Wayne State University

NIH Research Projects · FY 2025 · 2024-06

Somatic hypermutation (SHM) and class-switch recombination (CSR), are two molecular processes that are central to antibody maturation in mammals. A murine protein, mFAM72A, is expressed in germinal center B lymphocytes and plays a key role in both these processes. It interacts with the nuclear form of the DNA repair protein, mUNG2, and is required for optimal SHM and CSR. It causes inhibition of the enzymatic activity of the latter protein and promotes its degradation through a proteosome-dependent pathway. The enzyme activation-induced deaminase (AID) is essential for both SHM and CSR, and converts cytosines in DNA to uracil creating U•G mispairs. The uracils are processed by two distinct DNA repair pathways. The uracils may be excised by mUNG2 creating abasic sites that are processed by the base- excision repair machinery or the U•G mispairs may be recognized by a non-canonical mismatch repair (ncMMR) process that is not linked to replication. Together these repair pathways cause base substitution mutations and strand breaks that promote SHM and CSR. ncMMR works only when U•G pairs created by AID persist in the immunoglobulin genes and recent studies strongly suggest that mFAM72A helps with the persistence of U•G pairs through interference with mUNG2 stability and activity. However, it is unclear why the degradation of mUNG2 is necessary when the binding of mFAM72A to mUNG2 in itself causes substantial inhibition of enzymatic activity of mUNG2. It is possible that the degradation of mUNG2 is necessary to reduce the nuclear concentration of this protein changing its association with other proteins that are inhibitory towards SHM and CSR. To test whether the inhibition of UNG activity is sufficient for optimal SHM and CSR, we will synthesize a family of known chemical inhibitors of mammalian UNG and test them in the murine cell culture model for CSR, CH12F3 cells. These inhibitors bind within the active site of the enzyme, have micromolar to submicromolar IC50 and are able to inhibit the enzyme inside cells. We will also synthesize proteolysis-targeting chimera (PROTAC) versions of these inhibitors that should cause degradation of mUNG2. We will treat mFAM72A knockout (KO) CH12F3 cells with different concentrations of these inhibitors or PROTACs and determine the frequency of isotype switching from IgM to IgA in these cells. If inhibition of enzymatic activity of mUNG2 is sufficient for optimal CSR then CSR frequency should initially increase with increasing concentration of inhibitor reaching a maximum similar to that found in FAM72A+/+ cells. However, if degradation of mUNG2 is essential for optimal CSR, the PROTAC treatment- but not inhibitor treatment- should achieve maximal CSR. Following inhibitor and PROTAC treatment, we will also monitor mutations in the 5'Sµ region of CH12F3 genome as a proxy for SHM. Additionally, we will use a newly constructed RASH-1 cell line as a model for SHM. This RAMOS-derived human cell line contains an inducible AID gene and SHM in its genome can be conveniently monitored as loss of GFP fluorescence. mFAM72A promotes higher frequency of hypermutations in the variable region of the IGH gene in the B lymphocyte genome and hence we expect that the drug treatments will increase the SHM frequency. If the maximum SHM frequency is achieved using inhibitors alone, we will conclude that inhibition of UNG2 activity is sufficient for optimal SHM.

NIH Research Projects · FY 2025 · 2024-05

Project Summary The Hippo pathway is conserved from yeasts to humans. In mammals, this signaling pathway controls tissue development by balancing cell proliferation and death. The Hippo kinases, MST1 and MST2, transduce signals via phosphorylation of the scaffold protein MOB1 and the downstream kinase LAST1. Animal models show that tissue-specific Mst1/2 gene knockouts lead to abnormal organ development and tumor formation. In addition to its well-known functions in development and cancer, recent studies found the Hippo pathway is targeted and manipulated by bacterial pathogens, suggesting a role of this conserved host pathway in bacterial pathogenesis. Notably, individuals with loss-of-function mutations in the MST1 gene are immunocompromised. Consistent with this observation in humans, our lab knocked out both Mst1 and Mst2 genes in macrophage and found that these macrophages are highly susceptible to infection by the human pathogen Legionella pneumophila, highlighting an emerging role of the Hippo kinases in immunity against infection. Using the Mst1/2 double knockout cell model, we further discovered that MST1/2 are the key regulators for immune gene expression and programmed cell death during infection. Importantly, upon challenge of virulent L. pneumophila, MST1/2 full-length proteins are proteolytically cleaved, resulting in MST1/2 fragments (MST1/2-NT) that contain the kinase domains but are separated from the regulatory domains. In biochemical assays, MST1/2-NT, surprisingly, no longer phosphorylate their cognate substrate, MOB1, but exhibit altered substrate specificity for protein phosphorylation. In cell-based assays, MST1/2-NT production contributes to activation of apoptotic cell death that is critical for restricting virulent L. pneumophila. Based on the preliminary data collected in our lab, we propose a model that, upon infection, macrophages trigger cleavage of MST1/2 to activate an inflammatory Hippo signaling mediated by the proapoptotic MST1/2-NT as a defense mechanism. The objective of the proposed study is to dissect the importance of this novel inflammatory Hippo signaling. In Aim 1, we will use an innovative technique, Thiophosphorylation Proteome Screen (TPS), that allows us to screen more than 21,000 human proteins for direct substrates of full-length MST1/2 and MST1/2-NT. We will use the established substrate validation methods which include in vitro and cell-based assays to validate genuine substrates of these kinases. This aim will yield a comprehensive phosphoproteome controlled by the Hippo kinases that is fundamental for understanding both the canonical and inflammatory Hippo signaling. In Aim 2, we will focus on how MST1/2 influence gene expression and communication in immune cells. Specifically, we discovered that expression of the complement protein C1q is depleted in Mst1/2 double knockout macrophages. We will use genetic and cell-based assays to elucidate the molecular mechanism by which MST1/2 regulate C1q, the complement protein with a crucial role in bacteria killing. This study will provide insights for boosting innate immunity against infection through modulating the activities of the Hippo kinases.

NIH Research Projects · FY 2026 · 2024-04

Project Summary/Abstract Pancreatic cancer liver metastasis (PCLM) is present in 50% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and has a median survival of <6 months. Recent studies in mice found that modulating the gut microbiome can reduce PCLM tumor burden by augmenting anti-tumor immunity. The gut microbiome is constantly producing antigens the travel to the liver. This suggests that gut microbiome modulation induces PCLM anti-tumor immunity by altering the antigens presented in the liver, however the mechanism is unclear. Mucosal Associate Invariant T (MAIT) cells are an innate subset of T cells which can be classified into two phenotypes: an “anti-tumor” MAIT1 or a “pro-tumor” MAIT17 phenotype. MAIT cells closely interact with gut microbiome derived antigens through their activation mechanisms, and this can shift their phenotype. Direct activation leads to a MAIT17 phenotype, involving interactions with microbial riboflavin synthesis pathway- derived metabolite ligands presented on antigen presenting cells (APCs) via MHCI related protein (MR1). Indirect activation induces a MAIT1 phenotype by stimulating MAIT cell IL-12 and IL-18 receptors15. These cytokines are released by APCs upon stimulation by microbial-produced ligands. Consequently, the goal of this project is to harness the gut microbiome to modulate MAIT cell phenotypes towards treatment of PCLM. Preliminary data suggests that MR1 expression is upregulated in the PCLM tumor microenvironment (TME) which promotes a pro-tumor MAIT17 phenotype via direct MAIT cell activation. This suggests an abundance of gut microbiome derived MR1 ligands in the PCLM TME. In Aim 1, we hypothesize that gut microbiome dysregulation provides MR1 ligands to the PCLM TME which promotes PCLM via inducing a MAIT 17 phenotype. We will test this by 1) Correlating the abundance of MR1 ligands in the PCLM TME with the MAIT17 phenotype; 2) Determining the gut microbiome composition of mice with PCLM by shotgun metagenomic sequencings; 3) characterizing how a MAIT17 phenotype promotes PCLM tumor burden by single cell RNA sequencing. Further preliminary data shows that promoting a MAIT1 phenotype reduces PCLM tumor burden. Also, gut microbiome depletion by antibiotic treatment (ABX) promotes a MAIT1 phenotype, which likely promotes anti- tumor TH1 cells, in the liver. In Aim 2, we hypothesize that ABX reduces MR1 ligands in the PCLM TME which promotes a MAIT1 phenotype and anti-tumor immunity. We will test this by 1) Investigating the MAIT cell phenotype in PCLM+ABX mice and validating by fecal microbiome transfers; 2) Determining the gut microbiome composition following ABX; 3) Probing anti-tumor immunity in the PCLM+ABX TME by imaging mass cytometry. These studies will identify MAIT cells as the missing link between the gut microbiome and anti-tumor immunity and will affect how PCLM and other liver conditions are treated. This research plan and my outlined training plan are supported by exceptional institutional support and by an exceptional mentorship team. This will provide an unmatched multidisciplinary research and clinical training experience that perfectly aligns with my career goals.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY/ABSTRACT Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplasm with a wide range of clinical manifestations and severity caused by mutations that constitutively activate mitogen-activated protein kinase (MAPK) signaling in myeloid lineage cells- most commonly BRAFV600E. Constitutive MAPK activation endows pathological histiocytes with a senescence associated secretory phenotype (SASP)- leading to resistance to apoptosis, increased inflammatory cytokine and matrix metalloproteinase expression, and decreased ability to migrate to lymph nodes. These features allow LCH cells to persist in tissues and reshape the microenvironment leading to the characteristic granulomatous lesions harboring CD207+ cells. However, little is known about tissue- specific and time-dependent features of the LCH microenvironment (LCH-ME). Treatment of LCH remains unacceptable due to significant drug-induced toxicity and inability of current treatment strategies to eliminate LCH precursor cells leading to high-rates of disease recurrence. Recurrence often leads to life-threatening sequalae. Drugs targeting both development and homeostasis of LCH cells could revolutionize treatment for patients with LCH. Our lab showed histone deacetylase 3 (HDAC3) regulates multiple facets of myeloid cell biology, including development and homeostasis. This F30 proposal seeks to define the therapeutic potential of targeting HDAC3 to ameliorate LCH. Collectively, the data presented will further our understanding of the epigenetic regulation of pathological myeloid cells, provide novel insights to the LCH-ME, and provide preclinical data for HDAC3 inhibition (HDAC3i) as a treatment for LCH. In Aim1, we will us a mouse model of LCH to define tissue-specific and time-dependent features of the LCH-ME. Using flow cytometry, single-cell RNA sequencing, and imaging mass cytometry we will compare the composition, gene expression, and spatial organization of lung and liver LCH lesions over time. In Aim2, the efficacy of HDAC3i will be evaluated in a mouse model of severe-multifocal LCH. Using genetic ablation and pharmacological inhibition, we will test how HDAC3i influences development, homeostasis, and SASP properties of LCH cells, and the LCH-ME. These studies will address a major gap in our understanding of LCH disease development and define a potential new therapeutic that could be translated to treat LCH and other MAPK-driven histiocytic disorders. The training plan outlined in this proposal will be performed between Henry Ford Health System, Wayne State University School of Medicine, and Karmanos Cancer Institute, which presents a unique opportunity to leverage the advantages and expertise at all three institutions providing a robust clinical and research training. Executing this training plan in such a vibrant multi-institutional environment, with the guidance of dedicated sponsors, collaborators, and mentors with expertise in immunology, epigenomics, cancer biology, and hematology, will provide an unparalleled training for a successful career as a physician-scientist.

NIH Research Projects · FY 2025 · 2024-03

ABSTRACT Breast cancer is the most common cancer and the second leading cause of cancer mortality in women in the U.S. Approximately 75% of breast cancers are estrogen receptor α (ERα) positive. The current standard of care for postmenopausal women with ERα+ breast cancers involves the choice of an upfront aromatase inhibitor (AI), a tamoxifen (Tam)-AI, or AI-Tam switch strategy. Tam is the first line therapy for premenopausal women, second line for postmenopausal women, and a chemopreventive agent for all age groups. Thus, Tam continues to remain an important selective ER modulator for treatment of ERα+ breast cancer patients. However, ~ 30% of ERα+ patients do not respond to Tam because of de novo resistance, and most who do respond eventually acquire Tam resistance (TamR). Breast cancer patients who relapse on Tam retain functional ERα signaling, suggesting that endocrine resistance involves agonistic function of Tam or estrogen independent ERα activity. Crosstalk between ERα and growth factor receptor (GFR) signaling pathways is believed to contribute to endocrine resistance. However, clinical trials with GFR inhibitors have yielded mixed results, indicating complex ER biology and involvement of novel crosstalk or additional/alternate mechanisms in endocrine resistance. In this application, based on our data from isogenic models of acquired and de novo TamR, we propose a novel transcription reprogramming induced mechanism of TamR that ensues from ERα- dependent/Tam-induced upregulation of β-catenin/TCF-mediated transcriptional activity. This is further supported by our data that show ectopic β-catenin expression in Tam-sensitive ERα+ breast cancer cells transforms Tam from an antagonist to an agonist with resultant ERα-dependent Tam-induced transcriptional activation of β-catenin/TCF-responsive genes. Rad6B (a.k.a UBE2B), an ubiquitin conjugating enzyme (UBC), stabilizes and activates β-catenin by K63-linked polyubiquitination (polyUb) of its lysine 394 residue, a ubiquitination event that protects β-catenin from 26S proteasomal degradation. Rad6B is itself a β-catenin transcriptional target, thus creating a vicious positive feedback loop between Rad6B gene expression and β- catenin oncogenic activation. Based on these data, we hypothesize that Rad6B-mediated β-catenin polyubiquitination and transcriptional activation drive TamR by facilitating Tam-induced ubiquitinated-β- catenin/ERα complex formation and transcriptional reprogramming of β-catenin/TCF gene targets. Hence a hyperactive Rad6B/β-catenin axis would drive cells from a Tam-sensitive to a Tam-refractory state, whereas inhibition of Rad6B catalytic activity will inhibit β-catenin ubiquitination/activation and uncouple TCF/β- catenin/ERα coregulated transcriptional programs resulting in restoration of Tam sensitivity. We will test this hypothesis with two specific aims. Specific Aim 1. Establish the causative role of Rad6B in tamoxifen-induced β-catenin/TCF transcriptional reprogramming and TamR breast cancer development and progression. Specific Aim 2. Identify and characterize β-catenin/ERα complex regulated transcriptional networks and underlying alterations in epigenetic landscape associated with TamR and assess their vulnerability to Rad6B or β-catenin inhibition. This study will uncover the molecular underpinnings of a novel paradigm shifting hypothesis that has the potential to impact clinical management of endocrine resistant breast cancers. It will lay the foundation for testing a new nontoxic strategy for treating TamR breast cancers, as well as identify markers with diagnostic/prognostic and/or therapeutic potential.

NIH Research Projects · FY 2026 · 2024-03

PROJECT ABSTRACT Diabetic patients are known to have a higher incidence of infection, with increased disease severity and an increased rate of multi-drug resistance. In the diabetic (DM) cornea, this results in increased susceptibility to and the rapid progression of microbial keratitis. To date, the mechanisms underlying this are unknown. To address this critical and understudied question, Pseudomonas (P.) aeruginosa and Staphylococcus (S.) aureus keratitis models with Streptozotocin-induced type 1 (T1) and db/db type 2 (T2) DM mice were established. In our models, higher inocula of both P. aeruginosa and S. aureus were required to infect mouse corneas and bacterial keratitis progressed faster in DM, compared to normoglycemia B6 mice, mimicking human diabetic infectious keratitis. High-throughput RNA sequencing (RNA-seq), in combination with comprehensive bioinformatics, was used to identify differentially expressed genes (DEGs) and biological processes associated with the increased susceptibility and severity of diabetic BK. Our preliminary data show that P. aeruginosa infection resulted in many DEGs, some of which have not been linked previously to the pathogenesis of bacterial keratitis. Gene Ontology enrichment analysis revealed that programmed cell death (PCD) pathways, known to play a key role in homeostasis and in the pathogeneses of many human diseases, including DM and sepsis, were altered during P. aeruginosa infection. Our preliminary data showed that while noninflammatory apoptosis was decreased, lytic PCDs, including caspase-8, receptor-interacting protein family of serine/threonine protein kinases (RIPK)-1 and -3, mediated necroptosis, NETosis, a biological process to generate neutrophil extracellular traps (NETs), as well as efferocytosis that removes cell corpses, were elevated in DM, compared to NL corneas in response to P. aeruginosa infection. The hypothesis that DM causes lytic PCD pathways and impairs efferocytosis, resulting in increased susceptibility and severity of BK will be tested with three specific aims: (1) To test the hypothesis that DM skews PCD from apoptosis to necroptosis, resulting in hyperinflammation and tissue damage. This can be tested by assessing the levels of cleaved (activated) caspase-8 (apoptosis), phospho-RIPK1, and 3 (necroptosis), and by targeting Casp8 and RIPK1, 3 in both T1 and T2 DM mice. (2) To test the hypothesis that DM primes PMNs for NETosis, increasing bacterial burden, and keratitis severity in B6 mouse corneas. This can be tested by assessing the generation of Neutrophil extracellular traps containing neutrophil granule proteins and host DNA that were modified by peptidyl arginine deiminase (PAD)-4 and nuclear neutrophil elastase and blockade of DAD4 in T1 and T2DM mice. (3) To test the hypothesis that DM impairs efferocytosis, resulting in secondary necrosis and tissue deterioration in bacterial infected B6 mouse corneas. This can be tested by measuring macrophage efferocytosis of dead corneal epithelial cells and neutrophils and assessing the effectiveness of promoting efferocytosis on bacterial clearance and tissue preservation. This proposed study will allow for a better understanding of cornea immunity, identify factors and pathways responsible for the increased susceptibility of diabetic corneas to microbial infection, and lead to the identification of mechanism-based therapies for treating microbial keratitis, for both T1 and T2DM patients. CDC (1/22/2023) reported one death, three (five more recently) with permanent vision loss, and at least 50 people infected with antibiotic-resistant P. aeruginosa, linked to the use of EzriCare eye drops. Hence, our proposal is timely, necessary, and relevant.

NIH Research Projects · FY 2026 · 2024-02

SUMMARY Male factor infertility is responsible in as much as 50% of cases of couple infertility; moreover, male infertility has been observed to represent an early-life predictor of later-life disease risk. Conventional semen parameter analysis remains the most prevalent diagnostic tool for assessing semen quality, but has well documented limited ability to explain male factor infertility and poor reproductive success. Limitations are likely due to reliance on measures that do not identify the underlying biological influences of sperm function, and incomplete use of information from semen parameters. Development of novel biomarkers of biological determinants of male reproductive health is a critical step toward developing interventions to improve clinical care and public health. Our research to date suggests that sperm mitochondrial DNA copy number (mtDNAcn) and deletions (mtDNAdel) may fill this gap, and directly measure the physiological processes that determine male reproductive health. Mitochondria play key roles in sperm function and harbor their own genome, which is highly susceptible to damage. Our published data suggest that mtDNA biomarkers are related to male infertility, fertilization probability, clinical infertility treatment outcomes, and time-to-pregnancy (TTP). As a next step in this line of research, it is important to evaluate these potential relations in large study samples. Moreover, while conventional semen parameters remain controversial in predicting male factor infertility and reproductive success, little is known on how best to leverage the combination of semen parameter and novel mtDNA biomarker data to advance clinical care to understand the male contribution to reproductive success. We propose to evaluate these relationships using data and biospecimens from the NIH funded Folic Acid and Zinc Supplementation Trial (FAZST) and the Sperm Environmental Epigenetics and Development Study (SEEDS). Using these resources, we can evaluate hypotheses in large (n=2,570) preconception cohort that includes couples using a range of fertility treatments and provide opportunity to test mechanistic pathways. The proposed research represents an efficient approach to evaluate our hypothesis that mtDNA biomarkers are direct measures of the underlying biology of male reproductive health, and thus represent a biomarker of overall sperm fitness. For this proposed research, we will analyse associations of sperm mtDNA biomarkers with semen parameters, determine associations of sperm mtDNA biomarkers with clinical reproductive outcomes, and develop clinical prediction models integrating sperm mtDNA biomarkers and semen parameters using machine learning to optimize use of these measures to inform clinical care. The impact of the proposed research is expected to improve our understanding of the etiology of male reproductive health by evaluating sperm mtDNA as novel biomarkers of sperm function and overall fitness. This innovative proposal holds promise to positively impact clinical reproductive care and is a critical step toward developing interventions for male sub- and infertility.

NIH Research Projects · FY 2026 · 2024-02

Genetic testing for both germline and somatic mutations has improved our understanding of the basic biology of carcinogenesis, identified high-risk populations for targeted prevention and screening, identified targets for new treatment strategies, and has led to some of the most significant inroads in reducing cancer burden. Yet, there is still much to learn about the role of inherited genetic susceptibility and cancer, and the significant barriers to getting genetic counseling. We have assembled one of the largest populations of African American cancer survivors to date to study genetic susceptibility in this population. The Detroit Research on Cancer Survivors (ROCS) (U01CA199240) includes participants at high-risk due to family history, age at diagnosis or multiple primaries, and the infrastructure will be used to expand participation. The Program includes three projects and two cores with an overall goal of improving the identification and clinical management of hereditary and multiple primary cancers in African Americans. To do this we will: 1) Use bioinformatic analyses, family structure, gene expression, and somatic alterations to identify African American cancer survivors most likely to harbor high-penetrance genetic variants currently classified as pathogenic or having uncertain significance; 2) Characterize the spectrum of germline genetic variation in African Americans with multiple primary cancers in relation to known pathogenic mutations, VUS, race- and site-specific polygenic risk scores (PRS), environmental exposures, medical history and health behaviors; and 3) Develop a self-paced educational intervention to increase access to genetic counseling services among African Americans and increase informed decision-making about genetic testing so that we are poised to translate novel genetic discoveries into clinical practice.

NIH Research Projects · FY 2025 · 2024-01

PROJECT ABSTRACT There is a critical need to identify new mechanisms and signaling pathways promoting diabetic kidney disease. Intrarenal expression of renin and pro-renin are enhanced in diabetes and blockade of the renin angiotensin system improves diabetic kidney disease (DKD) and kidney function. Pro-renin is elevated in plasma of patients and mice with type-1 diabetes and plays a role in DKD, independently from its role in blood pressure. The main source of the circulating renin and pro-renin are Juxtaglomerular Cells (JG cells) located in the afferent arteriole. Pro-inflammatory cells infiltrate the diabetic kidney cortex, increasing the release of interleukins (IL). The specific inflammatory cells and their localization in the cortex in diabetic mice is not clear. Our preliminary data shows an increased number of T lymphocytes (Th17) in kidneys from diabetic mice. Our data also show that IL17 increases renin release from JG cells and that receptors for IL17 (IL17-R) are enhanced in JG cells from diabetic mice suggesting that this pathway could be in volved in activation of the kidney renin angiotensin system in diabetes. In this exploratory grant we will identify new subpopulations of inflammatory cells in the renal cortex of diabetic mice by single cell RNA seq and mass cytometry and study their localization in the juxtaglomerular area by CyToF imaging. In addition, we will develop a triple transgenic diabetic mouse with specific deletion of IL17R in JG cells to determine the role of this pathway in DKD.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Cancer-associated cachexia is a debilitated syndrome that has a dramatic impact on the quality of life and outcome of patients. Cancer cachexia occurs with a remarkably high incidence in pancreatic ductal adenocarcinoma (PDAC) patients. Cancer cachexia is characterized by the progressive depletion of muscle skeletal mass, which often culminates in general organ dysfunction and patient death. Therefore, a deeper understanding of the underlying mechanisms of cancer cachexia would ultimately lead to the identification of innovative therapeutics to improve the management of muscle wasting in cancer patients. Our recent studies have shown that pancreas-specific ablation of the Tgif1 gene in a KrasG12D background led to a dramatic acceleration of PDAC in mice. Quite intriguingly, despite having this severe PDAC phenotype, these mice do not display any prominent sign of cancer cachexia, such as progressive weight loss and decreased muscle mass and function. This contrasts with our previous study using two different mouse models of human PDAC, KPC (KrasG12D and heterozygous deletion of Trp53) and KICLuc (KrasG12D and homozygous deletion of p16Ink4a), which consistently manifest severe cancer cachexia during PDAC progression. These findings hint at the possibility that TGIF1 might function in PDAC cells to initiate events that culminate in muscle cachexia. In efforts to probe this possibility, we generated a new mouse model that allows for conditional overexpression of Tgif1, and found that enforcing TGIF1 expression in the pancreatic epithelium was sufficient to induce muscle cachexia. Subsequent mechanistic experiments revealed an ability of TGIF1 to induce expression of FN14, which functions as a transmembrane receptor in cancer cells to drive inflammation leading to muscle cachexia. Enforced TGIF1 expression also induced the expression of the membrane-bound form of transforming growth factor alpha (TGFa), whose overexpression in the pancreas also drives an inflammatory phenotype similar to what we observed in mice with conditional overexpression of TGIF1. Inspired by these intriguing findings, we created a new bispecific antibody (Bis-14a) to simultaneously neutralize the pro-cachectic activities of FN14 and TGFa. This tremendous progress prompted us to design a variety of innovative genetic and antibody-based pharmacological approaches to provide irrefutable proof-of-principle that targeting FN14 and TGFa downstream of TGIF1 could offer innovative therapeutic strategies to curb cancer cachexia. Overarching specific aims are: Specific Aim 1: Expand role and translational potential of TGIF1 in PDAC-driven muscle cachexia Specific Aim 2) Explore the mechanisms by which TGIF1 facilitates PDAC-driven muscle cachexia Specific Aim 3) Test the therapeutic efficacy of monoclonal and bispecific antibodies targeting FN14 and TGFa signaling in PDAC-mediated muscle cachexia As such, completion of this highly innovative proposal will likely culminate in a paradigm shift in our understanding and treatment of this lethal wasting syndrome.

NIH Research Projects · FY 2026 · 2023-12

Project Summary: DNA double strand breaks (DSB) pose harmful threats to genomic integrity. For this reason, cells evolved a diverse set of tightly regulated pathways dedicated to repairing these lesions. In human cells, two major pathways are responsible for repairing most DSBs, including Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ). Mediator of DNA Damage Checkpoint 1 (MDC1) is a key DSBR protein that facilitates HR and NHEJ by binding to γH2AX at DSB sites and promoting RNF8- and RNF168-dependent ubiquitination of H2A at K13 and K15. Considering that MDC1’s functions in HR do not require BRCA1 and its deletion only mildly increases cellular sensitivity to DSBs, the exact mechanism by which MDC1 promotes HR has remained unclear. Using live-cell single-molecule (LCSM) imaging, we recently found that MDC1 is constitutively tethered to chromatin by its intrinsically disordered PST repeat domain, which has unknown functions in DSBR. Compelling preliminary data suggests this domain is essential for MDC1-dependent HR and is dispensable for MDC1- dependent NHEJ. Interestingly, LCSM imaging studies reveal the PST domain may spatially restrict MDC1- dependent HR by directly tethering MDC1 to transcriptionally active chromatin. In addition, our data demonstrate the PST-chromatin interaction can be negatively regulated by phosphorylation, which may link phospho- dependent changes in the PST-chromatin interaction with MDC1’s distinct functions in HR and NHEJ. Therefore, I hypothesize that MDC1-dependent HR is a specialized pathway dedicated to resolving transcription-associated DSBs that depends upon a regulatable interaction of MDC1’s PST domain with chromatin. To test this hypothesis, I will integrate a variety of molecular, biochemical and biophysical approaches with training in ChIP- seq and genome-wide CRISPR screening during the K99 phase of this award to: 1. Identify the molecular mechanism underlying MDC1’s PST-dependent association with chromatin (Aim 1); 2. Elucidate a functional role for MDC1 and its PST domain in transcription-coupled HR (Aim 2); and 3. Define the molecular mechanism of the MDC1-dependent HR pathway (Aim 3). Not only will the Aim 1 and 2 studies uncover the molecular mechanism of MDC1’s PST domain in DSBR, but they will also lead to the identification of MDC1 mutants with pathway-specific DSBR functions. Therefore, in Aim 3 I will use these pathway-specific MDC1 alleles and genome-wide CRISPR screens to genetically define the MDC1-dependent HR pathway. These studies will significantly advance our understanding of the fundamental processes by which cells maintain their genomic integrity by revealing the mechanistic basis for MDC1’s functions in homologous recombination. In addition to the mentored scientific and professional development opportunities provided during the K99 phase, results from the proposed studies will be used as preliminary data for R-series grants to be submitted during the R00 phase of this award.

NIH Research Projects · FY 2025 · 2023-09

Summary Airborne particulate matter with a diameter of <10µm (PM10) is a major global airborne pollutant, with an irritant effect on mucous membranes, causing serious health (cardiovascular and respiratory) and economic outcomes. Pertinent to our studies, clinical evidence has shown that exposure to PM10 is linked to increased emergency room visits for keratitis and dry eye and conjunctivitis exacerbate the problem. Unfortunately, no studies have mechanistically investigated the effect of PM10 on the eye and the link/mechanisms leading to increased microbial infection. Therefore, the long-term goal of this study is to test the hypothesis that in the cornea, PM10 triggers reactive oxygen species (ROS), disrupts nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, leading to inflammation and that this in turn enhances the disease response to bacterial infection. A corollary to this is that inhibition of ROS by SKQ1, a novel mitochondrial targeted antioxidant, will reverse these changes. Preliminary in vivo data showed that airborne exposure of mice to PM10 vs ambient air results in disruption of the Nrf2 pathway, lower levels of reduced glutathione (GSH), and elevated mRNA levels of COX- 2, iNOS, IL-6 and TNF-α, decreased protein levels of Nrf2, and increased levels of malondialdehyde (MDA), the latter indicative of lipid peroxidation. We also showed that PM10 exposure exacerbates Pseudomonas aeruginosa (P. aeruginosa) infection in the mouse cornea with earlier perforation and corneal thinning compared with ambient air exposed mice. In vitro, human corneal epithelial cell cultures support these findings and show that PM10 adversely affects cell viability and that SKQ1 rescues it. Three aims are proposed: Specific Aim 1: Tests the hypothesis that PM10 exposure triggers ROS, disrupts the Nrf2 signaling pathway, decreases cytoprotective genes and leads to corneal inflammation; and that SKQ1, an antioxidant and inhibitor of ROS, reverses these effects. Specific Aim 2: Tests the hypothesis that PM10 exposure exacerbates bacterial keratitis and that SKQ1 alone or as an adjunct treatment to Moxifloxacin improves disease outcome. Specific Aim 3: Tests the hypothesis that PM10 exposure of human corneal epithelial cells parallels the mouse data in that it induces ROS, Nrf2 signaling, decreases cytoprotective genes and that SKQ1 reverses these effects.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Through the research and training described in this K01 proposal, the P.I. (Dr. El-Sabawi) will acquire the necessary skills to become an independent researcher who uses legal epidemiological methods to study and design evidence-based laws and policies that improve the health of persons who use drugs (PWUD). Overdose deaths are surging across the U.S., with 40 states reporting increases in mortalities for the 12 month period ending in May 2022. Some prosecutors have responded to local overdose crises by charging persons who distribute drugs that cause an overdose death with homicide (drug-induced homicide) (DIH), resulting in dramatic increases in DIH prosecutions since 2011. Prior research has demonstrated that increased police activity and enforcement of criminal laws are largely ineffective in decreasing drug use and often lead to poorer public health outcomes for PWUD. However, the effects of the prosecutorial implementation of DIH laws on public health remains largely unstudied —equally as unstudied is the relationship between prosecutorial actors and the local public health and treatment infrastructure. With this 5-year K01, the P.I. aims to examine the prosecutor’s role in addressing the overdose crisis by focusing on how policy implementation decision-making is affected by socio-ecological (S.E.) factors, including the presence of other organizations (public health departments and treatment infrastructure); organizational relationships between the criminal legal, public health, and treatment systems; and the perceived effect of such prosecutions on drug use and treatment- seeking behaviors. The P.I. will conduct exploratory semi-structured interviews of prosecutors (AIM 1), local public health administrators, members of local management entities, defense attorneys, and PWUD (AIM 2) in North Carolina to capture the S.E. factors present in localities where prosecutors have chosen to pursue DIH cases versus the factors present in localities where prosecutors have chosen not to pursue such charges. Using the data gathered in AIMs 1 & 2, the P.I. will develop a Socio-Ecological Model (SEM) of policy implementation explaining how S.E. factors (including public health and treatment infrastructure) influence the implementation of DIH laws and how such implementation is perceived to influence the behaviors and experiences of PWUD (AIM 3). The P.I. will work with an accomplished, multidisciplinary mentorship team (Dr. Taxman, Dr. Rudes, and Prof. Beletsky) to master four relevant areas of training: (1) policy implementation science, (2) advanced legal epidemiological evaluation, (3) qualitative interviewing, and (4) implementation study design. In doing so, this K01 award advances the development of the P.I. as an independent and productive researcher. The research presented in this K01 award addresses an important public health concern and has the potential to advance the field by developing a SEM that posits how criminal legal actors interact with the public health infrastructure in ways that impact the public health of people at risk of overdoses.

NIH Research Projects · FY 2025 · 2023-09

Project Summary / Abstract: Retinitis pigmentosa (RP) is a disease that leads to untreatable and irreversible cone death and blindness. A myriad of loss-of-function mutations, including in transducin 1 or phosphodiesterase 6 genes, underlie RP. Ex vivo studies from experimental models support abnormal mitochondria performance as a common pathogenic condition leading to RP pathology. However, evaluating such mitochondrial abnormalities in patients is not possible and a one-therapy-fits-all approach is unlikely to improve outcomes patient diversity. Addressing these major knowledge gaps will require a patient-friendly, non-invasive biomarker of mitochondria performance. Recently, we discovered a novel index of mitochondria performance based on a feature that is readily identifiable in optical coherence tomography (OCT), the inner segment ellipsoid zone (ISez). Our first-in-kind studies in wild-type mice show that the shape of the ISez profile changes from elongated during a low energy demand condition (light) to rounder during a high energy demand condition (dark). The underlying mitochondria etiology of the change in ISez profile shape is supported by electron microscopy and oxygen consumption rate measurements in two mice strains with distinct mitochondria activity. For example, OCT examination of cyclic-light reared 2-month-old mice with a mutation in the α subunit of transducin 1 (Gnat1rd17) shows modest rod loss with a rounder-than-normal ISez and higher rate of oxygen consumption than in the dark, biomarker evidence for early mitochondria overperformance. Also, at postnatal (P) day 23, dark-reared mice with a mutation in the rod phosphodiesterase 6b gene (Pde6brd10) show modest rod loss together with rounder-than-normal ISez when examined in the dark, biomarker evidence for mitochondria overperformance. When P23 dark-reared Pde6brd10 mice are exposed to room light for 1 hour they showed a more-elliptical-than-normal ISez shape suggesting rod mitochondria underperformance. This is notable because, whilst the 1 hour of light did not cause immediate additional rod death, accelerated rod loss reportedly occurs days later after continued dark- rearing. These considerations show that the ISez profile shape is sensitive to abnormalities in the rod energy landscape that precede later rod loss. The natural history of change of the ISez profile shape as it relates to rod atrophy in cyclic-light reared Gnat1rd17 or Pde6brd10 mice is unknown. Our working hypothesis is that restoring our mitochondria performance biomarker (the ISez profile shape) to wild-type-like levels predicts pro-survival treatment outcomes in experimental IRD. These studies introduce an innovative and clinically relevant imaging biomarker, the ISez profile shape, for assessing treatment efficacy in RP/IRD. Therapies that restore the ISez profile shape to normal are ultimately expected to prevent loss of sight in patients with IRD.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY / ABSTRACT In the height of the opioid crisis in our country, deaths from methamphetamine (METH) overdose are on the rise, and there is no FDA-approved medication for METH use disorder (MUD). New drug targets are needed, particularly for people who heavily use the drug because they have the most difficulty quitting METH use, suffer from a variety of serious neurological problems, and are at high risk to overdose on the drug. Our work with a rat model of heavy compulsive METH consumption (severe MUD) provided evidence that overexpression of neuroprotective protein parkin in the nucleus accumbens (NAc), a key reward-mediating brain area, plays a role in severe MUD. Parkin is a protein-ubiquitin ligase known to play a critical role in maintaining mitochondrial health and, therefore, in maintaining generation of cellular energy - adenosine triphosphate (ATP). Our preliminary proteomic data shows that upregulation of parkin in the NAc leads to upregulation of several Krebs cycle enzymes whereas parkin knockout leads to their downregulation. Dysfunctional mitochondria are the known consequence of METH use. This proposal investigates the novel hypothesis that parkin decreases METH cravings and addictive behaviors by the Krebs cycle function in the NAc from METH neurotoxicity. This helps to maintain ATP generation in METH-exposed NAc neurons, which otherwise would be impaired by METH- induced oxidative stress. In other words, we will test whether dysfunctional mitochondria are a cause of MUD. We will test the hypothesis by three independent specific aims. The specific aim 1 will establish whether and how parkin protects Krebs cycle function in rat NAc from neurotoxicity of self-administered METH. The specific aim 2 will determine whether parkin overexpression in the NAc of rats with developed severe MUD will decrease METH addictive behaviors. The specific aim 3 will determine whether overexpression of oxidative stress- sensitive Krebs cycle enzyme DLST (dihydrolipoamide S-succinyl-transferase) in the NAc of rats with developed severe MUD will decrease METH addictive behaviors. Given that parkin may serve a dual function of decreasing METH neurotoxicity and METH cravings, these studies will determine whether targeting parkin has a significant therapeutic potential in severe MUD. Given the limited indirect data suggesting that METH neurotoxicity in NAc mitochondria contributes to development of MUD, these studies will provide clarity about the role of METH- induced oxidative stress in mediating METH use and in development of motivation to seek METH during abstinence. Furthermore, the results will benefit opioid and alcohol use disorder research as these disorders induce mitochondrial dysfunction. Lastly, since chronic use of METH, particularly at high doses, predisposes to development of Parkinson’s disease and potentially also to Alzheimer’s disease, the results from this research will add information to these research areas.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT This project seeks to investigate the intersection of a rare neurometabolic disorder, sepiapterin reductase (SPR) deficiency, and a relatively rare childhood disorder, cerebral palsy (CP). Both present with motor deficits and sometimes the clinical presentation can be similar. Mutations in the SPR gene result in deficiency of tetrahydrobiopterin (BH4). BH4 is not only a cofactor of five important enzymes in the brain, but is also involved in the pathways of cell death, especially involving oxidants. Of the forms of non-apoptotic cell death, ferroptosis is caused by a slew of oxidants along with the involvement of BH4. Given our past interest in oxidant effects and that antioxidants reduce motor deficits, we propose to study ferroptosis in our rabbit model of CP. The exact role of BH4 will be investigated by comparing the changes in ferroptosis pathways between a knock-in of a human mutation in the rabbit and wild-type rabbits. Generating precise knock-in rabbits representing patient mutations has not been possible until recently, achieved by our multi-principal-investigator team. The knock-in of the human R150G mutation was done through the clustered-regularly-interspaced-short-palindromic-repeats (CRISPR) gene editing platform. The rabbit model of CP utilizes hypoxia-ischemia (H-I) akin to human acute placental insufficiency at preterm gestation, based on the human abruptio placentae. This fetal H-I model is the first to reliably lead to CP, allowing us to rigorously test not only mechanistic pathways but also possible therapies for motor deficits, for which there is none currently available. With the development of a surrogate marker of magnetic resonance imaging (MRI), we can predict which fetuses will develop postnatal motor deficits. This advance allows the early identification of critical pathways causing hypertonia, making an in-depth study of ferroptosis possible, as ferroptosis occurs early after the fetal insult. Our hypothesis is that cell death by specific pathways of oxidant stress related to BH4 determines the development of later CP motor deficits The first Aim will determine whether knock-in of the human mutation R150G in rabbits increases susceptibility to motor deficits. The second Aim will determine whether specific oxidants related to BH4 determine critical ferroptosis that leads to motor deficits. The underlying biochemical mechanisms will be studied utilizing the identification of fetuses destined to get postnatal hypertonia and studying entire brain, brain regions and cell suspensions. Innovations proposed are the systemic integration of MRI as a surrogate marker with flow cytometry techniques, fluorescent and electron microscopy, automated western blot, electron paramagnetic spectroscopy, high performance liquid chromatography, and mass spectrometry into the unique animal model to probe the biochemical basis of ferroptosis, with testing of possible future therapies. These studies will elucidate the early events around critical cell death that cause motor deficits. The clinical importance is that the proposed studies provide the mechanistic understanding for the systematic development of much-needed therapies for prevention of motor deficits from congenital BH4 deficiency and CP.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Development of a universal micro total analysis system (TAS) represents the pinnacle of measurement science. Integrating all aspects of sample preparation, analysis, and detection into an inexpensive, automated platform would streamline analyses and enable rapid validation of biomedical samples. The ideal TAS would not only validate the chemical composition of a biological sample, but also characterize higher order biomolecule structure (e.g. disulfide bonds, chirality, sulfation) to evaluate bioactivity. To date, though, these dreams have not been realized. Consequently, researchers must manually prepare samples for analyses that characterize sample purity, but assessments of biological activity often remain neglected. This time-consuming, incomplete sample validation risks biasing results of subsequent research studies. To help improve the rigor and reproducibility of NIH-sponsored projects, we propose to develop a universal TAS to provide researchers with a tool to rapidly validate biomedical samples, including evaluations of higher order biological structures that dictate activity. Thermal gel electrophoresis (TGE) will serve as the heart of the TAS. Our group developed TGE to enrich, separate, and detect biomolecules within a temperature-responsive gel, thus integrating multiple steps of an analytical method into an inexpensive microfluidic device. Building on our prior work, we propose to further expand our capabilities towards the ideal comprehensive TAS. Additional characterizations will be developed to screen the higher order structure of proteins, peptides, RNAs, and sugars with high selectivity and sensitivity that are inaccessible to other techniques (e.g. LC-MS). To streamline analyses, sample preparation capabilities will be integrated into devices to filter cells, desalt samples, and label analytes for detection. This approach will enable direct analysis of biological samples on-chip, obviating the need for external sample pretreatment by the user. Additionally, label-free detection schemes will be developed to further expedite analyses and simplify operational constraints. Collectively, the innovative analytical strategies developed here will provide a convenient, inexpensive means of characterizing biomedical samples that cannot be achieved by other techniques. Ultimately, we envision our TGE-based TAS platform will make robust sample validation accessible to researchers, which will increase reproducibility of biological studies in academic, government, and industry laboratories.

NIH Research Projects · FY 2025 · 2023-09

Significant progress in subcutaneous insulin administration (SIA) technology has been realized over the past two decades. Nonetheless, SIA technology failure and underlying tissue damage caused by insulin phenolic preservatives (IPP) present in all commercial insulin formulations could impede the progress of SIA technology. Limited wear time accompanied by SIA device site rotation are the current solutions to minimizing tissue damage and maintaining infusion or injection site integrity over time. These practices, while ultimately beneficial, will not allow for drug delivery devices to perform beyond the current recommended wear time of three days. Extending SIA technology to align with current continuous glucose monitoring sensors, approved for 10-14 days of wear, is a significant unmet need. Challenges to extending the lifespan of infusion pumps or injection ports involve surmounting the IPP-induced tissue reactions of inflammation and fibrosis at these devices’ location. Insulin formulations are also susceptible to mechanical and chemical stressors that lead to non-functional insulin molecules through polymerization designated as insulin fibril formation (IDF), even in the presence of IPP. Our published and preliminary data indicate that both, IPP and IDF, are pro-inflammatory. This pro-inflammatory response leads to cumulative cell/tissue toxicity, inflammation, and maladaptive wound healing. To overcome this challenge, we opine that optimum IPP reduction and IDF removal at the time of insulin dosing, in-line and just in time, rather than focusing on the preparation of new insulin formulations provides a more elegant solution. Thus, the objective of this proposal is to design, fabricate and validate an in-line ß-cyclodextrin-based adsorbents platform that 1) can reduce IPP levels in commercial insulin formulations, and 2) remove any IDF formation in-line and in a “just in time” mode, i.e., just before SIA. Commercial insulin formulations passed through this platform are able to mitigate blood glucose levels without triggering acute and chronic SIA-induced inflammation and fibrosis. This would achieve physiological euglycemia, while preserving long-term tissue integrity at SIA site. To achieve these goals, we have developed the following three specific aims: 1) Design and evaluate ß- cyclodextrin-based adsorbents in insulin phenolic preservative removal platforms, 2) Design and evaluate micro/ultrafiltration-based membranes (MFM) as IDF removal platforms, and 3) Preserve long-term tissue integrity and bioactivity during SIA through usage of ß-cyclodextrin-based adsorbent (beads and MFM filtration) platforms in a pre-clinical porcine model. Ultimately, the successful accomplishment of this project could result in transforming current diabetes management practices that would achieve the goals of increasing the lifespan of insulin infusion devices and most importantly, sustaining tissue site viability for future recurrent insulin administrations.

NIH Research Projects · FY 2026 · 2023-09

Therapeutically delaying the progressive decline in cognition in patients with Alzheimer’s disease (AD) would transform AD into a manageable morbidity, a goal that has not been achieved using drugs targeted to β-amyloid (Aβ) plaque deposition. Accumulating results indicate that cognitive loss (linked to circuit / synaptic dysfunction) and β-amyloid (Aβ) plaque deposition can occur independent of each other, with both driven by a cross-linked soluble amyloid β-peptide oligomer - neuronal hyperactivity “AD cycle”. Remarkably, the prediction that cognitive dysfunction can be restored without altering plaque deposition has been confirmed in several AD models, for example, by drugs that prolong the opening time of the endoplasmic reticulum (ER) ryanodine receptor type 2 (RyR2) calcium channel and suppress neuronal hyperactivity. Conventional biomarkers are unable to interrogate either part of the “AD cycle” in patients at cellular resolution, an unmet goal for evaluating treatment efficacy at the prodromal stage. Here, we propose a novel solution to this problem based on the retina, a readily accessible part of the nervous system with damage similar to that found in the brain of patients with AD. The retina develops soluble amyloid β-peptide oligomers and plaque deposition before their appearance in the brain, as well as phosphorylated tau and neurofibrillary tangles. Before overt AD pathology and cognitive decline are evident, patients report impaired contrast sensitivity (CS), a major risk factor for falls as well as decreased survival. CS is driven by photoreceptors. Our first-in-kind preliminary results in an AD model when there is sparse plaque deposition in the retina show early impairment of CS, and rod hyperactivity measured using three OCT mitochondria-driven biomarkers developed in our laboratory. We have also discovered that CS impairment and rod hyperactivity biomarkers in 5xFAD male C57BL6/J (B6J) mice occur faster than in 5xFAD male C57BL/6Tac (B6NTac) mice. In WT male B6J mice, rods showed a lower OCT energy signature than in age-matched WT male B6NTac mice, indicating strain differences in baseline mitochondria activity. We propose to test two working hypotheses with three Specific Aims. First, that impaired CS, a hyperactive rod energy signature, and/or synaptic dysfunction occur earlier B6J 5xFAD mice than in B6NTac 5xFAD mice. Second that in 5xFAD mice, RyR2-targeted treatments that delay cognitive declines mitigate changes in early CS and energy biomarkers, declines in rod synaptic activity, and later spatial memory deficits but do not change the rate of plaque deposition.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide, with healthcare costs in the U.S. exceeding $1 billion annually to treat frequent and unpredictable vaso-occlusive episodes (VOEs). Despite the same, monogenic disease that results in the production of hemoglobin S (HbS) causing mature red blood cells (RBCs) to sickle following deoxygenation, the frequency of VOEs amongst SCD patients is highly variable. Sickle reticulocytes (immature RBCs) contribute to VOEs by participating in a series of adhesive events mediated by cell surface adhesion molecules that delay blood flow in small blood vessels which promote sickling and entrapment of RBCs in the microvasculature. Hydroxyurea (HU), the mainstay therapy for SCD, reduces but does not eliminate VOEs, hence treated patients remain at considerable risk for debilitating VOEs. Initially, HU was administered to induce hemoglobin F (HbF) expression with anti-sickling effects, although HU also provides immediate clinical benefits by increasing nitric oxide (NO) and cGMP levels (NO 2nd messenger) and decreasing adhesion receptor expression and RBC-endothelial interactions. There is some compelling evidence that HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity; however, specific mechanisms are unclear. A better understanding of HU mechanisms that reduce adhesive interactions will reveal novel therapeutic targets to reduce VOEs effectively in SCD. My long-term goal is to identify cellular and molecular targets to aid in the development of effective therapies to improve the care for SCD patients. This project will enhance our knowledge of the pathobiological mechanisms underpinning SCD and will allow us to gain new insights into molecular pathways that influence the clinical manifestations and severity of SCD. Novel concepts proposed in the research proposal combined with a detailed training plan and mentorship from a highly accomplished team of basic science, translational, and clinical researchers will also facilitate my career development. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on reticulocytes and white blood cells (WBCs). Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands elevated in the SCD micro-environment. VLA-4 expressing reticulocytes and WBCs are elevated during VOEs and in SCD patients with severe disease phenotypes, but decreased in response to HU therapy. Utilizing our standardized, flow adhesion bioassay, we previously showed that VLA-4 binding can be used clinically to stratify SCD patients based on disease severity and predict impending VOEs. We have also shown that reticulocyte and HbF levels strongly correlate with VLA-4 binding and VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. More recently, preliminary data from my lab demonstrate that HU reduces VLA-4 activity/binding affinity in sickle reticulocytes. Others have shown that VLA-4 binding is enhanced by a kinase-dependent mechanism and reduced through NO/cGMP pathway signaling; yet, specific mechanisms in sickle RBCs are unclear. My central hypothesis is that HU reduces RBC-endothelial interactions in SCD by decreasing kinase activity through a NO/cGMP-dependent pathway. Using our standardized flow adhesion assay, optimized flow cytometry protocol, and mass spectrometric approach, I will test this hypothesis in the following specific aims: 1) Determine the effect of HU on VLA-4 activity/binding affinity; and 2) Elucidate HU mechanisms that modulate VLA-4 protein- protein interactions and post-translational modifications in sickle RBCs.

NIH Research Projects · FY 2025 · 2023-09

Herpes stromal keratitis (HSK) is a leading cause of infection-induced vision loss in the United States. The HSK develops in response to corneal herpes simplex virus-1 (HSV-1) infection. Recurrent corneal HSV-1 infection can cause the persistence of innate immune cells, such as monocytes, neutrophils, and effector CD4 T cells in the corneal stroma of the infected cornea. As the accumulation of neutrophils and effector CD4 T cells in the HSK cornea plays a pivotal role in orchestrating the tissue damage, the strategies to manipulate the stay of infiltrating immune cells in the HSK cornea are anticipated to have an effect in reducing the HSK severity. Chemokine receptors, such as CXCR4, can play an important role in the retention of inflammatory cells in the injured tissue. CXCR4 signaling is also reported to enhance hemangiogenesis in ischemic tissue. Our preliminary results showed an increased expression of CXCR4 and CXCL12 in HSV-1 infected cornea. We recently standardized a protocol to separate an intact corneal epithelium (CE) and corneal stroma (CS) from uninfected and HSV-1 infected cornea. Single-cell suspension prepared from individual CE and CS can be used for flow cytometry. Using this strategy, we detected the presence of CXCR4-expressing cells in uninfected CE of C57BL/6 mice. Our results showed that most CXCR4-expressing cells in CE of naïve cornea display the phenotype of Langerhans cells (LCs) or plasmacytoid dendritic cells (pDCs). Recently, monoamines have been shown to engage CXCR4 on pDCs and inhibit IFN-a production. Histamine is a monoamine that is released upon corneal HSV-1 infection. Experiments proposed in aim 1 will test the hypothesis that histamine released in HSV-1 infected cornea engages CXCR4 on pDCs, and downplays IFN-a production from CE pDCs, increasing viral load. Our results also showed that the number of LCs decreases in the CE but increases in the CS from 4- day to 9-day post-infection (p.i.), suggesting a possible migration of these cells from CE to CS in HSV-1 infected corneas. An increased expression of CXCL12 (CXCR4 ligand) was detected in CS than CE of the infected cornea at 9-day p.i. Furthermore, most CD4+ T cells in the CS of infected cornea displayed effector (Ly6Chi) phenotype at 9-day p.i. Experiments proposed in aim 1 will test the hypothesis that LCs-mediated restimulation of effector CD4 T cells in the CS of the infected cornea is essential for their retention and effector function. Aim 1 experiments will also test whether LCs migrating out of CE and neutrophils infiltrating in the CE of HSV-1 infected cornea impair corneal epithelial healing. In addition to corneal resident innate immune cells, our results also detected the expression of CXCR4 on infiltrated neutrophils and vascular endothelial cells from newly formed blood vessels in the CS of HSK cornea. Experiments proposed in aim 2 will test the hypotheses that blocking CXCR4 signaling in HSK cornea will cause reverse migration of neutrophils and inhibit the development of hemangiogenesis. The successful completion of the studies proposed in this application will underscore the pleiotropic effects of CXCR4 signaling in HSK cornea and document it as a potential therapeutic target in HSK.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Air pollution is a major environmental health threat and is associated with several adverse health outcomes in children and adolescents including asthma, obesity, and childhood cancer. Growing evidence indicates that air pollutants, including particulate matter (PM), can also negatively affect brain development and increase risk of poor mental health outcomes. Indeed, recent work has shown that exposure to air pollution, specifically PM2.5 (PM with an aerodynamic diameter < 2.5 μm) is associated with both the prevalence and severity of anxiety disorders in youth. Further, anxiety disorders commonly begin during adolescence and early-onset (vs. adult- onset) is associated with poor long-term outcomes, including more chronic disease and poorer treatment response. However, the neurodevelopmental mechanisms underlying environmental risk of anxiety are unknown. The proposed F32 will be the first to test the novel hypothesis that adolescents exposed to higher recent PM2.5 concentrations will exhibit poor fear extinction recall, lower frontolimbic activation, and higher anxiety symptoms. This project builds on prior research demonstrating that impaired fear extinction and frontolimbic dysfunction are neurodevelopmental markers of anxiety disorders, and our recent and preliminary data show that fear regulation and frontolimbic circuitry (i.e., hippocampus, ventromedial prefrontal cortex) develop during early adolescence and are sensitive to environmental insults (e.g., traumatic stress). Further, emerging preclinical and human neuroimaging studies suggest that fear-related learning and frontolimbic brain regions are susceptible to PM2.5 exposure, particularly during adolescence, a period of psychiatric vulnerability. The proposed study will recruit adolescents exposed to recent PM2.5 concentrations, estimated using state-of-the-art high resolution (0.74 km2) spatiotemporal models developed by Co-Sponsor Brokamp. Participants will complete a two-day fear extinction functional magnetic resonance imaging (fMRI) experiment developed and validated by Sponsor Marusak to probe fear regulation and frontolimbic circuitry. This paradigm uses virtual reality coupled with psychophysiological recordings and neuroimaging. This fellowship study provides an important first step towards identifying neurodevelopmental mechanisms underlying environmental risk of psychopathology, and will inform targeted early interventions to stem the etiology of anxiety in at-risk pollution-exposed youth. With key training in environmental impacts on brain development, psychophysiology and fMRI, and the neurobiology of pediatric anxiety, this project is ideally suited for the F32 mechanism. This project is supported by a team of mentors with complementary expertise, including Sponsor Marusak and Co-Sponsors Jovanovic, Ryan, Strawn, and Brokamp. This training project will provide PI Zundel with the critical data and training needed to expand on this work longitudinally, evaluating developmental trajectories in pollution-exposed youth. It will also prepare PI Zundel for a career committed to uncovering neurodevelopmental mechanisms contributing to environmental risk of neuropsychiatric disease.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Bone metastatic disease correlates with increased morbidity and mortality in prostate cancer (PCa) patients. Various studies, including our own, have determined that the bone marrow niche plays a supportive role during metastatic progression, which leads to increased tumor cell survival and escape from therapy, but the molecular mechanisms are not fully understood. Our previous studies have highlighted adipocytes, an abundant cell type in the bone marrow, as key modulators of tumor cell energetics and contributors to their aggressive phenotype. We have demonstrated that adipocyte-supplied lipids, released upon adipocyte-tumor cell crosstalk, induce pro- survival phenotype and promote ER and oxidative stress activation as potential cell adaptation mechanisms in PCa cells. My new preliminary data have now uncovered novel molecular consequences of tumor cell-adipocyte crosstalk, including the following: 1) Activation of an adaptive response to lipid peroxidation in PCa cells is mediated by adipocyte-supplied lipids; 2) Augmented expression of ER stress gene, ATF4, and lipid desaturation enzyme, SCD, in PCa cells is a possible defense mechanism against damaging effects of lipid peroxidation; 3) ER stress gene, ATF4, plays a potential role in regulating SCD levels and activity in PCa cells grown under lipid- rich conditions; and 4) Adipocyte-supplied lipids are likely involved in regulating the tumor metabolome. Stemming from these findings, the central hypothesis of this proposal is that adipocyte activation of the ATF4- SCD axis modulates tumor metabolism to promote the survival of PCa in bone. I propose a multi-faceted approach that includes cell culture and mouse models of lipolysis, in vivo models of intratibial tumor growth, as well as state-of-the-art RNAseq and metabolomics approaches to determine the role of previously unexplored ATF4-SCD axis in regulating tumor metabolism and promoting progression in bone. These studies will be performed in two aims. In Aim 1, I will utilize mice deficient in lipolysis and create human and murine PCa cells with stable knockdown of ATF4 to study the role of adipocyte-supplied lipids in modulating the ATF4-ER stress response pathway to promote progression in bone. In Aim 2, I will focus on the ATF4-SCD interaction and utilize RNA-seq and metabolomics approaches to determine how lipid-mediated activation of this axis alters tumor metabolome. Altogether, the results of this study will be used to delineate key molecular mechanisms by which metastatic PCa cells engage ATF4 and SCD to survive in the lipid-rich bone marrow. This work will also likely reveal potential novel options for targeting tumor metabolism for improved therapy and/or prevention of aggressive disease. This proposed work will have broad implications for numerous cancer types that metastasize to bone or those that arise from bone and are likely to be affected by the bone marrow adipose tissue.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The work in this proposal builds on our expertise in catalytic stereoselective glycosylations to result in efficient synthetic methodologies for construction of the challenging glycosidic bonds. We recently discovered that readily available phenanthrolines, rigid and planar organic compound with two pyridine rings fused to a benzene ring, effectively act as nucleophilic catalysts to promote stereoselective 1,2- cis glycosylation reactions of alcohol nucleophiles with both pyranosyl and furanosyl bromide donors. The phenanthroline catalytic system provides efficient access to a myriad of 1,2-cis pyranosides and furanosides bearing the C2-oxygen, -azido and -fluoro functional groups under mild and operationally simple conditions. The phenanthroline-catalyzed methodologies represent long standing synthetic challenges for highly desirable glycosylations to generate biologically important oligosaccharides and glycopeptides to advance an understanding of their biological functions. In this R35 grant, we continue uncovering the simplicity and versatility of phenanthroline catalysts to provide a new principle for the stereoselective construction of the challenging α-2-deoxy glycosides. Precisely tailored phenanthroline catalysts can activate both the alcohol nucleophile and the glycosyl halide electrophile simultaneously. We will also investigate the dual activation mechanism of the phenanthroline catalysts to control site- selective coupling of polyol nucleophiles and chemoselective coupling of the hydroxyl of serine residue in the presence of the thiol of cysteine-containing peptides. Further, we will apply the phenanthroline catalyst platform to the synthesis of heparan sulfate-like oligosaccharides as inhibitors of heparanase, which is a druggable target for anticancer therapy. We will also develop the library of heparan sulfate mimetics with all of the possible O- and N-sulfation motifs from readily available aminoglycosides to advance an understanding of the role of sulfated glycosaminoglycans in many biological systems. In particular, we will investigate these structurally well-defined heparan sulfate mimetics as the potential binders of clinically important fibroblast growth factors.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Positron Emission Tomography (PET) with fluorodeoxyglucose (FDG) has revolutionized molecular imaging and substantially improved diagnosis and monitoring response to treatment of many deadly diseases such as cancer. However, FDG-PET technology has a number of limitations including long examination time, long pre-scan fasting time, and the use ionizing radiation. Hyperpolarization of nuclear spins increases their alignment with the field of an MRI scanner by 4-6 orders of magnitude, resulting in corresponding gains in the MRI signal. As a result, it becomes possible to detect low-concentration metabolites in vivo. Furthermore, spectroscopic MRI enables detection of real-time metabolism of an injected exogenous hyperpolarized contrast agent because it can map the injected metabolic probe and its products. The entire hyperpolarized MRI scan is performed in approximately 1 minute. The leading hyperpolarized contrast agent is [1-13C]pyruvate, which probes the biochemical pathways of aberrant energy metabolism at the cellular level. This next-generation technology has the potential to revolutionize molecular imaging in the future. It is now being evaluated in nearly 30 clinical trials. The hyperpolarized state of [1-13C]pyruvate is currently produced at clinical-scale via dissolution Dynamic Nuclear Polarization (d-DNP) technology, which employs cryogenic temperature, high magnetic field, and high- power microwave irradiation. This technology is very slow: it takes approximately 1 hour to produce a clinical dose. Minor concerns are the high cost of over $2M and requirement for expensive cryogens for operation. Faster and more affordable approaches are needed to make hyperpolarized [1-13C]pyruvate accessible for widespread clinical use. In 2015, we have co-invented an alternative technology for low-cost production of metabolic probes called Signal Amplification by Reversible Exchange Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH). In 2019-2022, we and others have demonstrated that hyperpolarized [1- 13C]pyruvate can be produced using this new technique, which relies on the simultaneous exchange of parahydrogen gas (the source of nuclear spin hyperpolarization) and [1-13C]pyruvate on metal complexes. Unlike d-DNP, SABRE-SHEATH is highly scalable, rapid (1 min) potentially allowing to produce over 10 doses per hour. Moreover, our collaboration has demonstrated the feasibility of removing the SABRE catalyst from hyperpolarized solutions to prepare catalyst-free solutions of hyperpolarized compounds. This proposal focuses on addressing the key remaining aspects of SABRE-SHEATH to prepare bio-compatible formulations of hyperpolarized [1-13C]pyruvate contrast agent. Specifically, the investigators will develop and optimize the instrumentation (based on an already commercialized prototype) that will integrate (1) clinical-scale (~1 g dose) production; (2) SABRE-catalyst extraction; and (3) reconstitution in a biocompatible buffer, followed by feasibility studies in cells. We anticipate that our end product of this two-year award, i.e., the developed instrumentation (a.k.a. hyperpolarizer) will enter clinical trials and will be commercialized.