Mayo Clinic Rochester

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT Radiation dose is a major drawback of computed tomography (CT) imaging. While the long-term effects of radiation are difficult to quantify, there is a continued desire to reduce radiation dose through technical improvement. In theory, photon counting detectors (PCDs) provide several opportunities to reduce CT radiation dose. In practice, however, PCD CT scanners today have shown only modest gains in dose efficiency compared to scanners from prior generations for most scanning protocols. Several components of the CT scanner must be re-engineered to benefit from the dose saving potential of PCDs. The objective of this work is to perform this re-engineering by designing new components that can take advantage of radiation dose reduction opportunities with PCD CT. In Specific Aim 1, we will develop hardware that could improve quantum efficiency, or the fraction of incident photons that are successfully detected. Today, about 40% of the x-rays that arrive at the detector are never absorbed by the sensor. 30% of the x-rays are stopped by the anti-scatter grid, and 10% of the x-rays penetrate the sensor without interaction due to the relatively low stopping power of the sensor. First, we will experimentally demonstrate a lightweight, striped anti-scatter grid that will increase the number of primary photons detected while providing a means for estimating residual scatter. The geometry of this grid introduces striping in the detector signal, with stripe contrast proportional to the level of remaining scatter. Therefore, the images themselves will contain a means to subtract the remaining scatter. Second, we will develop a guardrail that can fluoresce a fraction of the punch-through photons and return them to the sensor. We will demonstrate the guardrail experimentally in a CdTe benchtop PCD. In Specific Aim 2, we turn to spectral nonidealities in PCD CT. PCDs suffer from pulse pileup and charge sharing. We will experimentally test a pileup detection circuit that reroutes suspected pileup events into separate counters. We show that this substantially reduces the impact of pileup and will simulate its use together with charge summing modes to reduce charge sharing effects. Historically, charge summing modes have not been used in CT applications because of their pileup penalty. We hypothesize that this mode could be enabled by stronger pileup corrections, such as the one we will demonstrate. Charge summing modes also improve dose efficiency for grayscale (non-spectral) tasks by eliminating double counting effects. Successful completion of these Aims could allow PCDs to unlock their dose reduction potential and could improve the dose efficiency of PCD CT by 40% for non-spectral imaging tasks and 200% for spectral imaging tasks.

NIH Research Projects · FY 2025 · 2024-06

Abstract. The choice of a delivery system for genetic manipulation or gene expression remains a topic of debate in the research community. The development of a safe, unique, and efficient delivery vehicle that could promote gene editing would significantly contribute to the field of gene therapy and regenerative medicine. Recently, we have demonstrated that the Measles virus can be developed as a single cycle vector for multiple gene delivery. We have shown that we can express the four reprogramming factors from on single measles genome and reprogram adult human fibroblasts into induced pluripotent stem cells. We further have evidence that MeV can express the gene-editing system CRISPR-Cas9 and lead to the correction of cells in the presence of a molecule of DNA template. The goal of this application is to develop new prototype measles vectors that can either genetically modify or correct a specific gene locus without relying on a DNA molecule using Prime Editors. As a proof of concept, we will develop prototype vectors based on the vaccine strain of the measles virus, a negative-strand RNA virus. The objectives of this application are (1) to establish the proof of principle that the MeV vector can express the Prime Editing system (MeV-PE2). To do this, we will express the two elements required for prime Editing: the Prime editor (PE2) and prime editor guide RNA (pegRNA), and test their functionality in producing modified cells for the correction of Sickle cell disease (SCD) (2) In the second aim, we will introduce the latest generation of Prime editor in MeV vector (MeV-PEmax), which consists of the PE2 and the introduction of (i) a standard gRNA targeting the non-edited template, (ii) the MLH1dn protein to reduce mismatch repair or (iii) a modified pegRNA to increase its stability. The proposed work is innovative, challenging, and significant, and if successful, it will lead to the production of novel prototypes of “all-in-one” non-integrating RNA viral vectors for cellular gene editing without the addition of an exogenous molecule of DNA. This work will open the future development of safe vectors for gene editing and the production of corrected cells for multiple genetic disorders. This new platform will have a significant impact in the field of gene therapy and regenerative medicine.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Around 80 million US adults will have arthritis by 2040, which causes a significant societal problem resulting in an annual healthcare cost of > $136.8 billion. These progressive degenerative diseases result in long-term functional disabilities and morbidities leading to activity limitations and productivity loss. Joint instability typically precedes before the symptom onset, but it is typically not detectable in standard imaging exams. Patients can therefore suffer for years, as symptoms worsen before an accurate diagnosis is made. There is a critical need for an accurate imaging technique for early diagnosis, which is crucial to providing the most effective medications, therapies, and surgical interventions and restoring joint function before the onset of disabling symptoms. Various limitations have hindered the accurate detection of joint instability in standard imaging exams: radiography and fluoroscopy cannot visualize 3-dimensional (3D) joint anatomy; ultrasound lacks visualization of bone structure; static MRI/CT cannot assess the effect of dynamic structural and biomechanical pathologies. Accurate diagnosis is therefore oftentimes acquired using invasive arthroscopy, which exposes patients to risks of the associated complications. Although there have been some recent advancements in 4D (3D + time) CT to provide dynamic imaging capability, its clinical use is limited due to (1) joint motion artifacts that affect accurate anatomical and functional analyses, and (2) poor soft-tissue visualization that prohibits robust assessment of connective tissue involvement. The specific goal of this application is to develop and validate a new diagnostic imaging technique – 6DCT (3D + time + spectral information + kinematics) in combination with custom deep-learning methods. The 6DCT will provide an accurate, informative, one-stop-shop diagnostic tool to assess early dynamic joint pathologies before the onset of arthritis, which will obviate the need for invasive tests and procedures, with improved patient care and associated cost savings. We will accomplish this goal through three specific aims: 1. Develop a robust motion correction technique for dynamic joint imaging. 2. Develop a spectral post-processing technique for characterizing connective tissue involvement. 3. Demonstrate the feasibility of 6DCT in imaging dynamic joint pathology. This work is the first to develop innovative 6DCT as a robust dynamic imaging tool for both bone and connective tissues, and to provide new anatomical and functional information to facilitate assessing dynamic joint pathologies. These capabilities have clinical significance as they will enable early diagnosis of joint instability, which will subsequently enable early interventions that will improve patient health. This proposal will develop the essential techniques and preliminary data for future large cohort prospective patient studies.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY: Triple-negative breast cancer (TNBC) represents ~15% of invasive breast cancer cases, with disproportionately high prevalence in African-American and Hispanic women, and is defined by the lack of expression of estrogen receptor, progesterone receptor, and HER2 (ER-/PR-/HER2-). Due to the absence of currently targetable molecular drivers, treatment for TNBC still relies on non-specific toxic chemotherapeutics and outcomes remain poor. As a result, there is a pressing need to develop molecularly-informed, targeted therapies to treat TNBC. In this proposal, we investigate MALT1 protease as a potential new therapeutic target in a subset of TNBC. MALT1 is the enzymatic effector protein of a signaling complex composed of proteins CARMA3, BCL10 and MALT1 (CBM signalosome) that functions downstream of specific G protein-coupled receptors (GPCRs) to drive oncogenic reprogramming in a subset of carcinomas including TNBC. GPCRs known to activate MALT1 in breast cancer include PAR1, AGTR1, and the LPARs (LPAR1-3) and overexpression of these GPCRs is associated with aggressive breast cancer behavior in experimental models and worse clinical outcomes for human patients. We recently found that the GPCR/MALT1 signaling axis drives a program of epithelial-to-mesenchymal transition (EMT) in TNBC. Since tumor cells undergoing EMT are known to promote a permissive, immune- suppressed microenvironment, we propose to investigate the role of the GPCR/MALT1 signaling axis in the regulation of the tumor immune microenvironment. Our preliminary data indicate that MALT1 is a key mediator of immune suppression induced by GPCR+ TNBC, suggesting that inhibiting MALT1 protease activity in TNBC cells may have a beneficial effect. Additionally, inhibition of MALT1 protease activity in immune cells has recently been found to preferentially impair Treg function, tipping the balance between regulatory and effector T cells to promote heightened immunoreactivity. Together, these observations in TNBC cells and immune cells lead us to hypothesize that pharmaceutic MALT1 inhibition may have dual therapeutic benefit in GPCR+ TNBC by (1) preventing MALT1-mediated tumor immune suppression from within cancer cells and (2) altering the composition of tumor infiltrating immune cells in favor of anti-tumor immunity. This proposal will evaluate this hypothesis via a combination of elegant in vitro and in vivo model systems. The overarching goal of our proposal is to rigorously evaluate the role of MALT1 as a pathogenic driver of TNBC and to test pharmaceutic MALT1 inhibition as a novel therapeutic strategy in this difficult-to-treat disease.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Potentially lethal cardiac channelopathies associated with pathogenic variants in the RYR2-encoded cardiac ryanodine receptor type 2 (RyR2)/calcium release channel (CRC) are the pathogenic basis for a significant portion of autopsy-negative sudden unexplained death in the young (SUDY). RYR2 gain- of-function (GOF) pathogenic variants account for 60% of autosomal dominant catecholaminergic polymorphic ventricular tachycardia (CPVT1), a potentially lethal heritable arrhythmia syndrome that classically manifests as exercise-induced syncope, sudden cardiac arrest (SCA), or sudden cardiac death (SCD). In 2020, we discovered a novel RYR2 loss-of-function (LOF) mechanism that we have termed calcium release channel deficiency syndrome (CRCDS). We identified a novel homozygous duplication (RYR2-DUP) involving ~26,000 bp of intergenic sequence, RYR2’s 5’UTR/promoter region, and exons 1-4 of RYR2 that is responsible for highly penetrant, exertion-related SCA/SCD in the Amish community without an overt phenotype to suggest RYR2-mediated CPVT1. Unlike typical CPVT1, individuals homozygous for the RYR2 duplication have displayed typically only intermittently prolonged QT intervals or prominent U-waves and typically had completely normal exercise/epinephrine stress tests and normal 24-hour Holter monitoring. Thus, cardiologic tests, such as ECG, stress testing, and echocardiogram, are currently unable to reliably identify or further risk stratify family members likely to be homozygous for the RYR2 duplication. Given the potentially lethal nature of these inheritable RYR2- CRCDS variants and the lack of a robust and measurable clinical phenotype, it is vitally important to better understand the spectrum and contribution of CRCDS-associated RYR2 variants in SUDY and to determine the underlying disease-associated mechanisms in patient-specific re-engineered heart cell models using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Here, we propose to 1) determine the frequency of CRCDS due to RYR2 coding region, non-coding region, and structural variants (SVs) in SUDY and 2) develop an international registry for CRCDS to identify genotype/phenotype correlates to assist in clinical diagnosis and management of patients with CRCDS. 3) determine the relative contribution of CRCDS (LOF)-associated RyR2 variants versus CPVT1 (GOF)- variants in SUDY using functional studies, 4) develop a function-based iPSC-CM platform for RyR2 VUS resolution, and 5) determine the compensatory mechanisms related to loss of RYR2 transcript/RyR2 protein expression and function as it relates to calcium handling components and heart cell function during early cardiac development.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY The alveolus is lined by two epithelial cell types: thin gas-exchanging alveolar type 1 (AT1) cells and cuboidal surfactant-producing AT2 cells. Both are selected from a common distal progenitor by FGF, Notch, and stretch signaling. Injury in the adult lung activates AT2 into a facultative progenitor state to regenerate lost AT1 and AT2 cells. While the inductive cues that select fate have been identified, it is less clear what role – if any – repressive cues play in fate maturation. Two gaps of understanding in the lung field are 1) when and how is stemness lost after fate selection and 2) once lost, how is stemness re-accessed by AT2s after injury to regenerate the alveolar epithelium. Our recent work indicates perinatal AT2s retain stemness for weeks after fate selection, despite being transcriptionally and functionally differentiated. This proposal focuses on a putative repressor of AT2 stemness, determining its target and modes of action, whether and how it is overridden in adult AT2s to re-access stemness, and investigating the extent to which it is dysfunctional in pulmonary fibrosis - wherein aberrant AT2 states are observed. Our first aim focuses on a putative repressor of stemness we identified, the CCAAT enhancer binding protein C/EBPα. Using transgenic mouse models, we will test the requirement of Cebpa for AT2 stemness and determine whether the targeted stemness program acts in a cell autonomous or non-autonomous manner by mosaic deletion. Next, scRNAseq, ATAC- and ChIP-seq will identify genes targeted by, as well as molecules interacting with, C/EBPα. Finally, we will use organotypic culture and transgenic mouse experiments to determine whether timing of Cebpa expression is itself regulated earlier in development by the polycomb repressive complex PRC2. The second aim of the proposal is to determine whether and how C/EBPα regulation is overridden following injury to re-access AT2 stemness and promote repair. We will determine whether C/EBPα downregulation and AT2 stemness is dependent on AT1 cell death in vivo by performing genetically targeted cell type specific ablation. Finally, we will compare the spatiotemporal program of C/EBPα regulation we identify during injury and repair to the fibrotic lung and investigate whether it is dysfunctional in its associated aberrant AT2 cells. Our approach is innovative, as no transgenic mouse experiments using mosaic deletion or lineage tracing have been performed for Cebpa in the lung, nor have precise cell ablation studies been conducted in the adult lung to study C/EBPα regulation. Further, the observation of retained stemness in perinatal AT2 cells is novel and thus no investigation has implemented a multi-omics approach to understand its underlying mechanism. Finally, little is known on the role repressive cues play in AT2 cells differentiation and maturation. If successful, we will have uncovered a novel mechanism of AT2 stemness regulation for the lung field which will be informative in understanding pulmonary diseases with aberrant epithelial differentiation. Further, our findings will guide development of novel diagnostics or therapeutics, either for direct use in patients or in culture to control stem cell differentiation through reprogramming patient’s adult AT2 cells.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY While most forms of dementia arise from irreversible, degenerative processes, normal pressure hydrocephalus (NPH) symptoms can often be alleviated by placement of a ventriculoperitoneal shunt to divert cerebrospinal fluid from the characteristically enlarged ventricles. However, due to overlapping clinical and biomarker phenotypes with other common age-related disorders, NPH remains substantially underdiagnosed and undertreated. Even when diagnosed, current methods are limited in predicting outcomes of shunt surgery, particularly when considering noninvasive methods. Since the disorder was first described, it has been hypothesized that the intracranial mechanical environment was involved in NPH pathogenesis. Consistent with this hypothesis, we recently reported that NPH is associated with a characteristic pattern of brain viscoelasticity, as measured by magnetic resonance elastography (MRE). Furthermore, the presence of this pattern is sensitive and specific for differentiating NPH patients from healthy volunteers and those with Alzheimer’s clinical syndrome. While MRE is an accurate diagnostic biomarker for NPH, we expect that with further technical development, MRE will also enable accurate prediction of treatment outcomes. In our preliminary data, patients with low brain stiffness are less likely to benefit from shunting. However, these predictions are not yet accurate enough to use clinically. We hypothesize that the existing discrepancies arise from an ambiguity that low brain stiffness, as measured by current technology, can result either from decreased stiffness in the solid tissue matrix or increased fluid content in the extracellular space. These two effects must be disentangled to identify the patients with preserved matrix stiffness as those most likely to benefit from shunting. Therefore, the overall goal of this work is to develop an MRE framework that jointly leverages strain and diffusion measurements to estimate the viscoelastic properties of the solid matrix while accounting for interspersed extracellular fluid. In Aim 1, we will modify the forward model of our machine learning-based inversion framework to account for subvoxel fluid elements. We will then evaluate the algorithm’s accuracy in simulation and phantom experiments, along with its repeatability in vivo. In Aim 2, we will compare the diagnostic accuracy of the new method against existing methods, confirming that this solid-fluid mixture framework can similarly discriminate patients with NPH from healthy controls and those with Alzheimer’s dementia. Finally, using objective measures of gait collected before and after surgery, we will test the overall study hypothesis that baseline solid matrix stiffness estimates (accounting for fluid) can predict the degree of gait improvement following shunt placement. The proposed technology represents a fundamental shift in the field of brain MRE toward biophysically inspired modeling. Most importantly, the success of this proposal will provide new insights into the pathogenesis of NPH with the potential to directly impact patient care.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY End-stage kidney disease (ESKD) affects a significant number of patients in the U.S. (>785,000 in 2018), and presents substantial medical, social, and economic challenges. Kidney transplantation is the preferred treatment for ESKD. However, there is a pressing unmet clinical need for better methods that allow for noninvasive, accurate, and frequent characterization and monitoring of kidney allograft injury. Such methods are critical for enhancing long-term allograft survival and improving the quality of life for the kidney transplant recipients. The change of kidney parenchymal microvasculature and perfusion has been shown to play a vital role in the progression of allograft injury, while noninvasive tools for imaging and quantification of allograft microvasculature are still lacking. In this project, we will develop a novel noninvasive, robust, and translatable super-resolution ultrasound imaging (SRUI) technology. This technology aims to provide imaging and quantification of parenchymal microvasculature to enable reliable assessment of allograft injuries. In our pilot patient study of transplant kidneys, parameters derived from SRUI correlate strongly with pathology (r ≥ 0.9). Aim 1: Technical development. We will advance and optimize the novel SRUI technology for kidney allograft imaging and quantification. New signal enhancement and localization methods will be developed to improve the overall performance of SRUI in clinical settings. We will advance the SRUI to 3D to enable more comprehensive assessments of the graft microvasculature. We will develop novel quantitative SRUI metrics, including microvascular density, tortuosity, flow speed, cortex perfusion and micro-resistive index. Aim 2: Clinical patient study. We will study 158 patients to investigate the value of SRUI for kidney allograft assessment using biopsy histology as validation. The association of SRUI metrics with histological injuries will be assessed. The ability of SRUI, conventional ultrasound, and clinical measures (eGFR, proteinuria) to distinguish between kidney allografts with varying degrees of histological injuries will be evaluated. We will also assess the inter-sonographer reproducibility of the SRUI technology in a subset of 46 patients. Aim 3: Longitudinal follow-up study. We will conduct a longitudinal follow-up study in 62 patients to assess the efficacy of SRUI in monitoring and predicting the progression of allograft injury, using biopsy histology as validation. The association of changes in SRUI metrics with the changes in histological injuries from 1-year to 2-year post-transplant will be assessed. We will study if SRUI metrics or combined metrics at 1-year post- transplant, or the changes of these metrics from 1-year to 2-year, can distinguish between allografts without and with histological worsening during this period. Successful completion of this project will lead to a noninvasive, accessible, cost-effective, and translatable tool to address the critical clinical need for reliable and frequent characterization of allograft in transplant recipients.

NIH Research Projects · FY 2026 · 2024-05

PROJECT DESCRIPTION/ABSTRACT This NIH K08 proposal describes a five-year career development plan for Dr. Aivi T. Nguyen to obtain the necessary research and professional skills and serve as transition to become a successful, independently funded physician-scientist. Dr. Nguyen completed her clinical training in Anatomic Pathology and Neuropathology at the Hospital of the University of Pennsylvania, after which she pursued post-doctoral training under the mentorship of Dr. Edward B. Lee in the Translational Neuropathology Research Laboratory, University of Pennsylvania. Currently, Dr. Nguyen is an assistant professor at Mayo Clinic Rochester, MN and contributing neuropathologist to the Alzheimer’s Disease Research Center (ADRC) and Mayo Clinic Study of Aging (MCSA). Dr. Nguyen’s long-term research focus is examining the role of microglia in cognitive resilience, normal aging, and aging-related CNS diseases through integration of neuropathology and translational research approaches to better understand neuroinflammation in human disease. This K08 award will provide protected time to acquire expertise in integrating antemortem Alzheimer’s disease (AD) biomarkers and neuropsychiatric data with postmortem neuropathology and to obtain expertise in two-photon in vivo imaging of microglia in AD mouse models. Research will be performed under the mentorship of Dr. Prashanthi Vemuri, an expert in cognitive resilience and neuroimaging, and Dr. LongJun Wu, an expert in microglial dynamics and neuroimmune interactions using two photon in vivo imaging. This award will also provide protected time for Dr. Nguyen to gain expertise through formal coursework, scientific seminars, and scientific meetings. AD, neuropathologically defined by β-amyloid plaques and neurofibrillary tangles, progresses in a spatio- temporally distinct fashion that can be studied in vivo by recent advances in neuroimaging and fluid biomarkers. Several ante- and postmortem studies have shown a discrepancy between AD pathology extent and cognition, forming the basis of cognitive resilience. A potentially protective microglial subpopulation termed amyloid-responsive microglia (ARM) was previously described in a single-nuclei RNA sequencing study of human AD brain. Whether microglial heterogeneity, specifically ARM, contribute to resilience is unclear. This proposal leverages antemortem AD biomarkers, cognitive data, and postmortem tissue from MCSA participants to address these specific aims: (1) Determine postmortem ARM tissue expression association with antemortem fluid and neuroimaging biomarkers, and whether ARM may be predicted by biomarkers; (2) Evaluate differential ARM tissue expression in cognitively resilient individuals versus not, and test antemortem predictors; and (3) Examine ARM phagocytosis and effects on downstream neuronal dystrophy in the setting of AD genetic risk. These studies will drive the development of an independent research program to study microglial heterogeneity and its role in resilience and AD and provide better understanding of cognitive aging.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Hypertension is the chief risk factor for cardiovascular (CV) disease and a major threat to population health. Given the burden of hypertension, it is critical to identify risk enhancers in populations susceptible to CV disease, for risk stratification. While family history of hypertension, especially parental hypertension, is well- known as a robust predictor of high blood pressure (BP) and CV disease in the offspring, the role of chronic sleep deficiency is being increasingly recognized. Evidence from epidemiological and laboratory-based studies converges to indicate that insufficient sleep increases BP and alters CV disease mechanisms, thus predisposing to adverse outcomes. Whether the detrimental impact of insufficient sleep on CV risk is potentiated in populations with preexisting vulnerabilities, such as the offspring of hypertensive families, is plausible yet unknown. To bridge this gap, we propose a randomized, controlled, crossover, 10-day inpatient study to investigate whether experimental sleep restriction increases BP and impairs CV function in individuals with a positive parental history of hypertension (+PHH), and whether the hypothesized adverse impact of shortened sleep is greater in this group compared to individuals with a negative PHH (−PHH). We propose the following Specific Aims comparing sleep restriction to normal sleep in the offspring of hypertensive parents relative to the offspring of normotensive parents. Aim 1. To compare the effects of sleep restriction on ambulatory BP in individuals with a +PHH compared to those with a −PHH. Aim 2. To determine the impact of sleep restriction on neural circulatory control and vascular function in individuals with a +PHH compared to those with a −PHH. Aim 3. To explore epigenetic and transcriptomic modifications in response to sleep restriction in individuals with a +PHH compared to those with a −PHH. This proposal builds upon important strengths, including an interdisciplinary team of investigators with unique experience and expertise; supporting preliminary data; a robust study design which will ensure rigorous and unbiased research; and integration of physiological, behavioral, and omics variables in a translational framework. This mechanistic study will inform on the role of sleep deficiency as a CV risk enhancer in individuals with familial predisposition to hypertension, and guide future preventative strategies targeting sleep to ameliorate CV risk in vulnerable populations.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Therapy-related myeloid neoplasms (tMNs), including myelodysplastic syndrome (MDS) and acute myeloid leuk- emia (AML), are severe, often rapidly fatal bone marrow disorders that occur in a small fraction of solid tumor patients who have received cytotoxic treatment. Ovarian cancer (OC) survivors have one of the highest inci- dences of tMNs among all cancer patients, reflecting both an increased risk due to platinum exposure and a recently recognized further increase in risk with PARP inhibitor (PARPi) therapy. Collectively, these PARPi-as- sociated myeloid neoplasms are approximately as frequent as acute promyelocytic leukemia, a rare AML sub- type that previously had a high fatality rate but has become among the most curable myeloid neoplasms through the concerted efforts of the research community. In this context, our team has contributed to studies showing that a disproportionate number of PARPi-associated tMNs in OC patients have biallelic inactivation of the TP53 tumor suppressor gene. Like other TP53-mutant myeloid neoplasms, tMNs in OC patients receiving PARPis respond poorly to conventional chemotherapy and relapse quickly, with a median survival of 4-8 months, high- lighting the need for improved therapies. In preliminary studies, we have shown that replication checkpoint mod- ulators, including inhibitors of the kinases CHK1, WEE1 and ATR, exhibit antileukemic activity in AML cell lines, xenografts, and primary AML samples, including TP53-mutant AMLs. This activity reflects TP53-independent induction of apoptosis at therapeutically achievable concentrations through at least two distinct apoptotic path- ways, one of which involves leukemia cell-intrinsic transactivation of the TNF gene followed by TNF receptor- dependent caspase activation and the other of which involves the mitochondrial apoptotic pathway but is less completely defined. These observations lead to the hypothesis that replication checkpoint modulators, admin- istered as monotherapy or in combination, will have antileukemic effects in the tMNs that develop in OC patients. To further advance the study of these agents, we now propose to define the second pathway by which replication checkpoint modulators induce apoptosis in TP53-mutant AML, thereby providing deeper insight into determi- nants of tMN sensitivity to this emerging class of drugs (Aim 1); determine the antineoplastic effects of replication checkpoint modulators in vitro, alone and in combination with hypomethylating agents and BH3 mimetics (two classes of current standard-of-care AML agents), in samples of PARPi-emergent tMNs from OC patients (Aim 2); and assess the efficacy of replication checkpoint modulators in vivo, alone and in combination with hypomethyl- ating agents or BH3 mimetics, in patient-derived xenografts generated from PARPi-emergent AMLs of OC pa- tients (Aim 3). Collectively, these studies are designed to provide new insight into the antineoplastic action of replication checkpoint modulators and simultaneously position our multidisciplinary team to perform a future early phase clinical trial of replication checkpoint modulator-based therapy in OC patients with PARPi-emergent tMNs.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal disease with limited therapies. IPF patient lungs display hallmarks of accelerated aging, including overabundance of senescent cells. In particular, senescent lung fibroblasts (sLFs) are key drivers of lung fibrosis. However, the signaling mechanisms that promote sLF accumulation and persistence in lung fibrosis remain poorly understood. This proposal seeks to fill these gaps with mechanistic studies that will enhance our capabilities to clear sLFs in IPF. Flavonoids are naturally occurring molecules which show potential to treat idiopathic pulmonary fibrosis (IPF). Their rise to prominence in recent years has been driven by the discovery that several of these compounds are can selectively induce apoptosis in senescent cells. In order to develop a better tool to investigate sLFs we designed and synthesized a novel, potent, senolytic flavonoid (F-4N). We present preliminary data shows that F-4N promotes senescence clearance and resolution of lung fibrosis in bleomycin-injured, aged mice with chronic fibrosis. We further observed F-4N selectively inhibits NF-κB signaling in sLFs without affecting resident pulmonary leukocytes. NF-κB activation is a hallmark of senescent cells, however its role in their apoptosis-resistance has not been explored, and senescence-unique mechanisms for targeting NF-κB have not been identified. NF-κB is critical for host defense, and systemic inhibition has very limited application. Based on these observations we propose to test the central hypothesis that sLF apoptosis resistance depends on NF-κB activity and clearance of sLFs with F-4N represents a viable strategy to treat IPF. This hypothesis will be tested with three specific aims: First, we will establish the molecular mechanism of action essential to F-4N senolytic activity. We will perform apoptosis quantification, ChIP-seq, and RNA-seq from cultured sLFs to identify changes in response to gain and loss-of-function studies. We will use proteomic analyses to identify global phosphorylation changes in sLFs under these conditions. Second, we will characterize the cellular specific impact of F-4N on NF-κB activity and apoptosis by quantifying the effect of F-4N across fibroblast, epithelial, endothelial, and leukocyte populations after acute administration to mice with bleomycin induced lung fibrosis. We will culture in vivo- derived low and high NF-κB activity fibroblasts and epithelial cells in a 3D co-culture model to investigate paracrine signaling and senescence propagation. Lastly, will determine the antifibrotic efficacy of F-4N in chronic experimental fibrosis using one-time bleomycin lung injury in aged mice (18 months) and repeated bleomycin injury in young mice (2 months). F-4N will be benchmarked against the antifibrotic efficacy of pirfenidone and nintedanib, and previously established senolytic strategies. We will also measure the clearance of senescence burden and collagen deposition in precision-cut lung slice (PCLS) ex vivo cultures derived from IPF patient lungs. Together these proposed studies will enhance our understanding of the molecular and cellular mechanisms essential to sLF accumulation and persistence.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease and Related Dementias (ADRDs) are major causes of death and disability in the United States, with the number of adults with ADRDs projected to reach 13 million by 2050. Among these, the frontotemporal degeneration (FTD) spectrum disorders are among the most common causes of dementia in younger individuals (<60y), resulting in high social and economic burden. Diagnosis of FTD is challenging, typically requiring subspeciality expertise that is not widely available in a timely manner. Many FTD spectrum disorders manifest in speech, and speech changes can help differentiate FTD subtypes. Prior research supports the clinical utility of speech-based prediction of FTD presence and subtype. Unfortunately, rigor of these prior studies is limited for several reasons, including small sample sizes, failure to follow predictive modeling best practices, use of research grade speech recordings, and lack of prospective validation. We propose a highly innovative approach to speech-based prediction of FTD that avoids the weaknesses of prior work. Central to our approach is the insight that FTD may be too rare to use powerful deep learning models, but the abnormal speech characteristics seen in FTD are also seen in other disorders. Training a model to recognize these characteristics in FTD does not require limiting the dataset to FTD patients. We plan use this to our advantage. In Aim 1 we will use a self-administered, web-based speech exam to create a large dataset of all disorders seen in our speech clinic. Recordings will be made in a standard exam room using mobile phones or tablets. Our expert speech and language pathologist will annotate the recordings with perceptual speech characteristics, such as abnormal rate or vocal strain. The large sample size will enable us to use deep learning for what it excels at – trainable feature extraction optimized for the task at hand. We will follow predictive modeling best practices, including use of a validation set. In Aim 2 we will apply these trained models in a large cohort spanning the FTD spectrum and extract the data from the last layer in the network, just before it makes its prediction. This is a low dimensional representation of the speech signal, but which contains the information necessary for predicting perceptual characteristics. We will use these representations to develop a nearest neighbor classifier for FTD. Essentially, the model matches a new case to similar ones in a labeled set based on the low-dimensional representation and uses the neighborhood to assign a label for the new case. Finally, in Aim 3 we will combine our self-administered speech exam and the models from Aims 1 and 2 into a single tool and perform a prospective validation study to test performance in a clinical setting. The predicted increase in ADRDs and lack of access to specialists will necessitate a shift in clinical practice from a few expert centers to a distributed system of non-expert providers. The digital tool we propose meets this challenge head on through scalable and easy to use automated speech analysis and prediction of FTD.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY The study of genomes and genomic medicine is an urgent and critical need and using population genetics data to inform construction of polygenic scores for biologically relevant quantitative traits is a scientific imperative. The Southeast Minnesota region represents a population offering access to a blend of urban and rural communities. Using a well-established research framework to guide efforts, we will engage with the regional population across age ranges.. Our prior research interactions have identified significant enthusiasm to engage in genomic medicine and other research. Therefore we see no difficulty in establishing a genomic medicine research program in this region. We propose the following specific aims: Specific Aim 1. (a) Establish a genomic medicine research program in the Southeast Minnesota region. We will seek input from an Advisory Board with broad representation including old, middle-aged and young subjects. We will conduct a series of engagement efforts and seek feedback regarding research priorities and expectations related to genomic medicine, informed consent for genomic research, data sharing, and return of actionable results; (b) Create a biobank of 1000 individuals that includes plasma, DNA, and RNA. Clinical variables, health drivers (including occupation, activity levels, sleep and diet) and non-clinical factors will be obtained from the electronic health record (EHR) and from surveys and measures that ascertain risk factors for cardiometabolic diseases, family history, and lifestyle factors. Specific Aim 2. (a) Perform population genetic analyses of WGS (30x) data, to investigate the demographic history of the Southeast Minnesota population. We will use a graph genome reference for more accurate variant calling, and will assess demographic history including ancient/recent admixture, population structure, and signals of polygenic adaptation, using state-of-the art methods; (b) Use population genetics analyses to inform the construction of polygenic scores (PGS) for biologically relevant quantitative traits, starting with lipid levels, height, and body mass index (BMI). Specific Aim 3. (a) Identify actionable variants in medically relevant genes (including the American College of Medical Genetics and Genomics Secondary Findings v3.0 list) as well as select pharmacogenomic variants; we will leverage ClinGen resources for variant curation; (b) Return actionable results based on participant choice and assess near term outcomes using a previously described framework. Long term impact. This grant application will enable a research partnership with the Southeast Minnesota population, create a biobank for ‘omic’ studies, generate insights into human evolutionary history, and catalog actionable genetic information. By conducting comprehensive integrated genotype-phenotype research in a regional sample, the proposed work will extend our knowledge of genomic medicine and provide novel insights that are relevant to using population genetics data to inform construction of polygenic scores for biologically relevant quantitative traits.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Androgen receptor (AR) signaling is a major driver of prostate cancer (PCa) progression. Persistent activation of AR due to aberrant intratumoral androgen synthesis (ITAS) plays pivotal roles in resistance to therapies with the next-generation AR signaling inhibitors (ARSI) such as abiraterone. In castrated males, besides androgens produced by de novo synthesis from cholesterol, adrenal steroids dehydroepiandrosterone (DHEA) and DHEA- sulfate (DHEA-S) are the major precursors for ITAS. 3β-hydroxysteroid dehydrogenase-1 (3βHSD1), encoded by HSD3B1 gene, is a key 3βHSD enzyme that catalyzes the conversion of the adrenal-derived steroids DHEA and DHEA-S to testosterone. CHD1 is an epigenetic ‘reader’ that selectively recognizes methylated histone H3 lysine 4 such as H3K4me3, a transcription active chromatin mark. The CHD1 gene is deleted in 9-11% of both primary and advanced PCa, stressing the relevance of loss of function of CHD1 in PCa pathogenesis and progression. It has been shown that loss of CHD1 promotes AR cistrome redistribution and antiandrogen resistance in PCa cells; however, the underlying mechanism remains elusive. Our preliminary data showed that CHD1 interacts with the SIN3A corepressor protein SIN3A and that loss of CHD1 induces elevation of the total level of pan histone acetylation, increased expression of steroidogenesis genes and ITAS. We also showed that upregulation of steroids genes including HSD3B1 induced by CHD1 loss associated with increased expression of transcription factor HOXC13. Furthermore, we demonstrated that abiraterone treatment partially inhibits growth of CHD1-deficient cells and this effect is abolished by concomitant depletion of HSD3B1. Based on our novel preliminary data, we hypothesize that CHD1 functions as a transcription repressor by interacting with the SIN3A corepressor, mediating transcription repression of the HOXC13 transcription factor and downregulation androgenesis genes. However, loss of CHD1 results in transcriptional de-repression of HOXC13, which in turn induces aberrant expression of androgenesis genes, abnormal intratumoral androgen synthesis, and resistance to ARSI therapies in PCa. Aberrantly elevated 3βHSD1 and increased androgenesis represent actionable vulnerabilities for effective treatment of CHD1-deficient PCa cells. To test these hypotheses, we will determine the molecular mechanism and extent to which CHD1 interacts with SIN3A and modulates histone acetylation and transcriptional outputs in PCa (Aim 1); determine the molecular mechanism and functional importance of HOXC13 in upregulation of steroidogenesis genes and castration-resistant growth of CHD1-deficient PCa (Aim 2); and determine clinical significance and ant-cancer efficacy of pre-clinical therapeutic targeting of the deregulated CHD1-HOXC13-3βHSD1 signaling axis in PCa (Aim 3). Findings from this application will shed new light on the novel transcription repression function of CHD1, the pivotal role of the HOXC13-HSD3B1 axis in aberrant androgen synthesis and castration resistance in CHD1-deficient prostate cancer, and identification of new actionable targets for effective treatment of this subtype of prostate cancer.

NIH Research Projects · FY 2025 · 2024-04

ABSTRACT Durable tumor regression by immune checkpoint blockade (ICB) has been observed in a small cohort of cancer patients, but ICB efficacy varies widely on an individual basis. ICB largely targets cytotoxic CD8+ T cells, but the mechanisms regulating CD8+ T cell responses to ICB are yet to be elucidated. CX3CR1+ CD8+ T cells remain highly cytotoxic and proliferative within the tumor microenvironment and single-cell RNA-sequencing of CX3CR1+ CD8+ T cells from ICB responders exhibited a consistent decrease in expression of Dual Specificity Phosphatase 2 (DUSP2), a nuclear phosphatase, compared to CX3CR1- CD8+ T cells. Peripheral human CD8+ T cells with DUPS2 knockdown (KD) increased tumor cytotoxicity in vitro, whereas overexpression (OE) trended towards decreased secretion of effector molecules, Granzyme B and Interferon gamma. Recent reports have additionally documented increased in vivo anti-tumor capacity in DUSP2 KO mice and have loosely made a connection between DUSP2 and CX3CR1 expression6, but the role of DUSP2 in CD8+ T cells and in ICB responses specifically, is a context not yet explored. Furthermore, the signaling pathways in human primary cells have yet to be revealed. Our preliminary data initiated a goal to uncover DUSP2’s mechanistic role in ICB response in hopes of pinpointing therapeutic targets that may improve ICB response rates. This proposal will investigate the hypothesis that DUSP2 expression in CD8+ T cells negatively regulates effector function in response to ICB. The proposed specific aims of this proposal are to 1) characterize the impact of DUSP2 expression in CD8+ T cells on ICB response and 2) determine the regulatory mechanisms of DUSP2 transcription in CD8+ T cells and the downstream pathways modulated by DUSP2. To determine the impact of DUSP2 on ICB response, tumor size and kinetics, as well as CD8+ T cell phenotype will be analyzed in CD8+ T cell Dusp2 conditional knockout (cKO) mice receiving tumor and ICB injection. The DUSP2 signaling network will be uncovered by promoter pull-down assay, single-cell profiling, and RNA-sequencing analyses of human CD8+ T cells with DUSP2 KD or OE. Identified pathways will be modulated (blocked or activated) in human CD8+ T cells by in vitro administration of pathway inhibitors and agonists. DUSP2 expression will be subsequently measured by quantitative reverse transcription PCR, western blot, and flow cytometry and CD8+ T cells will be phenotyped by flow cytometry and included in tumor co-culture cytotoxicity assays. The proposed in vivo experiments will expose DUSP2’s role in regulating ICB response. Uncovering the DUSP2 interactome in human CD8+ T cells can provide a novel and translationally significant opportunity to improve ICB response in oncology patients with limited therapeutic alternatives.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Prostate cancer (PCa) is the most commonly diagnosed malignancy and effective treatment of castration- resistant PCa (CRPC) remains the major challenge in clinic. SPOP is a substrate-binding adaptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex and the SPOP gene is mutated in 10-15% of primary and non- castrate metastatic PCa. Notably, approximately 50% of SPOP-mutated PCa remain resistant to androgen receptor (AR) pathway inhibitors such as enzalutamide (Enza) and therefore there is an urgent need to elucidate the underlying mechanism of resistance and devise new treatment strategies. Our preliminary studies demonstrated that SPOP mutation (SPOPmut) co-occurred with aberrant activation of the MAPK pathway (MAPKalt) such as genomic alterations in the components of the RAS/RAF/MEK/ERK signaling axis and that two lesions cooperated to induce prostate oncogenesis and progression in mice. We found that SPOPmut/MAPKalt PCa cells are castration-resistant in culture and in mice. Mechanistically, we demonstrated that SPOP mutation induced stabilization of GLP and G9a proteins and augmented MAPKalt-induced expression of DNA methyltransferase DNMT1, thereby promoting DNA hypermethylation and epigenetic silencing of a group of tumor suppressor genes (TSGs), including AR-interacting proteins such as KDM6A. We confirmed hypermethylation in KDM6A gene locus and its downregulation in TCGA and SU2C PCa patient samples and in cultured PCa cells with high-level ERK1/2 phosphorylation (MAPKalt). We further showed that KDM6A interacts with AR and that SPOP mutation increases AR protein methylation in a manner dependent on KDM6A. We showed that co-treatment with MEK inhibitor re-sensitized SPOPmut/MAPKalt PCa cells to Enza. Based on these novel preliminary data, we hypothesize that SPOP mutation cooperates with aberrantly activated MAPK pathway to induce aberrant GLP/G9a protein stabilization and DNMT1 upregulation, which in turn promote DNA hypermethylation at the loci of TSGs such as KDM6A, increased AR protein methylation, AR signaling reprograming, and castration-resistant progression of PCa, thereby representing a viable target to overcome the antiandrogen resistance in SPOPmut/MAPKalt PCa. We will determine the molecular mechanism and extent to which KDM6A regulates AR protein demethylation and AR activities in SPOPmut/MAPKalt PCa cells (Aim 1), determine molecular mechanism underlying the deregulations of the GLP/G9a/DNMT1-KDM6A axis and their impact on castration-resistant growth of SPOPmut/MAPKalt PCa cells (Aim 2), and determine the mechanism, disease relevance and preclinical therapeutic targeting of functional cooperation between SPOP mutation and aberrant activation of the MAPK pathway in PCa (Aim 3). Findings from this innovative proposal will not only shed new light on our understanding of the tumor biology of SPOP mutations and molecular mechanisms of antiandrogen therapy resistance in SPOP mutated PCa, but also lead to identification of new therapeutic targets or strategies for effective treatment of PCa, thereby influencing the PCa field both scientifically and clinically.

NIH Research Projects · FY 2025 · 2024-04

ABSTRACT Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers and is the 8th leading cause of cancer death in the United States. In addition to near ubiquitous loss of VHL, the landscape of ccRCC is dominated by mutations in epigenetic regulators SETD2, PBRM1, and BAP1. The epigenome is profoundly disrupted in ccRCC with defects in epigenetic marks on DNA, RNA, and histones, culminating in aberrant gene expression. Mutations in the SETD2 histone H3 lysine 36 trimethylase occur in ~25% of primary ccRCC but increase to >60% in ccRCC metastases. Completion of TCGA enabled identification of actionable mutations in virtually every solid tumor and those profiles now drive treatment decisions. One major exception, however, is RCC, where the current standard of care, checkpoint inhibitor and anti-VEGF therapy, does not account for ~50% of RCCs having mutations in chromatin regulators like SETD2. Many ccRCCs display heterogeneous and subclonal intratumor SETD2 mutations, but we do not understand how SETD2 loss deregulates cell growth and what pathways it works through. After first-line therapy, response rates are 20%, highlighting the need to understand and target drivers of metastasis like SETD2. A recent novel finding linked SETD2 activity to the targeting of N6-methyladenosine in mRNA, a key mechanism for controlling many RNA metabolic processes, which has also been shown to be deregulated in cancer and contribute to metastatic processes. Our preliminary data demonstrate that SETD2 loss leads to large-scale re-localization of m6A and an enhanced sensitivity to an m6A inhibitor that is entering clinical trials. This SETD2-m6A link has not been explored in a ccRCC context but could represent a novel SETD2 effector mechanism and drug target in ccRCC. We explore this relationship for the first time with this pilot R21 study, making it directly relevant to NOT-CA-23-060, RNA Modifications in Cancer Biology. Our proposal is significant because ~30% of patients diagnosed with clinically localized RCC experience metastatic progression, and once metastatic disease develops only ~7% survive >5 years. Our central hypothesis is that loss of SETD2 drives aggressive ccRCC in part through deregulated m6A targeting that promotes a pro-metastatic expression program. Our proposal is expected to develop relevant ccRCC cell line models and define the links between SETD2/H3K36me3 and METTL3/m6A in ccRCC through the following specific aims. In aim 1, we will map H3K36me3, m6A, METTL3, and expression patterns using RIP-/ChIP-/RNA-seq in novel SETD2 isogenic renal cell line models. In aim 2, we will perform integrative analysis of omics data to uncover genes/pathways impacted by SETD2 loss in an m6A-dependent manner. Finally, in aim 3 we will genetically and pharmacologically modulate m6A in SETD2 isogenic cell lines and define cancer-relevant cellular growth phenotypes. This work is expected to positively affect human health by yielding a new understanding of the mechanistic underpinnings of SETD2 driven cell growth deregulation and uncovering new ways to therapeutically target this mutation.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic cause of kidney failure. It affects ~1 in 500 to 1,000 live births, with ~50% of patients needing dialysis or renal transplantation by age 60 years of age. Mutations in PKD1 and PKD2 genes account for >90% of ADPKD cases, and involves reduction/loss of functional ADPKD proteins, polycystins (PCs), in the sensory organelle, primary cilium. Growing evidence suggests that enhancing the ciliary dosage and/or function of PCs could slow down ADPKD progression. Thus, understanding mechanisms regulating the compartmentalized availability and functionality of PCs in primary cilia is vital for developing more effective and safer treatments, as the current FDA-approved drug, Tolvaptan, has limited benefits and significant side effects. The long-term goal of our research is to understand how the anti-cystogenesis function of PCs is compartmentalized in primary cilia and how it is specifically regulated. We have identified novel players involved in the ciliary trafficking and function of PCs. The current proposal addresses three questions: Aim 1, Investigate how the HYLS1-PIPKIg axis regulates the ciliary base docking of vesicles carrying PCs. Aim 2, Delineate how the NEK8-centered inversin compartment enables the ciliary entry of PCs. Aim 3, Determine the pathogenic impact of GPR161-mediated cAMP synthesis and PKA activation in primary cilia when PCs are depleted. We will employ gain-of-function, loss-of-function, and suppression-rescue approaches by applying gene editing tools in renal epithelial cell models carrying ADPKD-related mutations. Fluorescence microscopy including super- resolution and live-cell imaging will be used to analyze protein and lipid localization, level, and dynamics in primary cilia. We will determine whether changing the ciliary functional level of PCs can affect renal cystogenesis in Pkd1RC/RC mice that mimic the adult onset ADPKD condition. With this proposal, we aim to identify key molecules that regulate the functional level of PCs in cilia and their roles in ADPKD pathogenesis. These results may uncover novel molecules or pathways that can be targeted for PKD treatment. In the long term, our studies will inspire future research on enhancing the functionality of PCs in cilia to counteract the pathogenic defects in ADPKD.

NIH Research Projects · FY 2025 · 2024-03

ABSTRACT Monoclonal B-cell lymphocytosis (MBL) is a precancerous state affecting 8-10 million adults with no existing cancer control strategies. As a precursor to chronic lymphocytic leukemia (CLL), MBL is characterized by the presence of monoclonal B-cell clones in the peripheral blood of otherwise healthy individuals. Early evidence supports that MBL increases risk of adverse clinical outcomes, including progression to CLL requiring therapy, development of other hematological and solid cancers, hospitalizations due to serious infections, and reduced humoral immune response to vaccinations. However, the scale and diversity of MBL studies is currently limited by the need for flow cytometric analysis of peripheral blood mononuclear cells to diagnose MBL. These biospecimens are rarely available in large biobanking or cohort studies that could otherwise allow more comprehensive and well-powered investigation of the causes and consequences of MBL, especially in populations of diverse ancestries. We propose to overcome these limitations by developing and validating a strategy to predict MBL status (Aim 1). In brief, we propose using existing genotyping array data, that is readily available in many biobanks, to identify mosaic chromosomal alterations (mCAs), which affect large segments of DNA and include gains, losses, and copy number-neutral loss of heterozygosity events. Recent work in biobank studies has established mCAs as a potent risk factor for lymphoid malignancies but MBL is also a major risk factor of lymphoid malignancies, and thus the question of how mCAs relate to MBL is unknown. We have collected the largest cohort of individuals with MBL and genetic data, and in our preliminary analyses, we found that autosomal mCAs are associated with risk of MBL with an odds ratio of 50.5 (95% confidence interval 36.5-70.6, P = 1.34x10-152) with high discrimination. Aim 1 will validate these results, develop a predictive model with other known risk factors for MBL, and then examine generalizability of the model to individuals with strong family history of lymphoid malignancy and to individuals of African ancestry. We further hypothesize that mCAs are drivers of B-cell clone growth. In Aim 2 we will be the first to quantify the incidence of mCAs and the change in cell fraction of mCAs over time from serial peripheral blood samples that have also been screened for MBL. We will then evaluate the relationship between mCAs and growth trajectories of B-cell clones in individuals with MBL. Successful completion of these aims will unlock the ability to study MBL in the largest cohorts in the world, enabling future studies of MBL at an unprecedented scale. As we continue to improve understanding of the consequences of MBL, risk stratification of individuals with MBL will be essential. This proposal will also begin to enable such stratification by characterizing MBL clonal trajectories. Together, these aims will catalyze future studies of MBL to improve understanding of to inform etiologic factors and risk stratification of this prevalent precancer for eventual cancer control.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Advanced age renders humans susceptible to cancer, fatal infection, neurodegeneration and cardiovascular disease. Age-related immunodeficiency combines loss of protective immunity with gain in tissue inflammation, indicating the complex restructuring of the innate and adaptive immune system with age. An informative model system for immune aging is the autoimmune disease rheumatoid arthritis, in which T cells age prematurely by about 25 years and are key effector cells in tissue-destructive inflammation. In patients with rheumatoid arthritis, CD4+ T cells transition into pro-inflammatory effector cells due to defective mitochondria, resulting in poor ATP production, citric acid cycle reversal, and mitochondrial DNA seepage. In preliminary studies, we have discovered that mitochondrial malfunction is associated with the leakage of N-terminal formyl-methionine (f-Met) from the mitochondrial matrix, eliciting a strong innate immune response in surrounding macrophages and stromal cells. f-Met is the first amino acid in all bacterial and mitochondrial proteins and, as a bacterial motif, is a powerful danger-associated molecular pattern (DAMP) triggering innate immunity. CD4+ T cells from older individuals leak f-Met due to inappropriate opening of the mitochondrial permeability transition pore (mPTP), a high conductance channel that secures the containment of solutes in the mitochondrial matrix. We found that inappropriate pore opening and f-Met leakage have profound consequences for T cell differentiation and effector functions. Specifically, we have placed f-Met release across the inner mitochondrial membrane upstream of the T cell’s proteolytic machinery; with cytosolic f-Met minimizing lysosomal degradation and optimizing exosome formation. Essentially, leaky mitochondria turn f-Met-secreting older T cells into exosome superproducers. Here, we propose that the mitochondrial DAMP f-Met is a biomarker of T cell aging and a drugable target to mitigate immune aging in older populations. To pursue this hypothesis, we will map the molecular defects causing N-formyl-methionine leakage in CD4+ T cells of older individuals (Aim1), determine how cytosolic f-Met regulates lysosomal function and exosome generation (Aim2), and define the pro- inflammatory functions of f-Met-induced exosomes in T cell-macrophage and T cell-fibroblast interaction (Aim3). To leverage molecular understanding of mitochondrial abnormalities in aged T cells, we have assembled a series of targeted small molecule reagents that we will test for their ability to suppress and reverse pro- inflammatory T cell effector functions in vivo (Aim4). This proposal aims to define and manipulate biological processes underlying age-related mitochondrial failure with the goal to ameliorate or delay aging-induced tissue inflammation and develop molecular cues of mitochondrial aging into actionable biomarkers of immune aging.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Millions of people worldwide are affected by liver diseases. Some of these individuals progress to acute or acute- on-chronic liver failure where liver transplant becomes the only recourse. Given the shortage of transplantable organs, over >40% of patients on the waitlist do not receive a liver transplant in a timely manner. This motivates a strong interest in developing bioartificial liver (BAL) systems that may be used as a bridge-to-transplantation or a bridge-to-recovery for patients with liver failure. BAL systems are populated with liver cells (hepatocytes) that perform a wide array of liver functions (e.g. protein synthesis and glucose regulation) in addition to detoxifi- cation. Ideally, BAL systems should contain human hepatocytes, however, because such cells are typically derived from cadaveric organs, their availability is limited. Use of xenogenic (e.g. porcine) hepatocytes is com- mon but less than ideal because of the potential for zoonotic transmission of diseases. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), generally known as human pluripotent stem cells (hPSCs), may be expanded indefinitely and differentiated into any desired cell type, including hepatocytes. hPSCs hold incredible promise as the source of cells for many therapeutic strategies, including BAL systems. There are, however, distinct challenges that remain to be addressed: 1) stem cell (SC)-hepatocytes need to be differentiated at scale to achieve sufficient liver mass (~ 200 gr) to treat a patient, and 2) functionality of SC- hepatocytes needs to be improved to approach that of adult hepatocytes. Our project will take a step toward addressing these challenges by developing bioactive heparin-containing microcapsules that will carry hPSCs, will be loaded with growth factors for local and sustained delivery, and will enable high-density suspension cul- tures. Broader Impact: This project will enable scalable differentiation of hPSCs through the use of microencapsula- tion. Microcapsules will be used to deliver inductive cues for stem cell differentiation and will protect cells from mechanical damage in a stirred bioreactor. The use of microcapsules will allow us to increase the rate of oxygen and nutrient delivery to the cells and will enable higher density cultures. While the present project focuses on using encapsulated SC-hepatocytes in a BAL system, immunoisolation and other survival advantages offered by microcapsules will also enable future applications for cell transplantation.

NIH Research Projects · FY 2026 · 2024-03

SUMMARY Prostate cancer (PCa) is the most frequent malignancy and a leading cause of cancer death in men in the United States. Once PCa progresses to an advanced metastatic therapy-resistant stage, it becomes a lethal disease. To date, the understanding of mechanisms involved in the pathobiology of lethal PCa remain limited. Transcriptional rewiring of the cancer cell plays a fundamental role in enabling disease progression. GATA2 is a master regulator transcription factor that participates in prostate development and PCa pathogenesis. In PCa, GATA2 levels increase during disease progression and are elevated in almost all (~100%) of metastatic therapy- resistant tumors. We and others have shown that GATA2 is essential to enable androgen receptor (AR) transcriptional activity, as well as control the transcription of other genes independently of AR that participate through distinct mechanisms in the pathogenesis of lethal PCa. Most relevant to this proposal, our preliminary studies point to a key role of GATA2 in disease progression and in conferring plasticity to PCa cells. Indeed, our computational and functional studies suggest that elevated GATA2 levels increase the metastatic potential of PCa cells by transcriptionally regulating a subset of genes regulating the motility and invasiveness of PCa cells. Moreover, our transcriptomic single-cell analysis and functional studies indicate that PCa cells expressing high levels of GATA2 develop an undifferentiated stem cell-like phenotype. In addition, we have identified a novel therapeutic that significantly disrupts GATA2 transcriptional activity and demonstrates in vivo anti-cancer efficacy. Based upon these novel preliminary data, we hypothesize that transcriptional rewiring caused by elevated GATA2 expression contributes to PCa progression to lethal disease stages and that this mechanism can be targeted. We will investigate this hypothesis as follows: In Aim 1, we will define the extent and mechanisms by which GATA2 elevation enhances PCa metastasis, focusing on defining a GATA2 motility regulated molecules, as well as determine the role of GATA2 in disease progression using a transgenic conditional knock-in mouse model. In Aim 2, we will determine the impact of GATA2 in PCa cell plasticity and disease progression at the single-cell level, focusing on functionally validating the cistrome changes induced by increased GATA2 levels. We will also assess in human samples the clinical relevance of the GATA2-induced stem-like phenotype during disease progression in circulating tumor cells. Finally, in Aim 3, we will investigate the efficacy of targeting GATA2 in combination with standard therapy in pre-clinical PCa models. At its completion, results from this innovative proposal will not only enhance our understanding of how elevated GATA2 expression transcriptionally rewires the cancer cell and contributes to the pathobiology of lethal PCa, but will also have significant impact by identifying new therapeutic strategies to treat this lethal disease.

NIH Research Projects · FY 2025 · 2024-03

SUMMARY Intraductal papillary mucinous neoplasm (IPMN) is one of the two most common precursor lesions leading to the development of pancreatic ductal adenocarcinoma (PDA). IPMNs comprise a heterogeneous group of tumors with a wide range of grades and histotypes, and the emergence of single-cell RNA sequencing (RNA- seq) and multiplex digital spatial profiling have characterized unique cell populations, including dysplastic epithelial and immune cells, within the heterogeneous tumor microenvironment that carry signature gene expressions, which could be used as markers for disease progression. However, further studies are needed to delineate the biological interaction between epithelial subtypes and immune cells that drive IPMN lesion development. Genetically engineered mouse models (GEMMs) for IPMN have been established using oncogenic mutations and disruption of tumor suppressors found in human IPMN samples. Analyses of these IPMN GEMMs have provided important mechanistic insight into the underlying progression of these precancerous lesions to invasive PDA. Our group has developed a novel KNGC model (KRasG12D, nuclear GSK-3β, PDX1-Cre) resulting in the development of IPMN through the retention of a ductal progenitor pool defined by being AGR2+/AQP5+/DBA-, which are also detected in the IPMN KGC (KRasG12D, GNASR201C, Cre) model and patient samples with IPMN. Additionally, KNGC animals show progressive desmoplasia beginning as early as 4 weeks of age with increased immune cell infiltration, which we hypothesize is due to the high expression of CX3CL1 (fractalkine), a chemokine involved in the recruitment of CX3CR1-expressing monocytes, which are known to promote fibrosis in various disease models upon differentiation into type 2 macrophages. Indeed, our preliminary data from CX3CR1 heterozygous (CX3CR1GFP/+) and homozygous (CX3CR1GFP/GFP) knockout in the KNGC model shows an enrichment of GFP signal in M2-like macrophages in the IPMN lesions of the KNGCXGFP/+ model, whereas KNGCXGFP/GFP animals had a paucity of M2-like macrophages, substantially reduced desmoplasia and impaired development of IPMN. It is our central hypothesis that the ductal expression of CX3CL1 results in the accumulation of M2-like macrophages, which facilitate the development and progression of IPMN in KNGC mice. We will address our hypothesis through the following specific aims: (1) Determine the role of CX3CL1 in promoting IPMN development and desmoplasia in KNGC mice, and (2) Determine the role of CX3CR1 in promoting immune suppressive environment in KNGC mice. Altogether, this project will provide a mechanistic understanding of the CX3CL1- CX3CR1 axis in the generation of an immune/stroma microenvironment facilitating IPMN development. The information obtained in pursuit of the aims might lead to the identification of novel biomarkers that could be used to monitor or treat patients with IPMN.

NIH Research Projects · FY 2026 · 2024-03

Abstract Autoimmune diseases can occur in nearly any part of the body, and pose a significant problem in human health, including both systemic autoimmunity and tissue-specific autoimmunity. 1.25 million Americans have type 1 diabetes (T1D), where the immune system destroys insulin-producing beta cells. Although exogenous insulin can control blood glucose levels in patients with diabetes, glucose homeostasis is difficult to maintain and serious long-term complications including nephropathy, retinopathy, and peripheral neuropathy are far too common. Since the first procedure using the Edmonton protocol in 1999, islet transplantation has established itself as a promising therapy for patients with longstanding T1D, and beta cell replacement using replenishable sources like human embryonic stem cell-derived beta cells has the potential to become curative. Islet transplantation requires a drug regiment to suppress the immune response from allorejection as well as autoimmune attack against pancreatic b cells, resulting in chronic immunosuppression with its own severe complications, exposing patients to risk of infection and malignancies. Immunotherapies that provide local inhibition of immune response to the graft, without systemic immune inhibition, are a critical need. Immune cells have receptors that can either activate or suppress an immune response. Sialic acids incorporated into glycans have the ability to bind to a family of predominantly inhibitory receptors on the surface of many immune cells. We have shown that the sialic acid transferase ST8Sia6 generates ligands for the inhibitory receptor Siglec-E, which is expressed on innate immune cells. In addition, our work demonstrated that ST8Sia6 expression on tumor cells leads to enhanced growth and protection from the immune response. Here, we generated a novel line of mice in our laboratory where ST8Sia6 is constitutively expressed in pancreatic b cells in the NOD mouse model of Type 1 diabetes (“NOD bST mice”). NOD bST mice are strongly protected from the development of diabetes; only 6% of NOD bST female mice became diabetic as compared to 60% of NOD female littermate controls. Strikingly, NOD bST mice possessed a durable tolerance towards b cells, as protection was maintained after ST8Sia6 expression was inhibited with doxycycline. The focus of this proposal is to understand the changes in the immune response that occur with ST8Sia6 expression in b cells, and whether ST8Sia6 expression on b cells provides enhanced protection from immune attack after transplantation. By doing these experiments in mice, important information will be gained that may be translated to the clinic for islet replacement therapy for patients with T1D.