Mayo Clinic Rochester

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Chronic pain and prescription opioid use are common in older adults, with prevalent opioid use in 10% of those greater than 65 years of age. Epidemiological investigations have noted accelerated rates of cognitive decline in older adults with chronic pain,1 and recent evidence suggests that opioid use may be a risk factor.2–4 Our preliminary data from the Mayo Clinic Study of Aging (MCSA) shows a more than 30% increase in the odds of prevalent mild cognitive impairment (MCI) or Alzheimer’s disease dementia in those utilizing prescription opioids at study enrollment. If the association between opioid use and clinically-relevant cognitive decline is confirmed, it would have profound consequences for the management of chronic pain in older adults. The long-range goal of the applicant is to become a successful independent translational clinician-scientist leading a multidisciplinary team to optimize analgesic, cognitive, and quality of life outcomes in older adults. The scientific objectives of this application are: 1) to evaluate the relationships between prescribed opioids and longitudinal changes in global and domain-specific cognitive function in a population-based study of older adults, 2) to assess the relationships between prescribed opioids and longitudinal changes in brain morphometry, and 3) to explore the perceptions of older adults with chronic pain on the impact of opioids on analgesia and cognition through qualitative analysis. The training goals of the applicant involve 4 areas: epidemiology, cognitive outcome assessment, structural neuroimaging, and qualitative methods. Aim 1: To evaluate the relationships between opioid prescriptions, longitudinal changes in global and domain- specific cognitive z-scores, and incident MCI or dementia in a population-based cohort of older adults. Utilizing the resources available through the MCSA, including longitudinal assessments of global and domain-specific cognition and diagnostic evaluations for MCI and dementia, we will assess the relationships between prescription opioid use and cognition with appropriate adjustment for confounding variables. Aim 2: To assess the relationships between prescribed opioids and longitudinal changes in brain morphometry in a broad population-based cohort of older adults enrolled in the MCSA. Utilizing longitudinal MRI data from the MCSA, we will assess the relationships between prescription opioids, changes in brain structure, and neuropsychological outcomes. Aim 3: To describe the experiences and perceptions of older adults with chronic pain on the impact of opioids on analgesia and cognition through qualitative analysis. We will employ qualitative methods to explore perceptions and experiences of opioid therapy for chronic pain management, including analgesic efficacy and concerns for short- and long-term changes in cognition.

NIH Research Projects · FY 2026 · 2021-01

PROJECT SUMMARY/ABSTRACT Ovarian cancer (OC) is the eleventh most common cancer and fifth deadliest among U.S. women. The low incidence, high fatality and molecularly broad range of tumor histotypes make OC challenging to study and to treat. Consequently, survival rates have scarcely changed over the past 35 years, largely because precision therapy lags behind most other cancers. Endometrioid (ENOC) and clear cell (CCOC) account for ~25% of all invasive OC. They are a heterogeneous and understudied group of tumors that are closely associated with endometriosis, but show few similarities to the more common high grade serous OC. ENOC or CCOC have variable or poor response to standard platinum-based chemotherapy. CCOC, in particular, is more likely to be platinum resistant at early stage and resistant to second line chemotherapy at advanced stage, resulting in worse survival than HGSOC. We hypothesize that molecular tumor subtypes exist for ENOC and CCOC that reflect differences in biological processes and risk factors and that might inform new treatment strategies. Our preliminary results using genomics analyses of 185 ENOC and 115 CCOC supports this hypothesis by showing that associations with survival and risk factors such as smoking and body mass index differ according to the tumor’s molecular profile, with some subgroups showing rapidly fatal outcome. In the current proposal, we intend to delve deeper into the genomic profile of ~1,100 ENOC and CCOC tumors to identify key molecular features of the tumor subtypes. Our approach uses a consortium effort that combines existing data from well-conducted epidemiologic studies of risk factors with corresponding clinical information among investigators with a strong history of collaboration. We will first characterize molecular subtypes, separately for ENOC and CCOC, by integrating sequencing and array data from gene expression, mutations and methylated regions from a training set (483 ENOC, 292 CCOC) using statistical clustering. Next, we will assess replication of the molecular subtypes in an independent test set (207 ENOC, 125 CCOC). To assess subtype-specific associations in the total sample (689 ENOC, 417 CCOC), we will relate molecular subtypes of ENOC and CCOC separately to risk factors and to survival. Impact: Less common OC such as ENOC or CCOC are often overshadowed by investigations of more common cancers, yet our data show that ENOC and CCOC can also be rapidly fatal in certain patient subsets or show more favorable outcome in others, directly impacting patients’ lives. Finding patterns with other cancers by using integrated analysis of ENOC and CCOC subtypes has high potential to inform new avenues for targeted therapy and to enhance understanding of ENOC and CCOC cancer biology. Future replication of our findings using an independent 1,400 ENOC/CCOC tumors from our unique consortia resources can lead to needed gains in biological, epidemiologic and therapeutic insights for these patients.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Breast cancer remains the most commonly diagnosed cancer in women and is the second leading cause of cancer-related deaths among women. Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of all breast cancer patients, is the most aggressive sub-type of breast cancer and accounts for a disproportionately higher fraction of cancer-related morbidities and mortalities. Treatment options are extremely limited for TNBC patients and the most commonly employed neoadjuvant and adjuvant chemotherapy drugs have existed for decades. De novo and acquired chemotherapy resistance remains a major problem and disease recurrence results in breast cancer-related death for the large majority of patients. Further complicating the clinical management of TNBC is the lack of FDA-approved targeted therapies that can be utilized to prevent disease recurrence in the adjuvant setting. Thus, the identification and validation of novel drug targets and more effective treatment options continues to represent a major unmet clinical need. We have demonstrated that Estrogen Receptor Beta (ERβ) is expressed in approximately 20% of TNBCs, and have shown that patients with ERβ positive tumors have improved long-term prognosis. In addition, we have shown that ligand-mediated activation of ERβ by estradiol, or ERβ selective agonists, inhibits TNBC cell line and patient derived xenograft proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. Importantly, we provide the first evidence that estradiol can elicit clinical benefit in a patient with ERβ positive metastatic and chemo-refractory TNBC. Mechanistically, we demonstrate that ERβ potently suppresses the nuclear factor kappa B (NFκB) pathway in TNBC cells, effects that are mediated through association of ERβ with EZH2/PRC2 leading to epigenetic modifications to histone residues at NFκB target gene loci. Furthermore, we have demonstrated that ERβ modifies chemotherapy responsiveness of TNBC cell line models and patient derived organoids and inhibits chemo-resistant cell lines. Based on these data, the central hypothesis of this proposal is that ERβ repurposes EZH2 to inhibit NFκB signaling in TNBC thereby eliciting anti-cancer effects and enhancing chemotherapeutic responsiveness. To test this hypothesis, the following Specific Aims are proposed: 1). Determine the molecular mechanisms by which ERβ suppresses NFκB signaling in TNBC and 2). Elucidate the biological importance and clinical significance of ERβ-mediated suppression of NFκB signaling in TNBC using novel genetically engineered mice, PDX/PDO models and patient specimens. To conduct these Aims, we will utilize multiple model systems, innovative approaches and molecular tools, to comprehensively address our focused hypothesis. Given the extremely poor outcomes in women with TNBC, the proposed studies are of critical importance towards the goal of improving treatment strategies to more effectively manage this disease.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Development of effective therapies is an urgent unmet medical need for patients with metastatic gastroesophageal adenocarcinoma (mGEA). The advent of immune checkpoint inhibitors such as anti- programmed death (PD)-1 antibodies has revolutionized treatment of some cancers but benefits only a minority of patients with GEA. Combination approaches are required to extend this benefit to more patients. Most studies of immunotherapy combinations in GEA initiate PD-1 blockade at the same time as cytotoxic therapy despite the possibility that cytotoxic agents may kill some of the very T cells invigorated by PD-1 blockade, as our group and others have shown. Our long-term goal is to develop immunotherapy combinations that avoid this problem through rational sequencing of immunotherapy with other anti-cancer agents to enhance tumor destruction and improve patient survival. Based on our preliminary data, we hypothesize that serial combination immunotherapy utilizing anti-PD-1 and anti-angiogenesis therapy with immunomodulatory chemotherapy in a predefined sequence leads to meaningful improvements in clinical outcomes in association with disruption of the immunosuppressive tumor microenvironment and promotion of antitumor systemic immune responses. To test this hypothesis, we will perform a phase II trial to determine the therapeutic efficacy of serial combination immunotherapy and examine its impact on local and systemic immune responses in patients with mGEA through the following specific aims: Aim 1 will determine the therapeutic efficacy of serial combination immunotherapy in patients with metastatic GEA. Aim 2 will interrogate patient samples collected longitudinally from the parent trial to identify the impact of serial immunotherapy on immunosuppressive, antitumor, and angiogenic components within the tumor environment, including tumor- related local and systemic immune responses. Our expected outcomes are to show improved clinical efficacy using an innovative immunotherapy combination in which PD-1 blockade is delivered in a predefined sequence and to gain critical knowledge on the impact of this serial immunotherapy approach on tumor-related local and systemic immune responses in patients with mGEA patients. Together, these new proof-of-concept data are expected to inform the design of future definitive clinical trials that can improve the survival of patients with immunotherapy-resistant metastatic GEA.

NIH Research Projects · FY 2025 · 2021-01

Osteoporosis is a disease of aging that leads to ~2 million fractures and ~$17 billion in healthcare costs annually. Although several drugs are FDA-approved for the treatment of osteoporosis, the potential for serious side effects (e.g., osteonecrosis of the jaw, atypical femur fractures) has led most physicians to use these drugs only for the treatment, but not the prevention, of osteoporosis. This has led to the current situation where most postmenopausal women must wait until they develop frank osteoporosis (i.e., fractures, or sufficiently high fracture risk) to begin drug therapy. As such, there is a compelling need for novel, relatively low-risk and low-cost pharmacological approaches to prevent osteoporosis. The current proposal aims to translate evidence from rodent studies showing that the sympathetic nervous system (SNS) is an important regulator of bone metabolism to a simple, cost-effective, and safe approach for osteoporosis prevention. In key Preliminary Data, we obtained multiple lines of evidence to establish clearly the role of the SNS in regulating human bone metabolism. A critical component of these data was a “proof-of- concept” interventional study demonstrating that β1-selective blockers (atenolol, nebivolol), but not a non- selective β-AR blocker (propranolol), have favorable effects on bone turnover and bone mineral density (BMD) in postmenopausal women. Based on these data, we will perform a randomized, double-blind, placebo- controlled 2 year clinical trial addressing the following Specific Aims: (1) Test the hypothesis that treatment with a widely used, inexpensive, and relatively β1-selective blocker (atenolol) will prevent bone loss at the lumbar spine and femur neck as assessed by dual-energy X-ray absorptiometry in 420 postmenopausal women without pre-existing osteoporosis (Aim 1a); and evaluate the tolerability and safety of atenolol when used for the prevention of bone loss (Aim 1b). (2) Evaluate the effects of atenolol on trabecular and cortical bone microarchitecture using high resolution-peripheral quantitative computed tomography (Aim 2a), on bone turnover markers (Aim 2b), and test whether baseline measures of bone turnover or of sympathetic activity (resting heart rate, plasma catecholamine levels) are predictive of the BMD response to atenolol over 2 years (Aim 2c). (3) In a subset of patients, explore the underlying molecular and cellular mechanisms for the effects of β1-selective blockade on bone in humans using analyses of osteoblast populations isolated from bone biopsies as well as tissue-level bone formation rates on quadruple-labelled bone biopsies (Aim 3). The proposed studies will rigorously test whether atenolol is efficacious and safe for the prevention of osteoporosis in postmenopausal women and also further define the mechanisms of SNS effects on bone in humans. If our proposed clinical trial demonstrates protection from bone loss in postmenopausal women by atenolol, this would fill a crucial clinical need, as these women currently have virtually no pharmacological options for osteoporosis prevention.

NIH Research Projects · FY 2025 · 2020-12

Abstract Targeting immune checkpoint signaling with blocking antibodies has reached a limitation in the treatment of advanced cancers. Although antibodies that bind programmed death ligand 1 (PD-L1) are effective in blocking PD-L1's extracellular interaction with PD-1 receptor on T cells, the potential adaptive upregulation of PD-L1 and its recycling from intracellular compartment to the cell surface may compromise their efficacy. Importantly, the discovery of PD-L1's intracellular functions in cancer cells to promote thier survival and metabolism also highlight a mechanism by which tumor cells can gain resistance to cytotoxic therapy and call for a new strategy to target PD-L1. There is therefore a critical need to design, test, and translate new agents that can simultaneously inhibit PD-L1's extracellular and intracellular functions. This application will utilize a new PD-L1 antibody (clone H1A) that can reduce the expression of PD-L1 in tumor cells through disrupting the association of PD-L1 with CMTM6 (a molecule that can stabilize PD-L1 recycling and expression) and subsequently directing PD-L1 for degradation. H1A-induced degradation of PD-L1 may not only disrupt PD-1/PD-L1 interactions due to the loss of PD-L1, thus removing PD-1's suppressive signals in T cells, but also disrupt PD-L1's cell-intrinsic functions within tumor cells and myeloid cells, thereby decreasing tumor resistance to chemotherapy and releasing the immune-stimulatory function of myeloid cells. Thus, H1A antibody may be a good candidate for targeting the dual functions of PD-L1 for cancer therapy. Based on preliminary data, the central hypothesis of this proposal is that intracellular signaling through PD-L1 results in tumor resistance to cytotoxic chemotherapy and limits the immune-stimulatory function of myeloid cells. Thus, targeted agents that result in degradation of PD-L1 and elimination of its intracellular signaling ability represent a novel therapeutic strategy that will both synergize with chemotherapy and improve the immune response. This hypothesis will be tested by pursuing two specific aims: (1) Determine how H1A antibody synergizes with chemotherapy to overcome tumor resistance; (2) Determine how H1A antibody promotes an enhanced T cell response to attack tumors. To further assess the future clinical use of H1A antibody, a fully humanized version of H1A and humanized PD-1 and PD-L1 mice have been produced, which will allow for evaluation of the therapeutic effects of H1A either alone or in combination with chemotherapy and exploration of H1A's new mechanism of action in vivo. The overall impact of the proposed research is high because it will provide a new therapeutic agent that is capable of targeting the dual functions of PD-L1, resulting in improved efficacy of cytotoxic chemotherapy and an enhanced immune response. This strategy represents a significant paradigm shift within the field in terms of how to target immune checkpoint molecules like PD-L1 for future clinical applications.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY/ABSTRACT With population aging, the burden of stroke is rising globally and is among the leading and potentially preventable causes of dementia, disability, and death, with disproportionally worse outcomes in women. Patients with rheumatoid arthritis (RA), a female-predominant disease, are at 60% greater risk of stroke and 40% greater risk of Alzheimer’s disease-related dementias (ADRD) than the general population. Systemic inflammation is a key risk factor for this excess risk, acting synergistically with cardio- and cerebrovascular disease (CVD) on amplifying the risk of ADRD in RA. Thus, targeting systemic inflammation along with CVD risk factors in RA may offer major additive benefits for ADRD risk reduction. Longitudinal studies evaluating the concept of inflammation-driven excess in cerebrovascular disease burden contributing to cognitive decline in RA by sex are lacking, resulting in a substantial knowledge gap. To address this knowledge gap, we propose to 1) quantify the cumulative burden of stroke and its association with ADRD in RA vs. non-RA overall and by sex; 2) identify sex differences in RA-specific risk factors and outcomes of stroke in RA and 3) define the burden of preclinical cerebral small vessel disease (CSVD) using neuroimaging, its biomarker correlates among measures of RA disease activity and immune aging, and its association with cognitive outcomes in RA. We are uniquely positioned to address these objectives by leveraging our established and well-phenotyped longitudinal population-based RA inception cohort linked to the Rochester Epidemiology Project (REP) medical records-linkage system, with the availability of matched comparators without RA from the same underlying population. Additionally, we will utilize the existing resources from Mayo Clinic Study of Aging (MCSA), including serial cognitive assessments, APOE genotyping, neuroimaging, and laboratory measures for non-RA comparators, and will prospectively enroll people with RA, obtaining similar robust clinical, laboratory, and neuroimaging assessments. Taken together, these resources provide a one-of-a-kind opportunity to investigate the incidence, reoccurrence, and risk factors for stroke in patients with RA overall and by sex over several decades of follow-up in a population-based setting, and to build a detailed picture of CSVD in association with cognitive outcomes in the chronic inflammatory setting of RA. Successful completion of this project will provide much-needed clinically applicable insights into RA-specific risk factors for cerebrovascular disease, as well as the potential for prediction and prevention of CSVD and associated adverse cognitive outcomes in RA with a focus on sex differences.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Alzheimer’s disease and related dementias are typically thought of in terms of progressive cognitive decline, and relatively little is known about episodes of lucidity that occur among people with dementia. Paradoxical lucidity is defined as spontaneous, relevant communication or connectedness in a person with dementia. Few studies have focused on paradoxical lucid episodes, and little is known about what may cause paradoxical lucidity, how common lucid events are, and how lucidity impacts family caregivers of persons with dementia. The broad goal of this study is to define paradoxical lucidity, determine the prevalence and predictors of paradoxical lucidity, and assess the impact that lucidity has on family caregivers. Our proposal will establish a definition of paradoxical lucidity based on survey data and interviews with caregivers, as well as input from a panel of clinical experts in Alzheimer’s disease and related dementias. Next, we will administer an online survey to 8,000 dementia caregivers across three time periods (baseline, 6-months, and 12-months) to measure how frequently paradoxical lucidity occurs across time. We will analyze survey data to establish what may predict paradoxical lucidity and how paradoxical lucidity is linked to caregiver well-being. The results of this work will help inform clinical practice, build capacity for Alzheimer’s Disease education and advocacy, and build knowledge on an understudied aspect of dementia.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY The aging population has led to an increase in cognitive impairment (CI), including mild cognitive impairment and dementia. Alzheimer’s disease is the most common cause of dementia, with more than 6 million people currently affected and an estimated increase to 15 million by 2060, causing significant public health concerns. However, a recent study indicated that clinicians are not aware of CI in more than 40% of their patients, which results in missed opportunities for appropriate care plans, leading to adverse clinical outcomes. The clinical diagnosis of CI requires an extensive evaluation with a battery of standardized tests and questions to patients and caregivers. However, these assessments are not routinely performed in the majority of healthcare institutions, resulting in a significant delay in diagnosis. Electronic health records (EHRs) contain significant amounts of relevant information that is routinely recorded as part of clinical care. Indeed, our preliminary study shows that early signals of CI exist in EHRs, several years before clinical diagnoses. However, little is known about systematically analyzing patient health data in EHRs and how their temporal trends are associated with the development of CI. To address this gap, we will utilize unique resources to identify EHR patterns that rapidly detect the development and risk of CI: (a) Mayo Clinic Study of Aging (MCSA) cohort with longitudinal cognitive assessments and extensive clinical characterization. This will provide an ideal gold standard to assess the validity of EHR-derived CI; and (b) Rochester Epidemiology Project (REP), which provides access to longitudinal EHRs from multiple healthcare institutions. The primary goal of this study is to develop an informatics tool to extract patient health conditions related to CI from EHR data from multiple healthcare institutions (Aim 1, informatics). We will then characterize temporal health trends of CI patients by mining routinely-collected longitudinal EHR data (Aim 2, population health); and will develop a predictive model to early identify patients at high risk of CI using temporal trends of patient health (Aim 3, clinical practice). The tools developed will be deployed in public to facilitate further clinical research. In summary, the proposed research opens up new avenues for utilizing the routine EHRs to facilitate early detection of CI by characterizing patient’s temporal health trends (a potential surrogate of assessment-based clinical diagnosis). Widespread adoption of informatics tools can potentially lead to early detection of CI in healthcare settings and the ability to improve treatment plans and health outcomes for these patients.

NIH Research Projects · FY 2024 · 2020-09

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in one of two genes, PKD1 or PKD2, whereas it cannot be fully understood in terms of the constrained genetic setting, especially, in families with the same genetic mutations but variable disease severity. Epigenetic regulation as a critical driver of cell fate and survival can occur even in genetically identical humans, which may be an alternative means of explaining PKD-associated alterations. Thus, the roles of epigenetic modulation of gene expression and protein functions in ADPKD should become the focus of scientific investigation. However, in addition to histone deacetylases (HDACs), the roles of DNA and histone methylation and the enzymes that mediate these processes in ADPKD remain largely unexplored. PKD1 is hypermethylated in gene-body regions and its expression is downregulated in ADPKD. By performing whole-genome bisulfite sequencing (WGBS) analysis, we have identified the genome-wide abnormal DNA methylation signatures in ADPKD kidneys compared to those in normal kidneys, suggesting that DNA methylation is one of the key mechanisms underlying cystogenesis. Within the five DNA methyltransferases, DNMT1 is the only enzyme that functions to maintain the DNA methylation patterns in human genome. DNMT1 was upregulated in Pkd1 mutant renal epithelial cells and tissues, implying its role in the maintenance of the abnormal DNA methylation signatures in ADPKD genome. We will investigate the roles and mechanisms of DNMT1 in regulating renal cyst progression in aim 1. Since we identified an interaction between DNMT1 and Smyd2, one of the SET- domain-containing histone (lysine) methyltransferases, it suggested that Smyd2 may be involved in DNMT1 mediated DNA methylation. We will investigate the crosstalk of DNMT1 and Smyd2 in the regulation of DNA methylation and further delineate Smyd2-mediated molecular mechanisms in the regulation of cystogenesis in aim 2, which may address if Smyd2 serves as a recruitment platform for DNMT1 on specific gene methylation, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems and providing a possible explanation of why the upregulation of DNMT1 in cancer and PKD only targets specific genes but not all genes in patients’ genome. Furthermore, we will test if de- methylation of hypermethylated DNA mediated by DNMT1 with Hydralazine and Smyd2 inhibitor delays cyst growth in vivo in aim 3. This is the first study that not only links DNMT1 and DNA methylation to ADPKD but also links the corresponding DNMT1 and Smyd2 signaling together in regulation of DNA methylation and gene expression. In addition, this study will produce information that will be therapeutically relevant with excelling potential for translation.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in the world. Comprehensive cardiac rehabilitation (CR) programs have repeatedly been shown to reduce morbidity and mortality while improving symptoms and quality of life for patients living with this chronic disease. While referral rates to CR programs for patients following a coronary event or procedure have slowly begun to increase in recent years, patient participation in these programs has not. Further, adherence and completion of CR programs for those patients enrolled also falls well short of target. There are a number of known barriers to participation in and completion of CR, which contribute substantially the widening gap between CR referral, enrollment, and completion of CR. The objective of this proposal is to determine the effectiveness of a mobile health (mHealth) remote case management platform for increasing CR adherence, improving CVD risk factors, and improving patient- centered outcomes. This prospective, randomized controlled trial of CR using a mHealth remote case management platform compared to conventional center-based CR has been developed through interaction with a number of key stakeholders including patients, clinical CR support staff, scientists, physicians, and healthcare leaders. All non-surgical patients who have experienced a myocardial infarction, acute coronary syndrome, percutaneous coronary intervention, have stable angina, or have heart failure will automatically be referred to CR and be eligible to participate in this trial. We anticipate that patients who utilize the mHealth remote case management platform will have improved clinical outcomes compared to conventional center-based CR, as evidenced by increased adherence to CR programming, improved CVD risk factors, and improved rehospitalizations/mortality.

NIH Research Projects · FY 2026 · 2020-09

Project Summary/Abstract Carpal Tunnel Syndrome (CTS) is an idiopathic, non-inflammatory, age-related fibrotic disorder resulting in compression of the median nerve that affects 10 million Americans annually. Despite the prevalence, cost, and societal impact of CTS, little progress in its treatment and prevention has been made in the past 50 years, primarily due to the lack of mechanistic understanding of disease etiology. Aging is a major risk factor not only for CTS, but also for fibrosis and cellular senescence. Senescent cells exhibit a senescence-associated secretory phenotype (SASP) that is important for wound healing; however, failure to limit this response appropriately leads to fibrotic disease phenotypes. Our preliminary data shows that markers of cellular senescence, including senescence associated β-galactosidase, p16Ink4a, p53; immune evasion markers, as well as SASP factors are increased in both the tissue and cells of the subsynovial connective tissue (SSCT) of CTS patients. Further we have found that type II interferon ɣ (IFNɣ) is expressed in the SASP, and that it induces immune evasion markers. Moreover, targeting senescent pathways in human SSCT cultures using the senolytics dasatinib + quercetin reduces markers of senescence and fibrosis. Thus, our central hypothesis is that senescent cell accumulation in the SSCT is causally implicated in pathological aging and fibrosis found in CTS and that elimination of these senescent cells in the SSCT will attenuate the progression of this pathological fibrosis and thus alleviate disease progression. Importantly, we have developed a rabbit model of CTS and progressive SSCT fibrosis that will allow us to study disease mechanisms in vivo. Therefore, to test this hypothesis, we propose the following specific aims: 1) elucidate the mechanism by which IFNɣ promotes senescence and immune evasion in the SSCT, 2) determine the contribution of senescence to fibrosis in CTS, and 3) evaluate senolytic therapy in vivo. This work will significantly and fundamentally advance our understanding of SSCT fibrosis in CTS, which is a disease of aging and is associated with multiple age-related metabolic co-morbidities. Our discovery of increased senescent fibroblast expression of type IFNɣ in fibrotic tissue and cells could provide a novel therapeutic target for resolution of tissue fibrosis. The innovative aspects of this project are: it will critically test the heretofore untested hypothesis that senescent cells promote CTS, address fundamental questions about disease-related senescent cells, test the novel hypothesis that targeting senescence cells will be a novel therapeutic strategy for CTS and for the first time explore the role of IFNɣ on CTS pathology. This work aligns well with the “Geroscience Hypothesis” which postulates that interventions that slow the aging process will simultaneously delay the appearance or severity of many chronic age-related diseases. CTS is associated with multiple co-morbidities and thus, in addition to advancing the field of aging and fibrosis biology, novel targeted strategies for CTS treatment and prevention derived from these studies could rapidly be translated into new, non-surgical therapies for the millions of people who suffer with CTS.

NIH Research Projects · FY 2025 · 2020-09

Breast cancer has a strong heritable component with approximately 15% of patients exhibiting a family history of the disease. My group recently established that inherited variants in 12 genes (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, NF1, PALB2, PTEN, RAD51C, RAD51D, and TP53) predispose to breast cancer (1, 2), that variants in all 12 genes increase risks of breast cancer in minority populations (3), and that variants in certain genes predispose only to estrogen receptor (ER) positive (ATM and CHEK2) or ER negative and triple negative breast cancer (TNBC) (BARD1, RAD51C and RAD51D) (4-6). Despite these major advances, clinical application of the information is still lacking. In addition, up to 50% of the familial risk of breast cancer remains unexplained. Under this award we plan to address clinically relevant issues, including improved application of genetic testing results for risk management of patients and improved selection of breast cancer therapy. In addition, we aim to identify new breast cancer predisposition genes that account for the missing heritability. The proposed studies are unified under a theme of advancing understanding of predisposition genetics. The studies are as follows: A. Age-specific and population-specific cancer risk assessment for predisposition gene variants. Results from hereditary multigene panel testing has limited clinical utility because only lifetime risk estimates of cancer by age 80 are available. Here we will estimate 5 and 10-year risks of breast cancer, so that patients can make decisions about medical management. In addition, we have evidence that specific genes have much higher penetrance in African Americans. We will determine the penetrance of predisposition gene variants using a large African American cohort study in order to modify risk management guidelines for this population. B. Functional characterization of predisposition gene variants. Variants of uncertain significance (VUS) identified by genetic testing remain a major problem for individuals receiving clinical genetic testing. We aim to combine high-throughput functional analysis of VUS in ATM, BRCA2 and PALB2 genes with genetic data from families in integrated models to determine the clinical relevance of many VUS alterations. C. Therapeutic response for breast cancer predisposition genes. The responsiveness of breast tumors associated with predisposition gene variants to standard or targeted therapy is only known for BRCA1 and BRCA2 mutation carriers. Here we aim to identify all patients with pathogenic variants in the commonly mutated BRCA1, BRCA2, PALB2, ATM and CHEK2 genes from a series of neo-adjuvant, adjuvant and metastatic breast cancer clinical trials and to assess response to therapy and outcome. D. Identification of novel breast cancer predisposition alleles. The common and rare risk alleles for breast cancer account for only 50% of the familial risk in the population. In an effort to identify the missing heritability we will collaborate with Regeneron Inc. through our SIMPLEXO consortium to identify common and rare alleles associated with breast cancer risk in 45,000 breast cancer patients.

NIH Research Projects · FY 2024 · 2020-08

Project summary/abstract: This training grant seeks funding to support the career development of Dr. Ryan Solinsky, a Physical Medicine & Rehabilitation physician-scientist at Spaulding Rehabilitation Hospital and Harvard Medical School, Boston, Massachusetts. Dr. Solinsky is establishing himself as an early career investigator in the clinical application and translation of autonomic neuroscience to improve functional outcomes for individuals after spinal cord injury. This K23 award will provide Dr. Solinsky the necessary support to accomplish the following training goals: 1) Broaden his understanding in computational neuroscience, with a focus on developing expertise in direct sympathetic nervous system recordings and spike train decoding, and in ambulatory monitoring of autonomic indicators; 2) Develop novel functional autonomic neuroimaging through collaboration with local experts 3) Grow his expertise in advanced statistical methods, appropriate for small-n studies of often heterogeneous populations such as those with spinal cord injury; and 4) Expand research project management, grantsmanship, and clinical research skills. To achieve these goals, Dr. Solinsky has developed a training plan with Dr. J. Andrew Taylor as his primary mentor. Dr. Taylor is a well-respected PhD scientist, with research expertise in autonomic control of the cardiovascular system as well as how this system is affected by exercise, specifically in individuals with spinal cord injury. Dr. Solinsky will have additional co-mentors: Dr. Teresa Kimberley, PT, PhD whose research focuses on translation of autonomic neuromodulation to improve outcomes after neurologic disease, and Dr. Roy Freeman, MBChB, a Neurologist and researcher specializing in clinical assessments of autonomic dysfunction. Further collaborators with expertise in functional neuroimaging, advanced statistical methods, and clinical spinal cord injury care are included in this proposal, which will be headquartered at Spaulding Rehabilitation Hospital in Boston, Massachusetts. Autonomic dysfunction following spinal cord injury is a significant clinical issue contributing to mortality and increased healthcare costs. Unfortunately, our understanding of autonomic dysfunction in this population is in its infancy. The overall objective of this research proposal is to characterize cardiovascular autonomic dysfunction after spinal cord injury using a battery of laboratory, ambulatory, and imaging-based tools. These will be focused on cardiovascular autonomic function, as this has the highest potential for clinical translation, with correlative structures to other components of the autonomic nervous system investigated. Research will specifically look to answer two questions: 1) Can discrete cardiovascular autonomic phenotypes be identified within those with spinal cord injury and correlated to clinical secondary autonomic complications? 2) Do those with the most dysregulated cardiovascular autonomic phenotypes demonstrate the highest rates of aberrant spinal cord functional connectivity on fMRI? Answering these questions will help us understand how autonomic regulation changes after spinal cord injury and how alterations translate to clinical secondary complications.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY / ABSTRACT Trauma to the spinal cord disrupts neural pathways that convey signals between the brain and spinal sensorimotor networks (SSN) that reside below the injury site, resulting in chronic paralysis. There is currently no cure for spinal cord injury (SCI); however, recent studies involving a small number of humans with SCI have shown that paralyzed functions can be restored by electrically stimulating the dorsal surface of the lumbosacral spinal cord. Last year, our team reported the use of lumbosacral epidural stimulation (ES) with intense rehabilitation enabled recovery of independent standing and stepping by a man with complete paralysis due to a mid-thoracic SCI that occurred several years prior. The stimulation systems that are implanted in humans with SCI were originally developed, and subsequently approved by the U.S. Food and Drug Administration, for use in humans to treat intractable neuropathic pain. The mechanism of action through which ES alleviates pain is thought to involve inhibition of pathologic signals transmitted through the dorsal sensory roots and ascending dorsal columns of the spinal cord. Contrary to pain treatment, computational modeling and electrophysiological studies indicate ES enables motor functions after SCI via excitation of dorsal root signaling to downstream SSNs. Assuming appropriate parameters of ES (e.g., pulse frequency, pulse width, pulse amplitude, location on the dura mater) are applied, SSNs are capable of producing robust motor outputs that result in functions such as weight bearing standing and/or walking. However, currently available scientific evidence does not explain how ES interacts with nearby spinal structures to produce functional gains in humans with chronic paralysis. To address this gap in knowledge, we will temporarily implant spinal electrodes in 32 humans with lower extremity paralysis to stimulate the dorsal sensory roots and/or dorsal surface of the spinal cord during 10 days of rehabilitation. Dorsal root stimulation (DRS) and ES waveforms will be independently-controlled to inhibit and/or activate nearby structures. Each stimulus pulse will be synchronized to electrophysiologic recordings of downstream neuromuscular activity in order to characterize SSN activity in response to DRS and/or ES stimulation. We hypothesize unilateral DRS during motor-enabling ES will result in ipsilateral suppression of SSN outputs. We further hypothesize that bilateral DRS alone will enable motor functions that are similar to those generated by ES. To investigate the role of rehabilitation during stimulation-enabled motor recovery, we hypothesize that stimulation-enabled motor performance will improve significantly across 10 motor rehabilitation sessions with DRS/ES. Completion of this work will generate new information on the interactions that occur during SSN facilitation via spinal stimulation. This information will be used to develop algorithms that correlate stimulation waveform properties to neuromuscular recordings and motor performance metrics in order to identify stimulation settings that facilitate optimal performance of stimulation-enabled motor function in humans with SCI.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Geroscience refers to the multi-disciplinary approach to understand, at the molecular level, the relationship be- tween aging-associated pathologies and aging. Unfortunately, there continues to be a fundamental knowledge gap in implicating how these mechanisms predispose to a myriad of diseases with advancing age, including neurodegenerative diseases (ND) like Alzheimer’s disease (AD). This lack of comprehension represents a sig- nificant problem because, until it is recognized, development of interventions that prevent or attenuate this de- bilitating disease will continue to be unattainable. Senescent cells (SnCs) accumulate with age and at sites of age-related pathology and have been demonstrated to actively drive tissue deterioration. As removal of these cells has largely beneficial consequences for aging and lifespan, these cells are particularly attractive candi- dates to test the geroscience hypothesis that attenuated ‘rates of aging’ may delay neurodegenerative diseas- es and other age-related conditions. The long-term goal of the laboratory is to exploit SnC clearance as a ther- apeutic strategy for a variety of age-related diseases, including AD. Cells with features reminiscent of senes- cence have been observed in post mortem AD patients, therefore the overall objective in this application is to determine whether SnC elimination from established ND models attenuates disease severity. The central hy- pothesis is that SnCs actively drive disease processes and that removal of these cells will prevent or delay AD progression and severity. This hypothesis has been formulated on the basis of unpublished preliminary data produced in the applicants’ laboratory included in this application. The rationale for the proposed research is that once it is known how senescence of specific cell types in the brains impacts pathology, it can be tested if novel pharmacological modulations of SnCs and/or their effects influences the disease process. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) Establish the therapeu- tic potential of SnC removal in ND; and 2) Evaluate how attenuated SnC accumulation impacts ND and normal cognitive aging. Under the first aim, various methods of SnC elimination will be used in established disease to attenuate severity using novel mouse models established as feasible in the applicants’ laboratory. Under the second aim, senescence in specific cell types will be prevented to determine how this influences disease se- verity and SnCs will be genetically removed from geriatric mice to determine if this impacts cognition. The ap- proach is innovative, in the applicant’s opinion, because it departs from the status quo by utilizing an entirely novel approach to counteract ND through modulation of SnCs. The proposed research is significant, because it will address key fundamental questions about SnCs in ND and test whether targeting SnCs is a potential ther- apeutic strategy for this disease. This knowledge will pave the way towards transformative clinical interventions for treating or preventing not only ND, but also a broad spectrum of human age-related diseases.

NIH Research Projects · FY 2024 · 2020-07

Neuroblastoma (NB), the most common extracranial solid tumor in children, typically presents at diagnosis with evidence of widespread metastasis, especially in patients with amplification of the MYCN oncogene. This subgroup has a very high risk of treatment failure and death despite receiving greatly intensified chemotherapy. Attempts to improve the treatment of disseminated NB have been impeded by the lack of a detailed understanding of the multistep cellular and molecular pathogenesis of this complex form of the tumor. Thus, new mechanistic insights leading to safer and more effective treatments for metastatic NB are urgently needed. This proposal grew from whole-genome sequencing analyses of primary NB samples from 135 patients, in which we identified a novel deletion of a growth arrest-specific gene 7 (GAS7, located on chromosome 17p13.1), in a subset of patients with high-risk NB defined by MYCN amplification. Further analysis showed that low levels of GAS7 expression are associated with advanced-stage, MYCN amplification and a poor clinical outcome, while knockout of gas7 in a zebrafish model of NB with MYCN overexpression promotes hematogenous metastasis of tumor cells to sites commonly seen in patients with this disease. We also found that MYCN can bind indirectly to the GAS7 promoter region, leading to its transcriptional repression, while reduced expression of GAS7 can increase MYCN protein levels. Hence, the central hypothesis of this application is that, in addition to cases with the heterozygous deletion of 17p, over- or amplified expression of MYCN can downregulate GAS7 expression and maintain at deficient levels of this adaptor protein. This, in turn, appears to maintain MYCN protein expression at high levels, facilitating dissociation of tumor cells from the primary tumor and their subsequent dissemination. This hypothesis will be tested in three specific aims: 1) To probe the interplay between MYCN overexpression and low levels of GAS7 expression in metastatic NB, 2) To dissect the MYCN-induced cascade of downstream transcriptional and signaling changes in the context of GAS7 deficiency that lead to metastasis in high-risk NB, and 3) To assess the contribution of GAS7 deficiency to the timing of NB dissemination and the ability of GAS7 overexpression to block or attenuate MYCN-driven tumor metastasis, and to evaluate in vivo the effect of MYCN overexpression-GAS7 deficiency on NB metastasis using different murine models. The major innovation of this proposal is the use of emerging technology with a forward-looking integration of host and tumor genomics in a high-throughput animal model to dissect the interplay between low levels of GAS7 expression and MYCN overexpression in NB metastasis. The significance of this study is threefold: it will (i) shed new light on the contributions of MYCN to early NB metastasis via its effects on adhesion, motility, and invasion, (ii) clarify the mechanism(s) that underlie MYCN’s cooperation with low levels of GAS7 expression in NB metastasis, and (iii) identify promising molecular targets within metastatic signaling cascades that could be exploited therapeutically.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT Heart failure with preserved ejection fraction (HFpEF) affects more than 3 million adults of the United States and has no effective therapy. The promising effect of general strategies for improving endothelial cell (EC) function in HFpEF patients has highlighted the crucial roles of EC dysfunction in the disease pathogenesis. However, the molecular mechanism of EC dysfunction and how it contributes to the pathogenesis of HFpEF are still poorly understood. A recent patient study identified serum neuropilin-1 (NRP1) as a prognostic marker in HFpEF patients. Our preliminary data indicate that endothelial NRP1 is increased in the cardiac tissues of murine HFpEF models and mice deficient of endothelial NRP1 show improved EC and cardiac diastolic functions in these models. Tumor necrosis factor-α (TNFα) is a major inducer of EC dysfunction and associates with the disease progression of HFpEF. Our preliminary data suggest that knockdown of NRP1 reduces TNFα-induced expression of adhesion molecules but enhances the levels of endothelial nitric oxide synthase (eNOS) in ECs. Computational docking model and protein binding assay collectively indicate the direct interaction between NRP1 and TNFα. Based on these results, this project will test the central hypothesis that endothelial NRP1, acting as a novel co-receptor with TNFR, leads to the pathogenesis of HFpEF by increasing cardiac inflammation and impairing myocardial nitric oxide bioavailability. Two Specific Aims are proposed: Aim 1: Delineate the crosstalk between NRP1 and TNFα/TNFR in EC dysfunction. We will characterize the interaction between NRP1 and TNFα/TNFR using microplate-based binding assay and cultured ECs. The role of NRP1 in TNFα-induced EC dysfunction will be examined in both mouse and zebrafish endothelial cell specific NRP1 knockout models. Aim 2: Reveal the role of endothelial NRP1 in the pathogenesis of HFpEF. We will use mouse HFpEF models to investigate how endothelial NRP1 cooperates with TNFR1 to control the disease initiation and progression of HFpEF.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT The mitochondrial apoptotic pathway plays a critical role in the response to various cellular stresses, including targeted anticancer therapies. This pathway is regulated by interactions between various members of the BCL2 family of proteins. In particular, BAX and BAK play an indispensible role in this pathway by permeabilizing the mitochondrial outer membrane (MOM). While BAX plays a predominant role in epithelial tissues, especially in postnatal life, BAK is particularly abundant in normal white blood cells, leukemia cell lines, and clinical leukemia specimens. Our previous studies have demonstrated that BAK activation is initiated by two distinct processes: i) Transient binding of BH3-only members of the BCL2 family in response to certain stimuli (e.g., transient binding of NOXA, which is upregulated in response to the NEDD8 activating enzyme inhibitor pevonedistat), and ii) concentration-dependent BAK autoactivation, a process we initially described. Once activated, BAK forms multimers that permeabilize the MOM. Our recent studies indicate that this MOM permeabilization involves the action of a C-terminal lipid binding domain that is externalized upon BAK activation and interacts with the MOM lipid cardiolipin. Counterbalancing this pro-apoptotic effect, however, BAK can be bound and neutralized by anti- apoptotic BCL2 paralogs in lymphohematopoietic cell lines and primary acute myeloid leukemia (AML) specimens. Importantly, the response of these cells to BH3 mimetics, proapoptotic small molecules that selectively bind and neutralize BCL2, BCLXL and/or MCL1, reflects which of the anti-apoptotic BCL2 family member(s) constitutively bind BAK. Collectively, these observations lead to the hypothesis that AMLs with higher BAK levels will harbor more constitutively activated BAK and will be particularly sensitive to BH3 mimetics as well as targeted therapies that activate BH3-only proteins. We now propose three aims that will test this hypothesis and provide additional insight into the action of BAK in AML during anti-leukemic therapy. First, we will assess the mechanisms responsible for high BAK expression in some AMLs but not others because high BAK expression contributes to BAK autoactivation. Second, we will determine the biochemical basis for BAK autoactivation and subsequent restraint by anti-apoptotic BCL2 family members because this partially- activated-and-then-restrained BAK is the species poised to kill leukemia cells upon exposure to BH3 mimetics and targeted therapies that upregulate BH3-only proteins. Third, we will assess the relationship between high BAK expression, BAK restraint by various anti-apoptotic BCL2 family members, and response of clinical AML to a novel pevonedistat-containing combination undergoing early phase clinical testing, thereby assessing the potential importance of constitutive BAK activation in the clinical setting. These studies, which build on our recent advances in understanding the action of BAK at the molecular level, are collectively designed to enhance current understanding of BCL2 family biology and simultaneously provide new insight into a potentially important determinant of AML sensitivity in the clinic.

NIH Research Projects · FY 2025 · 2020-07

We have recently demonstrated that, in humans, islet function is dependent on Glucagon-Like Peptide-1 (GLP- 1), presumably originating in the α-cell. Islet GLP-1 regulates insulin and glucagon secretion and its effects seem to be more important in type 2 diabetes. However, these observations raise additional questions which we seek to address in this application. The first is whether the isolated defects in the regulation of fasting or postprandial glucose tolerance, seen in prediabetes, represent selective failure of islet GLP-1 to support islet function. Accordingly, we will use GLP-1 receptor blockade with exendin 9-39 to better understand the role (or lack thereof) of islet GLP-1 in the pathogenesis of prediabetes. At present we do not know how the system is regulated. Rodent models suggest that factors secreted by the β-cell in response to agonism of the β-cell’s GLP-1 receptors regulate expression of the prohormone convertase enzyme necessary to convert proglucagon to GLP-1. This is testable in humans with and without functional β-cells. Their response to exendin 9-39 will be compared before and after 4-week treatment with a GLP-1 receptor agonist. The experimental design will also establish if the system is affected by glycemic control and weight loss. Finally, a common genetic variant in TCF7L2 predisposes to type 2 diabetes through effects on α- and β-cell function. However, the mechanism by which it does so remains obscure. TCF7L2 is a transcription factor that mediates proglucagon gene expression in endocrine-derived cells, driving GLP-1 expression in enteroendocrine cells. Therefore, we will test the hypothesis that the diabetes-associated variant at this locus (rs7903146) impairs islet production of GLP-1 thereby impairing islet function. The proposed experiments will help to understand the role and regulation of islet GLP-1, furthering our ability to prevent type 2 diabetes.

NIH Research Projects · FY 2025 · 2020-07

In the inaugural round of the Stimulating Access to Research in Residency (StARR) program, Mayo Clinic’s residency research training model achieved substantial success. With a focus on heart (and vascular), lung, hematologic (blood), and sleep (HLBS) disorders/diseases aligned to the priorities of the National Heart, Lung, and Blood Institute (NHLBI), our program has supported eight resident-investigators, four women and four men. Three have completed training, while five are currently at various stages of matriculation. With the objective to enhance the pipeline of clinician-scientists in HLBS fields, the Mayo Clinic StARR program has developed a model grounded on three straightforward aims. Aim 1: To attract and retain resident-investigators for academic careers in HLBS disorders/diseases research; Aim 2: To train and prepare resident-investigators for independent research careers; and Aim 3: To evaluate the effectiveness and success of the Mayo Clinic StARR Program. To achieve this model, we built a high supportive and comprehensive environment grounded on three significant strengths: 1) Experienced and expert faculty of preceptors and research opportunities representing a broad spectrum of basic, translational, and clinical science; 2) Didactic courses in research training able to address the needs of residents at this training stage; 3) Career Development scaffolds and guidance that supports progress toward achieving goals, including downstream career development funding such as the K38, and continuing on the physician-scientist pathway. Our program is also exceptionally well supported through recruitment of resident investigators through Mayo Clinic’s own Clinician Investigator Training Program— expanding a successful model with specific focus on HLBS diseases, an extensive research infrastructure, numerous resources to develop and maintain research career goals, and the financial commitment of Mayo Clinic leadership to buttress StARR’s comprehensive design and ensure an outstanding program. Moreover, this well-integrated model stands on Mayo Clinic’s strong team science culture where preceptors, education, and support structures all imbue the import of multidisciplinary perspectives to achieve discovery. In our next cycle, we will continue to enhance the Mayo Clinic StARR program, extending opportunities to residents in Laboratory Medicine and Pathology and adding experienced preceptors from the same department. The program will also enhance its recruitment and retention strategies to residents. We are also enthusiastic to cultivate junior faculty as preceptors, including several talented researchers in mentor training and tutelage under our more experienced investigators, ensuring a robust and sustainable StARR program into the future. Thus, an already successful program is poised for even more impressive outcomes in its second cycle.

NIH Research Projects · FY 2026 · 2020-05

PROJECT SUMMARY/ABSTRACT This is a renewal application to build on multidisciplinary predoctoral training in discovery and translational research at Mayo Clinic with a focus on Digestive Diseases and Gastroenterology including the inter-related fields of Diabetes/Metabolic Disease and Kidney Disease (DDK). The overarching objective remains to develop a pool of well-trained biomedical career scientists with the experience and skillsets to become leaders as the next generation of DDK researchers. Two senior and accomplished Mayo scientists with an extensive background in graduate education and mentoring will continue to co-direct this program centered upon four Mechanism-Centric Research Communities in 1) Bioengineering and Biophysical Analysis, 2) Cell-to-Cell Communication, 3) Genomic Regulation, and 4) Intracellular Signaling, with 27 interdisciplinary faculty members and $20 M in NIH funding. This core DDK faculty provides a robust infrastructure with a successful history of training career scientists in discovery-based and translational biomedical research. These laboratories offer a rich mentoring and training environment that is uniquely tailored to the individual student while also promoting collective experiences across multiple disciplines. The Specific Aims focus on fundamental aspects of reproducible and rigorous biomedical research aligned with NIDDK priorities. 1) Offer a solid foundation in hypothesis-based experimentation, research design, rigor, quantitative analysis of datasets, with objective interpretation of results. 2) Foster broad multidisciplinary understanding combined with critical independent thinking skills to identify outstanding biomedical questions and initiate new approaches to advance the field. 3) Develop effective oral and written communication skills. 4) Promote ethical conduct in biomedical research and team skills that facilitate collaborative research while promoting a respectful work environment. 5) Provide the background to identify and effectively transition into productive DDK investigators. The program encourages synergistic interactions between basic science departments and NIDDK-funded Centers in Digestive Disease, Translational PKD, and Pathobiology of the Enteric System at Mayo Clinic. Participation in national meetings and various programmatic activities, like journal clubs, seminar and works-in-progress series, career development events, annual retreat, plus Mayo-wide symposium and poster session are encouraged. These activities synergize with three related postdoctoral programs at Mayo in Digestive Diseases, Diabetes and Metabolism, and Kidney Disease, while offering a complete continuum from summer undergraduate research experiences through PhD and postdoctoral training. Requirements for completion of the PhD degree conform to those of Mayo Clinic Graduate School of Biomedical Sciences, including a first author publication. With a strong pool of 198 qualified eligible graduate students, the proposal requests expanded support for 6 predoctoral trainees per year.

NIH Research Projects · FY 2025 · 2020-05

Summary/Abstract When DNA is damaged, cells activate a complex network of proteins that sense, signal, and initiate repair. This DNA damage response (DDR) also encompasses mechanisms for DNA damage tolerance, enabling cells to replicate damaged DNA. In eukaryotes, the DDR operates within chromatin, consisting of repeating nucleosomes where histone proteins and DNA serve as docking surfaces for DDR proteins. Central to signaling DNA double- strand breaks (DSBs) is the mammalian E3 ubiquitin ligase RNF168. RNF168 catalyzes the ubiquitylation of histone H2A and variant H2AX at DSB sites, regulating the recruitment of several DDR proteins to chromatin, including those involved in homologous recombination or homology-directed repair (HDR). Despite progress in understanding RNF168-mediated signaling, key gaps remain, particularly regarding the function and mechanisms of action of E3 ubiquitin ligase RAD18, one of the effector proteins of RNF168. The central hypothesis of this proposal is that downstream effectors of RNF168 integrate DNA damage repair with DNA damage tolerance, focusing primarily on the E3 ubiquitin ligase RAD18. RAD18 is unique in its involvement in both HDR and DNA damage tolerance pathways. The planned research will investigate the role of RAD18 in HDR and other repair pathways activated by replication stress using integrative structural biology, including single-particle cryo-electron microscopy (cryo-EM), NMR spectroscopy, X-ray crystallography, biophysical approaches, chemical biology, and collaborative cell biology. Specifically, the nucleosome association of RAD18 and of chromatin maintenance proteins recruited in a RAD18-dependent manner will be characterized. Additionally, mechanisms through which RAD18 and cognate E2 ubiquitin-conjugating enzyme RAD6 promote DNA damage tolerance via the mono-ubiquitylation of DNA sliding clamp PCNA will be examined. The research will also explore a mechanism involving RAD18-RAD6 that integrates elements of both DSB repair and DNA damage tolerance through PCNA-ubiquitylation-directed homologous recombination. Overall, this work will contribute fundamental knowledge that enhances the understanding of DNA damage repair and tolerance mechanisms, including how they are integrated. Given the importance of the DNA damage response in maintaining genomic stability and preventing diseases such as cancer and neurological disorders, these studies are expected to yield new findings that will have long-term benefits for human health. During this research, it is probable that unexpected and intriguing new questions will arise, and some of these may be addressed under the flexibility afforded by the MIRA mechanism.

NIH Research Projects · FY 2026 · 2020-04

The Overall Objectives of this proposal are to define the mechanism of liver sinusoidal endothelial cell (LSEC) endotheliopathy and its pathogenic role in portal hypertension and liver inflammation in metabolic dysfunction-associated steatohepatitis (MASH). MASH pathogenesis involves both lipotoxicity (toxic lipid-induced cellular stress) and sterile inflammatory responses. Lipotoxicity in LSEC triggers aberrant signaling, resulting in LSEC dysfunction and a proinflammatory phenotype, which we refer to as endotheliopathy. Emerging data implicate LSEC endotheliopathy in liver inflammation. Furthermore, LSEC dysfunction leading to subclinical portal hypertension in non-cirrhotic MASH patients has been recognized as a driver of liver fibrosis\o "Baffy, 2022 #36". However, the molecular mediators of LSEC endotheliopathy and functional consequences in MASH are largely unknown. We identified glycogen synthase kinase (GSK)3β as the top kinase in LSEC phosphoproteomics and focal adhesion and transendothelial migration (TEM) as the major pathways of multiomics integration on LSECs from mice with MASH. Our preliminary data indicate that during lipotoxicity: 1) GSK3β phosphorylates myosin light chain 2, generating contractile stress fibers and increasing cellular stiffness, thereby augmenting vascular resistance; 2) GSK3β increases the LSEC expression of the adhesion molecule intercellular adhesion molecule 1 (ICAM1) and enhances myeloid cell adhesion and TEM; 3) pharmacological GSK3 inhibition reduces liver inflammation and fibrosis, and endothelial cell-specific Gsk3β deletion reduces portal pressure in mice with diet-induced MASH. Based on these novel observations, we formulated the CENTRAL HYPOTHESIS that lipotoxicity-induced GSK3β activation in LSEC increases vascular resistance and mediates TEM of myeloid cells, thereby promoting portal hypertension and liver inflammation in MASH. The proposal will test this hypothesis and provide insights into two independent and integrated specific aims. First, we will study the in vitro mechanism and functional outcome of GSK3β-mediated LSEC cytoskeletal rearrangement and increased cellular stiffness. Using mice with diet induced MASH, and a novel GSK3 inhibitor, or mice with conditional deletion of Gsk3β in endothelial cells, we will define the LSEC vs hepatocyte specific role of GSK3β in MASH, and the potential for therapeutic targeting. Second, we will define the mechanism and functional outcome of GSK3β-mediated ICAM1 transcriptional upregulation and the therapeutic potential of ICAM1 pharmacological inhibition and endotjelial cell genetic repression in mouse models of MASH. We will then validate the human relevance of LSEC endotheliopathy and myeloid cells TEM in MASH by spatial transcriptomics.

NIH Research Projects · FY 2026 · 2019-12

PROJECT SUMMARY/ABSTRACT In the first cycle of this RO1 (2019-2024), summarized in 62 MOGAD publications, we provided foundational knowledge that informed 2023 MOGAD diagnostic criteria and clinical trials. However, fundamental gaps in our knowledge of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) remain with limited epidemiology data, lack of prognostic biomarkers, incomplete understanding of pathogenesis and no proven treatments. These gaps in MOGAD have hindered prognostication, clinical trial design and recruitment, and development of novel treatments. The long-term goal is to better diagnose, treat, prognosticate, and understand MOGAD. The objective is to utilize epidemiology data, fluid, and imaging outcome biomarkers to inform prognostication, pathophysiology, and therapeutics. The central hypothesis is that MOGAD has a unique epidemiology, distinctive fluid biomarker profile in attacks and longitudinally with hallmark features on innovative imaging. The rationale is that our findings in MOGAD will inform prognostication, surrogate biomarkers for clinical trials, novel targetable therapeutic pathways, and mechanisms of damage and recovery. The hypothesis will be tested by pursuing three specific aims: 1) To determine MOGAD sero-epidemiololgy using unique population-based registries and use big data from our neuroimmunology lab to determine its age, sex, titer dynamics and seasonal distribution; 2) To evaluate the prognostic value of cytokines, chemokines, injury biomarkers, microglial signals, and MOG-specific humoral markers in attacks and longitudinally; 3) To assess microglial activity and myelin integrity including evidence of remyelination using 11C-ER176 microglia positron emission tomography (PET), 11C-PiB myelin PET, and advanced MRI. The disease registries from the USA and Sweden will be screened for MOG-IgG, big data from the neuroimmunology lab will be analyzed and Mayo Clinic patients will be recruited for serum/CSF analyses and advanced imaging studies. The approach is innovative because: 1) Our unique MOGAD biobank, the largest in the world, includes a population-registry of CSF; 2) Use of MOG epitopes, MOG-IgM, MOG-reactive B cells will offer new insight into MOGAD pathogenesis; 3) 11C-ER176 microglia PET and soluble triggering receptor expressed on myeloid cells 2 (sTREM2) offer novel in vivo measures of microglial activity; 4) 11C-PiB myelin PET and MRI neurite orientation dispersion and density imaging are new ways to detect microstructural injury, neurite integrity and remyelination. The proposed research is significant because: 1) MOGAD is an under-studied distinct demyelinating disease with potential for severe morbidity; 2) Determining which MOGAD patients will have a relapsing course will allow earlier treatment or enrollment in trials and prevent disability; 3) Determining the role of cytokines and microglia in MOGAD may assist prognosis and inform pathogenesis. We expect our findings will improve clinical trials, prognostication and mechanistic insight while identifying novel therapeutic targets.