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Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment. S.R.P.'s laboratory is partially funded by funds from the Spanish National Research and Development Plan, Instituto de Salud Carlos III and FEDER [PI17/02303 to S.R.P]; R.T.R. is supported by a fellowship from the AECC scientific foundation; J.M.B. is supported by Spanish Ministry of Education, Culture and Sport [FPU15/01978]; J.B.'s laboratory is partially funded by FEDER funds, H2020 BRIDGES project and the Spanish Network on Rare Diseases (CIBERER) [FIS PI16/00440]; Faculty of Medicine at the Norwegian University of Science and Technology and the Central Norway Regional Health Authority supports [46056921 to A.S. and H.E.K.]; Svanhild and Arne Must's Fund for Medical Research (to A.S. and H.E.K.); Norwegian Research Council (to T.V.); SIN-TEF SEP project 102020885 (to T.V.); Vinnova (to A.C.K and P.S.); the Torsten and Ragnar Soderberg Foundation (to T.H.) and the Helleday Foundation (to C.B.). Funding for open access charge and project support: European Union's Horizon 2020 research and innovation programunder the Marie Sklodowska-Curie grant agreement [722729 to A.P., B.M.F.H., T.H.]; European Research Council [ERC TAROX-695376 to T.H.]; Swedish Research Council (to T.H. and P.S.); Swedish Cancer Society (to T.H. and P.S.); Swedish Children's Cancer Foundation (to T.H.); Swedish Pain Relief Foundation (to T.H.). Sí

På tross av lovende fremskritt innen immunterapi, som anses som en ny pilar i kreftbehandling, responderer kun en liten andel pasienter som ønsket på spesifikke behandlinger. Utviklingen av forenklede metoder for studie av komplekse og spesifikke cellulære responser på bestemte behandlinger, vil kunne fungere veiledende i pasientutvalg og muliggjøre økt fremdrift innen forskning på immunterapi. Makrofager har en viktig rolle i kreft-relatert inflammasjon, og en økt andel tumor-assosierte makrofager (TAMs) i tumorer korrelerer ofte med en redusert total prognose. I hovedsak er det funnet at TAMs uttrykker en M2-liknende fenotype, som fremmer tumorprogresjon og undertrykker en effektiv adaptiv immunrespons. Flere strategier for manipulering av makrofager har blitt foreslått som potensielle immunterapeutiske behandlinger mot kreft. I dette studiet ble det utviklet en forenklet makrofag polariseringsmodell for å undersøke kjennetegn ved makrofager stimulert mot en M1- eller M2-liknende fenotype. Monocytter av THP-1 cellelinjen ble behandlet med phorbol 12-myristate 13-acetate (PMA) for å indusere differensieringen til makrofager. THP-1 makrofagene ble deretter enten eksponert for LPS og IFN-γ for å utvikle M1-liknende makrofager, eller IL-4 og IL-13 for å utvikle M2-liknende makrofager. Evaluering av THP-1 makrofagenes resulterende fenotype ble utført etter ulike eksponeringstider. Flowcytometri og ELISA ble brukt for å undersøke antatte M1 og M2 spesifikke markører på celleoverflaten og utskillelsen av karakteristiske cytokiner. Både flowcytometri- og ELISA-målingene indikerte en vellykket polarisering av THP-1 makrofager mot en M1-liknende fenotype. De M1 polariserte THP-1 makrofagene ble karakterisert ved en signifikant økning i antall celler med overflatemarkørene MHCII og CD80, og en signifikant økning i utskillelsen av TNF-α og CXCL10. I motsetning førte M2 polariseringen verken til den forventede uttrykkelsen av CD163 og CD206, eller til den antatte utskillelsen av IL-10 og IL-1Ra. Dataene indikerer et potensiale ved THP-1 monocytter til å utvikle en M1-liknende fenotype, mens aktiveringen og karakteriseringen av en M2-liknende fenotype i THP-1 makrofager krever videre undersøkelse. Med den oppnådde aktiveringen og karakteriseringen av M1 polariserte THP-1 makrofager, ble den forenklede makrofagmodellen brukt til å undersøke viktigheten av spesifikke aktiveringsspor for M1 polarisering. Effekten av å eksponere THP-1 celler for TH5487, BMS-345541 eller Rapamycin under M1 polarisering ble undersøkt ved å detektere følgende utskillelse av TNF-α. Både TH5487 og BMS-345541 er kjent for å hindre inflammatorisk genuttrykkelse ved å forstyrre NF-κB, mens Rapamycin er en anerkjent inhibitor av mTOR sporet. Verken TH5487 eller BMS-345541 endret utskillelsen av TNF-α. Resultatene antyder derfor at utskillelsen av TNF-α fra THP-1 makrofager under disse eksperimentelle betingelsene er uavhengig av NF-κB sporet. Likedan førte ikke tilstedeværelsen av Rapamycin under LPS- og IFN-γ-stimulering av THP-1 makrofagene til en endring i utskillelsen av TNF-α. Dermed viste forsøkene ingen tydelig kontroll over M1 polariseringen ved å inhibere mTOR sporet. Effekten av TH5487, BMS-345541 og Rapamycin, aktiveringssporene disse angriper, og den resulterende M1 polariseringsprofilen bør undersøkes nærmere ved å studere et utvalg av cytokiner, både på mRNA- og proteinnivå. Despite the promising advancements of immunotherapy, notifying a new pillar of cancer treatment, a limited portion of patients respond successfully to specific treatments. The development of simplified assays to investigate complex and specific cellular responses to particular treatments would aid in patient selection and advance the research on cancer immunotherapy. Macrophages play a prominent role in cancer-related inflammation, and the prevalence of tumor-associated macrophages (TAMs) within tumors often correlates with a reduced overall prognosis. Importantly, most TAMs have been found to resemble an M2-like phenotype, promoting tumor progression and suppression of an effective adaptive immune response. Several strategies of macrophage manipulation have been suggested as potential anti-cancer immunotherapeutic treatments. In this thesis, a simplified macrophage polarization model was developed to investigate the characteristics of macrophages stimulated towards either an M1- or M2-like phenotype. Monocytes of the THP-1 cell line were exposed to phorbol 12-myristate 13-acetate (PMA) to induce differentiation into macrophages, followed by incubation with LPS and IFN-γ to develop M1-like macrophages or IL-4 and IL-13 to develop M2-like macrophages. Evaluation of the resulting THP-1 macrophage phenotypes was performed after various exposure times by flow cytometry and ELISA to investigate putative M1 and M2 specific cell surface markers and secreted cytokines, respectively. Flow cytometry and ELISA measurements indicated a successful polarization of THP-1 macrophages towards an M1-like phenotype. The M1 polarized THP-1 macrophages were characterized by a significantly increased amount of cells expressing the surface markers MHCII and CD80, and a significantly increased secretion of TNF-α and CXCL10. In contrast, exposure to M2 polarizing stimuli did not induce the hypothesized surface expression of CD163 and CD206 or the secretion of IL-10 and IL-1Ra. Therefore, the data suggest a potential of THP-1 macrophages to develop into an M1 macrophage phenotype, whereas the activation and characterization of an M2 phenotype in THP-1 macrophages require further investigations. With the achieved activation and characterization of M1 polarized THP-1 macrophages, the simplified macrophage model was utilized to investigate the impact of specific pathways on M1 macrophage polarization. The effect of exposing the THP-1 cells to TH5487, BMS-345541, or Rapamycin during M1 polarization was investigated by detecting the consequent secretion of TNF-α. Both TH5487 and BMS-345541 are known to prevent inflammatory gene expression by perturbing NF-κB, whereas Rapamycin is a recognized inhibitor of the mTOR pathway. Unexpectedly, neither TH5487 nor BMS-345541 altered the secretion of TNF-α. Hence, the results suggest an NF-κB-independent mechanism of TNF-α secretion in THP-1 macrophages under the investigated experimental conditions. Similarly, exposure to Rapamycin during LPS- and IFN-γ-stimulation of THP-1 macrophages did not alter the secretion of TNF-α, showing no apparent sign to control the M1 polarization. However, the effect of TH5487, BMS-345541, and Rapamycin on their target pathways and the resulting M1 polarization profile should be further investigated by studying an assortment of cytokines, both at the mRNA and protein levels.

Novel anticancer therapies are needed to overcome drug resistance of chemotherapeutic drugs leading to tumor relapse. Combination therapy with target-specific drugs underlies great potential to increase the efficiency of chemotherapy. Increased DNA repair capacity is one of the resistance mechanism of chemotherapy drug cisplatin. Here, inhibitors against DNA glycosylase OGG1 were studied to overcome cisplatin resistance. Combination treatment of OGG1 inhibitor with cisplatin reduced the proliferation of cisplatin resistant bladder cancer cells, whereas sensitive cells did not respond. Increased OGG1 levels in resistant cells and decreased OGG1-GEP mobility upon cisplatin treatment suggests that OGG1 might support cisplatin resistance. Moreover, the activity of PGG1 upon MTH1 inhibition was studied as a side project as both of these enzymes are responsible for 8-oxoG repair, but the findings did not support a role of OGG1 in mediating DNA repair upon MTH1 inhibition. Overall, this study gives insights how OGG1 inhibition could be exploited in anticancer combination therapy.