title: Unraveling metabolic mechanisms underlying the folate pathway and targeting antifolate resistance via synthetic lethality
creator: Pirona, Anna Chiara
subject: ddc-000
subject: 000 Generalities, Science
subject: ddc-570
subject: 570 Life sciences
description: 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.
date: 2022
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/31501/1/20191208_thesis%20final%20Pirona%20COMPLETE.pdf
identifier: DOI:10.11588/heidok.00031501
identifier: urn:nbn:de:bsz:16-heidok-315018
identifier:   Pirona, Anna Chiara  (2022) Unraveling metabolic mechanisms underlying the folate pathway and targeting antifolate resistance via synthetic lethality.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/31501/
rights: info:eu-repo/semantics/openAccess
rights: Please see front page of the work (Sorry, Dublin Core plugin does not recognise license id)
language: eng