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Purpose: All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (G alpha(q)) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of G alpha(q) have shown promising preclinical results, but their therapeutic activity in distinct G alpha(q) mutational contexts and in vivo have remained elusive. Experimental Design: We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of G alpha(q) inhibition as a single agent and in combination with MEK inhibition. Results: We demonstrate that the G alpha(q) inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. Conclusions: These data suggest that the combination of G alpha(q) and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting G alpha(q) in uveal melanoma. See related commentary by Neelature Sriramareddy and Smalley, p. 1217

Background Blockade of tyrosine kinase 2 (TYK2) signalling has previously shown therapeutic potential in the treatment of psoriasis. The primary objective of this study was to assess the safety and tolerability of the TYK2 inhibitor PF-06826647. Methods This phase 1, randomised, double-blind, placebo-controlled, parallel-group study assessed once daily oral dosing of PF-06826647 in participants with plaque psoriasis, at a single clinical research site in the USA. Eligible participants (aged 18-65 years) had plaque psoriasis covering at least 15% of total body surface area and a psoriasis area and severity index (PASI) score of at least 12 at baseline. Participants received PF-06826647 (100 mg or 400 mg), or placebo once daily for 28 days. Using a computer-generated randomisation schedule with a block size of 3, participants were sequentially randomly assigned into two cohorts by the investigator; in the first cohort, participants were randomly assigned in a 2:1 ratio to receive either oral PF-06826647 400 mg or placebo once daily, whereas participants in the second cohort were randomly assigned in a 2:1 ratio to receive either oral PF-06826647 100 mg or placebo once daily. Site, investigator, Pfizer personnel, and participants, were masked to treatment. The primary endpoint was the safety of multiple-dose PF-06826647 in participants with plaque psoriasis. Secondary endpoints were the characterisation of the pharmacokinetics of multiple-dose PF-06826647 in plasma and the change in PASI score at day 28. Safety analysis was done in all participants who received at least one dose of study drug. Efficacy analysis was done in all participants who received at least one dose of randomised study drug, and had a baseline and at least one post-baseline measurement. This study is registered as a randomised, controlled trial with ClinicalTrials.gov, NCT03210961 and is completed. Findings The trial was done between July 14, 2017, and Jan 25, 2019. Overall from 91 participants assessed, 40 participants with moderate-to-severe psoriasis were randomly assigned to treatment (placebo 14 [35%] of 40; PF-06826647 100 mg, 11 [28%] of 40; PF-06826647 400 mg, 15 [38%] of 40). Treatment-emergent adverse events (TEAEs) were reported in 12 (80%) of 15 participants in the PF-06826647 400 mg group, seven (50%) of 14 in the placebo group and five (45%) of 11 in the 100 mg group. All TEAEs were mild in severity, except one moderate TEAE of vomiting reported in the placebo group. There were no deaths, serious TEAEs, severe TEAEs, dose reductions, or temporary discontinuations. Compared with placebo, the change from baseline in PASI score at day 28 showed a significant reduction in least squares mean difference for the PF-06826647 400 mg group (-13.05; 90% CI -18.76 to -7.35; p=0.00077) but not for the PF-06826647 100 mg group (-3.49; -9.48 to 2.50; p=0.33). Both the area under the concentration-time curve over the dosing interval and the maximum concentration increased in a less than dose proportional manner with increasing dose from 100 mg to 400 mg PF-06826647. Interpretation PF-06826647 showed significant improvement in disease activity within 4 weeks of dosing with an acceptable safety profile. PF-06826647 holds promise over conventional oral treatments for psoriasis that have shown limited efficacy or unfavourable safety profiles. Copyright (C) 2020 Elsevier Ltd. All rights reserved.

Background Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. Methods Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. Results Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. Conclusions Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Tuberculosis (TB), usually caused by Mycobacterium tuberculosis bacteria, is the first cause of death from an infectious disease at the worldwide scale, yet the mode and tempo of TB pressure on humans remain unknown. The recent discovery that homozygotes for the P1104A polymorphism of TYK2 are at higher risk to develop clinical forms of TB provided the first evidence of a common, monogenic predisposition to TB, offering a unique opportunity to inform on human co-evolution with a deadly pathogen. Here, we investigate the history of human exposure to TB by determining the evolutionary trajectory of the TYK2 P1104A variant in Europe, where TB is considered to be the deadliest documented infectious disease. Leveraging a large dataset of 1,013 ancient human genomes and using an approximate Bayesian computation approach, we find that the P1104A variant originated in the common ancestors of West Eurasians similar to 30,000 years ago. Furthermore, we show that, following large-scale population movements of Anatolian Neolithic farmers and Eurasian steppe herders into Europe, P1104A has markedly fluctuated in frequency over the last 10,000 years of European history, with a dramatic decrease in frequency after the Bronze Age. Our analyses indicate that such a frequency drop is attributable to strong negative selection starting similar to 2,000 years ago, with a relative fitness reduction on homozygotes of 20%, among the highest in the human genome. Together, our results provide genetic evidence that TB has imposed a heavy burden on European health over the last two millennia.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The "on-demand" induction of this machinery required gamma delta T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of gamma delta T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for gamma delta T cells in nutrient sensing.

Background Adalimumab, a tumor necrosis factor-alpha inhibitor, is a biologic used for the treatment of moderate-to-severe hidradenitis suppurativa (HS). It is well known that patients may experience loss of efficacy from its use in other conditions, and it is suggested that developing a strategy for therapeutic drug monitoring (TDM) may help secure optimal clinical outcomes. Objectives We sought to determine serum adalimumab concentrations and anti-adalimumab antibody (AAA) status in patients with moderate-to-severe HS. Methods A retrospective case series of 38 patients with suboptimal response to adalimumab 40 mg weekly was conducted at a community dermatology clinic. Adalimumab serum trough levels, AAA status, and inflammatory biomarkers were collected. Blood was drawn on identification of suboptimal response (after a minimum of 12 weeks) and was collected once prior to receiving the next scheduled dose. Kruskal-Wallis and Chi-squared tests were used for data analysis. Results A total of 38 patients had a median adalimumab trough concentration of 8.76 (interquartile range [IQR] 1.3-12.5) mu g/mL. The median duration of adalimumab therapy of all patients was 21 (IQR 12-24) months. AAAs were detected in nine patients (24%), and all had subtherapeutic serum concentrations (< 6 mu g/mL). Patients who were AAA+ had a significantly lower median adalimumab concentration than those who were AAA- (0.02 mu g/mL [range 0.02-0.81] vs. 10.14 [range 0.76-48.00]; p = 0.0006). Conclusion Patients with AAAs had significantly lower serum adalimumab levels. The current study suggests that TDM may identify underlying reasons for suboptimal response and detect patients who may benefit from dose optimization strategies.

The identification of discrete subclasses within the immunoglobulin G (IgG) isotype by Grey and Kunkel (1964. J. Exp. Med. https://doi.org/10.1084/jem.120.2.253) provided the framework for our current understanding of differential IgG subclass activity in protective and self-reactive immune responses.

Olivieri, Walker, Karamafrooz et al. show that the fusion of the dynamic J-domain to PKA-C (PKA-CDNAJB1) disrupts the internal allosteric network, attenuating the nucleotide/PKI binding cooperativity. This study suggests that the reduced allosteric cooperativity may contribute to the pathology that PKA-CDNAJB1 drives. An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-C-DNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic alpha subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-C-DNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-C-DNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.