Helicobacter pylori testing prior to or at gastric cancer diagnosis and survival in a diverse US patient population.

BACKGROUND: Gastric cancer (GC) accounts for the greatest disparity in cancer mortality between Black and White Americans. Although clinical trials have shown that Helicobacter pylori (Hp) treatment reduces risk of GC, Hp testing and treatment is not consistently performed in the US, and may offer an opportunity to improve survival. METHODS: In a diverse retrospective cohort of 99 GC cases diagnosed at Duke University from 2002-2020 (57% Black; 43% white), we examined the association of Hp testing and treatment prior to or at cancer diagnosis with overall survival using Cox regression analyses to calculate adjusted hazards ratios (HRs) and 95% confidence intervals (CIs). RESULTS: Overall, 62% of patients were tested for Hp prior to or at GC diagnosis. Of those, 25% tested positive and were treated < 1 year prior to or at diagnosis, 15% tested positive and were treated ≥ 1 year prior to diagnosis, 6% tested positive without evidence of treatment, and 54% tested negative. Compared to never tested, Hp testing and treatment < 1 year prior to or at diagnosis was associated with a significantly reduced likelihood of death (HR 0.21, 95% CI 0.08-0.58). The benefit of any Hp test and treat prior to or at GC diagnosis was significant even among stage IV patients only (HR, 0.22; 95% CI 0.05-0.96). CONCLUSIONS: These findings support Hp testing and treatment for patients at risk of or diagnosed with GC, and suggest Hp treatment may provide an opportunity to reduce GC mortality disparities in the US.

Type 2 Diabetes Mellitus and Helicobacter pylori Eradication in a Clinical Population.

OBJECTIVES: Eradication of Helicobacter pylori reduces the risk of gastric cancer (GC). Individuals with type 2 diabetes mellitus (T2DM) are known to be at increased risk for GC. In a cohort of H. pylori-positive individuals, we assessed whether those with T2DM were at risk of persistent infection following H. pylori treatment compared with individuals without T2DM. METHODS: A random subset of all individuals diagnosed as having H. pylori without intestinal metaplasia at endoscopy from 2015 to 2019 were stratified evenly by race (Black and White). After excluding those with T1DM and those without eradication testing after H. pylori treatment, logistic regression analysis was used to determine the association of T2DM with the risk of persistent H. pylori infection following treatment. RESULTS: In 138 patients, H. pylori eradication rates did not differ between the 27% of individuals with T2DM compared to those without (81.1% vs 81.2%). After adjusting for age, race, and insurance status, we found no significant increased risk of persistent H. pylori infection for individuals with T2DM (odds ratio 1.40; 95% confidence interval 0.49-3.99). CONCLUSIONS: H. pylori eradication rates do not differ by T2DM status, providing support for clinical trials of H. pylori eradication to reduce GC incidence among high-risk populations in the United States, such as individuals with T2DM.

Creation of an asynchronous faculty development curriculum on well-written narrative assessments that avoid bias.

BACKGROUND: The COVID-19 pandemic in parallel with concerns about bias in grading resulted in many medical schools adopting pass/fail clinical grading and relying solely on narrative assessments. However, narratives often contain bias and lack specificity. The purpose of this project was to develop asynchronous faculty development to rapidly educate/re-educate > 2000 clinical faculty spread across geographic sites and clinical disciplines on components of a well-written narrative and methods to minimize bias in the assessment of students. METHODS: We describe creation, implementation, and pilot data outcomes for an asynchronous faculty development curriculum created by a committee of volunteer learners and faculty. After reviewing the literature on the presence and impact of bias in clinical rotations and ways to mitigate bias in written narrative assessments, the committee developed a web-based curriculum using multimedia learning theory and principles of adult learning. Just-in-time supplemental materials accompanied the curriculum. The Dean added completion of the module by 90% of clinical faculty to the department chairperson's annual education metric. Module completion was tracked in a learning management system, including time spent in the module and the answer to a single text entry question about intended changes in behavior. Thematic analysis of the text entry question with grounded theory and inductive processing was used to define themes of how faculty anticipate future teaching and assessment as a result of this curricula. OUTCOMES: Between January 1, 2021, and December 1, 2021, 2166 individuals completed the online module; 1820 spent between 5 and 90 min on the module, with a median time of 17 min and an average time of 20.2 min. 15/16 clinical departments achieved completion by 90% or more faculty. Major themes included: changing the wording of future narratives, changing content in future narratives, and focusing on efforts to change how faculty teach and lead teams, including efforts to minimize bias. CONCLUSIONS: We developed a faculty development curriculum on mitigating bias in written narratives with high rates of faculty participation. Inclusion of this module as part of the chair's education performance metric likely impacted participation. Nevertheless, time spent in the module suggests that faculty engaged with the material. Other institutions could easily adapt this curriculum with provided materials.

C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways.

Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated G αi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G αi or G αs activity. ß-arrestin (ßarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in ßarr2 conformation. ßarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by ßarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of ßarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.

Prevalence of the cagA Virulence Factor Varies by Race Among Helicobacter pylori -Infected Patients Undergoing Upper Endoscopy.

INTRODUCTION: We designed a race-conscious study to assess the presence of Helicobacter pylori v irulence factor cagA in a retrospective cohort of patients with active H. pylori infection. METHODS: We compared cagA status by race in gastric tissue samples from 473 patients diagnosed with active H. pylori infection from 2015 to 2019. RESULTS: H. pylori + Black patients were 2 times more likely to be cagA + than H. pylori + White patients (82% vs 36%, P < .0001). DISCUSSION: Presence of cagA is common among endoscopy patients with active H. pylori infection; appropriate testing and treatment of H. pylori can both reduce gastric cancer risk and address health disparities.

Cancer Progress and Priorities: Gastric Cancer.

Gastric cancer, the fifth leading cause of cancer worldwide, is estimated to be responsible for approximately 1.4% of all new cancers and 1.8% of all cancer-related deaths in the United States. Despite declining incidence rates and improved survival rates, however, gastric cancer continues to disproportionately affect racial and ethnic minorities and individuals of lower socioeconomic status at higher rates than the general population. To improve outcomes globally and address disparities within the United States, continued improvements are needed in risk factor modification and biomarker development and to improve access to existing preventative measures such as genetic testing and H. pylori eradication testing, in addition to expanding upon current clinical guidelines for premalignant disease to address gaps in endoscopic surveillance and early detection.

Phosphorylation barcodes direct biased chemokine signaling at CXCR3.

G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or "phosphorylation barcodes." At chemokine receptors, ligands act as "biased agonists" with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered ß-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes.

Phosphorylation barcodes direct biased chemokine signaling at CXCR3.

G protein-coupled receptor (GPCR)-biased agonism, selective activation of certain signaling pathways relative to others, is thought to be directed by differential GPCR phosphorylation "barcodes." At chemokine receptors, endogenous chemokines can act as "biased agonists", which may contribute to the limited success when pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomics studies. Mutation of CXCR3 phosphosites altered ß-arrestin 2 conformation in cellular assays and was consistent with conformational changes observed in molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes, leading to distinct physiological processes.

Bioactivation of Napabucasin Triggers Reactive Oxygen Species-Mediated Cancer Cell Death.

PURPOSE: Napabucasin (2-acetylfuro-1,4-naphthoquinone or BBI-608) is a small molecule currently being clinically evaluated in various cancer types. It has mostly been recognized for its ability to inhibit STAT3 signaling. However, based on its chemical structure, we hypothesized that napabucasin is a substrate for intracellular oxidoreductases and therefore may exert its anticancer effect through redox cycling, resulting in reactive oxygen species (ROS) production and cell death. EXPERIMENTAL DESIGN: Binding of napabucasin to NAD(P)H:quinone oxidoreductase-1 (NQO1), and other oxidoreductases, was measured. Pancreatic cancer cell lines were treated with napabucasin, and cell survival, ROS generation, DNA damage, transcriptomic changes, and alterations in STAT3 activation were assayed in vitro and in vivo. Genetic knockout or pharmacologic inhibition with dicoumarol was used to evaluate the dependency on NQO1. RESULTS: Napabucasin was found to bind with high affinity to NQO1 and to a lesser degree to cytochrome P450 oxidoreductase (POR). Treatment resulted in marked induction of ROS and DNA damage with an NQO1- and ROS-dependent decrease in STAT3 phosphorylation. Differential cytotoxic effects were observed, where NQO1-expressing cells generating cytotoxic levels of ROS at low napabucasin concentrations were more sensitive. Cells with low or no baseline NQO1 expression also produced ROS in response to napabucasin, albeit to a lesser extent, through the one-electron reductase POR. CONCLUSIONS: Napabucasin is bioactivated by NQO1, and to a lesser degree by POR, resulting in futile redox cycling and ROS generation. The increased ROS levels result in DNA damage and multiple intracellular changes, one of which is a reduction in STAT3 phosphorylation.

Oncogenic KRAS Induces NIX-Mediated Mitophagy to Promote Pancreatic Cancer.

Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.

Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation.

The chemokine receptor CXCR3 plays a central role in inflammation by mediating effector/memory T cell migration in various diseases; however, drugs targeting CXCR3 and other chemokine receptors are largely ineffective in treating inflammation. Chemokines, the endogenous peptide ligands of chemokine receptors, can exhibit so-called biased agonism by selectively activating either G protein- or ß-arrestin-mediated signaling after receptor binding. Biased agonists might be used as more targeted therapeutics to differentially regulate physiological responses, such as immune cell migration. To test whether CXCR3-mediated physiological responses could be segregated by G protein- and ß-arrestin-mediated signaling, we identified and characterized small-molecule biased agonists of the receptor. In a mouse model of T cell-mediated allergic contact hypersensitivity (CHS), topical application of a ß-arrestin-biased, but not a G protein-biased, agonist potentiated inflammation. T cell recruitment was increased by the ß-arrestin-biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and ß-arrestin in T cells. In mouse and human T cells, the ß-arrestin-biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that ß-arrestin-biased signaling activated the kinase Akt, which promoted T cell migration. This study demonstrates that biased agonists of CXCR3 produce distinct physiological effects, suggesting discrete roles for different endogenous CXCR3 ligands and providing evidence that biased signaling can affect the clinical utility of drugs targeting CXCR3 and other chemokine receptors.

A Practical Guide to Approaching Biased Agonism at G Protein Coupled Receptors.

Biased agonism, the ability of a receptor to differentially activate downstream signaling pathways depending on binding of a "biased" agonist compared to a "balanced" agonist, is a well-established paradigm for G protein-coupled receptor (GPCR) signaling. Biased agonists have the promise to act as smarter drugs by specifically targeting pathogenic or therapeutic signaling pathways while avoiding others that could lead to side effects. A number of biased agonists targeting a wide array of GPCRs have been described, primarily based on their signaling in pharmacological assays. However, with the promise of biased agonists as novel therapeutics, comes the peril of not fully characterizing and understanding the activities of these compounds. Indeed, it is likely that some of the compounds that have been described as biased, may not be if quantitative approaches for bias assessment are used. Moreover, cell specific effects can result in "system bias" that cannot be accounted by current approaches for quantifying ligand bias. Other confounding includes kinetic effects which can alter apparent bias and differential propagation of biological signal that results in different levels of amplification of reporters downstream of the same effector. Moreover, the effects of biased agonists frequently cannot be predicted from their pharmacological profiles, and must be tested in the vivo physiological context. Thus, the development of biased agonists as drugs requires a detailed pharmacological characterization, involving both qualitative and quantitative approaches, and a detailed physiological characterization. With this understanding, we stand on the edge of a new era of smarter drugs that target GPCRs.