Zhang, Cheng

Abstract Background Acamprosate is an FDA-approved medication for the treatment of alcohol use disorder (AUD), however, only about 50% of patients will respond to acamprosate. Previously, we conducted an acamprosate trial (n=442), which is one of the largest acamprosate clinical studies ever conducted and currently the only study with multi-omics data. To date, no biological measures are utilized to predict response to acamprosate treatment. Aims & Objectives We set out to apply our established pharmaco-omics informed genomics strategy to identify potential biomarkers associated with acamprosate treatment response. Method Our open-label acamprosate clinical trial recruited 442 patients with AUD, all of whom were treated with acamprosate for three months. The primary outcomes were 1) relapse to alcohol use and 2) relapse to heavy drinking. Alcohol consumption was measured using the self-reported timeline follow back method. Plasma protein levels were measured using Olink proximity extension immunoassays. Results Our established “Pharmaco-Omics-informed genome-wide association study (GWAS)” research strategy identified 12 proteins, including interleukin-17 receptor B (IL17RB), that were associated with acamprosate treatment response. A GWAS for IL17RB concentrations identified several genome-wide significant signals. Specifically, the top hit single nucleotide polymorphism (SNP) rs6801605 with a minor allele frequency of 38% in the European American population mapped 4 kilobase (Kb) upstream of IL17RB, and intron 1 of the choline dehydrogenase (CHDH) gene on chromosome 3 (p: 4.8E-20). The variant genotype (AA) for the SNP rs6801605 was associated with lower IL17RB protein expression. In addition, we identified a series of genetic variants in IL17RB that were associated with acamprosate treatment outcomes. Furthermore, the variant genotypes for all of those IL17RB SNPs were protective against alcohol relapse. Finally, we demonstrated that the basal level of mRNA expression of IL17RB was inversely correlated with those of nuclear factor-κB (NF-κB) subunits, and a significantly higher expression of NF-κB subunits was observed in AUD patients who relapsed to alcohol use. Discussion & Conclusions This study illustrates that IL17RB genetic variants might contribute to acamprosate treatment outcomes. This series of studies represents an important step toward generating functional hypotheses that could be tested to gain insight into mechanisms underlying variation in acamprosate treatment response phenotypes.

Alcohol use disorder (AUD) is the most prevalent substance use disorder. Excessive alcohol consumption leads to a range of health issues. We set out to identify inflammatory markers linked to alcohol consumption, which might ultimately offer novel insight into genetic underpinnings and have implications for alcohol-associated disease. Alcohol consumption and blood-based multi-omics data were collected by The Mayo Clinic Center for Individualized Treatment of Alcohol Dependence study. Plasma samples from patients with AUD were used for proteomics analysis using the OLINK “Explore Inflammation” panel (n=410). Liver enzymes were also measured. A genome-wide association study (GWAS) was performed to explore the relationship between genetic variants and plasma TREM2 levels. Our findings show thatplasma triggering receptor expressed on myeloid cells 2 (TREM2), a key gene associated with neurodegenerative disease, was the most significant signal correlated with alcohol consumption, and has also been associated with liver enzyme levels in patients with AUD. We identified the rs7232 single nucleotide polymorphism (SNP) in MS4A6A as a key genetic variant associated with plasma TREM2 levels, with the minor allele (A) linked to higher TREM2 levels and increased alcohol consumption, particularly in men. Furthermore, MA4A6A is an ethanol-responsive gene in a SNP-dependent manner, and the variant genotype of the rs7232 SNP was associated with lower expression for MA4A6A due to proteasome-mediated protein degradation. In summary, this study provides insight into the relationship between plasma TREM2 levels, alcohol consumption, and liver function in AUD patients, shedding light on genetic factors underlying alcohol-related diseases.

ABSTRACT His domain protein tyrosine phosphatase (HD-PTP; also known as PTPN23) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, phosphoinositide 3-kinase (PI3K)/AKT and receptor tyrosine kinases (RTKs), such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. The ESCRT components Vps4 and Hrs have previously been implicated in cholesterol homeostasis; thus, these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with a dismal 5-year survival rate, highlighting the urgent need to improve drug response and overcome primary or acquired (secondary) resistance to conventional chemotherapy. Glycogen synthase kinase 3β (GSK-3β) is a target in PDAC and the GSK-3 inhibitor, elraglusib, has been shown to improve chemosensitivity of PDAC and survival in preclinical cancer models. In addition, data from phase II clinical trials in patients with previously untreated metastatic pancreatic cancer is demonstrating preliminary evidence of clinical benefit in patients receiving a combination of gemcitabine/nab-paclitaxel. While this early evidence is highly encouraging, the biological determinants of elraglusib response are unknown. Using genome wide CRISPR/Cas9 library screening, we identify aurora kinase A (AURKA), an oncogenic kinase with major roles in mitosis, as a critical regulator for elraglusib sensitivity. Elraglusib treatment activates AURKA by inducing a G2/M cell cycle arrest. We show that knocking out AURKA reduces the phosphorylation of polo like kinase 1 (PLK1), arresting cells in mitosis, and leading to mitotic catastrophe and significant apoptosis after elraglusib treatment. Strikingly, the AURKA inhibitor, alisertib, works synergistically with elraglusib to inhibit PDAC cell line growth and induce apoptosis. In summary, our results demonstrate that targeting AURKA is an effective approach to sensitize PDAC tumors to elraglusib. Citation Format: Li Ding, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Daniel Schmitt, Andrew Mazar, Hu Li, Daniel Billadeau. Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4658.

Background: Cellular senescence has been associated with neurodegenerative disease and clearance of senescent cells using genetic or pharmaceutical strategies (senolytics) has demonstrated beneficial effects in mouse models investigating individual disease etiologies of Alzheimer’s disease (AD). However, it has remained unclear if senescent cell clearance in a mouse model exhibiting both plaque and tau pathologies modifies the disease state (3xTg). Objective: To investigate the effects of senescent cell clearance in the 3xTg mouse model. Methods: 3xTg mice were treated with senolytics (ABT263 (navitoclax; NAVI), a combination of dasatinib and quercetin (D+Q)), or subjected to transgene-mediated removal of p16-expressing cells (via INK-ATTAC). Results: Senolytic treatments consistently reduced microgliosis and ameliorated both amyloid and tau pathology in 3xTg mice. Using RNA sequencing, we found evidence that synaptic dysfunction and neuroinflammation were attenuated with treatment. These beneficial effects were not observed with short-term senolytic treatment in mice with more advanced disease. Conclusions: Overall, our results further corroborate the beneficial effects senescent cell clearance could have on AD and highlight the importance of early intervention for the treatment of this debilitating disease.

Background: The existence and contribution of microglia with senescent-like alterations in the pathogenesis of age-related neurodegenerative diseases like Alzheimer’s disease (AD) have been suggested in recent years. However, the identification of this distinct microglial population in vivo has proven challenging, largely due to overlaps in the inflammatory phenotype of activated and senescent microglia. Furthermore, attempts at recapitulating senescence in microglia in vitro are limited. Objective: To identify and characterize senescent microglia that occur in vivo in an animal model of neurodegeneration driven by pathologic tau. Methods: We analyzed the RNA expression patterns of individual microglia from normal mice and the pathogenic tau P301 S PS19 mouse model. We have previously demonstrated that p16-expressing senescent microglia occur in these mice when neurodegeneration has occurred. Results: Here we identify a subset of disease-associated microglia with senescent features, notably characterized by the expression of Ccl4 . This signature overlaps with established markers of senescence from other cell types. Conclusion: Our characterization of senescent microglia can be used to better understand the role of senescent microglia in various age-related contexts, including whether clearance of senescent microglia represents a viable therapeutic option.

Abstract Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent “vulnerable spots” within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

Abstract Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were ‘unmasked’ only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

BACKGROUND A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS We report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures. CONCLUSION While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION Not applicable. FUNDING The Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).