Mayo Clinic Rochester

NIH Research Projects · FY 2026 · 2021-12

Project Summary The long-term goal of our research is to develop first-in-class, protein-based inhibitors against human bacterial pathogens by directly blocking efflux pumps. Drug resistant bacteria pose an urgent global health challenge by reducing the effectiveness of antibiotics used to treat infections in humans and animals. The broadest resistance mechanism against antibiotics are efflux pumps, which transport drugs out of the cytoplasm and reduce toxicity to the organism. While it is known that efflux pumps display broad specificity to structurally distinct compounds, the mechanisms of polyspecific drug binding and ion-coupled transport remain unanswered questions in the field. Given the promiscuity of efflux pump binding to structurally distinct drugs, it is also unclear whether potent and selective efflux pump inhibitors can be designed to target specific classes of efflux pumps. The specific goals of this project are to discover novel mechanisms of active transport in drug resistant Staphylococcus aureus and to harness this knowledge to design selective inhibitors toward efflux pumps. Our proposal is strongly motivated by our recent discovery of antibody fragments (Fabs) that bind the Staphylococcus aureus efflux pump NorA and successful determination of high-resolution cryoEM structures using the Fabs as fiduciaries. The structures revealed that the Fabs insert a loop into the substrate binding pocket from the extracellular side, which suggests a design path toward protein- and peptide-based inhibitors. This interaction is facilitated by an electrostatic interaction between a positively charged arginine on the Fab and two essential anionic residues within NorA. Building on these preliminary data, we propose to carry out four Specific Aims. Aim 1 will develop a hybrid approach of cryo-electron microscopy and NMR spectroscopy to comprehensively study the transport cycle of NorA. Aim 2 will seek to determine the molecular basis for polyspecific drug binding. Aim 3 will design and characterize protein-based inhibitors that target the accessible, outward-open conformation of NorA. Aim 4 will develop peptides that miniaturize the antibody loops observed in the binding pocket of NorA. We have assembled an interdisciplinary team with expertise in structural biology, protein engineering, microbiology, chemical synthesis, and computational chemistry to rapidly answer fundamental questions about multidrug transport and inhibition of efflux pumps. All of the approaches applied to NorA will be translatable to other transporter systems.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT The long-term objective of this application is to define the molecular and cellular mechanisms that drive the pathogenesis of nonalcoholic steatohepatitis (NASH), with the goal of identifying therapeutic strategies. NASH is a progressive form of nonalcoholic fatty liver disease characterized by sublethal hepatocyte lipotoxicity (i.e., toxic lipid-induced cellular stress, which does not induce cell death) and consequent liver tissue inflammation. The overall objective of this proposal is to examine: (a) how sublethal lipotoxicity in hepatocytes triggers the release of extracellular vesicles (EVs) to augment liver immune infiltration by pathogenic CD4+ T helper cells; and (b) how these abnormal processes during NASH can be therapeutically reversed. To this end, we have made several pivotal observations. First, we found that sublethal lipotoxicity in hepatocytes activates Rho- associated protein kinase (ROCK) 1 by a caspase-6-dependent mechanism. Moreover, genetic ablation of ROCK1 specifically in hepatocytes attenuates NASH-associated liver injury and fibrosis in mice. Mechanistically, ROCK1 is critical for the release of lipotoxicity-induced EVs and their enrichment in activated leukocyte cell adhesion molecule (ALCAM), a ligand for the CD6 receptor expressed by CD4+ T cells. Consistent with ALCAM enrichment, hepatocyte-derived lipotoxic EVs significantly enhance migration of CD4+ T helper cells in an ALCAM-dependent manner. Finally, activated CD4+ T cells are enriched in NASH livers, and their depletion attenuates murine NASH. Based on these preliminary observations, we propose the CENTRAL HYPOTHESIS that sublethal lipotoxic signals induce ROCK1 activation in hepatocytes, which results in the release of pathogenic EVs that promote CD4+ T cell-mediated hepatic inflammation during NASH. We will now employ in vitro experimental approaches, animal models, and human biospecimens to examine the HYPOTHESIS in three integrated SPECIFIC AIMS. First, we will test the hypothesis that sublethal lipotoxicity activates ROCK1 through: a) an incomplete mitochondrial outer membrane permeabilization; and b) non-lethal proteolytic activity of caspase-6. Second, we will investigate how sublethal lipotoxicity in hepatocytes results in: a) the release of ALCAM-bearing EVs; and b) the stimulation of CD6 receptor expressed on CD4+ T helper cells, leading to their recruitment into the liver. Third, we will test the hypothesis that in a murine model of NASH: a) CD6 inhibition attenuates NASH by preventing CD4+ T cell infiltration of the liver; and b) specific ROCK1 inhibition reverses NASH progression. Finally, the human disease relevance of our findings from the preclinical studies will be assessed using well-annotated human liver tissue specimens. This technically and conceptually innovative application is also significant because it provides mechanistic insights into hepatocyte-to-CD4+ T helper cell crosstalk in NASH, thus identifying viable therapeutic interventions, such as inhibition of ROCK1 kinase activity or CD4+ T cell-associated CD6 receptor.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Similar to other complications of diabetes mellitus (DM), diabetic gastroenteropathy (DGE) reflects organ- specific dysfunctions likely due to DM-related endocrine, metabolic and immune/inflammatory disturbances. Therapies for DGE are ineffective and address the symptoms, not the disease itself. Hence, and aligned with the mission of RFA-DK-20-030, this proposal seeks to uncover the cellular and molecular mechanisms of DGp and its relative, non-ulcer dyspepsia, in carefully phenotyped patients. We focus on the links between dysimmunity, ICC, glycemia, and mitochondrial dysfunction. The specific aims are as follows: Aim 1. To analyze the relationship between the transcriptome and epigenome of single gastric immune cells, gastric emptying (GE), and glycemia in DM and non-DM patients with upper GI symptoms. Aim 2. To determine the relationship between the transcriptome and epigenome of single circulating PBMCs and single gastric immune cells in DM and non-DM patients with upper GI symptoms. Aim 3. To investigate the relationship between the transcriptome and epigenome of single gastric ICC and immune cells and single circulating PBMCs in DM and non-DM patients with upper GI symptoms. These studies will be investigated with in vivo (i.e., assessment of the DM phenotype, gastrointestinal symptoms, and GE) followed by ex vivo assessments (i.e., transcriptome and epigenome of single circulating PBMCs, single gastric CD45+ cells, and single ICC) in 45 patients undergoing sleeve gastrectomy (i.e., 15 patients in each of 3 groups: non-DM + normal GE, DM + normal GE, DM + delayed GE). This proposal is anchored by, and builds on, an established bench-to-bedside collaboration between Drs. Bharucha and Ordog which, through their complementary expertise, has enhanced our understanding of diabetic gastroparesis. It seeks to better understand the pathogenesis, and through the studies in peripheral immune cells also identify novel biomarkers, of DGE.

NIH Research Projects · FY 2025 · 2021-09

Abstract Multiple myeloma (MM) is an incurable, plasma cell (PC) malignancy that progresses from the precursor conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Although MGUS and SMM PCs exhibit similar oncogenic mutations to MM PCs, they are considered benign and it is unclear what drives tumorigenesis of these pre-malignant PCs. Aging is the primary risk factor for cancers, including MM; it is therefore unsurprising that aging and tumorigenesis exhibit shared mechanisms. One of these shared mechanisms is cellular senescence. Cellular senescence is induced in response to cellular stress, such as oncogenic mutations, and activates growth arrest to prevent tumor formation. However, the accumulation of senescent cells with age contributes to aging pathologies, including cancer. Although deletion or inhibition of genes necessary for senescence induction leads to tumor formation, elimination of senescent cells reduces tumor incidence in aged mice. This suggests that senescent cell accumulation may create a pro- tumorigenic microenvironment; alternatively, pre-tumor cells may themselves be senescent and targeted by senescent cell elimination strategies. In support of this, we found that both MGUS and SMM PCs have enrichment of cellular senescence genes. In addition, SMM PCs show characteristics of late-senescence, including an interferon senescence-associated secretory phenotype (SASP) and the accumulation of cytosolic ssDNA and DNA:RNA hybrids that are associated with increased genomic instability. These findings support a role for PC senescence in monoclonal gammopathies, and suggest a potential mechanism that may ultimately drive tumorigenesis. Importantly, clonal PCs have also been shown to induce senescence in the bone marrow microenvironment (BMME), suggesting additional mechanisms by which senescence may promote a permissive niche for MM tumorigenesis. Thus, we hypothesize that senescence within PCs and their proximate BMME drives progression of MGUS/SMM to MM. In this application, we propose to use genetic mouse models and human patient-derived cells and bone biopsies to ascertain the mechanisms by which senescence drives tumorigenesis through both direct effects on pre-tumor cells and on the BMME. The results of these studies will define a role for senescence, a common aging mechanism, in tumorigenesis. We anticipate that these findings can be rapidly translated to clinical trials targeting the progression of monoclonal gammopathies to MM.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Alzheimer’s disease (AD) is a heterogeneous neurodegenerative disorder characterized by the abnormal accumulation of amyloid-β plaques and neurofibrillary tangles (NFT) composed of tau. Important preliminary data has shown that tau PET with quantitative analysis can distinguish Braak III vs IV NFT pathology. However, there is a gap in knowledge and only limited numbers of participants have been evaluated with early Braak NFT stages (I-IV). We will leverage our database of 1779 participants who have had tau PET over the past 6 years who provide a well characterized cohort for this study at substantial cost savings. Our hypotheses are that novel methods of assessing tau PET will be associated with early Braak NFT stages (I-IV) and cognitive test abnormalities. We will test our hypotheses in three aims: Aim 1: Characterize tau PET measurements that are associated with early NFT. We will use innovative tau PET analysis methods to predict early Braak NFT stage in a large sample of early Braak NFT stage participants. We will use a novel method of quantitative tau PET assessment to improve on prior methodologies and test tau PET for association with early Braak NFT stages. Aim 2: Characterize tau PET features associated with cognitive test findings. We will use tau PET to predict cognitive test findings in CU participants. Cognitive test findings correlate best with tau PET in this regard vs MRI or amyloid PET and tau PET could be an optimal therapeutic biomarker. We will use innovative tau PET imaging analysis methods to predict early cognitive test findings in a large sample of CU. Aim 3: Identify associations of different tau PET radiotracers with early neuropathologic tau hyperphosphorylation and cognitive testing. Alternative tau PET radiotracers may provide improved sensitivity for early NFT pathology and early cognitive findings in CU participants. We will test the differential ability of an alternative tau PET imaging drug to predict early Braak NFT stages and demonstrate association with cognitive tests. This work will provide a rigorous assessment of the biological meaning of tau PET signal. It will have a dramatic impact on treatment algorithms by identifying a subset with early tau deposition and therefore higher risk for developing AD dementia than those with only suspected amyloid pathology. Here we propose a multipronged approach to define tau PET imaging of early NFT and correlation with cognitive test findings in cognitively unimpaired individuals. This proposal is transformative, considering the novel concept of detecting NFT at early stages with state-of-art in vivo PET with novel methods and pathologic correlation that could provide insight for asymptomatic prevention trials.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT One in six adults in the U.S. suffers from chronic and often disabling symptoms of irritable bowel syndrome (IBS). Intestinal infections are an established risk-factor for development of post-infection IBS (PI-IBS). The intestinal tract contains a variety of proteases, and we have discovered that PI-IBS patients have significantly higher fecal proteolytic activity (PA) than controls. More importantly, PA associates strongly with loss of intestinal barrier function and worse symptoms for the patients. We found that patients who develop PI-IBS and have high PA have a significant loss of microbial diversity that starts soon after the infection. Key microbial taxa are lost, especially from the Alistipes genus. Using metaproteomics, we found that proteases driving PA in these patients are of human origin. In order to understand if loss of microbiota could be affecting host proteases, we used germ-free mice. Colonization of germ-free mice with healthy human microbiota (humanization) results in a significant decline of PA suggesting commensal microbes inhibit host proteases and thus have a role in maintaining intestinal health. However, the dysbiotic microbiota from the high PA patients that are missing specific microbes were unable to suppress PA. We hypothesize that Alistipes and other missing bacteria play a critical role in suppression of PA. We plan to test the candidate bacteria identified in the preliminary experiments and inter-species interactions in PA regulation in Aim 1. Next, we determined how loss of microbes result in poor inhibition of proteases. Unconjugated bilirubin is an inhibitor of serine proteases and microbial β-glucuronidases deconjugate bilirubin. We found that the PI-IBS patients with high PA have lower fecal microbial β-glucuronidase enzymatic activity. Additionally, they have lower levels of end products of bilirubin deconjugation. β-glucuronidases are a large family of microbial enzymes with varying sources, structures and catalytic efficacies for different substrates. We hypothesize that loss of specific microbial β-glucuronidases will result in impaired deconjugation of bilirubin. In Aim 2, we will analyze metagenomics data from our PI-IBS patients with high and low PA for presence of microbial β-glucuronidases as well as determine the efficacy of these fecal samples for bilirubin deconjugation. Additionally, we will generate purified β-glucuronidases from Alistipes and other bacterial taxa for assessing bilirubin deconjugation efficacy in vitro. Next, we have shown that fecal microbiota transfer using an Alistipes enriched low PA community can suppress PA in high PA humanized mice providing a rationale for using microbiota for protease suppression and correcting intestinal barrier function. In Aim 3, we will use cohousing strategies to allow microbiota transfer between humanized high and low PA mice and determine if barrier dysfunction associated with a high PA state can be reversed. Furthermore, we will determine changes in barrier pathways, ionic selectivity and expression of tight junction proteins upon engraftment of new microbiota. Together, these aims will examine microbial influence on PI-IBS pathophysiology via regulation of intestinal proteases. Identification of microbiota-based strategies that can result in protease inhibition and restoration of barrier function can be beneficial for IBS and other conditions associated with microbial dysbiosis.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT We have recently conducted and published a game changing phase 3 clinical North American Intergroup (NAIG) trial (E1912) for chronic lymphocytic leukemia (CLL) therapy which tested a combination of Ibrutinib and Rituximab (IR) vs. the prior gold standard chemoimmunotherapy (CIT): fludarabine, cyclophosphamide, and rituximab (FCR). This trial showed that both progression free survival (PFS) and overall survival (OS) are superior with IR and subsequently was the driving factor in FDA approval for frontline use of IR in progressive previously untreated CLL in the spring of 2020. While our work revealed distinct clinical advantages to non-CIT approaches, a number of new questions have emerged with respect to how best apply this advance. The durability of the response to first-line ibrutinib-based therapy is highly variable and requires indefinite treatment exposing patients to the risk of chronic toxicity and selective pressure that may foster resistant clones. The ability to more accurately predict the durability of response could help identify patients more likely to have long term remission with ibrutinib therapy (candidates for time limited therapy) and those more likely to have a short duration of response whom may benefit from intensive combination therapy with alternative novel agents. We wish to develop a unique model(s) incorporating multiple key prognostic factors that will have a high level of confidence in predicting patient outcomes to novel therapy combination. Our initial study on patients treated on IR arm of E1912 found a subset of patients on the IR arm with evidence for emerging mutations and changes in their clonal architecture predicting relapse. The exact mechanisms for relapse need to be defined as we predict that these patients will be difficult to treat and alternative strategies needed. We found that IR therapy was uniquely able to reactivate the previously exhausted T cell killing activity directed against the leukemic CLL cells. While we have some information on the mechanism(s) for this, much remains to be learned and also the exact timing for achieving the maximal restoration of T cell function or fitness. This beneficial impact on T cell function will also be studied as it relates to generation of CAR T cells as these cells are powerful inducers of immunotherapy which is itself capable of removing residual CLL tumor burden. We hypothesize that the outcome of the studies will add significant and important information on how to best select non-chemotherapy for CLL patients and also the treatment impact on the immune system. These goals will be accomplished through the following specific aims: Aim 1: Develop an Integrated Model to Predict Clinical Outcomes for CLL Patients Treated with Novel Agents. Aim 2: Determine the Genetic, Epigenetic and Transcriptomic Changes in Ibrutinib Treated CLL. Aim 3: Characterize the Impact of Ibrutinib Treatment on T-cell Fitness to Guide Application of Immunotherapy.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Therapies aimed at preventing development of cirrhosis-related complications are lacking. Statins have been shown to improve liver function and attenuate portal hypertension although definitive studies are lacking. Our investigative team has deep expertise in cirrhosis and portal hypertension including clinical, translational, and laboratory aspects of disease. Our aims in this proposal are: Aim 1. To conduct a prospective, multicenter, observational study of patients with compensated cirrhosis that will serve as the foundation for discovery of novel mechanistic and therapeutic targets. We will consolidate a longitudinal database to (a) provide unique information on the natural history and outcomes of cirrhosis and (b) support translational research (proposed in Aim 3). This robust multicenter dataset will be used for the development and validation of an artificial intelligence-derived algorithm for prediction of decompensation combining extensive clinical, laboratory and radiographic data, including magnetic resonance elastography and electrocardiogram. Aim 2. To perform a multicenter prospective randomized phase 3 clinical trial of simvastatin versus placebo to improve outcomes in patients with compensated cirrhosis. This aim will test the hypothesis that simvastatin is superior to placebo for reducing complications of cirrhosis and overall mortality. This phase 3 efficacy trial will randomize patients to Simvastatin 20 mg or placebo daily with median follow up of 36 months. The primary endpoint will be development of varices, decompensating events or death analyzed as ordinal outcomes based six prognostic stages. Secondary endpoints will include fibrosis regression, incidence of hepatocellular carcinoma, and cardiovascular complications. Aim 3. To identify novel pathogenic targets in cirrhosis progression through 2 Sub-aims: Sub-Aim 3a. To investigate the rate of telomere attrition in cirrhosis progression and decompensation. Telomere shortening has been observed in advanced cirrhosis and preclinical studies have shown reduced fibrosis with telomere length restoration. However, the rate of telomere attrition in cirrhosis and its association with disease progression remain unknown. Our proposal will serve as the basis for future studies assessing new therapies aimed at telomere length preservation in cirrhosis and effects of statins. Sub-Aim 3b. To measure circulating markers of neutrophil extracellular traps (NETs) and its correlation to development of hepatic decompensation. Anticoagulation has been shown to reduce hepatic decompensation in a small trial of patients with cirrhosis. Our group has recently shown that NETs drive intrahepatic thrombosis, and inhibition of NETs reduces portal pressure in preclinical studies. Thus, the results of this sub-aim will serve as the foundation for future human studies investigating novel therapies aimed at disrupting NETs in the liver. Our center and team have substantial expertise in clinical trials and therapeutics for advanced liver disease and are thus well poised to conduct these studies.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT This is Mayo Clinic’s application to participate in the NIH HeartShare Research Consortium as a HeartShare Clinical Center (CC). Our goal is to collaborate with the other HeartShare Investigators to elucidate the pathophysiology of heart failure (HF) with preserved ejection fraction (HFpEF) and discover novel diagnostic and therapeutic approaches. Multiple pathophysiologic processes may ultimately lead to different HFpEF phenotypes, though the specific mechanisms remain largely undefined. It is also not known whether standard clinical information can identify patients with different mechanistic etiologies, which is necessary to provide targeted therapies in clinical trials and eventually in clinical practice. Our proposal outlines four specific aims. In Specific Aim 1: We document that Mayo Clinic has the resources and the Mayo HeartShare Team has the expertise and track record of productivity in HFpEF and relevant related diseases, clinical research, patient recruitment and retention, data science, and collaborative team science to help drive the success of HeartShare Network. In Specific Aim 2: We propose a broad mechanistic phenotyping protocol providing quantitative variables reflective of senescence, systemic disease processes, and multi-organ integrity (L2 data), which are used as input variables in unsupervised machine learning (ML) models. We hypothesize that this approach will allow identification of unique HFpEF pathophysiologic phenogroups (clusters). We also propose invasive hemodynamic signatures, trans-cardiac gradients of circulating biomarkers and myocardial, adipose and skeletal muscle tissue characterization (L3 data) be obtained in a subset within each HFpEF pathophysiologic phenogroup. We hypothesize these L3 data will enhance identification of targeted therapeutic strategies. Lastly, we outline supervised ML using EHR data to develop automatable algorithms to accurately identify the HeartShare HFpEF pathophysiological phenogroups derived using L2 data. We hypothesize that if successful, this approach will enhance translation of HeartShare findings by allowing automated identification of patients in the different HFpEF phenogroups for enrollment in clinical trials of agents targeting their specific pathophysiology. In Specific Aim 3: We propose that use of circulating proteins alone (n=5000; defined by the SOMAScanTM Aptamer based platform) as input variables for unsupervised ML models will identify unique HFpEF pathophysiologic phenotypes (clusters). In Specific Aim 4: We outline the Mayo HeartShare Research Skills Development Program. Providing HFpEF clinical investigators a short-term intensive immersion experience by collaboration with a data scientist intern in the Mayo Cardiovascular Disease AI Internship or a long term dedicated program in data science as a Mayo Kern Center Scholar in Data Science will equip a new generation of HFpEF investigators with a robust data science toolbox to drive future discovery.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT The rapid growth of genomic medicine reflects increases in our understanding of how genomics impacts health. Advances in the clinical utility of patient genomic information to inform diagnostic and therapeutic decision-making are driving increases in testing and the subsequent reporting of genomic data to electronic health records (EHRs) and other clinical systems. Germline genomic data are presumed to be stable over a patient’s life, but the interpretation of those data and its clinical utility will continue to evolve as new discoveries are made and translated to clinical practice. Furthermore, modern interpretations of genomic data often include multi-step processes in which one interpretation is used to inform another. The temporally dynamic nature of genomic information and knowledge (GIK) and the inter-dependencies among interpretations make a formal knowledge model for those data essential. Existing approaches to represent and manage GIK are not sufficient to fully support the needs of genomic medicine and thus a new approach must be developed. The specific objective of this research program is to develop and evaluate the Genomic Interpretation and Knowledge (GenIK) Framework for capturing interpretations related to clinical genomic results as standardized, structured data. The GenIK framework will: 1) facilitate more robust integration of genomic data into EHRs; 2) enable scalable knowledge management; 3) enable utilization of GIK by other systems; 4) enable clinical, translational, and discovery research; and 5) enable meaningful data sharing of GIK. Moreover, because the framework will be based on a generalized, standards-based knowledge model, it will elegantly extend to support new types of genomic interpretations and uses for those data. The GenIK framework will fill a critical gap in the representation, management, and delivery of GIK, thereby advancing genomic medicine. The proposed research program will develop foundational resources that will enable more seamless integration of genomics within both research and clinical settings. Research Area 1 will focus on discovery activities that support the development of the GenIK framework. Research Area 2 will focus on the translation and application of the GenIK framework within clinical systems. Research Area 3 will focus on dissemination to and engagement with stakeholder communities. The main hypothesis of this research is that implementations based on the GenIK framework will more fully capture GIK than existing approaches, thereby improving the management, use, and sharing of GIK. The Genomic Innovator Award will enable, in team-science projects and in collaboration with genomic research consortia, the study of the requirements for and implementation of scalable GIK management within clinical systems. The resources developed by the proposed research program will allow the capture and exchange of GIK to take place at the speed of discovery, supporting research in precision medicine as part of a learning healthcare system and accelerating scientific and medical breakthroughs that improve human health.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY In this application we propose to build on our prior work on polygenic risk scores (PRSs) to extend these to be applicable to all individuals. By improving risk stratification, PRSs for common diseases have the potential to transform clinical practice. However, such PRSs must be available for all individuals to ensure implementation of genomic medicine. Our application aims to address the critical need to develop PRSs for all individuals and will focus on coronary heart disease (CHD) and its risk factors: hypertension, diabetes, obesity and hypercholesterolemia, collectively an enormous health burden world-wide. CHD is the prototypical complex disease for the use of PRSs given available validated risk prediction equations that bin individuals into risk categories and substantial reclassification across these categories by a PRS with consequent therapeutic implications. We will develop methods to generate PRSs for ancestry using existing and new datasets with genomic and phenotype data for CHD and its risk factors. We will harmonize data elements across these data sets. The methods we develop will be applicable towards the generation of PRSs for a broad range of common diseases. The investigative team is part of the Mayo eMERGE IV application and will serve as a bridge to this effort. To generate PRSs, we will use data from the eMERGE consortium, Million Veteran’s Program (MVP), the All of US (AoU) program, dbGAP, , UK Biobank, and collaborations with several international groups representing the Middle Eastern, South Asian and East Asian cohorts. Our application includes several innovations to enable the use of PRSs for risk stratification and prevention of CHD in individuals. Our specific aims are: Specific aim 1. Integrate and harmonize phenotype data from heterogeneous sources to enable cross platform phenotyping and generation of PRSs for common diseases. Specific aim 2. Develop PRSs for CHD and its major risk factors (hypertension, diabetes, obesity, hypercholesterolemia) in populations that represent all individuals. Specific aim 3. Develop novel statistical and computational methods to account for heterogenous data for models of polygenic risk. Specific aim 4. Develop ‘clinic ready’ PRSs by creating reference distributions of a PRS for CHD and integrate it with clinical information to compute absolute risk estimates.

NIH Research Projects · FY 2025 · 2021-09

PROJECT DESCRIPTION/ABSTRACT – OVERALL The development of effective therapies for glioblastoma (GBM) has been incredibly vexing with no new drug approvals in over a decade. Therapeutic resistance in any one patient with GBM can be related to multiple factors including extensive tumor cell infiltration into adjacent brain, molecular heterogeneity of tumor cell populations, and heterogeneity of drug distribution. In order to understand and overcome these challenges, we have built a highly productive, multi-disciplinary scientific team over the past decade with expertise spanning systems biology, pharmacology, tumor biology, and animal models of GBM. In the proposed U19 Center application, we will integrate this established cross-disciplinary translational science team with physician scientists in radiation and medical oncology, neurosurgery and neuroradiology with a collective focus of translating novel therapeutic strategies into highly effective therapies for patients with GBM. Impaired DDR enables the genomic instability required for tumorigenesis, and differences in DDR functionality between tumor and normal tissue provides the fundamental rationale for using radiation therapy or genotoxic drugs as anti-cancer therapies. Additional targeted pharmacologic disruption of DDR in tumors can markedly enhance the efficacy of these cytotoxic therapies and widen the therapeutic window. In this context, we have collaborated extensively with multiple pharmaceutical companies to evaluate various small molecule DDR inhibitors and have developed significant preliminary data demonstrating profound combinatorial efficacy for these drugs when combined with radiation or alkylating chemotherapy routinely used for GBM. Thus, the initial focus for our Center is to optimize the clinical deployment of DDR inhibitors in combination with cytotoxic therapies for GBM. A Pharmacology Core will support both pharmacokinetic (PK) and pharmacodynamic (PD) evaluations in animal models and human samples, and our Therapy Evaluation Core will support both pre-clinical and clinical testing of novel therapeutic strategies. The Project and Core teams will work in close collaboration to accomplish the goals of the Center, and this collaborative effort within the Center and across the broader Glioma Therapeutics Network (GTN) will be coordinated by the Administrative Core.

NIH Research Projects · FY 2024 · 2021-09

Summary The immune system plays an important role in protecting us from disease. Interstitial inflammation has been consistently reported in human and animal models of ADPKD, and it may become worse during cyst expansion which results in more damages in renal parenchyma. In addition to the increase of macrophages in the interstitium and pericystic areas, T lymphocytes are also increased in cystic kidneys. However, whether and how PKD mutant cystic renal epithelial cells escapes immune attacks in cystic microenvironment during cyst initiation and expansion remains elusive. In this study, we investigate the roles of programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1), a PD-1 ligand, in ADPKD. We found that, 1) PD-1 was upregulated on T cells in Pkd1 mutant kidneys; 2) PD-L1 was upregulated in Pkd1 mutant renal epithelial cells and tissues, and was increased in cystic cell derived exosomes; 3) knockout of Pd-l1 delayed cyst growth and increased the survival of Pkd1 knockout mice; 4) targeting PD1 and PD-L1 with antibodies delayed cyst growth in Pkd1 knockout kidneys; 5) treatment with exosomes isolated from cystic renal epithelial cells and urine of ADPKD patients increased Pkd1 wild type renal epithelial cell proliferation, and induced the activation of PKD associated signaling in these cells; 6) treatment with cystic renal epithelial cell derived exosomes promoted cyst growth in Pkd1 mutant kidneys; 7) renal epithelial cells (NRK-52E cells) treated with ADPKD urinary exosomes also developed cysts-like structures in collagen gels; and 8) inhibition of exosome secretion with GW4869 delays cyst growth in Pkd1 knockout kidneys. Our central hypothesis is that upregulation of PD-L1 on cystic renal epithelial cells and PD-1 on T cells results in immune evasion of cystic cells via inhibition of T cell function, and exosomes secreted by cystic renal epithelial cell regulate immunosuppression via adjacent T cells and the function of other neighboring cells, including renal epithelial cells and fibroblasts, contributing to cyst growth. We test this hypothesis with three specific aims. This study will determine for the first time whether PD-1 and PD-L1 are immune-suppressors in cystic kidneys, which helps cystic epithelial cells to escape immune attack in ADPKD, and whether exosomes secreted by cystic epithelial cells contribute to immune suppression and other cellular communication. In addition, we will determine whether PD1 and PD-L1 are effective targets to slow disease progression in preclinical setting. Accomplishing this study will lead to a better understanding of the mechanism of immune surveillance in renal cyst formation and the roles of cystic cell exosomes in regulating immunosuppression and other cell-to-cell communication, which will provide novel therapeutic strategy for ADPKD treatment.

NIH Research Projects · FY 2025 · 2021-09

PROJECT DESCRIPTION/ABSTRACT Programmed death ligand 1 (PD-L1), which promotes immune escape, is overexpressed in triple negative breast cancer (TNBC), an aggressive subtype of breast cancer characterized by poor prognosis. Clinically approved PD-L1 antibodies augment anti-tumor immunity by blocking extracellular PD-1/PD-L1 binding. However, the contribution of intracellular PD-L1 to anti-tumor immunity and therapeutic resistance has remained poorly understood. We have discovered a novel role for intracellular PD-L1 as an RNA binding protein that promotes the stability of target RNAs. This new intracellular PD-L1 function in regulating RNA expression was independent of the established extracellular role of PD-L1 as the ligand for PD-1. The activity of the anti-tumor immune response is governed by a balance between immune effector cells and immune suppressor cells. Regulatory T cells (Tregs) are a CD4+ T cell subpopulation that inhibit effector cell activity, suppress anti-tumor immunity and promote therapeutic resistance. A hallmark of Tregs is the expression of the transcription factor, Foxp3, which binds to the promoters of genes that support Treg activity. Foxp1 is a closely related family member of Foxp3. Emerging data has demonstrated that Foxp1 cooperates with Foxp3 to encourage Foxp3-mediated transcription and Treg function by maintaining Foxp3 occupancy at promoters of target genes. In our preliminary data, we have identified Foxp1 as a key PD-L1 target RNA. We have found that the PD-L1 cytoplasmic domain, but not the PD-L1 extracellular domain, interacts with Foxp1 RNA and promotes Foxp1 expression. In addition, we have discovered that intracellular PD-L1’s promotion of Foxp1 expression is necessary for proper Treg differentiation, Treg function, and TNBC progression. The Akt-mammalian target of rapamycin (mTOR) pathway regulates metabolic reprogramming for proper T cell maturation and function. Our preliminary data suggests that PD-L1 and Foxp1 are required for proper Akt-mTOR pathway activation and metabolism specifically in Tregs, but not effector T cells, suggesting that targeting the PD-L1-Foxp1 pathway may preferentially inhibit Tregs to address therapeutic resistance. Our overarching hypothesis is that intracellular PD-L1 stabilizes Foxp1 RNA to promote Treg immunosuppressive activity and therapeutic resistance. Further, inhibiting intracellular PD-L1 will promote anti-TNBC immunity by blocking Treg activity. This hypothesis will be tested in a series of three aims: Aim 1 will determine the influence of intracellular PD-L1 on Foxp1 mRNA stability and Treg differentiation; Aim 2 will determine the effect of intracellular PD-L1 on Treg function; Aim 3 will compare intracellular PD-L1 and extracellular PD-L1/PD-1 directed TNBC therapy. Clear delineation of the impact of intracellular PD-L1 on cancer therapy will provide important insight for optimizing combination strategies aimed at overcoming immune escape and therapeutic resistance, laying the foundation for the design of the next generation of TNBC clinical trials.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Sporadic cerebral amyloid angiopathy (CAA) is a small vessel disease caused by cerebrovascular deposition of amyloid-β (Aβ). CAA frequently overlaps with Alzheimer’s disease (AD), presumably because Aβ is considered major culprit in development of AD pathology. Importantly, there is no disease-specific treatment available to patients with CAA. Relevant to this project, molecular mechanisms underlying pathogenesis of CAA are incompletely understood thereby limiting our ability to prevent initiation and progression of this disease. Despite mechanistic differences between vascular-induced brain injury in CAA and neurodegenerative injury in AD, clinically, CAA overlaps with AD and it is associated with more severe cognitive impairment in AD patients. This application is designed to advance the concept that in early stages of CAA, deposition of endothelium-derived Aβ in cerebral blood vessel wall is an important mechanism contributing to pathogenesis of the disease. We performed extensive preliminary studies on cultured human brain microvascular endothelial cells (BMECs), mouse microvessels, and brain endothelial cells isolated by fluorescence activated cell sorting (FACS). Next generation sequencing (RNA-Seq) was used to determine global gene expression profiles in human and murine cerebrovascular endothelium. Genetically modified mice and a murine experimental model of CAA were used to validate and expand observations obtained in cultured human endothelium. We identified previously unrecognized (Aβ-independent) endothelial functions of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) and its homologue (BACE2). Consistency between findings in human and murine endothelium was in agreement with strong evolutionary conservation of BACE1 and BACE2. However, while endothelial BACE1 exerts detrimental vascular effects, endothelial BACE2 appears to be previously unrecognized and very important vascular protective molecule. Further preliminary analyses of BACE1 and BACE2 function and signaling in endothelium of mice vulnerable to development of CAA, suggested that dysfunctional BACE1 and BACE2 in endothelium promote elevated Aβ deposition in the cerebral blood vessels. Based on these preliminary findings our working hypothesis is that endothelial BACE1 and BACE2 play distinct roles in cerebrovascular homeostasis and pathogenesis of CAA. We anticipate that successful completion of this project will offer new opportunities to utilize endothelial BACE1 and BACE2 as molecular targets for therapeutic interventions designed to prevent detrimental effects of CAA on cerebrovascular and cognitive function.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Sepsis is the most frequent cause of death in United States hospitals, and the number one cause of death worldwide, responsible for 11 million deaths in 2017. Primary drivers of sepsis morbidity and mortality are immune exhaustion leading to deadly secondary infection, and organ tissue damage leading to multi-organ failure. Despite the critical importance of these processes in governing ultimate sepsis outcomes, we have an incomplete understanding of their kinetics and dynamics. We thus propose a two-pronged effort to shift our paradigm regarding sepsis dynamics through plasma cell-free DNA analysis. Specifically, we propose to 1) track the dynamics of organ-specific damage, and 2) track the dynamics of T cell exhaustion during sepsis. We will do this through analysis of daily blood samples acquired from patients admitted to the intensive care unit for sepsis. Methodologically, we will achieve these through genome-wide methylation sequencing of cell-free DNA. Bioinformatically, we will then perform CIBERSORTx deconvolution to delineate and quantify specific cell/tissue types contributing to plasma cell-free DNA, thus enabling us to infer organ tissues being damaged as well as T cell exhaustion levels from both blood and tissue sources. The methods we will utilize here are highly innovative yet feasible given recent literature and our own preliminary data. Our proposed work will form the basis for a rich sepsis-focused research program utilizing cell-free DNA analysis to answer major questions in the field with the potential to ultimately improve patient survival for the deadliest disease worldwide.

NIH Research Projects · FY 2026 · 2021-09

Project Summary: High frequency oscillations (HFOs) of intracranial EEG (iEEG) have the potential to identify the surgical resection area/seizure onset zone (SOZ) in patients with drug resistant epilepsy. However, multiple reports indicate that HFOs can be generated not only by epileptic cerebral tissue but also by non-epileptic sites often including eloquent regions such as motor, visual and language cortices. In this project, we present the initial evidence of a recurrent waveform pattern that may be sufficient to distinguish pathological HFOs from physiological ones. Specifically, we show that the SOZ repeatedly generates sets of stereotypical HFOs with similar waveform morphology whereas the events recorded from out of SOZ were irregular. This morphological pattern served as a robust neurobiomarker to isolate SOZ from other brain areas in multiple patients consistently. While these promising preliminary results are in place, the functional utility of stereotyped HFOs in a closed-loop seizure control system remains unknown. As of today, not much is known whether the stereotyped HFOs generated by the SOZ can be detected with an implantable system. If this can be achieved, then HFOs can be strategically translated as a neurobiomarker into closed-loop seizure control applications. We hypothesize that pathologic stereotyped HFOs can be captured with the implantable Brain Interchange (BIC) system of CorTec and spatial topography of these events can be utilized by the implantable system to deliver targeted electrical stimulation to achieve seizure control. Using an acute setup within the epilepsy monitoring unit (EMU), this project will investigate the feasibility of capturing stereotyped HFO events using the new BIC system and compare the detection results to those obtained with the commercially available amplifier. If the first phase (Aim-1) of our study becomes successful, later in the second phase (Aim-2), once again in the EMU, we will deliver targeted electrical stimulation to those brain sites associated with stereotyped HFOs using the BIC. We will investigate the modulatory effects of this closed-loop stimulation strategy by monitoring the changes in signature events such as spikes, epileptic discharges, ripples and fast ripples. If successful, this closed-loop system does not have to wait for a seizure to start in order to deliver the stimulation at its onset as done by the RNS system of Neuropace. In contrast, the system will monitor the spatial topography and rate of stereotyped HFOs and deliver targeted stimulation to these areas to prevent seizures from occurring. If the outcomes of our research in acute setting become successful, we will execute a clinical trial and run our methods with the implanted BIC system in a chronic ambulatory setting. 1

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT Secreted serine proteases are abundant in the intact CNS and become deregulated by injury and disease, yet we lack knowledge regarding their physiological functions and contributions to pathology. Several years ago, the discovery of a set of enzyme-activated G protein-coupled receptors, the Protease Activated Receptors (PARs), led to a new conceptual framework for understanding the physiological impact of proteases. PARs permit activating enzymes to signal in a hormone-like fashion to modulate key cellular functions, but when overactivated can contribute to pathology. The PI’s team recently discovered that mice with global PAR1 gene knockout exhibit significant improvements in locomotor recovery after spinal cord injury (SCI). Functional improvements were accompanied by reductions in inflammation and astrogliosis and improvements in the appearance of myelin and axons, all integral substrates to support restoration of function. We also documented that CNS injury relevant proteases, such as thrombin and kallikrein 6 elicit Ca2+, MAPK and STAT3 signaling linked to neuroinflammation and pro-injury responses across neurons and neuroglia in a PAR1-dependent manner. Together, these studies highlight the likely multifactorial roles played by PAR1 in key cellular and molecular events positioned to govern outcomes after SCI. These findings also highlight the potential to target PAR1 for neural protection and repair. Despite these encouraging findings the cellular mechanisms by which blocking PAR1 improves recovery after SCI have not been defined and this knowledge gap hampers progress towards translation of existing FDA approved and orally bioavailable PAR1 small molecule inhibitors. Additionally, whether blocking PAR1 therapeutically at acute or chronic time points after SCI are both capable of improving neural recovery is unknown. Based on recently published findings, taken with new preliminary results, we propose 3 integrated Aims to test the Central Hypothesis that PAR1 is an essential regulator of reactivity across the microglial- astrocyte compartments and can be selectively blocked to improve glial-neuronal trophic coupling, neuroprotection and repair after SCI. In Aim 1, we will determine the impact of pharmacologic PAR1 inhibition initiated at acute or chronic time points after injury on signs of neuroprotection, neural repair and recovery of sensorimotor function and use ribosomal mRNA capture techniques to document cellular and molecular mechanisms engaged across the astroglial, microglial/monocyte and neuron compartments. In Aim 2, we will determine whether conditional deletion of PAR1 selectively in astrocytes, microglia or peripheral monocytes is sufficient to enhance recovery. In Aim 3, we will use glial-neuron co-cultures as bioassays to establish PAR1- regulated glial-neural trophic coupling mechanisms relevant to neuroprotection and repair. The studies proposed address key mechanistic questions regarding the functional roles of PAR1 in neural injury and will provide new information needed to optimize therapeutic targeting strategies for recovery of function.

NIH Research Projects · FY 2025 · 2021-09

PROJECT DESCRIPTION/ABSTRACT Candidate: Dr. Tweet’s long-term goal is to become an independent clinical investigator in the field of coronary artery disease in women. Her short-term goal is to explore the vascular, autonomic and microvascular phenotypes in women with a history of spontaneous coronary artery dissection (SCAD). To achieve these goals, Dr. Tweet will: 1) acquire the expertise required for her scientific aims; 2) improve her data analysis skills via coursework and biostatistical support; 3) develop scientific leadership skills and collaborative relationships to move this field forward. Environment: The Mayo Clinic SCAD Registry is the largest international SCAD registry. Dr. Tweet’s primary mentor, Dr. Joyner and her Career Advisory Committee have the expertise and mentorship qualities to guide Dr. Tweet’s career development as a physician-scientist. Background: SCAD is a leading cause of nonatheroembolic acute coronary syndromes in women. Most patients do not have traditional risk factors such as tobacco use, hyperlipidemia, or diabetes. Rather, peripheral arterial abnormalities such as fibromuscular dysplasia are observed in the majority of patients. SCAD is associated with stress, exercise, and pregnancy. Recurrent SCAD occurs in 10-30% of patients. Specific Aims: We will aim to assess arterial structure and mechanics (Aim #1), neurovascular function (Aim #2), and microvascular function (Aim #3) in women with SCAD. Study Design & Outcome Measures: This is a prospective study of women evaluated in the Mayo Clinic SCAD Clinic. Measurements will include arterial thickness and stiffness, sympathetic baroreflex sensitivity and autonomic responses to sympathoexcitatory to standardized stressors, and myocardial perfusion echocardiography with dobutamine stress. Relationship to Career Goals of the Candidate: The projects in the application will be instrumental for the candidate’s career development by facilitating: scientific management and leadership skills, acquisition of new research techniques, and generating interdisciplinary and multi-institutional collaborative alliances. Thus it is the next logical step in her research career progression. Relationship to the NHLBI Mission: The proposed project is consistent with the NHLBI strategic vision for understanding underlying pathophysiological mechanisms of disease and for improving study of diseases affecting women. It is consistent with the NHLBI’s interest in hypertension, sympathoexcitation and cardiovascular disease risk. It is also consistent with broader NIH goals related to training the next generation of clinical investigators.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Hematopoietic stem cell transplantation (HSCT) offers an advanced treatment option for conditions that are otherwise incurable. Pulmonary complications are common following HSCT and severely compromise its overall success rate. The most severe end of this spectrum is acute respiratory distress syndrome (ARDS), which occurs in 5% of patients following HSCT, with a mortality >60%. As effective treatments for ARDS are lacking, developing new ways to prevent ARDS is likely to be more successful in improving patient outcomes. Two critical knowledge gaps make developing successful prevention strategies difficult: 1) an inability to reliably identify high-risk patients before ARDS has developed and; 2) an incomplete understanding of the mechanisms and subtypes of post-HSCT ARDS. The scientific objectives of this application are to address these critical knowledge gaps through the following Specific Aims: 1) Develop and validate a risk-prediction model incorporating pre-transplant, post-transplant and in- hospital risk factors that can help identify which patients are most likely to develop post-HSCT ARDS. 2) Prospectively evaluate metabolomics in patients at high-risk of developing post-HSCT ARDS and utilize clinical, biomarker and metabolomics data to explore endophenotypes of post-HSCT ARDS. Aim 1 leverages highly innovative data extraction techniques to develop a real-time continuous ARDS risk surveillance system for patients who undergo HSCT. Aim 2 evaluates the role of metabolomics, an emerging analytic tool, in advancing mechanistic understanding of why patients develop post-HSCT ARDS, as well as whether recently described ARDS endophenotypes can be identified post-HSCT ARDS. The applicant’s long-term goals are to: 1) become an independent translational clinician-scientist leading a multidisciplinary research team focused on improving outcomes of patients who develop respiratory complications after HSCT, and 2) develop effective strategies to prevent post-HSCT ARDS, one of the most significant post-transplant complications today. The training goals of the applicant are to develop advanced skills in statistics, biomedical modeling, machine learning, and metabolomics to facilitate these long-term goals. To address these training needs, the applicant has constructed a comprehensive career development plan that includes targeted didactic opportunities in the areas of training need above, with additional “hands-on” experience in all of these areas. The mentorship team and research environment is uniquely well suited to the applicant’s needs, combining expertise in critical care informatics and metabolomics at a high-volume academic transplant center. The proposal supports the NIH mission by striving to improve outcomes of patients who develop respiratory failure after transplantation, while training a junior investigator in the essential skills necessary for his transition to research independence.

NIH Research Projects · FY 2025 · 2021-09

Lung cancer is the leading cause of cancer-related death in the United States, and more than 150,000 Americans die of lung cancer each year. Despite our best treatment, efforts to cure lung cancer have failed in most cases, partly due to an insufficient understanding of the biology of metastasis, which almost inevitably leads to lung cancer death. Therefore, understanding better the mechanism regulating metastasis will allow us to gain deeper insights into the disease basis, on which novel therapeutic strategies for treating metastatic lung cancer can be developed and tested. On the basis of our preliminary studies, we posit that an autophagy related gene plays a critical role in the regulation of lung cancer metastasis through a novel mitochondria associated mechanism. The primary objectives of this proposal are to determine whether manipulating the expression of this gene or its mitochondria associated function can inhibit the dissemination of lung cancer. As such, our studies not only reveal a new basic mechanism for lung cancer, but have translational potentials. Because there are about 2 million new lung cancer cases each year globally (World Health Organization statistics), our studies may have a major impact on public health.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Glaucomatous optic neuropathy (GON) remains the world’s leading cause of irreversible blindness. At present time, the only reliable therapeutic target is the reduction of intraocular pressure (IOP), the most prevalent risk factor for GON, by means of pharmacologic, laser, or surgical intervention. Of these, pharmacologic therapy with topical eye drop monotherapy is generally the initial treatment of choice for patients with GON. Of the available medication classes, Prostaglandin F2 analogues (PGF2α) such as latanoprost are often used as first- line therapy. However, despite its success in reducing IOP in many patients, treatment non-response or side- effects limit its use in other patients. Furthermore, some studies estimate that less than half of patients use glaucoma eye drops as prescribed. Additionally, because of the pharmacokinetics and dosing regimens, fluctuation in IOP is common which also contributes to GON. We recently identified a peptide hormone, Stanniocalcin-1 (STC-1) that lowers IOP when applied topically and can be expressed in a sustained fashion with a viral vector to provide sustained IOP reduction. STC-1 was identified in our laboratory’s search of downstream effector molecules in latanoprost-mediated IOP reduction. To date, we have demonstrated that: 1) STC-1 is required for the IOP-lowering effects of latanoprost; 2) Topical STC-1 lowers IOP as a stand-alone drug and is equivalent to latanoprost for IOP reduction in normotensive mice; 3) IOP-lowering effects of STC-1 are independent of the FP receptor; 4) STC-1 lowers IOP in ocular hypertensive mice; 5) STC-1 lowers IOP in the domestic cat; and 6) STC-1 delivered by adeno-associated virus (AAV-STC-1) lowers IOP in a sustained fashion in normotensive mice. Our central hypothesis for this application is that expression of STC-1 with a viral vector will provide an effective, safe, and sustained treatment for IOP reduction that has potential to benefit the 80 million people worldwide afflicted by glaucoma. Aim 1 will confirm and optimize our preliminary data that STC-1 can be delivered with a viral vector to provide sustained IOP reduction in normotensive mice. The data will determine the optimal viral vector for IOP reduction and correlate with tissue expression, define minimal therapeutic dose of virus, and evaluate safety using histologic methods. Aim 2 will utilize the optimized viral construct and evaluate IOP reduction in models of ocular hypertension. A steroid- induced ocular hypertension model as well as the DBA/2J model of pigment dispersion will be used. Additional measures will include assessment of aqueous outflow parameters. Aim 3 will evaluate viral expression of STC-1 in domestic and primary congenital glaucoma cats as well as to evaluate aqueous humor outflow and safety. Combined with strong preliminary data and an expert mentorship panel, these aims will support our long- term goal of developing a novel, targeted, sustained delivery of an IOP-lowering agent for the estimated 80 million people worldwide with GON.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT: High-grade astrocytomas (HGA) are rare incurable cancers (combined USA prevalence < 30,000) with few treatment options. Mayo Clinic is collaborating with Wayshine Biopharma to develop WSD0922-FU (WSD) (IND 145566) – a novel oral, small molecule orphan drug which potently inhibits EGFR aberrations specific to high-grade astrocytoma (HGA) (EGFR amplification, EGFRvIII mutation). Our central hypothesis is that safe and tolerable doses of WSD achieve sufficient tumor concentrations in HGA to inhibit EGFR signaling and improve efficacy in patients with EGFR-amplified / EGFRvIII HGA. This is being examined in a first-in-human phase I basket trial which is the subject of this three-year R01 submission to the FDA (“Phase I Study to Evaluate Safety, Tolerability, Pharmacokinetics and Anti-Tumor Activity of WSD0922- FU”). The investigators have broad clinical trial experience in early drug development and neuro-oncology. Early input on trial design was obtained from patient advocates to ensure study feasibility and to reduce patient burden. We expect that study completion will advance available treatments for HGA with EGFR alterations by establishing the maximum tolerated dose (MTD) for WSD, while generating critical data on central nervous system (CNS) PK (pharmacokinetics), impact on tumor EGFR signaling, preliminary efficacy and relevant genetic biomarkers of response/resistance. These data will provide robust evidence to optimize phase II/III trial design, which may lead to a new indication for WSD in HGA. The central hypothesis is evaluated in three aims: Aim 1: Evaluate the safety, tolerability and systemic PK of WSD at MTD. Patients with EGFR-amplified / EGFRvIII HGA or non-small cell lung cancer with CNS metastases will be treated with WSD in a dose escalation cohort to define the MTD and evaluate the toxicity profile of WSD. Plasma PK will be quantified by LC-MS/MS to evaluate systemic exposure. A dose expansion cohort will also include a food-effect study prior to starting continuous dosing. Plasma PK will be quantified in both the fed (WSD dosed after a high fat meal) and fasted states (WSD dosed one hour before or two hours after eating) to evaluate the effect of food on systemic PK. Aim 2: Evaluate intratumoral PK of WSD and assess its pharmacodynamic (PD) impact on EGFR pathway signaling in HGA. Patients with EGFR-amplified / EGFRvIII HGA requiring a therapeutic surgical tumor resection as part of routine clinical care will preoperatively be dosed with WSD at MTD. During surgery, tumor tissue will be collected and flash frozen. Tumor PK will be quantified by LC-MS/MS and PD impact on EGFR pathway signaling will be evaluated by functional proteomics. Aim 3: Characterize the molecular biomarkers which influence efficacy of WSD in patients with EGFR aberrant HGA. Patients with EGFR- amplified / EGFRvIII HGA will be treated continuously with WSD at MTD and monitored for treatment efficacy. Plasma PK will be quantified by LC-MS/MS to confirm sufficient exposure. DNA/RNA sequencing will be performed on archived tissue to evaluate biomarkers of response and resistance (e.g. EGFRvIII, TP53, etc.).

NIH Research Projects · FY 2024 · 2021-09

Project Summary / Abstract The gastrointestinal (GI) tract is the only organ system that is capable of intrinsic neural reflexes. These are initiated by a unique neuron type called intrinsic primary afferent neurons (IPANs). IPANs are key to orchestrating neural reflexes that allow efficient processing of meals for nutrient uptake by rapidly adapting to changing luminal content to alter vascular, secretory and motor function. In the guinea pig, IPANs use multiple mechanisms of neuroplasticity to adapt to inflammatory, hormonal and neural stimuli. From these studies it is clear that IPAN neuroplasticity mediates digestive disease. Even though the mouse has become the vertebrate animal model of choice for digestive disease, murine IPANs have lacked consensus markers making precise studies of mouse IPANs inconceivable. These issues are now resolved by our recent transcriptome and morphological analysis of the ENS, which challenges the dogma that IPANs are a single class of neuron, and suggest that rather there are four classes of IPANs. In combination with recent advances in morphological (i.e. tissue clearing) and physiological approaches (i.e. genetically-encoded markers and activity indicators) we are now able to study mouse IPANs in a relatively high throughput manner. The objective of this proposal is to test the overall hypothesis that different classes of IPANs possess morphologies and physiology that uniquely contribute to intestinal function. This hypothesis will be tested in a series of experiments designed to address three specific aims: Specific Aim 1: determine the structure of receptive fields and connectivity of murine IPANs; Specific Aim 2: determine responses of murine IPANs to mechanical and chemical stimuli; Specific Aim 3: determine the role of IPANs in gastrointestinal physiology. Collectively, these studies address a critical gap in our knowledge on the basic neural control of gut functions. Deciphering sensory capabilities and functional responses of molecularly defined IPANs are likely to pave the way for future improvements in diagnostic and therapeutic strategies of digestive disease.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT PD-1 is a key immune checkpoint receptor that dampens T cell function. While extensive studies have investigated how PD-1 modulates T cell effector function in cancer settings, how PD-1 regulates systemic autoimmunity and humoral immunity remains poorly defined. Clinically, immune checkpoint inhibitors, including anti-PD-1, induces inflammatory arthritis immune related adverse events (IA-irAE), but the underlying mechanisms and the relationship between IA-irAE and classic rheumatic diseases are unexplored. Importantly, no autoimmune animal models exist that permit direct analysis of clinically-used anti-PD-1 antibodies because they do not interact with mouse PD-1. We utilize novel humanized PD-1 and PD-L1 mouse models to investigate the mechanisms by which clinically-used anti-PD-1 and anti-PD-L1 biologics modulate humoral immune response in a classic collagen induced arthritis model. Furthermore, through comparative analysis of clinical data and immunological profiles between anti-PD-1 induced IA-irAE and rheumatoid arthritis, we uncover remarkable similarity between IA-irAE and seronegative rheumatoid arthritis, a hitherto little understood form of systemic autoimmunity. Our central hypothesis is that anti-PD-1 and anti-PD-L1 biologics induces T cell-dominating and antibody-independent autoimmune toxicity, and impaired PD-1 signaling is a contributing factor to IA-irAE and seronegative rheumatoid arthritis (RA). To test this hypothesis, we will (1) To determine the immunopathology and molecular mechanisms of clinically-used PD-1 and PD-L1 blockade mediated autoimmune diseases, and (2) To investigate how dysregulation of PD-1 signaling contributes to IA-irAE and seronegative RA in human subjects. Building on our clinical and basic research expertise and innovations that integrate novel mouse models, innovative pharmacological interventions and comparative patient cohort analysis, our research will address fundamental questions in PD- 1 signaling, humoral immunity, and systemic autoimmunity, with direct relevance to the improvement of clinical practice and patients’ wellbeing. Additionally, our study will provide a valuable animal model as a research tool for the field of irAE.