Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with ...distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.
•There are higher rates of mutation in ERMS than in ARMS tumors•RAS pathway mutations are associated with intermediate- and high-risk ERMS•ERMS tumor cells have elevated oxidative stress•ERMS tumors are sensitive to drugs that target oxidative stress
With combination therapies becoming increasingly vital to understanding and combatting disease, a reliable method for analyzing combined dose response is essential. The importance of combination ...studies both in basic and translational research necessitates a method that can be applied to a wide range of experimental and analytical conditions. However, despite increasing demand, no such unified method has materialized. Here we introduce the Bivariate Response to Additive Interacting Doses (BRAID) model, a response surface model that combines the simplicity and intuitiveness needed for basic interaction classifications with the versatility and depth needed to analyze a combined response in the context of pharmacological and toxicological constraints. We evaluate the model in a series of simulated combination experiments, a public combination dataset, and several experiments on Ewing's Sarcoma. The resulting interaction classifications are more consistent than those produced by traditional index methods, and show a strong relationship between compound mechanisms and nature of interaction. Furthermore, analysis of fitted response surfaces in the context of pharmacological constraints yields a more concrete prediction of combination efficacy that better agrees with in vivo evaluations.
Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients ...with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.
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•Ewing sarcoma cells are defective in DNA damage repair•DNA damage potentiates the effect of PARP inhibitors in Ewing sarcoma cells•PARP inhibitors might be combined with irinotecan and temozolomide for EWS treatment
Stewart et al. now find that Ewing sarcoma cells are defective in DNA damage repair and sensitive to PARP inhibitors. The schedule of irinotecan used in pediatric patients is well tolerated and can potentiate PARP-inhibitor-mediated killing of Ewing sarcoma cells in vivo. These data raise the possibility that PARP inhibitors may be combined with irinotecan and temozolomide for the treatment of Ewing sarcoma.
Paediatric solid tumours arise from endodermal, ectodermal, or mesodermal lineages. Although the overall survival of children with solid tumours is 75%, that of children with recurrent disease is ...below 30%. To capture the complexity and diversity of paediatric solid tumours and establish new models of recurrent disease, here we develop a protocol to produce orthotopic patient-derived xenografts at diagnosis, recurrence, and autopsy. Tumour specimens were received from 168 patients, and 67 orthotopic patient-derived xenografts were established for 12 types of cancer. The origins of the patient-derived xenograft tumours were reflected in their gene-expression profiles and epigenomes. Genomic profiling of the tumours, including detailed clonal analysis, was performed to determine whether the clonal population in the xenograft recapitulated the patient's tumour. We identified several drug vulnerabilities and showed that the combination of a WEE1 inhibitor (AZD1775), irinotecan, and vincristine can lead to complete response in multiple rhabdomyosarcoma orthotopic patient-derived xenografts tumours in vivo.
Combination therapy is increasingly central to modern medicine. Yet reliable analysis of combination studies remains an open challenge. Previous work suggests that common methods of combination ...analysis are too susceptible to noise to support robust scientific conclusions. In this paper, we use simulated and real-world combination datasets to demonstrate that traditional index methods are unstable and biased by pharmacological and experimental conditions, whereas response-surface approaches such as the BRAID method are more consistent and unbiased. Using a publicly-available data set, we show that BRAID more accurately captures variations in compound mechanism of action, and is therefore better able to discriminate between synergistic, antagonistic, and additive interactions. Finally, we applied BRAID analysis to identify a clear pattern of consistently enhanced AKT sensitivity in a subset of cancer cell lines, and a far richer array of PARP inhibitor combination therapies for BRCA1-deficient cancers than would be identified by traditional synergy analysis.
The chemical scaffolds from which screening libraries are built have strong influence on the libraries' utility for screening campaigns. Here we present analysis of the scaffold composition of ...several types of commercially available screening collections and compare those compositions to those of drugs and drug candidates.
Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, ...high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2'-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate analytical tools are used.
Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their ...clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.
Abstract
Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes and their levels and/or cellular localization are often altered in ...human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27
Kip1
(p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27
in vitro
, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).
N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an ...untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.