Mayo Clinic Rochester

NIH Research Projects · FY 2024 · 2012-07

PROJECT SUMMARY/ABSTRACT Multiple myeloma (MM) is a life-threatening plasma cell malignancy. It is 2-3 times more common in blacks compared with whites. MM has a prolonged clinically detectable premalignant phase called monoclonal gammopathy of undetermined significance (MGUS) that can be identified by detection of the secreted monoclonal immunoglobulin (commonly referred to as a monoclonal protein). MM is a serious incurable malignancy, and the best approach for treatment is to prevent end organ damage by early intervention. This is best done by targeting patients with an intermediate asymptomatic stage referred to as smoldering multiple myeloma (SMM) that resides between MGUS and MM. Over the last 5 years of this grant we have extensively investigated the reasons why MM is more common in blacks compared with whites, demonstrated that first- degree relatives of patients with MM have a high risk of having the precursor MGUS lesion, and identified several biomarkers that predict risk of imminent progression in SMM. Our studies show that a principal reason for high risk of MM in African Americans is that they have a high risk of the precursor MGUS condition, which we also found is present at a much earlier in age in blacks compared with whites. More recently we made an important discovery using DNA sequencing based ancestry analysis that 3 specific cytogenetic abnormalities in MM account for most of the racial disparity. Our research has assumed greater urgency with recent findings that early intervention (in high-risk SMM stage) can prevent end-organ damage and prolong overall survival. The goals of this renewal are to further determine the mechanisms behind the racial disparity in incidence of MM, to identify new biomarkers for high risk SMM needing therapy, and to develop a feasible screening strategy to identify patients with SMM. In Aim 1 we will determine the age at onset of MGUS using sensitive, mass spectrometry (MS)-based detection of monoclonal protein in >12,000 NHANES samples, and identify new cytogenetic abnormalities that are associated with predisposition to MM in blacks. In Aim 2 we will identify new biomarkers that are associated with high risk of progression from SMM to symptomatic MM, specifically utilizing mass spectroscopic characterization of the monoclonal protein, measurement of circulating clonal plasma cells, and by studying clonal diversity and immune profile. In Aim 3, we will institute and determine the feasibility and impact of a screening approach for identification of SMM eligible for early intervention, by targeting high risk populations, specifically African Americans, first-degree relatives, and persons with high total protein levels. Our grant will have a major impact on the management of patients with SMM and MM, especially African Americans and first-degree relatives or persons with MM.

NIH Research Projects · FY 2025 · 2011-08

In phase IV of eMERGE, a single, Network-wide, combined risk assessment and management protocol was implemented across the various groups represented in the 25,000 patients recruited at 10 sites in the United States (US). Polygenic risk scores (PRS) were returned for 10 conditions, family history was ascertained using the MeTree or backup survey and monogenic basis was assessed for Tier 1 conditions. In this Supplement application we propose to finalize analysis-ready datasets, ascertain near term outcomes within 1 year after return of results (RoR), assess response of providers and participants to the return of Genome Informed Risk Assessment (GIRA). Our specific aims for the extension year are: Aim 1. Create a curated dataset of eMERGE IV participants for analyses. Using data from Common Data Platform (CDP), surveys, and the EHR, finalize an analysis ready data set for assessment of outcomes and genomic discovery. Along with genomic data from Broad and Invitae, phenotype dataset will be placed in ANVIL and we will deploy Tanagra and 12b2 to analyze data. Aim 2. Ascertain near-term outcomes after return of GIRA through automated and manual abstraction. We will use novel phenotyping methods described in Aim 1. We will compare the accuracy of EHR algorithms with and manual abstraction. Outcomes will include process, intermediate and clinical outcomes and will be assessed Network-wide as well in a condition-specific manner. We will explore phenotype abstraction using Large Language Models. Aim 3. a) Clinical informatics and EHR integration Continue to develop an ecosystem to store GIRA in the EHR and develop computable representation of the PRS report to enable automated generation of clinical decision support (CDS). b) Investigate ELSI aspects including assessing responses to PRS return among patients and providers. Aim 4. Genomic discovery using the EHR. We will compare predictive value of the PRS, family history and monogenic conditions for disease, stratifying by self reported race/ethnicity. We will also compare the predictive value of SDOH to PRS and jointly model both to improve risk assessment. Genomic data from Broad and Invitae will be placed on AnVIL will enable numerous analyses including computing updated PRS and also PRS for conditions that were not selected for clinical deployment, e.g., abdominal aortic aneurysm and deep vein thrombosis.

NIH Research Projects · FY 2026 · 2011-05

PROJECT SUMMARY/ABSTRACT Biofilm bacteria cause two-thirds of infections in modern clinical practice, including wound infections. Host defenses and most available antibiotics are inactive against biofilms, rendering the infections they cause challenging to treat. Given the failure of antibiotics in management of biofilm-associated infections, novel and innovative approaches are needed. Avoiding antibiotics will also decrease the dysbiosis and selection of genotypic antibiotic resistance linked to their use. Our team, which includes microbiologists and animal and human researchers at Mayo Clinic, alongside electrochemists and biofilm engineers at Washington State University, is developing novel, mechanistically precise, potentiostatically driven electrochemical anti-biofilm devices for wound infection prevention and treatment, and promotion of wound healing, which we call electrochemical bandages (e-bandages). During the current funding period, we designed and constructed a small prototype e-bandage controlled by a customized micropotentiostat that continuously generates non-toxic concentrations of H2O2. We tested the H2O2-generating e-bandage in vitro and in mice. in vitro activity was demonstrated against mono- and dual-species biofilms formed by 34 bacterial and 15 yeast isolates. The e- bandage prevented and treated methicillin-resistant Staphylococcus aureus (MRSA) biofilms in a porcine ear explant model and was then evaluated in vivo in a wound MRSA and/or Pseudomonas aeruginosa infection model in mice where, compared to non-polarized or no e-bandage treatment, polarized e-bandage treatment reduced bacterial counts in infected wounds, improved wound closure, and decreased wound purulence. Based on success developing a small, H2O2-producing e-bandage and micropotentiostat to control its operation, and demonstration of efficacy in treating wound infections in mice, we propose to scale up the device to sizes suitable for human wounds of varying dimensions. We will confirm infection treatment and wound healing activity and assess wound infection prevention in swine, and preliminarily evaluate safety on healthy human skin. e-Bandages will be designed to conform to varying wound dimensions and geometries while contacting uneven wound topologies to deliver H2O2 to the entire injury. We hypothesize that H2O2- producing e-bandages will prevent and treat MRSA and P. aeruginosa infections and improve wound healing in swine. We will preliminarily assess safety and tolerability of e-bandages when placed on normal human skin. We hypothesize that H2O2-producing e-bandages will be well-tolerated by humans. Our goal is to have a product ready for testing in human wounds at the end of our studies - that is, a product for infection prevention and treatment, and promotion of wound healing. The innovative H2O2-producing e-bandage strategy provides an original way to address wound infection prevention and treatment, avoiding conventional antibiotics and, therefore selective pressure on commensal microbiota and emergence of antibiotic resistance.

NIH Research Projects · FY 2026 · 2011-04

Project Summary Epicardium is a mesothelium layer that covers the surface of a vertebrate heart. Epicardial activation, which is reflected by increased expression of certain epicardial genes in the whole epicardium, has been reported to play a vital role during heart regeneration. However, whether epicardial activation occurs in cardiac aging or cardiac diseases such as cardiomyopathies remains unknown. Using a zebrafish bag3 cardiomyopathy model, we recently noted accelerated cardiac aging in this cardiomyopathy model, and discovered fatty acid binding protein 7 (fabp7) as a therapeutic modifier gene. Interesting, Fabp7 manifests remarkable activation in the epicardium in the bag3 cardiomyopathy model, prompting that epicardial activation and associated remodeling an important pathological event. We went on to screen for aging-associated genes and identified gpnmb, a seno-surface marker, as another therapeutic modifier. Different from fabp7 that is activated in epithelial epicardium, gpnmb is activated in mesenchymal epicardium, a different epicardial subset. Consistent with the hypothesis that epicardial remodeling is an aging-associated event, we noted epicardial remodeling during normative cardiac aging in killifish and mouse. Together, these preliminary data prompted us to propose the central hypothesis predicting that epicardial remodeling is a pivotal pathological event in bag3 DCM and cardiac aging, which can be harnessed to mitigate cardiomyopathy and to rejuvenate an aged heart. Our proposal will be divided into the following three specific aims. In specific Aim 1, we focus on fabp7 and propose to test hypothesis that activation of fabp7 in epithelial epicardium represents a pathological event in bag3 DCM that can be inhibited to exert cardioprotective effects. In our Specific Aim 2, we propose to study gpnmb as a mesenchymal epicardial marker for epicardial remodeling. Its relationship with fabp7 in epithelial epicardium will be deciphered by promoter analysis, lineage tracing and genetic interaction studies. In Specific Aim 3, we propose to directly test the hypothesis that epicardial remodeling is a hallmark of normative cardiac aging. To facilitate genetic studies of cardiac aging, we will integrate African turquois killifish, a vertebrate model with the shortest lifespan, into our study. Once successful, the proposed experiments would significantly advance cardiomyopathy and cardiac aging fields by establishing epicardial remodeling as a novel pathological event in bag3 DCM, as well as a hallmark for normative cardiac aging.

NIH Research Projects · FY 2026 · 2011-02

The primary microstructural attributes seen in “normal” kidneys are nephrosclerosis (arteriosclerosis, global glomerulosclerosis, and interstitial fibrosis/tubular atrophy), nephron number, and nephron size. However manual measures of these microstructures are impractical. Further, understanding their pathophysiology may lead to new interventions for kidney disease. Automated morphometry with deep learning (DL) networks may be able to rapidly measure nephrosclerosis, nephron number, and nephron size. Novel morphometry of structures impacted by glomerular hyperfiltration (podocyte and parietal epithelial cell (PEC) density, Bowman’s space, and the diameter of proximal and distal tubule) and of microvasculature are needed to better understand the pathophysiology of early disease. Proteomic analysis of specific microstructures that differ between kidneys that do versus do not develop CKD outcomes has the potential to identify prognostic or even pathogenic proteins for early kidney disease. The multi-discipline multi-site Aging Kidney Anatomy study has unique resources for the study of microstructure in “normal” kidneys. This includes data and specimens on living kidney donors including needle core biopsies at donation with digitized whole slide images (WSI) and long-term CKD outcomes in the donor and recipient. This also includes data and specimens on patients who had a radical nephrectomy for tumor including digitized WSI of kidney wedge sections and annual eGFR testing for CKD outcomes during follow-up. Aim 1 will determine if automated morphometry of nephrosclerosis, nephron number, and nephron size predicts CKD outcomes to test the hypothesis that DL tools allows for efficient quantification of these clinically relevant microstructural attributes. This aim will use both previously developed and new DL networks, develop models to predict CKD outcomes from automated morphometry, and compare prediction of CKD outcomes between automated and manual morphometry. Aim 2 will characterize novel microstructural attributes that associate with kidney function, CKD risk factors, and CKD outcomes to test the hypothesis that encoded in the kidney tissue are unexplored structural attributes that reflect the glomerular hyperfiltration and interstitial microvascular status that are prognostic for CKD. This aim will automatically quantify podocytes, PECs, peritubular capillaries (PTC), Bowman’s space (volume), and proximal and distal tubules (diameter) on WSI using previously developed and newly developed DL tools and associate these structures with kidney function, CKD risk factors, and CKD outcomes. Aim 3 will discover protein markers linked to the microstructural attributes that are prognostic for CKD outcomes to test the hypothesis that differentially expressed proteins contained within kidney microstructures predict CKD outcomes. This aim will use laser capture microdissection, mass spectroscopy-based proteomics (both discovery and targeted validation approaches), and immunohistochemistry to identify proteins on kidney biopsy sections that predict CKD outcomes and to determine their association with microstructural attributes.

NIH Research Projects · FY 2024 · 2010-09

ABSTRACT The Mayo Clinic BIRCWH program has been funded for two cycles since 2010. Over the 9 years of the BIRCWH program, we have demonstrated progress toward our long term goal of increasing the science workforce of interdisciplinary teams translating scientific discoveries into clinical practice to improve women’s health. Evidence of this success is provided by the fifteen scholars trained in the BIRCWH program who enhanced the interdisciplinary research in women’s health at Mayo Clinic. BIRCWH scholars received 61 grants as Principle Investigators with a return of investment of $2.91 per NIH dollar as Principle Investigators and $30.2 per NIH dollar as Principle Investigators or Co-Investigators. Our short-term goal in this cycle is to provide training to investigators who can assume leadership roles in interdisciplinary research teams in women’s health. In order to achieve these goals, our program plan is structured around three aims that align with the three pillars of the BIRCWH program: interdisciplinary research, mentoring, and career development. First, we aim to recruit a diverse group of early-stage investigators with an outstanding potential to lead interdisciplinary research teams in women’s health. Our second aim is to provide both structured and tailored educational experiences based on career development needs that include didactic programs and a mentored research experience in an interdisciplinary environment. And finally, we aim to partner with other BIRCWH programs, career development programs within Mayo Clinic, and nationally, to enhance training in women’s health research and foster a community of scholars. A strong community of fellow scholars and established investigators in women’s health research provides the networking opportunities and supports collaborations essential for sustaining and developing new interdisciplinary research programs. The rich environment of other career development programs at Mayo Clinic such as the Mayo Clinic Center for Translational and Clinical Science (CCaTS) and the Specialized Centers of Research Excellence (SCORE) on Sex Differences programs, training in incorporating sex as a biological variable in research, and our collaborations with the BIRCWH programs at other institutions provide outstanding networking opportunities for our scholars.

NIH Research Projects · FY 2026 · 2010-02

PROJECT SUMMARY In 2020, nearly 808,000 U.S. patients had end-stage kidney disease (ESKD) and the majority underwent hemodialysis (HD) using arteriovenous fistulas (AVFs). AVFs have ~62% 1-year patency due to venous neointimal hyperplasia (VNH) and venous stenosis (VS). The molecular mechanisms of VS/VNH after AVF are not understood. We completed a randomized, blinded phase 1 clinical trial where 21 patients (5 females, average 65 yo) underwent periadventitial delivery of autologous adipose derived mesenchymal stem cells (MSC, n=10) or placebo to the outflow vein at new upper extremity AVF creation. The primary endpoint was time to AVF maturation. It was significantly decreased in MSC treated patients versus controls (1.5 vs 2.5 m respectively, Log Rank: P<0.05). Several MSC- treated patients had AVFs with added procedures to maintain patency (‘non-responder MSCs’). Murine xenografts using human MSCs (hMSCs) from responders had PPARg reduction in CD68 (+) cells and this is protective for VS/VNH formation. Senescence alters cellular function leading to increased production of pro-inflammatory cytokines known as senescence-associated secretory phenotype (SASP). MSCs are anti-inflammatory and reduce VS/VNH by decreasing many of the SASP cytokines. MSCs isolated from patients with chronic kidney disease are senescent. We hypothesized that senescence is responsible for the dysfunction of MSCs from non-responders, obtunding the ability of MSCs to reduce inflammatory cues responsible for VS/VNH. Studies show senolytic drugs such as Dasatinib and Quercetin (D&Q) can decrease the senescent burden in cells and organs leading to improved function. So, we treated hMSCs from responder and non-responder with D&Q (D&Q hMSCs) and found it decreased Sensig scores (senescent gene expression) and increased proliferation. In vitro studies showed conditioned media (CM) from MSCs reduced PMA induced THP-1 (monocyte) cell differentiation with an increase in pSTAT3 with a decrease in PPARg expression. Next, D&Q hMSC xenografts in mice with AVFs were created and there was a decrease in Pparg gene expression, Fridman_Up (fibrosis gene), and Sensig scores accompanied with a reduction in inflammatory, SMCs, fibrosis, with increased endothelial cells (ECs), and less VS/VNH. Our central hypothesis is that arteriovenous fistulas treated with D&Q MSCs increase pSTAT3 and decrease PPARg in resident monocytes/ MΦs of AVFs leading to less pro- inflammatory cells resulting in positive vascular remodeling, less fibrosis, Sensig, Fridman-Up scores, and increased endothelization There are three specific aims: Aim 1. Investigate the role of CM from D&Q hMSCs from non-responders and responders on monocyte to macrophage differentiation, activation, pSTAT3/ PPARG signaling, functional changes, and Sensig score. Aim 2. Determine the effect of D&Q hMSC on pSTAT3/ PPARg signaling and VS/VNH formation in murine AVFs. Aim 3. Assess the efficacy of D&Q MSC in retarding VS/VNH in a pig AVF model.

NIH Research Projects · FY 2025 · 2009-09

PROJECT SUMMARY The overall objective of the Mayo Clinic Center for Cell Signaling in Gastroenterology (C-SiG) is to improve the health of patients with digestive diseases. We do this by facilitating discovery-translation-application paradigms driven by mechanistic insights, cell signaling pathways, cellular networks, and spatial biology of gastrointestinal tissue. C-SiG provides a robust infrastructure supporting technological advances, thematic platforms, and career development opportunities leading to the integration of diverse expertise and impactful discovery and translational science. Our Research Base consists of 59 scientists (16% increase since the center was founded in 2009) involving 15 departments/divisions and $22 million direct costs (64% growth since center was founded in 2009) in digestive diseases-related funding (47% from NIDDK). Responding to members’ evolving interests and scientific advances, we’ve realigned members into three interconnected Mechanistic Research Themes (cellular networks, intracellular signaling, and genetics/epigenetics), each intersecting with three Disease Focus Groups (liver pathobiology, dysmotility/metabolism, inflammation/transformation), a matrix that fosters both discovery and disease relevant investigation. Our ongoing CENTRAL HYPOTHESIS is that advances in care of patients with digestive diseases requires a facilitative infrastructure supporting meaningful interactions among multidisciplinary scientists investigating cellular mechanisms, pathways, and therapeutic targets to enhance rapid translation of basic discoveries into clinical trials. Our OVERALL SPECIFIC AIMS are to: i) Foster multidisciplinary research by integrating a diverse group of clinical and basic science investigators in a team- based approach to advancing knowledge and technical capabilities; ii) Offer access to cutting-edge, specialized technologies, resources and skilled technical expertise through core services (Microscopy and Microfluidics, Epigenomics and Spatial Biology, and Clinical Cores), with continually evolving service options and quality and project management oversight in response to member feedback; iii) Create opportunities to engage and nurture new GI investigators via a peer-reviewed Pilot and Feasibility (P/F) Program including structured mentorship, career development retreats, curricula, and structured (30/42, 71% of P/F recipients achieving federal funding); iv) Support a robust Enrichment Program facilitating collaboration and technology transfer; and v) Promote interactions between C-SiG with Mayo institutional partners (e.g., Center for Individualized Medicine) and existing DDRCCs, especially in the Midwest (i.e., Midwest DDRCC Alliance). Our global efforts have resulted in 160 manuscripts, with 46% percent intra- and 54% inter-thematic publications (77% involving ≥ 2 members). Importantly, we’ve made critical advances in understanding disease pathogenesis relevant to cellular networks, signal transduction, and genetics/epigenetics as evident by the academic and translational achievements of our research base.

NIH Research Projects · FY 2026 · 2009-08

Project Summary Pulmonary fibrosis (PF) remains a major and growing medical burden with unsatisfactory therapeutic options that fail to reverse established disease. Our prior work has identified that YAP/TAZ activation in fibroblasts is a central feature of the pathological feedback loop that propagates progression of PF. In the prior funding cycle, we identified Dopamine D1 receptor (D1R) agonism as a strategy to inactivate YAP/TAZ selectively in lung fibroblasts, leading to accelerated resolution of experimental pulmonary fibrosis in mice in part by switching lung fibroblasts from matrix depositing to matrix degrading state. Moreover, we demonstrated that the lungs of individuals with PF exhibit a deficit in expression of DOPA decarboxylase (DDC), the enzyme that catalyzes conversion of L-DOPA into bioactive dopamine. These studies lead us to propose that restoration of endogenous local dopamine levels in the lung is an essential trigger for the matrix degradation and fibrosis clearance that is essential to successful repair of the lung, but is impaired in PF. Our preliminary data show that Ddc transcripts are transiently depressed in lung tissue of young mice following bleomycin injury and rise during fibrosis resolution, whereas aged mice exhibited sustained reductions in Ddc that parallel persistent fibrosis. Moreover, small molecule inhibition of Ddc enzymatic activity or the D1R from day 21 to 42 post- bleomycin in young mice ablates the spontaneous resolution of lung fibrosis, demonstrating the essential role for dopamine signaling in fibrosis resolution. In addition, we find that dopamine is detectable in supernatants of precision cut lung slices and is diminished in slices cultured from fibrotic lungs, confirming the local synthesis of dopamine within the lung. Based on these findings, we propose to test the central hypothesis that epithelial dopamine synthesis is essential to fibrosis resolution and that restoration of normal dopamine levels in PF lung tissue can promote collagen resorption and repair of the lung. We will test this hypothesis in three aims spanning non-resolving mouse models of pulmonary fibrosis as well as ex vivo models of mouse and human lung tissues. To define the functional roles of dopamine signaling we will leverage both cell-specific conditional genetic models as well as well-characterized small molecule inhibitors and dopamine agonists in these systems. Together our studies will define the cellular sources and regulatory systems that control dopamine bioavailability during normal lung repair, and will delineate how this repair system fails in human PF. These studies may reveal new therapeutic approaches to promote fibrosis resolution and lung repair.

NIH Research Projects · FY 2026 · 2009-07

PROJECT SUMMARY The broad objective of the current application is to advance our understanding of 2 major clinical phenotypes of heart failure with preserved ejection fraction (HFpEF): 1) HFpEF with volume overload in the presence of chronic kidney diseases (HFpEF-CKD) and 2) HFpEF with exercise induced (HFpEF-EI) dyspnea, to elucidate the differences in the pathophysiological mechanisms, to identify biomarkers to differentiate the two clinical phenotypes and to develop novel therapies for individualization of treatment. 50% of patients with heart failure (HF) have preserved EF. Pathophysiological heterogeneity in HFpEF is substantial, ranging from chronic kidney diseases, diabetes, obesity, hypertension, etc. There is no FDA approved therapy for HFpEF (LVEF>55%) which may be due to the heterogeneous underlying pathophysiological causes. Recently, the NHLBI Research Priorities for HFpEF Working Group emphasized the need for phenotyping of patients with HFpEF so as to classify patients into phenotypically homogeneous subpopulations, to understand pathophysiological mechanisms and to facilitate individualization of treatment. Sacubatril/valsartan is a dual angiotensin receptor (AT1) blocker and neprilysin (NEP) inhibitor which is approved for management of HFrEF. However, the PARAGON Study failed to demonstrate significant clinical benefit in HFpEF patients. This may be because NP are very low in some subgroups of HFpEF, thus negating the actions of NEP inhibition and therefore, Sacubatril/valsartan effectively functions as an AT1blocker, which has previously been shown to be not beneficial in HFpEF. Therefore, we hypothesized that the endogenous NP levels (specifically ANP) are low in those with exercise induced dyspnea as compared to those with CKD and extravascular fluid overload. Hence, those with HFpEF-EI may not respond to Sacubatril/valsartan but will respond to exogenous NPs administration, while those with HFpEF-CKD will respond to Sacubatril/valsartan due to increased endogenous NPs. MANP is a novel particulate-guanylyl-cyclase A (pGC-A) receptor activator designed at the Mayo Clinic which is more potent than ANP in promoting natriuresis, inhibiting aldosterone with greater activation of cGMP and longer half-life. Our Specific Aims: Specific Aim 1: To perform high definition phenotyping of HFpEF-CKD and HFpEF-EI, defining the differential cardiorenal and humoral response to acute saline volume expansion (VE) Specific Aim 2: To determine the effects of neprilysin and angiotensin receptor inhibition with Sacubatril/valsartan on the cardiorenal and humoral response to acute VE in HFpEF-CKD and HFpEF-EI. Specific Aim 3: To determine the effects of MANP on the cardiorenal and humoral response to acute VE in HFpEF-CKD and HFpEF-EI. The impact of our proposed studies is high as it will advance our knowledge of the integrated cardiorenal and humoral physiology in patients with HFpEF-CKD and HFpEF-EI, and to test novel diagnostic and therapeutic strategies specific for HFpEF-CKD and HFpEF-EI, thus advancing a precision medicine approach in HFpEF.

NIH Research Projects · FY 2025 · 2009-07

PROJECT SUMMARY – OVERALL This revised application of the Mayo Clinic SPORE in Ovarian Cancer (OC) builds on translational research con- ducted during Years 6-10 of funding. Our accomplishments over the past five years include i) identification of a unique vaccine strategy that, in a phase I study, led to 39% recurrence free survival at 49 months in high grade serous OC, ii) new understanding of the genomic and biochemical changes that limit the action of PARP inhibitors (PARPis) in OC, iii) distribution of over 9600 biospecimens for OC research, and iv) publication of 220 articles. The Developmental Research Program (DRP) has contributed to two of the four projects in this renewal; and the Career Enhancement Program (CEP) contributed to leadership of two projects. The overall goal of the SPORE remains to support innovative, interactive, translational OC research that leverages the expertise of bas- ic and translational investigators. This renewal contains four translational projects designed to investigate OC biology and enhance therapeutic response, building on recent clinical advances and promising preclinical results: • P1 (Development of a Th17-Inducing Dendritic Cell [DC] Vaccine for OC): Based on our study showing that DCs pulsed with Folate Receptor α peptides and matured to a Th17-inducing phenotype induced immune re- sponses in all patients and long-term recurrence-free survival in 39%, we will identify determinants of long- term vaccine response in a phase II trial and elucidate mechanisms of immune escape from this vaccine. · P2 (Next Generation TOP1 Inhibition for the Treatment of OC): Building on our observation that TOP1 inhib- itors are active in PARPi-resistant OC models and this activity can be enhanced by PARPi treatment even in the face of PARPi resistance, this project will identify determinants of sensitivity to TOP1 inhibitor/PARPi combinations and conduct a phase II trial of the ultra-long acting TOP1i PLX038 with the PARPi rucaparib. · P3 (Repurposing Ceritinib for OC Therapy): Based on the finding that ceritinib, a kinase inhibitor used for ALK- rearranged lung cancer, inhibits mitochondrial respiration, increases reactive oxygen species, and sensitizes OC cell lines and PDXs to PARPis independent of ALK status, we will identify pathways that mediate these effects and conduct a phase I trial of the ceritinib/olaparib combination. · P4 (Treatment of Advanced OC Using Gene-Edited CAR NK Cells): Building on a prior developmental research project, this team located at the University of Minnesota will apply advanced cellular engineering techniques to generate activated NK cells with enhanced tumor homing and persistence, then test the safety and efficacy of administering this allogeneic adoptive immunotherapy in a phase I clinical trial in platinum-resistant OC. These impactful projects are supported by four highly interactive cores: Core A (Administrative), Core B (Biospec- imens/Patient Registry), Core C (Biostatistics/Bioinformatics) and Core D (Animal Models). A DRP and CEP will be used to nurture the next generation of translational OC investigators. Collectively, these SPORE activities will provide new insight into OC biology while examining potential therapeutic advances for this lethal disease.

NIH Research Projects · FY 2025 · 2009-05

PROJECT SUMMARY/ABSTRACT The goals of this Musculoskeletal Research Training Program are to provide trans-disciplinary research opportunities for postdoctoral fellows, graduate students, and medical students at Mayo Clinic and to train them to be future leaders of biomedical and musculoskeletal research. Musculoskeletal ailments such as osteoarthritis, osteoporosis, back pain, tendinopathies, skeletal muscle atrophy, sarcopenia and fractures are some of the most common reasons why people of all ages visit a doctor. They significantly impact quality of life, cause disabilities, and impose large personal and societal burdens. The repair, regeneration, or rejuvenation of musculoskeletal tissues and joints requires knowledge of complex and interconnected biomechanical, biological, and physiological processes. This program aims to train future biologists, engineers, physicians, and surgeons to solve orthopedic and musculoskeletal problems by providing outstanding research and educational opportunities within the setting of a state-of-the-art medical and research center. Forty faculty members, who are leaders in basic, translational, and clinical research of orthopedics and the musculoskeletal system, will mentor trainees by directing research projects and group discussions on timely topics. Peer-mentors provide new trainees with opportunities to receive advice and counseling from past trainees who successfully obtained independent fellowships. All trainees will receive training in the responsible conduct of research, data management, and in scientific rigor and reproducibility. Postdoctoral fellows (5 per year) and graduate students (2 per year) will engage in multiyear projects and receive training in grant writing and career development. They will be expected to submit applications for independent fellowships and to participate in a full array of programmatic activities, including but not limited to journal clubs, seminars, symposia, webinars, and scientific meetings. Medical students (2 per year) from fully accredited medical schools in the United States and her territories (i.e., Puerto Rico) will spend two to three months in the training program and are expected to complete a mentored research project and participate in programmatic educational activities. This blended musculoskeletal research training program values individuals from diverse educational and societal backgrounds. Our community benefits from the unique perspectives they bring to solving complex medical problems, identifying social determinants of musculoskeletal disease, and reducing the burden of musculoskeletal conditions on patients, their families, and our society.

NIH Research Projects · FY 2026 · 2008-04

Project Summary/Abstract This application is focused on probing how post-translational modifications (PTMs) contribute to the DNA damage response (DDR), a vital process for the maintenance of genomic integrity. The long-term goal is a basic mechanistic understanding of DNA damage repair that will facilitate the development of new therapeutic strategies to combat cancer. In this context, the proposed overall objective is to elucidate how the tumor suppressor E3 ubiquitin ligase BRCA1-BARD1 and DDR protein 53BP1 regulate the cell cycle dependency of DNA double-strand break (DSB) repair pathway selection. BRCA1-BARD1 promotes DSB repair by homologous recombination (HR) during S and G2 phases of the cell cycle whereas 53BP1 inactivates HR and promotes repair by non-homologous end-joining (NHEJ) mostly in G1 phase. Imbalance between HR and NHEJ—for example linked to mutations in BRCA1-BARD1—can result in chromosomal rearrangements that drive oncogenesis. BRCA1-BARD1 and 53BP1 can recognize a similar DSB-dependent PTM, the ubiquitylation of histone H2A at K15 in the nucleosome, the basic subunit of chromatin, suggesting that competition for chromatin association in response to DNA damage could account, at least in part, for the antagonistic activities of BRCA1- BARD1 and 53BP1 with respect to HR. The central hypothesis to be tested is that BRCA1-BARD1, through its ubiquitin ligase activity on the nucleosome, indirectly leads to 53BP1 displacement from damaged chromatin and directly prevents the establishment in newly replicated chromatin of a PTM (i.e., dimethylation of histone H4 K20) required for the chromatin recruitment of 53BP1. The rationale for this work is that it will provide fundamental knowledge about the mechanisms of action of BRCA1-BARD1 and 53BP1. Such knowledge is important for understanding how DSBs are repaired with implications for cancer prevention and the development of new therapies. Two specific aims will be pursued to test the central hypothesis. Aim 1: Probe how BRCA1-BARD1 associates with and modifies chromatin in response to DNA damage. Aim 2: Probe how 53BP1 associates with chromatin and is antagonized by BRCA1-BARD1. In both aims, structural biology approaches will be integrated to characterize BRCA1-BARD1, 53BP1 and a chromatin remodeler that may connect BRCA1-BARD1 and 53BP1 functionally. The structures will inform functional in vitro studies and collaborative cell biology to reveal the chromatin recruitment mechanisms of BRCA1-BARD1 and 53BP1. The proposed research is significant because it is expected to explain how BRCA1-BARD1 opposes the chromatin recruitment and retention of 53BP1 to promote HR DSB repair post-replication. This work will therefore explain how BRCA1-BARD1 and 53BP1 regulate DSB repair pathway selection in a cell cycle-dependent manner. Knowledge gained from this research is expected to stimulate the long-term development of new therapies to treat cancer.

NIH Research Projects · FY 2026 · 2008-04

Project Summary/Abstract The identification of cancer metastases to the bony vertebral column obligates the treating clinician to make a surgical decision. Current spinal stability decision-making is empirical, qualitative, and can be inaccurate. The consequences of that decision for the patient, however, are significant. If the spine is deemed at risk for fracture, then the patient will undergo a major spinal operation. Conversely, the patient whose spine is deemed stable risks fracture and possible paralysis if the analysis was incorrect. This research program addresses both the stability decision and the nature of the treatment. In this renewal application, we will continue our efforts to develop non-invasive, quantitative, and reliable methods to predict the fracture risk of vertebrae with metastatic cancer under physiologically relevant loading conditions, and to optimize minimally invasive techniques using novel biomaterials to reconstitute the load bearing capacity of an affected vertebra. In Aim 1, we propose a novel injectable polymer network that can be self-crosslinked via catalyst-free click chemistry into “click” organic-inorganic nanohybrid (click-ON) bone cement. Compared to our previous injectable system, the novel cement has improved biocompatibility, injectability, and crosslinking efficiency. In Aim 2, we will investigate the efficacy of the optimized click-ON bone cement to both prevent impending fractures and treat existing fractures in cadaveric models using the clinical vertebroplasty and kyphoplasty procedures, respectively (Aim 2a). Intact lumbar spines (L1-S1), spines with simulated lytic defects, and spines with biomaterial augmentation will be tested under accurate and biomimetic loading conditions using a novel robotic testing system. Our previously developed quantitative computerized tomography based finite element analysis (QCT/FEA) models will be expanded to include both kinematic motion evaluation and fracture risk prediction under physiological loading and boundary conditions and validated using the experimental results (Aim 2b). In Aim 3, We will develop a phantom-less calibration technique to account for the effects of QCT protocols on QCT/FEA results (Aim 3a). Using the powerful AnalyzeMD platform, we will implement an automated process to further advance the FEA technique for time efficiency and reproducibility (Aim 3b). We will apply the comprehensive QCT/FEA models in a retrospective cohort of spine metastasis patients and assess the virtual reconstruction using the click-ON bone cement as a first step towards clinical translation. The QCT/FEA technique developed in this work takes into consideration both the quality and quantity of bone and the degeneration status of the intervertebral discs. This technique allows the clinician to counsel her/his patient regarding activities of daily living that can be performed with a low risk of spinal fracture. Our future plans are to expand the clinical implementation of the spinal FEA analysis at Mayo Clinic. We will add FEA evaluation results in our discussion with the patients regarding our recommendations for their care. We will study the outcome results of those recommendations, adjust the decision parameters as necessary, and then extend the analysis to additional institutions.

NIH Research Projects · FY 2026 · 2007-08

The overall aim of this application is to better understand the pathogenesis of fasting hyperglycemia and its contribution to the development of type 2 diabetes. Insulin and glucagon are the most important glucoregulatory hormones. There is evidence that both α-cells and β-cells (which secrete glucagon and insulin, respectively) can directly regulate (stimulate or inhibit) each other. In addition, both hormone systems alter glucose concentrations through effects on endogenous glucose production (EGP). Through these actions, insulin can indirectly affect glucagon secretion and vice versa. Dysfunction of these regulatory networks leads to prediabetes and, subsequently, to type 2 diabetes. However, the relative importance of these abnormalities, and how they interact to differentially affect fasting vs. postprandial glucose tolerance remains unknown. There is also controversy as to whether minute to minute variation in insulin secretion can control glucagon secretion and whether these pulse characteristics can serve as biomarkers of islet function. In this series of experiments, we will examine how glucagon directly, and indirectly through insulin, affects glucose metabolism. Conversely, we will examine how insulin directly, and indirectly through glucagon, alters glucose metabolism. Subsequently, we will use our novel methodology to measure islet hormone pulse characteristics to identify early defects in islet cell function. The proposed experiments will help elucidate the mechanisms by which fasting hyperglycemia develops in different subtypes of prediabetes thereby providing opportunities to individualize intervention. In addition, we will develop new methods to quantify fasting islet function and identify new biomarkers allowing early prevention and treatment of type 2 diabetes.

NIH Research Projects · FY 2025 · 2005-08

Project Summary TITIN (TTN) truncating variants (TTNtvs) have been found to be the most common genetic factor for dilated cardiomyopathy (DCM). However, allelic heterogeneity (AH) of TTNtv DCM, i.e. TTNtvs are also found in reference populations, significantly confound diagnosis and therapeutic development of these patients. Pathogenic TTNtvs are mainly found in the C-terminal A-band region (TTNtv-As) but less in the N-terminal Z- disc region (TTNtv-Zs). Moreover, pathological signaling pathways for TTNtv DCM remain largely unknown. Here, we aim to leverage unique research opportunities enabled by zebrafish genetics to decipher underlying mechanisms of AH, discover pathological signaling pathways, and develop effective therapeutic avenues. Our preliminary studies showed that AH of TTNtv DCM can be recapitulated in both embryonic and adult zebrafish, opening the door for mechanistic studies of AH in vivo. From our screen of known cardiomyopathy signaling pathways, we identified mTOR, autophagy, MAPK and PDE1 as candidate signaling pathways that could be leveraged for therapeutic benefits. We also established a F0-based genetic assay using the Microhomology- mediated end joining (MMEJ) genome editing technology that enables us to rapidly discover new signaling pathways. Based on these preliminary studies, we proposed to leverage unique genetic and chemical genetic tools in zebrafish to prove that zebrafish is the first in vivo animal model for allelic heterogeneity of TTNtv DCM, which can be used to decipher primary damages incurred by TTNtvs, to discover sequential pathological signaling pathways, and to develop mechanism-based therapies. The proposal is organized into the 3 specific aims. In Specific Aim 1, we will decipher allelic heterogeneity of ttntv DCM via studying a panel of ttntv mutants and ttn null mutants. In Specific Aim 2, we propose to elucidate molecular basis of autophagy dysregulation in ttntv DCM and develop an autophagy-based therapy. In Specific Aim 3, we will confirm MAPK and PDE1 as candidate signalings and discover additional new genes and signaling pathways by carrying MMEJ-based F0 screens. Upon completion of the proposal, we anticipate the following deliverables: 1) provide in vivo evidence to clarify why TTNtv-As are more likely to cause DCM phenotypes than TTNtv-Zs; 2) obtain insights on autophagy, MAPK and PDE signaling pathways in ttntv DCM, and identify mechanism-based therapeutic avenues for ttntv DCM; 3) establish a F0-based genetic screening approach that is capable of systematically discovering new genes for ttntv DCM, opening an unprecedented opportunity for mechanistic studies of an inherited cardiomyopathy.

NIH Research Projects · FY 2025 · 2005-07

Program Summary/Abstract This third renewal application of the Mayo Clinic Breast Cancer SPORE is being submitted with the vision that the burden of breast cancer (BC) can be reduced through the performance of innovative translational research addressing issues of high significance for women. The science of the SPORE includes three translational research projects. Project 1: “The influence of variants in ER-positive BC predisposition genes on BC risk and response to therapy”. This project builds upon the work from the prior funding period, where the principal investigators (PIs) identified that certain BC predisposition genes (ATM, CHEK2 and PALB2) were associated mainly with the risk of ER+ BC. The Pis will assess pathogenic variants in ATM, BRCA2, CHEK2, PALB2 and determine a) age-related risks, the risks of contralateral BC and second cancers (ovarian, colorectal, pancreatic etc.), b) the prognostic effects of these PV and c) the clinical relevance of variants of uncertain significance in ATM, CHEK2, and PALB2. Project 2: “Improving the endocrine management of premenopausal ER+/HER2- BC” brings forward a new ER-targeting drug, Z-endoxifen (ENDX), for premenopausal ER+ BC based on new data that ENDX dually targets both ERα and PKCβ1, with potent protein downregulation of ERα, Cyclin D1, and E2F1. This dual targeting is not only unique to ENDX, but at the dosing proposed in the clinical trial, ENDX is able to completely block the stimulatory effects of premenopausal levels of estrogen. Project 3: “Development of a novel multi-antigen BC prevention vaccine for women with premalignant disease” is based on pioneering work of Mayo investigators to develop a novel vaccine that targets six antigens collectively expressed by all BC subtypes. The work in this project will provide critical information that will allow the investigators to a) understand the expression of target antigens during progression from normal breast tissue to BC; and, b) in the phase I study, evaluate blood and breast tissue biomarkers of immune response. Successful completion of this project will establish the parameters for a phase III clinical trial of the vaccine for BC prevention in women with benign breast disease (BBD), with the enormous potential to drive significant reductions in overall BC incidence and mortality. These research projects are supported by three highly interactive cores: Core A: Administrative Core, Core B: Biospecimen and Pathology Core, and Core C: Biostatistics, Bioinformatics, and Patient Registry Core. A Developmental Research Program will continue to identify and develop research projects that hold the greatest promise to advance to full SPORE projects, and a Career Enhancement Program will continue to identify and support faculty investigators in BC translational research that have the greatest potential to become future SPORE leaders. The investigators, cores, and the research programs in the SPORE are all integrated in the Mayo Clinic Cancer Center. Collectively, our SPORE will make discoveries and translate them into the clinic for the benefit of women with, or at risk of BC.

NIH Research Projects · FY 2026 · 2002-04

Polycystic kidney diseases (PKD) are a group of disorders associated with defects in primary cilia and often causing end stage kidney disease. They can be divided into disorders mainly involving just kidney and liver, nonsyndromic PKDs (NS-PKDs), and ones involving other organ systems, including the brain, skeleton, and sensory organs, syndromic PKDs (S-PKDs). Autosomal recessive PKD (ARPKD; PKHD1) is the main recessively inherited NS-PKD, whereas S-PKDs are a group of diverse, mainly recessive diseases, including: Meckel [MKS], Joubert syndrome [JBTS]; and short rib thoracic dysplasia (SRTD), with up to 80 different genes involved. S-PKDs have marked genetic complexity, for instance the gene, TMEM67, is associated with several different disorders, and although the underlying reason for the varied phenotypes is not well understood, allelic effects may be important. In addition, it is becoming clear that heterozygous carriers of recessive PKHD1 alleles can have a mild cystic kidney/liver phenotype, similar to very mild autosomal dominant PKD (ADPKD). We have recently found that IFT140, an S-PKD gene encoding an intraflagella transport protein (IFT140) that is required to generate a fully functional cilium, also has a heterozygous phenotype of mild kidney cyst development. The goal of this grant is to better understand the genetic complexity associated with recessive (and sometimes dominant) PKD, with the premise that understanding the effects of gene loss and reduction (gene dosage), including the role of allelic effects, will improve our understanding of the etiology and pathogenesis of PKDs. Aim 1, Mutation screen a PKD positive and PKD unknown population to identify the role of “recessive” PKD alleles to the etiology of S-PKD and NS-PKD, will conduct mutation screening using next generation sequencing methods. A PKD cohort and a population of individuals not known to have PKD (Mayo Clinic Biobank; n=53,220) will be screened to determine the etiology of the PKD population and evaluate the role of single “recessive” PKD alleles to manifest as mild cystic disease. Aim 2, Develop cell-based assays to evaluate NS-PKD (PKHD1) and S-PKD (TMEM67) alleles, will establish in vitro systems to determine the pathogenicity of variants of unknown significance (VUS) in an NS-PKD gene, PKHD1, and an S-PKD gene, TMEM67. Trafficking, maturation, and ciliary localization of products of these genes will be determined. Aim 3, Explore the disease mechanism of IFT140 pathogenic alleles, will generate animal models to better understand disease pathogenesis. Both conditional and hypomorphic allele approaches will be employed to generate viable models and characterize the renal and extrarenal phenotypes. Aim 4, Determine ciliary defects and genetic interactions associated with NS-PKD and S-PKD genes, will monitor ciliary trafficking and composition in cells from NS-PKD and S-PKD models, and explore genetic interactions between these genes. Overall, these studies will provide diagnosis, prognostic and mechanistic data, important steps toward developing novel therapeutics for this group of devastating diseases.

NIH Research Projects · FY 2025 · 2001-09

The Mayo Clinic Cancer Center (MCCC) is poised to provide an exceptional career development experience for clinician investigators and other translational cancer researchers who seek to establish a career in patient- oriented research. The MCCC is a National Cancer Institute (NCI)-designated comprehensive cancer center with 45+ years of continuous funding; it sits at the epicenter of high-quality, practice-changing research. Its funding portfolio has remained highly competitive with respect to the acquisition of R01 and R01-equivalent funding over the past 10 years; includes 3 Specialized Programs of Research Excellence ("SPORE's") in cancer of hepatobiliary system, breast cancer, and lymphomas (the latter is a 2-institution, shared SPORE); and remains an active contributor to the NCI’s National Clinical Trials Networks (NCTN’s), including the Alliance for Clinical Trials in Oncology. In this application, the MCCC seeks to renew the "Paul Calabresi Program in Clinical/Translational Research at Mayo Clinic," requesting 5 career development positions at any one time for a dual track, M.D. or Ph.D., career development program. The MCCC has been privileged to hold this award since 2001. To date, this cohort of 40+ scholars remains engaged in translational cancer research; includes many who hold leadership positions in academia and other venues; and, over the years, has cumulatively amassed 3000+ publications during and after their scholar tenure. In this application, scholars must focus on 1 of 5 research tracks that align with translational MCCC Programs: Gastrointestinal Malignancies, Hematology, Neuro-Oncology, Novel Therapeutics, or Women's Cancers. Multidisciplinary mentoring teams will work with each scholar as he/she embarks upon a tailored career development program of didactic instruction, hands-on patient-oriented research, and a path toward career independence. Administrative infrastructure consists of a principal investigator who has been committed to this training program since its inception and an internal advisory committee comprised of accomplished leaders/mentors within the MCCC, and expertise in training a multi-disciplinary academic work force in cancer research. Marking almost 20 years of this program’s existence, this application now strives to examine ways to integrate innovative methods to further improve on the successful track records of future scholars. In essence, the MCCC seeks to train the next generation of investigators who will conduct cutting edge, hypothesis-driven, practice-changing cancer research.

NIH Research Projects · FY 2026 · 2001-08

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that results in progressive renal insufficiency and often kidney failure and accounts for ~5% of US kidney transplant and dialysis patients. ADPKD is phenotypically and genetically heterogeneous with seven genes now associated with this disease, including the major loci, PKD1 and PKD2. Large patient populations collected at Mayo Clinic have been central to new gene identification, and newly available populations of normal individuals with whole exome sequencing and clinical data are providing insight into the penetrance of ADPKD genes and alleles. In Aim 1, using these populations we will determine the extent to which phenotypes within the ADPKD spectrum are dictated by novel genes, and explore the penetrance of known alleles. Allelic diversity and complexity, especially for PKD1, has made diagnostics for this disorder complex, with many patients not obtaining a definite genetic diagnosis following testing using the existing variant evaluation guidelines; only variants of uncertain significance (VUS) are defined. Functional, cellular assays and whole animal systems can help to determine pathogenicity and penetrance of variants classified as VUS. In Aim 2 we will employ cellular and in vivo studies to determine the mechanism(s) of disease causation for PKD1 and PKD2 pathogenic variants. These studies will improve diagnostics and generate models that together will unravel the pathogenic mechanism of many nontruncating variants and allow the associated disease to be modeled. Insights into the pathomechanism of disease can also highlight new treatment options that are proximal to the primary genetic defect, strategies that have been successful exploited for other monogenic disorders. Two variant types in particular, missense changes that result in folding and trafficking problems, and nonsense variants are common causes of disease in PKD1 and PKD2. In Aim 3 we will, explore allele-based treatment options for ADPKD. Specifically, chaperone treatments will be tested for missense variants where a folding/trafficking defect is the mutational mechanism, and the value of readthrough drugs for a variety of PKD1 and PKD2 nonsense variants will be tested, both with cellular assays and in vivo systems. Treatment options for ADPKD also depend on better understanding the mutational mechanism at the level of the cyst; there has long been controversy in ADPKD about the importance and timing of somatic second hits for cyst initiation and/or expansion. In Aim 4 we will investigate the role of somatic changes in cyst initiation and development using single cell DNA sequencing methods. Overall, our proposal will result in better understand the pathomechanisms associated with the genetic complexity of ADPKD, with the premise that improved understanding will result in new targeted therapeutic options.

NIH Research Projects · FY 2025 · 2001-04

PROJECT SUMMARY/ABSTRACT Our OVERALL OBJECTIVE is to clarify the pathogenesis of Primary Sclerosing Cholangitis (PSC) and identify new therapies. PSC accounts for 6% of liver transplants, costs ~$125 million annually, affects ~40,000 Americans, has a median survival of ~15 years, and is associated with inflammatory bowel disease and increased risk of colon, bile duct and gallbladder cancers. There are no regulatory approved drugs for PSC because its pathogenesis is obscure. We reported that stress-induced cholangiocyte senescence (cell cycle arrest, apoptosis resistance, bioactive secretome) is a feature of PSC, informing its pathogenesis and revealing new therapeutic targets. Experiments proposed here extending this observation address the concept that the cholangiocyte response to injury in PSC is not uniform: some cholangiocytes arrest in the cell cycle (become senescent) while others acquire a ductular reactive, proliferative phenotype (resist senescence). We discovered fundamental differences in epigenetic and downstream signaling pathways that influence this binary cholangiocyte injury response. Our preliminary data show: i) we can isolate enriched subpopulations of senescent-sensitive and senescent-resistant cholangiocytes with distinct epigenetic signatures from human and rodent tissue, and from cholangiocyte cell lines; ii) chromatin modifiers, AP1 (transcription factor) and p300 (histone acetyltransferase), establish an epigenetic profile promoting senescence; iii) destabilization of the effector protooncogene, c-Myc, via the kinase, GSK3, drives senescence and cell-cycle arrest; in contrast, stabilization of c-Myc via the kinase, AKT, promotes senescence resistance and cholangiocyte proliferation; and iv) the cholangiocyte subtypes exhibit distinct bioactive secretomes that differentially affect portal fibroblasts (PF) and hepatic stellate cells (HSC). The data support our CENTRAL HYPOTHESIS that, in PSC, the cholangiocyte epigenome drives kinase cascades that determine if cholangiocytes develop senescent or proliferative phenotypes that influence the periductal and bridging fibrogenic responses of PF and HSC. Our integrated SPECIFIC AIMS test 3 hypotheses. Aim 1 Hypothesis: The epigenetic modifiers, AP1 & p300, promote open chromatin and active transcription via histone acetylation at senescence-associated enhancers/promoters and drive cholangiocytes to stress-induced senescence. Aim 2 Hypothesis: The epigenetic profiles of senescent resistant or sensitive cholangiocytes establish kinase cascades that either promote proliferation via c-Myc stabilization (AKT) or senescence via c-Myc destabilization (GSK3) and activation of the senescence effector, ETS1. Aim 3 Hypothesis: Genetic or pharmacologic inhibition of c-Myc or p300 alters the secretomes of proliferative and senescent cholangiocytes, their communication with PF and HSC, and periductal and bridging fibrosis. Thus, we will clarify how the epigenome determines the cholangiocyte injury response, how this binary response drives fibrogenesis, and how pharmacologic modification of the cholangiocyte epigenome can inhibit PSC progression.

NIH Research Projects · FY 2026 · 2000-04

PROJECT SUMMARY Interstitial cells of Cajal (ICC) are one of the two known main classes of interstitial cells that regulate gastrointestinal (GI) motor functions in health and disease. Throughout the GI tract, ICC modulate smooth muscle excitability, facilitate efferent and afferent neural control, and generate electrical slow waves (SWs), which drive phasic contractile activity underlying peristalsis and segmentation. Pacemaker activity is compartmentalized in specific ICC classes in an organ-specific manner. Current models of SW generation involve spontaneous transient inward currents triggered by Ca2+ release from the endoplasmic reticulum, activation of other ion channels including voltage-dependent Ca2+ currents, Ca2+-activated Ca2+ release, and store-operated Ca2+ entry. Previous work supported by this grant demonstrated selective expression of the regulated isoform of the electrogenic Na+/HCO3− cotransporter SLC4A4 (solute carrier family 4 member 4; isoform NBCe1b) in pacemaker ICC classes and revealed a role for intracellular HCO3−/pH regulation in SW activity, adding a new component to the ICC pacemaker apparatus. However, while SLC4A4’s roles in acid- base regulation and renal tubular acidosis are well established, its significance to GI disease in humans remains unclear as is its precise role in SW generation and the mechanisms of regulation of its expression and function. Linking SLC4A4 to GI diseases is a major challenge because of the uncertainty about the phenotypes associated with its dysfunction or dysregulation. Furthermore, mechanistic evaluation of gene function and regulation in ICC is severely limited by the phenotypic instability of these cells in culture. Based on strong preliminary data, in this proposal we aim to overcome these difficulties by screening a large phenome-wide association study (PheWAS, which starts with SNVs and searches for associated traits) for SNVs associated with GI disease-related phenotypes that map within the topologically associating domain containing SLC4A4 (i.e., where most cis-regulatory interactions occur) (Specific Aim1). We will predict the significance of these SNVs by deep epigenomic profiling and validate the predicted functions by genome and epigenome editing (Specific Aim 2). To enable these experimental manipulations and the detailed analysis of the regulation of SLC4A4 functions in the context of SW activity, we have validated cell lines derived from gastrointestinal stromal tumors (GISTs) as models of human pacemaker ICC. In Specific Aim 3, we will subject GIST cells expressing or lacking SLC4A4 to genetic and pharmacological manipulations along with phosphoproteomics to dissect the contribution of SLC4A4 to SW activity in the context of cholinergic and KIT receptor tyrosine kinase signaling. SLC4A4’s role in GI motor functions will be investigated using Slc4a4-deleted mice. Results from this project will establish genotype-phenotype relationships for SLC4A4 and the mechanisms of epigenetic control of SLC4A4 expression in the context of GI disorders. We will also determine the contribution of SLC4A4 activity to SWs and the pathways regulating SLC4A4 function, setting the stage for future preclinical work.

NIH Research Projects · FY 2025 · 1998-07

ABSTRACT This proposal is a request for continued funding of the NIH-sponsored Clinical Pharmacology T32 Fellowship Training Program at the Mayo Clinic. The foundation for this long-running program is strong training in state-of-the-art biomedical research as applied to human-drug interactions, i.e., Clinical Pharmacology. The Mayo Clinical Pharmacology training experience includes a curriculum that exposes Trainees to the rapidly evolving science that underlies Clinical Pharmacology. However, beyond a strong curriculum that includes pharmacokinetics, drug metabolism and pharmacogenomics, at the heart of the Program are outstanding individual research experiences within a supportive mentoring environment. Clinical Pharmacology lies at the confluence of molecular pharmacology, multiple “omics” and, increasingly, exciting analytical techniques such as Artificial Intelligence and Machine Learning—techniques that this center has embraced and applied to studies of drug mechanisms and drug response. The union of these analytical techniques with the leadership that the Mayo Clinic Program has demonstrated over the years in the application of “multiple omics” to Clinical Pharmacology will help move us closer to the twin goals of truly “individualized” and “rational” drug therapy. We propose to take advantage of the opportunity represented by the dramatic advances occurring in biomedical and computational science to train the next generation of Clinical Pharmacologists by joining the latest laboratory-based pharmacologic science with modern computational techniques to enhance mechanistic understanding and the predictability of drug response. Comprehensive, highly integrated academic medical centers like the Mayo Clinic are ideally positioned to address this challenge. The Mayo Clinic has a history of performing and integrating outstanding basic and clinical medical research as well as a tradition of continuing contributions to the discipline of Clinical Pharmacology and decades of experience in recruiting and training both physician scientists and laboratory-based translational scientists in Clinical Pharmacology. During the next funding cycle, the Mayo Clinical Pharmacology Fellowship Training Program will continue to emphasize strong laboratory- based research training in a supportive mentored environment joined with a continually evolving curriculum and systematic exposure to advances in biomedical and computational science—all directed toward the goal of preparing each Fellow enrolled in the Program to become a future leader in Clinical Pharmacology in academia, in industry and in regulatory agencies.

NIH Research Projects · FY 2025 · 1997-09

PROJECT SUMMARY/ABSTRACT Coordinated gastrointestinal (GI) tract motility is fundamental for normal GI tract function. Several cell types combine to regulate GI motility, with the smooth muscle cell (SMC) as the workhorse required to provide the physical power for contractions. Disruptions in SMC function contribute to common GI disorders, may occur after infections and inflammation, and associate with rare but devastating GI motility disorders like visceral myopathies and pseudo-obstruction. The gut wall is a highly complex multilayered structure under mechanical stress at baseline and constantly moving. Therefore, cells in the GI tract experience a range of types and amounts of mechanical stimuli. The normal coordinated motility requires an ability to sense and adjust to forces. In multiple cycles of this grant, we have dissected mechanisms of smooth muscle mechanotransduction, have made discoveries that advanced GI physiology and pathophysiology, and provided novel drug targets. However, our current understanding of SMC mechanosensing remains incomplete. It is established that SMCs, even as single cells, adjust their contractions in response to force in a process called the myogenic reflex. In vascular SMCs, the myogenic reflex depends on mechanogated ion channels, but in the GI tract, cellular and molecular mechanisms remain poorly understood. Therefore, the overall objective of our research is to determine the primary mechanogated ion channels involved in GI SMC mechanosensitivity. For this proposal, we created novel animal models and used cutting-edge techniques to generate compelling preliminary data. Our preliminary studies show that a recently discovered mechanogated ion channel Tmem63a is expressed in a subpopulation of SMCs, which are optimized for force sensing and distributed across the tissue to detect force. Indeed mechanosensitive ionic currents in a population of primary mouse GI SMCs have unique biophysical properties consistent with Tmem63a, the activation of which by force leads to a Ca2+ increase, modulating small and large bowel contractions and whole gut transit time. Interestingly, our data also show that patients with slow transit constipation have a decrease in Tmem63a. Thus, the central hypothesis that a mechanogated ion channel Tmem63a significantly contributes to the myogenic reflex will be tested in two Aims. In Aim 1, we determine Tmem63a function, its response to force, and its role in GI SMCs using conventional and cutting-edge techniques electrophysiology and Ca2+ imaging approaches. In Aim 2, we propose experiments to define the Tmem63a+ SMC population and to determine the role of Tmem63a SMCs in regulating GI smooth muscle function. Since Tmem63a is found in a subpopulation of SMCs, we use single-cell and spatial transcriptomics, novel Ca2+ imaging, smooth muscle contractility assays and in vivo whole gut transit. Successful completion of the proposed innovative experiments has both basic significance and clinical impact, evaluating and establishing a novel SMC mechanogated ion channel which contributes to SMC function and the myogenic reflex and, in the long term, may provide a novel target for functional and motility GI disorders.

NIH Research Projects · FY 2025 · 1997-07

DESCRIPTION (provided by applicant): Many disease processes cause profound changes in the mechanical properties of tissue, providing motivation for developing technologies to measure these properties for diagnostic purposes. In addition, over the last decade there has been growing awareness of the importance of tissue matrix mechanics on cellular function. Cells react to the dynamic and static properties of their matrix environment through mechanotransduction and cytoskeletal remodeling. It is now known that mechanobiology has an important role in the origin and evolution of many disease processes, including fibrosis and cancer. The goal of this research is to develop advanced MRI-based technologies for quantitatively assessing the mechanical properties of tissue and to explore and translate high-impact clinical and research applications. MR Elastography (MRE) is based on the principle that propagating mechanical waves reflect the properties of their medium. Shear waves are generated in the body and imaged with MRI techniques that have the remarkable ability to depict cyclic motions as small as 100 nanometers. The data are processed with inversion algorithms to provide cross-sectional images quantitatively depicting mechanical properties such as the complex shear modulus. In the last grant cycle, the hepatic MRE technology developed under this grant was successfully translated into wide clinical practice and is now used in patient care at hundreds of medical facilities around the world. Liver fibrosis is an important health problem with a rising prevalence in the US population. For many patients, MRE provides a safer, more comfortable, and less expensive alternative to liver biopsy for diagnosing this condition. Research has revealed many other promising applications, including noninvasive diagnosis of fibrosis and inflammation in other organs, detection and characterization of malignancies, providing new biomarkers to assess brain disease, and as a tool in basic research mechanobiology at the tissue and organ scales. As in the last grant cycle, the primary focus of the work will continue to be advanced technology development, to enable further basic and clinical research in this promising field, as well as to conduct pilot studies to identify clincal applications, and to develop practical protocols that will allow validation and eventual translatio to MRE to clinical practice. The research plan involves theoretical work, basic MRI pulse sequence development, device engineering, and protocol testing studies with normal and patient volunteers. Innovative approaches will be implemented and evaluated for generating mechanical waves in tissue, acquiring image data, and processing to generate quantitative images depicting previously inaccessible biomarkers. These technologies will be integrated into protocols that can be shared with other investigators and used to explore the practicality and value of promising applications.