Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by limited treatment options and unfavorable clinical outcomes. Therefore, the research described in this thesis... Show moreTriple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by limited treatment options and unfavorable clinical outcomes. Therefore, the research described in this thesis focused on the exploration of novel targeted therapies for TNBC. Through high-throughput screening, we assessed the responsiveness of TNBC cells to kinase inhibitors, which indicated a notable sensitivity to cyclin-dependent kinase (CDK) inhibitors. Building upon these findings, this thesis systematically compares the efficacy of targeting various transcription-associated CDKs, with CDK9 and CDK12 emerging as highly potent targets for disrupting TNBC cell proliferation. Transcription-associated CDKs play multiple roles in regulating mRNA transcription. Yet, inhibitors of these CDK’s induced selective, rather than global, changes in gene expression. This provides insights into their mechanisms of action and suggesting potential opportunities for combination therapy. Furthermore, our research elucidated the mechanism underlying the synergistic effects of combining kinase inhibitors with transcriptional CDK inhibitors, by demonstrating that this is mediated through the inhibition of cellular drug efflux by ABCG2. In conclusion, this thesis highlights the potential of transcription-associated CDK inhibitors as a promising avenue for treating TNBC. The insights from this thesis will help to further steer the (pre)clinical development and strategy of using transcription-associated CDK inhibitors for the treatment of TNBC. Show less
The clinical potential of applying synthetic lethality to cancer treatment is famously demonstrated by the BRCA1/PARP1 paradigm: a tumor specific defect in BRCA1 – a component of the DNA double... Show moreThe clinical potential of applying synthetic lethality to cancer treatment is famously demonstrated by the BRCA1/PARP1 paradigm: a tumor specific defect in BRCA1 – a component of the DNA double-strand break (DSB) repair pathway homologous recombination (HR) – results in a remarkable sensitivity to PARP1 inhibition (PARPi). Despite spectacular initial responses in patients, resistance to PARPi treatment may develop and must be overcome to maximally exploit this interaction in the clinic. Genetically engineered (mouse) model systems have shown that PARPi resistance may arise through inactivation of the 53BP1 pathway. The 53BP1 pathway normally protects DSB ends from resection and the removal of this “brake” restores HR in the absence of BRCA1. However, how the 53BP1 pathway protects DSB ends from resection has remained elusive. In this thesis, advances in 3D tumor organoid culture protocols and CRISPR/Cas9 (screening) technology were applied to identify and validate new components of the 53BP1 pathway that render BRCA1 deficient cells resistant to PARPi upon their loss. Furthermore, a new acquired vulnerability that can be therapeutically exploited to deplete such PARPi resistant cells is described. Together, this thesis provides mechanistic insight in DSB repair and illustrates how such fundamental knowledge may stand at the basis to combat resistance. Show less
One of the challenges of nanotechnology is to improve the efficacy of treatments for diseases, in order to reduce morbidity and mortality rates. Following this line of study, we made a nanoparticle... Show moreOne of the challenges of nanotechnology is to improve the efficacy of treatments for diseases, in order to reduce morbidity and mortality rates. Following this line of study, we made a nanoparticle formulation with a small size, uniform surfaces, and a satisfactory encapsulation coefficient as a target for colorectal cancer cells. The results of binding and uptake prove that using the target system with folic acid works: Using this system, cytotoxicity and cell death are increased when compared to using free oxaliplatin. The data show that the system maximized the efficiency of oxaliplatin in modulating tumor progression, increasing apoptosis and decreasing resistance to the drug. Thus, for the first time, our findings suggest that PLGA-PEG-FA increases the antitumor effectiveness of oxaliplatin by functioning as a facilitator of drug delivery in colorectal cancer. Show less
He, J.; Wink, S.; Bont, H. de; Le Dévédec, S.; Zhang, Y.; Water, B. van de 2019
Enhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling... Show moreEnhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling networks. Evaluation of the effect of various kinase inhibitors on these networks is critical to understand kinase inhibitor efficacy in cancer therapy. The dynamic activation of some kinases can be monitored by fluorescence resonance energy transfer (FRET) biosensors with high temporal resolution. Here, we established a FRET biosensor-based high throughput imaging approach to determine ERK and AKT activity in two triple negative breast cancer (TNBC) cell lines HCC1806 and Hs578T. FRET functionality was systematically evaluated using EGF stimulation and different MEK and AKT inhibitors, respectively. Next, we assessed the effect of a kinase inhibitor library containing >350 different kinase inhibitors (KIs) on ERK and AKT kinase activity using a FRET high-throughput screening setting. Suppression of FRET-ERK activity was generally positively correlated with the proliferation phenotype against inhibitors targeting MAPK signaling in both cell lines containing FRET-ERK reporter. AKT inhibitor (AKTi) resistant HCC1806 showed decreased proliferation associated with downregulated dynamics of FRET-ERK when treated with KIs targeting protein receptor tyrosine kinase (RTK). Yet, MEK inhibitor (MEKi) resistant Hs578T showed positively correlated FRET-AKT and proliferative responses against different PI3K and AKT inhibitors. Altogether, our data demonstrate the feasibility to integrate high throughput imaging-based screening of intracellular kinase activity using FRET-based biosensors in assessing kinase specificity and possible signaling crosstalk in direct relation to therapeutic outcome. Show less
Venetoclax is an oral BCL2 inhibitor undergoing investigation for use in relapsed or refractory multiple myeloma (RRMM), particularly in combination with proteasome inhibitors (VPI)[1,2]. An... Show moreVenetoclax is an oral BCL2 inhibitor undergoing investigation for use in relapsed or refractory multiple myeloma (RRMM), particularly in combination with proteasome inhibitors (VPI)[1,2]. An interim analysis of a current phase 2 trial of venetoclax with carfilzomib in RRMM demonstrated an overall response rate of 78% with a very good partial response rate of 56%[3,4]. However, a separate ongoing phase 3 trial of venetoclax with bortezomib found a decrease in overall survival due to increased fatal infections in the venetoclax arm compared to placebo. Better describing these infections may give insight into the pathophysiology and prove useful in mitigating strategies for use with VPI therapy in RRMM. Show less
Triple-negative breast cancer (TNBC) constitutes a small subtype (~15%) of breast cancer, but causes the majority of breast cancer-related deaths. As defined by the absence of ER and PR expression... Show moreTriple-negative breast cancer (TNBC) constitutes a small subtype (~15%) of breast cancer, but causes the majority of breast cancer-related deaths. As defined by the absence of ER and PR expression and HER2 overexpression, TNBC is not curable by hormone receptor or HER2-targeted therapies. Furthermore, TNBC is highly heterogeneous and most aggressive. To date, cytotoxic chemotherapy remains the mainstay in the management of TNBC. Despite the initial response to the standard-of-care chemotherapy, TNBC often exhibits intrinsic or acquired drug resistance, and subsequently, recurs in local and distal organs. Targeted therapies have long been pursued for the treatment of TNBC, but rarely demonstrate satisfactory clinical outcomes. Therefore, improved understanding of the intricate biological basis underlying TNBC insensitivity to targeted agents and defining new therapeutic opportunities are of the upmost importance. The aim of the studies presented in this thesis was to systematically identify gene/kinase susceptibilities of refractory TNBC cells, and reveal novel potent targeted therapies for TNBC as monotherapy or in combination with approved kinase drugs. Show less
He, J.; McLaughlin, R.P.; Noord, V.E. van der; Foekens, J.A.; Martens, J.W.M.; Westen, G.J.P. van; ... ; Water, B. van de 2019
Owing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide... Show moreOwing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide. Combined targeted therapy strategies to block interactive oncogenic signaling networks are being explored for effective treatment of the refractory TNBC subtype.A broad kinase inhibitor screen was applied to profile the proliferative responses of TNBC cells, revealing resistance of TNBC cells to inhibition of the mammalian target of rapamycin (mTOR). A systematic drug combination screen was subsequently performed to identify that AEE788, an inhibitor targeting multiple receptor tyrosine kinases (RTKs) EGFR/HER2 and VEGFR, synergizes with selective mTOR inhibitor rapamycin as well as its analogs (rapalogs) temsirolimus and everolimus to inhibit TNBC cell proliferation.The combination treatment with AEE788 and rapalog effectively inhibits phosphorylation of mTOR and 4EBP1, relieves mTOR inhibition-mediated upregulation of cyclin D1, and maintains suppression of AKT and ERK signaling, thereby sensitizing TNBC cells to the rapalogs. siRNA validation of cheminformatics-based predicted AEE788 targets has further revealed the mTOR interactive RPS6K members (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1) as synthetic lethal targets for rapalog combination treatment.TOR signaling is highly activated in TNBC tumors. As single rapalog treatment is insufficient to block mTOR signaling in rapalog-resistant TNBC cells, our results thus provide a potential multi-kinase inhibitor combinatorial strategy to overcome mTOR-targeted therapy resistance in TNBC cells. Show less
The research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a... Show moreThe research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a dismal prognosis and limited therapeutic options. In particular, the work centred on reversing resistance of TNBC cells to EGFR inhibitors. High-throughput kinase inhibitor library-based screens were utilised to evaluate the potential of novel targeted agents in a panel of TNBC cell lines and subsequently identify TNBC-specific genetic dependencies using siRNA-based screening. The signal transduction pathways perturbed by drug treatment were delineated and subsequently scrutinised using transcriptomic profiling and western blotting. The impact of drug treatment or gene silencing on cell death, proliferation, cell cycle progression and migration was assessed simultaneously. This work demonstrated that TNBC cell lines resistant to both MEK and Akt inhibitors are sensitive to disruption of CDK function. Additionally, it revealed that novel CDK inhibitors with strong activity against P-TEFb/CDK9 are highly effective against TNBC cells as single agents and in combination with multiple targeted therapies. These agents provoked profound down-regulation of multiple oncogenic pro-proliferative pathways, the silencing of which was detrimental to TNBC cell proliferation, thus defining several genes as potential future drug targets. Show less
In this thesis, we used genetically engineered mouse models to identify genes and pathways that are involved in ILC formation and in the development of resistance to FGFR-targeted therapy. These... Show moreIn this thesis, we used genetically engineered mouse models to identify genes and pathways that are involved in ILC formation and in the development of resistance to FGFR-targeted therapy. These mice carry conditional alleles of Cdh1, which result in the inactivation of the cell-adhesion molecule E-cadherin when Cre-recombinase is expressed. As mice with mammary-specific inactivation of E-cadherin alone were not prone to develop mammary tumors, they were used to investigate the contribution of additional genetic mutations to the development of ILCs using different genetic approaches. Firstly, we used non-germline modeling to study the role of PI3K-AKT signaling in the development of ILCs by performing intraductal injections of high-titer lentiviruses. Secondly, we employed a Sleeping Beauty (SB)-based insertional mutagenesis screen in conditional Cdh1 knockout mice to identify novel genes and pathways involved in the development of ILCs. We show that active transposon mutagenesis drives ILC formation and analysis of common insertion sites in SB-induced tumors identified a mutually exclusive group of four genes, of which three are frequently aberrated in human ILCs. Lastly, we used active mobilization of transposons in transplanted mouse ILCs to identify genes involved in acquiring resistance to the FGFR inhibitor AZD4547. Show less
Due to their abilities to eliminate pathogens and modulate host__s immune responses, antimicrobial peptides are considered as potential alternatives for the treatment of infections with (multi-drug... Show moreDue to their abilities to eliminate pathogens and modulate host__s immune responses, antimicrobial peptides are considered as potential alternatives for the treatment of infections with (multi-drug resistant) pathogens. In this thesis the immunomodulatory actions of two peptides have been investigated in order to gain insight in their mechanism of antimicrobial action; human lactoferrin-derived antimicrobial peptide hLF1-11 and the human cathelicidin LL-37. As hLF1-11 is active against infections in animals within a short period, we hypothesized immunomodulatory effects of hLF1-11 on innate immune cells. Results described in this thesis show that hLF1-11 enhances the inflammatory response of monocytes and modulates monocyte-macrophage differentiation resulting in macrophages that display enhanced antimicrobial effector functions. In addition, hLF1-11 drives monocyte-dendritic cell differentiation toward DCs that promote antifungal responses and induce Th17 polarization. Myeloperoxidase was identified as the intracellular target of hLF1-11 mediating immunomodulatory effects of this peptide. We also found that LL-37 was able to modulate monocyte-macrophage differentiation, however different than hLF1-11 as this resulted in macrophages with a pro-inflammatory phenotype. Together, these data underscore the bright future of antimicrobial peptides with immune modulating activity as these peptides might be developed further into a novel class of anti-infectives to which microbial drug-resistance is unlikely to develop quickly. Show less
The studies in this thesis were performed as part of the AMRIN (Antimicrobial Resistance in Indonesia) study that addressed antimicrobial resistance, antibiotic usage and infection control in... Show moreThe studies in this thesis were performed as part of the AMRIN (Antimicrobial Resistance in Indonesia) study that addressed antimicrobial resistance, antibiotic usage and infection control in Indonesia. They are the first studies that give insight into the incidence of healthcare-associated infections, determinants for carriage of resistant bacteria in Indonesian individuals and the implementation of measures for the prevention of the spread of bacteria and nosocomial infections in Indonesian hospitals. Show less
