Mayo Clinic Rochester

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is characterized by the selective destruction of insulin producing β-cells due to infiltrating autoaggressive T-cells and a break in immune tolerance. As a consequence, the patient is met with a severe loss of β-cell function and mass thus requiring the need for exogenous insulin. Preventative immunomodulatory therapeutic strategies to improve disease outcome have been met with minimal success. Therefore, novel strategies to restore overall β-cell function and health are warranted. Recent evidence suggests that β-cells themselves facilitate their own complicit demise during the progression of T1D and thus may serve as potential therapeutic targets of intervention. Chronic exposure to pro-inflammatory mediators has been shown to induce β-cell dysfunction; however, the mechanisms mediating this process have yet to be fully elucidated. In this application, we propose to study the impact of pro-inflammatory mediators on the β-cell dialogue through exchange of circulating nanovesicles termed extracellular vesicles (EVs). We and others have noted significant content alterations in EV cargoes at the protein and miRNA level upon T1D pro- inflammatory cytokine induction. However, the direct physiological and mechanistic implications of altered pro- inflammatory β-cell EV cargo to recipient β-cells have yet to be fully elucidated. The long-term goal of the applicant is to establish an independent laboratory exploring the physiological and mechanistic role of EVs in the pathogenesis of T1D. The central hypothesis of the application is that β-cells contribute to their own demise through diabetogenic β-cell EV cargo exchange to induce β-cell dysfunction and enhancement of antigen processing and presentation. To test this hypothesis, two specific aims have been proposed which are backed by extensive preliminary data. In Aim 1, we will test the hypothesis that pro-inflammatory β-cell EVs activate the CXCL10:CXCR3 axis to induce β-cell failure. In Aim 2, we will test the hypothesis that pro- inflammatory β-cell EVs contribute to β-cell immunomodulation during the pathogenesis of T1D. Both Aims will use ex vivo and in vivo mouse models of T1D and human islets and cell lines to address the hypotheses. The applicant is part of the highly collaborative Islet Regeneration Center (Center for Regenerative Medicine) at Mayo Clinic, Rochester, MN. This outstanding research environment in addition to a carefully selected mentoring committee comprised of established NIH-funded investigators will provide the applicant with the necessary tools and support to establish herself as an independent research investigator.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Despite being the most common myopathy after the age of 50, inclusion body myositis (IBM) pathogenesis remains poorly understood. Traditionally, IBM is considered an inflammatory myopathy. However, unlike other inflammatory myopathies, IBM almost never happens before the age of 40, has histopathological features of rimmed vacuoles and protein aggregates reminiscent of other neurodegenerative diseases, and remains refractory to all forms of immunotherapy. Patients continue to progress relentlessly and most become wheelchair dependent within 20 years from onset. Therefore, there is a critical need to determine the underlying disease mechanisms that would offer a unifying holistic explanation of the involved downstream immune and degenerative processes, and help identify new therapeutic targets. Our central hypothesis is that declining mitochondrial function plays a primary role in IBM pathogenesis, which after reaching a critical threshold at a certain age, triggers downstream inflammatory and degenerative pathways. The overall objective of this application is to define the underexplored role of mitochondria in IBM pathogenesis, aiming in the long term to identify better diagnostic biomarkers and novel evidence-based therapeutic targets for clinical trials. Therefore, we aim to: 1) Characterize the mitochondria’s morphology, dynamics and interaction with other organelles in IBM, by using 3D Scanning Electron Microscopy. 2) Identify the specific molecular level of mitochondrial dysfunction in IBM by a series of cutting-edge techniques. 3) Define the disease-specific metabolomic and transcriptomic profiles of IBM. Our proposed approach will define the nature and the extent of mitochondrial dysfunction in IBM, and provide valuable insights into IBM pathogenesis focused on, but not limited to the mitochondrial pathways. The candidate is a neurologist with advanced training in Neuromuscular Medicine, Muscle Pathology and Electrodiagnostic Medicine. The goal for this application is to help him develop laboratory-based skills necessary for translational research, expertise in Electron Microscopy, and training in Systems Biology approaches. He will be guided by a multidisciplinary team of well-established researchers who have made significant contributions to the fields of mitochondrial biology, Neuromuscular medicine and Aging. These include: Jania Trushina PhD (primary mentor), Anthony J. Windebank MD, and Ian R. Lanza PhD. The career development plan combines the strengths of the candidate, the mentors and the research institution and will help enable the candidate to become a successful independent investigator.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT Right heart failure (RHF) is the leading cause of mortality in people with repaired tetralogy of Fallot (TOF), and the sequence of events leading to this suboptimal outcome begins with pulmonary regurgitation (PR) and right heart (RH) remodeling. Arrhythmias and impaired aerobic capacity are the most common presentations prior to the onset of RHF, but performing pulmonary valve replacement (PVR) after the onset of these symptoms is not associated with improved outcomes. Recent data show that elevated right atrial pressure (RA hypertension), as estimated by echocardiographic assessment of inferior vena cava (IVC) size and collapsibility (IVC hemo- dynamics), precedes the onset of arrhythmias and impaired aerobic capacity in TOF patients, and it is associ- ated with accelerated RH remodeling, symptomatic deterioration and mortality in this population. However, the mechanism linking RA hypertension, RH remodeling and symptomatic deterioration, and the extent to which performing PVR prior to the onset of RA hypertension improves clinical outcomes are unknown. The long-term goal is to prevent premature cardiovascular deaths in TOF patients by modifying the risk factors for mortality. The overall objective is to delineate the pathophysiologic mechanism linking RA hypertension, RH remodeling and onset of symptoms such as arrhythmias and impaired aerobic capacity, since symptomatic status is a risk factor for mortality in the TOF population. Our central hypothesis is that RA hypertension leads to accelerated RH remodeling and onset of symptoms (arrhythmias and impaired aerobic capacity), and that performing PVR prior to onset of RA hypertension is associated with RH reverse remodeling and improvement of symptoms. This hypothesis will be tested by pursuing two specific aims: (1) Determine the mechanism linking RA hyper- tension (assessed by IVC hemodynamics), RH remodeling and onset of symptoms (arrhythmias and impaired aerobic capacity) in TOF patients with moderate-severe PR; (2) Determine the extent to which performing PVR prior to the onset of RA hypertension is associated with RH reverse remodeling (improvement of imaging and biomarker indices of RH remodeling) and improvement of symptoms (less arrhythmias and improved aerobic capacity). Under the first aim, 150 asymptomatic subjects (75 in each arm) will undergo imaging, laboratory blood tests, exercise test, and patient reported quality of assessment at baseline, 12 months and 24 months. Under the second aim, 120 subjects (60 in each arm) undergoing PVR for clinical indications will be enrolled to undergo multi-domain assessments at baseline (prior to PVR), 12 months and 24 months similar to the first aim. This proposal is innovative because it will delineate the mechanisms responsible for symptomatic deterio- ration, and the impact of PVR on these mechanisms. The results will be significant because it will set the stage for future clinical trials to test the survival benefits of PVR performed at different stages of disease pathogene- sis, and development of novel therapies for the prevention and early treatment of RHF.

NIH Research Projects · FY 2025 · 2021-07

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the United States (US) with ~21,000 new cases diagnosed each year. CLL is still incurable despite the fact that there have been significant developments in therapeutic interventions with subsequent improvements in outcome. There are initial findings demonstrating differences in molecular and clinical characteristics of CLL patients across human populations. In particular, African American CLL patients have a younger age of onset, a more aggressive disease at diagnosis, shorter median time to therapy initiation, and reduced overall survival compared to other CLL patients, even after controlling for therapy, suggesting that additional factors may exist that may be driving these observed differences. Despite these differences, little is known about the relationship between genomics and CLL pathogenesis across patients in the US. The goal of this application is to expand our understanding of the genetic basis of CLL with the overall hypothesis that genomic features exist that may be associated with the observed differences of incidence, morbidity, and mortality in CLL patients. To test this hypothesis, we will leverage our extensive experience in CLL and applying it to our unique cohort of CLL patients representative to that seen in the US. In Aim 1 we will perform a multi-omic study in our CLL patients and compare the findings with publicly available sequencing data in CLL. With these data, we will be able to characterize the tumor variability and identify novel somatic findings in our unique cohort. In Aim 2, we will evaluate the known CLL susceptibility loci identified through genome wide association studies (GWAS) in our CLL cases and controls in order to provide insight into the generalizability of the CLL known variants. Finally, in Aim 3, we will evaluate the generalizability of our findings that the genomic summary measure, the tumor mutational load, defined as the number of recurrently mutated CLL driver genes, is prognostic in our unique cohort of CLL patients. The knowledge gained from this application may provide novel insight into the biological variability in leukemogenesis, as well as provide understanding of the generalizability of the genetic findings.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Hand flexor tendon injuries are common and often occur in a young working-age population resulting in considerable disability and economic impact. Surgical direct repair immediately after tendon injury is the clinical standard in practice. However, clinical and functional outcomes following tendon repair remain unsatisfactory due to restrictive adhesions and poor digital motion, often resulting in multiple surgical revisions, such as tenolysis or tendon grafting. It is known that both intrinsic and extrinsic healing mechanisms are involved in flexor tendon healing. Intrinsic healing is accomplished by cellular productivities from the cells within the tendon resulting in fewer adhesions and better function. In contrast, extrinsic healing relies on the healing from outside tissues, leading to adhesion and scar formations that bond to the tendon with surrounding tissues and diminish hand function. Therefore, research strategies to improve clinical outcomes have focused on either enhancing intrinsic healing or eliminating extrinsic healing, or a combination of both. It is also recognized that some intrinsic healing elements (IHE) involve tendon intrinsic healing capacity including flexor vinculum (FV) for tendon blood supply and epitenon cells (ECs) within the tendon for tendon regeneration. However, it is still unknown if and how these IHEs would affect the intrinsic healing ability. Since the IHEs can be damaged during tendon injury, it is critical to better understand the intrinsic healing associated with IHEs, which not only help to bridge the scientific gap between clinic and research in this field, but also improve the intervention strategy development. Recently, we have successfully developed a novel turkey animal model, which is similar to the human flexor tendon in size, anatomy, structure, function, and most importantly the intrinsic healing capacity. This unique animal model provides an ideal opportunity to investigate the effects of the IHE on tendon intrinsic healing. Furthermore, we have recently explored a purified exosome product (PEP) developed by Mayo Center for Regenerative Medicine in the ISO-5 Good Manufacturing Practice (GMP) Facility to improve tendon intrinsic healing with promising results. We have also developed a lubricating barrier material using carbodiimide derivatized synovial fluid plus gelatin (cd-SF-G) to reduce adhesions in the tendon graft; but it has not been tested in flexor repair model. Therefore, Aim 1 of this proposal is to determine the role of two major factors of IHEs including FV and ECs on tendon healing and functional restoration using our novel turkey flexor tendon injury model. This specific aim, if successful, we will address a critical barrier for the understanding of flexor tendon intrinsic healing mechanism and advance the current knowledge in hand surgery. Aim 2 will define the effectiveness of our novel interventions using PEP for enhancing intrinsic healing ability and cd-SF-G for preventing extrinsic healing to reduce scar and adhesion formations using our new turkey animal model. If successful, we will have developed and validated the clinically translational interventions to improve functional outcomes following flexor tendon repair, since both our therapeutics, GMP grade PEP and native SF based material, are one step close to a clinical trial. Thus, the proposal has a significant impact on both basic science research and clinical translation.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Acute myeloid leukemias (AMLs) are a genetically heterogeneous group of clonal hematopoietic disorders characterized by accumulation of immature non-lymphoid marrow progenitors. While there have been notable therapeutic advances over the past 5 years, many AML subtypes continue to have case fatality rates of >50%. Despite the introduction of a number of targeted therapies, conventional cytotoxic drugs – alone or in combination with the targeted agents – remain the mainstay of AML therapy. Among the conventional cytotoxic drugs used to treat AML, several act by increasing unique types of DNA lesions known as DNA-protein crosslinks (DPCs). In particular, topoisomerase poisons increase the number of DPCs containing TOP2 (daunorubicin, mitoxantrone, etoposide) or TOP1 (topotecan) covalently bound to DNA. In addition, the hypomethylating agents decitabine and azacitidine increase the number of DPCs containing DNA methyltransferases covalently bound to DNA. The mechanisms involved in DPC repair are at present incompletely understood. To facilitate the further development of topotecan and other TOP1 poisons, as well contribute to the study of TOP1-containing DPCs, we have generated an antibody that specifically recognizes TOP1-DNA covalent complexes (TOP1ccs). Using this antibody, we have shown that the nuclear metalloproteinase SPARTAN and the serine protease FAM111A, acting upstream of the phosphodiesterase TDP1, play important roles in the repair of TOP1ccs in some tissues. Importantly, loss of SPARTAN, FAM111A or TDP1 leads to accumulation of TOP1ccs in the absence of drug treatment as well as enhanced sensitivity to the prototypic TOP1 poison camptothecin. More recently, we have also observed that a variety of malignant myeloid cells, including AML cell lines and primary AML specimens, contain readily detectable TOP1ccs in the absence of drug treatment and are slow to repair TOP1ccs upon exposure to the TOP1 poison topotecan. In contrast, the vast majority of tissues, including normal and malignant lymphoid cells as well as normal marrow, contain very few TOP1ccs in the absence of drug treatment. These results lead to the hypothesis that many myeloid neoplasms have previously unsuspected defects in TOP1cc repair that might affect their therapeutic sensitivity. To test this hypothesis, we now propose to define the biochemical basis for the constitutive increase in TOP1ccs in myeloid neoplasms (Aim 1), examine the impact of low TOP1cc repair on leukemia cell sensitivity to agents that stabilize DPCs (Aim 2), and assess the relationship between TOP1cc levels (constitutive and drug-induced) and clinical response of myeloid neoplasms to a topotecan-containing regimen currently undergoing NCI-sponsored phase II clinical testing in high risk AML (Aim 3). Collectively, these studies will provide important new insight into a previously unsuspected DPC repair defect in myeloid malignancies and begin to determine whether this repair defect has therapeutic implications that can be used to guide improvements in AML therapy.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT High-grade serous ovarian cancer (HGSOC) remains the deadliest form of ovarian cancer, in part because most patients develop recurrent disease that is resistant to standard treatment, including platinum. Poly(ADP- ribose) polymerase (PARP) inhibitors (PARPis) have recently been approved as an important therapy for HGSOCs, especially for HGSOCs with defects in homologous recombination (HR) DNA repair due to mutations in BRCA1 or BRCA2. However, over 70% HGSOCs that initially respond to PARPis later develop resistant disease. Unfortunately, the underlying mechanisms of PARPi resistance are poorly understood. This project is designed to understand acquired PARPi resistance mechanisms and associated therapeutic vulnerabilities in HR-defective HGSOC. Our preliminary studies using HR-deficient HGSOC cell lines and patient derived xenograft (PDX) models show that acquired PARPi resistance is associated with high levels of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). Our results also show that NMNAT1 upregulation results in an increase in NAD+ levels, which restores HR and creates a unique metabolic dependency in PARPi-resistant cells. These findings led to our central hypothesis that HR-deficient HGSOC acquire PARPi resistance by upregulating NMNAT1 that induces NAD+ levels leading to HR restoration as well as causing a metabolic dependency that may be therapeutically tractable. Guided by strong preliminary data, we propose three specific aims to: 1) examine how NMNAT1 is upregulated in HGSOC cells; 2) determine how NMNAT1 induces PARPi resistance; and 3) test whether NMNAT1-induced metabolic dependency in PARPi- resistant tumors can be targeted in preclinical models of HGSOC. These studies will unveil a previously unknown mechanism by which HGSOC cells become resistant to PARPis and may identify a potential new therapeutic option for PARPi-resistant HGSOC. The proposed work comprises an essential step toward our long-term goal of developing effective therapy for patients with HGSOC.

NIH Research Projects · FY 2025 · 2021-06

Abstract Age-related bone loss puts individuals at risk for debilitating osteoporotic fractures. Current osteoporosis therapies primarily target bone-resorbing osteoclasts to prevent further bone loss; however, because of the coupling of osteoclasts and osteoblasts, these therapies are limited by a concomitant decrease in bone formation. Thus, new treatments are needed to reduce bone loss while protecting or stimulating new bone formation. Osteoclasts are multinucleated cells derived from the myeloid lineage. While most well-known for bone resorption, osteoclasts exhibit a range of functions, including stimulating bone formation by osteoblasts (coupling activity). Increasing evidence shows that osteoclasts exhibit functional heterogeneity. Our data presented herein confirm that not all osteoclasts are actively resorbing or coupling, and others have documented heterogeneity in osteoclast resorptive activity itself. With aging, there is a shift in osteoclast functional distribution, with an increase in the percentage of actively resorbing osteoclasts and a decrease in osteoclasts positive for coupling factor expression. Aging also leads to an increase in an aggressive subpopulation of osteoclasts, which move while resorbing bone leading to trench formation. Our data support that these aggressive, trench-forming osteoclasts, which exhibit greater acidification of the resorption lacunae and increased protease activity, have impaired ability to recruit osteoprogenitors to sites of resorption. Therefore, we hypothesize that the increase in aggressive, trench-forming osteoclasts with age is linked to reduced osteoclast coupling activity, leading to overall bone loss. To test this hypothesis, we propose to 1) Determine whether inducible activation of osteoclast coupling activity prevents age-related bone loss.; 2) Evaluate whether differential osteoclast resorptive behaviors impact coupling; and 3) Test the role of matrix-derived TGF-β as a feedback mechanism to modulate osteoclast activity. Altogether, these studies will reveal the distribution of osteoclast behaviors and how these are dysregulated during aging. In addition, understanding the mechanisms by which changes in resorption contribute to altered coupling activity will reveal potential new therapeutic targets for shifting osteoclast activities to treat osteoclast-mediated bone disease.

NIH Research Projects · FY 2025 · 2021-06

Project Summary The main goal of our proposal is to investigate the role of astrocytes in the dorsal striatum (DS), which regulates goal-directed and habitual reward-seeking behaviors in mice. The DS has a critical role in shaping goal-directed and habitual actions, which are the main determinants for the reward-dependent decision-making process. Astrocytic processes in close proximity to the synaptic milieu clear glutamate, which protects neurons from excitotoxicity. Our recent studies revealed that chemogenetic activation of dorsomedial striatum (DMS) astrocyte enhances the activities of indirect medium spiny neurons (iMSNs), but not dMSNs in the DMS. The DMS is known to regulate goal-directed actions as lesions or inactivation of DMS render actions habitual instead of goal-directed. Conversely, the dorsolateral striatum (DLS) is necessary for habitual actions as lesions or temporary inactivation of DLS bias behavior towards goal- directed actions. Since GABAergic iMSNs project to external globus pallidus (GPe), we also examined the role of astrocyte in the GPe. Interestingly, both chemogenetic astrocyte activation of DMS and GPe promotes transition from habitual to goal-directed ethanol-seeking behaviors. However, the precise role of astrocytes in the DMS-GPe or DLS-GPe circuits in regulating habitual ethanol seeking behavior has not been explored. Based on our findings, we hypothesize that astrocyte activities differentially regulate MSNs activities in the DMS and DLS, thereby determine goal-directed and habitual ethanol-seeking behaviors. To investigate this hypothesis, we propose three aims. First, we will determine how activation of astrocyte activities differentially regulates the alcohol-induced changes in glutamatergic and GABAergic signaling of the DMS and DLS. Second, we plan to examine the role of DMS and DLS astrocyte activation in goal- directed or habitual ethanol-seeking behaviors. Finally, we will investigate the effect of ablation of DMS- GPe and DLS-GPe circuits in goal-directed or habitual ethanol-seeking behaviors. Our study will elucidate the neural mechanisms encoding goal-directed and habitual ethanol-containing reward-seeking behaviors. We will provide a rational path for the development of new therapeutic methods for the treatment of AUD.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract Anemia is a common but underappreciated complication of critical illness. While increased tolerance of anemia has not been associated with negative short-term outcomes such as mortality, the impact on long-term functional outcomes (physical function, cognition, mental health, quality of life) remains unknown. Impairments in these functional domains are highly prevalent and debilitating in survivors of critical illness, and the identification of modifiable risk factors has been recognized as a key priority in critical illness research. The long-range goal of the applicant is to become a successful independent translational clinician-scientist leading a multidisciplinary team to optimize patient recovery after critical illness. The scientific objectives of this application are to: 1) identify patients at highest risk for impaired recovery from anemia after critical illness; 2) assess the relationships between persistent post-hospitalization anemia and functional outcomes; and 3) to test the feasibility and impact of a novel anemia prevention and treatment intervention on post- hospitalization functional outcomes. The training goals of the applicant are to obtain training and expertise in biostatistics and data science, multi-dimensional functional outcome assessment, and pragmatic clinical trial design and implementation science. Aim 1. To assess patterns and consequences of anemia recovery in the first year after critical illness • Recovery from anemia after critical illness remains incompletely characterized. In this aim, epidemiologic data from a large cohort of ICU survivors will be utilized to evaluate hemoglobin recovery after critical illness. Advanced statistical and data science approaches will be used to identify unique profiles of hemoglobin recovery, predict patients at high risk for impaired recovery, and assess the relationships between hemoglobin recovery and functional outcomes in the first year after hospitalization. Aim 2. To perform a pilot pragmatic clinical trial testing a multi-faceted anemia intervention (optimized phlebotomy practice, clinical-decision support, targeted pharmacologic anemia treatment) to attenuate anemia development and promote functional recovery in the setting of critical illness • The impact of anemia treatment interventions on functional outcomes after critical illness has not been defined. This aim will employ a pilot clinical trial testing the feasibility and efficacy of a novel multi-faceted anemia intervention aimed at attenuating and treating anemia during critical illness for the modification of hemoglobin levels and post-hospitalization functional outcomes (i.e. 3 and 6 months after hospitalization).

NIH Research Projects · FY 2025 · 2021-06

Abstract Homologous recombination (HR) is an important DNA repair mechanism for DNA damage caused by PARPi and platinum. The HR pathway is closely associated with ovarian cancer development and chemoresistance. Recent clinical studies showed that PARP inhibitors and platinum are effective in treating ovarian cancers with mutations of BRCA1 or BRCA2, and other genes encoding proteins involved in HR. Conversely, factors involved in HR promote the repair of DNA lesions caused by PARP inhibitors and platinum, and this enhanced HR capability contributes to chemotherapy resistance. Therefore, targeting HR pathway may be a powerful strategy to overcome resistance to DNA damage-based therapy. Here we propose to study a new ATM-SYK-CtIP pathway that regulates HR. We found the tyrosine kinase SYK plays an important role in HR. SYK’s function in immune regulation is well established, however, its function in DNA repair has not been shown. We found that SYK phosphorylates CtIP and regulates CtIP function in HR. SYK itself is also phosphorylated in an ATM dependent manner and get recruited to the sites of DNA damage. Interestingly, SYK is overexpressed in recurrent ovarian cancers; high expression of SYK is related to poor outcome of OCs. Furthermore, inhibition of SYK in OC cell lines renders OC cells sensitize to PARPi or cisplatin. Taken together, we hypothesize that the ATM-SYK-CtIP pathway is a new regulatory mechanism for HR. Inhibiting of SYK sensitizes OC cells to cisplatin or PARPi, suggesting SYK as the novel potential therapeutic targets or biomarkers for ovarian cancer therapy. To test this hypothesis, we propose the following Specific Aims: 1. Investigate the regulation of HR by SYK; 2. Investigate the regulation of SYK by the DNA damage signaling; 3. Determine the inhibition of SYK in chemo-response in OCs using organoid and mouse models. Our studies will reveal the novel function of SYK in DNA repair and response to chemotherapy. In addition, it will reveal the new therapeutic targets and biomarkers in OC therapy.

NIH Research Projects · FY 2024 · 2021-06

PROJECT SUMMARY Pediatric high-grade gliomas including Diffuse Intrinsic Pontine Gliomas (DIPG) are aggressive brain tumors that occur in children. The lifespan for these patients after diagnosis is about one year with no cure in sight. Countless clinical trials have been performed without success and treatment remains palliative despite extensive research over the past decade. It is therefore critical to identify new therapies for these deadly diseases. Among pediatric patients, one of the most devastating brain tumor types is diffuse midline gliomas with the H3K27M mutation, which includes the previously named Diffuse Intrinsic Pontine Glioma (DIPG). Recently, somatic mutations in the H3F3A gene, one of the 16 genes that encode Histone H3, have been detected in the majority of high-grade pediatric glioma cases including DIPG. This mutation leads to an amino acid change at lysine (K) 27 residue of H3.3 to methionine (M). The H3K27M mutation is a striking example of a genetic alteration that drives tumorigenesis by modifying the epigenome. H3K27 is modified post- translationally by either acetylation or methylation. H3K27 trimethylation (H3K27me3) plays an important role in gene silencing during stem cell differentiation and maintenance. The major pathologic finding in H3K27M tumors is a global loss of H3K27me3. The changes in H3 Lysine methylation patterns dramatically change gene expression and are likely to function as drivers of malignancy in these tumors. Our goal is to develop new therapies for treating children with H3K27M tumors by developing a toolbox of primary and secondary assays and to identify small molecule compounds that increase the suppressed H3K27me3 levels in tumors with the H3K27M mutation. Our preliminary data along with others show increasing H3K27me3 leads to tumor death in H3K27M mutant tumors. Building on these exciting results we hypothesize that a disease-relevant high- throughput screening (HTS) assay can be developed and executed leading to novel therapeutic agents for brainstem tumors with the H3K27M mutation.

NIH Research Projects · FY 2025 · 2021-05

MODIFIED PROJECT SUMMARY/ABSTRACT SECTION Rural areas, where ~60 million Americans live, have a higher burden of type 2 diabetes mellitus (T2DM) compared to urban areas, yet, there is sparse information on T2DM risk factors and interventions that improve T2DM primary prevention. Dr. Dugani, an academic hospitalist and early-stage investigator, is committed to improving the primary prevention of T2DM in rural areas. His long-term goal as an independent clinical investigator is to reduce health disparities for T2DM in rural populations by developing patient-centered interventions for better T2DM primary prevention. The short-term goals of this proposal are to acquire proficiency in data science, qualitative/mixed methods, health disparities research, and clinical trial study design through a Master of Public Health degree; mentorship; and, leadership skills to build and lead an independent research program to advance T2DM care. This research will be conducted at Mayo Clinic, which has robust infrastructure and facilities for patient-oriented research. Dr. Dugani will leverage data from the Centers for Disease Control and Prevention (Aim 1) and Mayo Clinic Biobank (Aims 2 and 3) to advance the research aims and also rely on a dedicated team of mentors and advisors with expertise in T2DM, epidemiology, health disparities research, and health services research. Dr. Dugani will accomplish his short-term goals through the novel Rural Patient Risks and Exposures for Diabetes ConTrol (Rural PREDICT) study, which he developed to investigate the higher burden of T2DM in Midwest rural compared to urban populations. At present, there is sparse information on risk factors for T2DM in Midwest rural populations and no T2DM risk prediction score for Midwest rural populations. Through Rural PREDICT, Dr. Dugani will address these knowledge gaps. Specifically, in Aim 1, he will characterize the incidence rate and temporal trend of diabetes in the rural Midwest. In Aim 2, he will evaluate the associations of traditional risk factors (e.g., lifestyle, socioeconomic status) and novel ruralenriched risk factors (e.g., organic chemicals, pesticides) with incident T2DM in the rural Midwest. He will directly engage rural patients to explore their perspectives regarding the high burden of risk factors. In Aim 3, he will develop a non-invasive (i.e., non-laboratory) 5-year T2DM risk prediction score for rural populations and obviate the need for laboratory testing, which may be a barrier for rural populations. He will engage patients to develop a meaningful, non-stigmatizing questionnaire for the risk score and explore barriers to the use of this risk score. These studies will form the basis for an NIH R01 (years 4-5) application to advance T2DM primary prevention in Midwest rural populations. His research goals align with NIMHD’s priority to reduce health disparities in minority groups, including rural communities.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Aging is the strongest risk factor for cognitive decline and dementia. Targeting fundamental aging mechanisms offers promising new strategies to counter brain dysfunction. Recent breakthroughs have demonstrated that proteins in aged blood circulation mechanistically contribute to accelerated brain aging, and senescent cells (SCs) accumulate in aging and may drive tissue deterioration, in part, through the proinflammatory senescence associated secretory phenotype (SASP). This research is designed to test whether progeronic SASP proteins produced by systemic SCs mechanistically contribute to accelerated brain aging through blood circulation. This premise is based on published findings establishing that aged blood circulation and/or direct administration of progeronic plasma proteins that are putative SASP factors are sufficient to transfer an accelerated aging phenotype to young mouse brain and our research demonstrating that systemic SC clearance attenuates the SASP in circulation, which is associated with improvements in brain inflammatory parameters and cognitive decline. To test our central hypothesis, we will combine circulatory exchange methods with mouse models in which SCs can be eliminated or production of the SC proteome can be precisely monitored, which will enable us to study whether reducing the circulating SASP is sufficient to ameliorate the adverse influence of aged blood on brain homeostasis. We will develop an innovative transgenic mouse model that will enable bioorthogonal labeling of the nascent p16+ SC proteome. This will empower our discovery of the age- and tissue-specific p16+SC-proteome, its contribution to the circulating progeronic proteome, and its responsivity to SC clearance. Administration of bioorthogonally labeled aged plasma +/- SC clearance to young mice will facilitate discovery of candidate SASP proteins responsible for accelerated aging brain phenotypes for further mechanistic interrogation. Modifying aged blood composition and targeting SCs are therapeutics actively being pursued for ameliorating age-related decline. This project is designed to mechanistically synergize and advance these two promising concepts. Our research may preclinically implicate systemic SC clearance as an option to deplete the progeronic influence of aged blood, ultimately revealing a novel approach for treatment or prevention of age-dependent cognitive decline and dementia.

NIH Research Projects · FY 2025 · 2021-05

PROJECT ABSTRACT Portal hypertension (PHTN) is a common final pathway of multiple forms of chronic liver disease which accounts for significant morbidity and mortality among patients with liver disease. However, there is a paucity of therapies available to ameliorate PHTN. The overall objective of this proposal is to elucidate the contribution of neutrophils and platelets to the pathogenesis of PHTN, with the therapeutic goal of facilitating the design of therapies to decrease portal pressure. The pathophysiology of PHTN is complex and is regulated at multiple levels, including paracrine signaling within sinusoids, formation of microvascular thrombosis, and endothelial dysfunction. We have previously identified a novel but critical role of neutrophils in the pathogenesis of PHTN. We found that cyclic stretch imposed by congestive hepatopathy (CH) induces activation of mechanosensitive Piezo channels within liver sinusoidal endothelial cells (LSECs). Piezo channels activate mechanocrine signaling pathways which culminate in secretion of the neutrophil chemotactic cytokine CXCL1. CXCL1 induces infiltration of neutrophils into liver sinusoids. Neutrophils form complexes with platelets which lead to the formation of neutrophil extracellular traps, or NETs. We found that genetic and pharmacologic inhibition of NET formation significantly decreases portal pressures in murine models of CH and PHTN. Although platelets have been implicated in NET formation in certain settings, the mechanisms which recruit platelets and regulate their interactions with neutrophils to generate sinusoidal NETs require further investigation. Weibel-Palade bodies (WPBs) are endothelial organelles which generate extracellular vesicles (EVs) containing inflammatory and hemostatic factors. The impact of WPB-derived EVs on platelet recruitment to liver sinusoids has not been studied. We have formulated the central hypothesis that platelets activated by WPB-derived EVs interact with the CD11b/CD18 integrin receptor on neutrophils to stimulate NET formation and PHTN. We will test this hypothesis through the following independent but integrated specific aims which are both technically and conceptually innovative. First, we will test the hypothesis that Piezo channels serve as master mechanosensors whose activation regulates the generation of both neutrophil- and platelet- chemotactic factors which modulate portal pressures. We propose that Piezo activation generates platelet chemotactic factors within EVs derived from WPBs in LSECs. Finally, using murine models and clinically- relevant forms of platelet inhibition, we will test the hypothesis that interaction of the neutrophil integrin receptor CD11b/CD18 with the platelet glycoprotein receptor GPIbα drives NET formation. Our proposal is significant because it has the potential to elucidate novel therapeutic targets to better manage PHTN, a devastating and prevalent disease which is currently curable only with liver transplantation.

NIH Research Projects · FY 2025 · 2021-05

Enter the text here that is the new abstract information for your application. The advancements of genomic technologies and assemblies of large disparate sets of biological and health data have outpaced the ability to integrate these different sources of information. Powerful statistical methods and software are needed to fill this gap in order to provide novel understandings of biological processes, as well as provide better predictions of human diseases to achieve the vision of personalized medicine. The broad goals of this project are to advance genetic epidemiology studies of human traits and diseases by expanding our development of statistical analytic methods and software encompassing four main areas: 1) multivariate methods to decipher genetic contributions; 2) statistical fine-mapping of genetic variants; 3) causal mediation methods; 4) polygenic risk scores (PRS) for predicting disease. Although these areas might appear broad and disparate, there is pressing need to build more integrative methods across these domains. For example, because molecular pleiotropy is pervasive, multivariate analysis is essential to identify shared genetic factors acting through common biological mechanisms of multiple traits, and when using PRS to predict disease, complex traits are often better predicted when multivariate correlated traits are used. And, the methods used for statistical fine-mapping, including use of annotation, are relevant for creating PRS to predict disease. Our team, involving statistical geneticists, computational biologists, genetic epidemiologist and clinical investigators, has decades of experience and will capitalize on the extensive resources and collaborations we have developed. Our novel methods will be applied to a broad range of diseases, with ultimate aims to better understand disease etiology and improve disease prediction to achieve earlier diagnosis. User-friendly software will be distributed with open access to the scientific community. We will take advantage of rapidly evolving technologies, biologic and computational insights from multiple fields, and evolving public health and clinical unmet needs to inform our science.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY / ABSTRACT Neurodegeneration markers (N markers) are associated with cognitive impairment in Alzheimer's disease (AD) and related dementias. Since they are more proximally related to cognitive decline than AD-specific biomarkers (amyloid and tau measures), they have been proposed as surrogate endpoints in clinical trials and even for prognosis in the clinical setting. Despite the promise of N markers, several barriers to their implementation exist. The major limitations include i) only validation in convenience samples that exclude those with significant cerebrovascular disease rather than the general population, ii) lack of systematic comparison of the frequently used N markers across modalities (i.e., imaging, CSF, blood) to predict cognitive outcomes at varying levels of AD and cerebrovascular disease pathology, and iii) lack of understanding of the pathological profile associated with each N marker. In the current application, our overall goal is to understand the unique information each N marker (imaging, CSF, blood) provides with regards to cognitive decline and underlying pathology to optimize their use in clinical practice and clinical trials. We will utilize the Mayo Clinic Study of Aging (MCSA) a longitudinal population-based study where participants undergo psychometric, neurologic, and neuroimaging investigations (MRI, amyloid and tau PET, FDG PET), and blood draws; a subset also have CSF and/or post- mortem data as the primary dataset for this grant. In Aim 1, we will compare N markers from neuroimaging, CSF, and blood in MCSA in relation to demographics (including socioeconomic status), in vivo AD (amyloid and tau PET) and cerebrovascular disease biomarkers, and cognition. We will validate some of these relationships in a biracial Mayo Clinic Jacksonville sample and in ADNI. In Aim 2, we will conduct an ante- mortem N marker – post-mortem validation study to determine the pathological profiles associated with (each and combination of) N markers. The findings of the grant will lead to better understanding of N markers associated with longitudinal cognitive decline and the pathologies associated these N markers. This knowledge will inform clinical trial design and guidance of which N marker(s) to choose for future clinical use for AD and ADRD.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Vertebral fracture is the most common type of osteoporotic fracture. Spine is also the most common site of bone metastasis, leading to pathologic vertebral fractures. While performing activities of daily living is an essential part of healthy aging, pathologic and non-pathologic vertebral fractures can occur during these activities in metastatic or osteoporotic spines. These fractures cause pain and neurologic manifestations, affecting quality of life. Currently, there is no objective clinical technique that can assess bone fracture risks associated with physical activities. To fill the gap, we propose a patient-specific computational technique to quantitatively evaluate spine injury risks associated with physical activities. This novel approach will enable clinicians to reliably recommend safe and individualized activities to elderly populations. To achieve this, we will obtain motions and muscle activity outcomes from an elderly patient cohort using video motion analysis. These data will be used as input for kinematic motion analyses and mechanical testing on cadaveric lumbar spines, to create and validate our computational models. The rationale for this project is that a QCT/FEA process that can mimic physical activities will be able to reduce vertebral fractures and improve quality of life in elderly patient populations. Our long-term goal is to develop reliable computational techniques to enable earlier injury risk predictions in elderly patients with musculoskeletal diseases. Our overall objective, in this application, is to develop a patient-specific quantitative computed tomography-based finite element analysis (QCT/FEA) method that can assess both kinematic motions and fracture characteristics of the spine, to estimate fracture risks of physical activities. To achieve the overall objective, the following three independent specific aims will be accomplished: 1) to obtain lumbar range of motion and muscle response outcomes in an elderly patient population during five physical movements; 2) to perform kinematic testing on cadaveric spines to measure intradiscal pressures –using a novel approach– during physical movements; and also mechanical testing on spine segments to measure intradiscal pressure at fracture; and 3) to develop and validate QCT/FEA models of the lumbar spine to estimate spine injury risks. This research will lead to the development of a computational tool that can assign a risk score associated with physical activities. Further, this work will provide preliminary data for future R01 grant proposals to predict fracture risks associated with physical movements and exercises in patient populations.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract Around 15% of the US population suffers from a motility or functional GI disorder (FGID), like irritable bowel syndrome (IBS). The pathophysiology of FGIDs remains poorly understood, leading to poorly targeted treatments. Enteroendocrine cells (EECs) in the GI epithelium release signaling molecules that control many processes affected in FGID patients, like motility and secretion. A large proportion of FGID patients have abnormalities in mechanosensation. A population of mechanosensitive EECs in the GI epithelium exhibits structural and functional similarities with specialized sensory epithelia, such as light touch receptors in the skin. The mechanosensitive EECs sense physical forces and convert them into hormone release. Thus, mechanosensitive EECs are primary mechanotransducers in the GI epithelium, and they may be targets for the treatment of FGIDs. They are characterized by expression of Piezo2, an ion channel that opens in response to force. Piezo2 generates a receptor current that initiates EEC mechanotransduction. Piezo2 is not the only mechanosensitive protein in these cells: actin fibers and tight junctions are also critical in epithelial force transmission. Understanding Piezo2 localization and its functional interactions with other mechanosensitive proteins is important for understanding sensory epithelial mechanotransduction. The overall goal of this proposal is to uncover mechanisms by which mechanosensitive proteins work together in EECs to make them efficient force sensors. The hypothesis is that the actin cytoskeleton plays a critical role in Piezo2+ EEC mechanotransduction by linking the channel to other mechanosensors and directly changing channel currents. Aim 1 investigates the assembly of mechanosensory proteins in the EEC by superresolution imaging and co- immunoprecipitation studies. These experiments explore direct and indirect interactions between the mechanosensors Piezo2, actin fibers and claudin-4. Aim 2 investigates how these mechanosensors affect overall EEC mechanotransduction by tracking force-induced calcium transients and electrophysiological studies of Piezo2. These experiments explore how force is transmitted in epithelial sheets to initiate the receptor current. The results of this work are poised to bridge knowledge gaps in Piezo2+ EEC mechanotransduction, as well as inform broader mechanosensing mechanisms in sensory epithelia. The proposed work will be carried out in a supportive environment that provides cutting edge tools and expert knowledge towards achieving the specified goal, including collaborations with experts in cytoskeletal biology,and an imaging core with vast experience in the proposed imaging techniques. The proposal includes a comprehensive training plan with physician-scientist mentors, by which the PI will gain valuable skills in the study of molecular mechanotransduction on clinically relevant questions. Along with research activities, the plan also includes clinical training and shadowing activities to prepare the PI for his transition to the next stage of training as a future surgeon-scientist.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Vasomotor symptoms (hot flashes) associated with menopause consist of rapid and intense sweating, peripheral vasodilation, and an exaggerated feeling of internal heat. Over 70% of women experience hot flashes at some point during menopause, which significantly impacts day to day life. Furthermore, hot flash frequency is related to increased risk of hypertension, cardiovascular disease (CVD), sleep, and mood disorders--even when controlling for traditional cardiovascular disease risk factors (obesity, hypertension, lipids, etc.). As such, the overall goal of this application is to determine autonomic neurovascular function in midlife women with and without vasomotor symptoms (hot flashes). For the purposes of this application we will define neurovascular function as a concept that encompasses sympathetic outflow, the responses of blood vessels to neurotransmitters released by the sympathetic nerves and the intrinsic properties of the vascular endothelium and smooth muscle. It is thus related to the regulation of tissue blood flow. In this context, neurovascular function is critically important to women’s health and aging. Aim 1 will compare microvascular function in women with objectively assessed low and high frequency hot flashes. Aim 2 will determine autonomic function and reactivity in women with objectively assessed low and high frequency hot flashes. Finally, Aim 3 will characterize cerebrovascular function in women with objectively assessed low and high frequency hot flashes. Importantly, this work will provide the first comprehensive and mechanistic evaluation of autonomic and neurovascular function in midlife women including those undergoing menopause. Understanding how the neurovascular physiology of women relates to hot flashes will inform hypertension and cardiovascular disease (CVD) treatment and prevention options, as the presence of hot flashes are a CVD risk factor beyond traditional CVD risk factors and as such, is likely to be an important determinant for a woman’s health. Thus, the Applicant’s long-term goal is to develop interventions to reduce the incidence of cardiovascular disease in women with hot flashes. The proposed studies will build on the Applicant’s previous training and the mentorship team, formal course work on running clinical trials, and technical training plan will provide opportunities to gain additional knowledge in a new area and learn new experimental techniques and approaches. Finally, this work will generate an investigative niche that will complement the Applicant’s intellectual and technical skills that will facilitate the launch of her independent career.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD), defined by the presence of beta-amyloid and tau, is the most common cause of dementia worldwide. Neuropathological studies have, however, revealed that in the majority of AD cases, beta- amyloid and tau are accompanied by the presence of other abnormal proteins. One such protein is the TAR DNA binding protein of 43 kDa (TDP-43), which is present in over 60% of AD cases. It has now become apparent that TDP-43 plays an important role in the AD neurodegenerative process. As such, our team has found strong associations between the presence and burden of TDP-43 and: (1) episodic memory loss, (2) smaller hippocampal volumes at death, (3) faster rates of hippocampal atrophy 10 years prior to death, and (4) the apolipoprotein epsilon 4 genotype, all which support TDP-43 as one of the important players in AD neurodegeneration. Recently, we reported two different types of TDP-43 deposited in the brains of patients with AD that we termed TDP-43 type-α and type-β. TDP-43 type-α is similar to TDP-43 seen in the brains of patients with frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) while type-β, is different, and is associated with neurofibrillary tangles, and hence tau. What is currently lacking though is an understanding of the mechanism/s underlying TDP-43 neurodegeneration in AD. TDP-43 is an RNA-binding protein known to regulate many facets of RNA metabolism; thus, TDP-43-associated toxicity in AD may result from the accumulation of key aberrant transcripts as a result of TDP-43 mislocalization to the cytoplasm, abnormal post- translational modifications to TDP-43 and/or TDP-43 sequestration into inclusions. One of these key aberrant transcripts may be the recently discovered truncated transcript variant of a microtubule-associated protein that is involved in axonal regeneration, termed stathmin-2 (STMN2). It has been demonstrated that decreased levels of TDP-43 lead to the accumulation of a truncated variant of STMN2 that lacks exons 2 through 5 (tSTMN2). Our exciting preliminary data shows that in FTLD-TDP, tSTMN2 is significantly elevated in frontal cortex where it correlates not only with TDP-43 burden but also with age at onset, yet not with survival after onset, suggesting that increased tSTMN2 RNA accelerates disease onset but does not alter the rate of disease progression. Therefore, given similarities between TDP-43 type-α in AD and TDP-43 in FTLD-TDP could the accumulation of tSTMN2 be involved in the mechanism of TDP-43-induced neurodegeneration in AD? Furthermore, given the link between TDP-43 type-β and neurofibrillary tangles, are there other unique aberrant transcripts that are specific towards toxicity in AD and which contribute to the pathogenesis of AD? We will leverage our knowledge and experience with clinical, pathological, neuroimaging, molecular and biological aspects of TDP-43 in AD and FTLD, by assessing relationships between STMN2, TDP-43 (including type-α and type-β), and clinical and neuroimaging features in a large cohort of 756 clinically and pathologically well- characterized cases with AD neuropathologic changes.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT - Alzheimer’s disease (AD) and other neurodegenerative conditions are characterized by heightened inflammation, neurodegeneration, and CNS vascular permeability, including microhemorrhage formation. The role of specific immune cell types in the underlying neuropathology associated with AD and other neurologic diseases remains an active area of research. Significant attention has been given to the role of innate immune cells and microglia in the development of AD. However, the role of adaptive immune cells, including CD8 T cells, has not been defined despite their presence in brain parenchyma of AD patients. These findings were further accentuated by the recent analysis of CD8 T cell repertoire and correlation with disease severity in human AD patients. APP/PS1 mice revealed significant brain infiltration of CD8 T cells of effector phenotype. Similarly, our Co-investigator, Dr. John Fryer of Mayo Clinic Arizona, has also observed significant CD8 T cell brain infiltration in his novel rAAV initiated tauopathy mouse model. Using our novel MHC class I conditional knockout mice, we have determined that macrophages and dendritic cells prime non-equivalent CD8 T cell responses in response to PbA infection. While both antigen presenting cells prime CD8 T cell response that infiltrate the brain, only CD8 T cells raised by dendritic cells induce lethal blood-brain barrier disruption. Our central hypothesis that clonally expanded CD8 T cells engage brain vasculature and migratory antigen presenting cells during infiltration which contributes to neuropathology and cognitive deficits in AD and Tauopathies. We plan to test this central hypothesis through execution of the following specific aims: Specific Aim 1 – Define the CD8 T cell repertoire and phenotype(s) generated in APP/PS1 and Tauopathy mice through transcriptional profiling. Specific Aim 2 – Determine critical role of residential and migratory APCs in priming and enabling CNS infiltration of CD8 T cell responses in APP/PS1 and Tauopathy mouse models Specific Aim 3 – Dissect the critical MHC class I expressing CNS cell type required for CD8 T cell induced neuropathology and cognitive deficits The proposed work is innovative because it capitalizes on our unique transgenic mouse models, novel imaging methodology, and new core facilities available to our research program at Mayo Clinic. Our goal is to define mechanistically the contribution of CD8 T cells in human dementia through knowledge gained using leading experimental models. Beyond the innovative methodology employed, the concept that antigen presenting cells raise differential CD8 T cell responses is highly novel and warrants further investigation to a mechanism which is therapeutically targetable. This is especially important if CD8 T cell priming and engagement of antigen presented by specific cell types is promoting neuropathology and behavioral deficits.

NIH Research Projects · FY 2025 · 2021-04

The goal of this career development award is for the applicant to expand her knowledge and technical expertise in tumor immunology and bioinformatic analyses in order to apply these new skills to investigation of the childhood cancer Ewing sarcoma. Relapsed Ewing sarcoma is nearly uniformly fatal, and thus new treatment approaches are desperately needed. A growing number of immune-based therapies are now being incorporated into pediatric cancer treatment protocols. Thus, in order to be a successful clinician scientist directing a cutting- edge research program in Ewing sarcoma, it is essential that the applicant gain expertise in tumor immunobiology. Currently, very little is understood about regulation of the Ewing sarcoma tumor immune microenvironment. This proposal addresses the interplay of two broadly important facets of pediatric cancer research: DNA damage repair and tumor immune response. This proposed work specifically examines the ability of DNA damage to activate Ewing tumor cell immunoregulatory pathways. The proposed aims will utilize a combination of in vitro cell co-culture analyses, in vivo tumor animal modeling and human specimen analyses in order to address this important topic. The applicant has chosen a primary mentor who is an expert in immune/inflammatory signaling in cancer, and a co-mentor with expertise in T-cell biology and advanced immunology techniques. Additionally, the applicant has constructed a physician-scientist career mentoring committee and network of collaboartors including individuals with expertise in Ewing sarcoma, humanized mouse models, DNA damage repair, the tumor immune-microenvironment, and bioinformatic analyses. As a team, the mentor, co-mentor, committee members and collaborators are dedicated to providing the applicant with technique training in tumor immunology and advanced cancer biology, as well as critical feedback on grants and manuscripts and career guidance. The primary mentor and co-mentor are invested in guiding the applicant toward a highly successful career as physician scientist by transitioning her to senior authorship on manuscripts, preparing her to start an independent laboratory and encouraging her to directly mentor scientists and clinicians at various levels of training. The University of Pittsburgh is an outstanding environment in which to develop a career and expand a research program in tumor immunobiology. The applicant’s career development plan takes full advantage of the many unique offerings at Pittsburgh including face-to-face career development workshops with seasoned scientists, participation in the Pittsburgh Women in Science forum, graduate level tumor immunobiology and bioinformatics courses and opportunities for presentation of work-in-progress at the University of Pittsburgh Cancer Immunology Program meetings. With the enthusiastic support and exceptional scientific expertise of her mentor and co-mentor, the applicant is eager to gain specific training in tumor immunobiology and bioinformatic analyses and to launch an independent research career as a pediatric oncologist physician scientist studying Ewing sarcoma at the University of Pittsburgh.

NIH Research Projects · FY 2025 · 2021-03

Project Summary Glioblastoma (GBM) prognosis remains dismal with a median survival of 16-18 months despite the use of multimodality treatment. Immunotherapy attempts have been unsuccessful in GBM treatment, including negative phase III trials of immune checkpoint inhibitors and vaccines. Based on strong preclinical data, we hypothesize that we can develop an effective immunotherapy approach against GBM by employing an immunostimulatory measles virus strain (MV-s-NAP) expressing the Helicobacter Pylori neutrophil-activating protein (NAP), a toll-like receptor 2 agonist. We also hypothesize that MV-s-NAP induced changes in the tumor microenvironment, resulting from immunogenic cell death, can increase efficacy and lead in synergy when combined with immune checkpoint inhibitors. We propose to further enhance the efficacy of this approach by blocking the inhibitory effect of IDO upregulation. We also propose to optimize viral replication in glioblastoma by blocking the interferon response pathway, a known mechanism of mammalian cell resistance to oncolytic viruses, with JAK inhibitors. This project has three specific aims: In specific aim 1, we plan to evaluate the efficacy, optimal sequence and mechanism of action of MV-s-NAP virotherapy in conjunction with antibody blockade of the PD-1/PD-L1 axis and IDO inhibitors in immunocompetent GBM models, including GL261, CT2A, as well as genetically engineered models of spontaneous gliomagenesis. In specific aim 2 we will evaluate the impact of modulating expression of interferon stimulated genes on the efficacy of MV-s-NAP virotherapy and immunovirotherapy by inhibiting the interferon response pathway, which has been shown to decrease viral permissiveness and replication. In specific aim 3 we will test the safety of the optimal efficacy approach identified in specific aims 1 and 2 by conducting toxicology and biodistribution studies in measles replication permissive Ifnarko CD46 Ge mice (an FDA approved model of measles virus replication) in order to determine the safe dose of the combination prior to clinical translation. Safety of the recommended human dose will be further confirmed in a second primate (Rhesus macaques) model. Overall, this work will introduce an innovative multipronged immunovirotherapy approach in the treatment of glioblastoma that has the potential to overcome the lack of efficacy observed with other strategies.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Molecular tau PET imaging has been available for the past four years and is able to detect paired helical filament (PHF) tau in Alzheimer’s disease. The most extensively utilized tau PET ligand is flortaucipir ([18F]AV- 1451). Over the past four years, however, since the development of flortaucipir, the field has been stumped by the observation of flortaucipir uptake occurring in patients with a frontotemporal lobar degeneration (FTLD) syndrome who would be expected to have underlying TDP-43 or 4R tau pathology, but not PHF tau. This observation has not been addressed, mainly due to the lack of autopsy cohorts, which is necessary to address this issue. We have been accumulating autopsied cases over the past four years and were able to assess the relationship between flortaucipir PET uptake and histopathology in an autopsy cohort of 26 patients (25 with Alzheimer’s disease and 1 with FTLD). Using a validated meta-ROI (region of interest) cut-point that we developed to determine whether PHF tau is present or absent in Alzheimer’s disease, we found flortaucipir PET to have great sensitivity and specificity to PHF tau, and to the Braak neurofibrillary tangle staging scheme. Interestingly, however, the single FTLD patient in that autopsy cohort with 4R tau deposition was classified as positive, suggesting that flortaucipir PET may indeed be detecting 4R tau. To date, there are no other autopsy studies, and no FTLD autopsy cohort has been described. Hence, there remain important knowledge gaps in the field. We still do not know whether flortaucipir uptake is indeed reflecting underlying 4R tau, what flortaucipir uptake is reflecting in FTLD TDP-43 where there is no tau, and how our meta-ROI cut-point would perform in an FTLD cohort. We have now amassed a large autopsy cohort of 80 cases with antemortem flortaucipir PET (FTLD with TDP-43 or 4R tau, n = 42 and Alzheimer’s disease, n=38). In this R01, we will study the relationship between antemortem flortaucipir uptake and pathological protein immunohistochemistry. Specifically, we will evaluate the association of flortaucipir PET with pathological diagnoses, association between flortaucipir PET uptake and the Braak neurofibrillary tangle stage in an FTLD cohort, and assess for associations between regional quantitative flortaucipir uptake and quantitative protein immunohistochemistry, across all 80 cases, to determine whether flortaucipir uptake is influenced by non-PHF tau targets, such as β- amyloid, 4R tau, TDP-43, microglia and astroglia, and whether uptake is influenced by the presence of multiple non-PHF tau protein deposition. This multi-PI proposal is led by two expert PIs with almost 40 years of combined research experience in FTLD neuroimaging, including with flortaucipir PET (Professor Whitwell), and in degenerative neuropathology (Professor Josephs). If the aims of the grant were achieved the field would not only have a better understanding of flortaucipir PET behavior, but better understanding of tau ligand behavior in general, which would aid with second and third generation tau ligand development. Development of sensitive and specific future tau PET ligands is important for biomarker utility in clinical trials.