developed by the tumour. In this thesis, I have attempted to explore the nature of this balance in two cancer types, which are potentially susceptible to immune attack, in order to develop immunotherapeutic strategies for the treatment of these disorders. Alveolar rhabdomyosarcoma (ARMS) is an aggressive paediatric solid tumour associated with the translocation t(2;13)(q35;ql4). The resultant PAX3-FKHR fusion protein is a desirable target antigen for directed anti-tumour immunotherapy. We have explored the use of dendritic cells (DCs) loaded with PAX3-FKHR antigen as a cellular vaccine for the treatment of ARMS patients. We developed a murine ARMS model by stably transfecting a mouse rhabdomyosarcoma cell line (76-9) with PAX3-FKHR cDNA (clones called C23 and C24). We found that immunisation with C23 tumour lysate-pulsed DCs or non-pulsed DCs protected C57BL/6 mice from a subsequent C23 tumour challenge. The small tumours that did form in DC-immunised mice were shown to contain large numbers of infiltrating CD8+ and CD4+ T lymphocytes. However, ELISpot assays revealed that the DC vaccines were unable to initiate PAX3-FKHR-specific immune responses. We identified one peptide, derived from the N-terminal PAX3 portion of PAX3-FKHR, that binds with intermediate affinity to human leukocyte antigen (HLA)-A*0201, and could be loaded onto DCs as an alternative immunotherapeutic approach. In another project, we attempted to identify the target genes of PAX3-FKHR by comparing the global mRNA profile of C23 and C24 cells with empty vector transfected 76-9 cells, using oligonucleotide microarray expression profiling. We found that PAX3-FKHR up-regulated 31 genes and down-regulated 69 genes, by more than 2 fold, Interestingly, a large proportion of the genes repressed by PAX3-FKHR are known to be stimulated by interferon (IFN)-γ (21 out of the 44 genes with known function), including the major histocompatability complex (MHC) class I genes H2-K and H2-D1, and plasminogen activator inhibitor-1. Results from flow cytometric and Western blot analyses suggest that PAX3-FKHR exerts some of its effect on the ARMS phenotype, including the down-regulation of MHC class I which might act as an immune escape mechanism, by negatively interfering with IFN-γ signalling. In a separate study using patient blood samples, we assessed the feasibility of developing an immunotherapeutic strategy for the treatment of juvenile myelomonocytic leukaemia (JMML). Using granulocyte macrophage-colony stimulating factor and interleukin-4, we successfully differentiated JMML cells in vitro into immunostimulatory DCs. A cytotoxicity assay revealed that the leukaemic DCs were able to activate allogeneic T lymphocytes that specifically lysed leukaemic targets. Our novel findings suggest that JMML-derived DCs could be used for the in xivo or ex vivo generation of anti-leukaemia immune responses. Our findings suggest that DC-based immunotherapeutic approaches can be used to manipulate the balance between host immunosurveillance and tumour immune escape, resulting in effective anti-tumour immunity

The development of pulsed dendritic cell therapeutic vaccine strategies for the treatment of paediatric leukaemias and solid tumors

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