Indiana University Indianapolis

NIH Research Projects · FY 2026 · 2025-06

Project Summary/Abstract Alzheimer’s disease (AD) is the most common form of dementia often presenting with vascular contributions to cognitive impairment and dementia (VCID). Growing evidence has highlighted pathological contributions from an immune cell within the peripheral immune system: neutrophils. In cases of hyperhomocysteinemia (HHcy), a common risk factor for VCID, neutrophils cause vascular damage by oxidative stress and releasing products for matrix degradation as well as increasing blood hypercoagulability via release of neutrophil extracellular traps (NETs), any of which can be a confounding factor in current therapeutics. This proposal aims to investigate the mechanisms by which neutrophils are contributing to worsened cerebrovascular outcomes in AD and VCID, and the impact of depleting neutrophils on disease pathology to give a broader picture of immune-mediated neuropathologies. To answer these questions, we propose to examine the neutrophil response to APP, VCID, and APP-VCID comorbidities (Aim 1) and determine whether chronic depletion of neutrophils reduces neuropathology for each case (Aim 2). We hypothesize that mechanisms unique to each pathology will be additive while overlapping mechanisms across pathologies will be exacerbated. For Aim 1, cohorts of wild-type or Tg2576 mice will be placed on control diet or diet to induce HHcy. In vivo multiphoton microscopy imaging, flow cytometry, single- cell RNA sequencing, and intravascular labeling of leukocytes prior to tissue collection will be used to examine upregulated mechanisms. In Aim 2, mice from Aim 1 will be given isotype control antibody or murinized anti- Ly6G antibody for chronic neutrophil depletion at the start of diet to assess changes in physiological and cognitive outcomes. We will test the hypothesis that chronic depletion reduces the severity of comorbid disease pathology by longitudinal imaging using MRI, multiphoton imaging, and immunohistochemical assays for neuroinflammation and vascular damage. These aims will provide important information on an overlooked factor to vascular dementias. Achievement of these aims will aid in my own goals of becoming an independent researcher by improving my scientific knowledge of VCID and AD as well as supplement my available research skills in MRI, immunohistochemistry, and single-cell transcriptomics. To complete these aims, I have the resources at Indiana University – Indianapolis such as the Alzheimer’s Disease Research Center as part of the NIH, a program for developing new mouse models (MODEL-AD) to be used for investigating new treatments (TREAT- AD), as well as a well-equipped environment for dementia-related studies. My mentor, Dr. Wilcock, and mentoring committee will guide my professional development by providing assistance with my written and communicative skillsets as well as expanding upon my academic network. Overall, this proposal will provide both scientific and professional development training while adding important knowledge to the AD/VCID field.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder characterized by β-amyloid (Aβ) plaques, neurofibrillary tau tangles (NFTs), and cognitive decline. Despite recent approvals of anti-amyloid antibodies, there remains a significant unmet need for more effective, safer, and more convenient therapeutics. Recent human genetic evidence highlights the critical role of microglia in AD etiology. INPP5D, identified as a risk gene for AD, encodes SHIP1, a phosphatase that regulates pathways downstream of immune receptors like TREM2 expressed on microglia. Our research has shown a positive correlation between INPP5D levels and Aβ plaques in human AD cases, findings that we replicated in an amyloidosis mouse model of AD. Additionally, we demonstrated that reduced Inpp5d expression improves cognition, reduces plaque burden, and lowers cytokine levels in this mouse model of AD. We have developed and validated a library of fully modified oligonucleotides optimized for broad and efficient RNAi knockdown of INPP5D. Our long-term goal is to slow or halt AD progression by modifying microglial function through siRNA-mediated knockdown of INPP5D. However, various transcriptional isoforms of Inpp5d identified in mouse models of AD have complicated efforts to validate INPP5D as a therapeutic target. To address this gap in knowledge, we will identify and characterize Inpp5d transcriptional variants in murine cellular and animal models using genetic analysis tools. In parallel, we will design and validate self-delivering siRNA oligonucleotides, suitable for animal studies, that efficiently and specifically knockdown Inpp5d isoforms in mouse microglia. These siRNAs will be tested in AD mouse models to demonstrate that Inpp5d knockdown shifts microglial states, reduces Aβ plaque levels and cytokines, and promotes a resilient brain resistant to neurodegeneration. Ultimately, by validating INPP5D as a therapeutic target with an siRNA design suitable for clinical studies, we aim to shift microglial states in humans into an overall protective phenotype that clears harmful proteins and supports brain health, potentially leading to a new treatment that harnesses the brain's immune cells to resist neurodegeneration and offer hope to patients affected by this debilitating disease.

NIH Research Projects · FY 2025 · 2025-06

SUMMARY/ABSTRACT The goal of this study is to elucidate how Kalirin, a multifunctional GTP/GDP exchange factor, regulates osteocyte function and bone mass across various skeletal sites, with a particular focus on periodontal bone. Osteocytes, the most abundant bone cell type, play a key role in bone remodeling, yet the specific mechanisms governing their function remain unknown. Mice with global Kalirin deletion (Kal-KO) display low bone mineral density, attributed in part to dysfunctional osteocytes as shown by decreases in osteocyte morphology and dendrite length, altered expression of osteocyte genes and an increase in osteocyte apoptosis in cortical and trabecular bone. We found dental, craniofacial and alveolar deficiencies in global Kal-KO mice. Given that osteocyte viability is linked with the pathology of periodontitis, we hypothesize that osteocyte specific deletion of Kalirin will decrease bone mineral density across different skeletal sites and exacerbate bone loss in a mouse model of experimental periodontitis. Using osteocyte-targeted Kalirin knockout mice (Kal-Dmp1Cre), we will examine changes in bone mass and architecture as well as osteocyte morphology, viability, and function across different skeletal sites, and in experimental periodontitis induced by ligation of maxillary molars. In Specific Aim 1, we will characterize the effects of osteocyte-targeted Kalirin deletion on bone mass and architecture at craniofacial, periodontal, spine, and femoral sites using µCT, histology, and histomorphometric analysis. Changes in osteocyte morphology and function will be investigated through histological staining of tissue and analysis of primary osteocyte morphology and gene expression in vitro. In Specific Aim 2, we will investigate if Kalirin deficiency in osteocytes exacerbates periodontal bone loss during experimental periodontitis. We will perform a ligature-induced bone loss study in osteocyte-targeted Kalirin deletion (Kal-Dmp1Cre) compared to control (Kal- FF) mice. Bone changes will be analyzed by µCT, histology, and histomorphometric methods. In addition, in situ changes in the expression of osteocytic genes and inflammatory cytokines in LIP maxillae and control maxillae from these mice will be performed by RNAscope and complemented with analysis of gingival crevicular fluid. Both male and female mice will be used for all studies. These studies aim to enhance the overall understanding of Kalirin’s role in osteocyte function and bone health at different skeletal sites, potentially leading to new therapeutic targets for preventing bone loss and improving patient care, particularly in the context of periodontitis and other bone loss diseases. The mentorship, training, resources and facilities available to me during my journey towards my DDS/PhD will ensure a positive trajectory towards my future career as an independent dental clinician scientist.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract: Our primary goal continues to be the provision of answers to questions that clinicians, governments, programs and the U.S. consider central to the evolution and sustainability of their long-term HIV care and treatment strategies for achieving epidemic control targets, amid changes in global health funding. Our central hypothesis is that retention in the HIV care cascade and treatment outcomes are influenced by dynamic factors at the level of the patient (including demographic, clinical, developmental, and behavioral factors); the ambient health care system; and the broader contextual environment. We will continue to leverage our strengths, including robust working relationships with HIV treatment programs, a substantial harmonized regional, as well as broad experience in machine-learning methodologies and novel analytical approaches. During this two-year extension, we will continue to pursue our original aims: SA-1: Describe movement through the HIV care cascade with a focus on identifying health care environment and broader contextual factors that influence optimal retention in care and viral suppression, in the face of global changes in donor funding priorities. SA-2: Examine the impact of developmental stage and behavioral factors on retention in the cascade and subsequent outcomes. SA-3: Examine the immediate and long-term outcomes of people diagnosed with Tuberculosis (TB) with a focus on identifying and addressing factors associated with patient outcomes. SA-4: Explore the use of new technologies, including eHealth and machine (deep) learning to diagnose and manage HIV-associated cancers with a focus on Kaposi’s Sarcoma (KS) and Cervical Cancer. SA-5: Examine the epidemiology of non communicable diseases (NCD) comorbidities and antiretroviral therapy (ART) complications with a focus on the oldest and youngest age groups affected by HIV. Kenya, Uganda and Tanzania (East Africa IeDEA countries) all rank among the top 15 countries with the highest HIV prevalence in the world (ranging 3.7-5.4%), making it imperative that we understand the dynamics of ART rollout and its impact on HIV control in this region. By meeting the above aims of this proposal, East Africa IeDEA will continue to contribute to the body of knowledge on factors influencing retention in the HIV care cascade and the outcomes of people treated with ART. Findings from these aims will contribute to informing and enhancing the US government’s impact on the HIV response globally and provide invaluable knowledge that can be adapted to improve the care and outcomes of people with HIV in the US.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract Impulsivity has been identified as both a risk factor for and consequence of an Alcohol Use Disorder. To identify novel interventions to reduce alcohol use disorder development in at-risk populations, it is critical to clearly define how neural circuits that underlie impulsivity are altered by familial risk. Thus, the overarching goal of this work is to identify the neural circuit mechanisms underlying impulsivity, and how they are altered by genetic risk for excessive drinking. The dorsomedial prefrontal cortex (dmPFC) is a brain region that controls impulsive decision-making. Our lab has observed that dmPFC ensembles are required for strategy updates during delay discounting (DD), a task which models impulsive choice, but the distinct roles of specific cell types within dmPFC remain undetermined. This proposal will dive deeper into the contributions of specific cell-types and provide conceptual training in translational behavioral models, PFC microcircuitry, computational techniques, and technical training in high-density electrophysiology. Parvalbumin inhibitory interneurons (PV) are one of the most abundant interneuron subtypes in dmPFC and are capable of precisely regulating dmPFC activity. In particular, dmPFCPV-mediated gamma (ɣ) oscillations are known to be important for strategy updating and flexible behavior. Alcohol-preferring (P) rats are a well-validated preclinical model of behavioral genetic risk for excessive alcohol drinking. P rats drink much more alcohol than non-genetic risk progenitor strain controls (Wistars) and exhibit similar behavioral phenotypes due to familial risk that match clinical findings (i.e., increased impulsive choice). Preliminary data from our lab indicates that markers of PV function (PV protein and perineuronal net expression) are innately altered in P rats as compared to Wistars in dmPFC. This proposal will test the hypothesis that genetic risk for excessive drinking is associated with reduced dmPFCPV interneuron ɣ oscillatory activity, which results in deficits in strategy updating and increased impulsive choice. Aim 1 of this proposal will combine high-density in vivo electrophysiology and optotagging techniques to characterize 1) whether dmPFCPV interneuron-mediated ɣ oscillations facilitate strategy updating during DD and 2) whether dmPFCPV activity is disrupted in P rats. Aim 2 of this proposal will determine whether restoring dmPFC inhibitory tone via optogenetically inducing dmPFCPV ɣ oscillations is sufficient to reduce impulsive choice in P rats. Performing these experiments in conjunction with my professional development activities will enhance my conceptual training in the alcohol field and will provide the computational neuroscience skills critical for my research and necessary to transition to independence. Further, these results are expected to increase our understanding of the neurobiological and behavioral changes that underlie genetic risk for alcohol misuse and assist in the development of novel approaches to treat alcohol use disorder.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY/ABSTRACT Type 1 Diabetes (T1D) develops following autoimmune-mediated destruction of pancreatic β-cells and is caused by a combination of genetic and environmental factors. However, the mechanisms that lead to autoimmune targeting of the β-cell remain incompletely understood. One potential contributing factor is defective β-cell autophagy. The autophagy pathway scavenges and degrades cellular proteins and organelles under conditions of stress. Recently, our lab discovered that the pathway of autophagy is impaired in β-cells before clinical onset of T1D. Based on this discovery, and other preliminary results, I hypothesize that dysfunctional β-cell autophagy contributes to autoimmune targeting of β-cells by altering protein degradation and antigen production leading to a change in β-cell–immune cell dialogue. Experiments in aim 1 will assess how β-cell specific deletion of an essential autophagy enzyme (ATG7) in mice affects β-cell survival, stress response, immune cell recruitment/activation, and neoantigen formation. In aim 2, experiments will determine how disrupted autophagy alters β-cell peptide processing and islet immunogenicity. Completion of these aims will determine how impaired β-cell autophagy alters β-cell health/survival, as well as pathways of protein degradation and antigenic peptide formation. These results will help determine whether the autophagy pathway is a useful therapeutic target to modulate disease course. This project and opportunity for fellowship training will also serve as an important launch point for my future career as a physician-scientist. The methods learned and applied to answer these questions will cultivate a well-rounded skillset of biochemical and molecular biology techniques to apply in future translational focused research. Furthermore, my expanding knowledge on mechanisms of β-cell stress pathways, β-cell autoimmunity, and disease development will help prepare me to become a successful contributor in the field of diabetes research and pediatric endocrinology clinical care. This F30 award entails a 4-year training plan designed to achieve 4 main objectives: 1) develop technical skills and knowledge of important concepts in diabetes research, 2) train in the use and handling of mouse models, 3) enhance written and oral scientific communication, and 4) integrate clinical development with research training. In addition, the collaborative research environment provided by the Center for Diabetes and Metabolic Diseases at the Indiana University School of Medicine (IUSM), and the integrated clinical and research training received in the IUSM Medical Scientist Training Program, will provide an excellent training environment. An advisory committee consisting of carefully selected, well-established investigators with complementary expertise across relevant fields, together with a supportive laboratory research environment, will aid achievement of the proposed goals.

NIH Research Projects · FY 2026 · 2025-05

Abstract Tauopathies are a group of devastating neurodegenerative diseases, such as frontotemporal lobar degeneration (FTLD)-tau or Alzheimer’s disease (AD), that are characterized by the presence of pathological hyperphosphorylated and misfolded aggregates of the microtubule-associated protein tau. There are no effective treatments for tauopathies. Interestingly, it has been reported that higher intake of niacin (also known as nicotinic acid) correlates with reduced risk of AD, which suggests that niacin may protect against pathogenic tau-induced neurodegeneration and could be of therapeutic utility. Niacin can cross the blood brain barrier and directly activate HCAR2 (GPR109A), a GPCR that is expressed in the brain selectively by microglia. HCAR2 activation by niacin and other ligands have been demonstrated to elicit a protective microglial response and overall salutary effects in different models of neuronal injury or neurodegeneration. However, an HCAR2-directed therapeutic strategy has not been tested for tauopathies. We hypothesize that HCAR2 activation with niacin may be a promising therapeutic strategy for tau pathology. Importantly, there are FDA-approved formulations of niacin, which could potentially be rapidly repurposed for tauopathies in the clinical context. We provide preliminary evidence that pathological tau induces HCAR2 expression by microglia. Inactivation of Hcar2 in tauopathy mouse models leads to exacerbation of the disease, suggesting a protective effect of HCAR2. Conversely, treatment with niacin reduced neuronal loss, motor deficits, tau phosphorylation, propagation and seeding activity in these models. Aim 1) Study the therapeutic potential of HCAR2 in tauopathy preclinical models We aim to establish an effective therapeutic strategy for the treatment of tau pathology by activating HCAR2. The HCAR2 agonist niacin will be delivered to pre-clinical tauopathy mouse models by enriched diet or oral gavage using the FDA- approved formulation Niacin-ER. Furthermore, HCAR2 will be selectively inactivated in microglia cells in tauopathy mouse models after disease onset. The effects of niacin treatment and Hcar2 deletion on tau pathology will be assessed in PS19 mice and the effects on tau spreading will be analyzed in mice injected with an AAV encoding pathogenic tau. Aim 2) Determine the cellular mechanism subserving HCAR2 actions on tau pathology. We propose to establish the cellular mechanisms and pathways underlying the role of HCAR2 on tau clearance using a combination of pharmacological, genetic, and omics approaches. We postulate activation of HCAR2 induces the clearance of pathogenic tau species by microglia, overall reducing the propagation of tau pathology. Microglia cultures will be used to assess the role of HCAR2 on tau uptake, intracellular degradation, and release of pathogenic forms of tau. Elucidating the mechanisms subserving HCAR2 actions will allow us to understand how this therapeutic strategy can be further improved and identify novel potential pharmacological targets.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY This proposal describes a five-year plan to develop Christopher Collier, MD into an independent orthopaedic- surgeon scientist whose career goal is to drive bench-to-bedside research that addresses the musculoskeletal effects of cancer. His prior education, research experience, and expertise in orthopaedic oncology prepare him for this opportunity. This career development award will catalyze his growth by providing complementary training in musculoskeletal biology, with an emphasis on muscle and bone biology, cancer cachexia, and laboratory leadership. Dr. Collier will receive exceptional mentorship and instruction at the Indiana University School of Medicine, an institution with a world-class environment for the study of musculoskeletal health and cancer. He will also benefit from strong institutional support, including independent laboratory space and protected research time, to enact his research plan. Cachexia is a syndrome of systemic muscle wasting and bone loss that is responsible for 20 to 40% of cancer-related deaths. Recent evidence suggests a direct link between cachexia and metastatic bone disease. The objective of this proposal, which addresses a critical need, is to define the contribution of metastatic bone disease to cachexia in kidney cancer and identify key mediators. Kidney cancer was selected because metastatic bone disease and cachexia are common disease features. Dr. Collier has established a mouse model of metastatic kidney cancer to bone, which develops cachexia and elevated serum IL-6. He hypothesizes that metastatic bone disease in kidney cancer increases systemic muscle wasting and bone loss by IL-6 mediated activation of JAK/STAT3 signaling in muscle. This hypothesis will be tested by pursuing three specific aims. Aim 1 will determine the impact of metastatic bone disease on cachexia in kidney cancer in vivo. Tumor cells will be implanted into mouse kidney, lung, or bone and cachexia outcomes compared. Aim 2 will determine the drivers of IL-6 signaling in kidney cancer and the potential of IL-6 blockade to reduce cachexia. IL-6 levels in serum and JAK/STAT3 signaling in muscle will be measured in tissues from Aim 1. IL-6 will also be deleted and/or pharmacologically neutralized in mice to determine the effect on cachexia. Finally, Aim 3 will establish the contribution of metastatic bone disease to cachexia and the IL-6 pathway in kidney cancer patients. Human serum and muscle will be collected from patients with localized kidney tumors or metastatic bone disease and compared. Collectively, these studies will describe the impact of metastatic bone disease and IL-6 on cachexia in kidney cancer. This research is conceptually innovative because it is the first to investigate the mechanisms of cachexia in metastatic kidney cancer and is technically innovative because it uses new preclinical models, a unique biorepository of human tissues, and cutting-edge approaches. This research is significant because it will likely provide strong scientific justification for or against new cachexia treatments targeting metastatic bone disease and the IL-6 pathway. It will also provide a pathway to scientific independence for a promising early-career orthopaedic-surgeon scientist.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Colorectal cancer (CRC), in particular metastatic CRC is frequently accompanied by the development of cachexia, a multi-organ wasting syndrome affecting musculoskeletal health. Cachexia is directly responsible for worsened quality of life in cancer patients and for over 30% of all cancer-related deaths. We and others have shown that cancer is accompanied by metabolic and genomic perturbations of the liver, and further, that CRC liver metastases (LM) exacerbate musculoskeletal wasting. Unfortunately, there is no available cure for cachexia, and investigations into how metastatic disease exacerbates musculoskeletal wasting are sparse; hence, there is a critical need to identify targetable mediators of skeletal muscle and bone loss in advanced CRC. In this regard, our preliminary findings suggest that FGF21, an endocrine hormone belonging to the fibroblast growth factor (FGF) family of proteins, plays a causative role in cancer-induced musculoskeletal wasting. In our preliminary studies, circulating FGF21 was elevated in CRC patients and in MC38 tumor hosts, which present with losses of skeletal muscle and bone. Deletion of FGF21 from MC38 cells reduced myotube atrophy and osteoclastogenesis in vitro and preserved musculoskeletal health in vivo. Though suggestive that FGF21 is largely tumor-derived in CRC cachexia, we also found elevations of hepatic FGF21 in tumor hosts. Supporting a causative role for host-FGF21 in the development of cachexia, muscle mass, muscle torque, bone mass and bone strength were protected in FGF21 knockout mice bearing CRC. Of interest, mice bearing CRC LM, which display exacerbated musculoskeletal wasting, also have heightened circulating FGF21, further linking FGF21 to the development of cachexia. Follow-up studies also revealed greater FGF21 in hepatocyte-CRC mixed cultures and in the livers of mice with advanced CRC, suggesting a role of FGF21 in tumor metastasization. Altogether, these observations suggest a novel role of FGF21 in the development of cachexia in advanced CRC. The objective of this proposal is to interrogate the FGF21-dependent effects in metastatic CRC-associated muscle and bone loss. Our central hypothesis is that metastatic CRC exacerbates FGF21 production, which in turn mediates musculoskeletal wasting in advanced CRC. In Aim 1, we will investigate the FGF21-dependent effects on bone loss in advanced CRC. The working hypothesis is that FGF21 determines bone loss in advanced CRC, by promoting osteoclastogenesis. In Aim 2, we will determine the mechanism(s) by which FGF21 triggers muscle wasting in advanced CRC. The working hypothesis is that FGF21 is participates in the development of muscle atrophy. In Aim 3, we will examine the metastatic niche and its role in exacerbating CRC cachexia. The working hypothesis is that tumor metastasization to the liver alters hepatocyte and cancer cell gene expression, consistent with elevated pro-cachectic signaling and aberrant expression of FGF21. Completion of this study will identify FGF21 as a novel therapeutic target for the treatment of musculoskeletal wasting in CRC and will facilitate new avenues for cachexia research.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Mesoderm specification is a critical event at the gastrulation stage of embryogenesis, ultimately giving rise to various mesoderm-derived tissues, including blood and heart. Over the past decades, mechanistic studies on mesoderm specification have predominantly centered on conserved genes, such as transcription factors, epigenetic regulators, and signaling pathways. Particularly, Wnt/β-Catenin pathway critically contributes to the induction of mesodermal cells towards cardiac and hematopoietic fate during gastrulation. However, the existence of human-specific molecular mechanisms underlying mesoderm specification remains enigmatic. Recently, accumulated evidence underscored the essential roles of long non-coding RNAs (lncRNAs) in human cardiovascular development, metabolism, and disease. Using human embryonic stem cells (hESCs) to recapitulate human cardiogenesis, we identified a novel human lncRNA HBL1, which is highly expressed in hESCs and critically regulates human cardiogenesis (Dev Cell 2017,4:333-8; Development 2021.13: dev199628). In this proposal, we discovered another novel human lncRNA termed Mesoderm Commitment Regulatory LncRNA 1 (MCRL1). MCRL1 is a 735bp nuclear lncRNA conserved in non-human primates but absent in rodents. During cardiac differentiation from hESCs, MCRL1 transiently expresses in the mesoderm precisely at the mesoderm specification stage. Notably, MCRL1 deficiency (MCRL1KO) does not affect mesoderm formation, but prominently switches mesoderm specification from cardiac to hematopoietic fate. Transgenic overexpression of MCRL1 in mouse mesoderm leads to embryonic lethality with enlarged heart. Intriguingly, MCRL1 oppositely modulates β-Catenin occupancy on cardiogenic and hematogenic genes. Furthermore, MCRL1-β-Catenin interaction repressed BMI1, a crucial subunit of the PRC1 epigenetic complex. BMI1 ablation in MCRL1KO hESCs restored the aberrant cardiac and hematopoietic commitment from MCRL1KO mesoderm. Thus, we hypothesize that MCRL1 plays a pivotal role in human mesoderm specification towards cardiac and hematopoietic fates by modulating β-Catenin downstream gene expressions at both transcriptional and epigenetic levels. Two specific aims are outlined. Specific Aim 1. Deciphering the transcriptional mechanisms governed by MCRL1-β-Catenin interaction in regulating mesoderm specification. Specific Aim 2. Investigating the epigenetic mechanisms driven by the MCRL1-β-Catenin→BMI1 axis in modulating mesoderm specification.

NIH Research Projects · FY 2024 · 2025-05

ABSTRACT Intermittent fasting and caloric restriction are newly identified therapeutic interventions against cardiometabolic disease. Our laboratory discovered that activating the hepatic glucose fasting response is sufficient to convey several of the key therapeutic effects of generalized caloric restriction. This is clinically relevant because targeting hepatic glucose transport is highly amenable to small-molecule and nutraceutical therapy. Therefore, our long-term goal is to understand adaptive liver glucose metabolism during fasting to produce new therapies that leverage these pathways against cardiometabolic disease. Intermittent fasting in rodents blocks pathological remodeling and infarct expansion after myocardial infarction, and treating mice with FGF21 – a liver-derived peptide hormone secreted in response to fasting – prevents experimental cardiac left ventricular hypertrophy (LVH) and LV dysfunction. We demonstrated that blocking hepatic glucose transport using the naturally occurring disaccharide, trehalose, recapitulates the hepatic adaptive fasting response. Our new data now demonstrate that oral trehalose recapitulates the effects of intermittent fasting on cardiac protection against pathological remodeling. Specifically, trehalose induces hepatic FGF21, and prevents pathological LVH and LV dysfunction in response to chronic pressure overload. We also identified a novel trehalose analog that resists degradation by host and microbial metabolism, and which activates hepatic fasting-like signal transduction to a greater extent than native trehalose. This study’s objective is thus to define mechanisms and contexts of cardioprotection by trehalose-class compounds as a prelude to the use of these compounds in human trials. Our central hypothesis is that hepatic GLUT inhibition blocks LVH and LVD by activating canonical hepatic fasting signals to the myocardium. We propose three Specific Aims to test this hypothesis. In Aim 1, we will delineate mechanisms by which trehalose prevents LVH and LVD. In Aim 2, we define pathophysiological contexts in which trehalose attenuates secondary cardiomyopathies. In Aim 3, we examine the impact of trehalose catabolism on its efficacy against secondary cardiomyopathies. The innovation of this proposal is that we our team has identified and will examine further: 1) a novel and tractable cardioprotective pathway, and 2) a novel compound class that activates this cardioprotective pathway. Completing these aims will define how hepatocyte fasting responses protect from pathological remodeling and dysfunction; and nominate specific clinical contexts in which the adaptive hepatic fasting response is cardioprotective. The impact of this work is that it will mechanistically inform next-generation glucose fasting- mimetics, which also leverage the adaptive fasting response against cardiac disease, and will justify further efforts toward clinical trials that utilize trehalose-class compounds to ameliorate secondary cardiomyopathies.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT. Metastasis is the primary cause of breast cancer deaths and many therapies for metastatic patients are more effective when immune cells are within the tumor. However, metastatic breast tumors have less immune cells compared to primary tumors, especially CD8+ T cells, whose absence is a predictor for worse outcomes. This creates a problem wherein tumors exclude antitumor immune cells that would make therapies more effective. A gap in knowledge is the regulation of immune cell infiltration to metastatic tumors, which could be harnessed to increase immune trafficking to make therapies more effective. Our work identified that heat shock factor 1 (HSF1) can suppress the presence of CD8+ T cells in primary breast tumors. This relationship was confirmed in vivo as HSF1 loss reduced tumor volume and evoked an influx of CD8+ T cells and myeloid cells. Depletion of CD8+ T cells rescued tumor growth with HSF1 knockdown suggesting HSF1 protects breast tumors from immune-mediated killing. Attraction of CD8+ T cells with HSF1 knockdown was regulated by suppression of CCL5, a chemokine for CD8+ T cells. Our preliminary studies also showed HSF1 has increased activity in matched metastatic tumors compared to primary tumors and HSF1 activity was negatively correlated with CD8+ T cells in primary and metastatic human tumors. We hypothesize that HSF1 activity in metastatic breast tumors reduces attraction of CD8+ T cells, leading to decreased levels of other antitumor immune cells, thereby making metastatic tumors less responsive to treatments. To test this hypothesis, we propose three aims. Aim 1: Determine the role of HSF1 in the recruitment of immune cells to metastatic breast tumors. We will utilize an inducible system to knockdown or overexpress HSF1 in immune-competent mouse models and perform single cell RNA sequencing to identify changes in cell populations. We will correlate active HSF1 in human primary and metastatic tumors using multiplex tissue immune phenotyping. We will also determine the importance of CD8+ T cells to metastatic tumor growth and the importance of CCL5. Aim 2: Assess the efficacy of HSF1 inhibition on metastatic tumor response to chemotherapy and immune checkpoint therapy. We will utilize immune competent models to genetically reduce HSF1 or two HSF1 small molecule inhibitors (SISU- 102 & NXP800) with therapies that benefit from an influx of immune cells (paclitaxel, capecitabine, anti-PD-L1). Aim 3: Determine the role of HSF1-regulated myeloid cell populations in breast tumors. We will determine which immune populations are dependent on CD8+ T cells and IFN-γ for the influx into HSF1-depleted tumors, whether CD8+ T cells primed from HSF1-depleted tumors adoptively transfer anti-tumor immunity, and the effect of myeloid cells from HSF1-depleted tumors on CD8+ T cell and cancer cells. These studies will have a significant impact in metastatic breast cancer with a greater understanding of metastatic tumor-immune interactions and potentially develop a therapeutic strategy to enhance the efficacy of therapies for this patient population that would decrease mortality and extend the lives of these vulnerable patients.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY / ABSTRACT Type 2 Diabetes (T2D) is a global health pandemic that results from a combination of defective insulin secretion and impaired insulin action in the peripheral tissues. T2D typically arises in the context of obesity and overnutrition; however, genetic and physiologic data suggest that inadequate insulin secretion from the  cell is the chief determinant predicting the development of overt T2D. Recent studies have identified  cell dedifferentiation, in which  cells downregulate key identity markers, lose insulin secretory function, and differentiate into other endocrine cell types, as one potential mechanism leading to  cell failure in T2D; however, the molecular mechanisms leading to  cell dedifferentiation in T2D are not well understood. Calcium (Ca2+) plays a vital role in regulating  cell health and function. In the  cell, the endoplasmic reticulum (ER) serves as the dominant Ca2+ store, and store-operated Ca2+ entry (SOCE) functions to maintain robust ER Ca2+ levels. When ER Ca2+ is depleted, SOCE is activated by stromal interaction molecule 1 (STIM1), which promotes Ca2+ influx from the extracellular space to refill depleted ER Ca2+ stores. Recently our laboratory found that obese female mice with reduced SOCE due to  cell-specific genetic deletion of STIM1 had evidence of  cell dedifferentiation. Further, islets isolated from these mice had reduced G protein coupled estrogen receptor (GPER) mRNA and protein expression with no change in Estrogen Receptor  or  expression, suggesting a potential interaction between SOCE, GPER signaling, and  cell identity. My preliminary data has extended these observations and linked loss of GPER and loss of estradiol signaling through GPER with loss of  cell identity and potential  cell dedifferentiation. Against this background, I hypothesize that decreased SOCE leads to reduced GPER signaling and loss of  cell identity and function, thereby contributing to the pathophysiology of T2D in females. I will test this hypothesis through two specific aims. In Aim 1, I will define the role of GPER in  cell differentiation status. In Aim 2, I will define the relationship between impaired SOCE and GPER function and expression/subcellular localization. Completion of this project has the potential to identify a novel mechanism linking SOCE and GPER signaling to  cell failure during T2D. With this F30 Predoctoral Fellowship, I will have the support to complete this research and master the objectives of my training plan: 1) Create a strong foundation of techniques and concepts critical in diabetes research and islet biology, 2) Build strong technical skills in super-resolution nanoscopy, 3) Develop and refine my clinical skills, and 4) Create a strong foundation of written and oral scientific communication. My training will be supported by the experienced faculty provided by the Center for Diabetes and Metabolic Diseases at the Indiana University School of Medicine and the Purdue University Weldon School of Biomedical Engineering. In summary, this comprehensive research strategy and training plan will provide me with the skillset needed to reach my long-term career goal of becoming a physician scientist trained to perform clinically translatable, innovative, and rigorous diabetes research.

NIH Research Projects · FY 2025 · 2025-02

PROJECT SUMMARY At least 15.4 million opioid prescriptions in the United States are provided each year during surgical care. Excessive and risky perioperative opioid prescribing patterns are common and increase the risk of opioid overdose, addiction, diversion, and persistent opioid use. To mitigate these harms, policymakers and payers in most states have enacted policies that restrict opioid prescribing for acute pain or mandate clinicians to review prescription drug monitoring program databases before prescribing opioids (PDMP use mandates). To date, few studies have rigorously assessed the intended and unintended effects of these policies in the context of surgical care. In this 4-year study, we will use quasi-experimental methods to examine the impact of opioid prescribing limits and PDMP use mandates on perioperative opioid prescribing, high-risk prescribing, opioid-related adverse events, and patient-reported outcomes. First, we will use commercial, Medicare, and Medicaid claims databases to evaluate the effect of state opioid prescribing limits and to assess variation in effects by policy feature, patient population, procedure, and prescriber (Aim 1). Second, we will evaluate the effect of state PDMP use mandates and examine heterogeneity in effects using the same claims databases (Aim 2). Finally, we will determine the impact of a major Michigan insurer’s opioid prescribing limit and Michigan’s PDMP use mandate on opioid prescribing and patient-reported outcomes after surgery, using a novel linkage between a statewide registry of surgical patients and the state PDMP database (Aim 3). Our findings will directly inform efforts to mitigate morbidity from perioperative opioid prescribing and close critical knowledge gaps needed to optimize future policy design. For example, if opioid prescribing limits and PDMP use mandates have reduced perioperative opioid prescribing with minimal unintended effects, policymakers should consider implementing these policies more broadly. However, if the policies have not reduced perioperative opioid prescribing or have had substantial unintended effects, other approaches may be needed. Ultimately, this proposal will contribute to the development of well-designed policies that balance the need for safe opioid prescribing with the need for effective postoperative pain management.

NIH Research Projects · FY 2026 · 2025-02

ABSTRACT My long-term career goal is to develop more effective treatments for brain and spinal cord tumors in children and young adults with cancer predisposition syndromes. Spinal ependymoma (SP-EPN) is a molecularly distinct tumor that arises predominantly in the upper spinal cord. Most SP-EPN have biallelic genetic perturbations affecting the NF2 gene, and patients with NF2-associated schwannomatosis are predisposed to SP-EPN development. There is currently no effective medical therapy for SP-EPN. Surgical resection of late-stage symptomatic lesions is associated with high morbidity and mortality. There is a critical need to identify the molecular mechanisms underpinning pre-neoplastic lesions to halt progression in NF2 patients at an early stage. In our preliminary studies, we leveraged a unique spinal neuroepithelial stem (NES) cell model to show failure of differentiation at the radial-glia cell stage in NF2 knockout (NF2-/-) NES cells and the formation of pre- neoplastic growths in vitro and in vivo. Further, although most of the SP-EPN tumor display ependymal cell signatures, we identify a rare population within the tumor with radial-glia stem cell markers. The specific objectives of this study are: (i) to identify the cellular hierarchy in NF2-/- NES cell derived preneoplastic cells and patient-derived SP-EPN; (ii) to define the dysregulated signaling pathways in NF2-/- NES cells that fail to differentiate and (iii) to identify activated kinase targets that may rescue this differentiation failure. We hypothesize that derailed radial glia cells in the developing spinal cord are the source of SP-EPN formation and that targeting the aberrant signaling pathways in these cells will be important to prevent growth. To test this hypothesis, we will perform single cell RNA sequencing (scRNA seq) of NF2-/- NES cells during in vitro differentiation and xenografts (Aim 1). We will compare the molecular signatures of cells in pre-neoplastic growths with that of patient samples to validate the target pre-neoplastic cells in SP-EPN development. We will perform bulkRNA seq and multiplexed kinase inhibitor bead affinity chromatography and mass spectrometry (MiB/MS) to define the activated kinome profile of these pre-neoplastic cells compared to patient SP-EPN (Aim 2a) and the key signaling pathways that are disrupted in these cells. In Aim 2b we will test candidate kinase inhibitors to test in our NF2-/- NES cell model to determine if the failure of differentiation can be rescued pharmacologically. We expect to identify the cellular identity of NF2-/- preneoplastic cells that fail to differentiate, and the dysregulated signaling pathways that may be governing these cells. We aim to identify novel, rational therapeutic targets to stop the progression of tumor formation in children with NF2. To achieve research independence, I have identified three key areas in which I need mentorship and additional experience: (1) advanced cell engineering; (2) single cell analytics; (3) kinome profiling for drug discovery. I will accomplish this work under the mentorship of Dr Wade Clapp, a world expert in neurofibromatosis. Ultimately, I want to become an RO1 funded independent investigator directing a research program in brain and spinal cord tumorigenesis.

NIH Research Projects · FY 2025 · 2025-02

ABSTRACT SCYL1 deficiency syndrome is a rare, autosomal recessive, pediatric, multisystem disorder cause by inactivating mutations in the SCYL1 gene. The disease is characterized by recurrent episodes of liver failure, growth retardation, skeletal defects, as well as a range of neurological abnormalities including cognitive defects, ataxia, tremor, motor dysfunction, and neurodegeneration. SCYL1 encodes a protein whose function remains elusive Consequently, no treatments or cures have been developed or made available to treat the disorder. The genetic nature of the mutations, however, suggest that gene replacement therapy may represent an effective approach for its treatment. To test this hypothesis, we propose to engineer mouse models allowing for the inducible restoration of SCYL1 expression. Mice represent an excellent model to study SCYL1 deficiency syndrome as targeted disruption of the Scyl1 gene in mice recapitulates virtually all phenotypic changes seen in humans. In all, we propose to engineer two mouse models. The first model will make use of a loxP-flanked gene trap cassette containing a strong splice acceptor site followed by sequences encoding the enhanced green fluorescent protein followed by the bovine Growth Hormone polyadenylation signal. The cassette will be inserted within intron 1 of the mouse Scyl1 gene such that in the absence of the recombinase Cre, the cassette will act as a gene trap, preventing expression of the Scyl1 gene. In the presence of Cre, the cassette will be removed, allowing for expression of Scyl1 from its endogenous promoter. The second model will be engineered using a newly developed approach in our laboratory which makes use of short inverted artificial introns (iAI). In this system, a small DNA cassette – which contains sequences encoding a splice donor site, essential intronic sequences running antisense of the target gene and a strong splice acceptor site flanked by head-to-head lox71 and lox66 sites, followed by a weak splice acceptor site – is inserted within an exon of a target gene. In the absence of Cre, the DNA cassette is not recognized as an intron and remains within the transcript of the targeted gene. A string of stop codons in all three frames within the cassette, causes early translation termination, and prevents expression of the protein. In the presence of Cre, the iAI is flipped via recombination between the head-to-head lox71 and lox66 sites. The inversion of the cassette allows intronic sequences to be recognized by the splicing machinery which removes them, allowing for the normal expression of the Scyl1 gene. Both models will be characterized at the genomic level as well as functionally by crossing the mice with mice ubiquitously expressing CreERT2, a tamoxifen inducible Cre. The generation of these models and the successful application of gene replacement therapy in mice will provide the necessary evidence to move forward with more translational approaches for the treatment of the disorder.

NIH Research Projects · FY 2026 · 2025-02

Project Summary Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant neoplasia with an extremely low 5-year overall survival (OS) rate (~10%). Its current standard first-line treatment is the combination of Gemcitabine/nano albumin-bound-Paclitaxel (Gem/NabP), but with a median OS of only ~8.5 months and considerable toxicity. The poor therapeutic outcomes underscore the urgent need for innovative PDAC treatment strategies. Gem/NabP have been reported to activate the nuclear factor (NF)-κB and drive their therapeutic resistance in PDAC. Moreover, aberrant expression of NF-κB is a pivotal driver of PDAC. We were the first to discover that PRMT5 methylates NF-κB to activate genes crucial to PDAC progression. Moreover, Kaplan-Meier plots show that PDAC patients with high PRMT5 expression have dramatically worse OS than those with low PRMT5 expression. Thus, effective NF-κB inhibitors and their combination with Gem/NabP are expected to enhance PDAC treatment. Recently, the protein arginine methyltransferase 5 (PRMT5) was identified as a synthetic lethality combinatorial target with Gem in PDAC via CRISPR screening, further supporting the promise of PRMT5 inhibitor and Gem/NabP combination. To target PRMT5 in PDAC, our lab has identified a potent small molecule inhibitor of PRMT5, PR5-LL-CM01 (abbreviated CM01) (US Patent No. 11,034,689). We showed that CM01 exerts an anti-cancer effect in PDAC in vitro and in vivo. Preliminary data also revealed no overt systemic toxicity in mice. Importantly, CM01 was more potent than GSKi and JNJi in killing PDAC cells, avoiding the concern of myelosuppression toxicity of GSKi and JNJi. CM01 possesses the patented unique chemical structure, and thus, is a highly promising PRMT5 inhibitor warranted for further development. Despite the therapeutic potential, CM01 has low water solubility, which significantly compromises its bioavailability. In this proposal, we aim to improve the efficacy of CM01 by formulating it with our patented albumin-coated nanocrystal (NC) (or Abxtal) technology, which does not require solvents or chemical surfactants for solubilizing hydrophobic CM01. In a pilot experiment, we produced Abxtal-CM01 of <100nm (~87nm). In this study, we hypothesize that nanocrystal Abxtal-CM01 will maintain biological functions of CM01, improve its tumor delivery and PDAC inhibition efficacy, and show synergy with Gem/NabP in PDAC treatment in vivo. To test this central hypothesis, we propose to pursue the following aims: Aim 1. Scale-up production and characterization of NC form of the PRMT5 novel inhibitor, Abxtal-CM01. Aim 2. Determine the biological functions of Abxtal-CM01 in vitro and its antitumor efficacy alone or in combination with Gem/NabP in PDAC model in vivo. IMPACT: Upon successful completion, this research will have established that NC formulation of PRMT5 inhibitor Abxtal-CM01 can provide an effective treatment of PDAC, a disease in urgent need of effective treatments.

NIH Research Projects · FY 2025 · 2025-02

SUMMARY Housed at the Indiana University School of Medicine (IUSM), the country’s largest medical school, the Indiana Stimulating Access to Research in Residency (I-StARR) program aims to train residents in research methods focused on understanding and reducing health disparities in cardiovascular and pulmonary diseases. We focus on primary care residency programs because their graduates enter clinical practice at the frontline of prevention and treatment of highly prevalent cardiovascular, pulmonary, and related diseases. Despite the availability of postdoctoral research training programs for clinician-investigators, programs tend to be linked to subspecialty fellowships and do not engage trainees from primary care. Consequently, very few primary care physicians purse research careers. I-StARR will recruit and train resident-physicians from residencies in Family Medicine, general Internal Medicine, general Pediatrics, Medicine-Pediatrics, and general Obstetrics- Gynecology. Over the five-year period of the award, I-StARR will train up to 12 resident-investigators, to be recruited among the 446 residents of the five participating residencies. Our goal is to provide high quality research skills training to place early-career clinicians on a structured pathway from resident-investigator to clinician-investigator, equipping them with research skills and grant writing expertise along the way. We will accomplish this by (1) enrolling residents in existing didactics in research methods, (2) providing career development experiences, including a new grant writing curriculum for I-StARR residents, and (3) matching resident-investigators with research preceptors for hands-on mentored research experiences for a minimum of 12 months at 80 percent effort. We designed I-StARR in three interdisciplinary tracks: clinical and translational research, health services research, and community-engaged research. With a strategic focus on recruiting residents with diverse backgrounds and experiences, we will train research preceptors in Culturally Aware Mentoring. Grant-writing coaching and mentoring training will follow evidence-based best practices of the NIH- funded National Research Mentoring Network. The evaluation arm of I-StARR will continuously monitor all components and characteristics of the training program and incorporate feedback for quality improvement from internal and external advisory boards. Applying a health disparities lens to the focus on cardiovascular and pulmonary diseases, I-StARR will enable clinician-investigators to devise more effective intervention strategies to improve population health especially for disadvantaged and marginalized populations.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY/ABSTRACT Oropouche virus (OROV) is an emerging arbovirus and a significant public health issue in Central and South America. Since the early 1960s, over half a million infections have been documented. However, the actual numbers are likely much higher due to the lack of an adequate differential diagnostic system. OROV has a tri- segmented negative-sense RNA genome made up of Small (S), Medium (M), and Large (L) segments. S encodes the nucleoprotein, M encodes the glycoproteins, and L encodes the RNA-dependent RNA polymerase. The glycoproteins are responsible for viral entry into host cells and are targets for neutralizing antibodies. The segmented nature of the genome allows OROV to exchange segments with co-infecting viruses through a process known as reassortment. Reassortment is responsible for antigenic shifts in influenza viruses, which is critical for the emergence of pandemic strains. Such effects on bunyaviruses, including OROV, are poorly understood. This is a deficit since three known OROV reassortants are circulating in South America. These reassortants differ in their glycoproteins, which could influence host tissue tropism, immune evasion, and vector competency. The goal of this project is to investigate how reassortment modulates OROV virulence. Aim 1 will use in vitro methods to identify phenotypic changes within the reassortants due to the different glycoproteins. Aim 2 will investigate the reassortants' virulence compared to OROV using a murine model. These studies are significant as they will provide critical data for future studies to (a) assess the threat of OROV and OROV reassortants to public health and (b) develop pan-OROV antiviral therapeutics and vaccines.

NIH Research Projects · FY 2026 · 2025-01

Vaping has become popular among teens and young adults. It has been reported that an acetate conjugate of vitamin E, α-tocopherol-acetate (α-T-acetate), has been added to some base e-liquids for vaping. α-T-acetate in e-liquids is linked to E-cigarette, or vaping associated lung injury (EVALI) and deaths. There were 2,807 illness and 68 deaths (by October 2020) associated with vaping. α-T-acetate is in the bronchoalveolar lavage of EVALI patients, and vaping α-T-acetate and other acetate conjugates is still occurring. Patients with EVALI have lung symptoms of cough, shortness of breath, or chest pain, as well as fever, nausea, vomiting, diarrhea, fatigue or weight loss. EVALI patients have elevated systemic inflammatory mediators and lung remodeling. The CDC suggested that the lipid α-T-acetate not be added to vaping products until mechanistic studies are done. We propose novel mechanisms for manifestations of EVALI from vaping α-T-acetate. Our novel preliminary data indicate that a novel high molecular weight compound is generated from vaping α-T-acetate and that this compound would not be detected in the LC-MS approaches in previous reports on vaped compounds. The novel compound was toxic to epithelial cells, induced eosinophilia, fever and overt lung remodeling in mice, but did not lipid-load lung macrophages. In contrast, non-vaped α-T-acetate in base e-liquids do lipid-load lung macrophages but this does not induce fever. Although, we show that a high molecular weight toxic compound is generated and we have separated the compound, the structure of the toxic compound, and the mechanisms for induction of fever, lung damage and lung inflammation by this novel compound are not known. Our novel concept is that a novel high molecular weight compound is generated from vaping α-T-acetate that is highly toxic to epithelial cells, without lipid-loading of macrophages. Our long term goal is to identify mechanisms for effects of vaping α-T-acetate on lungs and fever in youth and adults with EVALI. As a step towards our long- term goal, our central HYPOTHESIS is that vaping α-T-acetate generates a novel high molecular weight toxic compound that induces lung inflammation, lung damage and fever in youth and adults, without lipid-loading lung macrophages. We will test this with the following aims: Aim 1. Test the hypothesis that a novel high molecular weight compound is generated from vaping α-T-acetate as assessed by mass spectrometry (MS), chemical structure modeling and NMR. Aim 2. Test the hypothesis that a novel compound, generated by vaping α-T- acetate, induces fever, lung damage, lung inflammation and reduces lung function, without lipid-loading of lung macrophages. We will also assess regulation of these manifestations by the early recruitment of lung eosinophils. Completion of these studies 1) will have a significant impact on our understanding of mechanisms of vaped α-T- acetate-induced lung damage and inflammation and 2) may lead to design of future human studies for interventions that significantly impact risk for EVALI and death from vaping in youths and adults.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to better define the role of GATA2 in the pathogenesis of acute myeloid leukemia (AML) and the MonoMAC syndrome, and to use this information for therapeutic benefit. Somatic GATA2 mutations occur in -3-4% of AML cases, and inherited mutations are the cause of the MonoMAC syndrome, which is associated with the development of AML in most patients. We recently showed that Gata2 can act as a tumor suppressor in AML. However, the mechanisms by which GATA2wr and GATA2 mutations influence AML pathogenesis are currently unclear. In preliminary studies, we found that AMLassociated GA TA2 mutations dysregulate gene expression, and alter GAT A2 protein interactions. Based on these findings, we hypothesize that GATA2wr normally acts as a tumor suppressor as part of an interacting set of proteins that regulate a specific set of target genes; GA TA2 mutations may promote AML and MonoMAC by altering these functions, We will test these hypotheses via the following aims: Specific Aim 1: We will define the genes that are regulated by Gata2wr, and how their expression is altered by AML- and MonoMAC-associated Gata2 mutations, We generated a novel Gata27354M knock-in mouse- the first to model a MonoMAC- and AML-associated GATA2 mutation. In preliminary studies, we used single-cell RNA sequencing (scRNA-seq) and flow cytometry to find that these mice have decreases in key hematopoietic populations similar to that of the MonoMAC syndrome. ScRNA-seq also allowed us to identify 346 genes that are differentially expressed in myeloid progenitor cells from Gata2'354M mice vs. littermates. The Gata27354M mice will be fully characterized for changes in hematopoiesis and AML development We will also use an overexpression model to define alterations in gene regulation caused by other AML-associated Gata2 mutations including A318V, L321 F, T354M, and R362G in human and mouse hematopoietic stem/progenitor cells (HSPCs). If successful, these studies will allow us to identify the genes that are regulated by Gata2wr in primary HSPCs, and to determine pathways that are disrupted by Gata2 mutations. Specific Aim 2: We will identify the GATA2 protein "interactome", and how it is altered by AML- and MonoMAC-associated GATA2 mutations, We developed a proximity labeling system in which the Gata2 cDNA is fused to an enhanced biotin ligase, called "TurbolD". GATA2-TurboID biotinylates proximal proteins, allowing for the identification of interacting proteins by mass spectrometry. In preliminary studies, we identified the protein "interactome" of GA TA2wr in primary mouse and human HSPCs, and determined that GATA2T354 M alters many of these interactions. We will apply this system to study how other GATA2 mutations (A318V, G320D, L321 F, R362G, and R398W) affect protein interactions. Co-immunoprecipitation assays in HSPCs from Gata27354M mice will be used to orthogonally validate our results. If successful, these studies may reveal mutant GAT A2 protein interactions that can be targeted with novel therapeutic approaches to treat AML.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is a fatal neurodegenerative condition characterized by cognitive decline, β- amyloid (Aβ) plaques, and tau-containing neurofibrillary tangles (NFTs). Recent human genetic evidence supports an important role for microglia in the etiology of AD. Microglia are the resident immune cells in the brain and play important roles in maintaining neuronal health and proper immunomodulation of neighboring glial cells. Microglia clear neurotoxins, Aβ oligomers, and Aβ plaques, and thereby mitigate an inflammatory microenvironment that is toxic to neurons. Lower expression of TREM2, a cell surface microglial immune receptor, and hypomorphic variants (e.g. R47H), are correlated with an increased risk of developing of AD. Conversely, enhanced signaling downstream from TREM2 via the hypermorphic P522R variant of PLCγ2 reduces risk for AD. This human genetic evidence suggests that microglia that are more sensitive to TREM2- mediated signaling protect against neurodegeneration. Src homology 2 domain containing inositol polyphosphate 5-phosphatase 1 (SHIP1) is a member of the inositol polyphosphate-5-phosphatase (INPP5) family of phosphatidylinositol phosphatases. INPP5D, the gene that encodes SHIP1, has also been identified as a risk gene for AD. SHIP1, plays a key role regulating pathways downstream from immune receptors, including TREM2 and FcγRIIB. SHIP1, binds immunoreceptor tyrosine-based inhibition motifs (ITIMs) where it competes with kinases and modulates phosphatidylinositol-dependent signaling. We hypothesize that inhibition of SHIP1 will improve TREM2-mediated microglial responses to neurotoxins, and promote an overall neuroprotective phenotype to microglial states , and thereby slow cognitive decline. Furthermore, since recently approved disease modifying anti-Aβ antibodies depend in part on Fc receptor activation of microglia, SHIP1 inhibitors may also be combined with anti-Aβ antibodies to improve their efficacy. To test this hypothesis, we will optimize the potency and drug-like properties of SHIP1 inhibitors from identified chemical scaffolds (Aim 1). We will identify the effect of SHIP1 inhibition on microglial states in mouse brain, define pharmacokinetic (PK) and pharmacodynamic (PD) relationships, and determine the level and duration of SHIP1 inhibition required for efficacy in a mouse model of AD (Aim 2). With an understanding of the target engagement required for efficacy, SHIP1 inhibitors with sufficient human potency and drug-like properties will be used to develop target engagement and translational biomarker assays for human studies (Aim 3). Collectively, these studies will support the translation of new molecular entities into the clinic that will reduce neuroinflammation and amyloid burden and improve cognition, thus advancing the NIH/NIA mission to develop novel therapies for AD.

NIH Research Projects · FY 2026 · 2025-01

ABSTRACT Clotting factor replacement therapy to manage bleeding in patients with hemophilia can lead to the development of neutralizing anti-drug antibodies called inhibitors, which complicate therapy. Current immune tolerance induction (ITI) protocols eradicate inhibitors only in a subset of patients and necessitates frequent intravenous (IV) infusions of clotting concentrates, placing a heavy burden on patients and generating high treatment costs. Further, patients with hemophilia B can develop anaphylactic reactions to chronic clotting factor exposure, discouraging healthcare providers from attempts at tolerization. Therefore, there is high interest in developing novel approaches to promote tolerance to the replacement protein. Foxp3+ regulatory T cells (Tregs) are essential for establishing and maintaining immune tolerance to inhibitor development against clotting factor replacement therapy in hemophilia. The cytokine IL-2 engages transmembrane signaling receptors in Tregs to mediate proliferation, differentiation, and expansion, thus promoting this tolerance effect. However, effector T cells and NK cells also express components of the IL-2 receptor complex, resulting in harmful off-target effects and toxicities. Additionally, the short half-life of cytokines necessitates frequent repetitive dosing for therapeutic effect, which increases the burden on patients. In this R33 product definition application, we will test a novel biotherapeutic IL-2 based immunocytokine, F5111- IC, which uses structure-based design to detarget IL-2 pleiotropy from non-Tregs. Pre-clinical assessments of F5111-IC has shown highly promising targeted immunosuppressive effects in models of autoimmune disease. We propose to investigate the F5111-IC treatment platform to suppress the formation of inhibitors in hemophilia. Our proposal seeks to validate this targeted immunomodulatory technology in multiple preclinical models of hemophilia A and B as part of our clinical translation strategy. We outline measurable milestones in the identification of an optimal treatment protocol for durable tolerance with acceptable safety profiles in small and large animal models of hemophilia. If successful, this technology is likely to be relevant to other biologic treatments that are complicated by anti-drug antibody formation and can also be applied other conditions such as antibody mediated hyperacute rejection in transplantation.

NIH Research Projects · FY 2026 · 2024-12

Takotsubo Cardiomyopathy (TCM) is an acute, life-threatening condition triggered by an intense physical or emotional stressor and characterized by cardiac dysfunction, apical ballooning of the left ventricle, and thinning of the ventricular wall. With maximal medical care, patients may recover, but they often live with permanent sequelae. Patients who have experienced an episode of TCM are at increased risk of an additional episode, increased risk of additional cardiovascular events, and reduced life expectancy. Importantly, there are no current formal recommendations for treatment or patient care post-TCM episode. Recent studies have demonstrated that TCM is associated with a robust immune response in both human patients and in preclinical rodent models, particularly an infiltration of macrophages into the cardiac tissue. In a similar manner, immune activation has been demonstrated to play a causal role in preclinical models of hypertension, or an elevation in blood pressure. Our laboratory has particular expertise in interrogating the immune contribution to hypertensive pathology and recently demonstrated a role for macrophages in hypertensive heart failure. My preliminary data confirm that mice treated with isoproterenol (ISO), a catecholamine that mimics the intense the stress response that occurs in the human condition, develop TCM with macrophage infiltration into the heart. Furthermore, I have found that the expression of the enzyme ROCK2 (Rho associate coiled-coil containing protein kinase 2), is elevated in left ventricle of these mice, concurrent with the observed increase in macrophages. Inhibition of ROCK2 using the novel pharmaceutical KD025 (Belumosudil) prevented the full development of TCM in mice. I have also found that KD025 treatment halts the progression of hypertension- induced cardiac fibrosis and in vitro, KD025 attenuates macrophage activation. Lastly, I have found that mice who have experienced an episode of TCM have a potentiated hypertensive response to low dose Angiotensin II (Ang II). These recent studies and my preliminary data lead me to the hypothesis that ROCK2 in macrophages promotes cardiac dysfunction in the acute phase of TCM and contributes to the elevated cardiovascular risk after a TCM episode. To test this hypothesis, I will use models of TCM and hypertension, cell culture, and transcriptomics to: Aim1 (K99): Determine the role of ROCK2 in isoproterenol-induced macrophage activation in TCM and Aim 2 (R00): Determine the mechanisms by which a history of TCM potentiates future hypertension through reprogramming of myeloid cells. During the K99 phase, I will receive training in RNA sequencing analysis, immune training assays, and assessing cardiac remodeling. Determining how ROCK2 contributes to TCM and whether ROCK2 contributes to increased cardiovascular risk post-TCM will greatly advance the field and result in new therapeutic opportunities. These proposed studies, along with my proposed career development plan, will provide a foundation for my career as an independent investigator in an outstanding environment.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Obesity promotes heart disease and diabetes; both causes of early mortality. Most adults in the United States are overweight, and obesity adds cost to healthcare. Mechanisms regulating energy homeostasis are not completely understood. Recently, small cell appendages in the brain called cilia were shown to be essential to prevent overeating and obesity. Primary cilia are critical for proper cell-to-cell communication. The best understood cilia-mediated signaling pathway is Hedgehog (HH) signaling. During development, HH is essential for the patterning of many tissues. Our lab has discovered that the components of the HH pathway continue to be expressed in the adult hypothalamus and both feeding status and body composition dynamically regulate hypothalamic HH signaling. Moreover, we found that genetically activating HH signaling in specific cell types causes hyperphagia and obesity. Thus, HH signals are redeployed after embryonic development to influence adult feeding behavior and energy homeostasis. I seek to build on these insights to determine how HH signaling in the hypothalamus controls long-term energy homeostasis. I have chosen to focus on a specific cell type in the hypothalamus, called tanycytes. Tanycytes line the third ventricular space extending their processes deep within the hypothalamus. Interestingly, tanycytes undergo cell proliferation during early postnatal development. They also proliferate in response to extreme changes in feeding status and body composition. My preliminary data shows that tanycytes express and localize several HH pathway components to their cilia. Thus, I hypothesize that HH signals regulate tanycyte proliferation during postnatal development which is required for proper adult energy homeostasis. I will test these hypotheses using complementary in vitro (Aim 1) and in vivo (Aim 2) approaches. Together, the experiments in this project test whether ciliary HH signaling induces tanycyte proliferation needed for regulating long-term energy homeostasis.