Clofarabine Has a Superior Therapeutic Window as compared to Gemcitabine in Preclinical Bladder Cancer Models.

Current standard-of-care systemic therapy options for locally advanced and metastatic bladder cancer (BC), which are predominantly based on cisplatin-gemcitabine combinations, are limited by significant treatment failure rates and frailty-based patient ineligibility. We previously addressed the urgent clinical need for better-tolerated BC therapeutic strategies using a drug screening approach, which identified outstanding antineoplastic activity of clofarabine in preclinical models of BC. To further assess clofarabine as a potential BC therapy component, we conducted head-to-head comparisons of responses to clofarabine versus gemcitabine in preclinical in vitro and in vivo models of BC, complemented by in silico analyses. In vitro data suggest a distinct correlation between the two antimetabolites, with higher cytotoxicity of gemcitabine, especially against several nonmalignant cell types, including keratinocytes and endothelial cells. Accordingly, tolerance of clofarabine (oral or intraperitoneal application) was distinctly better than for gemcitabine (intraperitoneal) in patient-derived xenograft models of BC. Clofarabine also exhibited distinctly superior anticancer efficacy, even at dosing regimens optimized for gemcitabine. Neither complete remission nor cure, both of which were observed with clofarabine, were achieved with any tolerable gemcitabine regimen. Taken together, our findings demonstrate that clofarabine has a better therapeutic window than gemcitabine, further emphasizing its potential as a candidate for drug repurposing in BC. PATIENT SUMMARY: We compared the anticancer activity of clofarabine, a drug used for treatment of leukemia but not bladder cancer, and gemcitabine, a drug currently used for chemotherapy against bladder cancer. Using cell cultures and mouse models, we found that clofarabine was better tolerated and more efficacious than gemcitabine, and even cured implanted tumors in mouse models. Our results suggest that clofarabine, alone or in combination schemes, might be superior to gemcitabine for the treatment of bladder cancer.

Combination of Drug Delivery Properties of PAMAM Dendrimers and Cytotoxicity of Platinum(IV) Complexes-A More Selective Anticancer Treatment?

Based on their drug delivery properties and activity against tumors, we combined PAMAM dendrimers with various platinum(IV) complexes in order to provide an improved approach of anticancer treatment. Platinum(IV) complexes were linked to terminal NH2 moieties of PAMAM dendrimers of generation 2 (G2) and 4 (G4) via amide bonds. Conjugates were characterized by 1H and 195Pt NMR spectroscopy, ICP-MS and in representative cases by pseudo-2D diffusion-ordered NMR spectroscopy. Additionally, the reduction behavior of conjugates in comparison to corresponding platinum(IV) complexes was investigated, showing a faster reduction of conjugates. Cytotoxicity was evaluated via the MTT assay in human cell lines (A549, CH1/PA-1, SW480), revealing IC50 values in the low micromolar to high picomolar range. The synergistic combination of PAMAM dendrimers and platinum(IV) complexes resulted in up to 200 times increased cytotoxic activity of conjugates in consideration of the loaded platinum(IV) units compared to their platinum(IV) counterparts. The lowest IC50 value of 780 ± 260 pM in the CH1/PA-1 cancer cell line was detected for an oxaliplatin-based G4 PAMAM dendrimer conjugate. Finally, in vivo experiments of a cisplatin-based G4 PAMAM dendrimer conjugate were performed based on the best toxicological profile. A maximum tumor growth inhibition effect of 65.6% compared to 47.6% for cisplatin was observed as well as a trend of prolonged animal survival.

Highly Cytotoxic Molybdenocenes with Strong Metabolic Effects Inhibit Tumour Growth in Mice.

A series of six highly lipophilic Cp-substituted molybdenocenes bearing different bioactive chelating ligands was synthesized and characterized by NMR spectroscopy, mass spectrometry and X-ray crystallography. In vitro experiments showed a greatly increased cytotoxic potency when compared to the non-Cp-substituted counterparts. In vivo experiments performed with the dichlorido precursor, (Ph2 C-Cp)2 MoCl2 and the in vitro most active complex, containing the thioflavone ligand, showed an inhibition of tumour growth. Proteomic studies on the same two compounds demonstrated a significant regulation of tubulin-associated and mitochondrial inner membrane proteins for both compounds and a strong metabolic effect of the thioflavone containing complex.

The copper transporter CTR1 and cisplatin accumulation at the single-cell level by LA-ICP-TOFMS.

More than a decade ago, studies on cellular cisplatin accumulation via active membrane transport established the role of the high affinity copper uptake protein 1 (CTR1) as a main uptake route besides passive diffusion. In this work, CTR1 expression, cisplatin accumulation and intracellular copper concentration was assessed for single cells revisiting the case of CTR1 in the context of acquired cisplatin resistance. The single-cell workflow designed for in vitro experiments enabled quantitative imaging at resolutions down to 1 µm by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS). Cisplatin-sensitive ovarian carcinoma cells A2780 as compared to the cisplatin-resistant subline A2780cis were investigated. Intracellular cisplatin and copper levels were absolutely quantified for thousands of individual cells, while for CTR1, relative differences of total CTR1 versus plasma membrane-bound CTR1 were determined. A markedly decreased intracellular cisplatin concentration accompanied by reduced copper concentrations was observed for single A2780cis cells, along with a distinctly reduced (total) CTR1 level as compared to the parental cell model. Interestingly, a significantly different proportion of plasma membrane-bound versus total CTR1 in untreated A2780 as compared to A2780cis cells was observed. This proportion changed in both models upon cisplatin exposure. Statistical analysis revealed a significant correlation between total and plasma membrane-bound CTR1 expression and cisplatin accumulation at the single-cell level in both A2780 and A2780cis cells. Thus, our study recapitulates the crosstalk of copper homeostasis and cisplatin uptake, and also indicates a complex interplay between subcellular CTR1 localization and cellular cisplatin accumulation as a driver for acquired resistance development.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Quantification in bioimaging by LA-ICPMS - Evaluation of isotope dilution and standard addition enabled by micro-droplets.

This study explores quantitative bioimaging as enabled by laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS), designing standardization methods based on robotic micro-droplet dispensing. The potential of producing controlled and highly precise pL-volume droplets was exploited to establish on-tissue isotope dilution and standard addition. Both strategies eliminate matrix effects and offer high metrological order traceable to SI units. The absolute quantity was obtained for µm-sized regions of interest in tissue samples, as defined by the extension of the deposited pL-volume droplet. While the gold standard isotope dilution (ID) was restricted to the accurate quantification of a single element, i.d. platinum in different tissue samples (mouse liver, spleen and tumor tissue), multiplexed matrix-matched calibration was obtained by on-tissue standard addition by depositing a dilution series of certified multi-element standards. Here, the working range was determined by the heterogeneity of the tissue samples and the background levels of elements intrinsically present and/or artificially introduced during sample preparation. Both methods, ID and standard addition served as reference methods for validation of external calibration using gelatin-based micro-droplet standards. Given full ablation, these external standards revealed a high dynamic range together with an excellent repeatability. Where applicable, the cross-validation revealed consistent quantitative results for the three quantification approaches. The comparable sensitivity obtained for standard addition and external standardization, respectively expressed as slope of the calibration function, provided proof that gelatin-based micro-droplets could serve as matrix-matched calibrations. Therefore, gelatin micro-droplets offer a valid tool for multiplexed matrix-mimicking standardization at high-throughput.

Molecular and Pharmacological Bladder Cancer Therapy Screening: Discovery of Clofarabine as a Highly Active Compound.

BACKGROUND: The heterogeneity of bladder cancers (BCs) is a major challenge for the development of novel therapies. However, given the high rates of recurrence and/or treatment failure, the identification of effective therapeutic strategies is an urgent clinical need. OBJECTIVE: We aimed to establish a model system for drug identification/repurposing in order to identify novel therapies for the treatment of BC. DESIGN, SETTING, AND PARTICIPANTS: A collection of commercially available BC cell lines (n = 32) was comprehensively characterized. A panel of 23 cell lines, representing a broad spectrum of BC, was selected to perform a high-throughput drug screen. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Positive hits were defined as compounds giving >50% inhibition in at least one BC cell line. RESULTS AND LIMITATIONS: Amongst >1700 tested chemical compounds, a total of 471 substances exhibited antineoplastic effects. Clofarabine, an antimetabolite drug used as third-line treatment for childhood acute lymphoblastic leukaemia, was amongst the limited number of drugs with inhibitory effects on cell lines of all intrinsic subtypes. We, thus, reassessed the substance and confirmed its inhibitory effects on commercially available cell lines and patient-derived cell cultures representing various disease stages, intrinsic subtypes, and histologic variants. To verify these effects in vivo, a patient-derived cell xenograft model for urothelial carcinoma (UC) was used. Well-tolerated doses of clofarabine induced complete remission in all treated animals (n = 12) suffering from both early- and late-stage disease. We further took advantage of another patient-derived cell xenograft model originating from the rare disease entity sarcomatoid carcinoma (SaC). Similarly to UC xenograft mice, clofarabine induced subcomplete to complete tumour remissions in all treated animals (n = 8). CONCLUSIONS: The potent effects of clofarabine in vitro and in vivo suggest that our findings may be of high clinical relevance. Clinical trials are needed to assess the value of clofarabine in improving BC patient care. PATIENT SUMMARY: We used commercially available cell lines for the identification of novel drugs for the treatment of bladder cancer. We confirmed the effects of one of these drugs, clofarabine, in patient-derived cell lines and two different mouse models, thereby demonstrating a potential clinical relevance of this substance in bladder cancer treatment.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance.

Clinical efficacy of oxaliplatin is frequently limited by severe adverse effects and therapy resistance. Acquired insensitivity to oxaliplatin is, at least in part, associated with elevated levels of glutathione (GSH). In this study we report on an oxaliplatin-based platinum(IV) prodrug, which releases L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis. Two complexes bearing either acetate (BSO-OxOAc) or an albumin-binding maleimide (BSO-OxMal) as second axial ligand were synthesized and characterized. The in vitro anticancer activity of BSO-OxOAc was massively reduced in comparison to oxaliplatin, proving its prodrug nature. Nevertheless, the markedly lower intracellular oxaliplatin uptake in resistant HCT116/OxR cells was widely overcome by BSO-OxOAc resulting in distinctly reduced resistance levels. Platinum accumulation in organs of a colorectal cancer mouse model revealed higher tumor selectivity of BSO-OxMal as compared to oxaliplatin. This corresponded with increased antitumor activity, resulting in significantly enhanced overall survival. BSO-OxMal-treated tumors exhibited reduced GSH levels, proliferative activity and enhanced DNA damage (pH2AX) compared to oxaliplatin. Conversely, pH2AX staining especially in kidney cells was distinctly increased by oxaliplatin but not by BSO-OxMal. Taken together, our data provide compelling evidence for enhanced tumor specificity of the oxaliplatin(IV)/BSO prodrug.

Development of a cobalt(iii)-based ponatinib prodrug system.

Receptor tyrosine kinase inhibitors have become a central part of modern targeted cancer therapy. However, their curative potential is distinctly limited by both rapid resistance development and severe adverse effects. Consequently, tumor-specific drug activation based on prodrug designs, exploiting tumor-specific properties such as hypoxic oxygen conditions, is a feasible strategy to widen the therapeutic window. After proof-of-principal molecular docking studies, we have synthesized two cobalt(iii) complexes using a derivative of the clinically approved Abelson (ABL) kinase and fibroblast growth factor receptor (FGFR) inhibitor ponatinib. Acetylacetone (acac) or methylacetylacetone (Meacac) have been used as ancillary ligands to modulate the reduction potential. The ponatinib derivative, characterized by an ethylenediamine moiety instead of the piperazine ring, exhibited comparable cell-free target kinase inhibition potency. Hypoxia-dependent release of the ligand from the cobalt(iii) complexes was proven by changed fluorescence properties, enhanced downstream signaling inhibition and increased in vitro anticancer activity in BCR-ABL- and FGFR-driven cancer models. Respective tumor-inhibiting in vivo effects in the BCR-ABL-driven K-562 leukemia model were restricted to the cobalt(iii) complex with the higher reduction potential and confirmed in a FGFR-driven urothelial carcinoma xenograft model. Summarizing, we here present for the first time hypoxia-activatable prodrugs of the clinically approved tyrosine kinase inhibitor ponatinib and a correlation of the in vivo activity with their reduction potential.

Landomycins as glutathione-depleting agents and natural fluorescent probes for cellular Michael adduct-dependent quinone metabolism.

Landomycins are angucyclines with promising antineoplastic activity produced by Streptomyces bacteria. The aglycone landomycinone is the distinctive core, while the oligosaccharide chain differs within derivatives. Herein, we report that landomycins spontaneously form Michael adducts with biothiols, including reduced cysteine and glutathione, both cell-free or intracellularly involving the benz[a]anthraquinone moiety of landomycinone. While landomycins generally do not display emissive properties, the respective Michael adducts exerted intense blue fluorescence in a glycosidic chain-dependent manner. This allowed label-free tracking of the short-lived nature of the mono-SH-adduct followed by oxygen-dependent evolution with addition of another SH-group. Accordingly, hypoxia distinctly stabilized the fluorescent mono-adduct. While extracellular adduct formation completely blocked the cytotoxic activity of landomycins, intracellularly it led to massively decreased reduced glutathione levels. Accordingly, landomycin E strongly synergized with glutathione-depleting agents like menadione but exerted reduced activity under hypoxia. Summarizing, landomycins represent natural glutathione-depleting agents and fluorescence probes for intracellular anthraquinone-based angucycline metabolism.