A mechanical study of cancer drug-receptor interactions, specifically in G-Quadruplex DNA and Topoisomerase I enzymes

Computational methods are becoming essential in drug discovery as they provide information that traditional drug development methods lack. Using these methods to understand drug-receptor interactions in detail, researchers are able to efficiently design promising drug candidates. In this study, extra precision Glide docking, molecular dynamics simulations and MMGBSA binding energy calculations provided information about the binding behavior of small molecules to two specific targets for current cancer therapeutics: G-quadruplex DNA and Topoisomerase I enzyme. The first study focuses on the compound Telomestatin, which induces apoptosis of various cancer cells with a relatively low effect on somatic cells due to its high selectivity toward G-quadruplex over duplex DNA. Three major binding poses were discovered: top end stacking, bottom end stacking and a groove binding. A high resolution structure of this complex does not yet exist, so this is the first time Telomestatin binding modes have been reported. The second study focuses on 8 Camptothecin class Topoisomerase I inhibitors, which have been reported to effectively treat multiple types of cancer, however are limited by their drug resistance. Recent computational studies have indicated that the mutations near the active binding site of the drug can significantly weaken the drug binding and may be a major cause of the drug resistance. Here, a complete study of each Camptothecin analog in each mutated complex in the active binding site is presented. On this set of mutant complexes, Topotecan and Camptothecin have much smaller binding energy decrease than a set of new Camptopthcin derivatives (Lurtotecan, LESN-38, Gimatecan, Exatecan and Belotecan) currently under clinical trials. Lucanthone, a non-Camptothecin, shows comparable results to Topotecan and Camptothecin, indicating that it may exhibit the least drug resistance and is therefore a promising candidate for future studies as a Topoisomerase I inhibitor. In addition, a trend is observed from our binding energy data that the shorter the distance of a mutant to a ligand, the greater the decrease in binding energy (with one exception). The results found in each of these binding studies will be utilized to further advance effective cancer therapeutics in the future

Rationalising Multi-Agent Chemotherapy through Systems Analysis of Drug-Induced Apoptosis for High-Risk Neuroblastoma

High-risk neuroblastoma is an aggressive and invasive paediatric malignancy, with few actionable somatic mutations. As such attempts to implement precision medicine based approaches have been limited and intense multi-agent chemotherapy remains the standard-of-care. This project applied a systems biology approach to perform integrated proteomic and functional analysis of chemotherapy induced apoptosis to identify and define the emergence of synergy between relevant chemotherapy drug combinations. Firstly, a functional genomics screen was performed on a high content cellomics platform with a siRNA library of 200 apoptotic genes with current standard-of-care chemotherapy and preclinical drugs. Multi-dimensional analysis of this dataset elegantly demonstrated that synergy between any two chemotherapy drugs is proportional to the magnitude of divergence in apoptotic signalling between individual drugs. In particular, romidepsin was identified as an apoptotically distinct chemotherapy with universal drug synergy within the investigative drug panel. Identified key drug-specific apoptotic signalling nodes underlying this synergy were validated in vitro using genetically incorporated fluorescent biosensors, endogenously tagging and multiplexed IF. These tools allowed us to perform high-throughput kinetic live cell analysis at the single cell resolution. Optimised drug combinations were further validated in vivo using matched PDXs of treatment naïve and relapsed tumours. Collectively, this project has demonstrated that synergistic combinations emerge from the differential utilization of apoptotic signaling pathways by each single agent, regardless of the direct molecular target of each drug. This is contrary to the current dogma of utilising drugs with different molecular targets in combination chemotherapy. The results of this work contribute to developing non-traditional precision medicine approaches to rationalise multi-agent chemotherapy by characterising single agent apoptotic signalling

Découvertes de nouveaux mécanismes de résistance au Topotecan, un inhibiteur des topoisomérases, chez Leishmania infantum

Le protozoaire Leishmania est à l’origine d’une maladie tropicale négligée qui peut s’avérer mortelle si elle n’est pas traitée adéquatement. Dans les dernières années, l’efficacité des médicaments utilisés dans le combat de la leishmaniose a grandement diminué en raison de la résistance du parasite à ceux-ci. Bien que le Topotecan (TPT), présentement utilisé comme antitumoral, ait démontré une activité antileishmaniale puissante (EC50 de l’ordre des nanomolaires), son efficacité pourrait être compromise par l’émergence de la résistance du parasite d’autant plus que des résistances tumorales cliniques au TPT ont déjà été rapportées. Dans cette étude, les mécanismes de résistance au TPT par Leishmania infantum ont été caractérisés d’un point de vue moléculaire. Le génome complet des parasites sélectionnés résistants au TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) a été séquencé. Le rôle dans la résistance des différents éléments génétiques identifiés a été confirmé à l’aide d’une complémentation par nucléofection épisomale dans le parasite sauvage et a été étudié avec des simulations computationnelles. Aucune amplification ni délétion n’a été identifiée et seulement une variation mineure du nombre de chromosomes a été observée. Cependant, un polymorphisme d’un seul nucléotide non synonyme a été identifié dans le gène de la grande sous-unité de la topoisomérase IB (TOP IB), la cible du TPT, chez chacun des parasites mutants résistants conférant des niveaux de résistance variables (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > sauvage). De plus, des modélisations in sillico ont permis d’illustrer la proximité de ces substitutions d’acides aminés au site catalytique de TOP IB ainsi qu’au site de liaison du TPT. En conclusion, ces résultats suggèrent qu’une mutation ponctuelle dans la grande sous-unité TOP IB est suffisante pour engendrer des hauts niveaux de résistance (environ 24 x EC50) chez TPT700.1F187Y, TPT700.2G191A et TPT700.3 W232R. TPT pourrait être considéré comme un modèle pharmacologique pour l’étude de la résistance chez Leishmania.Leishmania, a protozoan parasite, causes a neglected tropical disease that is fatal if left untreated. In recent years, the effectiveness of the drugs used to tackle leishmaniasis has decreased dramatically due to the emergence of drug resistant parasites. Even though Topetecan (TPT), currently employed as an anti-tumoral drug, has shown strong anti-leishmanial activity (its EC50 being measured in nanomoles), its efficiency may be compromised by the resistance developped by the parasites, similarly to the resistance already recorded by tumoral cells to the drug. In this study, the mecanisms of resistance to TPT by Leishmania infantum were caracterised at the molecular level. The whole genome of parasites resistant to TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) was sequenced. The role of various genetic elements in the resistance mecanisms was confirmed via a complementation by episomal nucleofection in the wild type and was studied with the help of computational models. Neither amplications nor deletions were identified and only a minor variation in the chromosome number was observed. However, a non-synonymous single nucleotide polymorphism was identified in the gene coding the large subunit of topoisomerase IB, TPT’s target, within each of the resistant mutant parasites confering variable levels of resistance (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > wild type). Furthermore, in sillico models highlighted the proximity of these amino acid substitutions to the catalytic site of topoisomerase IB and also to the binding pockets of TPT. In conclusion, these results suggest that a point mutation in the large subunit of TOP IB is sufficient to confer high levels of resistance (about 24 x EC50) to TPT700.1F187Y, TPT700.2G191A and TPT700.3 W232R. Therefore, TPT can be considered a pharmacological tool to study resistance in Leishmania

PRECLINICAL AND CLINICAL DEVELOPMENT OF THE LIPOPHILIC CAMPTOTHECIN ANALOGUE AR-67

AR-67 is a lipophilic third generation camptothecin analogue, currently under early stage clinical trials. It acts by targeting Topoisomerase 1 (Top1), a nuclear enzyme essential for DNA replication and transcription and is present in two forms, the pharmacologically active lipophilic lactone and the charged carboxylate. In oncology patients participating in a phase I clinical trial, AR-67 lactone was the predominant species in plasma. Similarly to other camptothecins, the identified dose-limiting toxicities for AR-67 were neutropenia, thrombocytopenia and fatigue. In addition, in vitro metabolism studies indicated AR-67 lactone as a substrate for CYP3A4/5 as well as the UGT1A7 and UGT1A8 enzymes localizing in the liver and the gut. Numerous studies have demonstrated the over-expression of transporters in certain tumor types. Here, the effect of interactions between AR-67 and efflux or uptake transporters on the antitumor efficacy of AR-67 in vitro was studied. We showed that BCRP and MDR1 overexpression confers resistance to AR-67. Moreover, we demonstrated the therapeutic superiority of protracted dosing over more intense dosing regimens of AR-67 using xenografts models. Our studies indicated the schedule-dependent expression of Top1 and the preferential partitioning of AR-67 in the tumor tissue. We reason that these are factors that need to be taken into consideration when designing dosing schedules aiming to maximize efficacy. As most cytotoxic drugs, AR-67 has a narrow therapeutic window. Thus, it is essential to identify the variables influencing exposure to this camptothecin analogue. A thorough compartmental pharmacokinetic analysis was performed on the patient data obtained in a phase 1 clinical trial on AR-67. Moreover, sources of intersubject variability associated with obtaining pharmacokinetic parameter estimates were identified and a population covariate pharmacokinetic model was developed. In conclusion, the drug development of AR-67 is a work in process. Findings presented above provide an insight on the factors contributing to its efficacy and toxicity when given to cancer patients

The Use of Preclinical Models to Improve the Treatment of Retinoblastoma

Rodent models play an essential role in the development of new chemotherapeutics and dosing regimes. It is often difficult to carryout a clinical study for pediatric cancers due to the small patient population. Retinoblastoma, a pediatric cancer of the eye, is one example of a pediatric cancer that can benefit from preclinical studies. Over the years various retinoblastoma rodent models have been developed used to test various combination of broad-spectrum systemic chemotherapy. It was found form these studies that the combination of topotecan and carboplatin was effective. However both drugs cause myelosuppression and therefore administrating both of these drugs systemically is not possible. An alternative effective therapy in the clinic was the use of a subconjunctival administration. We thought if we could administer both drugs, one by systemic and one by a subconjunctival injection, perhaps we could decrease the systemic exposure with good tumor response. Detailed pharmacokinetic studies were conducted to understand the subconjunctival injections of topotecan and carboplatin. It was found that both drugs could successfully penetrate the eye and increase drug exposure. In addition, in the presence of a tumor, drug exposure to the vitreous was greater. Additionally comparative pharmacodynamic studies combining topotecan subconjunctival injection with carboplatin intraperitoneal or carboplatin subconjunctival injection with topotecan intraperitoneal were conducted. The tumor response, systemic toxicity and local toxicity were studied. There was tumor response in both combinations and no ocular toxicity was seen with a single eye subconjunctival injection for either drug. However, rats that received the combination with topotecan subconjunctival injection and carboplatin intraperitoneal experienced great toxicity and morbidity. The data and observations suggest the death is due to dehydration. Therefore it was concluded that the alternative combination was better. The above data suggested an appropriate drug combination and schedule for a preclinical study. However, the noninvasive methods to follow tumor progression and choosing the correct genetic model needed to be determined. This was essential to ensure the preclinical study could be easily translated for future clinical studies. A characterization study of five modalities, retina camera, optomotry, tonometer, ultrasound and MRI, was done with retinoblastoma mice. We determined the feasibility of each technique. It was found that the retina camera could detect the tumor the earliest in a high throughput manner. Additionally, the tonometer and optomotry machines could assess ocular health. While the ultrasound and MRI could image the eye and tumor in one field of view, MRI could capture the posterior chamber in more detail along with the extraocular space. With different software programs, the tumor to eye ratio volume measurement were determined and compared to the gold standard of enucleation, embedding, serial sectioning and hand tracing. It was found that there was a better correlation between the ultrasound and hand tracing histological sections. Concurrently, the tumor progression of six different genotypes was assessed. The tumor progression depended on the number and different genes deleted. Additionally, based on genotypes, it was determined there was not a strong genotypic trend in the increase in IOP or the loss of vision. From the studies of tumor progression we have learn more about the influence of genes on tumor progression, which will benefit additional genetic studies in mouse model systems and human tumors

Evaluation of the complexity of cell cycle effects induced by anticancer drugs

The time- and dose-dependence of the blocking activity, cell loss and recycling from block that follow a treatment with anticancer agents is not always easy to interpret, especially because of the superimposition of cytostatic and cytotoxic effects. Their separate quantification, in terms of the cellular control mechanisms that regulate the response to a drug treatment, is important to deeply understand the mode of action of an anticancer compound. We investigated cell cycle effects induced by different drugs on a cell line growing in vitro with an approach including experimental data coming from cell counting and different flow cytometric techniques and a mathematical model reproducing the perturbed cell growth after drug treatment. In this way we could quantify the effects induced by treatments performed with topotecan, doxorubicin and melphalan on IGROV1 cells, evaluating separately cytostatic and cytotoxic effects. For each one of the considered drugs we obtained and compared a set of parameters describing the time- and dose-dependence of cell cycle effects. These results were compared with those previously published by our group (Sena et al., 1999; Montalenti et al., 1998). Besides the classical flow cytometric techniques, we used the method of carboxyfluorescein diacetate succinimidyl ester staining to quantify and appreciate the heterogeneity of the effects induced by a treatment on the cells of the same population. The results obtained could be simulated with our mathematical model. In the last part of the project, the previously listed tools were used to obtain two sets of parameters describing the effects induced by a short treatment of cisplatin on HCT-116 and HCT-116 p53-/-. This allowed the quantification of the role of p53 in the cell response to cisplatin treatment, finding that this protein has a more important role in the activation of the apoptosis than in the cytostatic effects induced by this drug

STRATEGIES TO MODEL AND CIRCUMVENT ACQUIRED RESISTANCE TO THERAPEUTICS IN SMALL CELL LUNG CANCER

Small cell lung cancer (SCLC) is an extremely aggressive, pulmonary malignancy linked to lifetime smoking of cigarette tobacco. Most SCLC is diagnosed in the metastatic setting and surgical resection is rarely performed. However, SCLC is one of the most chemosensitive tumors, with >50% objective responses observed in de novo disease. These responses are impressive, but brief. Median progression free survival remains less than 5 months in current clinical trials. Recent studies have characterized the genome and epigenome of primary, untreated SCLC tumors. These studies have revealed near universal inactivation of the tumor suppressors TP53 and RB1, with frequent alterations in chromatin modifying enzymes, Notch signaling and amplification of MYC family members. Little in known about what factors permit acquired resistance and enable such a rapid shift in chemosensitive to chemorefractory disease within the same patient. No studies to date have comprehensively characterized paired sensitive and resistant disease states. The research presented below describes three studies aimed at determining how acquired resistance to chemotherapy is generated in vivo and targeted therapeutic approaches to chemoresistant disease. The first study details the discovery of genetic and epigenetic mediators of acquired chemoresistance in vivo through the development and characterization of paired chemosensitive and chemoresistant patient derived xenograft (PDX) models of SCLC. This work establishes a central role for EZH2 in promoting resistance to DNA damaging agents by silencing the gene SLFN11 and is highlighted by the impressive efficacy of EZH2 inhibition with first or second line standard of care chemotherapy. The second study describes a targeted approach to overcoming resistance to programmed cell death (apoptosis) by chemically inhibiting a protein-protein interaction between BCL-2 family members in SCLC. This study relied on the small molecule ABT-263 and attempts to explain disappointingly transient responses in SCLC patients observed in clinical testing. Finally, the third study builds on the second study, highlighting the importance of combinatorial strategies to improve the efficacy of this targeted agent in vivo, with a focus on potential mechanisms of acquired resistance, notably genomic loss of BAX. Research presented in these studies is directly informing the design of proposed clinical trials in SCLC

Quantitative proteome landscape of the NCI-60 cancer cell lines

Here we describe a proteomic data resource for the NCI-60 cell lines generated by pressure cycling technology and SWATH mass spectrometry. We developed the DIA-expert software to curate and visualize the SWATH data, leading to reproducible detection of over 3,100 SwissProt proteotypic proteins and systematic quantification of pathway activities. Stoichiometric relationships of interacting proteins for DNA replication, repair, the chromatin remodeling NuRD complex, β-catenin, RNA metabolism, and prefoldins are more evident than that at the mRNA level. The data are available in CellMiner (discover.nci.nih.gov/cellminercdb and discover.nci.nih.gov/cellminer), allowing casual users to test hypotheses and perform integrative, cross-database analyses of multi-omic drug response correlations for over 20,000 drugs. We demonstrate the value of proteome data in predicting drug response for over 240 clinically relevant chemotherapeutic and targeted therapies. In summary, we present a novel proteome resource for the NCI-60, together with relevant software tools, and demonstrate the benefit of proteome analyses

Patient-individual models of gliomas: Establishment, characterization and applications

Glioblastomas are the most common and aggressive brain tumors. The aim was establishing tumor models from individual patients (in vitro and in vivo). Modeling was successful from fresh and of note also from vitally frozen patient tumors. The models were characterized in direct comparison to the original tumors. Molecular features as mutations, amplifications and epigenetic alterations were highly maintained. Extensive drug sensitivity screens were performed. These well-defined patient individual models are ideal for establishment of individualized therapies and even immunological strategies.Ziel dieser Arbeit über Glioblastome, der häufigsten und aggressivsten Form von Hirntumoren, war die Etablierung von Patienten-individuellen Tumormodellen in vitro und in vivo. Eine neue Technik zur vitalen Kryo-Asservierung konnte etabliert werden. Molekulare Eigenschaften wie Mutationen, Amplifikationen und epigenetische Veränderungen waren in den Modellen im Vergleich zu den Primärtumoren zumeist erhalten. Neuronaler Ursprung, Malignität und Sensitivität auf Therapeutika wurden analysiert. Diese Modelle sind hervorragender Ausgangspunkt zur Etablierung individualisierter Therapien