The experience of point-of-care testing for influenza in Scotland in 2017/18 and 2018/19 - no gain without pain.

BackgroundDuring the 2017/18 and 2018/19 influenza seasons, molecular amplification-based point-of-care tests (mPOCT) were introduced in Scotland to aid triaging respiratory patients for hospital admission, yet communication of results to national surveillance was unaccounted for.AimThis retrospective study aims to describe steps taken to capture mPOCT data and assess impact on influenza surveillance.MethodsQuestionnaires determined mPOCT usage in 2017/18 and 2018/19. Searches of the Electronic Communication of Surveillance in Scotland (ECOSS) database were performed and compared with information stored in laboratory information management systems. Effect of incomplete data on surveillance was determined by comparing routine against enhanced data and assessing changes in influenza activity levels determined by the moving epidemic method.ResultsThe number of areas employing mPOCT increased over the two seasons (6/14 in 2017/18 and 8/14 in 2018/19). Analysis of a small number of areas (n = 3) showed capture of positive mPOCT results in ECOSS improved between seasons and remained high (> 94%). However, capture of negative results was incomplete. Despite small discrepancies in weekly activity assessments, routine data were able to identify trend, start, peak and end of both influenza seasons.ConclusionThis study has shown an improvement in capture of data from influenza mPOCT and has highlighted issues that need to be addressed for results to be accurately captured in national surveillance. With the clear benefit to patient management we suggest careful consideration should be given to the connectivity aspects of the technology in order to ensure minimal impact on national surveillance.

Dual direction CRISPR transcriptional regulation screening uncovers gene networks driving drug resistance.

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.

Increasing the performance of pooled CRISPR-Cas9 drop-out screening.

Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.

Identification of approved and investigational drugs that inhibit hypoxia-inducible factor-1 signaling.

One of the requirements for tumor development is blood supply, most often driven by hypoxia-induced angiogenesis. Hypoxia induces the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which induces expression of an angiogenic factor, vascular endothelial growth factor (VEGF). The purpose of this study is to validate a new screening platform combined with orthogonal assays to rapidly identify HIF-1 inhibitors and to evaluate the effectiveness of approved drugs on modulating HIF-1 signaling. We generated an endogenous HIF-1α-NanoLuc luciferase reporter allele in the human HCT116 colon cancer cell line using genome editing and screened a panel of small interfering RNAs (siRNAs) to 960 druggable targets and approximately 2,500 drugs on a quantitative high-throughput screening (qHTS) platform. Selected compounds were further investigated with secondary assays to confirm their anti-HIF activity and to study their mode of action. The qHTS assay identified over 300 drugs that inhibited HIF-1α-NanoLuc expression. The siRNA screening results supported the effectiveness of several target-specific inhibitors. Moreover, the identified HIF-1 inhibitors, such as mycophenolate mofetil, niclosamide, and trametinib, were able to suppress cancer cell proliferation and angiogenesis. Our study indicates that blocking the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways effectively inhibits hypoxia-induced HIF-1α accumulation and HIF-1α transactivation and that proteasome inhibitors induce accumulation and decrease transcriptional activity of HIF-1α. These findings underline the importance of developing a battery of robust assay platforms and confirmation studies that focus on endogenous protein targets so that only relevant and reliable data will be taken into pre-clinical and clinical studies.

Tumour cell responses to MEK1/2 inhibitors: acquired resistance and pathway remodelling.

The Raf/MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2]/ERK1/2 signalling pathway is frequently activated in human tumours due to mutations in BRAF or KRAS. B-Raf and MEK1/2 inhibitors are currently undergoing clinical evaluation, but their ultimate success is likely to be limited by acquired drug resistance. We have used colorectal cancer cell lines harbouring mutations in B-Raf or K-Ras to model acquired resistance to the MEK1/2 inhibitor selumetinib (AZD6244). Selumetinib-resistant cells were refractory to other MEK1/2 inhibitors in cell proliferation assays and exhibited a marked increase in MEK1/2 and ERK1/2 activity and cyclin D1 abundance when assessed in the absence of inhibitor. This was driven by a common mechanism in which resistant cells exhibited an intrachromosomal amplification of their respective driving oncogene, B-Raf V600E or K-RasG13D. Despite the increased signal flux from Raf to MEK1/2, resistant cells maintained in drug actually exhibited the same level of ERK1/2 activity as parental cells, indicating that the pathway is remodelled by feedback controls to reinstate the normal level of ERK1/2 signalling that is required and sufficient to maintain proliferation in these cells. These results provide important new insights into how tumour cells adapt to new therapeutics and highlight the importance of homoeostatic control mechanisms in the Raf/MEK1/2/ERK1/2 signalling cascade.

A correction to the research article titled: "Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells" by A. S. Little, K. Balmanno, M. J. Sale, S. Newman, J. R. Dry, M. Hampson, P. A. W. Edwards, P. D. Smith, S. J. Cook.

The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal­regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal­regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF600E or KRAS13D, due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF600E. Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF600E or KRAS13D) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS13D leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.

Amplification of the driving oncogene, KRAS or BRAF, underpins acquired resistance to MEK1/2 inhibitors in colorectal cancer cells.

The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF(600E) or KRAS(13D), due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF(600E). Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF(600E) or KRAS(13D)) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS(13D) leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.