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Abstract

In this thesis we investigate acquired antifolate-resistance, with particular interest to folypolyglutamate synthase (FPGS) -dependent drug resistance. Our study model is T-cell acute lymphoid leukemia (T-ALL), already known to show methotrexate FPGSdependent resistance1. In particular, we use two different cell lines: wild type CCRFCEM and resistant MTA-C3, the latter one showing afunctional FPGS. Key of our study is the one carbon metabolism pathway, that takes part in the processing of (anti)folates in the cell. We first tried to get a better picture of one carbon metabolism and folate pathway in our model of study. We especially focused on the effect that different folate concentrations would have on the cytosolic vs mitochondrial flux usage, and our findings show that the capacity of retaining folates, dependent on RFC levels, is at the bases of it. Moreover, further investigations proved SHMT1 to be synthetic lethal to RFC. This is a novel discovery that can have considerable impact on clinical level. This part of the work contributed in establishing proper culturing conditions for our cell line models. This was essential in order to perform an unbiased CRISPR screening with the aim of finding synthetic lethal candidates to FPGS, downregulated in MTA-C3. To this aim we first established a new efficient process for cell transduction, that was necessary to not introduce subpopulation selection bias during the screening. In fact, T-ALL cell lines are well known for being quite difficult to manipulate and initial transduction trials showed 3-5% efficiency. The CRISPR library used in this screening had 260.000 sgRNA, therefore, not only to avoid experimental bias, but also to be able to handle practically wise such experiment, it was essential to establish a more efficient method. We aimed to reach 30% transduction rate, and we were able to achieve such result by using a new mixture of lipofectamines combined with protein columns for virus production, and cell pelleting for the transduction process. We then performed a CRISPR Cas9 screening and combined the results with three different protein screenings that were also conducted to better understand pathways involved in antifolate resistance. Out of these screenings, we selected 6 candidates based on common hits and possible relation with one carbon metabolism pathway. AHCY showed the most promising phenotype during validation, and our metabolomics analysis confirm the hypothesis that AHCY could act synthetic lethal to FPGS. However, further experiments need to be performed to unravel the complex puzzle and get a complete view.