Effects of hyperlinks on navigation in virtual environments

Hyperlinks introduce discontinuities of movement to 3-D virtual environments (VEs). Nine independent attributes of hyperlinks are defined and their likely effects on navigation in VEs are discussed. Four experiments are described in which participants repeatedly navigated VEs that were either conventional (i.e. obeyed the laws of Euclidean space), or contained hyperlinks. Participants learned spatial knowledge slowly in both types of environment, echoing the findings of previous studies that used conventional VEs. The detrimental effects on participants' spatial knowledge of using hyperlinks for movement were reduced when a time-delay was introduced, but participants still developed less accurate knowledge than they did in the conventional VEs. Visual continuity had a greater influence on participants' rate of learning than continuity of movement, and participants were able to exploit hyperlinks that connected together disparate regions of a VE to reduce travel time

Integrated Genomic, Epigenomic, and Expression Analyses of Ovarian Cancer Cell Lines.

To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer

Concise total syntheses of (–)-jorunnamycin A and (–)-jorumycin enabled by asymmetric catalysis

The bis-tetrahydroisoquinoline (bis-THIQ) natural products have been studied intensively over the past four decades for their exceptionally potent anticancer activity, in addition to strong gram-positive and -negative antibiotic character. Synthetic strategies toward these complex polycyclic compounds have relied heavily on electrophilic aromatic chemistry, such as the Pictet-Spengler reaction, that mimics their biosynthetic pathways. Herein we report an approach to two bis-THIQ natural products, jorunnamycin A and jorumycin, that instead harnesses the power of modern transition-metal catalysis for the three major bond-forming events and proceeds with high efficiency (15 and 16 steps, respectively). By breaking from biomimicry, this strategy allows for the preparation of a more diverse set of non-natural analogs

Concise total syntheses of (–)-jorunnamycin A and (–)-jorumycin enabled by asymmetric catalysis

The bis-tetrahydroisoquinoline (bis-THIQ) natural products have been studied intensively over the past four decades for their exceptionally potent anticancer activity, in addition to strong gram-positive and -negative antibiotic character. Synthetic strategies toward these complex polycyclic compounds have relied heavily on electrophilic aromatic chemistry, such as the Pictet-Spengler reaction, that mimics their biosynthetic pathways. Herein we report an approach to two bis-THIQ natural products, jorunnamycin A and jorumycin, that instead harnesses the power of modern transition-metal catalysis for the three major bond-forming events and proceeds with high efficiency (15 and 16 steps, respectively). By breaking from biomimicry, this strategy allows for the preparation of a more diverse set of non-natural analogs

High-throughput gene discovery in the rat

The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3′ ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to ∼50,000 rat UniGene clusters. We have identified, arrayed, and derived 5′ ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

Abstract Introduction Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines. Methods Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response. Results Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade. Conclusions These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991

Identification of Genomic Predictors of Response to the CDK4/6 Inhibitor Palbociclib using the UCLATORL Panel of Human Cancer Cell Lines

The landscape of genetic alterations that can lead to cancer is vast and complex. Precancerous cells accumulate mutations that affect the various molecular pathways involved in cancer progression. In the last two decades, hundreds of novel therapeutics designed to inhibit different molecular targets within these oncogenic pathways have recently entered clinical development. The success or failure of these compounds will depend on the ability to correctly identify subpopulations of cancer patients likely to be sensitive or resistant to these therapies. Patient-specific sensitivity to treatment is likely to be determined, at least in part, to the underlying genomic alterations that occurred during the development of the patient's particular cancer. Palbociclib is a highly specific small molecule inhibitor of cyclin-dependent kinases 4 and 6 currently in clinical development by Pfizer. It is designed to inhibit the cell cycle at the G1/S transition via preventing the phosphorylation of Rb by the Cyclin D/CDK4/6 enzyme complex. Our lab previously identified the estrogen receptor positive subpopulation of breast cancer patients as distinctively likely to benefit from treatment with palbociclib. This observation spurred the initiation of a Phase II clinical trial in this patient population where remarkable efficacy was observed. Given this translational success, we wished to investigate the molecular determinants of response to palbociclib in several additional cancer types. To this end, we assayed the in vitro sensitivity to palbociclib across a panel of 416 cancer cell lines derived from 12 distinct cancer types. We observed highly differential response to treatment both within and between cancer types. IC50s (the concentration of palbociclib required to inhibit fifty percent of population doublings) ranged from the low nanomolar range to above the highest dose tested (1�M). This response distribution was much broader than was observed in similar screens performed by the Broad and Sanger institutes, where the vast majority of cell lines assayed were listed as having IC50s above 1uM. Our ability to identify a higher proportion of palbociclib -sensitive cell lines can likely be credited to methodological innovations aimed at optimizing our screening protocol for the detection of longer-term cytostatic effects, as opposed to shorter-term cytotoxic effects of treatment. The generation of this highly differential response dataset allowed for a unique opportunity to explore the possible genetic mechanisms underlying differential sensitivity to treatment with palbociclib in vitro. We interrogated two large genomics datasets for genotype-response associations. The first of which consisted of whole exome point mutation data downloaded from the Cancer Cell Line Encyclopedia's hybrid capture sequencing database. This data was restricted by various criteria to enrich for functional, somatic point mutations that are known to be causally involved in carcinogenesis. From this restricted dataset we identified three proto-oncogenes (MLL, TSHR, SMO) where presumptive activating mutations were associated with resistance to treatment with palbociclib across our cell line panel. We further identified five recessive cancer genes where likely loss-of-function point mutations were significantly associated with palbociclib response. Mutations in two of these genes (CDH1, TOPBP1) associated with palbociclib sensitivity, while mutations in the other three (RB1, FANCA, NBN) associated with resistance. The other dataset interrogated for genotype-response associations was a copy number alteration dataset derived from comparative genomic hybridization arrays. This dataset was organized by gene and also restricted by various criteria to enrich for amplifications or deletions of genes likely to be causally involved in carcinogenesis. From this dataset we identified three chromosomal regions (17q12-21, 11q13, 1q32) where amplification was associated with sensitivity to palbociclib. Two amplified regions (19q13, 8q13) were found to be associated with resistance. Homozygous deletions of the 13q14 chromosomal region were found to be strongly associated with resistance to palbociclib. Following the identification of these candidate palbociclib response biomarkers from the crude, semi-supervised screens, several post-hoc analyses were performed to strengthen the argument for causation for each of the genotype-response associations. These analyses included: (1) a comprehensive literature search to investigate the possible causal mechanism of each biomarker, (2) Pearson correlation analysis to identify inter-biomarker associations followed by multiple regression to isolate the independent effect of each variable, (3) control of confounding by cell line growth rate and histology, and (4) analysis of misclassification in the genomics datasets. Following these extensive post-hoc analyses, we pruned our original set of fourteen biomarkers down to the eight most likely to play a causal role in determining sensitivity or resistance to treatment with palbociclib. The final set of candidate sensitivity biomarkers included: loss-of-function point mutations in CDH1, loss-of-function point mutations in TOPBP1, chromosomal amplification of 17q12-21 (ERBB2) and chromosomal amplification of 11q13 (CCND1). The final set of candidate resistance biomarkers included: activating point mutations in SMO, chromosomal amplification of CCNE1, loss-of-function point mutations in RB1 and chromosomal deletion of 13q14 (RB1). This final set of eight candidate biomarkers was analyzed by strata representing each of the 12 cancer types in our cell line panel. The four candidate sensitivity biomarkers were sufficiently frequent and associated with sensitivity in 6 of the 12 cancer types in our panel. These were the breast, colon, head/neck, lung, ovarian and upper gastrointestinal strata. The four candidate resistance biomarkers were sufficiently frequent and associated with resistance in 6 of the 12 cancer types in our panel. These were the breast, colon, kidney, lung, ovarian and upper gastrointestinal strata. The identified palbociclib response biomarkers represent good candidates for clinical translation, as we have shown them to be independently associated with palbociclib response in vitro and have a strong biologic rationale for causality. Follow-up experiments can further elucidate the molecular biology behind these associations and further validate these biomarkers before they are applied to the clinical setting. Ultimately, these biomarkers may be clinically implemented across a wide range of cancer types to identify patient subpopulations most likely to benefit from treatment with palbociclib

Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs–G protein signaling in osteoblasts

G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active G(s)-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced G(s) signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1 (2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated G(s) G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of G(s)-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of G(s) signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to G(s) signaling in mature OBs. Published by Elsevier Inc

Targeting activated PI3K/mTOR signaling overcomes acquired resistance to CDK4/6-based therapies in preclinical models of hormone receptor-positive breast cancer.

BackgroundCombined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2- breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance.MethodsIn this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance.ResultsWe show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2- breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2- breast cancer models.ConclusionsThese data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2- breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition