Motivation: High-throughput screens (HTS) by RNAi or small molecules are among the most promising tools in functional genomics. They enable researchers to observe detailed reactions to experimental perturbations on a genome-wide scale. While there is a core set of computational approaches used in many publications to analyze these data, a specialized software combining them and making them easily accessible has so far been missing.Results: Here we describe , a flexible software to build integrated analysis pipelines for HTS data that contains over-representation analysis, gene set enrichment analysis, comparative gene set analysis and rich sub-network identification. interfaces with commonly used pre-processing packages for HTS data and presents its results as HTML pages and network plots.Availability: Our software is written in the R language and freely available via the Bioconductor project at http://www.bioconductor.org.Contact: florian.markowetz@cancer.org.uk

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To investigate the kinetics of Cas9-mediated double strand break generation and repair in vivo, we developed two new tools. The first, chemically inducible Cas9 (ciCas9), is a rapidly-activated, single-component Cas9 variant engineered using a novel domain replacement strategy. ciCas9 can be activated in a matter of minutes, and the level of ciCas9 specificity and activity can be tuned. The second tool, DSB-ddPCR, is a droplet digital PCR-based assay for double strand breaks. DSB-ddPCR is the first assay to demonstrate time-resolved, highly quantitative and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally produce cleavage within minutes and repair within an hour or two. However, we observe distinct kinetic profiles, even for proximal sites, suggesting that target sequence and chromatin state modulate cleavage and repair kinetics.

We studied mechanisms of resistance to the novel taxane cabazitaxel in established cellular models of taxane resistance. We also developed cabazitaxel-resistant variants from MCF-7 breast cancer cells by stepwise selection in drug alone (MCF-7/CTAX) or drug plus the transport inhibitor PSC-833 (MCF-7/CTAX-P). Among multidrug resistant (MDR) variants, cabazitaxel was relatively less cross-resistant than paclitaxel and docetaxel (15 vs. 200-fold in MES-SA/Dx5 and 9 vs. 60-fold in MCF-7/TxT50, respectively). MCF-7/TxTP50 cells that were negative for MDR but had 9-fold resistance to paclitaxel were also 9-fold resistant to cabazitaxel. Selection with cabazitaxel alone (MCF-7/CTAX) yielded 33-fold resistance to cabazitaxel, 52-fold resistance to paclitaxel, activation of ABCB1, and 3-fold residual resistance to cabazitaxel with MDR inhibition. The MCF-7/CTAX-P variant did not express ABCB1, nor did it efflux rhodamine-123, BODIPY-labeled paclitaxel, and [3H]-docetaxel. These cells are hypersensitive to depolymerizing agents (vinca alkaloids and colchicine), have reduced baseline levels of stabilized microtubules, and impaired tubulin polymerization in response to taxanes (cabazitaxel or docetaxel) relative to MCF-7 parental cells. Class III β-tubulin (TUBB3) RNA and protein were elevated in both MCF-7/CTAX and MCF-7/CTAX-P. Decreased BRCA1 and altered epithelial-mesenchymal transition (EMT) markers are also associated with cabazitaxel resistance in these MCF-7 variants, and may serve as predictive biomarkers for its activity in the clinical setting. In summary, cabazitaxel resistance mechanisms include MDR (although at a lower level than paclitaxel and docetaxel), and alterations in microtubule dynamicity, as manifested by higher expression of TUBB3, decreased BRCA1, and by the induction of EMT.

Extrachromosomal DNAs (ecDNAs) are prevalent in human cancers and mediate high oncogene expression through elevated copy number and altered gene regulation1. Gene expression typically involves distal enhancer DNA elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs, comprised of ~10-100 ecDNAs clustered in the nucleus of interphase cells, drive intermolecular enhancer input for amplified oncogene expression. Single-molecule sequencing, single-cell multiome, and 3D enhancer connectome reveal subspecies of MYC-PVT1 ecDNAs lacking enhancers that access intermolecular and ectopic enhancer-promoter interactions in ecDNA hubs. ecDNA hubs persist without transcription and are tethered by BET protein BRD4. BET inhibitor JQ1 disperses ecDNA hubs, preferentially inhibits ecDNA oncogene transcription, and kills ecDNA+ cancer cells. Two amplified oncogenes MYC and FGFR2 intermix in ecDNA hubs, engage in intermolecular enhancer-promoter interactions, and transcription is uniformly sensitive to JQ1. Thus, ecDNA hubs are nuclear bodies of many ecDNAs tethered by proteins and platforms for cooperative transcription, leveraging the power of oncogene diversification and combinatorial DNA interactions. We suggest ecDNA hubs, rather than individual ecDNAs, as units of oncogene function, cooperative evolution, and new targets for cancer therapy.

Further data are presented concerning the unusual hemodynamic effects of 1 -hiydraziniophthalazine. Previous observations demonstrating a marked increase in cardiac output in noimotensive subjects are confirmed in hypertensive patients. The splanchnic vascular bed is one of the sites of increased blood flow. The similarity between the hemodynamic effects of 1-bydr.azinophthalazine and pyirogens is pointed out and the pharmacologic basis for the clinically observed additive effects of 1-1yd)rdazinol)lpthalazine an(d hexamethonium is (liscusse(l. G ROSS and his co-workers, in animals, ' and Reubi, in man,2 were the first to demonstrate that 1-hydrazinophthalazine (Apresoline) produces a reduction of arterial pressure and simultaneously an increase in renal blood flow. Since then considerable attention has been directed toward the further elucidation of the hemodynamic effects of this agent. Moyer and his associates, working with dogs, noted a marked increase in cardiac output and decrease in total peripheral resistance following administration of 1-hydrazinophthalazine.3 This observation was confirmed in normal and hypertensive pregnant women by Assali and his co-workers using the ballistocardiographic method4 and in normal subjects by Wilkinson and his associates using the intra-

HE STUDY of responses to drugs is rewarding to the cardiovascular physiologist. Many agents evoke integrated responses that involve the heart, the pulmonary circulation, the systemic arteries, and the veins. The challenge is to dissect these responses into their component parts-to discover the contributions to the overall change made by alterations in cardiac function and vasomotor activity in various regions. In this way, a clearer view is obtained of the ways in which the cardiovascular system can adjust itself to new demands, both physiologic and pathologic. In addition, these particular stimuli, drugs, can be understood more fully and used for their specific properties.Norepinephrine and angiotensin are important, naturally occurring, potent pressor agents. While both raise the systemic arterial pressure, the integrated responses they evoke are quite different. These differences can be appreciated by examination of figures 1 and 2. The tracings represent the results of single rapid intravenous injections of these drugs into the femoral vein of an anesthetized, openchest dog. Systemic arterial pressure, pulmonary arterial pressure, and left atrial pressure are recorded with strain gauges. Blood flow in the descending thoracic aorta is measured with an inserted ultrasonic flowmeter.Let us assume, for purposes of this discussion, that thoracic aortic blood flow is pro-From the

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance—random identity by descent—is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.