687 research outputs found

    Development of Novel Therapeutic Strategies to Target Therapy Resistance and Cancer Stem Cells

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    This thesis focuses on the core issues of multidrug resistance (MDR) in cancer, a process that hinders the success of chemotherapeutic treatments. MDR involves various mechanisms, including the upregulation of ABC transporter pumps, like MRP1, and increased cancer stemness, which contributes to malignancy and recurrence. The thesis comprises seven chapters: a literature review (Chapter 1), methodology (Chapter 2), results (Chapters 3-5), and discussions on findings and future studies (Chapters 6) and final discussion and overall summary (Chapter 7). Chapter 3 delves into the novel roles of MRP1 in cellular iron metabolism and proliferation, its interaction with c-Myc, and the effects on cellular proliferation. Silencing and inhibition studies reveal MRP1's role in regulating iron regulatory proteins through c-Myc. Chapter 4 investigates the role of ABC transporters in cancer stemness, revealing their connection with stemness states in different tumor types. Chapter 5 explores strategies for targeting drug-resistant cancer cells, demonstrating how doxycycline reduces the stemness marker SOX2 across multiple tumor types through a unique pathway. Chapter 6 examines the alteration of metabolism and stemness in drug-resistant cancer cells and strategies for targeting the cysteine metabolism pathway. The findings provide insights into cancer stemness regulation and potential therapeutic strategies, improving the efficacy of chemotherapeutics. The work reported in this thesis reveals an underlying and unique mechanism in regulation of SOX2-mediated cancer stemness. Moreover, the use of DXC to remove stemness was demonstrated to be a promising therapeutic strategy in combination with other common chemotherapeutics agents. These findings presented in this thesis enables us to understand cancer stemness better and improve the efficacy of current chemotherapeutics, which ultimately improve overall quality of life

    Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases

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    Countless biological processes allow cells to develop, survive, and proliferate. Among these, tightly balanced regulatory enzymatic pathways that can respond rapidly to external impacts maintain dynamic physiological homeostasis. More specifically, redox homeostasis broadly affects cellular metabolism and proliferation, with major contributions by thiol/disulfide oxidoreductase systems, in particular, the Thioredoxin Reductase Thioredoxin (TrxR/Trx) and the Glutathione Reductase-Glutathione-Glutaredoxin (GR/GSH/Grx) systems. These cascades drive vital cellular functions in many ways through signaling, regulating other proteins' activity by redox switches, and by stoichiometric reductant transfers in metabolism and antioxidant systems. Increasing evidence argues that there is a persistent alteration of the redox environment in certain pathological states, such as cancer, that heavily involve the Trx system: upregulation and/or overactivity of the Trx system may support or drive cancer progression, making both TrxR and Trx promising targets for anti-cancer drug development. Understanding the biochemical mechanisms and connections between certain redox cascades requires research tools that interact with them. The state-of-the-art genetic tools are mostly ratiometric reporters that measure reduced:oxidized ratios of selected redox pairs or the general thiol pool. However, the precise cellular roles of the central oxidoreductase systems, including TrxR and Trx, remain inaccessible due to the lack of probes to selectively measure turnover by either of these proteins. However, such probes would allow measuring their effective reductive activity apart from expression levels in native systems, including in cells, animals, or patient samples. They are also of high interest to identify chemical inhibitors for TrxR/Trx in cells and to validate their potential use as anti-cancer agents (to date, there is no selective cellular Trx inhibitor, and most known TrxR inhibitors were not comprehensively evaluated considering selectivity and potential off-targets). However, small molecule redox imaging tools are underdeveloped: their protein specificity, spectral properties, and applicability remain poorly precedented. This work aimed to address this opportunity gap and develop novel, small molecule diagnostic and therapeutic tools to selectively target the Trx system based on a modular trigger cargo design: artificial cyclic disulfide substrates (trigger) for oxidoreductases are tethered to molecular agents (cargo) such that the cargo’s activity is masked and is re-established only through reduction by a target protein. The rational design of these novel reduction sensors to target the cell's strongest disulfide-reducing enzymes was driven by the following principles: (i) cyclic disulfide triggers with stabilized ring systems were used to gain low reduction potentials that should resist reduction except by the strongest cellular reductases, such as Trx; and (ii) the cyclic topology also offers the potential for kinetic reversibility that should select for dithiol-type redox proteins over the cellular monothiol background. Creating imaging agents based on such two-component designs to selectively measure redox protein activity in native cells required to combine the correct trigger reducibility, probe activation kinetics, and imaging modalities and to consider the overall molecular architecture. The major prior art in this field has applied cyclic 5-membered disulfides (1,2 dithiolanes) as substrates for TrxR in a similar way to create such tools. However, this motif was described elsewhere as thermodynamically instable and was due to widely used for dynamic covalent cascade reactions. By comparing a novel 1,2 dithiolane-based probe to the state-of-the-art probes, including commercial TrxR sensors, by screening a conclusive assay panel of cellular TrxR modulations, I clarified that 1,2 dithiolanes are not selective substrates for TrxR in biological settings (Nat Commun 2022). Instead, aiming for more stable ring systems and thus more robust redox probes, during this work, I developed bicyclic 6 membered disulfides (piperidine fused 1,2 dithianes) with remarkably low reduction potentials. I showed that molecular probes using them as reduction sensors can be mostly processed by thioredoxins while being stable against reduction by GSH. The thermodynamically stabilized decalin like topology of the cis-annelated 1,2 dithianes requires particularly strong reductants to be cleaved. They also select for dithiol type redox proteins, like Trx, based on kinetic reversibility and offer fast cyclization due to the preorganization by annelation (JACS 2021). This work further expanded the system’s modularity with structural cores based on piperazine-fused 1,2 dithianes with the two amines allowing independent derivatization. Diagnostic tools using them as reduction sensors proved equally robust but with highly improved activation kinetics and were thus cellularly activated. Cellular studies evolved that they are substrates for both Trxs and their protein cousins Grxs, so measuring the cellular dithiol protein pool rather than solely Trx activity (preprint 2023). Finally, a trigger based on a slightly adapted reduction sensor, a desymmetrized 1,2 thiaselenane, was designed for selective reduction by TrxR’s selenol/thiol active site, then combined with a precipitating large Stokes’ shift fluorophore and a solubilizing group, to evolve the first selective probe RX1 to measure cellular TrxR activity, which even allowed high throughput inhibitor screening (Chem 2022). The central principle of this work was further advanced to therapeutic prodrugs based on the duocarmycin cargo (CBI) with tunable potency (JACS Au 2022) that can be used to create off-to-on therapeutic prodrugs. Such CBI prodrugs employing stabilized 1,2 dichalcogenide triggers proved to be cytotoxins that depend on Trx system activity in cells. They could further be exploited for cell-line dependent reductase activity profiling by screening their redox activation indices, the reduction-dependent part of total prodrug activation, in 177 cell lines. Beyond that, these prodrugs were well-tolerated in animals and showed anti-cancer efficacy in vivo in two distinct mouse tumor models (preprint 2022). Taken together, I introduced unique monothiol-resistant reducible motifs to target the cellular Trx system with chemocompatible units for each for TrxR and Trx/Grx, where the cyclic nature of the dichalcogenides avoids activation by GSH. By using them with distinct molecular cargos, I developed novel selective fluorescent reporter probes; and introduced a new class of bioreductive therapeutic constructs based on a common modular design. These were either applied to selectively measure cellular reductase activity or to deliver cytotoxic anti cancer agents in vivo. Ongoing work aims to differentiate between the two major redox effector proteins Trx and Grx, requiring additional layers of selectivity that may be addressed by tuned molecular recognition. The flexible use of various molecular cargos allows harnessing the same cellular redox machinery by either probes or prodrugs. This allows predictive conclusions from diagnostics to be directly translated into therapy and offers great potential for future adaptation to other enzyme classes and therapeutic venues.Die zelluläre Redox-Homöostase hängt von Thiol/Disulfid-Oxidoreduktasen ab, die den Stoffwechsel, die Proliferation und die antioxidative Antwort von Zellen beeinflussen. Die wichtigsten Netzwerke sind die Thioredoxin Reduktase-Thioredoxin (TrxR/Trx) und Glutathion Reduktase-Glutathion-Glutaredoxin (GR/GSH/Grx) Systeme, die über Redox-Schalter in Substratproteinen lebenswichtige zelluläre Funktionen steuern und so an der Redox-Regulation und -Signalübertragung beteiligt sind. Persistente Veränderungen des Redoxmilieus in pathologischen Zuständen, wie z. B. bei Krebs, sind in hohem Maße mit dem Trx-System verbunden. Eine Hochregulierung und/oder Überaktivität des Trx-Systems, die bei vielen Krebsarten auftreten, unterstützt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen für die Entwicklung neuer Krebsmedikamente macht. Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer Aktivität nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das Verhältnis reduzierter/oxidierter Spezies in zellulärem Umfeld oder spezifisch ausgewählte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind außerdem zur Validierung chemischer Hemmstoffe für TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von großem Interesse. Bislang gibt es keinen selektiven zellulären Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschließend bewertet. Ziel dieser Arbeit ist die Entwicklung niedermolekularer, diagnostischer und therapeutischer Werkzeuge, die selektiv auf das Trx-System abzielen und auf einem modularen Trigger-Cargo Design basieren. Hierzu werden zyklische Disulfid-Substrate (Trigger) für Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknüpft, dass dabei die Wirkstoffaktivität maskiert, und erst nach Reduktion durch ein Zielprotein wiederhergestellt wird. Diese neuartigen, synthetischen Reduktionssensoren basieren auf den folgenden Grundprinzipien: (i) Zyklische Disulfide sind thermodynamisch stabilisiert und können nur durch die stärksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische Reversibilität der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollständige Reduktion verhindert. Die meisten früheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fünfgliedriges Disulfid (1,2 Dithiolan) als Substrat für TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit für dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden für TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulären TrxR Aktivität und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate für TrxR, da sie labil sowohl gegen die Reduktion durch andere Redoxproteine, als auch gegen den Monothiol Hintergrund in Zellen sind (Nat. Commun. 2022). Als alternatives Strukturmotiv wird in dieser Arbeit ein bizyklisches sechsgliedriges Disulfid (anneliertes 1,2 Dithian) etabliert. Durch sein niedriges Reduktionspotenzial, also seine hohe Resistenz gegen Reduktion, werden molekulare Sonden basierend auf diesem 1,2 Dithian als Reduktionssensor fast ausschließlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die Enzymspezifität, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt für die flexible Verwendung weiterer funktioneller Einheiten ergänzt werden. Obwohl zelluläre Studien ergaben, dass diese neuartigen 1,2 Dithian Einheiten in Zellen sowohl Trx als auch das strukturell verwandte Grx adressieren, sind die daraus resultierenden diagnostischen Moleküle wertvoll, um den katalytischen Umsatz zellulärer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023). Begünstigt durch das modulare Moleküldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-Aktivität in Zellen vor. Es basiert auf der Verwendung eines zyklischen, unsymmetrischen Selenenylsulfid-Sensors (1,2 Thiaselenan), der selektiv von dem einzigartigen Selenolat der TrxR angegriffen wird, und dadurch letztlich nur von TrxR reduziert werden kann. RX1 eignete sich zudem für eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022). Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde für therapeutische Zwecke weiterentwickelt und nutzt dabei den einzigartigen Wirkmechanismus der Duocarmycin-Naturstoffklasse (CBI) (JACS Au 2022) zur Entwicklung reduktiv aktivierbarer Therapeutika. CBI Prodrugs basierend auf stabilisierten Redox-Schaltern (1,2 Dithiane für Trx; 1,2 Thiaselenan für TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darüber hinaus durch das Referenzieren ihrer Aktivität gegenüber nicht-reduzierbaren Kontrollmoleküle für die Erstellung zelllinienabhängiger Profile der Reduktaseaktivität in 177 Zelllinien genutzt. Schließlich waren diese neuen Krebsmittel im Tiermodell gut verträglich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b). Zusammenfassend präsentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten für das zelluläre Trx-System zur Entwicklung neuartiger, selektiver Reporter-Sonden, sowie eine neue Klasse reduktiv aktivierbarer Krebsmittel auf Basis eines adaptierbaren Trigger-Cargo Designs. Diese fanden entweder zur selektiven Messung zellulärer Proteinaktivität oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl für TrxR als auch für Trx/Grx identifiziert, wobei deren zyklische Natur eine Aktivierung durch GSH verhindert. Eine weitere Differenzierung zwischen den beiden Redox-Proteinen Trx und Grx und anderen Proteinen der Trx-Superfamilie erfordert eine zusätzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten. Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die „Pipeline-Entwicklung“ von Diagnostika und Therapeutika, die von der zellulären Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie übertragbar machen. Dies birgt großes Potenzial für künftige Entwicklungen bei einer potenziellen Übertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete

    Radiosensitization: Studies and Modern Approaches to Cellular Radiosensitivity

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    Even though radiation therapy has achieved great success, there is still an unsolved task of increasing radiation damage to tumor tissue and reducing side effects on healthy tissues. There is a wide variety of obstacles that reduce the efficiency of radiotherapy. Mechanisms of radioresistance involve tumor-specific oncogenic signalling pathways, tumor metabolism and proliferation, tumor microenvironment/hypoxia, and genomics. Radiosensitizers are promising agents that enhance injury to tumor tissue by accelerating DNA damage. Several strategies have been used recently to develop highly effective radiosensitizers with low toxicity. In this review, we considered the use of radiosensitizers, including small molecules and nanomaterials, in various malignant tumors and the problems and prospects for their clinical use in cancer therapy

    Tumor biomarkers that identify molecular subtypes and best responders to chemotherapy in patients with PDAC

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    Das duktale Adenokarzinom der Bauchspeicheldrüse (PDAC) ist eine tödliche Erkrankung, die auf die derzeitigen Therapien nur begrenzt anspricht. Es hat sich gezeigt, dass die molekulare Subtypisierung von PDAC mit dem klinischen Ansprechen auf Medikamente und der Prognose der Patienten zusammenhängt. Der Classical-like Subtyp ist mit einer relativ guten Prognose verbunden, während der Basal-like/QM-PDA-Subtyp mit einer schlechten Prognose und paradoxerweise mit einem guten Ansprechen auf die Chemotherapie korreliert ist. Die Untersuchung repräsentativer Biomarker für jeden molekularen Subtyp befindet sich noch in einem frühen Stadium der Entwicklung. In dieser Studie wurde das Transkriptom Profile von PDAC mit Hilfe der Laser-Capture-Mikroskopie in chemo-naiven Tumoren verbessert und die kanonischen Subtypisierungsschemata von Moffitt, Collisson, Bailey und Notta bestätigt. GATA6, CYP3A5 und HNF1A wurden als Biomarker auf mRNA-Ebene für Tumoren des Classical-like Subtyps identifiziert und erwiesen sich als prognostische Indikatoren. Die Expression von KRT81 auf mRNA-Ebene korrelierte mit dem Moffitt Basal-like Subtyp, war aber in diesem Fall kein signifikanter prognostischer Indikator. Nach einer neoadjuvanten Chemotherapie hatten PDAC Patienten, die viel GATA6 und CYP3A5 Proteine exprimieren, tendenziell ein relativ schlechteres Ansprechen, insbesondere nach einer FFX (FOLFIRINOX) Behandlung. Diese GATA6+ und CYP3A5+ exprimierenden Zellen, die nach der Chemotherapie im Tumorgewebe angereichert waren, könnten persistierende Krebszellen darstellen, die möglicherweise zu einem schlechten Ansprechen auf Medikamente und zu einer Medikamentenresistenz sowie zur Förderung von Tumormetastasen beitragen. Die Entdeckung weiterer repräsentativer Biomarker für molekulare Phänotypen wird zur einer Verbesserten Bemühungen um eine präzisere und stärker personalisierte Behandlung beitragen. Ein besseres Verständnis der Beschaffenheit der Persistenzzellen des Bauchspeicheldrüsenkrebses nach einer Chemotherapie sollte zur Entwicklung wirksamerer therapeutischer Strategien führen und so dazu beitragen, dass die betroffenen Patienten mit dieser Krankheit länger überleben

    Molecular mechanisms of resveratrol as chemo and radiosensitizer in cancer

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    One of the primary diseases that cause death worldwide is cancer. Cancer cells can be intrinsically resistant or acquire resistance to therapies and drugs used for cancer treatment through multiple mechanisms of action that favor cell survival and proliferation, becoming one of the leading causes of treatment failure against cancer. A promising strategy to overcome chemoresistance and radioresistance is the co-administration of anticancer agents and natural compounds with anticancer properties, such as the polyphenolic compound resveratrol (RSV). RSV has been reported to be able to sensitize cancer cells to chemotherapeutic agents and radiotherapy, promoting cancer cell death. This review describes the reported molecular mechanisms by which RSV sensitizes tumor cells to radiotherapy and chemotherapy treatment

    Expanding the toolbox of metabolically stable lipid prodrug strategies

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    Nucleoside- and nucleotide-based therapeutics are indispensable treatment options for patients suffering from malignant and viral diseases. These agents are most commonly administered to patients as prodrugs to maximize bioavailability and efficacy. While the literature provides a practical prodrug playbook to facilitate the delivery of nucleoside and nucleotide therapeutics, small context-dependent amendments to these popular prodrug strategies can drive dramatic improvements in pharmacokinetic (PK) profiles. Herein we offer a brief overview of current prodrug strategies, as well as a case study involving the fine-tuning of lipid prodrugs of acyclic nucleoside phosphonate tenofovir (TFV), an approved nucleotide HIV reverse transcriptase inhibitor (NtRTI) and the cornerstone of combination antiretroviral therapy (cART). Installation of novel lipid terminal motifs significantly reduced fatty acid hepatic ω-oxidation while maintaining potent antiviral activity. This work contributes important insights to the expanding repertoire of lipid prodrug strategies in general, but particularly for the delivery and distribution of acyclic nucleoside phosphonates

    The landscape of combination therapies against glioblastoma:From promises to challenges

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    We demonstrate in this thesis how new targets can be identified and highlight the challenges that lie in front of us when trying to translate these steps toward the clinic. We conclude that the blood-brain barrier, PD/PK of drugs, and therapy resistance are still major challenges and explain the limited improvement in treatment options for patients with GBM. First, GBM is a diffuse glioma located in the brain where the blood-brain barrier prevents the crossing of drugs and thereby limits the efficacy of treatment. Second, inter- and intratumoral heterogeneity have been observed in GBM leading to different cellular subpopulations with distinctive genetic profiles. Hence, treating these subpopulations with targeted drugs allows until now still survival of certain subpopulations that are not sensitive to this treatment. Lastly, therapy resistance is often seen in GBM patients and is probably related to intratumoral heterogeneity, but the intrinsic molecular mechanism is still not fully understood. Together they lead to the inevitable recurrence of the tumor

    The effect of bromalin and N-Acetylcysteine (NAC) on chemotherapeutic drug uptake by cancer cells in relation to their mucin and human nucleoside transporter profiles

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    Pancreatic cancer is a deadly disease with a five-year survival rate of less than 5%. Gemcitabine is the standard of care in the treatment of pancreatic cancer, however, limitations in effectiveness are widespread and need drastic improvement. This project aimed to develop a novel drug combination capable of enacting cytotoxic effect, eliminating the protein levels of mucin glycoproteins while having minimal reductions on human nucleoside transporters in pancreatic cancer cells. Consequently, I investigated the growth inhibitory, cell viability, mucin depleting and transporter depleting effects of bromelain, NAC, and gemcitabine as single agents or in combination against a panel of pancreatic cancer cell lines in vitro. I observed that bromelain and NAC exert cytotoxic effect as single agents, though combination treatments significantly increase potency. The addition of bromelain and BromAc to gemcitabine results in significant inhibition of cell proliferation, survival, and viability. The levels of hENT1/2, hCNT1/3 and MRP5 protein showed no significant reductions following treatment with bromelain, NAC, Gem/bromelain, and Gem/NAC, while BromAc treatments displayed significant reductions in hCNT1/3 protein levels. All transporters increased in protein levels following gemcitabine treatment with significant reductions observed in hENT2 and, hCNT1/3. Bromelain and NAC treatments displayed no significant changes in the levels of mucin, while BromAc displayed significant reductions in MUC1/4/5AC/13/16. All mucins displayed an increase in protein levels following Gem treatment and decreases following treatment with Gem/bromelain and Gem/NAC, apart from MUC13 which displayed increases. The Gem/BromAc treatment resulted in significant decreases in protein levels for all examined mucins. Correlations were identified between the transporters hENT2, hCNT1/3 and mucins MUC1/4/13/16. In addition, all examined mucins, hCNT1, and hCNT3 were seen to correlate with cell proliferation and viability though insufficient data was obtained to accurately predict synergy. To summarize, the addition of bromelain and NAC to gemcitabine results in a significant increase in potency and decrease in toxic dose. This is due in part to the dissolution of mucin glycoproteins encompassing pancreatic cancer cells, thus exposing the human nucleoside transporters required for the uptake of gemcitabine

    Expanding the Toolbox for Positron Emission Tomography – Radiotracer Development from PARP to Reporter Genes

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    Cancer is a life-threatening disease. Its treatment is challenging due to the disease’s heterogenous nature, and there is no universal therapy. In the last decades, different features of cancer have been explored for individualized treatment. One of those attributes is elevated levels of cellular stress, in particular replicative stress, within the tumors. Although treatments targeting replicative stress are available, to date, there is no specific biomarker available for the use with standard-of-care non-invasive imaging methods like position emission tomography utilizing radiolabeled molecules. Thus, therapy approaches are based on ex vivo information like histopathology or are applied without functional guidance. In this work, we evaluated PARP enzymes for their potential as biomarker for replicative stress. PARPs are heavily involved in the repair of single-strand DNA breaks, and their inhibition leads to synthetic lethality in tumor entities that lack alternative repair mechanisms. First, we synthesized five different PARP radiotracers, small molecules radiolabeled with the β+-emitting isotope 18F, for comparison of their biodistribution in the same mouse model and to determine the best application. We synthesized [18F]FPyPARP, a logD-optimized variant of [18F]PARPi, to shift the clearance route towards renal excretion, as high liver uptake hampers [18F]PARPi application for liver imaging. Compared to the gold-standards [18F]PARPi and [18F]FTT, [18F]FPyPARP presented with improved liver clearance, and might be an alternative to [18F]PARPi for liver imaging. [18F]Olaparib, an isotopologue of the first approved PARP inhibitor olaparib, was synthesized for direct comparison with the 100-fold more effective second-generation isotopologue [18F]talazoparib. The difference in efficacy here is attributed to the improved trapping capacity of PARP on the damaged DNA that prevents replication restart. In a xenograft model, [18F]olaparib and [18F]talazoparib showed similar biodistribution and PARP targeting, suggesting that the PARP trapping capacity does not influence radiotracer performance. In the overall comparison, target engagement was comparable but the radiotracers differed in non-target tissues; Thus, the choice of radiotracer is solely dependent on the envisioned application. To evaluate PARP as a biomarker for replicative stress, four in vitro models were probed for correlation of PARP radiotracer uptake with levels of stress. In myc overexpression models, the results were heterogeneous, and another attempt for a mIDH expression-based cell model did not indicate increased uptake. We then set out to induce replicative stress chemically, and did not observe significant differences in PARP radiotracer uptake compared to controls. We concluded that PARP is not a suitable biomarker as the expression is not upregulated but more likely the protein is activated on an enzymatic level upon replicative stress. Several other potential biomarkers were tested for changes in expression levels with Western blot, but did not result in a clear specific biomarker. As a surrogate, we developed novel reporter gene systems to compensate the lack of a specific replicative stress biomarker for preclinical therapy development and research on biomarkers and animal models. A reporter gene could be used to quantify promotor activity or other biological processes that can not be visualized directly. We designed, characterized and evaluated HaloTag, SNAPTag and CLIPTag and novel radiotracers designed to target the respective proteins in vitro and in vivo in a pilot xenograft study. All three presented with excellent target engagement and favorable pharmacokinetics. Interestingly, the HaloTag and CLIPTag radiotracers showed unspecific uptake in the naïve rodent brain, indicating that they are able to cross the intact blood-brain barrier. The blood-brain barrier is a recurrent obstacle in brain radiotracer development and thereby hampers global visualization of biological processes. Further evaluation in a murine model of viral gene transfer to the brain confirmed specific brain uptake and paves the way for future applications in the whole body. Thereby, our novel reporter gene systems are suitable to be used for future development of replicative stress specific radiotracers. The potential to stratify patients according to levels of replicative stress would ultimately aid selecting appropriate therapy regimen, and pave the way for new treatments targeting replicative stress
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