Ut Southwestern Medical Center

NIH Research Projects · FY 2026 · 2023-05

Project Summary Obesity is a leading risk factor for type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular diseases. A central driver of pathogenesis in obesity-associated disorders is the insufficient lipid-storing capacity of adipocytes and subsequent lipid deposition in extra-adipose organs. The lipid droplet (LD) is the organelle responsible for lipid storage and mobilization in adipocytes. It remains to be elucidated whether proteins and pathways regulating LD structure and function constitute limiting factors governing the lipid-storing capacity of adipocytes, and thus play an essential role in determining one’s susceptibility to obesity-associated disorders. We observed that mice deficient in CLSTN3B, a mammalian adipocyte-specific protein, are more prone to high-fat diet-induced metabolic disorders compared with body weight-matched wild-type mice, whereas the adipose-specific clstn3b transgenic mice display the opposite phenotype. Preliminary evidence shows that CLSTN3B localizes to endoplasmic reticulum (ER)/LD contact sites and ablation of CLSTN3B results in an impaired coating of LDs by phospholipids and proteins. Our overall objectives are to (i) establish the significance of CLSTN3B expressed in white adipocytes to the metabolic phenotype; (ii) reveal the molecular mechanism of CLSTN3B action at the ER/LD contact sites. The central hypothesis is that CLSTN3B enhances the structural and functional integrity of LDs, improves white adipocyte lipid-storing capacity, and contributes to the maintenance of metabolic health under obese conditions; mechanistically, this is achieved by replenishing LD surface phospholipids and promoting the binding of LD-targeting proteins. We will test this hypothesis by pursuing three specific aims: 1) Show that CLSTN3B expressed in white adipocytes is the main contributor to the metabolic benefits upon high-fat diet feeding; 2) Show that CLSTN3B promotes phospholipids transfer between ER and LD; 3) Probe the role of the C-terminal ER luminal segment of CLSTN3B in the formation of ER/LD contacts. For the first aim, we will construct genetic models allowing specific assessment of white adipocyte-derived CLSTN3B. For the second aim, we will design in vitro reconstituted phospholipid transfer assays and examine the functional significance of LD surface phospholipid density. For the third aim, we will use biochemical approaches to identify potential binding partners of the ER luminal C-terminal fragment of CLSTN3B, followed by assessing the significance of such interactions using cellular and animal models. The proposed research is innovative because it dissects the molecular mechanism of a novel protein and explains susceptibility to obesity-associated disorders from a novel perspective. The proposed research is significant because it aims to establish an integrated understanding encompassing interorganelle communication and metabolic physiology at the organismal level. Our long-term goal is to use CLSTN3B as a molecular handle to derive a thorough understanding of ER/LD interactions in the specific context of adipocytes and identify novel therapeutic targets for obesity-associated diseases.

NIH Research Projects · FY 2026 · 2023-05

Interstitial fibroblasts drive prostate branching morphogenesis Douglas W. Strand, PI Chad Vezina, Co-I UT Southwestern Medical Center, Dallas, TX Summary The paracrine factors that drive prostate development and disease are still unknown. We recently discovered a population of interstitial fibroblasts that surround the prostatic urethra of the normal prostate and are sparse in the transition zone. We showed that the characteristic nodules that form in human benign prostatic hyperplasia (BPH) are an expansion of interstitial fibroblasts in the transition zone. Key preliminary spatial transcriptomic analysis of interstitial fibroblasts inside BPH nodules revealed the enrichment of mitogens and morphogens that are normally only expressed during organogenesis. These data represent a breakthrough in our understanding of stromal-epithelial interactions in human BPH and could help solve the long-held hypothesis that BPH is a reawakening of the embryonic signaling that induces prostate development. We will test the hypothesis that paracrine signaling from interstitial fibroblasts drives prostate branching morphogenesis in development and disease with three critical lines of inquiry: 1) spatial transcriptomics analysis of interstitial fibroblasts in human prostate development and BPH; 2) mechanistic analysis of paracrine morphogens and mitogens on adult prostate epithelial proliferation and branching ex vivo; and 3) in vivo analysis of autocrine regulation of prostate epithelial proliferation by the receptors of interstitial fibroblast ligands. Successful completion of our aims will establish a new mechanism of prostate growth that is actionable in clinical trials. Relevance Establishing the paracrine factors responsible for prostate growth holds great promise for identifying novel approaches to medical therapy for the reversal or prevention of hyperplasia.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY / ABSTRACT Rhabdomyosarcomas (RMS) are the most common childhood soft-tissue sarcomas affecting hundreds of patients in the United States annually. Current standard treatments for rhabdomyosarcoma (RMS) patients include chemotherapy, surgery, and /or radiation. However, even with these combinations of therapeis, significant subsets of patients suffer tumor recurrence, relapse, and metastasis, associated with extremely worse prognosis and dismal 5-year survival rate. This remains the major hurdle to improve the patient outcomes with rhabdomyosarcomas. To better understand the mechanisms such as tumor-propagating cells, critical molecular regulators that drives therapy resistance and tumor-relapse in RMS, researchers has employed RMS cell lines, transgenic animal models, and xenograft studies to study the potential tumor-propagating cells (TPCs) for RMS. Yet, little is known about the tumor heterogeneity and cancer cell evolution dynamics in RMS. To dissect the inter-tumoral and intra-tumoral heterogeneity, I have used the single-cell transcriptomics to profile patient-derived samples of RMS. I uncovered distinct cell states in RMS tumors, including proliferation and a mesenchymal-like subpopulations that have higher TPC potential, whereas the differentiated muscle subpopulation that barely transits towards other cell states. With this knowledge, and the innovation of barcode tracing techniques, I propose to dissect molecular mechanisms that contribute to cell state transitions, and concurrently assess the cell phenotypes changes along with its transcripts, proteins, and epigenetics alterations. One class of important and challenging molecules in regulating cancer stemness, evolution post therapies is chromatin regulators, which requires deep sequencing in limited cell line models. The technical innovation of single-cell multiomics, including single-cell RNA, single-cell ATAC, single-cell CUT&Tag, and cell lineage barcode tracing largely decrease the cost and time needed to profile cancer cell evolution along with epigenetic modifications at single-cell levels. With effective collaboration with computational biologists, I hypothesize that EZH2 and its catalytic product H3K27me3 lock RMS cells in the proliferative cell state and inhibit their transition into other differentiated states. To test this hypothesis, I will first assess the role of EZH2 in regulating cell state transitions with barcode tracing and functional stem assays in the context of EZH2 knockdown (Aim 1). Independently, I will also profile the direct targets of EZH2 and histone H3 lysine 27 trimethylation by performing single-cell CUT&Tag, and interrogate mechanism that controls cell state transition (Aim 2). In addition, I will also assess the EZH2 inhibitors in collaboration with chemotherapy and radiation utilizing the unique immune-compromised zebrafish models along with cell line and mouse xenograft studies (Aim 3). The goals of the proposed research are to investigate chromatin regulators in rhabdomyosarcoma samples while also acknowledging the tumor-heterogeneity and cell plasticity in cell state transitions. By achieving these aims, I will illustrate a comprehensive mechanism as to how RMS tumors evolve and how chromatin regulators play critical roles in controlling this process.

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract: Our pioneering research in adolescents and adults born preterm has identified 3 distinct characteristics of the preterm heart that could increase risk for heart failure. These include (1) reduced cardiac size contributing to a blunted cardiac reserve during exercise, (2) increased left ventricular (LV) cardiac fibrosis, and (3) right ventricular (RV) dysfunction relative to the underlying pulmonary vascular disease, or impaired RV-pulmonary vascular (PV) coupling. However, how these findings may progress across the early lifespan is unknown. The objective of this proposal is (1) to develop cardiac growth and function curves using mixed effect quantile regression models to predict cardiovascular trajectories in children and adults born preterm, and (2) to identify risk factors and biomarkers for impaired growth and function. We propose cross-sectional repeated biventricular and pulmonary vascular assessments obtained at baseline and repeated after 2 years in children and young adults born <32 weeks preterm (age 8-30 years; n=150), compared to age-, sex-, and racially-matched term- born controls (n=150). Multivariate models of the natural history of disease will be developed using mixed effect quantile regression to predict growth and function trajectories. Aim 1: Identify novel characteristics that impair cardiac growth from childhood through early adulthood after preterm birth. We will use repeated measures of cardiac structure (e.g. LV end diastolic volume index and LV mass index by MRI) obtained at baseline and after 2 years. Multivariable models using mixed effect quantile regression will be used to develop cardiac growth curves for each sex, adjusting for effects of neonatal and common cardiovascular health modifiers. We hypothesize that preterm females will have a lower cardiac growth trajectory defined as growth at a consistent but lower growth percentile, while preterm males will have a growth failure defined as a progressive fall from a term growth curve. Aim 2: Determine whether biventricular cardiac fibrosis is associated with neonatal characteristics and progressive with chronological age after preterm birth. Using cardiac MRI with late gadolinium enhancement (LGE) and native T1 mapping, we will assess biventricular cardiac fibrosis. We hypothesize that fibrosis scores are elevated in preterm-born children and adults, associate with neonatal resuscitation, and progress with age. Aim 3: Assess whether RV-PV coupling declines with age due to worsening RV function. We will use serial noninvasive measures of RV-PV coupling (MRI RV stroke volume/end systolic volume), RV function (MRI ejection fraction, strain), and pulmonary vascular disease (ECHO tricuspid regurgitant jet velocity, pulmonary artery acceleration time, and pulmonary vascularization) to establish the trajectories of RV and PV disease after preterm birth. We hypothesize that preterm-born individuals demonstrate worsening RV-PV coupling with age due to worsening RV function rather than rising afterload (worsening PV disease), most notable in males and those with bronchopulmonary dysplasia. Study results will provide justification and potential targets for future early intervention studies to promote cardiac growth and preserve cardiac function after preterm birth.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Nuclear envelope proteins are essential for maintaining nuclear architecture, gene expression and chromatin organization. Mutations in nuclear envelope proteins and nuclear lamins cause numerous human diseases, many of which involve in skeletal muscle defects such as Emery-Dreifuss muscular dystrophy (EDMD). Although the genetic mutations responsible for these diseases are known, the molecular mechanisms whereby perturbations in the nuclear envelope cause disease are still not well understood. Moreover, it is still unclear why mutations in ubiquitously expressed nuclear envelope proteins lead to tissue-specific pathogenesis such as striated muscle-specific defects. Our lab recently showed that the nuclear envelope transmembrane protein 39 (NET39) is a muscle-specific regulator of nuclear envelope structure and function. NET39 is downregulated in EDMD patient muscle biopsies, and deletion of Net39 in mice caused nuclear envelope deformations, congenital myopathy and juvenile lethality. Within the nuclear envelope, NET39 interacts with several components of the nuclear envelope such as LEMD2. Our studies of NET39 provide an entry point to unravel the long-standing puzzle of why striated muscle is specifically affected in nuclear envelope related diseases. We hypothesize that NET39 plays a pivotal skeletal muscle-specific role in the pathogenesis of EDMD. The overall goals of this project are to define the functions of NET39 and its interactions with other nuclear envelope proteins in regulating nuclear envelope integrity and gene expression during skeletal muscle homeostasis and disease, and to explore the role of NET39 in EDMD and other laminopathies. Ultimately, we hope to use these insights to develop new therapeutic strategies for EDMD and related laminopathies.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY In recent years, there have been groundbreaking discoveries in the identification and therapeutic targeting of the PD-1/PD-L1 immune checkpoint axis. Lung cancer cells express high levels of Programmed Death Ligand 1 (PD-L1), a critical ligand for PD-1 on T cells. The PD-1/PD-L1 interaction allows tumor cells to directly suppress anti-tumor T cell activity, resulting in immune escape and tumor progression. Despite these advances, there remains a disconnect in patient expression of PD-L1 and treatment response. This underscores the critical need to understand mechanisms of PD-L1 upregulation, identify mechanisms of resistance to PD-1/PD-L1 therapy, and identify other immune checkpoints or pathways to pursue clinically in combination with this therapy. In response to tumor microenvironment stresses, such as hypoxia, heme deprivation, and amino acid starvation, cancer cells activate the integrated stress response (ISR). ISR activation allows cancer cells to escape these stresses through inhibition of global protein synthesis and increased translation of select mRNAs. The ISR has been shown to promote tumorigenesis, yet the role of the ISR in the translational control of immune checkpoint proteins has not been fully investigated. We recently demonstrated that ISR activation leads to potent induction of PD-L1 in non-small cell lung cancer (NSCLC) and suppression of anti-tumor immunity in vitro and in vivo, and we have new evidence that another immune checkpoint, CD155 (Cluster of differentiation 155), is induced upon ISR activation in NSCLC cells simultaneously with PD-L1. Our central hypothesis is that ISR activation causes tumor cell immune escape through translation of both PD-L1 and CD155. Guided by strong preliminary data, we will test this hypothesis by pursuing three specific aims: 1) Elucidate the mechanisms through which ISR activation promotes translation of CD155; 2) Determine the effect of ISR modulation on immune cell responses; 3) Examine the therapeutic efficacy of ISR inhibition in combination with PD-1 blockade and/or TIGIT (CD155’s immune cell receptor) blockade in mouse models. We will employ translational studies including luciferase reporter assays and ribosome profiling to dissect the mechanisms of ISR mediated PD-L1 and CD155 translational control in NSCLC cells. To determine the impact of ISR activation on immune cell responses, we will measure immune cell responses in co-culture studies and immunocompetent mouse models upon ISR activation. Finally, we will utilize mouse models and ISR inhibitors to determine whether ISR inhibition can suppress tumorigenesis by promoting an immune response and whether this can synergize with existing immune checkpoint therapies (Fig 1, model). Our proposed research is significant, because it will 1) uncover new regulatory circuits that govern immune checkpoint protein expression, 2) illuminate how insults experienced by cancer cells in the tumor microenvironment modulate the responses of immune cells, and 3) our studies will provide proof-of- concept for the development of new combination therapies for lung cancer.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Nonalcoholic steatohepatitis (NASH), an aggressive form of nonalcoholic fatty liver disease (NAFLD), is characterized by hepatic lipid buildup, liver damage, inflammation, and fibrosis. The prevalence of NASH has skyrocketed during the past decade, making it the leading cause of liver-related morbidity and mortality worldwide and a primary reason for liver transplantation. Dietary obesity, the trigger of NAFLD, induces excessive lipid accumulation in the liver, causing hepatocyte death and subsequent release of host-derived damage-associated molecular patterns that in turn activate liver macrophage to ignite hepatic inflammation. Such inflammation is featured by chronic production of proinflammatory cytokines, including TNF, IL-6, and IL-1b. Several landmark studies in the past decade have collectively shown that chronic liver inflammation is the key switch mediating simple steatosis transition into NASH. However, how dietary obesity promotes the establishment of chronic inflammation in the liver remains elusive. Recently, multiple single-cell transcriptomic studies revealed the emergence of a triggering receptor expressed in myeloid cell 2 (TREM2)-expressing macrophage population that is highly enriched in patients with NASH, cirrhosis and hepatocellular carcinoma. To study the role of macrophage TREM2 in NASH pathogenesis, we generated myeloid cell-specific Trem2 knockout mice and subjected them to a western diet-induced NASH model. We discovered that macrophage TREM2 protects mice against NASH development. Of note, we unexpectedly found that despite its mRNA being continuously upregulated throughout NASH progression, TREM2 protein only increases in simple steatosis but almost gets eliminated at NASH. We further demonstrated that the dramatic decline of TREM2 protein in NASH is due to proteolytic cleavage of full-length TREM2 present on macrophage surface. The overall objective of this proposal is to comprehensively investigate (1) how TREM2 expression is regulated during NASH pathogenic progression, (2) what TREM2 does in macrophages to restrict NASH development, and (3) whether blocking TREM2 cleavage can inhibit NASH progression. To achieve this goal, we will pursue the following three specific aims. In Aim 1, we will decipher the molecular mechanism by which TREM2 is dynamically regulated during NASH progression. Specifically, we will identify key signaling pathways responsible for TREM2 up- and down- regulation at simple steatosis and NASH stages, respectively. In Aim 2, we will test whether TREM2 plays a key role in macrophage efferocytosis of lipid-laden apoptotic hepatocytes and thereby restrict chronic liver inflammation and NASH development. Lastly, in Aim 3, by utilizing a cleavage-resistant Trem2 knock-in (Trem2- IPD) mice, we will perform a proof-of-concept in vivo test to determine if blocking TREM2 cleavage to restore macrophage efferocytosis can inhibit NASH. Completion of this study will not only provide much-needed mechanistic insights explaining how prolonged hypernutrition results in chronic liver inflammation, but also will establish a concrete foundation for designing anti TREM2 cleavage approaches to treat NASH.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract This application seeks to establish a T35 NIAAA Summer Research Program to enhance medical student research training in alcoholism and the effects of alcohol abuse at UT Southwestern Medical Center. The proposed program will provide short-term training opportunities for medical students in basic and clinical biomedical research focused in areas supported by the NIAAA. Based on our extensive experience gained from other NIH Research Training grants, we will develop a short-term training program for those medical students enrolled at UT Southwestern as well as outstanding applicants from medical schools across the country. Building upon our collective experience in the identification and pairing of students and mentors, we anticipate that these experiences will continue to enrich the research background of participating medical students with an interest in psychiatry and alcohol abuse. Besides the mentor-based teaching within the laboratories and clinics, trainees will also receive a comprehensive course in Research Methodology with NIAAA-specific journal clubs and participation in a works-in- progress psychiatry lab seminar series. The program described in this application will employ the outstanding faculty and resources already in place at UT Southwestern that are focused in areas of NIAAA interest. This program will serve as a focal point to further enrich medical student research activities at UT Southwestern and will enhance existing elements of NIAAA-funded research on campus.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMARY/ABSTRACT This proposal presents a five-year research and training program to establish Laurel Y. Lee, M.D., D.Phil. as an independent, R01-funded physician-scientist in academic cardiology with expertise in immune modulation of endothelial metabolism in atherosclerosis. This unique scientific focus combines Dr. Lee’s doctoral training in T- cell immunology with her subsequent clinical and research fellowships in cardiovascular medicine at the Brigham and Women’s Hospital (BWH) and Harvard Medical School (HMS). She is currently an Associate Physician in the Division of Cardiovascular Medicine and an Instructor in Medicine at BWH/HMS. Coronary artery disease remains a leading cause of mortality and morbidity worldwide. While endothelial dysfunction is known as a precursor to atherosclerosis, how altered endothelial metabolism contributes to atherogenesis remains incompletely understood. The principal investigator’s long-term goal is to define how local immune activation alters endothelial metabolism and contributes to atherogenesis. As a first step toward achieving this goal, she recently discovered that interferon gamma (IFN-γ), a T-cell cytokine abundant in human atheroma, impairs endothelial glucose metabolism and activates fatty acid oxidation in primary human coronary artery endothelial cells (Lee et al., Circulation, 2021). These metabolic derangements were associated with proatherogenic endothelial phenotypic changes, raising the central hypothesis that IFN-γ-induced endothelial metabolic reprogramming forms a novel mechanistic basis for accelerated atherosclerosis. This hypothesis will be tested through the following aims: (1) Define the effect of IFN-γ on endothelial fuel utilization, (2) Establish the mechanistic link between endothelial metabolic reprogramming and endothelial phenotypic changes, and (3) Define the changes in endothelial metabolism in a mouse model of immune exacerbated atherosclerosis in vivo. Using the cutting-edge approaches including metabolomics, vascular phenotyping, single-cell technology, and a mouse model of atherosclerosis, the principal investigator will acquire new skills and expertise in quantitative analyses of metabolism, lipid biology, and in vivo analysis of immune-endothelial interaction in experimental atherosclerosis. These studies, if successful, will establish immune mediated endothelial metabolic perturbations as a novel mechanistic basis for linking pathologic T-cell activation and atherosclerosis and may open new therapeutic strategies. Dr. Joseph Loscalzo, a distinguished vascular biologist with expertise in vascular metabolism, redox biochemistry, and systems biology will serve as the principal investigator’s primary research mentor. An advisory committee of physician-scientist experts in cellular metabolism and atherosclerosis research will provide further scientific and professional development guidance and assessment of her progress. In summary, Dr. Lee has created a superb environment and mentoring team to develop her unique niche in immune modulation of endothelial metabolism. The proposed research, training plans, and outstanding environment at BWH, HMS, and MIT will propel her transition to an independent investigator and a leader in vascular research.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY This K23 application supports Dr. Sheila Hegde, a highly promising cardiac imager and early-stage investigator who is committed to investigating the underlying mechanisms by which physical activity (PA) favorably impacts the risk of heart failure (HF) in late-life. Her long-term goal is to be an independent investigator studying modifiable biomarkers of aging and HF risk that will ultimately inform therapeutic intervention. The overall scientific objective of this proposal is to identify biologic pathways underlying the beneficial effects of PA on cardiac function, functional capacity, and HF risk using high dimensional ‘-omic’ data. The main hypothesis is that PA delays age-related changes in cardiac structure and function and mitigates HF risk through biologic pathways that can be identified through high throughput circulating proteomic profiling. The specific aims are to: 1) Employ high-throughput plasma proteomics and genomic data to identify novel molecular pathways underlying longitudinal PA-related changes in cardiovascular structure and function and incident HF (NHLBI ARIC cohort); and 2) Identify molecular pathways underlying the beneficial effect of a structured PA intervention on functional capacity with the use of plasma proteomics in older sedentary adults at high risk of HF. (Brigham and Women’s Hospital-based cohort). These aims will also serve as a vehicle to achieve the following career and learning objectives: 1) Attain advanced skills in novel, data science driven methods for high-dimensional data, particularly in proteomics analysis; and 2) Develop the necessary skills to design, implement, and conduct a clinical trial in order to transition to an independent clinical investigator. These objectives will be accomplished through: 1) structured interactions with a committed and expert multidisciplinary panel of mentors and advisors; 2) focused didactic coursework in study design and statistical approaches relevant to ‘-omic’ data; and 3) progressive participation in multi-institutional working groups and committees for national multicenter studies, dedicated mini-courses and meetings relevant to PA and multi ‘-omic’ analyses, and national cardiovascular professional organizations. These activities will also act as a framework to grow her leadership skills and to develop external collaborations. The results of the proposed early career development award will provide further insight into the underlying cardiovascular mechanisms by which physical activity impacts age-related changes in cardiac structure and function, functional capacity, and heart failure (HF) risk and serve as the necessary foundation for Dr. Hegde to transition to an independent investigator.

NIH Research Projects · FY 2026 · 2023-03

Abstract Preterm birth due to an ascending infection accounts for 25-40% of spontaneous preterm births. Disruptions in the cervical epithelia that perturb its barrier and immune function is a risk factor for an ascending infection. Using single cell transcriptomic and spatial approaches, epithelia subpopulations were identified in cervices from nonpregnant mice and mice at gestation days 6, 12, 15, 18 and in labor. Unique to pregnancy was the expansion of two populations of secretory goblet cells which produce a distinct mucus network and immune surveillance factors. In the current proposal, we aim to identify the lineage of these secretory cells which express markers of both squamous and columnar epithelia, and to define the progesterone and estrogen dependent gene regulatory networks that regulate proliferation and differentiation of the two goblet subtypes. Using single cell datasets from mice with epithelial barrier defects or mice with exposure to lipopolysaccharide to induce inflammation, we will define perturbations in epithelial subtype functions that contribute to ascending infection risk. Finally, the expression of olfactomedin 4, one immune surveillance factor upregulated in the goblet cells in pregnancy, will be measured in cervico-vaginal fluid from women in each trimester of a term or preterm pregnancy to determine its utility to monitor cervical epithelial cell health and risk of spontaneous preterm births.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT The Mechanisms of New-Onset Autoimmunity-Longitudinal Immune Systems Analysis (MONA-LISA) will use clinical data and biospecimens obtained during performance of the clinical trial, Study of Anti-Malarials in Incomplete Lupus Erythematosus (SMILE, NCT03030118) to investigate immunological mechanisms that propel individuals who have asymptomatic or minimally symptomatic autoimmunity towards a definite diagnosis of systemic lupus erythematosus. These data and specimens are a unique resource, representing longitudinal clinical assessments, patient-reported outcomes, DNA, RNA, serum, plasma, peripheral blood mononuclear cells and urine obtained in a standardized manner before, during, and after the progression from Incomplete Lupus Erythematosus to Systemic Lupus Erythematosus (SLE) – an observation that occurred in twenty percent of the SMILE participants available for study. The overall objective of the study is to learn the underlying immunological, genomic, and metabolic differences present in individuals who progress to SLE compared to those that do not progress with goals to both develop better diagnostic and prognostic tools for health care providers as well as describe novel therapeutic targets so that individuals with early features of autoimmunity can be prevented from progressing to a state of organ damage and disability. The Specific Aims of MONA-LISA include: 1) Obtain a comprehensive, multiplex analysis of the peripheral blood immunophenotype of progressors and non-progressors in the SMILE cohort using a novel 137-plex CITE-seq analysis, sn-ATAC-seq, sc-RNA-seq and T and B cell repertoire determination to create a robust, quantitative atlas of cell-specific mRNA and epigenomic profiling in PBMC of these individuals. 2) Perform targeted genomic sequencing to categorize regulatory and structural variants of SLE risk loci to tie together the transcriptomic and epigenomic data and create novel risk assessments based on gene regulation. 3) Explore plasma and serum metabolic and lipid components that can serve as novel features that classify the risk of SMILE participants who progress towards classification with lupus. 4) Lastly, because SMILE enrolled fewer Black individuals than are represented in the epidemiology of prevalent lupus patients, we will perform a focused recruitment of non-European individuals with ILE and compare their multi-omic characterization of immunophenotypes described in Aims 1-3. This will allow more complete and generalizable conclusions to be drawn across ethnic groups and identify any ancestry-specific variations in the risk of autoimmunity progression that can explain the observed differences in lupus epidemiology. When completed, MONA-LISA will provide researchers and health care providers with better tools to predict who will develop lupus and create more effective interventional trials for disease prevention.

NIH Research Projects · FY 2026 · 2023-03

Project summary Anemia is a nearly universal diagnosis in preterm infants, caused primarily by phlebotomy essential for medical care, though also exacerbated by a variety of factors inherent to immaturity in the ex utero environment. When severe enough to be treated with RBC transfusion, clinicians must be aware of the risk of critical adverse effects such as necrotizing enterocolitis (NEC), an inflammatory bowel necrosis characterized by infiltration of macrophage precursor(s), and a leading cause of mortality in those born between 22- and 28-weeks’ gestation. We have recently elucidated the connection between anemia and NEC, specifically, the “leaky gut” presentation characterized by monocytic infiltration, RBC transfusion-associated activation of infiltrated monocytes, and the resulting intestinal mucosal injury. Our long-term objective is to study the anemia-induced immunity changes in the neonatal liver and their contribution to gut mucosal injury during RBC transfusion. Our preliminary studies using our existing pre-clinical murine model of anemia demonstrate that anemia is associated with intestinal recruitment of a unique population of monocytes (CD11bhiLy6Cmid) expressing triggered myeloid receptor 1 (trem1), similarly to monocytes developing in the neonatal liver but unlike those in the bone marrow or spleen. Consistent with this, neonatal anemic liver monocytes displayed greater inflammatory activation to heme (found in stored RBC) than did bone-marrow derived cells. This inflammatory response could be dampened either by the use of anti-trem1 antibody treatment or by silencing monocyte trem1 expression. Taken together, the investigators propose a novel hypothesis that in the setting of anemia, a gut-liver-gut boomerang effect takes place as the leaky gut and associated bacterial translocation during anemia communicate via the portal vein to the liver, triggering the expansion of hepatic leukocyte populations developing in situ which proceed to infiltrate the anemic intestine, predisposing to RBC-associated gut injury. To test our central hypothesis, we will pursue the following specific aims: Aim 1: Elucidate the ontogeny of monocytes recruited to the neonatal intestine during anemia. Aim 2: Define the role of trem1 signaling on the migration of hepatic monocytes into the anemic intestine, and on inflammatory activation during RBC transfusion. Aim 3: Determine whether therapeutic targeting of hepatic trem1+ monocytes during anemia can prevent/attenuate RBC-transfusion associated NEC-like injury. Accomplishment of the proposed aims will develop an effective therapeutic strategy of inhibiting the hepatic response during anemia without suppressing protective innate immune mechanisms.

NIH Research Projects · FY 2026 · 2023-03

Abstract The innate immunity is the first line of defense of our body against invading pathogens such as viruses and bacteria. The cGAS/STING pathway is a recently discovered innate immunity pathway that plays critical roles in eliminating cytosolic DNA virus. Viral DNA in the cytosol is detected by the DNA sensor cyclic-GMP-AMP synthase (cGAS), which becomes active and synthesizes the second messenger cyclic-GMP-AMP (cGAMP) using ATP and GTP as the substrates. cGAMP binds and activates adaptor protein Stimulator of Interferon Genes (STING), a transmembrane protein normally residing on the endoplasmic reticulon (ER) membrane. cGAMP-bound STING polymerizes and translocates to the Golgi apparatus, where it activates the downstream signaling proteins the TBK1 kinase and the transcription factor IRF3. This signaling pathway ultimately leads to a plethora of anti-viral responses, including the production of interferons, induction of inflammatory responses and autophagy. The cGAS/STING can also launch immune responses to self-DNA, such as damaged DNA leaked into the cytosol in cancer cells. In fact, mounting evidence in the past few years have demonstrated the cGAS/STING pathway plays a critical role in immune responses to cancer through several mechanisms. cGAS/STING signaling can boost the effects of immune checkpoint inhibitors such as anti-PD1 and anti-PD-L1 antibodies in cancer therapy, whereas loss of function of the cGAS/STING pathway leads to severe immuno- deficiency and impaired response to immune checkpoint inhibitors. STING therefore has become a major target for cancer therapy in recent years, with several agonists undergoing clinic trials at present. One major goal of this project is to advance our understanding of the fundamental regulatory mechanisms of STING by using cryo-EM structural analyses in combination with biophysical and cell-based functional assays. In addition, our preliminary data revealed a novel cryptic agonist binding site in STING, opening the door to the development of a completely new class of STING agonists. The second major direction of the project is therefore to further characterize this new binding site in STING, and design more potent and specific agonists based on the structures. New STING agonists will be synthesized and tested in biophysical and functional assays, and structurally characterized using cryo-EM. The new agonists could be used as chemical tools for further mechanistic studies, and may be developed into anti-cancer drugs in future.

NIH Research Projects · FY 2026 · 2023-02

Project Summary/Abstract This K23 proposal will complete Anna Tavakkoli, MD, MSc’s training towards her long-term career goal of becoming an independent clinician investigator and leader in advanced endoscopy. Dr. Tavakkoli is an advanced endoscopist in the Division of Digestive and Liver Diseases at the University of Texas Southwestern (UTSW) with Master’s training in health services research principles. This proposal builds on Dr. Tavakkoli’s prior experience, leveraging advanced statistical methods, utilizing complex datasets, developing a prospective patient cohort, and the rich training environment at UTSW to improve our understanding of ERCP outcomes and costs throughout the United States. The research will be completed under the guidance of her primary mentor, Amit G. Singal MD, MSc, and co-mentor, B. Joseph Elmunzer MD, MSc, with a planned mentor-the-mentor strategy and additional input from an advisory board of physician-investigators. The 5-year plan includes formal coursework, professional development, and mentored research, with defined milestones to ensure productivity and a successful transition to independence. This mentored research has 3 Specific Aims: • AIM 1 Characterize variation in, and identify patient-, provider, and system-level covariates for post-ERCP outcomes. • AIM 2a Characterize 30-day post-ERCP variations in healthcare utilization, Medicare expenditures and drivers of expenditures across US health systems. • AIM 2b Enumerate out-of-pocket and indirect costs associated with post-ERCP adverse events. • AIM 3 Model the impact of potential intervention strategies to improve post-ERCP pancreatitis. Inherent to completing these high-level aims, Dr. Tavakkoli will also (1) characterize variation in post-ERCP outcomes across Medicare beneficiaries; (2) identify key patient-, provider-, and system-level correlates associated with variation in post-ERCP outcomes; (3) characterize Medicare expenditures as it relates to ERCP; (4) characterize healthcare utilization, and correlates that contribute to variations in utilization, as it relates to post-ERCP outcomes; (5) characterize indirect costs associated with post-ERCP adverse events through direct patient contact; (6) utilizing decision analytic modeling to understand the clinical impact that various intervention strategies, such as a selective referral approach to high-volume endoscopists, could have on rates of post-ERCP pancreatitis. This work will build to an R01 proposal focused on: 1) Building a multi-center prospective cohort to identify granular clinical factors associated with post-ERCP adverse events; 2) Evaluating patient and provider acceptance of intervention strategies; 3) Cost-effectiveness of intervention strategies from our decision analytic model. Beyond establishing a foundation for a programmatic line of research to improve endoscopic care for patients, this proposal will support and accelerate the career development activities of Dr. Tavakkoli, allowing her to successfully launch into the next phase of her career as an independent investigator.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease and other tauopathies are tau protein conformation diseases that impact millions of people. There is currently no means to diagnose patients based on tau conformation. The microtubule associated protein tau can convert into distinct pathogenic shapes each causing different human diseases. High-resolution cryo- Electron Microscopy structures of tau fibrils isolated from different diseases reveal the diversity of structures that tau can adopt in each disease. Fibril structures highlight the central role of amyloidogenic motifs forming stabilizing nonpolar contacts to determine the distinct folds. Our model predicts that the propensity of a tau monomer to adopt structural polymorphs is linked to perturbation of local structures that expose different patterns of amyloidogenic motifs. Detection of tauopathy-derived tau conformations will be essential to develop accurate disease diagnoses. To understand the structure and origins of tau amyloid assembly in tauopathies, we propose to: (i) develop predictive algorithms for tau structure based on functional genetics, (ii) test role of local motifs in control of tau assembly, (iii) test role of pathogenic mutations on local structure. We anticipate that our approach will allow diagnosis of tauopathies based on tau conformation in premortem, and possibly presymptomatic, patients samples, and also provide fundamental insight into the rules that govern tau assembly in disease. Our long-term goal is to develop diagnostic and therapeutic approaches to treat neurodegenerative diseases. This project is in alignment with the mission of the NIA to support biological and clinical research on aging.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY/ABSTRACT ETV6 is a transcriptional repressor involved inhematopoietic stem cell maintenance and terminal differentiation of megakaryocytes. ETV6 conditional knockout mice demonstrate a marked decrease in peripheral platelet counts and a compensatory increase in immature megakaryocytes. In concordance with these findings, in recent years, a number of germline mutations in ETV6 that result in mislocalization of the protein from the nucleus to the cytoplasm have been associated with inherited thrombocytopenia. Carriers of these mutations are also at an increased risk of hematologic malignancies as ~30% have gone on to develop myelodysplastic syndrome or leukemia. Most of these germline mutations are found in the DNA-binding domain (DBD) of ETV6. Functional studies of these DBD mutations demonstrate a loss of DNA-binding capacity in vitro and a loss of transcriptional repression in cells. However, one mutation, the Pro214Leu missense mutation identified in 5 families thus far, occurs in the long intrinsically disordered central domain of ETV6. It too demonstrates a loss of transcriptional repression in vitro, but the mechanism explaining this loss has not yet been established. Preliminary data I have gathered demonstrates that this Pro214Leu missense mutation creates a de novo nuclear export signal (NES) leading to exportin 1 (XPO1) mediated nuclear export. This constitutes the first described instance of a point mutation creating a de novo NES. We intend to develop cellular and animal model systems to probe the effects of this unexpected disease mechanism on thrombopoiesis. We are developing a homologous ETV6 P214L transgenic mouse line will validate its suitability as an animal model of ETV6-related thrombocytopenia. This will allow us to use genetic and chemical tools to study the effects of ETV6 P214L nuclear relocalization on megakaryocyte and platelet development. Lastly, a preliminary bioinformatics search utilizing ClinVar, a publicly available database of genetic variation, and an NES prediction server has yielded additional candidate mutations that may also create de novo NESs. We intend to show that missense mutation dependent nuclear export is a general mechanism of disease, and characterization of candidate NESs may yield novel biomarkers of disease.

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract Deep learning (DL) has been widely applied across all life sciences to construct predictive models. However, it relies on the assumption that training samples are independent and identically distributed. This is frequently violated in the life sciences, where data is “grouped” by measurements from the same sample (patient, cell, tissue), by the same observer, or at the same site. This leads to clusters of correlated data (random effects), and when the models are fit to such data, the model parameters can be severely biased, leading to type I and II errors. Proper accounting for such dependencies in DL models has gone unsolved. The objective of this proposal is to develop the appropriate DL modifications to separately model global fixed effects and random effects that increase model interpretability and performance for precise unbiased predictions related to human disease. Our proposal is based on a novel, model-agnostic framework to transform conventional DL models into proper mixed effects DL (MEDL) models. This affords capabilities of statistical linear mixed effects models, including the separation of cluster-invariant fixed effects from cluster-specific random effects, while preserving the ability of DL to learn data-driven nonlinear associations. The core premise is that proper MEDL models 1) are more resilient to confounding effects and more attentive to true predictive features, 2) can capture, quantify, and visualize random effects to enhance interpretability, and 3) attain better generalization to new clusters. We propose to incorporate MEDL into three of the most important DL model types including dense feed-forward neural networks (DFNNs), convolutional neural networks (CNNs), and autoencoders. Our preliminary results demonstrate multiple advantages of MEDL over conventional DL in both accuracy and interpretability. MEDL outperforms previous clustered data approaches including: domain adversarial models, meta-learning, and the inclusion of cluster membership as an input covariate. We developed an ME-DFNN to predict conversion from mild cognitive impairment to Alzheimer’s Disease (AD) from tabular data, an ME-CNN to diagnose AD from MRI, and an ME-autoencoder to compress and classify live cell images. Across these test cases, MEDL models were the most discriminative between known confounded and real features; they were able to quantify or visualize the random effects and outperformed other models on clusters both seen and unseen during training. This proposal further develops the methods to handle complex architectures and hierarchical effects, with external validation, through these aims: 1) Develop ME-DFNNs for classification and regression. 2) Develop 3D ME-CNNs and multi- modal 3D ME-CNNs for medical image classification. 3) Develop convolutional and vector ME-autoencoders for image and omics data. We describe the innovative incorporation of an adversarial classifier to constrain the base model to learn fixed effects, a Bayesian random effects subnetwork, and an approach to apply random effects to unseen clusters. All these solutions will be released as open source software that improve existing DL models to ultimately support precision biomedicine for the study and treatment of human disease.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY/ABSTRACT Glucose is an essential fuel for cancer cell proliferation in serving both as a substrate for ATP production and as an irreplaceable carbon source for biomass accumulation. Cancer cells are especially addicted to glucose but only to secrete the majority as lactate (known as aerobic glycolysis or Warburg effect), thereby creating an inhospitable glucose-poor and lactate-rich microenvironment that would otherwise be lethal to most cells. However, cancer cells can efficiently use the limiting glucose and excess lactate for unlimited growth through unclear mechanisms. My preliminary data revealed that in low glucose conditions, extracellular lactate enhances cancer cell proliferation. Mechanistically, I found that lactate preferentially enters the mitochondria TCA cycle over glucose to increase oxidative phosphorylation (OXPHOS) activity, which in turn suppresses glycolysis to conserve extracellular glucose, suggesting cancer cells rely on lactate-induced OXPHOS for optimal growth. The proposed studies are aimed at mechanistically dissecting the metabolic interplay between lactate-mediated mitochondrial OXPHOS and glycolysis (Aim 1 & 3), and assessing therapeutic potential of targeting lactate oxidation in cancer (Aim 2). The following specific aims are being pursued: Aim 1. Determine how lactate-mediated increase in OXPHOS suppress glycolysis; Aim 2. Assess the in vivo therapeutic potential of targeting lactate oxidation using Phenformin; Aim 3. Mechanistically dissect how cells distinguish and preferentially use extracellular lactate over glucose for entry into TCA cycle. The knowledge and scientific expertise gained through these studies will facilitate my transition to independence, with my long-term goal to study and target metabolic vulnerabilities in cancer as a physician scientist. In addition to the scientific goals, I have also outlined a detailed career development plan in this application to obtain skills that are necessary for leading an independent research laboratory. The proposed research and training plan will be conducted under the mentorship of Dr. Craig Thompson. Memorial Sloan-Kettering Cancer Center, along with the nearby Rockefeller University and Weill Cornell Medical College will provide the ideal academic environment to achieve these goals for me to transition to independence.

NIH Research Projects · FY 2026 · 2022-12

Schistosomes infect 200 million of the world’s poorest people. Although these parasites kill 250,000 people annually, and rob millions more the ability to lead healthy and productive lives, treatment of schistosome infection relies on a single drug (Praziquantel). Unfortunately, Praziquantel is not a magic bullet. Indeed, praziquantel is not equally effective against all intramammalian stages of the parasite’s life cycle and it suffers from a variable cure rate in some endemic settings. This later point, raises the specter that praziquantel-resistant parasites could emerge as organizations ramp up mass drug administration programs. Indeed, several studies have demonstrated that strains with reduced sensitivity to praziquantel can be rapidly selected in the laboratory. Therefore, efforts to develop the next generation of antischistosome therapies to replace praziquantel are key to a sustained effort to eradicate this disease. Drug development efforts against schistosomes suffer from two important limitations. First, the large size of the adult parasites (~1cm in length) makes screening thousands of compounds for activity against the worm impractical. The second important limitation is that we only know the function of a small number of proteins in these worms making it challenging conduct informed target-based drug discovery efforts. To address these issues, we have conducted the first large-scale loss-of-function RNA interference (RNAi) screen in adult schistosomes. Using this platform, we have performed over 2000 individual RNAi knockdown experiments in adult Schistosoma mansoni and identified nearly two hundred genes, a large fraction of which are potentially druggable, that are essential for parasite survival and/or neuromuscular function both in vitro and in vivo inside mice. To capitalize on these studies we will execute three specific aims to discover and experimentally prioritize druggable targets in the schistosome. In Specific Aim 1, we will utilize RNAi and in silico approaches to identify and prioritize druggable targets that are essential in vitro and in vivo in three medically relevant schistosome species. In Specific Aim 2, we will experimentally validate the druggability of the highest priority targets from Aim 1 by assessing the performance of these targets in small-scale high throughput small molecule screens. These studies will identify both validated druggable targets in the schistosome that can form the basis of future drug discovery campaigns and compounds poised for lead optimization efforts.

NIH Research Projects · FY 2026 · 2022-12

Project Summary Distinct histone modifications, on N or C-terminal tails, act as a “histone code” to elicit downstream events. Histone subunit post-translational modification (PTM), such as methylation, acetylation and phosphorylation regulates epigenetic regulation and gene expression in diseases such as cancer. Here we identified a new PTM on H3 called hydroxylation on proline 16, which is catalyzed by proline hydroxylase EglN2. Our preliminary data show that EglN2-mediated H3 Pro16-OH leads to increased Lysine (K)-Specific Demethylase 5A (KDM5A) binding corresponding with decreased H3K4me3 in breast cancer. We hypothesize that H3 prolyl hydroxylation mediated by EglN2 recruits KDM5A therefore controlling gene expression important in breast cancer. This is the first study reporting H3 Prolyl hydroxylation and its potential role in epigenetic regulation and gene expression in cancer. In Specific Aim 1, we will determine the effet of H3 prolyl hydroxylation on epigenetic regulation and gene expression on a genome wide scale in breast cancer cells. In Specific Aim 2, we will elucidate the molecular mechanism by which H3 prolyl hydroxylation regulates epigenetic reprogramming by recruiting KDM5A. In Specific Aim 3, we will elucidate the molecular mechanism by which H3 prolyl hydroxylation regulates Wnt/b-Catenin signaling in breast cancer cells. Successful completion of this proposal will characterize a new H3 post-translational modification in its epigenetic regulation and gene expression important in breast cancer.

NIH Research Projects · FY 2025 · 2022-12

Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal disease characterized by extensive desmoplasia caused by the rapid expansion of cancer-associated fibroblasts (CAFs), resulting in the formation of dense stroma. CAFs stimulate cancer progression by secreting a variety of factors that support cancer cells and facilitate immunosuppression. In addition, they also secrete extracellular matrix (ECM) that provides survival and invasion cues to cancer cells and impairs drug delivery. Recently, several populations of CAFs with distinct functions have been characterized in PDA by our group and others using single cell RNA sequencing (scRNA seq). One population is characterized as myofibroblastic CAFs (myCAFs), another population is characterized as inflammatory CAFs (iCAFs), the third population was first identified as antigen-presenting CAFs (apCAFs), which express MHC II molecules and can effectively present antigen to T cells. My preliminary data demonstrated that apCAFs are mesothelial cells. Mesothelial cells form a continuous layer of epithelial cells known as mesothelium. The mesothelium is traditionally thought to be a membrane providing a non-adhesive surface covering organs and tissues. However, until the description of apCAF population, mesothelial cells have been neglected as a potential functional constituent of the tumor microenvironment. My preliminary data suggest that during PDA progression, mesothelial cells go through a mesothelial-fibroblastic transition (MFT), in which they down-regulate MHC II molecules that are required for CD4+ T cells activation, and up-regulate fibroblast genes that have been known to prevent T cell infiltration and activation. Peripheral T cell exclusion is a major immune evasion phenotype in PDA, and my preliminary data show that this exclusion occurs at the region where mesothelial cells are transitioning to CAFs. Therefore, MFT might be an important mechanism of immune evasion and understanding this process is critical. In this proposal, I will test the hypothesis that the fibroblastic transition of mesothelial cells promotes immune evasion in PDA and identify potential strategies to inhibit this process. I propose the following two aims: Aim 1. Determine the fate of mesothelial cells during PDA progression. Aim 2. Determine the functions of MFT on immune evasion. The outcome of the proposed study has the potential to shift the paradigm of tumor microenvironment studies, identify novel strategies to target CAFs and overcome resistance of immune therapies in PDA and other tumor types.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY/ ABSTRACT Hepatocellular carcinoma (HCC) is ranked as the second most common cause of cancer-related death globally. Transarterial chemoembolization (TACE) remains the only first-line treatment for unresectable intermediate- stage HCC, despite the fact that this stage is comprised of a heterogeneous group of patients with a wide range of liver function, variable tumor number and size. In clinical practice only 50-60% of patients with intermediated HCC benefit from TACE, thus repeated rounds of TACE therapy are performed to achieve maximum tumor recession. The critical factors that impact the effectiveness of TACE therapy are the worsening of liver function and tumor recurrence. The former arises from progressive off-target embolic ischemic injury to the liver, while the latter results from hypoxia-induced angiogenesis, epithelial-to-mesenchymal transition (EMT) and tumor growth triggered by TACE. These processes inevitably dominate the course of this disease resulting in poor long term survival, with a 5-year survival rates <12%. Novel therapies against HCC are urgently needed as the incidence of HCC is steadily increasing in the United States. In recent years the natural omega-3 fatty acid, docosahexaenoic acid (DHA) has been shown to possess promising anticancer properties and its consumption has been implicated in reducing the risk of HCC. The effects of dietary DHA on established solid tumors is nominal. To address this issue, our lab has engineered a novel low-density lipoprotein (LDL) based biologic that is reconstituted with unesterified DHA (herein referred to as LDL-DHA). Therapeutically, we have shown in a syngeneic rat model of HCC, that transarterial delivery of LDL-DHA is able to induce extensive necrosis (>80%) of HCC tumors and impede the tumor growth (3 fold) without injury to surrounding normal liver. Moreover, repeated intra-arterial LDL-DHA treatments was shown to provide sustained regression of HCCs. Furthermore, the uptake of LDL-DHA in the normal liver was shown to be not only safe but potentially hepatoprotective. In addition, recent preliminary data from our group has documented that LDL-DHA is able to downregulate HIF-1α and EMT signaling in HCC cells, thus inhibiting tumor angiogenic/regrowth activity. The goal of the present proposal is to evaluate the utility image-guided locoregional LDL-DHA therapy for intermediate-stage HCC. To address this goal we will examine the following specific aims: 1) evaluate the safety of intra-arterial LDL-DHA delivery in rat models of cirrhosis; and 2) compare the therapeutic efficacy of LDL-DHA versus conventional TACE methods to provide sustained tumor control in a patient derived-xenograft rat model of HCC. We expect that the combined work of these Aims will validate the safety of LDL-DHA treatment in preserving liver function in settings of cirrhosis and demonstrate the efficacy of this therapy to provide sustained tumor eradication over TACE. The LDL-DHA treatment strategy will be significant because it offers a new method of effectively treating HCC while preserving liver function. Ultimately it is our endeavor to bring this technology to the clinic, where it is anticipated to provide safe and efficacious approach to managing of unresectable HCC.

NIH Research Projects · FY 2026 · 2022-12

Project Summary/Abstract The overarching goal of our research program is to develop small-molecule anticancer drugs. We focus herein on a new chemical method to control aberrant Wnt/β-catenin signaling that drives tumorigenesis and metastasis of many cancers, in particular, colorectal cancer (CRC). CRC affects about 4% of the population and caused ~60,000 deaths in 2021. Despite decades of effort, drugging this oncogenic pathway has not been successful. In 2009, we reported for the first time that Wnt/β-catenin signaling can be intercepted by small molecules. Catalytic inhibition of tankyrases prevents the turnover of the Axin, which leads to a rapid accumulation of Axin. The accumulated Axin then stabilizes the β-catenin destruction complex (DC) to facilitate the degradation of β-catenin. However, using this strategy to treat cancer has not been successful. Recent studies suggest that tankyrases can, paradoxically, support Wnt/β-catenin signaling through molecular scaffolding. The unexpected dichotomous mode of action potentially explains the unsatisfactory outcomes of various preclinical studies of tankyrase inhibitors. Although the mechanism by which tankyrases sustain Wnt/β- catenin signaling is not clear, mounting evidence suggests that the tankyrase aggregation is responsible for it. In this study, we will develop a chemical strategy to control the catalysis-independent function of tankyrases. We will then use this new tool to study how tankyrases affect the dynamic assembly of DC. We will further use a proteomic approach to delineate the catalytic and scaffolding functions of tankyrases with detailed characterization of their mode of action. This work will help us understand how tankyrases control multiple signaling pathways important to cancer. Additionally, we will compare the responsiveness of a large panel of immortalized human colonic epithelial cell lines and CRC cell lines toward tankyrase inhibition and depletion. We will then corroborate the results with in vivo studies. Correlating the cellular sensitivity with their genetic background will provide potential biomarkers and inform therapeutic strategies for cancers. Overall, this project will address the unsolved issue in drugging the Wnt/β-catenin pathway and improve our understanding of how tankyrases control Wnt/β-catenin signaling.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT/NARRATIVE Cancer cachexia (CCX), wasting of muscle and/or adipose, is associated with 20-30% of all cancer related deaths.1 Our clinical studies have shown that the presence of CCX is associated with a 50% decrease in median survival (14 months vs 28 months, p<0.001) independent of tumor-directed therapies.2-3 There are no FDA- approved CCX regimens, with a majority of trials focused on limiting sarcopenia. Using multiple established murine CCX mouse models, we consistently observed significant adipose tissue loss compared to muscle atrophy. Furthermore, blocking adipocyte lipolysis using global lipase null mice limited both adipose wasting and sarcopenia in murine models of CCX. Understanding upstream mechanisms cancers use to provoke adipose lipolysis and wasting could offer novel therapeutic targets to reverse CCX syndrome. The complex intracellular (stromal, vascular, immune, and adipocyte) interactions within adipose tissue ultimately regulate CCX wasting by altering the relative signals of adipocyte triglyceride lipolysis and synthesis. To understand the convergence of these interactions, we developed an in vitro CCX adipocyte assay to screen secreted factors from CCX lines that increase adipose inflammation and wasting by inducing adipocyte lipolysis and identified the cytokine leukemia inhibitory factor (LIF).5 Through the JAK-dependent inflammatory reprogramming of adipose tissue in mice, recombinant LIF caused a decrease in adipose mass by >50%, lean mass, and body weight by >10%, recapitulating CCX. LIF also altered the adipose expression and systemic levels of other cyto/adipokines to amplify this inflammation and alter food intake. Use of JAK inhibitors in murine CCX models led to decreased adipose inflammation (decreased STAT3 phosphorylation), adipocyte lipolysis, and adipose/muscle wasting, all increasing survival. To understand the contributions of adipose intracellular signaling in the regulation of CCX adipose inflammation, we selectively silenced the LIF receptor (LIFR) or STAT3 in adipocytes. Both mouse models doubled their adipose mass compared to littermate controls during development highlighting an inverse CCX phenotype. When allotransplanted with CCX tumors, both models still demonstrated adipose inflammation with persistent STAT3 phosphorylation, resulting in a partial suppression of CCX and defining the non-adipocyte cellular contributions of adipose to CCX wasting. FACS analysis verified longitudinal enrichment of immune cells during CCX progression, offering additional tumor/cytokine targets supporting CCX adipose inflammation and wasting. We hypothesize that CCX adipose inflammation occurs via a JAK-dependent Tumor-Cytokine-Adipose Axis that reprograms adipose through JAK/STAT signaling of multiple cellular subtypes to increase adipocyte lipolysis and alter secretion of cyto/adipokines, resulting in wasting. SA1-2 will dissect the multiple cellular/molecular signaling components of this axis facilitating adipose inflammation in support of this CCX wasting. SA3 will validate associations between JAK/STAT signaling events in human adipose to CCX induction in patients.