Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug–target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.

We read with interest the recent Gut study by Zuo and colleagues showing that a good outcome of faecal microbiota transplantation (FMT) in Clostridium difficile infection (CDI) was associated with bacteriophage transfer.1 FMT is an established treatment to restore a balance in disturbed intestinal microbiotas of patients with CDI,2 3 and recent multicentre randomised, double-blind, placebo-controlled trials showed its effectiveness in inducing clinical and endoscopic remission in patients with UC.4 We examined the gut virome dynamics of nine patients with UC who were undergoing FMT (64 patient and 8 healthy donor samples).5 Two patients with UC achieved long-lasting complete remission with mucosal healing (>2 years; figure 1: patient 1 and 3), and one patient reported temporary remission for 6 weeks (figure 1; patient 4).5 The remaining six patients did not respond to treatment. Using the NetoVIR protocol,6 we analysed the faecal virome (both RNA/DNA genomes of phages and eukaryotic viruses) using tailored bioinformatics approaches (see online supplementary data). Irrespective of their outcome, no statistical differences were observed in phageome richness between patients with UC and healthy donors (figure 2A) at any time point, in contrast …

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Glycoprotein acetylation [GlycA] is a novel nuclear magnetic resonance [NMR] biomarker, measured in serum or plasma, that summarizes the signals originating from glycan groups of certain acute-phase glycoproteins. This biomarker has been shown to be robustly associated with cardiovascular and short-term all-cause mortality, and with disease severity in several inflammatory conditions. We investigated GlycA levels in a cohort of healthy individuals [HCs], patients with Crohn’s disease [CD] and patients with ulcerative colitis [UC] prior to and after therapeutic control of inflammation. Serum samples of 10 HCs, 37 CD patients and 21 UC patients before and after biologic therapy were subjected to high-throughput NMR analysis by Nightingale Health Ltd. Paired C-reactive protein [CRP] and fecal calprotectin [fCal] measurements were used to characterize baseline differences, treatment effects and post-treatment association with endoscopic response [50% SES-CD decrease at Week 24] and mucosal healing [SES-CD ≤ 2 for CD, Mayo endoscopic score ≤ 1 for UC]. GlycA levels were significantly higher in patients with active inflammamtory bowel disease [IBD] compared with those in healthy controls, and accurately reflected the mucosal recovery to a ‘healthy’ state in both CD and UC patients achieving mucosal healing. In CD patients who experienced an endoscopic response without achieving full mucosal healing, GlycA levels also decreased but did not normalize to HC levels. Overall, GlycA correlated well with CRP and fCal, and accurately tracked disease activity in CRP-negative patients [<5 mg/dL]. GlycA holds promise as a viable serological biomarker for disease activity in IBD, even in patients without elevated CRP, and should therefore be tested in large prospective cohorts.

Retinoic acid‐related drugs have shown promising pre‐clinical activity in Adult T‐cell Leukemia/Lymphoma, but RORC signaling has not been explored. Therefore, we investigated transcriptome‐wide interactions of the RORC pathway in HTLV‐1 and ATL, using our own and publicly available gene expression data for ATL and other leukemias. Gene expression data from ATL patients were analyzed using WGCNA to determine gene modules and their correlation to clinical and molecular data. Both PBMCs and CD4⁺ T‐Cells exhibited decreased RORC expression in four different ATL cohorts. A small subset of RORC^hi ATL patients was identified with significantly lower pathognomonic CADM1 and HBZ levels but similar levels of other ATL markers (CD4/CD25/CCR4), hinting at a less aggressive ATL subtype. An age‐dependent decrease in RORC expression was found in HTLV‐1‐infected individuals, but not in healthy controls, suggesting an early molecular event predisposing to leukemogenesis. Genes upstream of RORC signaling were members of a proliferative gene module (containing proliferation markers PCNA/Ki67), whereas downstream members clustered in an anti‐proliferative gene module. IL17C transcripts showed the strongest negative correlation to PCNA in both ATL cohorts, which was replicated in two large cohorts of T‐ and B‐cell acute lymphoid leukemia (ALL). Finally, IL17C expression in purified CD4 + CCR4 + CD26‐CD7‐ “ATL‐like” cells from HTLV‐1‐infected individuals and ATL patients was negatively correlated with clonality, underscoring a possible antileukemic/antiproliferative role. In conclusion, decreased RORC expression and downstream signaling might represent an early event in ATL pathogenesis. An antiproliferative IL17C/PCNA link is shared between ATL, T‐ALL and B‐ALL, suggesting (immuno)therapeutic benefit of boosting RORC/IL17 signaling.