Exploring novel targeted therapeutic strategies against Acute Myeloid Leukemia cells based on inhibition of bromodomain proteins and CDC20 activity

Acute myeloid leukemia (AML) is a haematological malignancies arising from the accumulation of undifferentiated myeloid progenitors with an uncontrolled proliferation. The genomic landscape of AML revealed that the disease is characterized by high level of heterogeneity and is subjected to clonal evolution driven by selective pressure of chemotherapy. In this study, we investigated the therapeutic effects of the inhibition of BRD4 and CDC20 in vitro and ex vivo. We demonstrated that inhibition of BRD4 with GSK1215101A in AML cell lines was effective under hypoxia. It induced the activation of antioxidant response both, at transcriptomic and metabolomic levels, driven by enrichment of NRF2 pathway under normoxic and hypoxic condition. Moreover, the combined treatment with Omaveloxolone, a drug inducing NRF2 activation and NF-κB inhibition, potentiated the effects on apoptosis and colony forming capacity of stem progenitor cells. Lastly, gene expression profiling data revealed that combination treatment induced major changes in genes related to cell cycle, together with enrichment of cell differentiation pathways and negative regulation of WNT, in normoxia and hypoxia. Regarding CDC20, we observed its up-regulation in AML patients. Treatment with two different inhibitors, Apcin and proTAME, was effective in primary AML cells and in AML cell lines, through induction of apoptosis and mitotic arrest. The lack of correlation between proliferation markers and CDC20 levels in AML cell subpopulations supports the idea of alternative CDC20 functions, independent from its essential role during mitosis. CDC20-KD experiments conducted in AML cell lines revealed a mild effect on apoptosis induction, but no significant change in cell cycle progression. In summary, these results allowed the identification of a new strategy combination to improve the effects of BRD4 inhibition on LSC residing in the BM hypoxic niche, and provide some new evidence regarding the potential role of CDC20 as a new target for AML treatment.

Microbiome and resitome and foods of animal origin

To ensure food safety and to prevent food-borne illnesses, rapid and accurate detection of pathogenic agents is essential. It has already been demonstrated that shotgun metagenomic sequencing can be used to detect pathogens and their antibiotic resistance genes in food. In the studies presented in this thesis, the application shotgun metagenomic sequencing has been applied to investigate both the microbiome and resistome of foods of animal origin in order to assess advantages and disadvantages of shotgun metagenomic sequencing in comparison to the cultural methods. In the first study, it has been shown that shotgun metagenomics can be applied to detect microorganisms experimentally spiked in cold-smoked salmon. Nevertheless, a direct correlation between cell concentration of each spiked microorganism and number of corresponding reads cannot be established yet. In the second and third studies, the microbiomes and resistomes characterizing caeca and the corresponding carcasses of the birds reared in the conventional and antibiotic free farms were compared. The results highlighted the need to reduce sources of microbial contamination and antimicrobial resistance not only at the farm level but also at the post-harvest one. In the fourth study, it has been demonstrated that testing a single aliquot of a food homogenate is representative of the whole homogenate because biological replicates displayed overlapping taxonomic and functional composition. All in all, the results obtained confirmed that the application of shotgun metagenomic sequencing represents a powerful tool that can be used in the identification of both spoilage and pathogenic microorganism, and their resistome in foods of animal origin. However, a robust relationship between sequence read abundance and concentration of colony-forming unit must be still established.