Person:

Haber, Daniel

Background: Desmoplastic Small Round Cell Tumor (DSRCT) is a highly aggressive malignancy that affects mainly adolescents and young adults. A defining characteristic of DSRCT is a specific chromosomal translocation, t(11;22)(p13;q12), that fuses EWS with WT1, leading to a production of two isoforms of chimeric transcription factor, EWS/WT1(−KTS) and EWS/WT1(+KTS). The chimeric proteins are thought to play critical roles in various stages of oncogenesis through aberrant transcription of different genes, but only a few of these genes have been identified. Methodology/Principal Findings: We report the identification of a new target of EWS/WT1, ENT4 (equilibrative nucleoside transporter 4) which encodes a pH-dependent adenosine transporter. ENT4 is transcriptionally activated by both isoforms of EWS/WT1 as evidenced by promoter-reporter and chromatin immunoprecipitation (ChIP) analyses. Furthermore, ENT4 is highly and specifically expressed in primary tumors of DSRCT as well as in a DSRCT cell line, JN-DSRCT-1. Treatment of JN-DSRCT-1 cells with adenosine analogs, such as 2-chloro-2′-deoxyadenosine (2-CdA), resulted in an increased cytotoxic response in dose- and pH-dependent manner. Conclusions/Significance: Our detailed analyses of a novel target of EWS/WT1 in DSRCT reveal an insight into the oncogenic mechanism of EWS-fusion chromosomal translocation gene products and provide a new marker for DSRCT. Furthermore, identification of ENT4 as a highly expressed transcript in DSRCT may represent an attractive pathway for targeting chemotherapeutic drugs into DSRCT.

Targeted cancer therapy requires the rapid and accurate identification of genetic abnormalities predictive of therapeutic response. We sought to develop a high-throughput genotyping platform that would allow prospective patient selection to the best available therapies, and that could readily and inexpensively be adopted by most clinical laboratories. We developed a highly sensitive multiplexed clinical assay that performs very well with nucleic acid derived from formalin fixation and paraffin embedding (FFPE) tissue, and tests for 120 previously described mutations in 13 cancer genes. Genetic profiling of 250 primary tumours was consistent with the documented oncogene mutational spectrum and identified rare events in some cancer types. The assay is currently being used for clinical testing of tumour samples and contributing to cancer patient management. This work therefore establishes a platform for real-time targeted genotyping that can be widely adopted. We expect that efforts like this one will play an increasingly important role in cancer management.

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer.

Background: Preclinical studies have documented antitumor activity of PARP inhibition both in vitro and in vivo, against Ewing sarcoma cells. This study aimed to translate that observation into a clinical trial to assess the efficacy and tolerability of olaparib, a PARP inhibitor, in patients with advanced Ewing sarcoma (EWS) progressing after prior chemotherapy. Methods: In this nonrandomized phase II trial, adult participants with radiographically measureable metastatic EWS received olaparib tablets, 400 mg orally twice daily, until disease progression or drug intolerance. Tumor measurements were determined by CT or MRI at 6 and 12 weeks after starting olaparib administration, and then every 8 weeks thereafter. Tumor response determinations were made according to RECIST 1.1, and adverse event determinations were made according to CTCAE, version 4.0. A total of 22 participants were planned to be enrolled using a conventional 2-step phase II study design. If no objective responses were observed after 12 participants had been followed for at least 3 months, further accrual would be stopped. Results: 12 participants were enrolled, and all were evaluable. There were no objective responses (PR/CR), 4 SD (duration 10.9, 11.4, 11.9, and 17.9 wks), and 8 PD as best response. Of the SD, 2 had minor responses (−9% and −11.7% by RECIST 1.1). The median time to disease progression was 5.7 weeks. Further enrollment was therefore discontinued. No significant or unexpected toxicities were observed with olaparib, with only a single case each of grade 3 anemia and grade 3 thrombocytopenia observed. Conclusions: This study is the first report of a prospective phase II trial to evaluate the safety and efficacy of a PARP inhibitor in patients with advanced Ewing sarcoma after failure of standard chemotherapy. Olaparib administration was safe and well tolerated when administered to this small heavily pre-treated cohort at the 400 mg BID dose, although the median duration of dosing was for only 5.7 weeks. No significant responses or durable disease control was seen, and the short average interval to disease progression underscores the aggressiveness of this disease. Other studies to combine cytotoxic chemotherapy with PARP inhibition in EWS are actively ongoing. Trial registration ClinicalTrials.gov Identifier: NCT01583543

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of Map3k7 (Tak1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed enrichment for Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.

Alterations in cancer genomes strongly influence clinical responses to treatment and in many instances are potent biomarkers for response to drugs. The Genomics of Drug Sensitivity in Cancer (GDSC) database (www.cancerRxgene.org) is the largest public resource for information on drug sensitivity in cancer cells and molecular markers of drug response. Data are freely available without restriction. GDSC currently contains drug sensitivity data for almost 75 000 experiments, describing response to 138 anticancer drugs across almost 700 cancer cell lines. To identify molecular markers of drug response, cell line drug sensitivity data are integrated with large genomic datasets obtained from the Catalogue of Somatic Mutations in Cancer database, including information on somatic mutations in cancer genes, gene amplification and deletion, tissue type and transcriptional data. Analysis of GDSC data is through a web portal focused on identifying molecular biomarkers of drug sensitivity based on queries of specific anticancer drugs or cancer genes. Graphical representations of the data are used throughout with links to related resources and all datasets are fully downloadable. GDSC provides a unique resource incorporating large drug sensitivity and genomic datasets to facilitate the discovery of new therapeutic biomarkers for cancer therapies.

SUMMARY Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6–48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.

Purpose: Amplification of the MET proto-oncogene in gastroesophageal cancer (GEC) may constitute a molecular marker for targeted therapy. We examined a GEC cohort with follow-up and reported the clinical response of four additional patients with MET-amplified tumors to the small molecule inhibitor crizotinib as part of an expanded phase I cohort study. Patients and Methods: From 2007 to 2009, patients with GEC were genetically screened as a consecutive series of 489 tumors (stages 0, I, and II, 39%; III, 25%; IV, 36%; n = 222 esophageal, including n = 21 squamous carcinomas). MET, EGFR, and HER2 amplification status was assessed by using fluorescence in situ hybridization. Results: Ten (2%) of 489 patients screened harbored MET amplification; 23 (4.7%) harbored EGFR amplification; 45 (8.9%) harbored HER2 amplification; and 411 (84%) were wild type for all three genes (ie, negative). MET-amplified tumors were typically high-grade adenocarcinomas that presented at advanced stages (5%; n = 4 of 80). EGFR-amplified tumors showed the highest fraction of squamous cell carcinoma (17%; n = 4 of 23). HER2, MET, and EGFR amplification were, with one exception (MET and EGFR positive), mutually exclusive events. Survival analysis in patients with stages III and IV disease showed substantially shorter median survival in MET/EGFR-amplified groups, with a rank order for all groups by median survival (from most to least aggressive): MET (7.1 months; P < .001) less than EGFR (11.2 months; P = .16) less than HER2 (16.9 months; P = .89) when compared with the negative group (16.2 months). Two of four patients with MET-amplified tumors treated with crizotinib experienced tumor shrinkage (−30% and −16%) and experienced progression after 3.7 and 3.5 months. Conclusion: MET amplification defines a small and aggressive subset of GEC with indications of transient sensitivity to the targeted MET inhibitor crizotinib (PF-02341066).

Metastasis-competent circulating tumour cells (CTCs) experience oxidative stress in the bloodstream, but their survival mechanisms are not well defined. Here, comparing single-cell RNA-Seq profiles of CTCs from breast, prostate and lung cancers, we observe consistent induction of β-globin (HBB), but not its partner α-globin (HBA). The tumour-specific origin of HBB is confirmed by sequence polymorphisms within human xenograft-derived CTCs in mouse models. Increased intracellular reactive oxygen species (ROS) in cultured breast CTCs triggers HBB induction, mediated through the transcriptional regulator KLF4. Depletion of HBB in CTC-derived cultures has minimal effects on primary tumour growth, but it greatly increases apoptosis following ROS exposure, and dramatically reduces CTC-derived lung metastases. These effects are reversed by the anti-oxidant N-Acetyl Cysteine. Conversely, overexpression of HBB is sufficient to suppress intracellular ROS within CTCs. Altogether, these observations suggest that β-globin is selectively deregulated in cancer cells, mediating a cytoprotective effect during blood-borne metastasis.

Innovative microfluidic technology has enabled massively parallelized and extremely efficient biological and clinical assays. Many biological applications developed and executed with traditional bulk processing techniques have been translated and streamlined through microfluidic processing with the notable exception of sample volume reduction or centrifugation, one of the most widely utilized processes in the biological sciences. We utilize the high-speed phenomenon known as inertial focusing combined with hydraulic resistance controlled multiplexed micro-siphoning allowing for the continuous concentration of suspended cells into pre-determined volumes up to more than 400 times smaller than the input with a yield routinely above 95% at a throughput of 240 ml/hour. Highlighted applications are presented for how the technology can be successfully used for live animal imaging studies, in a system to increase the efficient use of small clinical samples, and finally, as a means of macro-to-micro interfacing allowing large samples to be directly coupled to a variety of powerful microfluidic technologies.