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Reversible protein phosphorylation, catalyzed by kinases and phosphatases, is arguably one of the most prominent signaling mechanisms in the human cell. Protein Phosphatase 2A (PP2A) is a major family of Serine/Threonine protein phosphatases of which the members are involved in a broad array of cellular processes, such as mitogenic signaling, growth, cell division, DNA transcription, protein translation, DNA damage signaling and repair, cell death, migration and differentiation, among others. PP2A is mainly composed as trimeric holoenzymes, consisting of a catalytic C subunit, a structural A subunit and one of a large family of regulatory B subunits, which determine activity, substrate specificity and subcellular localization. The holo-enzyme assembly of PP2A is a closely monitored process, and here, the Protein Phosphatase TwoA Activator (PTPA), is indispensible for proper assembly of PP2A holo-enzymes into functional trimers. Interestingly, PTPA has been shown to be dysregulated throughout multiple cancer types, and is associated with dismal survival perspectives. Seen the implication of PP2A in many cellular signaling pathways that are targeted by cancer therapeutics, we will investigate the consequence of PTPA inactivation on the therapeutic efficiencey of approved and experimental cancer therapies, specifically in the setting of the hard-to-treat KRAS mutant non-small cell lung cancer (NSCLC). With this project, we aim to identify therapies that would benefit KRAS mutant NSCLC patients, based on the PTPA status as a stratification marker for treatment response.Across non-small-cell lung cancer (NSCLC) patient databases we identified multiple point mutations in the gene (PPP2R4 ) that encodes for the PTPA protein, which affect the functionality of the PTPA as they result in loss of PP2A reactivating capacity. Moreover, common heterozygous loss of PPP2R4 was associated with a decrease in survival of KRAS-mutant NSCLC cancer patients. Loss of PTPA resulted in increased anchorage growth and xenograft growth in KRAS-mutant A549 cells, which was associated with increased expression of the oncogenic transcription factor c-MYC. Importantly, homozygous and heterozygous loss of Ppp2r4 in a LSL-KrasG12D mouse model was associated with accelerated onset of tumorigenesis at twelve weeks post. Furthermore, loss of PTPA in A549 cells caused resistance against the MEK inhibitor selumetinib, while it sensitized cells towards temsirolimus, both in vitro and in vivo, which was confirmed in the low PTPA expressing KRAS-mutant NSCLC cell line A427, in vitro.An unbiased MS and phospho-MS analysis revealed that loss of PTPA resulted in changes in DNA-damage signaling processes, among others, as witnessed by Panther Enrichment Analyses. In line with our findings, low-PTPA expressing KRAS-mutant NSCLC cells displayed increased resistance against gemcitabine, whereas PTPA-low cells were more vulnerable to gamma-irradiation. Furthermore, given the central role of PTPA in PP2A holoenzyme assembly, we wanted to verify whether the novel PP2A-activating drugs (Small Molecule Activators of PP2A, SMAPs; improved Heterocyclic PP2A Activators; iHAPs) were affected by loss of PTPA. Interestingly, we observed that for long term in vitro and in vivo treatments, loss of PTPA levels was associated a increased sensitization towards several of the tested compounds. However, both for DNA-damage agents as well as PP2A activators, the exact mechanism of action remains to be fully determined.To conclude, we have validated the role of PTPA with regard to tumorigenesis and therapy response of KRAS-mutant NSCLC in a preclinical setting, and are confident our findings have paved the way for follow-up investigations in a clinical setting.