To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaliana , laser microdissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in previous whole-leaf global expression analyses. For example, 67 transcription factors exhibited altered expression at the powdery mildew (PM) infection site, with subsets of these playing known or inferred roles in photosynthesis, cold/dehydration responses, defense, auxin signaling, and the cell cycle. Using integrated informatics analyses, we constructed putative regulatory networks for a subset of these processes and provided strong support for host cell cycle modulation at the PM infection site. Further experimentation revealed induced host endoreduplication occurred exclusively at the infection site and led us to identify MYB3R4 as a transcriptional regulator of this process. Induced endoreduplication was abrogated in myb3r4 mutants, and G. orontii growth and reproduction were reduced. This suggests that, by increasing gene copy number, localized endoreduplication serves as a mechanism to meet the enhanced metabolic demands imposed by the fungus, which acquires all its nutrients from the plant host.

BackgroundSelective patient recruitment can produce discrepancies between clinical trial results and real-world effectiveness.MethodsA systematic literature review and meta-analysis were conducted to assess vedolizumab real-world effectiveness and safety in patients with ulcerative colitis (UC) or Crohn’s disease (CD). MEDLINE, MEDLINE In-Process, EMBASE, and Cochrane databases were searched for real-world studies of vedolizumab in adult patients with UC/CD reporting clinical response, remission, corticosteroid-free remission, UC/CD-related surgery or hospitalization, mucosal healing, or safety published from May 1, 2014–June 22, 2017. Response and remission rates were combined in random-effects meta-analyses.ResultsAt treatment week 14, 32% of UC patients [95% confidence interval (CI) 27–39%] and 30% of CD patients (95% CI 25–34%) were in remission; and at month 12, 46% for UC (95% CI 37–56%) and 30% for CD (95% CI 20–42%). For UC, the rates of corticosteroid-free remission were 26% at week 14 (95% CI 20–34%) and 42% at month 12 (95% CI 31–53%); for CD they were 25% at week 14 (95%, CI 20–31%) and 31% at month 12 (95%, CI 20–45%). At month 12, 33–77% of UC and 6–63% of CD patients had mucosal healing. Nine percent of patients reported serious adverse events.ConclusionsVedolizumab demonstrated real-world effectiveness in patients with moderate-to-severely active UC or CD, with approximately one-half and one-third of patients, respectively, in remission at treatment month 12. These findings are consistent with clinical trial data and support the long-term benefit–risk profile of vedolizumab.Electronic supplementary materialThe online version of this article (10.1007/s00535-018-1480-0) contains supplementary material, which is available to authorized users.

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G 2 -M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G 2 phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924. Cancer Res; 73(1); 225-34. Ó2012 AACR.

We assessed the performance of two US Food and Drug Administration ( FDA ) 510(k)‐cleared wearable digital devices and the operational feasibility of deploying them to augment data collection in a 10‐day residential phase I clinical trial. The Phillips Actiwatch Spectrum Pro (Actiwatch) was used to assess mobility and sleep, and the Vitalconnect HealthPatch MD (HealthPatch) was used for monitoring heart rate ( HR ), respiratory rate ( RR ), and surface skin temperature ( ST ). We measured data collection rates, compared device readouts with anticipated readings and conventional in‐clinic measures, investigated data limitations, and assessed user acceptability. Six of nine study participants consented; completeness of data collection was adequate (> 90% for four of six subjects). A good correlation was observed between the HealthPatch device derived and in‐clinic measures for HR (Pearson r = 0.71; P = 2.2e‐16) but this was poor for RR ( r = 0.08; P = 0.44) and ST ( r = 0.14; P = 0.14). Manual review of electrocardiogram strips recorded during reported episodes of tachycardia > 180 beats/min showed that these were artefacts. The HealthPatch was judged to be not fit‐for‐purpose because of artefacts and the need for time‐consuming manual review. The Actiwatch device was suitable for monitoring mobility, collecting derived sleep data, and facilitating the interpretation of vital sign data. These results suggest the need for fit‐for‐purpose evaluation of wearable devices prior to their deployment in drug development studies.

MLN4924 is an investigational small-molecule inhibitor of the Nedd8-activating enzyme currently in phase I clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standard-of-care agents and may affect the clinical development of MLN4924. As such, we studied its interaction with other DNA-damaging agents. Mitomycin C, cisplatin, cytarabine, UV radiation, SN-38, and gemcitabine demonstrated synergy in combination with MLN4924 in vitro. The combination of mitomycin C and MLN4924 was shown to be synergistic in a mouse xenograft model. Importantly, depletion of genes within the ataxia telangiectasia and Rad3 related (ATR) and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. In addition, comet assay demonstrated increased DNA strand breaks with the combination of MLN4924 and mitomycin C. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924 results in frequent replication fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may provide useful information for the clinical development of these combinations. Mol Cancer Ther; 13(6); 1625-35. Ó2014 AACR.

Wearable digital devices offer potential advantages over traditional methods for the collection of health-related information, including continuous collection of dense data while study subjects are ambulatory or in remote settings. We assessed the utility of collecting continuous actigraphy and cardiac monitoring by deploying two US Food and Drug Administration (FDA) 510(k)-cleared devices in a phase I clinical trial of a novel compound, which included the use of an amphetamine challenge. The Phillips Actiwatch Spectrum Pro (Actiwatch) was used to assess mobility and sleep. The Preventice BodyGuardian (BodyGuardian) was used for monitoring heart rate (HR) and respiratory rate (RR), via single-lead electrocardiogram (ECG) recordings, together with physical activity. We measured data collection rates, compared device readouts with conventional measures, and monitored changes in HR measures during the amphetamine challenge. Completeness of data collection was good for the Actiwatch (96%) and lower for the BodyGuardian (80%). A good correlation was observed between device and in-clinic measures for HR (r = 0.99; P < 0.001), but was poor for RR (r = 0.39; P = 0.004). Manual reviews of selected ECG strips corresponding to HR measures below, within, and above the normal range were consistent with BodyGuardian measurements. The BodyGuardian device detected clear HR responses after amphetamine administration while subjects were physically active, whereas conventional measures collected at predefined timepoints while subjects were resting and supine did not. Wearable digital technology shows promise for monitoring human subjects for physiologic changes and pharmacologic responses, although fit-for-purpose evaluation and validation continues to be important prior to the wider deployment of these devices.Wearable digital technologies have been the subject of strong commercial promotion in recent years and have been adopted by many consumers. Although several devices have been approved by regulatory agencies and are increasingly used in healthcare settings, wearable devices have yet to find widespread application in industry-sponsored drug development studies.Despite significant progress, 1-3 there are few published studies that include critical analysis of the technology at

Traditional drug discovery practice usually follows the “one drug – one target” approach, seeking to identify drug molecules that act on individual targets, which ignores the systemic nature of human diseases. Pathway-based drug discovery recently emerged as an appealing approach to overcome this limitation. An important first step of such pathway-based drug discovery is to identify associations between drug molecules and biological pathways. This task has been made feasible by the accumulating data from high-throughput transcription and drug sensitivity profiling. In this paper, we developed “iPaD”, an integrative Penalized Matrix Decomposition method to identify drug-pathway associations through jointly modeling of such high-throughput transcription and drug sensitivity data. A scalable bi-convex optimization algorithm was implemented and gave iPaD tremendous advantage in computational efficiency over current state-of-the-art method, which allows it to handle the ever-growing large-scale data sets that current method cannot afford to. On two widely used real data sets, iPaD also significantly outperformed the current method in terms of the number of validated drug-pathway associations that were identified. The Matlab code of our algorithm publicly available at http://licong-jason.github.io/iPaD/

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.