Dynamic telomerase gene suppression via network effects of GSK3 inhibition

<b>Background</b>: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. <b>Methodology/Principal Findings</b>: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3′-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFκB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. <b>Conclusions/Significance</b>: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting

LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

Comparative analysis of the transcriptome across distant species

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Development of a High-Throughput Screening Method for LIM Kinase 1 Using a Luciferase-Based Assay of ATP Consumption

Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)–binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases

Evaluating and comparing immunostaining and computational methods for spatial profiling of drug response in patient-derived explants

Patient-derived explants (PDEs) represent the direct culture of fragments of freshly-resected tumour tissue under conditions that retain the original architecture of the tumour. PDEs have advantages over other preclinical cancer models as platforms for predicting patient-relevant drug responses in that they preserve the tumour microenvironment and tumour heterogeneity. At endpoint, PDEs may either be processed for generation of histological sections or homogenised and processed for ʻomic’ evaluation of biomarker expression. A significant advantage of spatial profiling is the ability to co-register drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. Spatial profiling of PDEs relies on the utilisation of robust immunostaining approaches for validated biomarkers and incorporation of appropriate image analysis methods to quantitatively and qualitatively monitor changes in biomarker expression in response to anti-cancer drugs. Automation of immunostaining and image analysis would provide a significant advantage for the drug discovery pipeline and therefore, here, we have sought to optimise digital pathology approaches. We compare three image analysis software platforms (QuPath, ImmunoRatio and VisioPharm) for evaluating Ki67 as a marker for proliferation, cleaved PARP (cPARP) as a marker for apoptosis and pan-cytokeratin (CK) as a marker for tumour areas and find that all three generate comparable data to the views of a histomorphometrist. We also show that Virtual Double Staining of sequential sections by immunohistochemistry results in imperfect section alignment such that CK-stained tumour areas are over-estimated. Finally, we demonstrate that multi-immunofluorescence combined with digital image analysis is a superior method for monitoring multiple biomarkers simultaneously in tumour and stromal areas in PDEs