Mayo Clinic Jacksonville

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Title: Acetylation modulates T cell receptor signaling PA-25-301 NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) Suboptimal TCR signaling is one of the major immune resistance mechanisms hampering the efficacy of cancer immunotherapy. Accordingly, restoration of optimal TCR signaling is of crucial importance to overcome immunologic barriers within the tumor microenvironment. We have recently identified a previously unrecognized role for acetylation as an important post-translational process that regulates TCR signaling. Our preliminary data demonstrate that Sirt2-/- mice mount superior anti-tumor immune responses in vivo and in vitro. Strikingly, we have observed amplified calcium flux and TCR signaling in Sirt2-/- T cells. Further mechanistic studies revealed that increased acetylation of Lck, drove enhanced T cell activation and effector functions to overcome T cell exhaustion. Based on these preliminary data, we will test the central hypothesis that acetylation of Lck by Sirt2 abrogation during cellular immunotherapy endows resistance to T cell exhaustion by amplified proximal TCR signaling within the TME. In Aim 1, we will define the role of acetylation in Lck activity and proximal TCR signaling. In Aim 2, we will dissect the intersection between acetylation and other known mechanisms regulating Lck in the context of T cell exhaustion. In Aim 3, we will optimize Sirt2 abrogation in melanoma and NSCLC TIL therapy and TCR-T therapy. These studies will establish acetylation as a new post-translational mechanism modulating TCR signaling and validate Sirt2 as an actionable target to overcome T cell exhaustion distinct from existing immune checkpoint pathways. Ultimately, we propose to improve the efficacy of T cell therapeutics via genetic manipulation of Sirt2 thus opening new frontiers for combinatorial immunotherapeutic strategies. Justification for Use of Vertebrate Animals: Our studies are focused on the role of Sirt2 in TCR signaling and effector functions. The immune system is complex and studies involving interactions between distinct cell types of the immune system can only be performed with appropriate animal models. Because we will evaluate the impact of Sirt2 on cancer, animal models that appropriately model the human disease are required. An in vivo model is an established procedure that provides valuable biological information for the potential of cancer therapy. As a major focus of this proposal is to translate findings to the clinic, in vitro models alone are inadequate. No in vitro or theoretical approaches can mimic the complex cellular interactions that drive immune-mediated responses. The easy handling of mice compared with other types of animals will allow for thorough experiments to fulfill statistical considerations. The results from our proposed studies may identify Sirt2 as appropriate targets for cancer immunotherapy to restore T cell responsiveness to suboptimal tumor antigen presentation.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT Loss-of-function mutations in the genes PINK1 and PRKN are the main cause of early-onset Parkinson’s disease (PD) and likely contribute to other, related neurodegenerative disorders too. The encoded enzymes, a ubiquitin kinase-ligase pair, cooperate to identify, label, and eliminate selectively damaged mitochondria through degradation in the autophagy-lysosome system. This quality control pathway (mitophagy) is critically important to maintain a healthy and functional mitochondrial network and is thought to broadly protect against neuronal demise and neuroinflammation. Missense mutations in, or elevated expression of, the SNCA gene lead to build-up, aggregation, and toxicity of the encoded protein alpha-synuclein (aSYN). aSYN deposits and matures into pathognomonic inclusions called Lewy bodies (LBs). Pathological aSYN is thought to seed and spread throughout the brain during progression of disease leading to widespread neurodegeneration and inflammation. Besides aSYN, LBs also seem to contain membranous remnants of mitochondrial and autophagic origin, but mechanisms leading to their formation and propagation as well as functional role(s) of these structures remain unclear. Loss of PINK1 or PRKN and elevated levels of aSYN seem to aggravate each other, but the interaction of these two major disease pathways is not well understood and the specificity is uncertain. There is considerable potential for crosstalk, convergence, or even synergism between the mitophagy and LB processes. The current project will employ a recently discovered PINK1 gain-of-function variant (PINK1G411A) that results in increased ubiquitin kinase activity, greater mitophagy rates, and enhanced resilience to stress and damage. As proof-of- concept, this new genetic model will help determine whether, how, and to which extent increasing mitophagy can also provide protection against different aspects of aSYN related disease. Toward this end, the project will interrogate the specific ability of enhanced PINK1 ubiquitin kinase activity to protect against toxicity and aggregation of aSYN, to modify seeding and spreading of the pathology, and/or to generally improve neuronal resistance to damage and inflammation. These different aspects will be evaluated in three specific aims in iPSC-derived neurons and in vivo in mouse brain using complementary models of viral aSYN overexpression or exposure to patient brain-derived pathological aSYN seeds. It is expected that the study will highlight the beneficial effects of increasing PINK1 activity and mechanistically determine how aSYN interferes with mitophagy, contributing to a better understanding of the pathobiology of LBs and future therapies.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT We seek to elucidate the phenotypic heterogeneity seen in people with an expanded repeat in C9orf72, the most frequent genetic cause of two devastating neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Remarkably, even within families, individuals can develop ALS, FTD, or both diseases. Although symptoms may appear when someone is about 20 years of age, others are 90 years of age. Similarly, survival after symptom onset ranges from several months to over 30 years. Presently, this substantial phenotypic heterogeneity remains largely unexplained. The need for a deeper understanding of this intriguing expansion is further substantiated by the recent failure of antisense oligonucleotide trials targeting C9orf72 transcripts. The C9orf72 repeat expansion is basically ill defined, even though it was discovered over a decade ago. In fact, a well-established pathogenic threshold is still lacking, resulting in uninterpretable size estimates of up to about one hundred repeats. This knowledge gap can probably be attributed to technical limitations of methods commonly used to characterize this expansion. Importantly, sequencing advances now enable an in- depth assessment of expanded repeats. As such, in our present application, we propose to employ a cutting- edge targeted long-read DNA sequencing technology to accurately obtain the repeat length, methylation profile, and sequence content of the C9orf72 expansion. We postulate that variability in these characteristics of the expansion might serve as disease modifiers, contributing to the reported phenotypic heterogeneity. To this end, we will include longitudinally collected ante-mortem blood specimens from C9orf72 expansion carriers who are symptomatic and belong to the ALS/FTD spectrum, pre-symptomatic, or phenoconverters (Aim 1). By capturing the length, methylation, and purity of their expansion at a single-molecule level, we can uncover vital differences between groups and changes over time, which might precede the occurrence of symptoms. Additionally, we will assess post-mortem brain specimens from C9orf72 expansion carriers who received a neuropathological diagnosis of ALS or frontotemporal lobar degeneration (FTLD; Aim 2). Because we will use cell sorting to specifically investigate major cell populations in multiple brain regions, we are equipped to find differences between diseases, regions, and cell types. Thus, our thorough assessment of the C9orf72 repeat expansion in a precious collection of ante-mortem and post-mortem specimens using a pioneering sequencing technology may aid in resolving this unusual expansion, paving the way for tailored treatment strategies.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT Loss of upper motor neurons (UMNs) and lower motor neurons (LMNs) is characteristic for classic amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that generally results in death within two to five years after the onset of symptoms. The motor phenotypic spectrum of ALS ranges from primary lateral sclerosis (PLS) with pure UMN dysfunction to progressive muscular atrophy (PMA) with pure LMN dysfunction. It is important to distinguish patients with PLS and PMA, because they exhibit a slower disease progression, resulting in a longer survival after disease onset, than patients with classic ALS. Even though it has been shown that RNA-binding proteins and splicing events (e.g., cryptic exons) play a critical role in these diseases, little is known about contributing transcripts. Hence, we will set out to identify relevant transcripts and dysregulated pathways that may drive UMN versus LMN pathology. As such, we will acquire the transcriptome of patients belonging to the ALS spectrum (Aim 1), assessing the motor cortex from post-mortem specimens. Because we will use both bulk and single-nuclei approaches, employing long-read sequencing technologies, we can accurately quantify genes and individual transcripts, comprehensively interrogate cell-type-specific gene expression and splicing changes, and reveal vulnerable cell populations. Additionally, since most cases remain genetically unexplained, despite the fact that numerous genes have been implicated in ALS pathogenesis, we will complete thorough genomic studies (Aim 2). Because we postulate that genetic causes or risk factors may have stayed hidden due to sequencing technologies utilized, we are proposing to perform long-read whole-genome sequencing, which gives us the opportunity to deal with highly repetitive or complex regions, thereby assisting in unraveling the genetic architecture of ALS and its spectrum disorders. Taken together, our innovative multi-omic studies can potentially uncover genetic drivers that may determine the degree of UMN and LMN involvement, possibly nominating biomarker candidates and promising therapeutic targets for these debilitating diseases.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT Disruption of carotid atherosclerotic plaque can cause atheroembolism and ischemic stroke. Treating the plaque with surgery or stenting reduces the long-term risk of atheroembolism but with an upfront risk of causing stroke during the revascularization procedure. Compelling evidence supports a net benefit from revascularization in patients with symptomatic carotid stenosis. However, there is a clinical need to improve selection of patients with asymptomatic high-grade (≥70% luminal reduction) carotid stenosis for endarterectomy or stenting in the context of modern intensive medical management (IMM) of atherosclerosis risk factors. CREST-2 (NCT02089217) is a pair of multicenter, randomized trials of IMM with or without revascularization for preventing stroke in patients with high-grade asymptomatic carotid stenosis. CREST-2 completed enrollment of 2,480 patients. Trial follow-up ends on July 31, 2025. CREST-2 specifies baseline pre-randomization carotid duplex ultrasound (DUS) and annual follow-up studies. DUS studies are performed at a lab trained by the CREST-2 Vascular Imaging Core (VIC). DUS blood flow measurements are used to confirm degree of carotid stenosis. The DUS studies also generate standardized B-mode images depicting carotid plaque. The VIC has archived these images. Using standardized image analysis algorithms on the first 503 trial DUS studies, we have successfully assessed the plaque morphologic characteristics we plan to study in this proposal. We will analyze 2000 baseline CREST-2 DUS images of the randomized artery and evaluate relationships between baseline plaque morphologic traits and periprocedural stroke or death plus subsequent ipsilateral stroke. We will also characterize the effectiveness of IMM in changing DUS plaque morphologic features over time in 500 patients followed longitudinally (1000 arteries total: 500 arteries on the randomized side along with 500 arteries on the contralateral side). The directed approach we propose overcomes difficulties inherent in non-randomized cohort studies, including observer bias and confounding by indication. This proposal will, for the first time in a multicenter randomized trial, determine the clinical utility of assessing morphological traits for selecting asymptomatic patients for revascularization and testing new therapies.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Metabolism is a key driver of T cell functions, and the switch from oxidative-phosphorylation to aerobic glycolysis is a hallmark of T cell activation. Unfortunately, tumor reactive T cells often display a compromised metabolic status due to metabolic competition with cancer cells within the tumor microenvironment (TME). Therefore, strategies to enhance metabolic fitness of T cells within the metabolically challenging tumor bed may rescue resistance to existing cancer immunotherapies. In this context, we have focused on the regulation of T cell metabolism by Sirt2, an NAD-dependent histone deacetylase. Our preliminary data demonstrate that Sirt2 functions as a metabolic checkpoint that harnesses T cell effector functions and impairs anti-tumor immunity. Specifically, upregulation of Sirt2 expression in human tumor-infiltrating T lymphocytes (TILs) negatively correlates with response to Nivolumab and TIL therapy in non-small cell lung cancer. Mechanistically, Sirt2 suppresses glycolysis and oxidative-phosphorylation by deacetylating key metabolic enzymes. Accordingly, Sirt2-deficient T cells manifest increased glycolysis and oxidative-phosphorylation, display enhanced proliferation and effector functions, and have superior anti-tumor activity. Importantly, pharmacologic inhibition of Sirt2 endows human TILs with these superior metabolic fitness and enhanced effector functions. These findings indicate targeting Sirt2 may allow reprogramming of T cell metabolism to augment a broad spectrum of cancer immunotherapies. Guided by this scientific premise we propose the overall hypothesis that Sirt2 activity governs the metabolic fitness of T cells at tumor beds, and therefore controls the magnitude of immune pressure against malignant progression. We will test this hypothesis in the following specific aims: Aim 1 will investigate the precise molecular mechanisms via which Sirt2 regulate glycolysis, TCA cycle, glutaminolysis and fatty acid oxidation, and the post-translational mechanisms that govern Sirt2 expression and function in tumor-reactive T cells. Aim 2 will explore the metabolic functions of Sirt2 in physiologic contexts of T cells during activation, differentiation, maturation and as they are subjected to metabolic constraints within the tumor beds. Aim 3 will determine the metabolic and immunologic consequences of Sirt2 inhibition in human TILs for clinical translation, and correlate Sirt2 expression in TILs with response to immunotherapy. These aims will be achieved by employing a variety of experimental strategies involving in vitro and in vivo metabolic and immunologic analyses of various genetically engineered animals, complemented by molecular biology and genetic studies using primary human T cells and patient-derived TILs from lung cancer and melanoma. Collectively, our proposed studies will provide a comprehensive view of the role of Sirt2-regulated metabolic processes in tumor-reactive T cells. The results from our proposed studies will validate Sirt2 as an actionable metabolic and immunologic target, and Sirt2 inhibition will be a tractable means to improve cancer immunotherapy.

NIH Research Projects · FY 2026 · 2026-02

ABSTRACT Colony stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is a devastating genetic neurodegenerative disease characterized by abnormal glial responses, marked white matter pathology in the brain, and severe motor and cognitive symptoms. Currently, there are no effective therapies against CSF1R- related leukoencephalopathy, and its etiology remains poorly understood. Despite the limited consensus regarding its underlying cellular mediators and mechanisms, CSF1R loss-of-function and deficient microglial function are thought to contribute to the disease pathogenesis. However, little is known about the immune changes at the border tissues of the brain, namely at the meninges, and how changes in meningeal immunity and lymphatic drainage might contribute to the initiation and progression of brain pathology in CSF1R- related leukoencephalopathy. Unexpectedly, our preliminary data shows a decrease of lymphatic vessels in the dura of the established model of CSF1R-related leukoencephalopathy (Csf1r+/- mice) and the Fms-intronic regulatory element (FIRE) knockout mice (Csf1rΔFIRE/ΔFIRE), a new mouse model of CSF1R- related leukoencephalopathy. We hypothesize that a deleterious innate immune response at the brain-meningeal interface might affect the pathophysiology of CSF1R- related leukoencephalopathy by dampening lymphatic drainage of the CNS. In this project, we will use the Csf1rΔFIRE/ΔFIRE mouse model to further explore the changes in the central nervous system (CNS) associated immune cells, namely in meningeal myeloid cells, and its association with impaired lymphatic drainage of the brain. I will determine the cellular mechanisms and immune mediators linked to the absence of meningeal lymphatic vasculature in Csf1rΔFIRE/ΔFIRE mice (Aim 1). Secondly, I will use different experimental approaches to enhance meningeal lymphatic drainage in Csf1rΔFIRE/ΔFIRE mice and determine its therapeutic impact on exacerbated glial activation, white matter degeneration, and behavior deficits (Aim 2). This proposal will establish whether impaired meningeal lymphatic vasculature represents a previously unappreciated pathological phenomenon, and a potential therapeutic target, in CSF1R-related leukoencephalopathy.

NIH Research Projects · FY 2025 · 2025-09

Ehlers-Danlos syndromes (EDS) comprise a heterogeneous group of heritable genetic connective tissue/ collagen disorders characterized by fragile soft connective tissue and widespread distribution that manifests in the skin, ligaments, joints, blood vessels and internal organs. Hypermobile Ehlers-Danlos syndrome (hEDS) and hypermobility spectrum disorders (HSD) are the most common form of EDS comprising 80-90% of cases and are believed to affect an estimated 3% or 255 million people worldwide. Although the mutations responsible for most types of EDS have been identified, the genes responsible for hEDS and HSD are unknown. Comorbidities associated with hEDS/HSD include subluxations/dislocations, pain, fatigue, anxiety, fibromyalgia, migraine/headaches, inflammatory bowel disease, mast cell activation syndrome, and many other conditions leading to reduced quality of life. As an autosomal dominant genetic condition hEDS/HSD would be expected to display a 1:1 sex ratio, but several studies have revealed a sex ratio of 9:1 women to men. Although sex differences are known to be important factors in the natural history of HSD and hEDS, no studies have systematically examined serum biomarkers, immune phenotype and/or comorbidities in hEDS/HSD according to sex. Because of the current gaps in knowledge, the average time from onset of symptoms to diagnosis is 14 years. Thus, there is a critical need to research sex differences in hEDS/HSD to better understand the pathogenic mechanisms and to provide a more comprehensive multi-disciplinary treatment approach to relieve the suffering of this large population of understudied patients. The overall goal of this proposal is to examine biological sex differences in immune defects that lead to higher hEDS in females compared to males and mitochondrial defects that lead to higher HSD in females compared to males.

NIH Research Projects · FY 2025 · 2025-09

Abstract: The 5-year relative survival of pancreatic ductal adenocarcinoma (PDA) patients is only 8%. PDA is predicted to be the second-leading cause of cancer related death in the U.S. by 2030. Understanding the key signaling mechanisms of tumorigenesis is critical for developing life-saving interventions. Hereditary pancreatitis (HP), an autosomal-dominant disorder with recurrent episodes of acute pancreatitis (AP) which eventually develops into chronic pancreatitis (CP), has a cumulative risk of pancreatic cancer of 44% by age 70 years. Cationic trypsinogen gene (or PRSS1) mutations are the most common causes of HP. Unfortunately, the development of targeted preventive or therapeutic interventions for HP has been hampered by gaps in our understanding of its pathophysiology, which is mainly due to the practical difficulties in obtaining tissues from human pancreas at early stages of the disease and the lack of animal models that recapitulate the human form of this disease. Recently we have developed a novel model of HP by expressing a common mutant of human PRSS1 (PRSS1R122H) in mice (J Clin Invest. 2020 Jan 2;130(1):189-202). Transgenic expression of mutant PRSS1 caused severe AP which progresses to CP, precancerous PanIN lesions, and pancreatic cancer. This model of HP will provide us with a powerful tool to fulfill our long-term goal of understanding the initiating events of HP and developing specific strategies to prevent its progression to pancreatic cancer. In this proposal, we will use our unique humanized pancreatitis model to test our central hypothesis that etiological factors and PRSS1 gene mutation cooperatively cause pancreatic tumorigenesis by intra-acinar cell stress signaling pathways and a trypsin receptor-mediated constant inflammatory milieu. We will characterize these signaling pathways in this newly developed HP model and investigate their roles in pancreatic cancer tumorigenesis by both pharmacological and genetic approaches. We expect these studies will significantly improve our understanding of the pathogenesis of HP, its progression to pancreatic cancer, and provide new insights for developing/testing novel preventive and therapeutic interventions.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disorder leading to progressive renal cyst growth, affects 12 million people globally. Arginine vasopressin (AVP) is a key driver of cystogenesis, and in rodent PKD models, the absence of AVP completely prevents cyst formation. Although V2 receptor (V2R) antagonists effectively inhibit cyst growth, their use is limited by significant polyuria (>6 Liters/day). An alternative strategy involves enhancing renal water reabsorption to suppress central AVP release. Promising approaches include regulating the water channel aquaporin-2 (AQP2) through pathways independent of V2R. The urate transporters GLUT9b and ABCG2, located in the collecting duct, may play roles in water homeostasis. Notably, hyperuricosuria is associated with hyponatremia and Glut9 deletion in mice lead to severe polyuria. Additionally, ABCG2 inhibitors might be associated with improved kidney function in ADPKD. Our preliminary findings suggest that intracellular urate accumulation promotes aquaporin-2 (AQP2) exocytosis via AMP-activated protein kinase (AMPK) phosphorylation, independent of V2R/PKA pathways. The overarching hypothesis posits that elevating intracellular urate in collecting ducts promotes AQP2 exocytosis and water reabsorption, suppressing central AVP release and reducing renal cystogenesis in ADPKD. Aim 1 investigates the impact and mechanisms by which urate controls AQP2 trafficking, evaluating the impact of ABCG2 inhibition by Ko143 analog and probenecid on urinary concentration in V2R knockout mice, assessing the impact of ABCG2 deletion on urinary concentration capacity and AQP2 phosphorylation, and exploring mechanisms by which increased intracellular urate, due to GLUT9b overexpression and ABCG2 inhibition, controls AQP2, cyclic AMP levels, Protein Kinase A (PKA), and AMPK activities in V2R-knockdown cell lines. Aim 2 determines the impact and underlying mechanisms of ABCG2 inhibition and genetic deletion in reducing renal cyst growth and mitigating tolvaptan-induced aquaresis in ADPKD, assessing the impact of ABCG2 inhibition and deletion on PKD amelioration in Pkd1RC/RC mice, investigating the long-term impact of ABCG2 inhibition and genetic deletion on the anti-cystogenic and aquaretic effects of tolvaptan, and exploring effects of elevated intracellular urate, ABCG2 inhibition, and AMPK inhibitor on in vitro cystogenesis, cAMP levels, PKA, AMPK, and PDE activities in WT and Pkd1 mice and human collecting duct cells. These investigations aim to elucidate the novel interplay between intracellular urate and AQP2-mediated water transport regulation in ADPKD, potentially leading to innovative treatments to slow cystogenesis.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Alpha-Synuclein (aSyn) is an intracellular neuronal protein which is implicated in a variety of neurodegenerative diseases including Lewy body (LB) dementias (consisting of Dementia with Lewy Bodies and Parkinson’s disease dementia), Parkinson’s disease, and Multiple Systems Atrophy. Endogenous healthy aSyn is involved in synaptic function; in pathogenic states, aSyn is believed to misfold, accumulate into oligomers and fibrils, and ultimately become part of large insoluble aggregates such as Lewy bodies and Lewy neurites. It is still unknown why aSyn aggregates begin to form. It is also unclear whether pathogenic aSyn exists in primarily monomeric or multimeric form, with the field currently believing that oligomeric aSyn may be toxic. Thus, there is great need to better understand cellular mechanisms involved in aSyn aggregation in disease pathology. The proposed study focuses on understanding the protein interactors of alpha-Synuclein during early aggregate formation and propagation. Protein-protein interactions and related mechanisms identified in this study have potential to facilitate biomarker development for early LB dementia diagnosis and/or promote new candidates for successful disease-modifying therapies. I will employ an in vitro proximity-dependent biotin identification (BioID) system to characterize the aggregate interactome of aSyn. This model is an unbiased antibody-free method for tagging proteins of interest; a mutant R118G BirA* biotin ligase enzyme will be fused to aSyn. Upon exogenous addition of biotin to in vitro assays, proteins within a 10nm radius will be biotinylated. Proteomic analysis of enriched biotinylated peptides will identify potential interactors. Aim 1 will characterize intracellular changes in the aSyn interactome in seeded and non-seeded states. Aim 2 will uncover proteins involved in aSyn secretion and propagation in seeded and non-seeded states. Validation of potential interactors will be performed using in vitro models such as split VenusYFP, split luciferase, siRNA knockdown, and co- immunoprecipitation assays. Analysis of post-mortem LB dementia patient tissue will investigate the presence of a protein target in late-stage LBs – suggesting true involvement of a protein target in pathological processes. Novelty of the proposed project comes from (1) the application of split BirA* fragments fused to aSyn to identify interactors of multimeric aSyn and (2) the use of LB dementia patient-derived pathogenic seeds to increase physiological relevance of this study. The proposed experiments will be carried out at Mayo Clinic, a research institution with state-of-the-art lab facilities and expert scientists leading advancements in neurodegeneration. With these resources, the long-term objective of this project is to reduce the burden of synucleinopathies for patients and their caregivers.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Drug allergy is the leading cause of fatal anaphylaxis in the US, with antibiotics being the most common cause of allergic reactions. For immediate penicillin allergy, evidence supports specific antigenic determinants and an immunoglobulin (lg)E-mediated mechanism. For other antibiotics, the antigenic determinants and mechanisms are not known. For some antibiotic classes, such as cephalosporins and sulfonamides, positive skin testing with non irritant concentrations supports an lgE mechanism, but for other antibiotics, such as vancomycin and fluoroquinolones, current data suggest immediate hypersensitivity reactions occur through primarily non-lgEmediated processes. Currently available tests and biomarkers for antibiotic allergy have limited utility. While fatal anaphylaxis has been associated with intravenous administration, the mechanisms supporting these observations have not yet been defined as large database studies lack accurate reaction phenotyping and structured causality assessments. Although both lgE and lgG antibodies are likely to be involved in immediate-onset allergic reactions to antibiotics, biological pathways need defining in advance of biomarker discovery given the complexity of antibiotic structures, potential epitopes, and protein and cellular interactions. In this UG3/UH3 application, we apply clinical epidemiology and translational immunology methods to enhance knowledge of immediate antibiotic allergy through extending the work of an established multi-site network of drug allergy specialists, the United States Drug Allergy Registry (USDAR) Consortium. USDAR studies include a multi-site database of participants evaluated for drug allergy (n=2,432) and a biorepository from specialistconfirmed antibiotic-allergic patients (n=28). Our overall goal is to determine the phenotypes, endotypes, and mechanisms of antibiotic allergy, including investigation of mechanistic differences according to drug route through these specific aims: 1) To describe the phenotypes and endotypes of immediate-onset allergic reactions to antibiotics, and 2) To define how delivery route impacts antibiotic anaphylaxis. We will achieve these aims through leveraging USDAR's existing research infrastructure and participants and leadership by two allergist/immunologist physician scientists with complementary methodologic expertise. This project aligns with NIH/NIAID goals to advance drug allergy research and RFA-Al-24-002 to support research that enhances understanding of the mechanisms and management of antibiotic drug allergy.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT We have shown that in addition to being irreplaceable for glioblastoma (GBM) invasion and mitosis, members of the non-muscle myosin II (NMII) family of molecular motors also regulate oncogenic signaling, mitochondrial homeostasis, and reactive oxygen formation. This suggests that targeting the two predominant NMII isoforms in GBM (NMIIA and IIB) will synergize with signal transduction inhibitors and with therapies that depend on reactive oxygen, including radiation. We find that MT-125, a non-toxic, CNS permeant small molecule inhibitor of NMIIA and NMIIB prolongs median survival by ~30% as a single agent. Furthermore, when it is combined with sunitinib, a multi-RTK inhibitor, MT-125 prolongs median survival nearly 2-fold over vehicle and produces long term, tumor-free remissions in nearly half of mice. In addition, MT-125 sensitizes GBMs to radiotherapy. Our central hypothesis is that by uncovering the mechanisms for these remarkable forms of synergy between MT-125, signaling inhibitors, and radiation therapy, we can optimize the use of this highly novel therapeutic in studies that should lay the groundwork for first-in-human clinical trials.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Cancer immunotherapy represents a major paradigm shift in cancer care. Despite such breakthrough, most cancer patients remain refractory to these therapies. Metabolism is a key driver of T cell functions, and the metabolic switch from oxidative phosphorylation to aerobic glycolysis is a hallmark of T cell activation. Unfortunately, tumor-reactive T cells often display a compromised metabolic activity due to nutrient competition with cancer cells at tumor bed. Therefore, strategies to rewire the metabolic fitness of T cells within the unfavorable microenvironment of tumors are expected to rescue the resistance to existing cancer immunotherapies. In this context, this proposal aims to understand the epigenetic mechanisms priming the metabolic switch in T cells under metabolic stress via Sirt6, an NAD+-dependent histone deacetylase. The preliminary data of this study establish Sirt6 as an epigenetic silencer of T cell metabolism that impairs T cell effector functions. Specifically, Sirt6 expression is upregulated in murine melanoma tumor-infiltrating T lymphocytes (TILs), and Sirt6 blockade increases their glycolytic activity and enhances their effector functions. Mechanistically, Sirt6 deacetylates histone H3K9 to repress the transcription of the glycolytic genes, thus directing glucose away from the glycolytic pathway, the metabolic program that is required for effective anti- tumor immune response. Accordingly, Sirt6 deficiency in murine T cells leads to an upregulation of glycolytic genes’ transcription with increased glycolytic rate, subsequently resulting in superior anti-tumor activity following tumor challenge in vivo. Importantly, Sirt6 blockade endows human TILs from non-small cell lung cancer patients with superior metabolic fitness and enhanced effector functions. These findings indicate that targeting Sirt6 may lead to boosting T cell metabolism to augment a broad spectrum of cancer immunotherapies. Guided by this scientific premise, the overall hypothesis of this study states that Sirt6 restrains the metabolic activity and effector response of T cells at tumor bed via chromatin remodeling, thus facilitating cancer immune escape. This hypothesis will be evaluated by the following specific aims: Aim 1 will investigate the precise epigenetic mechanisms via which Sirt6 regulates chromatin remodeling and metabolic reprogramming in T cells. Aim 2 will determine the immunologic consequences of Sirt6 inhibition in T cells against tumor challenge in Patient-Derived Xenograft mouse models for translational application. These aims will be reached by employing a multitude of experimental strategies involving in vitro epigenetic, metabolic, and immunologic analyses using primary mouse and human T cells, complemented by in vivo studies using genetically engineered patient-derived TILs and their autologous tumors. Collectively, the proposed studies will provide a comprehensive view of the epigenetic role of Sirt6 in metabolic processes in tumor-reactive T cells. The results from these studies will validate Sirt6 as an actionable metabolic and immunologic target with tractable means to improve cancer immunotherapy.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT Frontotemporal lobar degeneration (FTLD), which underlies frontotemporal dementia (FTD), encompasses a group of disorders with significant genetic, clinical, and neuropathological heterogeneity. FTLD is also genetically and pathologically associated with the motor neuron disease amyotrophic lateral sclerosis (ALS), with some patients developing both disorders. Understanding the diverse mechanisms governing FTLD pathogenesis is a fundamental area of interest of my research program, and we pursue this goal by asking impactful questions and applying innovative techniques. To accelerate scientific discovery, we have adopted a comprehensive approach that investigates multiple FTLD mechanisms driven by key molecular players like C9orf72, TDP-43, progranulin, tau and, more recently, TMEM106B. We also place great emphasis on translational research geared towards identifying much needed biomarkers and therapies, an area of particular importance given that there exists no treatment for FTLD. Since the funding of my current R35 at the end of 2016, my group has uncovered seminal findings related to the pathomechanisms mediated by FTLD-associated mutations in C9orf72 and GRN and shed crucial insight into the consequences of pathogenic TDP-43 and tau deposition in the brain. These findings have garnered high-impact publications in Science, Nature, and Cell and inspired new and ongoing avenues of research in my lab. The flexibility afforded by the R35 funding opportunity also allowed us to branch into other related topics and tackle urgent issues in the broader neuroscience field, including the need for biomarkers and mouse models for distinct repeat-associated disorders like spinocerebellar ataxias and the recent pressing need for tools to study and understand COVID-19 and its impact on the brain. Our productivity is influenced by the excellent research environment fostered at Mayo Clinic, which brings together highly interactive and devoted neurobiologists, geneticists, neuropathologists and physician scientists, the diversity of my team, and the numerous collaborations we have forged with world-renowned experts in the field, as well as our dedication to stewardship and the sharing of information and resources with the scientific community at large. Drawing from our past work on FTLD, we now propose to explore current cutting-edge questions related to: (1) the molecular underpinnings of TDP-43 localization and function and the downstream consequences of its dysfunction in disease, (2) the mechanisms underlying cryptic splicing in TDP-43 proteinopathies and the role of cryptic RNA and proteins in FTLD, (3) the role of the endo-lysosomal system in the development of TDP-43 pathology and neurodegeneration, and (4) the emerging role of TMEM106B fibrillogenesis in diverse neurodegenerative diseases including TDP-43 proteinopathies and tauopathies. We will use a combination of mouse and induced pluripotent stem-cell modeling, transcriptomics, proteomics, histology, and human tissue analyses to carry-out our proposed studies and address new and potentially transformative ideas as they emerge.

NIH Research Projects · FY 2025 · 2024-12

ABSTRACT Background: We recently revealed that glioblastoma (GBM) contain cell populations with distinct metabolic requirements, with fast-cycling cells (FCCs) harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. How the different tumor cells interact with immune cells and how this metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood. Objectives/Hypothesis: The objectives of this study are to understand the mechanisms of communication in the tumor microenvironment, specifically to characterize the metabolic interactions between SCCs (a therapeutically resistant population that drive disease progression and recurrence) and the immune compartment. Here, we will investigate a model of intercellular communication within GBM where SCCs shape an immunosuppressive tumor milieu, which in turn assume metabolic support to SCCs by providing them with lipids, which are essential for SCC metabolism and function. Importantly, we will test multiple genetic and clinically amenable pharmacological approaches disrupting this metabolic interplay to antagonize GBM. Specific aims: Our specific aims will be 1) Dissect the relationship of SCCs with the tumor microenvironment, 2) Delineate how recruited immune suppressive cell mediate SCC-driven tumor progression, and 3) Establish that immune infiltrates provide metabolic support to SCCs by providing lipids. Study design: The link between tumor heterogeneity and tumor immune landscape in GBM will be deciphered with specific investigations of the metabolic interplay taking place between these cellular compartments. In aim 1, we will delineate the cell lineage (SCC vs FCC) relationship with immune infiltrates by investigating their genomic profile and spatial organization, using single cell RNA sequencing technology and GeoMx Digital Spatial Profiling, respectively. We will also evaluate the role of the specific adipokine, Lipocalin-2, in shaping the immune microenvironment. In aim 2 we will employ multiple approaches disrupting the macrophage, myeloid- derived suppressor cell, and regulatory T cell compartments, and compare the effect on survival, growth and chemotherapy sensitivity of SCCs and FCCs. In aim 3 the use of fluorescently labeled lipids combined with flow cytometry and time lapse imaging will enable the comparison of lipid transfer between immune cells, FCCs and SCCs. Finally, in vivo experiments will test the hypothesis that targeting lipid trafficking (inhibition of FABP3 or ApoE) or lipogenesis (statin treatment) provide therapeutic benefits by affecting SCCs and rendering the overall tumor more responsive to chemotherapy. Based on the recently reported synergistic effect of statins with immune checkpoint inhibitors, we will also evaluate the combination of statins with anti PD-1 therapy. Impact: Successfully completed, this project will validate therapeutically amenable approaches targeting metabolic communication to improve brain tumor associated morbidity and mortality.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT Epithelial Cell Transforming Sequence 2 (ECT2) is a guanine nucleotide exchange factor that activates the function of RHO family GTPases. ECT2 expression is induced and prognostic in pancreatic ductal adenocarcinoma (PDAC), one of the deadliest cancer types. Our goal is to define the role of ECT2 in PDAC transformation. Two major ECT2 mRNA variants (ECT2-Ex4+ and ECT2-Ex4-) have been shown to express in cells through alternative splicing events that include or exclude exon 4 of the ECT2 gene. Our preliminary data indicate that: 1) ECT2 expression is elevated in PDAC precancerous lesions and PDAC tumors; 2) the ratio of ECT2-Ex4+ to ECT2-Ex4- expressed is elevated in primary PDAC tumors and cell lines when compared to normal pancreas cells and tissues; 3) an elevated ECT2-Ex4+ to ECT2-Ex4- ratio correlates clinically to poorer survival rates in PDAC patients; and 4) ECT2 is required for PDAC cell transformation. We hypothesize that the ECT2-Ex4+ isoform is oncogenic in PDAC and may serve as a biomarker of PADC progression and novel therapeutic target. This hypothesis will be tested through completion of three interrelated specific aims designed to: 1) determine the status of ECT2-Ex4+ in PDAC precursor lesions and PDAC tumors in situ; 2) define the specific role of ECT2-Ex4+ in normal pancreas cells and PDAC tumor growth and metastasis; and 3) determine the role of ECT2 exon 4 on ECT2 oncogenic signaling in PDAC cells. Successful completion of these studies will provide new insight into the role of ECT2 in PDAC transformation and enhance our understanding of PDAC progression. In addition, our studies will facilitate the development of a novel biomarker for early PDAC diagnosis. Finally, our studies may reveal an ECT2 isoform switch as a novel therapeutic vulnerability in PDAC.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Patients with rapidly progressive dementia (RPD) experience accelerated declines in cognition resulting in dementia within 1 year or complete incapacitation within 2 years of symptom onset. Although the medical literature has long emphasized the contributions of Creutzfeldt-Jakob disease to RPD, rapidly progressive forms of Alzheimer disease and AD-related dementias (rpAD/ADRD) account for most cases of RPD in older individuals. The rapid rate of decline in patients with rpAD/ADRD presents a great clinical challenge for the assessing clinician but also a unique opportunity to study the biological factors that underlie this extreme endophenotype across a shortened symptomatic period. The Biomarkers and Rates of Progression in Dementia (Bio-RaPID) study will capitalize on this opportunity by enrolling and evaluating 120 patients with rpAD/ADRD. Recruitment will be supported by a 9-site referral network engaging 7 Alzheimer Disease Research Centers with expertise in recruitment of underrepresented groups. Structured clinical evaluations will be conducted at baseline, 12-, and 24-months at Mayo Clinic Jacksonville (FL) or Rochester (MN). Interval telemedicine assessments with remote blood collection at 3- and 6-months will minimize participant burden while enabling serial measures of cognitive function and blood-based biomarkers. In this way, Bio-RaPID will combine clinical expertise, validated diagnostic approaches, long-read whole genome sequencing, and gold-standard in vivo measures of amyloid (A: CSF ptau181/Aβ42), tau (T: flortaucipir PET), neurodegeneration (N: MRI), cerebrovascular disease (V: MRI), pathologic aggregates of α-synuclein (CSF seed amplification assay), and biofluid biomarkers of neuroinflammation to determine the patient- and disease-specific factors that contribute to rpAD/ADRD. Use of ADRC protocols and coordination with ADRC Cores will enable comparison with existing patients with typical AD/ADRD and broad sharing of Bio-RaPID data. This “whole patient, whole brain” approach will inform the influence of patient-specific factors on rpAD/ADRD susceptibility, including age, sex, medical history, structural and social determinants of health, genetic variants, and ATN(V) and α-synuclein pathology (Aim 1); and the contributions of disease-specific factors to disease progression, including burden and topology of neuropathology and the mediating effects of neuroinflammation (Aim 2). Finally, Bio-RaPID will leverage unbiased proteomic analyses in CSF from an independent cohort of patients with autopsy-confirmed rapid and typical AD/ADRD (n=120, each) to validate findings from Aim 1 and 2 and identify cellular/protein pathways that are uniquely expressed or altered in rpAD/ADRD (Aim 3). Bio-RaPID aims will inform the cumulative contributions of demographics, concurrent health issues, structural and social determinants of health, genetics, biofluid biomarkers, and ATN(V) neuropathology to cognitive decline and synaptic dysfunction in rpAD/ADRD. In turn, these findings will be extended to identify biomarkers and novel treatment targets with the potential to arrest or slow pathologic progression in patients with rapid and typical progressive AD/ADRD.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Behavioral and social science research (BSSR) is instrumental in comprehending Alzheimer's Disease and related dementia (ADRD) and its far-reaching implications for individuals, families, and communities. BSSR investigates how behavioral and social determinants, encompassing factors like physical activity, cognitive engagement, diet, and social interactions, influence the risk of developing AD. This exploration aims to uncover non-pharmacological interventions that can manage symptoms and enhance the well-being of individuals with AD/ADRD, including cognitive stimulation programs, behavioral therapies, social engagement initiatives, and caregiver training. Moreover, BSSR delves into the social, cultural, and environmental elements contributing to health disparities, informing tailored interventions and policies for various populations, especially underserved and minority communities. Collectively, this research enriches our understanding of ADRD and guides the development of interventions, support systems, and policies to enhance the lives of those affected by the disease. Yet, there are challenges impeding the integration of ADRD-related BSSR data. A critical issue is the absence of formal representations for BSSR data and limited tools to link comprehensive BSSR information from diverse sources. This hampers the holistic consideration of BSSR factors in AD-related research, undermining evidence-based care and support. In response to PAR-23-182, we propose pioneering ontology-based approaches to formally represent ADRD-related BSSR factors in a standardized manner. We will develop natural language processing (NLP) methods to extract and normalize BSSR data from Electronic Health Records (EHRs) and literature. Our project aims to integrate structured and unstructured data across various research silos, culminating in a comprehensive and normalized knowledge graph incorporating BSSR factors for ADRD cohorts. More specifically, in Aim 1, we will develop the Behavioral Social Data and Knowledge Ontology for ADRD (BSO-AD) to standardize BSSR factors. We will also assess the BSO-AD for correctness and suitability, refining it based on evaluation scores. Aim 2 employs NLP technologies, including state-of-the-art large language models, to extract and normalize BSSR-related information from clinical notes and literature. This NLP system will ensure semantic interoperability and consistency in entity recognition and normalization. In Aim 3, we will create a knowledge graph (KG) to integrate annotated BSSR factors from structured and unstructured sources, supporting ADRD-related research and applications. We will evaluate the ontology and KG through demonstration studies and disseminate these resources to the research community, promoting collaborative research efforts. In summary, our project aims to bridge the gap in ADRD-related BSSR data integration by standardizing representation, enabling efficient extraction, and fostering collaboration within the research community. This endeavor will advance our understanding of ADRD and contribute to evidence-based care and support for affected individuals and their communities.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is the most common, aggressive and proliferative primary brain tumor in adults despite current therapeutic strategies that combine surgery, radiation, and chemotherapy. The high invasive capacity of GBM makes total surgical resection virtually impossible, resulting in an extremely high recurrence rate. The ability to form a new tumor resides in a subpopulation of cells within the GBM called brain tumor initiating cells (BTICs). BTICs are undifferentiated cells with self-renewing and pluripotential capacity, similar to neural stem cells (NSCs), but with the added ability of forming tumors in vivo. Our group and others have reported that among primary GBMs, those that are located in close proximity to the lateral ventricles (LV) present multiple factors, including increased recurrence at distant locations, that negatively affect patients’ survival. Possible explanations may involve the proximity of these tumors to the cerebrospinal fluid (CSF) and neurogenic niche in the subventricular zone (SVZ). We have previously reported that GBM tumors infiltrating the LV disrupt SVZ homeostasis, inducing signs of senescence in NPC and allowing CSF infiltration. The ensuing interaction with CSF heightens the proliferation and migration capacity of GBM-BTICs, fostering a more aggressive phenotype. However, the mechanisms used by GBM cells to modify the SVZ niche and the LV wall are not understood. In our preliminary studies we have observed extracellular vesicles (EVs) play an important role in the intercellular communication between BTICs and NPCs. The proposal's primary objective is to explore the role of EVs in mediating communication between GBM cells and the tumor microenvironment, specifically within the SVZ. Additionally, the research aims to evaluate EVs as potential biomarkers for monitoring brain tumor response to treatment and predicting recurrence.

NIH Research Projects · FY 2025 · 2024-09

TARGETING SIGNALING BETWEEN GLIOBLASTOMA AND THE SUBVENTRICULAR ZONE NICHE Glioblastoma (GBM) is the most malignant and proliferative primary brain tumor in adults. It remains a therapeutic challenge despite current treatment strategies that include surgery, radiation, and chemotherapy. Due to the tumor's high invasiveness, complete surgical resection is often impossible, leading to an almost 100% recurrence rate. Brain tumor initiating cells (BTIC), a subpopulation of undifferentiated cells within GBM, are responsible for tumor initiation and maintenance, exhibiting self-renewing and pluripotent properties in vivo, similar to those of Neural Progenitor Cells (NPC). Recent studies by our group and others have demonstrated that GBMs located in proximity to the lateral ventricles (LV) exhibit multiple factors that negatively impact patient survival, including increased proliferation and recurrence at distant locations. The underlying reasons for these worse outcomes in LV-proximal GBMs are not well understood but may involve the influence of the sub-ventricular zone (SVZ) neurogenic niche in the LV. Leveraging a GBM animal model that recapitulates the effects of LV-proximity on tumor malignancy, we have observed that neural progenitor cells (NPC) increase the proliferation and migration capacity of GBM-derived BTIC. Additionally, using image-guided biopsies of brain tumor samples in patients, we have identified intratumoral transcriptional differences induced by LV-proximity. Thus, we propose to investigate the specific protein components that drive the increased malignancy of LV-proximal tumors, utilizing novel cell-specific proteomic and transcriptomic approaches. The completion of this study will enhance our understanding of the mechanisms underlying the interaction between brain tumors and the neurogenic niche in the SVZ. Ultimately, our findings will identify novel therapeutic targets to improve the survival of patients with GBM.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Current treatment options for amyotrophic lateral sclerosis (ALS) are inadequate, and many patients living with ALS seek access to experimental therapies with the hope that they will be effective. Since most patients are excluded from participation in interventional trials based on restrictive inclusion and exclusion criteria, expanded access programs (EAPs) can bridge an important gap. As such, through an EAP, we are now proposing to offer a promising drug – ibudilast – that is presently in a phase 2/3 trial for ALS. Ibudilast has a well-established favorable safety record and has been approved for asthma and post-stroke symptoms in Japan. Most relevant to our application is the fact that it is able to penetrate the central nervous system and has beneficial pharmacological effects for ALS, such as phosphodiesterase inhibition, increased autophagy, and amelioration of TDP-43 pathology. This EAP will offer ibudilast as an experimental treatment to 200 ALS patients for 6 months. Our project will develop a network of ALS physicians, to be managed by Mayo Clinic and WideTrial, which features self-activation and streamlined workflows with e-consent and enrollment, home-health visit ordering, drug supply ordering, and oversight of safety events and outcomes data capture. Evaluations will be done either in a physician’s office or virtually in the patient’s home to minimize the burden of participation. Safety monitoring will be conducted by the treating physician and through blood drawn in the patient’s home, processed in a central laboratory. We will determine the effect of ibudilast on ALS progression using the gold standard primary clinical trial outcome measure – the ALS functional rating scale revised (ALSFRS-R) – with the emerging biomarker neurofilament light (NfL) as co-primary outcome. Given the urgent need for reliable biomarkers, we will also test other candidates (e.g., TDP-43 markers). Furthermore, to aid in differentiating between ibudilast responders and non-responders, we will use cutting-edge multi-omic sequencing methods. Our usage of long-read whole- genome sequencing enables us to span the full range of genomic variation, and by generating complementary transcriptomic data, we can capture RNA signatures and disease-relevant cell populations. Thus, we strongly believe that our datasets will provide a valuable resource and can possibly assist in patient stratification. In summary, our EAP will offer a promising drug, bring together novel web-based infrastructures, implement novel biomarkers, and obtain multi-omic profiles of responders, thereby allowing tailored personalized therapies and setting the stage for future ALS clinical trials.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY In the United States, beta-lactam antibiotics are the leading cause of allergic reactions. Cephalosporin antibiotics, in particular, are the most common cause of drug-induced anaphylaxis and perioperative allergy. For penicillin allergy, the mechanism of allergy and the antigenic determinants are known; validated penicillin skin testing followed by drug challenge has a 100% negative predictive value to exclude an immunoglobulin (Ig)E-mediated reaction. For cephalosporin allergy, the antigenic determinants and mechanism are not known, and skin testing is not validated. The diagnostic test characteristics of skin testing with native cephalosporins remain unclear with no sensitivity nor specificity reported. Although beta-lactam cross-reactivity has been hypothesized to be from the similarity of the R1 side chains, rather than the beta-lactam ring, cross-reactivity estimates among beta-lactams vary. Furthermore, it is not known whether the variance in cross-reactivity is due to true allergy versus sensitization based on positive skin testing, given that drug challenges were not performed on skin-test-positive patients. While an IgE mechanism is assumed for cephalosporin allergy and supported by skin testing that has been positive, the biology has yet to be characterized, and some cephalosporin anaphylaxis can occur on the first exposure, which is inconsistent with an IgE mechanism. Given the complexity of cephalosporin structures and potential epitopes, there may be several distinct biologic pathways involved in cephalosporin allergy. Future diagnostics in cephalosporin allergy are reliant on determination of these biological pathways and finding key haptens. Current national practice guidelines related to cephalosporin allergy assessment are considered conditional and based on low-quality evidence. Our overall goal is to identify the optimal diagnostic approach to cephalosporin allergy and determine beta-lactam cross-reactivity, while discovering the mechanism and antigenic determinants of cephalosporin allergy to advance future diagnostics. We will do this through a clinical trial that will generate empirical evidence through novel trial procedures, double-blind skin testing, and double- blind placebo-controlled drug challenges. Our specific aims are: 1) To determine the optimal approach to cephalosporin allergy evaluation; 2) To assess beta-lactam cross-reactivity in cephalosporin-allergic individuals; and 3) To investigate the antigenic determinants and mechanism of cephalosporin allergy. We will achieve these aims through collaboration with an established network of drug allergy specialists. Our study is the first clinical trial in drug allergy that investigates diagnostic strategies and mechanisms for a common and important antibiotic class. This project aligns with NIH/NIAID goals to advance drug allergy research and PAR-21-083 to support high-risk clinical trials with mechanistic studies.

NIH Research Projects · FY 2024 · 2024-07

Project Summary Glioblastoma (GBM) is the most devastating brain neoplasm with high morbidity and mortality. Despite multimodal treatment including surgery, radiotherapy, and chemotherapy, the disease recurs and is fatal. Given its public health importance, there is an unmet need for new treatment strategies to prolong patient survival while improving the quality of life. SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase) are stress-sensing kinases that phosphorylate and activate NKCC and KCC ion co-transporters which are key players in restoring intracellular Cl- and cell volume in response to cell stress. This is of importance because cell volume decrease and reduction of total intracellular ionic strength (via Cl- and K+) are the earliest stimuli that lead to apoptosis. Cancer cells can counteract pro- apoptotic signals by using regulatory volume increase through SPAK/OSR1/NKCC1 activation. Therefore, these kinases can serve as a resistance mechanism to cell death. On the other hand, cell volume regulation is an essential mechanism for cell migration in the confined spaces in the brain. Our preliminary data using genetic and pharmacological inhibition of SPAK/OSR1 shows that these kinases decrease GBM cell migration, proliferation and tumor growth in vivo and could serve as a mechanism of therapy resistance to chemoradiotherapy. However, SPAK/OSR1 function, mechanism, and therapeutic role in GBM remains unexplored. We hypothesize that SPAK/OSR1 exerts oncogenic functions as a cell-stress resistance mechanism allowing cancer cells to survive hostile environments and enhance GBM malignancy. The goal of this proposal is to evaluate if SPAK/OSR1 serves as a mechanism of resistance to the standard of care. We will evaluate the therapeutic efficacy of SPAK/OSR1 inhibition using a novel small molecule inhibitor in combination with chemo-radiotherapy and describe the role of SPAK/OSR1 in tumor resistance. Our long-term goal is to set the foundation for a research program that will study the relevance of the SPAK/OSR1 pathway in GBM progression identifying druggable signaling pathways that regulate GBM cell invasion and proliferation.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY The proposed studies seek to investigate the underlying disease mechanisms of Lewy body dementia (LBD) associated with the apolipoprotein E4 (APOE4) isoform. APOE4 is a major genetic risk factor for LBD, and our recent research has shown a direct influence of APOE4 on α-synuclein (αSyn) pathology in the human brain and mouse models. However, the mechanistic relationship between APOE4 and LBD remains unclear. APOE, primarily produced by astrocytes, mediates cell-to-cell lipid transport in an isoform-dependent manner. APOE4 is known to disrupt brain lipid homeostasis and compromise the endosomal-lysosomal system, which are pathways also associated with other LBD hits, GBA and BIN1. This suggests that these pathways may be key mechanisms linking APOE4 to LBD. Moreover, APOE4 is associated with the risk and severity of LBD, but not necessarily Parkinson’s disease (PD). This leads to the speculation that the effects of APOE4 on αSyn pathology may manifest more in the neocortex where LBD pathologies are found and have minimal impact on subcortical or brainstem structures where Lewy pathology is predominant in PD. Therefore, the proposed research aims to investigate the role of APOE4 in αSyn pathogenesis and define the underlying molecular pathways and brain region vulnerability using human postmortem brains, astrocyte-specific APOE3 or APOE4 deletion mouse models, and human induced pluripotent stem cell (iPSC)-derived organoid models. In Aim 1, we will perform multi-omics profiling (bulk RNA iso-seq, single nuclei RNA-seq, proteomics, and lipidomics) of the superior temporal cortex and midbrain of human LBD cases from APOE3/3 and APOE4/4 individuals. We will conduct network analysis to uncover lipid and endosomal-lysosomal related dysfunction, and other pathways associated to LBD and APOE4. In Aim 2, conditional mouse models will be used to delete APOE3 or APOE4 in astrocytes by crossing human APOE knock-in mice, where murine Apoe is replaced with floxed APOE3 or APOE4 gene, with Aldh1l1-CreER mice and human SNCA-overexpression mice. Deletion of APOE will be induced by tamoxifen treatment at different stages of αSyn pathogenesis. The αSyn pathology related phenotypes will be evaluated and multi-omics profiling will be conducted in the experimental mice. In Aim 3, we will validate the cellular mechanisms of APOE4 on affecting αSyn pathogenesis, lipid homeostasis, and endosomal-lysosomal functions using iPSC-derived cortical and midbrain-like organoid models from patients carrying SNCA triplication and different APOE genotypes edited by CRISPR/Cas9 technology. Together, this innovative proposal will utilize unique human brain resources, mouse models, and human iPSC- derived models, and combine state-of-the-art approaches for multi-dimensional integration of molecular profiling to comprehensively investigate the pathomechanisms of APOE4 in LBD. These efforts will contribute to our understanding of the APOE4-mediated pathways involved in αSyn pathology, providing valuable insights for the development of future therapeutic strategies targeting APOE in the treatment of LBD.