Research An Wouters

Abstract

Cell and gene therapies are revolutionizing the way cancer is treated. CAR-T cell therapies have demonstrated the ability to induce durable remissions or even achieve cures in patients with certain blood cancers that were in final stages, including leukemia and lymphoma. Despite these hopeful results in hematological malignancies, the application of CAR cellular therapies has been more challenging in solid tumors. Solid tumors have a complex tumor microenvironment (TME) that creates barriers to immune cell infiltration and function. This immune-suppressive niche is low in nutrients, highly acidic and hypoxic (lacking oxygen) due to the aberrant metabolism of tumor cells and growth pattern of the tumor. Accumulating data indicates that T cells and NK cells entering this hostile environment are rendered dysfunctional. Up to 90 percent of adult cancers consist of solid tumors, presenting a big need for new therapies. With our research, we aim to enhance the therapeutic potential of NK cell therapies for solid tumor patients in need of new treatment options. NK cells are cytotoxic cells from the innate immune system that can recognize and eradicate tumor cells swiftly. Their distinct advantages over T cells, including a superior safety profile and potential for allogeneic therapy, make NK cell treatments more economically feasible and logistically appealing, thereby broadening patient accessibility. Until now, efforts to enhance NK cell efficacy in cancer therapy have mainly focused on blocking inhibitory receptors, CAR engineering, or cytokine stimulation. However, recognizing the profound impact of the TME on NK cell metabolism, recent approaches aim to combine immunotherapy with metabolic manipulation to bolster cellular function. This growing awareness underscores the need for innovative engineering and gene editing to develop resilient cellular products capable of thriving in the challenging TME. In this project, we seek to (1) unravel metabolic interventions that boost the metabolic and functional resilience of NK cells in a hypoxic environment; (2) employ CRISPR-Cas9 to stably engineer NK cells based on our discoveries, thereby unlocking their potential in hostile tumor environments; and (3) validate the improved killing capacity and fitness of NK cells in hypoxic conditions using state-of-the-art assays. Driven by the need for novel therapeutic strategies that enhance the overall outcome for cancer patients, we aim to create an innovative NK cellular therapy product that is metabolically rewired to withstand the harsh and suppressive TME.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: De Waele Jorrit
• Co-promoter: Smits Evelien
• Fellow: Verhezen Tias

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type worldwide, with a majority of the patients progressing towards recurrent/metastatic HNSCC with limited treatment options, underscoring an unmet clinical need. The observation that HNSCC is one of the most inflamed and immune infiltrated tumor types, with an exceptionally high infiltration of natural killer (NK) cells, supports the potential of NK cells as a therapeutic agent for this indication. Importantly, immunometabolism is pivotal to immune responses, including for NK cells, yet, how the tumor microenvironment affects NK cell metabolism is largely unknown and identifying NK cell evasion mechanisms could unlock both current and new therapies for HNSCC. We hypothesize that the deregulated metabolism of HNSCC cells is a pleiotropic hallmark in evasion to NK cells. We postulate that these metabolic resistance mechanisms are dual. On the one hand, metabolic reprogramming of HNSCC cells could mediate intracellular resistance to killing by NK cells. On the other hand, the metabolically deregulated HNSCC cells modify the metabolic composition of the tumor microenvironment, resulting in suppression of NK cells due to metabolite disbalance. This ultimately promotes immune evasion and jeopardizes survival of HNSCC patients. Unravelling these resistance mechanisms bear the potential to push NK cell therapies forward. To this end, we will perform multi-omics analyses, target identification, genetic engineering and pharmacological targeting, 2D and 3D in vitro experiments, in silico validation and in vivo testing. First, we will elucidate the linchpins of intracellular metabolic pathways in HNSCC cell driving resistance to NK cell-killing by conducting a CRISPR knockout screen using a metabolism-focused library and the top targets will be validated using patient datasets and functional assays. Second, we will unravel the effect of extrinsic metabolites, secreted by HNSCC cells, on NK cell-mediated killing. The key metabolites responsible for reduced NK cellular killing capacity will be identified and validated correspondingly. Finally, the potential of metabolic interference of these targets to boost the next generation NK cell therapy for HNSCC, i.e. chimeric antigen receptor (CAR) NK cells, will be determined in vitro and validated in vivo. Concluding, the proposed project will enrich our understanding of evasion mechanisms to NK cells and will expedite the breakthrough of next-gen NK cell therapy for head and neck squamous cell carcinomas, with high potential for implementation in additional solid tumors types.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: De Waele Jorrit
• Fellow: de Beukelaar Julie

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. Despite advances in treatment options, conventional chemotherapy remains a pivotal part of NSCLC treatment, regardless of stage, even though it is accompanied with serious side effects and therapy-induced senescence (TIS). Cellular senescence is a durable cell cycle arrest and is characterized by the secretion of a strong pro-inflammatory senescence-associated secretory phenotype (SASP). Evidence indicates that TIS induces deleterious long-term effects including therapy resistance, disease progression, metastasis and recurrence. Thus, TIS acts as a barrier to complete eradication of the tumor, indicating the importance of targeting senescent cells during cancer therapy. Therefore, I will investigate a novel combination treatment in this project, specifically designed to eliminate therapy-induced senescent cells. Senescent tumor cells will be targeted by two strategies: senolytics to specifically kill these cells and senostatics to suppress or modulate the SASP. Moreover, I will identify the core senescent secretory profile of NSCLC, that will be used as a blood-based biomarker to identify and select patients that would benefit from our new senescence-focused therapy. The successful completion of my project will ultimately improve overall survival of NSCLC patients with a tumoral senescence signature, regardless of stage, by enhancing treatment efficacy and tumor eradication.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: Deben Christophe
• Co-promoter: Lardon Filip
• Fellow: Verswyvel Jasper

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type worldwide, with a majority of the patients progressing towards recurrent/metastatic HNSCC with limited treatment options. Despite the high natural killer (NK) cell infiltration, the efficiency of newly developed adoptive cellular therapies in clinical trials is limited. Therefore, I hypothesize that HNSCC cells secrete immunosuppressive metabolites in the tumor microenvironment (TME), exaggerated by the high level of hypoxia, inducing evasion to NK cells. Using physiologic and conditioned media at different oxygen levels, metabolic alterations in the TME are characterized by gas chromatography-mass spectrometry, providing high-value candidate metabolites that are later evaluated in a high-throughput screen to determine their effect on the NK cell killing capacity. Intracellular metabolic and functional changes of NK cells induced by exposure to the interfering metabolites are identified together with phenotypic profiling. Using an orthotopic humanized mouse model, NK cell functionality is investigated after modification of the TME and restoration of NK cell cytotoxicity combined with standard-of-care HNSCC treatment is evaluated. Concluding, this project will obtain fundamental insights into the suppressive role of hypoxia-induced metabolites on NK cells and will provide valuable knowledge for adoptive cellular therapies in development.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: De Waele Jorrit
• Co-promoter: Smits Evelien
• Fellow: Melis Jöran

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Volatile and semivolatile chemicals are recognised as byproducts of disease, boosting volatile analysis as paramount instrument to monitor health and disease, personalize health care and objectively establish the effect of different treatment strategies. Next to volatile organic compounds (VOCs), semivolatile compounds (SVOCS) are present in the environment and in biological matrices, but most of them need to be chemically and structurally identified and their role in health and disease is yet to be explored. In this proposal, we describe the set-up of a highend GCxGC-QTOF-MS facility for analysis of VOCs and SVOCs in biological samples like breath, blood, urine, faeces of humans and animals, and in the headspace of cells. The goal is to set up an infrastructure that allows to assess and investigate multiple biological sample types and their headspace for monitoring health and disease, to identify disease biomarkers, to intensify research on the environmental health issues of modern life, and to tackle the hurdles presently encountered in the metabolomics analysis of steroids and small organic acids. By this means, we intend to team up and complement with international volatomics research groups. In Flanders, such a specialised facility is lacking, and will be unique. It combines high sensitivity, ultralow detection limits for analysis and validation of the molecular composition of biological and headspace samples, with specific sampling devices and advanced data processing.

Researcher(s)

• Promoter: De Winter Benedicte
• Co-promoter: Covaci Adrian
• Co-promoter: Lamote Kevin
• Co-promoter: Lapperre Therese
• Co-promoter: Laukens Kris
• Co-promoter: Samson Roeland
• Co-promoter: van Meerbeeck Jan
• Co-promoter: van Nuijs Alexander
• Co-promoter: Wouters An

Research team(s)

• Laboratory Experimental Medicine and Pediatrics (LEMP)

Project type(s)

• Research Project

Abstract

FLASH-RT, based on ultra-high dose rate irradiation (instantaneous dose rate above 104 Gy/s), recently became a hot topic in the field of radiation oncology. Several studies have recently shown that classical pathogenic patterns observed in normal tissues exposed to radiation delivered at conventional dose rate radiation therapy (CONV-RT) were not induced by single fractions of FLASH-RT, collective observations that we have since defined as the "FLASH effect". Published data forms a growing body of literature, documenting the marked normal tissue sparing found in multiple tissues (brain, lung, skin, gut) and in multiple mammalian species (mouse, cat, pig, rat) subjected to FLASH-RT (Favaudon 2014; Montay-Gruel 2017, 2018, 2020; Vozenin 2018; Alaghband 2020; Wilson 2020). These findings, along with data showing fully conserved anti-tumour efficacy and the first human trial (albeit for skin cancer) (Bourhis 2019, 2020) point to the exciting clinical promises of delivering FLASHRT in a large number of tumour sites. This previous research highlights the feasibility of translating preclinical studies into clinical trials for the treatment of cancers while limiting normal tissue toxicities. Therefore, the Iridium Kankernetwerk and the University of Antwerp is the first radiation oncology centre in Belgium, and the second in the world, to be equipped with the ElectronFlash irradiator developed by S.I.T. (Sordina IORT Technologies S.p.A., Italy - hereafter "S.I.T.") and thereby able to deliver ultra-high dose rate irradiation to attain the FLASH effect in preclinical circumstances. Currently, S.I.T. is developing a new accelerator for IOeRT, capable to deliver both CONV-RT and FLASH-RT. We should be the first centre in the world where this revolutionary machine for clinical applications will be installed, in the course of 2023, including CE-marking for clinical use. The preclinical research described in this project will consist in developing and using in vitro and in vivo tumour models to validate and optimize the treatment of breast tumour types using FLASH-RT. Therefore, our project aims at investigating the effect of FLASH-RT (compared to CONV-RT) on breast cancer and on radiation-induced skin, soft tissue, lung and heart toxicities. Up to now, while FLASH-RT has been found to prevent the development of radiation-induced toxicities on many different organs while keeping a good antitumour effect, no study has focused on its use for breast cancer treatment. The preclinical data obtained from this project is essential to ensure a safe and efficacious transfer of FLASH-RT to clinical breast cancer practice at the Iridium Kankernetwerk and University of Antwerp. Recent studies have suggested that the optimal FLASH effect was obtained with single dose exposures and with hypo-fractionated regimens (Bourhis 2019; Montay-Gruel 2020). This is compatible with IOeRT in both partial and whole breast irradiation settings, as this entails routinely the delivery of a single high dose of radiation (21 Gy for partial breast irradiation in low-risk patients; 9 Gy for a boost combined with routine whole breast irradiation in high-risk patients.

Researcher(s)

• Promoter: Meijnders Paul
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project website

Project type(s)

• Research Project

Abstract

Both targeted and immunotherapies are the key to precision medicine for the treatment of cancer patients. Deregulated signalling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumourigenesis of multiple cancer types. Furthermore, it is well established that immune checkpoints are crucial for the tumour cell's escape from the immune system. The presence of drug resistance and/or immune evasion is a major obstacle to progress in the field. In our project, we will concentrate specifically on head and neck squamous cell carcinoma (HNSCC), a highly relevant tumour type with poor prognosis that is intensively studied at the Centre for Oncological Research (CORE) Antwerp. To date, there is still an urgent need to enhance the response to cetuximab treatment in recurrent/metastatic (R/M) HNSCC. Over the last years, cetuximab-related resistance mechanisms have been extensively studied at CORE. Based on our results and reports in literature, we hypothesize that inhibiting oncogenic bypass pathways responsible for cetuximab resistance, by a novel treatment strategy can lead to elimination of HNSCC cells that are resistant to treatment with cetuximab alone. In the proposed project, we will investigate the potency of a novel triple combination strategy in order to enhance the response to cetuximab therapy in HNSCC patients. To achieve this, cetuximab will be combined with buparlisib, a selective PI3K inhibitor, and an immune checkpoint inhibitor. Importantly, we will investigate the role of human papilloma virus (HPV) in this response, as HPV positive HNSCC patients represent a biologically distinct group. Furthermore, the nature of our project is translational, as from the beginning, we will use patient-derived HNSCC tumour organoids to validate our results from cell line experiments. These patient-derived tumour organoids are a very innovative and reliable model to identify effective treatment strategies and can actually be considered as a 'patient in the lab'. We are convinced that precision medicine using combinations of targeted therapies with immunotherapy may achieve the much-needed progress in HNSCC treatment. As reported in literature, both cetuximab and buparlisib treatment are able to promote anti-tumour immune response. Therefore, in the first work package, we will characterize the anti-tumour activity and immunomodulating effects of cetuximab in combination with buparlisib in HNSCC cell lines and patient-derived HNSCC organoids. Secondly, we will investigate the immunomodulating effects of cetuximab in combination with buparlisib on immune cells. In parallel, the effect of this combination treatment on the immune checkpoint profile will be assessed. Finally, the novel triple combination therapy consisting of cetuximab, buparlisib and an immune checkpoint inhibitor will be investigated in a humanized, PBMC engrafted HNSCC mouse model. This preclinical work will ultimately guide the start-up of a clinical trial to demonstrate feasibility of the novel triple combination therapy to treat HNSCC patients. Given the extensive preclinical (both in vitro and in vivo) and translational work packages to optimise the novel triple combination strategy, we are confident that the data generated in this project will provide insight into how therapeutic response to cetuximab treatment can be optimized, thus favouring the setup of a successful clinical trial with the newly identified triple combination therapy.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Pauwels Patrick
• Co-promoter: Peeters Marc
• Co-promoter: Prenen Hans
• Co-promoter: Smits Evelien
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Despite significant advancements in oncological treatments, many patients still succumb to the cancer, which remains the second leading cause of death worldwide. Novel treatment options are therefore urgently needed. The breakthrough of immunotherapy has revolutionized the field of oncology, however, the majority of patients remains unresponsive to immune checkpoint inhibitors. Armoured cellular therapies such as chimeric antigen receptor (CAR)-engineered T cells are now generating success in haematological malignancies, but comes with safety issues. Their lymphocytic counterpart, natural killer (NK) cells, are now presenting themselves as highly promising alternatives. Nonetheless, also NK cells remain ineffective against solid tumours. This is at least in part due to the hostile solid tumour microenvironment which impairs the metabolic and cytotoxic function of NK cells. Manipulating NK cells to withstand the vicious rigors of the tumour microenvironment could therefore be a gamechanger in their use as cell therapy against solid tumours. We discovered critical metabolic impairments in NK cells induced by tumour microenvironmental factors. We have found an actionable metabolic target to manipulate NK cells either pharmacologically or genetically, resulting in protection of their cellular health and cytotoxic function. With this IOF-POC project, we aim to validate and expand our patented claims in multiple solid tumour models on different levels and find industrial partners to proceed its valorisation route. Ultimately, our strategy to protect the fitness of NK cells in the tumour microenvironment could enable the efficacious application of CAR-engineered NK cell products for solid malignancies, and as such impact many cancer patients.

Researcher(s)

• Promoter: De Waele Jorrit
• Co-promoter: Smits Evelien
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. Despite advances in treatment options, conventional chemotherapy remains a pivotal part of NSCLC treatment, regardless of stage, even though it is accompanied with serious side effects and therapy-induced senescence (TIS). Cellular senescence is a durable cell cycle arrest and is characterized by the secretion of a strong pro-inflammatory senescence-associated secretory phenotype (SASP). Evidence indicates that TIS induces deleterious long-term effects including disease progression, metastasis and recurrence. Thus, TIS acts as a barrier to complete eradication of the tumor, indicating the importance of targeting senescent cells during cancer therapy. Therefore, I will investigate a novel combination treatment in this project, specifically designed to eliminate therapy-induced senescent cells. Senescent tumor cells will be targeted by two strategies: senolytics to specifically kill these cells and senostatics to suppress or modulate the SASP. Moreover, I will identify the core senescent secretory profile of NSCLC, that will be used as a blood-based biomarker to identify and select patients that would benefit from our new senescence-focused therapy. The successful completion of my project will ultimately improve overall survival of NSCLC patients with a tumoral senescence signature, regardless of stage, by enhancing treatment efficacy and preventing relapse.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: Deben Christophe
• Co-promoter: Lardon Filip
• Fellow: Verswyvel Jasper

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Acute myeloid leukemia (AML) is a hematological cancer that arises and spreads from the bone marrow. It has a very dismal prognosis, characterized by a high relapse rate, despite initial complete molecular remission. Residual leukemic stem cells (LSC) are believed to be to origin of this relapse. LSC reside in a tumoral bone marrow that features a heightened state of hypoxia. Immunotherapeutic strategies are on the rise and are promising approaches to go hunt-and-destroy LSC. However, they will have to overcome the hypoxic burden in the leukemic bone marrow. Indeed, hypoxia is nowadays recognized as a barrier for immunotherapy. In this project, we will focus on natural killer (NK) cells as a born killer with great potential as adoptive cell product. While cytokines and chimeric antigen receptors (CAR) have improved the cytotoxic potency and targetability of NK cell products, their effectiveness at the tumor site is incapacitated by hypoxia. To address this conundrum, we will investigate several approaches to metabolically sustain their killing capacity in hypoxia in order to elicit potent elimination of both LSC and differentiated AML cells in the leukemic bone marrow. This will open up opportunities to develop next-generation CAR NK cells as an available off-the-shelf product for the treatment of AML patients.

Researcher(s)

• Promoter: De Waele Jorrit
• Co-promoter: Smits Evelien
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Natural killer (NK) cells are potent cytotoxic cells from the hematopoietic system, playing an important role in the control of infection and malignancy. NK cells often operate under harsh conditions, such as in deprived oxygen or hypoxia. Hypoxia stabilizes its primary regulators hypoxia-inducible factors (HIF), more specifically HIF-1? and HIF-2?. While hypoxia reduces NK cell-mediated cytotoxicity, data on other NK cell features is conflicting. In addition, the role of HIF in NK cells has been neglected, as only one mouse study demonstrated that HIF-1? ablation in NK cells indirectly reduces tumor load via angiogenic rather than cytotoxic effects. However, murine NK cells show quite some disparities to human NK cells. In addition to hypoxia, also stimuli such as cytokines can stabilize HIF. Hence, HIF could play a pivotal role in NK cells. Therefore, this project aims to gain fundamental insights in the role and regulation of HIF in human NK cells via combining omics with functional assays. First, we will unravel the effect of hypoxia on (stimulated) NK cells. Next, we will dissect the divergent roles of HIF-1? and HIF-2? in the functioning of NK cells in response to hypoxic and stimulatory conditions. HIF isoform-specific knockout NK cells will be created using CRISPR-Cas9. Finally, we will elucidate how HIF isoforms regulate their effects in NK cells. The obtained knowledge could prove extremely valuable in rational guidance of rising novel NK-cell based immunotherapies.

Researcher(s)

• Promoter: Smits Evelien
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Development of therapeutic resistance poses a challenging problem and limits the success of cancer therapies in the clinic. Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), is currently used for the treatment of locally advanced head and neck squamous cell carcinoma (HNSCC) as well as recurrent/metastatic HNSCC. However, 5-year survival rates remain low. In this project, we will focus on the identification of novel combination therapies to overcome cetuximab resistance. Targets for the combination will be identified through genetic and proteomic analysis of the molecular profile of the tumour. We hypothesize that inhibiting oncogenic bypass pathways responsible for cetuximab resistance, by a novel treatment strategy combining (i) cetuximab with (ii) radiotherapy and (iii) an additional molecularly targeted agent, can lead to elimination of HNSCC cells that are resistant to treatment with cetuximab alone. We will investigate the role of human papilloma virus (HPV) in this response, as HPV positive HNSCC patients represent a biologically distinct group. The potential of these combination therapies will be investigated in vitro on a well-established, comprehensive panel of HNSCC cell lines with different sensitivities to cetuximab. Synergistic combinations will be validated under hypoxia and in 3D spheroids. Next, the most promising combination strategy will be investigated in vivo in HNSCC patient-derived xenograft (PDX) mouse models. The biological and therapeutic implications of our study hold great promise for clinical translation in patients with HNSCC.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour, however it remains a rare disease (incidence: 3.20/100,000). Tumour progression is fast and recurrence inevitable. The added value of the current standard of care (SOC: surgical resection, radiation and chemotherapy) is only limited, leading to a median survival of less than 15 months and a five-year survival of less than 5%. In addition, undesired side effects impact on the quality of life. Hence, new effective treatment modalities represent a highly unmet need. While scientific advances have generated clinical breakthroughs in other cancer types, this has remained a standstill in GBM for nearly 15 years. Immunotherapy has generated remarkable clinical success in the past decade, in particular with immune checkpoint blockade (ICB). Recent preclinical evidence has suggested that combination therapy can render GBM sensitive to ICB. In this project, we will develop an immunometabolic therapy in murine GBM models in vivo as innovative treatment option. We hypothesize that our combination strategy will ameliorate clinical outcome while improving quality of life.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: Lardon Filip
• Co-promoter: Peeters Marc
• Co-promoter: Smits Evelien
• Co-promoter: Specenier Pol

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Both targeted therapies and immunotherapies are now at the forefront of personalized cancer medicine. Aberrant signalling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of multiple cancer types, making it a compelling drug target. In addition, it is well established that natural killer (NK) cells possess natural anti-tumour activity and can mediate antibody dependent cellular cytotoxicity (ADCC) upon binding with monoclonal antibodies, such as the EGFR inhibitor cetuximab. However, the presence of drug resistance and/or immune evasion is a major obstacle to progress in this field. In our project, we will concentrate specifically on head and neck squamous carcinoma (HNSCC), a highly relevant tumour type with poor prognosis that is intensively studied at the Center for Oncological Research (CORE) Antwerp. In this research project, we hypothesize that increasing the NK cell activity by cetuximab in combination with targeting NK cell immune checkpoint molecules can synergistically generate immune mediated elimination of HNSCC cells that are resistant to treatment with cetuximab alone. Importantly, we will investigate the role of human papilloma virus (HPV) in this response, as HPV positive HNSCC patients represent a biologically distinct group. By characterizing NK cell functionality and, by extension, the whole immune checkpoint profile in HNSCC, we aim to rationally design new combination strategies to overcome cetuximab resistance, with the ultimate goal to improve the prognosis and life quality of HNSCC patients. Hereby, we will focus on HPV status and the hypoxic microenvironment as important mediators of treatment response. Therefore, the nature of our project is translational, as from the beginning, the link with clinical data is considered to be imperative before moving on to further preclinical investigation of the identified combination strategies. Successful combinations will be validated in animal studies, which will ultimately guide the start-up of a clinical trial to demonstrate feasibility of the most promising combination therapy to treat HNSCC patients. Given the extensive preclinical (both in vitro and in vivo) and translational work packages to optimise the novel combination strategy, we are confident that the data generated in this project will favour the setup of a successful clinical trial with the newly identified combination regimen.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Pauwels Patrick
• Co-promoter: Peeters Marc
• Co-promoter: Smits Evelien
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Non-small cell lung cancer (NSCLC), accounting for an estimated 85% of all lung cancers, retains its position as the most lethal type of cancer worldwide, with a 5-year survival rate for newly diagnosed cases below 20%. Despite the remarkable progress that has been made in the development of new treatment modalities, chemotherapy consisting of platinum-based doublets remains the standard first-line treatment for NSCLC patients. Anti-mitotic drugs are well-established components of the current combination treatment schedules in NSCLC patients. Nevertheless, serious adverse effects remain the dose-limiting factor. New approaches target cardinal regulatory proteins of mitosis, with Polo-like kinase 1 (Plk1) as one of the most promising targets in this research field. Our previous research showed Plk1 overexpression in 65% of NSCLC patients while no or weak Plk1 expression was noted in normal lung tissue, making it a compelling target for the treatment of NSCLC. Volasertib, at present the lead agent in category of Plk1 inhibitors, has been shown to be highly effective in multiple carcinoma cell lines and xenograft models, with minimal toxicity in normal cells. However, only modest anti-tumour activity was reported for volasertib monotherapy in patients with solid tumours, including NSCLC. Remarkably, an encouraging percentage of these patients reaches stable disease, providing an intriguing window for improving patient outcome. Based on promising results of our recent preclinical research at the Center for Oncological Research (CORE, UA), this proposed project will focus on (i) the identification of predictive biomarkers for Plk1 inhibition; and (ii) novel, rationally designed combination strategies with Plk1 inhibitors to improve therapeutic benefit. We previously identified p53 and hypoxia as potential biomarkers for response to Plk1 inhibition. However, no conclusive evidence could be found yet. As such, in the first objective of the proposed study, we will gain conclusive insights in the predictive role of p53 and hypoxia for the response to Plk1 inhibition. Therefore, we will investigate the effect of Plk1 inhibitors in a panel of isogenic cell lines with a different p53 background, under both normoxic and hypoxic conditions. Our second objective is to identify promising combination strategies with Plk1-inhibitors. In this regard, we will especially focus on drugs that eliminate senescent cells upon Plk1 inhibition. Recently, preclinical research by us has identified cellular senescence as an important outcome of Plk1 inhibition. Senescent cells are irreversibly growth-arrested, but remain metabolically active, thereby secreting multiple tumour-promoting factors to adjacent tumour cells. In-depth evaluation of the molecular pathways involved in induction of senescence after Plk1 inhibition will lead to the identification of potential targets to kill senescent NSCLC cells after Plk1 inhibition. At the time of writing this application, no investigation has been performed yet on the molecular pathways important for the survival of senescent cells after treatment, making this project challenging yet essential to enhance anti-tumour responses after Plk1 inhibition. Lastly, our third objective is to evaluate a novel combination therapy of Plk1 inhibitors with agents eliminating senescent cancer cells, in both vitro and in vivo models of NSCLC. We hypothesize that the anti-cancer effect of Plk1 inhibitors is synergistic with agents eliminating senescent cancer cells, so that this innovative combination strategy will ultimately result in improved survival and quality of life for patients with NSCLC. The proposed research project has the exciting potential to create a breakthrough in the optimization of Plk1 inhibition for patients with advanced NSCLC. Moreover, since Plk1 overexpression is found in multiple tumour types, our study results might also pave the way for improved treatment options for other malignancies.

Researcher(s)

• Promoter: Wouters An
• Co-promoter: Lardon Filip
• Fellow: Domen Andreas

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

Developing novel neuroprotective and/or immune-modulating therapeutic strategies for almost every neurological disease or trauma requires, both for academia and pharmaceutical industry, the existence of robust in vitro cell culture models to mimic disease-associated pathological events. Unfortunately, a complex interplay between multiple central nervous system (CNS) cell types and multiple cell types from the body's peripheral immune system, cannot be easily recapitulated by currently used 2-dimensional (2D) co-culture assays. It is exactly therefore that successful pre-clinical experimental efficacy has proved to be very difficult to translate into clinical benefit, and as a consequence there is an increasing gap in knowledge and progress between bench and bed side. One highly promising novel approach to improve the predictive power of in vitro human neuro-immune research consists in developing modular 3D brain organoids that resemble brain tissue at the structural, cellular and functional level. Within this project we aim to develop and optimize a new method for generating isogenic 3D brain organoids, comprising human pluripotent stem cell (hPSC)-derived neurons, astrocytes and microglia. Furthermore, hPSC-derived astrocytes and endothelial cells will be used to create a blood-brain-barrier model for physical separation of hPSC-derived macrophages from the generated human 3D brain organoids. Together, this integrated cell system will represent a powerful new 3D human neuro-immune cell culture paradigm. Within this multidisciplinary IOF-SBO project, the methodological approach to generate 3D brain organoids, combined with the experience in the field of clinical research and the availability of patient samples, is truly unique and will - in first instance - highly contribute to the field of in vitro cerebrovascular disease modelling and treatment validation. Furthermore, our aims to install an integrated 3D brain organoid technology platform at the University of Antwerp, will - given the current scientific and economic interests – allow for both short-term and long-term valorisation of our combined efforts, with both intellectual (PhD-theses, A1 publications) as well as financial (contract research) revenues.

Researcher(s)

• Promoter: Ponsaerts Peter
• Co-promoter: De Vos Winnok
• Co-promoter: Jorens Philippe
• Co-promoter: Timmermans Jean-Pierre
• Co-promoter: Wouters An

Research team(s)

• Laboratory for Experimental Hematology (LEH)

Project type(s)

• Research Project

Abstract

Both targeted therapies and immunotherapies are now at the forefront of personalized cancer medicine. Aberrant signaling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. In addition, it is well established that natural killer (NK) cells possess natural antitumor activity and can mediate antibody dependent cellular cytotoxicity (ADCC) upon binding with monoclonal antibodies, such as the EGFR inhibitor cetuximab. However, the presence of drug resistance and/or immune evasion is a major obstacle to progress in this field. In this research project, we hypothesize that increasing the NK cell activity by cetuximab in combination with targeting of NK cell immune checkpoint molecules can synergistically generate an immune mediated elimination of HNSCC cells that are resistant to treatment with cetuximab alone. Importantly, we will investigate the role of human papilloma virus (HPV), as HPV positive HNSCC patients represent a biologically distinct group. By characterizing NK cell functionality and, by extension, the whole immune checkpoint profile in HNSCC, we aim to rationally design new combination strategies to overcome cetuximab resistance, with the ultimate goal to improve the prognosis and life quality of HNSCC patients. Hereby, we will focus on HPV status and the hypoxic microenvironment as important mediators of treatment response.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Smits Evelien
• Co-promoter: Wouters An
• Fellow: Baysal Hasan

Research team(s)

Project type(s)

• Research Project

Abstract

Currently, there is an explosive interest in novel molecular targeted agents for cancer therapy and new approaches to mitosis inhibition target cardinal regulatory proteins, like polo-like kinase 1 (Plk1). Based on our previous promising findings with the Plk1 inhibitor volasertib, the overall objective is to further decipher the Plk1 pathway as a target for drug development. As such, this project aims to draw conclusions on the therapeutic potential of Plk1 inhibition, which will be investigated in vitro and in vivo, with emphasis on the impact of a hypoxic microenvironment, the role of combination therapy and the molecular pathways involved in NSCLC.

Researcher(s)

• Promoter: Lardon Filip
• Fellow: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

The introduction of targeted therapies is now at the forefront of personalised medicine in cancer treatment. After the initial promise of targeted therapies, drug resistance is however emerging as the major obstacle to progress in this field. In this project, we will focus on identification of new combination therapies to overcome intrinsic and acquired resistance to cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR). Hereby, we will concentrate specifically on head and neck squamous carcinoma (HNSCC), a highly relevant tumour type with poor prognosis that is intensively studied at the Center for Oncological Research (CORE) Antwerp. First, we will screen for new drug combinations by next-generation whole-exome sequencing and tumour kinome profiling of cetuximab-sensitive versus -resistant (intrinsic and acquired) HNSCC cell lines. Next, based on an integrative analysis of both the genetic profile and the kinome profile of cetuximab resistance, new combination treatments can be designed rationally to overcome cetuximab resistance. The molecular pathways underlying the cytotoxic effects of the selected compounds, in combination with chemotherapy and/or irradiation, will be investigated thoroughly, with focus on the hypoxic microenvironment as an important additional cause of therapy resistance. In conclusion, based on our screening results, new combination therapies will be designed rationally in order to thwart resistance to EGFR-targeting agents. Successful combinations will be forwarded into animal studies and ultimately into a clinical trail to demonstrate feasibility of the most promising combination therapy to treat HNSCC patients.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Pauwels Patrick
• Co-promoter: Peeters Marc
• Co-promoter: Wouters An

Research team(s)

• Center for Oncological Research (CORE)

Project type(s)

• Research Project

Abstract

The introduction of targeted therapies is now at the forefront of personalised medicine in cancer treatment. However, after the initial promise of targeted therapies, drug resistance is emerging as the major obstacle to progress in this field. In the proposed project, we will focus on unravelling and overcoming intrinsic and acquired resistance to cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR). Hereby, we will concentrate specifically on two highly relevant tumour types with poor prognosis, i.e. head and neck squamous carcinoma (HNSCC) and colorectal cancer (CRC). We will be the first to unravel drug resistant mechanisms and identify functional biomarkers by tumour kinome profiling. Using state-of-the-art PamGene technology, microarrays with kinase peptide substrates, mainly representing tyrosine residues, will be applied to analyse cetuximab-sensitive versus -resistant (intrinsic and acquired) HNSCC and CRC cell lines. As such, kinase activity (rather than presence) will be analysed, which is crucial to elucidate the underlying signal transduction pathways responsible for drug resistance. Afterwards, the in vitro kinase signature predicting intrinsic/acquired cetuximab resistance will be validated using HNSCC and CRC tumour patient material. Importantly, unravelling the molecular pathways underlying cetuximab resistance could have important implications not only regarding patient selection, but also regarding identification of new drug targets. Based on results from the above-mentioned kinome profiling, new (combination) treatments can be designed to overcome cetuximab resistance. In addition, the ongoing challenge of therapy resistance has already prompted a new approach to treat cancer patients, notably multiple inhibition of ErbB receptors simultaneously or irreversible inhibition, for example with the highly innovative, dual targeting agents afatinib and MEHD7945A. The molecular pathways underlying the cytotoxic effects of the selected compounds, either as monotherapy or in combination with chemotherapy and/or irradiation, will be investigated thoroughly, with focus on the hypoxic microenvironment as an important additional cause of therapy resistance. In conclusion, the strength of the proposed project lies in our multidisciplinary approach of drug resistance. The proposed model offers an attractive platform to investigate therapy resistance and action mechanisms of additional molecular targeted agents.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Peeters Marc
• Co-promoter: Wouters An
• Fellow: De Pauw Ines

Research team(s)

Project type(s)

• Research Project

Abstract

This project represents a research contract awarded by the University of Antwerp. The supervisor provides the Antwerp University research mentioned in the title of the project under the conditions stipulated by the university.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Peeters Marc
• Co-promoter: Wouters An
• Fellow: Van den Bossche Jolien

Research team(s)

Project type(s)

• Research Project

Abstract

In this project, we specifically wish to focus on two highly relevant tumour types, non-small cell lung cancer and pancreatic cancer. Firstly, the clinicopathological significance of Plk1 expression as a prognostic marker will be evaluated in a retrospective study investigating Plk1 gene amplification, Plk1 mRNA and protein expression. Secondly, the integration of a small-molecule Plk1 inhibitor with radiotherapy and chemotherapeutic agents for improving chemoradiation protocols will be studied. The interactions and underlying molecular biological pathways (p53 status, cell cycle progression, apoptosis, DNA repair, hypoxia-related signalling) will be investigated under both normal and reduced oxygen conditions, in parallel in an in vitro and in vivo setting. Elucidating these mechanisms could enable us to individually tailor the use of molecular targeted drugs in order to fully utilise their high potential in cancer therapy. Moreover, the proposed model offers an attractive platform to investigate the interactions and action mechanisms of additional molecular targeted agents in combination with chemoradation.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Peeters Marc
• Fellow: Wouters An

Research team(s)

Project type(s)

• Research Project

Abstract

In order to improve the efficacy of cancer therapy, the development of molecularly targeted agents has received a lot of attention. Their effects under hypoxia or in combination with radiotherapy are however largely unknown. Therefore, the integration of 2 types of EGFR inhibitors (cetuximab, a monoclonal antibody and erlotinib, a tyrosine kinase inhibitor) with gemcitabine and radiotherapy seems a promising strategy for improving chemoradiation.

Researcher(s)

• Promoter: Wouters An

Research team(s)

Project type(s)

• Research Project

Abstract

Objectives: 1) Study of the interactions between chemotherapy and radiotherapy under normoxic versus hypoxic conditions 2) Study of the molecular mechanisms underlying the radiosensitizing effect, under normoxic versus hypoxic conditions 3) Study of survival and apoptotic signal transduction pathways underlying the radiosensitizing effect, under normoxic versus hypoxic conditions

Researcher(s)

• Promoter: Wouters An

Research team(s)

Project type(s)

• Research Project

Abstract

It is well established that solid tumours frequently contain regions of hypoxia. Tumour hypoxia may induce resistance or a reduced sensitivity to radiation and chemotherapy. In that respect, it is very important to investigate new therapies in preclinical research under hypoxic conditions. However, an efficient in vitro hypoxia model is not available so far. Therefore, it seems very desirable to develop and optimize an hypoxic model. In this way, it will be possible to study the interaction between cytotoxic agents (for example cisplatin, gemcitabine) and irradiation under normoxic and hypoxic conditions in vitro. In addition, the hypoxia model can be used to analyse factors that contribute to radiosensitization under normoxia and hypoxia.

Researcher(s)

• Promoter: Lardon Filip
• Fellow: Wouters An

Research team(s)

Project type(s)

• Research Project

Abstract

It is well established that solid tumours frequently contain regions of hypoxia. Tumour hypoxia may induce resistance or a reduced sensitivity to radiation and chemotherapy. In that respect, it is very important to investigate new therapies in preclinical research under hypoxic conditions. However, an efficient in vitro hypoxia model is not available so far. Therefore, it seems very desirable to develop and optimize an hypoxic model. In this way, it will be possible to study the interaction between cytotoxic agents (for example cisplatin, gemcitabine) and irradiation under normoxic and hypoxic conditions in vitro. In addition, the hypoxia model can be used to analyse factors that contribute to radiosensitization under normoxia and hypoxia.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Vermorken Jan
• Fellow: Wouters An

Research team(s)

Project type(s)

• Research Project

Abstract

It is well established that solid tumours frequently contain regions of hypoxia. Tumour hypoxia may induce resistance or a reduced sensitivity to radiation and chemotherapy. In that respect, it is very important to investigate new therapies in preclinical research under hypoxic conditions. However, an efficient in vitro hypoxia model is not available so far. Therefore, it seems very desirable to develop and optimize an hypoxic model. In this way, it will be possible to study the interaction between cytotoxic agents (for example cisplatin, gemcitabine) and irradiation under normoxic and hypoxic conditions in vitro. In addition, the hypoxia model can be used to analyse factors that contribute to radiosensitization under normoxia and hypoxia.

Researcher(s)

• Promoter: Lardon Filip
• Co-promoter: Vermorken Jan
• Fellow: Wouters An

Research team(s)

Project type(s)

• Research Project