The tumour suppressor p53 is often disrupted by missense mutations that can result in p53 protein accumulation and acquisition of novel oncogenic activities. Various studies have demonstrated that DNA-damaging drugs currently used in the clinic aimed at activating wild type p53, can also stabilise and activate mutant p53 oncogenic functions and thereby paradoxically enhance tumour progression, resulting in poor response to the treatment. In this study we aimed to investigate whether, like in wt p53, post-translational modifications (PTMs) drive such drug-induced mutant p53 accumulation and activation. For this purpose, we generated plasmids expressing non-phosphorylatable and phospho-mimic versions of R175H mutant p53 and tested them in different cell line models. We demonstrated that in response to DNA damage mutant p53 is accumulated and phosphorylated and these phenomena appeared to be mediated by ATM and ATR kinases. DNA-damage induced acetylation was also observed and occurred in a S15 phosphorylation-dependent manner. This suggested a role of the HAT p300, which is recruited by phosphorylated S15. Of note, other works have shown that p300 is required to trigger some oncogenic functions of mutant p53. We then aimed at developing systems to explore mutant p53 functions and their dependence on PTMs. Although we showed that cell growth is compromised upon endogenous mutant p53 depletion, exogenous expression of mutant p53 or its phosphorylation-site forms did not result in a successful rescue in our experimental conditions, thus we were unable to use this strategy to test the effect of PTMs. Ectopic expression of R175H mutant p53 or its phosphorylayion-site versions did not interfere with the growth rate and response to chemotherapy of the p53-null cell line H1299. We also found that mutant p53 phosphorylation does not affect subcellular localisation of mutant p53 and mutant p53-mediated inhibition of p63. Interestingly, ectopically expressed mutant p53 enhanced cell migration in H1299 cells. Notably, our results suggested an apparent threshold effect of mutant p53 levels required to induce migration. Due to the difficulty of obtaining cell lines expressing similar levels of the different phosphorylation-site mutants, the determination of the role of phosphorylation in mutant p53-induced migration was not conclusive. Remarkably, we found that, while S15 and S20 phosphorylation decreased MDM2-dependent degradation, only phosphorylated S20 interfered with CHIP-induced turnover in H1299 cells. Overall our data suggest that, despite exhibiting opposite biological effects, mutant and wt p53 can share upstream regulatory mechanisms and thus present phosphorylation as a promising target to prevent mutant p53 stabilisation and activation and improve response to therapy. Our results also highlight the challenge of developing a good system for determining the effects of the mutant p53 protein and its regulation by PTMs.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.