Mayo Clinic Rochester

NIH Research Projects · FY 2025 · 2022-06

For most individuals living with epilepsy, seizures are relatively infrequent events occupying a small fraction of their life. Despite spending as little as 0.01% of their lives having seizures (typically only minutes per month), people with epilepsy take anti-seizure drugs (ASD) daily, suffer ASD related side effects, and spend their lives dreading when the next seizure will strike. The apparent randomness of seizures is associated with significant psychological consequences. In addition, despite daily ASD, approximately 1/3 of patients continue to have seizures. We hypothesize that epilepsy can be more effectively treated, both the seizures and their psychological impact, by providing patients with real-time seizure forecasting. There is strong evidence that focal epilepsy is associated with a variable seizure risk that may enable adaptive therapy targeting periods of high seizure probability. Periods of low seizure probability could require lower ASD doses, reducing exposure and side effects. We propose that high seizure probability states will respond to adaptive electrical brain stimulation (aEBS). In addition, patients could alter their activities during periods of high seizure probability to reduce injury and manage their ASD and activities. The hypotheses driving this proposal are that 1.) seizures can be prevented (reduced incidence) by targeted EBS therapy during the pre-ictal state 2.) seizures are not random events, and that brain states associated with low and high seizure probability can be reliably classified using machine learning methods applied to physiologic signals and used to adaptively change EBS parameters. 3.) Furthermore, we propose forecasting can be improved using multi-modal features beyond passive iEEG recordings, including active brain probing with electrical stimulation (impedance & evoked potentials), core temperature, ECG and serum immunological markers. Goal: Develop reliable seizure forecasting (>90% sensitivity) with few false positives (<1% time in warning) and demonstrate modulation of seizure risk and reduction of focal seizures using aEBS.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY/ABSTRACT We propose to create a multidisciplinary Mayo Clinic Center for Clinical Proteomics with the overarching goal of identifying and validating proteomic biomarkers for treatment response in multiple myeloma. This center is a unique alliance comprising of two teams within the Mayo Clinic umbrella and one team at Brigham Young University. This team of physicians and scientists at Mayo Clinic with expertise in multiple myeloma diagnosis, treatment, clinical trials and basic research as well as technologies such as mass spectrometry, genomics, transcriptomics, bioinformatics and clinical assay development will be joined by a world expert in novel instrument/platform development at Brigham Young University to create a unique center. Multiple myeloma is a complex disease with several distinct cytogenetic subtypes. A recently developed class of drugs designated immunomodulatory imide drugs (IMiDs) has become a mainstay of treatment of multiple myeloma although relapses among patients is high mainly due to drug resistance. The primary target of IMiDs is cereblon (CRBN), which is absolutely required for its anti-cancer and immune activity. IMiDs activate the enzymatic activity of the CRBN E3 ubiquitin ligase complex leading to ubiquitylation and degradation of transcription factors IKZF1 and IKZF3, thereby regulating tumor survival and immune response through downregulation of IRF4 and MYC. For the preclinical arm, our interdisciplinary team will undertake discovery studies involving comprehensive proteogenomic characterization (proteome, phosphoproteome, ubiquitylome, genome, transcriptome) of multiple myeloma models (genetically engineered cell lines, humanized mouse models and patient samples) to identify molecular markers of IMiD resistance. Given the centrality of CRBN-mediated pathways in IMiD resistance, we will jumpstart our targeted proteomics efforts by focusing on developing targeted assays for CRBN and its downstream effectors. In parallel, we anticipate identifying additional candidate proteins through our discovery studies, for which targeted assays will also be developed. Finally, Dr. Vincent Rajkumar, a co-investigator on this proposal, will provide access to samples from three NCI-sponsored clinical trials specifically designed to look at effects of IMiDs enabling validation of candidates through a targeted approach. By incorporating continuous development of multiple technology platforms including CyTOF, we will ensure that we maintain agility over the duration of the proposal. With an established advanced infrastructure, personnel experienced in the development of CAP/CLIA assays, dedicated instrumentation, high analytical capacity and existing pipelines for QC, data handling and bioinformatics, the proposed Mayo Clinic Center for Clinical Proteomics is poised for success to discover and validate proteomic markers of IMiD resistance in multiple myeloma.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Self-management abilities reflect patients’ capacity to manage emotions, social roles, and chronic medical conditions. In fact, self-management abilities are strongly associated with lung transplant candidates’ emotional health. Both self-management abilities and emotional health while waiting for lung transplant predict post- transplant quality of life. Additionally, a candidate’s self-management abilities and emotional health pre- transplant are associated with post-transplant adherence to immunosuppressive medications. Given the important role of these pre-transplant factors, the objective of this study is to optimize pre-transplant self- management abilities and emotional health with the long-term objective of improving post-transplant quality of life and transplant outcomes. Health coaching is an effective strategy to improve emotional health and self- management abilities in other populations and in non-transplant candidates with chronic obstructive pulmonary disease. We used semi-structured, qualitative interviews of lung transplant stakeholders to develop a relevant health coaching intervention for adult lung transplant candidates on the waiting list. In our small, single-center pilot trial we showed telephonic health coaching demonstrated improved pre-transplant self-management abilities, emotional health, and quality of life in comparison to usual care. In this application, we propose a randomized trial of a 30-minute telephonic health-coaching intervention once weekly for 12 weeks (compared to usual care) in 230 lung transplant candidates on the waiting list at the Mayo Clinic in Florida or Minnesota or at UW Medicine in Washington state. Patient-reported outcome instruments measuring emotional health, self- management, and quality of life will be measured at baseline, 12-16 weeks following enrollment, and post- transplant. Our primary outcomes will be differences between groups in Chronic Respiratory Questionnaire (CRQ) domains of Mastery (a measure of self-management) and Emotional function adjusted for baseline values. Secondary outcomes will compare the differences between groups of other psychometric questionnaire scores, adjusted for baseline, as well as short-term post-transplant outcomes. The long-term goal of this application is to optimize post-transplant outcomes through effective pre-transplant interventions. Our central hypothesis is that health coaching will improve lung transplant patients’ self-management abilities, emotional health, and quality of life pre-transplant. We further hypothesize that future studies will demonstrate that these pre-transplant improvements in self-management abilities and emotional health will translate into improved post- transplant quality of life. The results of this study will be used to refine the intervention and plan for a larger, multi-center trial designed to optimize dissemination and implementation in diverse settings. This proposal supports the NIH mission by striving to improve outcomes in lung disease patients while supporting a new investigator in her to transition to research independence.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY This application is to advance a paradigm-shift in particulate guanylyl cyclase A receptor (pGC-A) and 3’, 5’ cyclic guanosine monophosphate (cGMP) therapeutics with the development of a first-in-class small molecule targeting the pGC-A/cGMP pathway for cardiovascular disease (CVD). Our CV focus is on the unmet clinical need for novel therapeutic targets for hypertension (HTN), specifically resistant hypertension (RH), for which there are no approved drugs. The applicants have advanced the concept that the heart is an endocrine organ, which synthesizes ANP and BNP. Upon release, ANP and BNP bind to pGC-A , which is highly expressed in the heart, kidney and vasculature, and generates its second messenger, cGMP. The significance of the pGC-A/cGMP pathway in BP and CV homeostasis is supported by its biological actions which includes vasodilation, natriuresis, diuresis, suppression of hypertrophy, fibrosis, apoptosis and inflammation as well as inhibition of aldosterone. As RH patients are challenging to treat and have the highest risk adverse outcomes, the pleiotropic actions render pGC-A as an novel molecular target for CV therapeutics. To date, there are no small molecule pGC-A stimulators in existence. Through prior R01 funding, we discovered for the first time, pGC-A/cGMP small molecule scaffolds which function as positive allosteric modulators (PAMs) of which a potent derivative of our hit scaffold, MCUF-651, was engineered. Preliminary studies revealed that MCUF-651: 1) potentiates ANP/pGC-A mediated cGMP generation and reduces cardiomyocyte hypertrophy in vitro; 2) enhances ANP binding of pGC-A; 3) elevates cGMP and lowers BP in spontaneous hypertensive rats (SHRs) and 4) is orally bioavailable. Herein, we propose to advance our biological understanding of the cellular protective and BP lowering actions via small molecule pGC-A positive allosteric modulation utilizing the prototype, MCUF-651 and to pursue a drug discovery strategy to identify an optimized small molecule pGC-A PAM clinical candidate, building off MCUF-651. Aim 1: To define, in vitro, MCUF-651's cellular protective effects on pGC-A/cGMP mediated suppression of apoptosis and proliferation in human cardiorenal cells, inhibition of aldosterone in human adrenal cells, reduction in human coronary artery endothelial cell permeability and vasorelaxation in arteries. Aim 2: To establish, in vivo, the chronic cardiorenal protective, RAAS suppressing and BP lowering actions of orally administered MCUF-651 in SHRs. Aim 3: To perform lead optimization of MCUF-651 to improve potency and pharmacological properties, using iterative cycles of medicinal chemistry, selectivity profiling, functional potentiation and in vitro absorption, distribution, metabolism and excretion studies. Aim 4: To evaluate metabolic liabilities of MCUF-651 and subsequently, to advance prioritized optimized lead(s) to in vivo dose-dependent pharmacokinetic measurements and a chronic oral efficacy study in SHRs and to declare a first-in-class small molecule pGC-A stimulator for IND-enabling studies.

NIH Research Projects · FY 2026 · 2022-05

SUMMARY Our proposal will develop the largest cohort of Adenosquamous cancer of the pancreas (ASCP) models and characterize the genomic and epigenomic landscapes of this devastating tumor in comparison with that of pancreatic ductal adenocarcinoma (PDAC), with the expectation of identifying epigenetic features that can be exploited to selectively impair growth of cells of one or both subtypes. ASCP is a rare subtype of pancreatic cancer representing 2-4% of all pancreatic cancers with an incidence rate of < 1 case per 100,000 people per year. Strikingly, ASCP displays a higher metastatic potential and a worse clinical outcome than the more common (90-95% of cases) PDAC. Yet, a large population-based analysis did not detect any differences in tumor stage at the time of diagnosis between PDAC and ASCP. Despite aggressive surgical management for those few patients who present with resectable disease, the median survival has been reported to be consistently less than 1 year. Furthermore, no standard adjuvant therapy, or first line therapies for metastatic patients, has been established for this aggressive subtype of pancreatic cancer. Our preliminary observations suggest a unique ASCP genomic and epigenomic landscape and demonstrate how our molecular studies can identify candidate therapeutic targets and strategies for this dismal cancer that we now aim to validate using unique preclinical ASCP models. It is our HYPOTHESIS that ASCP evolve from the same lineage as PDACs yet contain distinct epigenomic features driving the aggressive phenotype of ASCP. We propose that modulating ASCP epigenome will sensitize ASCP cells to existing chemotherapies (e.g., gemcitabine and irinotecan) used as first line of treatments for PDAC and other solid malignancies. Further, we will evaluate the effects of depletion of key epigenetic proteins on the maintenance of the ASCP epigenome and cell viability, and the impact of epigenetically targeted drugs in combination with chemotherapeutic agents on ASCP tumor growth with the aim of identifying precision treatments for ASCP. Our studies will provide insight into the epigenetic mechanisms that maintain the ASCP and PDAC phenotypes and will be relevant to squamous carcinomas from other tissues (e.g., colon, lung and stomach). Further, the successful completion of this proposal will serve as a foundation for new treatment options for ASCP patients and potentially other cancers with mixed histologies.

NIH Research Projects · FY 2026 · 2022-05

Hepatic stellate cell (HSC) activation encompasses aphenotype that includes enhanced migration, proliferation, and matrix deposition. Migration is critical for coordinately situating HSC for matrix deposition and development of cirrhosis. Our Long-Term Objectives are to understand the molecular underpinnings of HSC biology that lead to cirrhosis with the goal of identifying therapeutic targets. Recently,we identified a critical role of synectin in the process of HSC migration and fibrosis. Synectin is a cytosolic protein that mediates signal transduction, vesicle trafficking, and ultimately gene expression. Synectin is chosen for this proposal as a significant protein warranting detailed investigation because, as we demonstrate, it is upregulated in human cirrhosis and is required for murine fibrogenesis. Mechanistically, we implicate synectin inHSC migration through both short term receptor signaling as well as longer term epigenetic regulation of gene expression networks. Our preliminary data show that depletion of HSC synectin reduces migration signaling downstream of the receptor tyrosine kinase, platelet derived growth factor receptor alpha (PDGFRα); attenuates transcription of a set of HSC activation genes including one that encodes the multifunctional signaling protein, IGFBP3 (insulin growth factor binding protein-3); and abrogates murine fibrosis in vivo. These important observations have led us to propose the central hypothesis that synectin increases HSC migration and fibrosis by promoting PDGFRα signaling and by regulating a network of genes that include IGFBP3. This hypothesis leads to the following Specific Aims: 1) Synectin promotes HSC migration by regulating PDGFRα targeting and signal activation. Aim 1a will determine how synectin recruits and binds specific vesicle trafficking proteins that maintain PDGFRα protein levels, target the protein to endosomes and activate migration signaling. Aim 1b will uncover how disruption of synectin function leads to autophagic degradation of PDGFRα and attenuated HSC migration. 2) Synectin epigenetically controls IGFBP3 gene expression to promote HSC migration. Aim 2a will identify how synectin regulates a specific histone methyl transferase, EZH2 and how this governs IGFBP3 gene expressionthrough histone methylation. Aim 2b will determine how IGFBP3 production stimulates HSC migration. 3) Synectin regulates fibrosis in vivo. Aim 3a will use a novel fibrosis regression model in mice with HSC selective modifications to synectin and PDGFRα to further ascertain the proposed role of these proteins in vivo. Aim 3b will use a synectin neutralizing peptide that selectively targets HSC in coordination with magnetic resonance elastography (MRE) imaging, and mice with genetic deletion of IGFBP3 to elucidate how synectin promotes fibrosis in vivo. In total, this proposal will utilize conceptually and technically innovative approaches and concepts to test a novel hypothesis pertaining to synectin as a “master regulator” of HSC signals that lead to migration and fibrosis. RELEVANCE (See instructions): Liver injury from alcohol and other etiologies can culminate in cirrhosis with significant associated morbidity and mortality.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY In Alaska (AK), smoking prevalence of Alaska Native and American Indian (ANAI) people is more than double that of non-Native adults (37% vs. 17%). Unlike other Indigenous populations, tobacco is not used in traditional ceremony. Current cessation strategies have not reduced smoking prevalence among ANAI people. AK geography (largely frontier and road-less) and climate further limit treatment access and reach. Based on behavioral economic theory, financial incentives to promote cessation is a promising approach in other populations, with smoking abstinence rates more than doubling one year after discontinued rewards compared with no incentives. Community input for this study indicate family members are essential in supporting smoking cessation; consistent with the ANAI cultural value of interdependence, defined as relationship-based, collaborative, and reliance on family systems rather than individuals. Our research and others’ document the influence of naturally occurring social support networks on smoking cessation. Family-based incentive interventions have not been evaluated for smoking or other addictions. To fill this gap, the proposed study will evaluate the effectiveness and implementation of a culturally-adapted, ANAI family-based incentives intervention for smoking cessation with the following Specific Aims: (1) Adapt an effective 6-month financial incentive intervention for ANAI people who smoke and family members; (2) Conduct a randomized controlled trial (RCT) to evaluate reach and effectiveness of the intervention compared with the control condition on confirmed, prolonged smoking abstinence at six and 12 months post-intervention; and (3) Evaluate key process indicators relevant to intervention adoption, implementation, and maintenance, and conduct a cost- effectiveness analysis to support further adaptation and dissemination. RCT-enrolled dyads (adult ANAI person who smokes [index participant] and adult family member) will be stratified by index participant’s sex, rural/urban location, residence with family member, and by family member’s smoking status, then randomized to a no incentives control condition (n=328 dyads) or a 6-month incentive intervention (n=328 dyads). All dyads will receive materials on evidence-based cessation resources, social support strategies, and family wellness. We will measure index participant smoking status in both groups weekly for four weeks, and at three and six months. Intervention index participants will receive rewards for verified smoking abstinence at each time point, up to $750; and the family member will receive rewards (e.g., fuel or other needs) equal to the value earned by the index participant. We will explore potential moderators (e.g., index participant sex) and mediators (e.g., interdependence) of intervention effects. To enhance reach, we will recruit participants statewide, primarily with social media, and deliver the intervention virtually. If successful, our results will inform Tribal policy to adopt the intervention within the AK Tribal Health System; and possibly, for use within ANAI communities beyond AK.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT Monoclonal gammopathy of undetermined significance (MGUS), is a benign plasma cell disorder, common in the population (3-5% ≥50 years) and characterized by an asymptomatic clonal plasma cell expansion. Although MGUS precedes multiple myeloma (MM) with progression rates of 1% per year, most (>75%) MGUS never progress. MGUS is also associated with increased risk of infection, fracture, osteoporosis, renal impairment, and thrombosis, with resultant morbidity and mortality. Few risk factors are identified for either the development of MGUS or MGUS progression to MM, with African American (AA) ancestry being one of the strongest. Identifying individuals who are more likely to progress is important given that individuals with MGUS who are closely monitored prior to development of MM have better outcomes. Prior work identified genetic variations associated with MM through family studies and, more recently, through genome wide association studies (GWAS). However, no well-powered genetic epidemiology studies of MGUS have been performed, particularly in AAs; and to our knowledge, none have investigated whether detection of genetic variation improves the identification of high-risk MGUS, including those that progress to MM. Therefore, we propose a comprehensive evaluation of genetic susceptibility to MGUS (Aim 1) and MGUS progression to MM (Aim 2) using established epidemiologic and genomic studies among European Americans (EA). Given the racial predisposition for MGUS and MM among AAs, for the first time, we propose to evaluate the known MM susceptibility variants and novel variants identified in Aims 1-2 in AAs (Aim 3). Using both GWAS and transcriptome-wide association studies (TWAS), we will answer the questions: (1) What are the genetic variations and genes predisposing to MGUS (Aim 1)? (2) How do these compare to genetic predisposition associated with progression from MGUS to MM (Aim 2)? (3) Do these identified genetic factors (and consequent polygenic risk scores, PRS) differentiate between MGUS patients that do and do not progress to active MM (Aim 2)? and finally, (4) Are these genetic factors for MGUS risk and progression similar across populations of EA and AA ancestries (Aim 3)? We will utilize established EA and AA studies of MGUS and MGUS progression to MM (MM vs. MGUS), the majority with GWAS, to allow for discovery and validation. We will also leverage genomic data, including RNA-sequencing of both whole blood (peripheral blood mononuclear cells, PBMCs) and sorted CD138+ bone marrow plasma cells (BMPCs) from MGUS patients to inform gene expression for the TWAS. Further, we will perform functional characterization of the new genes or variants validated in both EA and AA populations. Upon completion, our study will validate and characterize germline genetic factors associated with risk of MGUS and progression to MM in both EAs and AAs. It will have high impact, providing insight to MGUS and MM etiology and informing genetic contributions to clinical risk models for progression for the millions of people living with MGUS.

NIH Research Projects · FY 2025 · 2022-04

ABSTRACT: The 21st century workforce is experiencing increasing job demands while employers optimize job resources to meet regulatory, fiscal and productivity standards. This is perhaps most apparent in today’s healthcare system, wherein the workforce is under constant stress to cope with rapidly changing care delivery approaches, widespread adoption of electronic health records, and increased reliance on publicly reported quality metrics. In May 2019, the World Health Organization defined burnout as an occupational phenomenon. Unfortunately, burnout is underrecognized by those who suffer from it, and it typically goes undetected until employees’ performance deteriorates or catastrophes occur in workplace. Therefore, this project’s overarching goal is to develop a data-driven technology for predicting impending burnout before its effects on health and work performance become manifest. As a case study, this project will establish predictability of burnout in registered nurses (RNs). In hospital settings, 35%-45% of RNs report burnout primarily driven by increased work demands (higher patient acuity), work inefficiencies, interpersonal conflict, moral distress, and low level of control over decisions that affect their work. Burnout in RNs is associated with poor patient outcomes (increased risk of medical errors, hospital-acquired infections), lower quality of care, increased absenteeism and poor patient satisfaction. Within this context, the proposed project’s vision and aims are presented. This project’s vision is to develop a technology to predict burnout in RNs (as a case study) by combining workplace, psychological, and physiological factors, and exploring the barriers to adopting such a technology. This effort focuses on the following aims: Aim1. To create a unique, open- access, de-identified dataset that transforms the science of burnout internationally and informs the interaction of continuous physiological measures (measured from smart watches) and repeated (quarterly) psychological (measured using validated rating scales) and work-related factors (administrative databases) for predicting burnout (Aim 2) in RNs at Mayo Clinic’s Florida (Cohorts-A&B) and Rochester (Cohort-C) sites. Aim 2. To develop an analytical framework combining probabilistic graphical models (PGMs) and multitask learning (MTL) to derive interpretable predictions of burnout. PGMs addresses the challenge of inherent stochasticity of burnout manifestation across individuals, and MTL will identify common burnout factors predictive of burnout risks (high, medium and low). Predictability established using Cohort-A will be validated in Cohorts-B&C. Aim 3. Explore barriers (bioethics and administrative) to adopting burnout prediction technologies by assessing perspectives of RNs, nurse supervisors and hospital administrators.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Quantification of liver steatosis has weighty implications in management of Nonalcoholic Fatty Liver Disease (a condition affecting 75-100 million Americans), diabetes mellitus, and cardiovascular disease. Serum biomarkers, computed tomography, and existing ultrasound methods have low sensitivity or specificity for steatosis staging. Proton Density Fat Fraction (PDFF) measured by MRI has high accuracy, but is limited by accessibility and cost. Here we propose a novel ultrasound technology, Spectrum Normalization Attenuation Imaging (SNAI), to quantify ultrasound attenuation coefficient for accurate liver steatosis staging. SNAI does not require a calibration phantom, and is robust to rib shadowing as well as phase aberration and reverberation clutter from the body wall. Accuracy of SNAI for steatosis staging is demonstrated by a promising correlation coefficient of 0.91 with MRI-PDFF in 50 patients. In this project, we will prototype, optimize, and evaluate SNAI on a low-cost, pocket-sized, wireless ultrasound probe, which can be conveniently used for screening and follow-up at the point-of-care setting such as the office of a family doctor or hepatologist. Specific Aim 1: Technical Development. We will use phantom and patient studies to advance and optimize SNAI on the the wireless ultrasound probe. Acquisition parameters and post processing algorithms will be optimized for both fundamental and harmonic SNAI imaging. We will suppress reverberation clutter and correct for sound speed mismatch in liver for more accurate steatosis quantification. Specific Aim 2: Patient study. We will use the SNAI prototypes optimized in Aim 1 to study 250 steatosis patients with clinically indicated MRI-PDFF to investigate the efficacy of SNAI for steatosis quantification. Correlation analysis will be performed to assess the association of SNAI with MRI-PDFF. Steatosis will also be categorized as S0, S1, and S2/S3 according to PDFF. Receiver operating characteristic analyses will be performed to evaluate performance of SNAI for detecting ≥S1 and ≥S2. The agreement between SNAI and PDFF classification will be evaluated using the Kappa statistic. Fibroscan CAP will be used for benchmarking. Specific Aim 3: Reproducibility study. Two sonographers and two hepatology residents will repeatedly scan a subset of subjects (50 patients) studied in Aim 2. Intraclass correlation coefficients will be used to evaluate the inter-operator agreement for SNAI measurements. The within patient variance component from the model will provide an estimate of the inter-operator variance, which represents a lower bound for the minimum detectable difference for longitudinal follow-ups. Successful completion of this project will result in a safe, cost-effective, and easily accessible ultrasound solution for accurate quantification of liver steatosis for diagnosis and frequent follow-up of this very large patient population at point-of-care settings such as the office of a hepatologist or family doctor.

NIH Research Projects · FY 2025 · 2022-04

Project Summary/Abstract Breast and ovarian cancer are among the most common cancers in women worldwide.PARP inhibitors have shown potential for the treatment of BRCA-linked cancer, via a synthetic lethality mechanism that exploits the HR defect. However, tumors often develop resistance to these and other drugs. Therefore, there is a pressing need to find new, targeted treatments for BRCA-linked cancer. Genomic instability is a hallmark of cancer cells and a potential source of tumorigenesis. A major cause of genomic instability is replication fork stalling at sites of DNA damage or abnormal DNA structure. A limitation in the study of mammalian stalled fork repair has been a dearth of tools with which to analyze this process in molecular detail. The Scully lab solved this problem by adapting the Escherichia coli Tus/Ter replication fork barrier (RFB) to induce site-specific replication fork stalling on a mammalian chromosome. Tandem duplications (TDs) in primary cells lacking BRCA1 are induced specifically by a Tus/Ter block but not by a conventional double strand break (DSB), indicating specificity for the stalled fork response. Intriguingly, breast and ovarian cancers lacking BRCA1 similarly acquire large numbers of small (~10 kb) TDs, which we have termed “Group 1” TD. Thus, the Tus/Ter system recapitulates the BRCA1-specific regulation of Group 1 TD formation observed in human breast and ovarian cancer. I found that the stalled fork motor protein—FANCM (product of the Fanconi anemia [FA] group M gene) acts synergistically with BRCA1 to suppress Tus/Ter-induced TDs. Further, I discovered a novel synthetic lethal interaction between Brca1 and Fancm loss in mouse embryonic stem (ES) cells and in breast and ovarian cancer cells. These findings suggest that FANCM may be a promising therapeutic target in BRCA1-linked breast and ovarian cancer. My goals in this proposal are to delineate the novel FANCM-BRCA1 synthetic lethal interaction in cancer cells and to determine the mechanism of synthetic lethality (Aim1). Further, I will explore the chromatin environment and protein dynamics at the stalled fork and will study how alterations in these processes contribute to the FANCM-BRCA1 synthetic lethal interaction (Aim2). I observe an epistatic role of Fancm and its downstream target Fancd2 at Tus/Ter in promoting error free repair and suppressing error-prone repair.This critical role of Fancm and Fancd2 in repair pathway choice at stalled forks raises the possibility that they might share similar genetic interactions with Brca1. will identify how individual domains of FANCD2 function in repair pathway choice at stalled forks and their genetic interaction with Brca1 (Aim 3). This holistic approach will provide a full picture of the mechanism of FANCM-BRCA1 synthetic lethal interactions and might identify in FANCD2 a new synthetic lethal target for cancer therapy. I

NIH Research Projects · FY 2026 · 2022-03

ABSTRACT IDH-mutant gliomas are the most common gliomas of young adults. Despite initial sensitivity to chemotherapy and radiation, they invariably progress as treatment resistant lesions to become ultimately fatal. The unique metabolic phenotype of IDH-mutant glioma leaves malignant cells potentially vulnerable to several candidate therapies or therapeutic combination. There remains an urgent unmet need for a reliable quantitative monitoring biomarker to accelerate translational progress Since disease course frequently extends over several years, patient-centric models of therapeutic discovery could leverage reliable surrogate outcomes toward iterative refinement of individualized therapies. This project utilizes a phased, milestone-driven feasibility, discovery (R61; Aims 1-2) and validation analysis (R33; Aims 3-4) of D2-HG as a candidate biomarker of IDH-mutant glioma. This study takes advantage of neurosurgical access to the CNS, wherein CSF access devices utilized for clinical management may be deployed for longitudinal CSF access as an adjunct to lumbar puncture. Moreover, it utilizes tumor-based benchmarks for D2-HG content and production within the tumor based on analysis of tumor tissue and microdialysate. We hypothesize that CSF D2-HG represents a useful monitoring biomarker for IDH- mutant glioma to help quantify response to therapy and identify disease recurrence. To test this hypothesis, we propose the following aims: Aim 1: Determine the technical and biological performance characteristics of CSF D2-HG as a biomarker of IDH-mutant glioma. Detailed and rigorous analyses will be performed for D2-HG and its mass spectroscopy assay including stability, precision, accuracy, interference, and technical as well as biological variance upon repeated measurements and correlates to tumor properties based upon gold-standard benchmarks. Aim 2: Determine a baseline threshold value of CSF D2-HG diagnostic for IDH-mutant glioma and define the minimal percent change indicative of altered disease burden. Appropriate ROC models will be built with and AUC analysis to define a threshold diagnostic of IDH-mutant glioma. Cross-sectional patient cohorts will be used to evaluate responsiveness of D2HG to therapy and disease progression Aim 3: Validate CSF D2HG as a biomarker of therapeutic response. CSF D2-HG will be evaluated longitudinally in to validate responsiveness to therapy as benchmarked modified RANO criteria. Aim 4: Evaluate CSF D2HG as a biomarker of disease progression. A cumulative cross-sectional cohort of patients with verified IDH-mutant gliomas will be followed longitudinally during disease monitoring for recurrent disease to validate the defined threshold indicative of disease progression.

NIH Research Projects · FY 2026 · 2022-03

Project Summary The broad and long-term objective of this project concerns the development of novel quantitative methods and biostatistical tools for microbiome data analytics to aid in microbiome-based discovery sciences. The microbiome, also called the second genome of the human, has received much attention in the past few years. Due to its critical roles in human health and disease, the human microbiome has now been recognized as an integral part of the individualized medicine approach because it not only accounts for inter-individual variability in all aspects of a disease but also represents a potentially modifiable factor that is amenable to targeting by therapeutics. Despite those fruitful and promising findings from microbiome studies, there is no consensus in the current field as how to appropriately analyze the data, let alone the optimality and efficiency issues that have yet to be addressed. Several challenges amount to this predicament, including complex experimental designs of microbiome studies, an unknown interplay between microbiome and host, extremely sparsity and high dimensionality of the data, phylogenetic relatedness of the microbial taxa, and compositional structure of microbiome. As a result, although quite a few analytical methods and tools have been developed for microbiome data analysis, several specific gaps exist in the methodological toolbox, hindering the advance of microbiome-based biomedical sciences. To fill these gaps, this proposal aims to develop robust and powerful quantitative methods and tools for microbiome data analysis. Specifically, Aim 1 focuses on developing robust and powerful methods for differential abundance analysis in complex study designs. It will develop new methods to address zero-inflation, compositional effects and correlations in microbiome data. Aim 2 focuses on strategies to increase the power of microbiome-wide multiple testing. It proposes two new multiple testing procedures, which address confounders and phylogenetic relatedness, respectively. Aim 3 proposes to develop compositional canonical correlation analysis methods for integrating microbiome data with other omics data. Specifically, it will develop an efficient and flexible framework for integrating heterogeneous omics data with microbiome data, accounting for compositional effects and phylogenetic relatedness. Aim 4 will develop user-friendly and efficient software packages so the community can benefit maximally from methodological and scientific advances resulting from this application. The proposed methods will be evaluated using simulations, and more importantly, applications to several ongoing microbiome studies in the Center of Individualized Medicine at Mayo Clinic. The proposed quantitative methods and open-source software packages will contribute to microbiome biomarker discovery and microbiome-based mechanistic studies. All methods and tools developed under this grant will be made available free of charge to interested researchers and the public.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Prostate cancer (PCa) is the second leading cause of cancer death in American men. The TMPRSS2-ETS related gene (ERG) fusion (termed T2-ERG), juxtaposing the strong androgen-responsive TMPRSS2 gene promoter with the coding region of the putative oncogene ERG, occurs in approximately 50% of all PCa in patients. It has been inferred that overexpressed truncated T2-ERG is a key player in PCa pathogenesis. Notably, a number of studies show that overexpression of T2-ERG protein alone is insufficient to trigger formation of PCa in mice unless at very advanced age (> 24 months), implying the requirement of additional lesions in T2-ERG overexpression-induced PCa pathogenesis. By analyzing the NGS data from > 1500 cases of PCa patient specimens, we showed that T2-ERG fusion and p53 inactivation (loss of p53 tumor suppressor function due to TP53 gene deletion and/or mutation) co-occurred in both primary and metastatic PCa, supporting the notion that T2-ERG fusion and TP53 inactivation cooperate to drive PCa pathogenesis and progression. We demonstrated that prostate-specific T2-ERG transgene and Trp53 knockout (T2-ERG+/p53-) not only largely enhanced ERG target gene expression, but also induced high-grade prostatic intraepithelial neoplasia (HGPIN) and cancerous lesions in mice at 12-15 months of age. Mechanistically, we identified LSD1 as a direct binding partner of ERG protein and showed that cyclin-dependent kinase-2 (CDK2) phosphorylates LSD1 at threonine 59 (T59-p), which disrupts LSD1 interaction with HP1γ, an epigenetic `reader' of H3K9me2/3 and induces demethylation of these transcription repressive chromatin marks at ERG target gene loci. Based on these novel preliminary data, we hypothesize that in p53-proficient cells the oncogenic potential of T2-ERG is constrained under a transcription repressive chromatin state through association with the LSD1-HP1γ complex. However, p53 inactivation induces loss or downregulated expression of p21WAF1, aberrant activation of CDKs and accelerated cell cycle progression, which in turn triggers LSD1 T59-p-mediated disassociation of HP1γ from LSD1, HP1γ eviction from chromatin due to accelerated cell cycle progression-associated H3S10 phosphorylation (H3S10-p), thereby promoting H3K9me demethylation at ERG target gene loci, oncogenic reprogramming of ERG transcriptome, and PCa pathogenesis and progression. To test this hypothesis, we will determine the molecular basis and regulation of LSD1 phosphorylation-mediated inhibition of LSD1-HP1γ interaction in T2-ERG+/p53- PCa cells (Aim 1), define the mechanism and extent to which LSD1 enzymatic activity and T59 phosphorylation regulates ERG transcriptional program in T2-ERG+/p53- PCa cells (Aim 2), and determine the clinical significance of the interplay between ERG and p53 signaling and anti-cancer efficacy of targeting LSD1 and ERG pathways in T2-ERG+/p53- PCa (Aim 3). Findings from the proposed studies will shed new light on molecular mechanisms of PCa pathogenesis and could lead to development of novel therapeutics for the treatment of T2-ERG+/p53- PCa.

NIH Research Projects · FY 2025 · 2022-02

Lower urinary tract symptoms are highly prevalent in adult men and women, with an estimated prevalence of up to 50%, and can have a significant impact on quality of life. Urodynamic studies (UDS) are routinely performed in the assessment of lower urinary tract symptoms. Presently, UDS is used in the clinic to detect detrusor overactivity. Detrusor overactivity is caused by unintentional transient contraction of the bladder detrusor muscle, increasing the internal pressure in the bladder. Detrusor overactivity is a common problem. It is estimated that 20% of the total population suffer from overactive bladder, and of those with overactive bladder who are tested by UDS, 65% have detrusor overactivity. Another application of UDS is to assess bladder function in men with benign prostatic hyperplasia (BPH). BPH is a common clinical entity. Millions of men develop lower urinary tract symptoms and seek treatment for this entity. Surgery to de-obstruct the urinary tract may be pursued to treat these patients. Clinically, a UDS test includes the use of multiple pressure catheters (placed in the bladder and rectum/vagina), retrograde bladder filling for an assessment of urinary storage function, and a pressure-flow study to assess for abnormalities of the voiding phase. UDS is limited by its invasiveness, patient discomfort, testing artifacts, and the risk of complications. Adverse outcomes, including urinary retention, catheter trauma, or hematuria, are reported in up to 19% of men and 1.8% of women. Considering UDS limitations, there is a need for a noninvasive alternative to UDS. To overcome the limitations of UDS, we propose a noninvasive ultrasound technique, Quantitative Ultrasound Bladder Vibrometry (QUBV). This technique measures changes in the detrusor’s pressure due to detrusor overactivity. Since this method does not require catheterization, it does not expose the patients to the risks and discomfort associated with UDS. Other advantages of QUBV include a direct measure of the detrusor pressure without the influence of bowl contractions, low cost, and portability. Also, since QUBV is noninvasive and low cost, it is suitable for repeated longitudinal tests for monitoring and evaluating response to treatment. The goal of this project is to evaluate the efficacy of QUBV applications in detrusor overactivity and BPH. To achieve our goal, we propose the following Specific Aims: (1) Evaluate the performance of quantitative ultrasound bladder vibrometry (QUBV) in diagnosing detrusor overactivity; (2) Prognostically determine and monitor patients with BPH that may benefit from surgery to de-obstruct the urinary tract. Each Aims will be tested in a population of patients. Our team has over a decade of experience in developing new technology for application in urology, thus has the expertise and resources to achieve the goals of this project. Also, the project benefits from the world-class clinical research and facilities at the Mayo Clinic. Successful completion of this project may have a significant impact on the diagnosis and management of bladder dysfunction.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Amino acids are crucial nutrients that are also important to support immunity. Yet, we have limited understanding with regard to how immune challenges modulate amino acid availability, and how immune cells sense amino acid and transduce the signals to execute immune reponses. Rag-GTPase has recently been identified as a key amino acid sensor that mostly transduce signals from amino acids to mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) in non-hematopoietic cells. However, Rag-GTPase also modulates transcription factor TFEB, a member of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors, whose functions in B cells remain unknown. Moreover, Rag-GTPase independent mTORC1 activation has been identified. How Rag-GTPase and mTORC1 coordinates to regulate humoral immunity has not been addressed. We compared the functions of Rag-GTPase and mTORC1 in B cell response in vivo using genetic knockout models. Our data showed that while both Rag-GTPase and mTORC1 are required for systemic immune challenges, Rag-GTPase, but not mTORC1, is critical for humoral immune response towards respiratory influenza infection. This divergent requirement between Rag-GTPase and mTORC1 is associated with differential amino acid availability between systemic immunization and airway influenza infection. Furthermore, we showed that Rag-GTPase suppresses TFEB and promotes autophagy, which is associated with ERK activation, but largely independent of mTORC1. Thus, we hypothesize that reduced availability of specific amino acids during respiratory viral infection renders B cells dependent on Rag- GTPase-TFEB pathway, for GC reaction and anti-influenza antibody production. In Aim 1, we will first test whether the respiratory route of live virus immune challenge is the determining factor for Rag-GTPase dependent, but mTORC1 independent, humoral immunity. Second, we will further investigate the temporal and spatial dynamics of amino acid availability during immune challenges. Finally, we will test whether dietary amino acid intervention can improve humoral immunity against respiratory viral infection. In Aim 2, we will utilize complementary loss-of-function and gain-of-function approaches to elucidate the downstream signaling mechanisms by which Rag-GTPase promotes GC reaction and humoral immunity. We will further characterize the Rag-GTPase interactome in B cells using unbiased proteomics approach. Our study will define a novel Rag-GTPase-ERK-TFEB signaling axis that respond to amino acid availability to promote B cell activation and antibody production against airway viral infection.

NIH Research Projects · FY 2025 · 2022-02

Project Summary The apparently random nature of seizures is one of the most significant factors affecting quality of life for patients with epilepsy. Accurate seizure forecasting could be transformative for patients with epilepsy, allowing patients to modify activities to avoid risk, take fast-acting medications to stop seizures before they develop, and provide a sense of empowerment over their disease. Successful seizure prediction has now been established using ambulatory intracranial EEG devices. Unfortunately, no such device is currently available to patients, as no commercial vendor has been successful to date at obtaining approval for clinical use of such a device. Furthermore, invasive intracranial implants may not be appropriate for or acceptable to many patients given the associated risk of infection and hemorrhage. Subscalp EEG recording has recently emerged as a viable means for long-term monitoring of patients with epilepsy. A device is commercially available in the EU, but does not yet have FDA clearance in the US. Recently published studies show the device to be reliable and robust, and recordings longer than 6 months have been reported. In addition cycles of seizure risk have been identified in wearable device physiological signals, and these long-term cycles may be capable of contributing to the accuracy of seizure forecasts. This project will develop the ability to prospectively forecast seizures from simultaneous subscalp EEG and wrist-worn wearable signals, and will assess the safety and feasibility of administering a fast-acting supplemental medication with seizure forecasts to prevent seizures.

NIH Research Projects · FY 2025 · 2022-02

Once prostate cancer progresses to an advanced castration resistant stage it becomes an incurable deadly disease. To date the understanding of the intrinsic tumor cell biology processes involved in the pathobiology of lethal prostate cancer remains limited. Our research proposal aims to elucidate actionable molecular mechanisms that sustain the aggressiveness of lethal tumors by using clinically significant human prostate cancer models. Work from our laboratory and others have revealed that master regulator transcription factors regulate actionable mechanisms that dynamically reprogram the cancer cell by regulating the expression of key nuclear growth factor receptors of prostate cancer such as the Androgen receptor. Through the comprehensive analysis of human transcriptomic prostate cancer public databases that include primary and metastatic tissue samples, coupled with in vitro and in vivo patient derived experimental models, we have identified transcriptionally regulating mechanisms associated with the pathobiology of lethal prostate cancer. Most relevant to this proposal, our studies point to a key role of the Microphthalmia Transcription Factor (MITF) in advanced lethal prostate cancer. Low MITF levels are associated with lethal prostate cancer and disease relapse in primary prostate cancer patients. Notably, functional studies suggest that in prostate cancer MITF regulates the growth and confers resistance to androgen deprivation therapy by controlling a distinct clinically relevant gene network. Indeed, computational studies suggest that MITF regulates the protein synthesis of specific key oncoproteins and prostate specific growth factors, such as MYC and AR. Thus, based on these results, we hypothesize that MITF contributes to the pathogenesis of lethal prostate cancer by regulating both transcriptional and translational mechanisms that reprogram the cancer cell into a therapy resistant lethal state. We will investigate this hypothesis as follows: In aim 1 we will explore the MITF regulated transcriptional mechanisms associated with lethal prostate cancer, focusing on distinct MITF isoforms to delineate their individual functions, as well as determine the clinically relevant MITF regulated downstream effector genes. In aim 2 we will examine how MITF regulates protein synthesis, focusing on specific translation initiation subunits to determine their function in regulating the translation of specific mRNAs, as well as regulatory crosstalk between MITF and MYC. Finally, in aim 3 we will study the clinical relevance of these findings in circulating tumor cells from lethal prostate cancer patients along with testing the in vivo efficacy in preclinical models of targeting protein synthesis together with androgen deprivation therapy as a combined therapeutic strategy to delay the development of castration resistance in low MITF prostate tumors. Ultimately, these studies are poised to broaden our understanding of how master regulator transcription factors govern an intricate complex signaling network that rewires the cancer cell, in this case through regulation of translation, which may offer novel druggable therapeutic opportunities for prostate cancer patients.

NIH Research Projects · FY 2026 · 2022-02

Fundamental and applied studies of nucleic acids For 30 years the Maher laboratory has been investigating unresolved problems in nucleic acids biology and biophysics. We will continue our collaborative efforts related to two major challenges in the field. These challenges are deliberately diverse and multidisciplinary, spanning fundamental and applied aspects of nucleic acid structure and function. This work brings unique opportunities for synergy and creates an exceptional training environment. Challenge 1: Can principles learned from studies of bacterial DNA looping be applied to artificial DNA looping for gene regulation? Hypothesis 1.1: We hypothesize that small DNA loops form more easily in vivo than in vitro because a) proteins bridge DNA loops to reduce required DNA bending, b) DNA supercoiling pre-bends DNA, and c) architectural DNA binding proteins kink DNA. Molecular biology experiments will be performed in vitro and using elements of the lac operon in living E. coli cells. Hypothesis 1.2: We hypothesize that designed sequence-specific DNA binding proteins can be used to create artificial gene regulatory loops in bacterial and eukaryotic systems. Our studies will implement novel Transcription Activator-like Effector (TALE) proteins controlled by chemically-induced heterodimerization to regulate model and endogenous genes by creating tight DNA loops that exclude RNA polymerase from gene promoters. This work will impact synthetic biology efforts. Challenge 2: Can we identify naked DNA aptamers that home to sub-cellular compartments? Hypothesis 2.1: We hypothesize that the mechanism of nucleus-homing DNA aptamers we previously identified using Ligase Proximity Selection can be understood by proteomics and this selection concept extended to discover naked DNA aptamers that exhibit tissue-specific nuclear homing in mice. Hypothesis 2.2: We hypothesize that our new Peroxidase Proximity Selection will identify homing DNA aptamers specific for different sub-cellular compartments of interest. We will study peroxidase biotinylation reward chemistry, undertake selections in live cultured cells, and explore the mechanism of discovered homing aptamers as possible delivery agents for proteins and drugs. Support for this proposal will sustain the Maher laboratory's productive research program addressing these two important and unresolved challenges. The laboratory's track record shows that this investment will trigger further innovations with impact beyond these two problems.

NIH Research Projects · FY 2025 · 2022-02

Project Summary Hepatocellular Carcinoma (HCC) is a devastating and prevalent cancer of the liver with high rates of mortality. Major risk factors include chronic viral infection (hepatitis B or C), fatty liver disease, alcohol use, and exposure to environmental toxins. An independent risk factor is a family history of HCC, which raises the risk by more than 2.5-fold. However, despite evidence of familial risk, the inherited component remains unknown. It also remains unexplored whether inherited gene variants play a pathogenic role in HCC development, or whether they could be used to guide treatment with targeted therapies. We have pioneered an investigation into inherited (i.e. germline) genetic factors associated with HCC. We have completed a pilot analysis of 217 patients with HCC prospectively enrolled from our medical center for clinical-grade multigene panel genetic testing. We have captured details about their personal and family cancer history, risk factors, and outcomes. In our pilot analysis, we found a surprisingly high rate of pathogenic germline variants in cancer-associated genes in patients with HCC, including numerous pathogenic and likely pathogenic variants in genes required for homologous repair, DNA damage response (HR-DDR). We hypothesize that inherited loss-of-function variants in specific genes are enriched in HCC, and that carriers can be treated with targeted therapies. In Aim 1, we will conduct genetic association studies that are powered to detect clinically meaningful germline variants linked to HCC, and we will examine predictors of hereditary cancer syndromes in HCC including age of onset and family history of cancer. In Aim 2, we will explore the mechanism of HCC arising from defects in HR-DDR genes, and determine the implications for targeted therapies. These innovative studies of the hereditary genetics of HCC have the potential to personalize therapies for the subset of patients with hereditary cancer syndromes. We have assembled a team of experts in HCC, hereditary genetics, and animal models to complete this investigation. This study has the potential to impact on the care of patients with HCC in the US and worldwide.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Incidence of autoimmune encephalitis exceeds that of infectious cause, but 50% percent (half of approximate 4000/year in the US) are IgG biomarker negative. Seronegativity hinders diagnosis and pathophysiologic understanding in autoimmune encephalopathies (encephalitis, cerebellar ataxias and other movement disorders). Novel IgG characterization, which attempts to address this gap, is linear and slow occurring at a rate of 1-2 year (using standard tissue-based immunofluorescence assay [IFA] followed by western blots, immunoprecipitation, and mass spectrometry). The long-term goal is to molecularly, clinically and mechanistically characterize autoimmune encephalopathies, and develop targeted autoimmune therapies. The overall objectives in this application, are to: 1) identify disease-specific biomarkers for seronegative autoimmune encephalopathies; 2) molecularly validate novel biomarkers, and 3) clinically, radiologically and immunologically deep-phenotype autoimmune encephalopathies. The central hypothesis is that seronegative autoimmune encephalopathies are characterizable though biomarker discovery. The rationale for this project is that biomarker discovery and translation could occur expeditiously, by leveraging complementary techniques in parallel, that novel biomarkers could be molecularly validated, and that disorders associated with new biomarkers could be neurologically and immunologically deep-phenotyped. To test the central hypothesis the following three specific aims will be pursued: 1) Identify novel biomarkers, initially in 3 partly characterized seronegative autoimmune encephalopathies; 2) determine validity of novel biomarkers; and 3) assess for differentiated clinical phenotypes, and cytokine profile accompaniments. Under the first aim, serum and CSF from seronegative autoimmune encephalopathy patients will be interrogated for neural antibodies using native full-length protein microarrays, as principle technique, complemented by tissue and cell-based IFA for initial IgG screening, and, as needed, phage immunoprecipitation sequencing, and mass spectrometry techniques for verification. For the second aim, newly characterized IgGs will be validated in autoimmune encephalopathy patients and controls by multiple antigen-specific methods (confocal indirect immunofluorescence, and recombinant protein assays [western blot, cDNA-transfected cell-based]). For the third aim, patients with autoimmune encephalopathies with characterized and validated neural IgGs will be deep-phenotyped clinically, radiologically and immunologically, by evaluating for IgG effects in live neuron assays, and cytokine-chemokine profiles. Remaining seronegative cohorts will be immunologically profiled by cytokine-chemokine assays. The research proposed is innovative in the applicant’s opinion, as it focuses on multiplexed biomarker identification, validation, and deep phenotyping. The proposed research is significant because disease-specific biomarkers, with in-depth characterization, allow earlier diagnosis and treatment, and support disease mechanism studies.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY/ABSTRACT Congenital heart disease (CHD) is the leading cause of cardiovascular death in people less than 50 years of age, and most of the premature cardiovascular deaths occur in people with complex CHD. Although the Fontan operation is an effective palliation for complex CHD, it creates a unique physiology characterized by systemic venous congestion and end-organ dysfunction, which leads to premature death from circulatory failure (median survival ~40 years). One of the mechanisms leading to this suboptimal outcome begins with impaired nitric oxide signaling, leading to endothelial dysfunction and pulmonary vascular disease (PVD), and in turn, end- organ dysfunction and death. The therapeutic benefits of chronic pulmonary vasodilator therapy for PVD in people with Fontan palliation have not been consistent across trials, and this may be related to the lack well- defined criteria for PVD diagnosis in Fontan physiology, and lack of understanding regarding the underlying mechanisms. Recent data show that assessment of pulmonary vascular reserve during exercise improves detection of PVD, and that impairment in pulmonary vascular reserve correlates with severity of endothelial dysfunction and end-organ dysfunction. However, it remains unclear whether pulmonary vasodilators can improve pulmonary vascular reserve in these patients, or what the mechanisms might be. Such data will be critical for the development novel therapies for prevention and treatment of end-organ dysfunction due to PVD, an important risk factor for mortality in the Fontan population. The long-term goal is to delay the onset of end- organ dysfunction and mortality from systemic venous congestion, through early diagnosis and treatment of hemodynamic derangements in Fontan physiology. The overall objective for this application is to determine the mechanisms by which enhancement in nitric oxide signaling might improve pulmonary vascular reserve and end-organ function in Fontan physiology. The central hypothesis is that enhancement in nitric oxide signaling through treatment with phosphodiesterase-5 inhibition (PDE5i) will improve pulmonary vascular reserve, and endothelial and end-organ function. This hypothesis will be tested by pursuing two specific aims: (1) Determine the mechanism of response to pulmonary vasodilator therapy in Fontan physiology; (2) Determine the mechanism of improvement in end-organ function, aerobic capacity and quality of life (QOL) outcomes after pulmonary vasodilator therapy. For the first aim, 80 subjects will be randomized 1:1 to PDE5i or placebo, and invasive exercise test and peripheral artery tonometry will be performed before and after 52 weeks of therapy. For the second aim, multi-domain outcome assessment (liver, kidney, gut, aerobic capacity, and QOL metrics) will be assessed before and after therapy. This proposal is innovative and significant as it will delineate the mechanism of response to pulmonary vasodilators, and in turn, enable targeting of these mechanisms with current and novel therapies to delay the onset of end-organ dysfunction and circulatory failure.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY This competitive renewal application focuses on advancing the field of intracranial flow diversion (FDs), that currently constitutes approximately one-third of the treatment of unruptured intracranial aneurysms. There remain key gaps in the knowledge that hinder expansion of the clinical application of these transformational devices, which to date are limited in scope to unruptured, proximal aneurysms along the internal carotid artery. We envision that, with our proposed discovery system, we will facilitate application of novel, next-generation devices in ruptured aneurysms and in aneurysms distal to the Circle of Willis, and will allow customization of approaches to minimize thromboembolic risk in individual patients. We will break down these barriers to expanded utility by 1) understanding of key aspects of aneurysm occlusion, such as the role of acute and appropriate fibrin deposition across the aneurysm neck, 2) unraveling the mechanisms underlying side branch occlusion (i.e. the impact of hemodynamic, or neointimal growth and endothelizalization across the side branch ostia, or both), and 3) identifying the potential risk factors that cause elevated risk of thromboembolic complications, such as hemodynamical variable, device malapposition, platelet function, and untoward fibrin deposition beyond the neck of the aneurysm, among others. We propose to employ innovative approaches in in vivo intravascular fibrin molecular imaging, computational fluid dynamics modeling, and improved animal modeling, and finally biomarker discovery in clinical studies. These approaches can improve the outcome of not only FD, but other devices in treating aneurysms by better understanding of the mechanisms of both aneurysm healing and complications. Our robust and reproducible methods of statistical evaluations will directly assess 1) the role of fibrin deposition rapidity in the device at the neck of the aneurysm aids robust aneurysm, 2) the suitability and validity of the superior mesenteric artery branches to simulate the patency of the small perforating vessels covered by FDs, and 3) correlate biological and imaging data with delayed ischemic events following FD therapy.The discoveries from this hypothesis-driven, multidisciplinary, multimodality, clinical-translational research will provide a robust understanding of not only the mechanism of action of FDs in aneurysm healing, but also the development of device-related complications. These discoveries can provide guidance to clinicians using current technologies to optimize outcomes and minimize complications, as well as investigators and engineers to develop improved devices. Ultimately, this information will allow neurointerventionalists to make better informed decisions on device choice, leading to improved patient care.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Despite the impressive activity of chimeric antigen receptor (CART) T cell therapy in the treatment of B-cell malignancies, the therapy is limited by the development of cytokine release syndrome (CRS) and neurotoxicity, as well as by lower rates of durable responses. While CRS is related to extreme elevation of cytokines associated with T cell expansion, the exact etiology of neurotoxicity is unknown and no options for treatment of neurotoxicity are available. It has, however, become apparent that inhibitory myeloid cells and cytokines contribute to both CART cell toxicities and resistance. We have identified granulocyte-macrophage colony- stimulating factor (GM-CSF) as a dominant driver for CART cell toxicity and inhibition of their functions. Our robust preclinical data indicate that GM-CSF inhibition reduces monocyte activation, enhances CART cell functions and prevents the development of both CRS and neurotoxicity in a novel xenograft model for CART cell-associated toxicities. Our additional studies suggest that GM-CSF disruption in CART cells ameliorates their apoptosis, independent of its effect on myeloid cells. These findings were corroborated when we utilized GM-CSF depletion as a therapeutic strategy in patients with cytokine storm and severe Coronavirus Disease 2019, COVID-19. Based on this work, a Phase 1/2 multi-center study of GM-CSF neutralization after CART19 cell therapy was launched. Our central hypothesis is that depletion of GM-CSF results in modulation of myeloid cell behavior, amelioration of CART cell activation, reduction of CART cell associated toxicities, and enhancement of their efficacy. We will leverage our laboratory tools, novel preclinical models, and samples from this clinical trial to test our hypothesis. In Aim 1 of this project, we will examine the interactions between GM-CSF and monocytes after CART cell therapy. In Aim 2 of this project, we will study the effect of GM-CSF directly on CART cells, and Aim 3 will test how these changes affect toxicity and efficacy of CART19 cell therapy in the novel Phase 1/2 clinical trial. Completion of these Aims will identify novel insights into the toxicity and activity of CART cells and will develop a new strategy to prevent CART cell associated neurotoxicity and CRS, potentially enabling the outpatient administration of CART cell therapy.

NIH Research Projects · FY 2025 · 2021-12

Project Summary The prevalence of obesity has increased worldwide to epidemic proportions. Dysfunctional white adipose tissue (WAT) and brown adipose tissue (BAT) have been implicated in the pathogenesis of obesity and its related metabolic syndrome. BAT is densely packed with mitochondria and requires fatty acid (FA) oxidation to support thermogenesis. In comparison, WAT serves as a main energy storage organ and the FA oxidation in WAT is relatively low at ambient temperature. Chronic cold exposure induces browning of subcutaneous WAT to become thermogenic beige AT with increased FA oxidation capacity. Although FA oxidation is known to be a critical and fundamental metabolic end point in both humans and rodents, it is not completely clear how adipocyte FA oxidation is regulated post-translationally and how it contributes to whole-body energy metabolism in an autonomous manner. In this regard, our preliminary studies provide compelling evidence that a protein encoded by Apolipoprotein 6 (ApoL6) is a selective regulator of mitochondrial trifunctional protein (TFP), the key enzyme catalyzing FA β-oxidation. ApoL6 is highly expressed in WAT and differentiated adipocytes. In contrast, BAT normally expresses low levels of ApoL6, which increases during obesity or thermoneutrality-induced whitening. We found that in the basal condition, ApoL6 is localized to mitochondria, resulting in attenuation of mitochondrial FA oxidation. Transgenic mice expressing ApoL6 in BAT exhibited a lower thermogenic capacity upon cold exposure, and decreased energy expenditure along with increased adiposity. Conversely, adipose ApoL6 knockout mice on HFD showed a decreased body weight and fat mass gain. Based on these preliminary data, we hypothesize that by inhibiting mitochondrial FA oxidation and thereby decreasing energy expenditure, ApoL6 plays a key role in (1) maintaining fat storage in WAT and whole-body energy balance, and (2) impeding non-shivering thermogenesis in whitened BAT. There are three aims: In aim 1, we will determine the mechanistic role of mitochondrially localized ApoL6 in adipocyte FA oxidation by using gain- and loss-of-function cell models. In Aim 2 we will determine how adipose specific ApoL6 ablation in mice affects diet-induced obesity and its related metabolic changes including insulin resistance. In Aim 3 we will use both loss- and gain-of-function mouse models to define the role of ApoL6 in the regulation of BAT thermogenesis during obesity- and thermoneutrality-induced whitening. Evidence derived from this project should provide novel insight into the molecular basis for the regulation of FA oxidation and energy metabolism in adipose tissue, thereby advancing the possibilities for the development of novel therapeutic approaches to combat obesity and type 2 diabetes.