Cabozantinib in patients with platinum-refractory metastatic urothelial carcinoma: an open-label, single-centre, phase 2 trial.

BACKGROUND: Cabozantinib is a multikinase inhibitor of MET, VEGFR, AXL, and RET, which also has an effect on the tumour immune microenvironment by decreasing regulatory T cells and myeloid-derived suppressor cells. In this study, we examined the activity of cabozantinib in patients with metastatic platinum-refractory urothelial carcinoma. METHODS: This study was an open-label, single-arm, three-cohort phase 2 trial done at the National Cancer Institute (Bethesda, MD, USA). Eligible patients were 18 years or older, had histologically confirmed urothelial carcinoma or rare genitourinary tract histologies, Karnofsky performance scale index of 60% or higher, and documented disease progression after at least one previous line of platinum-based chemotherapy (platinum-refractory). Cohort one included patients with metastatic urothelial carcinoma with measurable disease as defined by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Two additional cohorts that enrolled in parallel (patients with bone-only urothelial carcinoma metastases and patients with rare histologies of the genitourinary tract) were exploratory. Patients received cabozantinib 60 mg orally once daily in 28-day cycles until disease progression or unacceptable toxicity. The primary endpoint was investigator-assessed objective response rate by RECIST in cohort one. Response was assessed in all patients who met the eligibility criteria and who received at least 8 weeks of therapy. All patients who received at least one dose of cabozantinib were included in the safety analysis. This completed study is registered with ClinicalTrials.gov, NCT01688999. FINDINGS: Between Sept 28, 2012, and Oct, 20, 2015, 68 patients were enrolled on the study (49 in cohort one, six in cohort two, and 13 in cohort three). All patients received at least one dose of cabozantinib. The median follow-up was 61·2 months (IQR 53·8-70·0) for the 57 patients evaluable for response. In the 42 evaluable patients in cohort one, there was one complete response and seven partial responses (objective response rate 19%, 95% CI 9-34). The most common grade 3-4 adverse events were fatigue (six [9%] patients), hypertension (five [7%]), proteinuria (four [6%]), and hypophosphataemia (four [6%]). There were no treatment-related deaths. INTERPRETATION: Cabozantinib has single-agent clinical activity in patients with heavily pretreated, platinum-refractory metastatic urothelial carcinoma with measurable disease and bone metastases and is generally well tolerated. Cabozantinib has innate and adaptive immunomodulatory properties providing a rationale for combining cabozantinib with immunotherapeutic strategies. FUNDING: National Cancer Institute Intramural Program and the Cancer Therapy Evaluation Program.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

Synthesis of a Fluorescent Analogue of Paclitaxel That Selectively Binds Microtubules and Sensitively Detects Efflux by P-Glycoprotein.

The anticancer drug paclitaxel (Taxol) exhibits paradoxical and poorly understood effects against slow-growing tumors. To investigate its biological activity, fluorophores such as Oregon Green have been linked to this drug. However, this modification increases its polarity by approximately 1000-fold and reduces the toxicity of Taxol towards cancer cell lines by over 200-fold. To construct more drug-like fluorescent probes suitable for imaging by confocal microscopy and analysis by flow cytometry, we synthesized derivatives of Taxol linked to the drug-like fluorophore Pacific Blue (PB). We found that PB-Gly-Taxol bound the target protein ß-tubulin with both high affinity in vitro and high specificity in living cells, exhibited substantial cytotoxicity towards HeLa cells, and was a highly sensitive substrate of the multidrug resistance transporter P-glycoprotein (P-gp).

Autocrine signaling by receptor tyrosine kinases in urothelial carcinoma of the bladder.

Comprehensive characterizations of bladder cancer (BCa) have established molecular phenotype classes with distinct alterations and survival trends. Extending these studies within the tyrosine kinase (TK) family to identify disease drivers could improve our use of TK inhibitors to treat specific patient groups or individuals. We examined the expression distribution of TKs as a class (n = 89) in The Cancer Genome Atlas (TCGA) muscle invasive BCa data set (n >400). Patient profiles of potentially oncogenic alterations (overexpression and/or amplification) clustered TKs into 3 groups; alterations of group 1 and 3 TKs were associated with significantly worse patient survival relative to those without alterations. Many TK pathways induce epithelial-to-mesenchymal transition (EMT), which promotes tumor invasiveness and metastasis. Overexpression and/or amplification among 9 EMT transcriptional activators occurred in 43% of TCGA cases. Co-occurring alterations of TKs and EMT transcriptional activators involved most group 1 TKs; 24% of these events were associated with significantly worse patient survival. Co-occurring alterations of receptor TKs and their cognate ligands occurred in 16% of TCGA cases and several BCa-derived cell lines. Suppression of GAS6, MST1 or CSF1, or their respective receptors (AXL, MST1R and CSF1R), in BCa cell lines was associated with decreased receptor activation, cell migration, cell proliferation and anchorage independent cell growth. These studies reveal the patterns and prevalence of potentially oncogenic TK pathway-related alterations in BCa and identify specific alterations associated with reduced BCa patient survival. Detection of these features in BCa patients could better inform TK inhibitor use and improve clinical outcomes.

Antibody-Drug Conjugate that Exhibits Synergistic Cytotoxicity with an Endosome-Disruptive Peptide.

Antibody-drug conjugates are an important class of cancer therapeutics. These agents generally bind a specific cell surface receptor, undergo receptor-mediated endocytosis, and enter the endosomal-lysosomal system, where the environment in these organelles facilitates the release of a membrane-permeable cytotoxin. By using a membrane-impermeable cytotoxin, we describe here a method that allows the cytotoxicity of an antibody conjugate to be triggered by co-administration with an endosome-disruptive peptide that exhibits low toxicity. This approach was validated by conjugation of an anionic derivative of the tubulin-binding cytotoxin colchinol methyl ether to lysine residues of the HER2-targeting antibody trastuzumab (Herceptin) via a disulfide. When this antibody binds HER2 on SKBR3 breast cancer cells and undergoes endocytosis, the membrane-impermeable cytotoxin is released, but it becomes trapped in endosomes, resulting in relatively low cytotoxicity (IC50 > 1 µM). However, co-administration with an essentially nontoxic (IC50 > 10 µM) cholesterol-linked endosome-disruptive peptide promotes the release of this small molecule into the cytoplasm, conferring subnanomolar cytotoxic potency (IC50 = 0.11 ± 0.07 nM). Studies of a structurally related fluorophore conjugate revealed that the endosome-disruptive peptide does not substantially enhance cleavage of the disulfide (t 1/2 = 8 ± 2 h) within endosomes, suggesting that the mechanism of endosomal escape involves the efflux of some small molecules without facilitating substantial influx of reduced glutathione.

Quantification of Small Molecule-Protein Interactions using FRET between Tryptophan and the Pacific Blue Fluorophore.

We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid. This route was used to synthesize fluorescent derivatives of biotin, as well as lower affinity thiobiotin, iminobiotin, and imidazolidinethione analogues that bind the protein streptavidin. Compared with previously published FRET acceptors for tryptophan, PB proved to be superior in both sensitivity and efficiency. These unique properties of PB enabled direct quantification of dissociation constants (Kd) as well as competitive inhibition constants (Ki) in the micromolar to nanomolar range. In comparison to analogous binding studies using fluorescence polarization, fluorescence quenching, or fluorescence enhancement, affinities determined using Trp-FRET were more precise and accurate as validated using independent isothermal titration calorimetry studies. FRET between tryptophan and PB represents a new tool for the characterization of protein-ligand complexes.