Title Promoter Affiliations Abstract "Evaluating the anti-tumor potential of PARP inhibitor treatment in combination with dendritic cell-based therapies in triple negative breast cancer" "Luna Fredericq, Eva Hadadi" "Department of Bio-engineering Sciences" "Treatment options for aggressive triple negative breast cancer (TNBC) are still limited. Only patients with germline BRCA1/2 mutations can currently benefit from targeted therapy with PARP inhibitors (PARPi). However, even these patients relapse and hence combination strategies with PARPi are being investigated in patients with and without BRCA1/2 mutations. One strategy to increase the response to PARPi is to improve the anti-tumor immune response. Dendritic cells are at the forefront when mounting an anti-tumor immune response. Therefore, the goal of the current project is to evaluate the anti-tumor potential of combining PARPi treatment and DC-based therapies. Using CITE-seq, flow cytometry and functional assays, we first characterize the DC compartment in models of BRCA1 or BRCA2 deficient TNBC upon PARPi treatment. Then, by depleting DCs from the tumor using genetic models or by increasing their influx/activation in the tumor by combining PARPi with cDC cell therapy, CD40 agonist or Flt3L therapy, we will respectively establish if DCs are required for PARPi response and if enhancing DCs can increase the response to PARPi in TNBC. Overall, this project will therefore significantly improve the understanding on the impact of PARPi on tumor-DCs as well as uncover novel combination strategies for treatment of BRCA proficient or deficient TNBC." "Targeting radiotherapy resistance in Head and Neck Cancers: the potential biomarker role of TIPRL1 and CIP2A, two cellular inhibitors of Protein Phosphatase 2A" "Veerle Janssens" "Laboratory of Protein Phosphorylation and Proteomics, Laboratory of Experimental Radiotherapy" "Being one of the main post-translational modifications, reversible protein phosphorylation is of major importance in signal transduction regulation. Over the years, the kinases phosphorylating many substrates have been extensively studied, while the protein phosphatases have been lagging behind. The PP2A-like phosphatases PP2A, PP4 and PP6, all have been shown to play a role in the regulation of tumorigenesis and the DNA damage response (DDR). Since many cancers including head and neck squamous cell carcinoma (HNSCC) are treated with DNA damaging agents, those phosphatases and their regulators could serve as attractive therapeutic targets improving treatment response.In this thesis, we focused on two cellular inhibitors of PP2A, CIP2A and TIPRL1, of which the latter is also known to inhibit PP4 and PP6. First, we showed TIPRL1 expression is increased in HNSCC tumor tissues compared to normal tissue. Patients with TIPRL1-high expressing tumors showed lower locoregional control and survival upon radiotherapy. This observation was further validated in an HPV-negative HNSCC cell line (SQD9), which showed radiotherapy sensitization after TIPRL1 depletion. This sensitization correlated with faster cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Moreover, more γH2AX foci remained in the TIPRL indel cell lines 24h after irradiation, although this phenotype was not rescued by reintroduction of TIPRL1. Next, we identified a role for TIPRL1 in ATM signaling, as we found that TIPRL1 is phosphorylated by ATM kinase at Ser265 upon radiotherapy or upon treatment with cisplatin or a PARP inhibitor. This phosphorylation was required for TIPRL1-mediated radiotherapy resistance. Furthermore, two new TIPRL1 interaction partners, DNA-PKcs and RAD51, were identified using mass spectrometry analysis and validated by immunoblotting. Both interactions were induced by radiotherapy and remained unaffected by TIPRL1 phosphorylation. In contrast, the nucleosomal histones were also identified as new TIPRL1 interaction partners upon irradiation, but their interaction was adversely affected by TIPRL1 phosphorylation. As expected, the PP2A-like phosphatases belonged to TIPRL1’s interactome as well, but their interaction was not influenced by radiotherapy nor by TIPRL1 phosphorylation. Interestingly, we found that PP6 could bind TIPRL1 both in the presence and the absence of PPIs, while PP2A and PP4 could only interact with TIPRL1 in the absence of PPIs, suggesting a different binding mode and, potentially, a distinct mechanism of inhibition/regulation, which should be further investigated.We also found increased radiosensitization upon CIP2A depletion from SQD9 cells. CIP2A indel cells showed lower cell survival and plating efficiency without treatment, which was even more pronounced when treating the cells with 4 and 6 Gy irradiation. In contrast to TIPRL1 depletion, CIP2A depleted cells showed less γH2AX foci shortly (1 to 3h) after 2 Gy irradiation and lower checkpoint kinase phosphorylation, corresponding with a PP2A activating effect. As reactivation of PP2A is an attractive new target, we aimed to validate whether bortezomib and erlotinib decreased cell viability through CIP2A downregulation; however, both showed similar efficiency in CIP2A-depleted cells, suggesting that their effects occurred independently of CIP2A downregulation.Finally, we showed that three alleged small molecule activators of PP2A, ATUX792, SMAP2 and iHAP, also decreased cell viability independently of CIP2A and TIPRL1 expression, except for ATUX792, which showed a little less efficiency in the absence of TIPRL1. As a next step, a combination treatment of ATUX792 and radiotherapy could be tested in HNSCC cells to improve radiotherapy response.Overall, our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in HNSCC RT resistance through modulation of DNA damage checkpoint activation and repair. Also, CIP2A mediates RT resistance in HNSCC via a role in the DNA damage response. Our research will help to guide better, personalized treatments for HPV-negative HNSCC patients, and by extension, for other cancer patients treated with DNA damaging therapies." "Serine auxotrophy: a novel metabolic vulnerability of platinum resistant ovarian cancers?" "Frédéric Amant" "Gynaecological Oncology, Laboratory of Cellular Metabolism and Metabolic Regulation (VIB-KU Leuven)" "Ovarian cancer is the eight most common type of cancer in women and accounts for the second highest number of gynecological cancers associated deaths in the developed world. While for many cancer types the overall survival increased over the last years, this is unfortunately not true for ovarian cancer, showing a stagnation over the last decades. For over 40 years standard of care consists out of platinum-based chemotherapy, with or without taxanes. The high mortality rate is mainly attributed to the development of resistance to conventional platinum-based therapies. Although a lot of research has been done on chemoresistance, translation into the clinic has been failed. In addition, there are no trustworthy biomarkers to distinguish platinum resistant from platinum sensitive tumors, often leading to ineffective platinum treatment. That is why the identification of resistance biomarkers and development novel therapeutic strategies is of crucial importance in translational cancer research.In recent years it has become clear that cellular metabolism is of major importance in the regulation of cell survival and cell death signaling, that is why we in this PhD project we aimed to investigate metabolic changes that are potentially linked to the resistant phenotype. To underline the clinical importance of our findings we combined state-of-the-art molecular technologies and relevant pre-clinical patient derived xenograft (PDX) mouse models and patient derived data.By applying unbiased metabolomics and isotope labeled tracer analysis in vitro, we managed to identify changes in serine metabolism in platinum resistant cells compared to its platinum sensitive counterpart. Moreover, we found that resistant cancer cells down regulate serine biosynthesis by reducing the intracellular amounts of its rate limiting enzyme phosphoglycerate dehydrogenase (PHGDH).Based on this finding we hypothesized that PHGDH can potentially be used as biomarker for platinum sensitivity. In addition, we also showed that platinum resistant cells are auxotroph for serine in vitro.Next, we investigated the underlying mechanism of PHGDH downregulation by integrating our metabolic findings and we found that serine synthesis down regulation is a part of global metabolic changes in central carbon metabolism towards a NAD+ generating phenotype. We found that NAD+ availability is required to sustain poly-(ADP)ribose-polymerase (PARP) activity under platinum treatment. PARPs are enzymes needed for many cellular functions, including DNA repair. By combining PARP inhibitors and carboplatin we managed to overcome platinum resistance in vitro.  To further investigate if our findings are clinically relevant we used patient-derived xenograft mouse models (PDX), their derived organoid cultures and patient data. We showed that PHGDH are also reduced in resistant models in vivo. These findings are in concordance with TCGA derived data. In addition, we showed that PHGDH gradually decrease and PARP1 levels increase after platinum-based treatment in different PDX models, suggesting that this is an adaptive mechanism to platinum exposure. Finally, we were able to acquire matched primary and recurrent tumor biopsies from patients from the clinic and we showed that, also in patients, PHGDH levels decrease when tumors became resistant to platinum after different cycles of platinum exposure. The identified changes in PHGDH can subsequently potentially be used as biomarker to distinguish platinum sensitive from resistant tumors. Our data provide evidence that combining platinum-based chemotherapy and PARP inhibitors might be a relevant method to overcome platinum resistance. Eventually we validated these results in PDX derived organoid models where we observed a synergistic effect of combining platinum with olaparib.To conclude, with this PhD project we managed to identify a novel potential biomarker for platinum resistance. We showed that resistant cancer cells down regulate serine biosynthesis by specifically decreasing PHGDH levels compared to sensitive cells. Investigation of the underlying mechanism led to a novel potential metabolic target, moreover PARPs, to overcome platinum resistant. We were able to overcome platinum resistance by combining PARP inhibitors with carboplatinum in vitro and in different ex vivo organoid models. These findings need further confirmation in clinical settings in future studies." "Deciphering the dendritic cell compartment in ovarian cancer to assess their potential as tumor vaccines." "An Coosemans" "Laboratory for Tumor Immunology and Immunotherapy, Vrije Universiteit Brussel" "Advanced stage ovarian cancer (OC) patients only have an overall 5-year survival of 20%. While targeted therapies such as PARP inhibitors have improved progression free survival, immunotherapy has so far not resulted in clear patient benefit. Importantly, the role of tumor-dendritic cells (DCs) as a key player in mounting an adaptive immune response has not been investigated yet in OC. Indeed, our lab has uncovered that vaccination with tumor-cDCs can elicit a therapeutically relevant immune response.Therefore, in this project, we will evaluate the cDC heterogeneity in OC and the potential to use tumor-cDCs as vaccine to treat OC. More specifically, we will identify and characterize different cDC populations at the transcriptomic, proteomic and functional level in both a murine OC model and patient samples using state-of-the-art technologies. As in OC, BRCA1/2 mutations are present in 20% of the patients and determine patient outcome, their role in defining cDC function will also be studied. On top, we will investigate the impact of chemotherapy and PARP inhibitors on tumor-cDC function and assess the role of immunosuppressive cells thereon. As last, in the murine OC model, the effectiveness of the different cDC populations as OC vaccine will be assessed.The results of this project will provide new insights into the role of cDCs in OC and will propose a novel therapeutic approach for OC that overcomes the currently witnessed barriers to effective therapeutic responses." "HBOC (Hereditary Breast and Ovarian Cancer), a paradigm shift for counseling and treatment of patients with defects in homologous recombination - an explorative study" "Gert Matthijs" "Department of Human Genetics" "Our knowledge about genes, associated with an increased risk of hereditary breast and ovarian cancer, has increased significantly. However, the exact risks associated with germline mutations, in these recently detected genes, are not yet known. This creates a complex situation in communicating genetic test results to persons who have a mutation in these genes. To get more insight into the risks, we will develop a national database in this inter-university project to gather relevant information to better estimate the risks.All known breast cancer genes play a role in the repair of fractures in DNA via a specific recovery mechanism, homologous recombination (HR). This is an interesting issue as it can be therapeutically intervened by treatment with PARP inhibitors. The purpose of this project is therefore to perform a clinical trial phase 2 with PARP inhibitors in patients with a mutation in a gene involved in HR. Furthermore, we will investigate whether patients with a mutation in an HR gene can be identified by studying the DNA that circulates in plasma from these patients. This would allow patients to benefit from treatment with PARP inhibitors based on a simple blood decrease rather than through a tumor biopsy. Through this interuniversity cooperation we expect to be able to contribute strongly to the clinical utility of genetic tests of breast cancer predisposition genes, both in the context of cancer prevention and therapy." "Unravelling The Tumour Immune-Biology Of Ovarian Cancer" "An Coosemans" "Laboratory for Tumor Immunology and Immunotherapy, NanoHealth and Optical Imaging Group" "Ovarian cancer is the fifth most lethal cancer for woman. It is a silent killer, metastasizing throughout the abdomen before causing symptoms. Consequently, over 70% of patients are diagnosed in advanced disease stages (FIGO stage III to IV). Cytoreductive surgery in combination with platinum-based chemotherapy remain the cornerstones in first-line therapy. However, risk of recurrence is high. Recently approved targeted therapies (anti-VEGF and PARP inhibitors) have improved the situation slightly for some patients, but cannot offer a cure for all patients. Five years after diagnosis, only 70% of the women will be alive. Therefore, the search for adequate treatment options is wide open.It has been well established that the immune system is an important player in the onset and development of cancer. The immune system controls once neoplastic cells arise. Based on both the adaptive and innate immune response, tumor cells will be eliminated. In some cases, elimination is not entirely successful and an equilibrium phase is established. Neoplastic cells are still in place, but in a dormant state, side-by-side with the immune system. During this process, tumor cells can transform and consequently escape tumor control. The immune systems fails and tumor proliferation cannot be stopped. Immunosuppressive cells are attracted towards the tumor side (MDSC (myeloid derived suppressor cells), Treg (regulatory T cells), M2 macrophages, …) and will even promote tumor growth.The aim of this thesis was to further characterize the tumor immuno-biology of ovarian cancer. Based on this information, we next aim to test different immunotherapy-based treatment combinations. Lastly, we provided a proof-of-concept study to facilitate the implementation of regular screening of the immune status of a patient in a clinical setting. " "Study of DDR/PARP1 signaling in the pathogenesis and treatment of soft tissue mineralization, using pseudoxanthoma elasticum as a model." "Olivier Vanakker" "Department of Biomolecular Medicine, University of Angers, University of Modena and Reggio Emilia" "DNA damage response (DDR) mechanisms - in particular PARP1 signaling - are suggested to be important in the emergence of soft tissue mineralization (STM). A hallmark STM disorder is pseudoxanthoma elasticum (PXE), caused by mutations in the liver transporter ABCC6, with a complex and incompletely understood pathogenesis. Several dysregulated mediators in PXE show remarkable resemblance with PARP1 signaling cascades; our preliminary data show activated PARP1 signaling in PXE patients, reduced mineralization in vitro and in vivo after PARP inhibition, and identify miR-204 as a regulator. Now, we will perform a more profound study of the pathophysiological role of PARP1 signaling in PXE and evaluate its use as a biomarker and therapeutic target for this intractable disease. First, we will study the mechanisms involved in activating the DDR/PARP1 cascade in PXE fibroblasts and the regulatory role of miRNAs. Second, we will evaluate possible correlations between PARP1 activity, circulating miRNAs and PARP1-related genomic variants and the clinical severity of PXE. Finally, we will evaluate several known PARP1 inhibitors as potential treatment in PXE, using in vitro and in vivo models. Taken together, this project will lead to major advances in the complex pathogenesis of PXE, will delineate non-invasive (miRNA) biomarkers for the disease and will lead to novel therapies by drug-repurposing for this disorder. As such it will significantly improve patient management and treatment." "Oxidative stress as a selective anticancer agent: investigation of a targeted combination strategy for mutant p53 non-small cell lung cancer and other solid tumors." "Christophe Deben" "Center for Oncological Research (CORE), Molecular Imaging, Pathology, Radiotherapy & Oncology (MIPRO)" "Despite many efforts, non-small cell lung cancer (NSCLC) has a dismal 5-year survival rate of less than 20% due to frequently occurring therapy resistance. In addition, currently available targeted therapies are only applicable to limited subgroups of patients. The presence of TP53 mutations is associated with resistance to a wide array of therapeutics that are currently used as first-line treatment in NSCLC, including platinum-based therapies and EGFR tyrosine kinase inhibitors. Since TP53 mutations occur in over 50% of all NSCLC patients, there is a pressing medical need for more effective treatment strategies to improve survival of these patients.In this project, we propose an innovative combination strategy which exploits the presence of mutant p53 by targeting the cellular redox balance. Increased oxidative stress is a hallmark of cancer cells, which makes them more vulnerable to induction of reactive oxygen species (ROS). P53 plays a crucial role in sensing and removing oxidative damage to DNA, and inactivating mutations in the TP53 gene attenuate this function. In addition, it was shown that mutant p53 is able to suppress the function of major antioxidant factors. Therefore, mutant p53 renders cancer cells even more susceptible to the induction of oxidative stress. Besides p53, the poly (ADP-ribose) polymerase 1 (PARP-1) protein plays and important role in the repair of ROS-induced DNA-damage. This led us to explore the potential of combining oxidative stress induction, using the compound APR-246, with the targeted inhibition of the PARP-1 protein, using olaparib. In our lab, this combination strategy showed promising in vitro results in NSCLC cell lines, resulting in strong synergistic interactions in the presence of mutant p53.Following our promising data, this project aims to translate this novel and selective combination strategy to the clinic. In this preclinical study we will explore the combination of two oxidative stress-inducing compounds, APR-246 and auranofin, in combination with the PARP-1 inhibitor olaparib. We will study the predictive value of mutant p53 and the role of ROS in the synergistic cytotoxic effects in NSCLC cell lines. Since oxidative stress and mutant p53 are characteristics that are also frequently observed in other tumor types, we will expand our study to pancreatic ductal adenocarcinoma in vitro." "Pharmacogenetic profiling of VEGF-inhibitors forcancer therapy and assessing the vascular effects of VEGF during neurodegeneration." "Diether Lambrechts" "Laboratory of Translational Genetics (VIB-KU Leuven)" "Mismatch repair (MMR) deficiency represents a well-established cause ofLynch syndrome, which is an autosomal dominantly inherited disorder of cancer susceptibility triggered by loss-of-function mutations in MMR genes (MLH1, MSH2, MSH6 or PMS2).(URL:#_ENREF_1) Lynch syndrome is responsible for 2-5% of endometrial (EM) or colorectal (CRC) tumors. Additionally, epigenetic silencing of MLH1 contributes to another 15-28% of these tumors.(URL:#_ENREF_2) Deficiency of the MMR machinery leads to DNA replicationerrors in the tumor tissue, but not in the normal surrounding tissue. In particular, errors often accumulate as insertion/deletion (indel) mutations in mono- and di-nucleotide repeats - a phenomenon referred to as microsatellite instability (MSI).(URL:#_ENREF_3)MMR-deficient tumors constitute a specific subtype due to their different prognosis and clinical outcome.(URL:#_ENREF_4) For instance, untreated CRC cancer patients with MMR-deficient tumors have a modestly better prognosis, but do not seem to benefit from 5-fluorouracil-based adjuvant chemotherapy, which is the first-choice chemotherapy for CRC cancer. In particular, in MMR-deficient tumors, mismatches induced by 5-fluorouracil are tolerated, leading to failure to induce cell death.(URL:#_ENREF_5) MMR-deficient tumors are also resistant to cisplatin and carboplatin, which are frequently used chemotherapies in EM cancer.(URL:#_ENREF_5) Furthermore, MMR-deficient tumors can be resistant to targeted therapies, because they acquire secondary mutations in genes that activate alternative or downstream signaling pathways (e.g. PIK3CA). Another possibility is that epigenetic silencing of MLH1 coincides with particular mutations, such as the BRAF V600E mutation(URL:#_ENREF_6), which represents an established negative predictor of response to targeted anti-EGFR therapies in advanced CRC cancer.(URL:#_ENREF_7) There have been some efforts to individualize the treatment of MMR-deficient cancers. These focused on identifying synthetic lethal interactions with the MMR pathway or targeting secondary mutations occurring as a result of MMR-deficiency. However, until now, these efforts failed to translate into clinically effective treatment options.The objectives of this thesis were to generate a more comprehensive picture of the mutation spectra arising in MMR-deficient tumors and to more rationally predict their response to therapies. Firstly, whole-exome sequencing was applied to characterize the unique somatic mutation patterns underlying MMR-deficient tumors. Each MMR-deficient tumor exhibited a clear hypermutator phenotype, containing on average 50 times more novel somatic mutations than MMR-proficient tumors. The mutations in MMR-deficient tumors were mostly occurring as indels in homopolymers. Indels were clearly less frequent in exons, suggesting that they are loss-of-function mutations undergoing negative clonal selection during tumorigenesis. On the other hand, a large set of recurrent indels affecting MMR-deficient tumors was identified implying positive clonal selection. Secondly, a novel MSI panel was designed to detect MMR-deficiency in clinical tumor samples by high-throughput profiling of the selected recurrent indels. This Sequenom-based panel was tested on more than 400 tumor samples, fromendometrial, colorectal and ovarian cancer patients, and demonstrated that it outperforms the standard diagnostic panel (i.e. the revised Bethesda panel). Thirdly, pathway analyses revealed that the recurrent indels in MMR-deficient tumors preferentiallyaffect the DNA double-strand break repair (DSBR) by homologous recombination (HR) pathway, containing on average 3.3 indels per tumor. The biological relevance of these secondary mutations was validated by performing functional studies in 11 primary tumor cultures. Double immunostaining for γH2AX and RAD51, as a measure of DSBs and ongoing HR, respectively, showed that MMR-deficient tumors mobilized less RAD51-positive foci, after DSB formation. BrdU and PI cell cycle analysis using FACS, as well as Western blots were used to exclude a reduced S/G2 phase or a reduced RAD51 expression as confounding factors for areduced DSBR by HR activity. Thereby, it is confirmed that the DSBR by HR pathway is also functionally affected in MMR-deficient tumors. Moreover, DSB inducers, such as the PARP inhibitor olaparib, triggered a dose-dependent reduction in proliferation in primary cultures of MMR-deficient tumors, identifying a synthetic lethal interaction. In addition, to more accurately measure the level of HR-deficiency in MMR-deficient tumors, the level of knock-down of BRCA1, BRCA2 and ATR needed to achieve an olaparib sensitivity similar to that observed in MMR-deficient cells was assessed. It is established that the loss of DSBR by HR activity in MMR-deficient tumors corresponds to a loss of about 70-80% BRCA1 or -2 expression, indicating that the sensitivity to olaparib that is observed in MMR-deficient tumors is intermediateto that of cells heterozygous and homozygous-deficient for BRCA1.Insummary, this thesis not only provides the first comprehensive catalogue of somatic mutations accumulating in MMR-deficient tumors, but also demonstrates the relevance of these mutations in the detection of MSI within the diagnostic setting, and potentially also the targeted treatment of these tumors." "Predictive genetic markers in gynaecological cancers: how to improve targeted therapy." "Toon Van Gorp" "Gynaecological Oncology" "The development of more targeted therapies opened up a completely new era in treatment of gynaecological malignancies. However, little is known about the ideal position and timing in the treatment strategy and which patients in particular will benefit from treatments such as poly (ADP-ribose) polymerase inhibitors (PARPi) and immune checkpoint blockade (ICB). The aim of this proposal is to find (epi-)genetic markers that predict response to these treatments. 1) PARPi proved to be a successful treatment in BRCA mutated ovarian cancer. However, other functional defects in the homologous recombination DNA repair pathway seem to cause a good  response to PARPi. The first aim of this project is to develop and validate an inhouse homologous recombination repair deficiency (HRD) test that can predict response to PARPi in BRCA mutated and non-mutated ovarian cancer. 2) The current predictive biomarkers that are being used in clinical practice do not seem to be reliable enough to predict response to ICB in both ovarian and cervical cancer. To achieve a blueprint of the tumor’s ecosystem at single cell resolution, we will perform single cell RNA-sequencing (scRNA-seq) and single cell T-cell receptor sequencing (scTCR-seq), and evaluate these in the light of clinical response of our patients. Furthermore, besides the tumor microenvironment, we aim to analyse the role of methylation in the resistance of cervical cancer cells to immune therapy. "