University Of Nebraska Medical Center

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY Base excision repair (BER) is a critical mechanism for preventing the mutagenic and lethal consequences of DNA damage generated by endogenous reactive chemical species or exposure to environmental hazards. BER is multi-step pathway that requires a tight coordination between the repair proteins. The downstream steps of BER pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing by DNA ligase (ligase I or IIIα). This step-to-step coordination is orchestrated by non-enzymatic scaffolding protein X-Ray Repair Cross Complementing 1 (XRCC1) that plays a key role in assembling repair proteins. Although the roles of the individual enzymes are largely studied, how the multi-protein BER complex coordinates while maintaining the repair efficiency remains unclear. Though often considered an accurate process, the BER can contribute to genome instability if normal coordination breaks down. For example, the mutations in the polβ gene that have been found in many human cancers result in the modifications in its repair functions that impair BER efficiency. Similarly, XRCC1 cancer-associated variants with a defective scaffolding role predispose the cell to genomic instability and transformation. Failure in the BER pathway coordination could result in the formation of strand-break repair intermediates that are more mutagenic or toxic than the initial DNA lesions. My research program will fill the important gap of knowledge in the BER field by elucidating the molecular components of multi-protein BER complex that are necessary for accurate repair and define the ramifications of defective pathway coordination during DNA ligase I and IIIα activities. We are in a unique position to advance this scientific front based on our strong track record and our multidisciplinary approach. In Project 1, we build off our substantial prior work using biochemical and biophysical approach to define the molecular mechanism by which polβ, DNA ligases I and IIIα execute the repair pathway coordination. Our studies will also elucidate cancer-associated XRCC1 and polβ variants with altered BER functions as an important determinants of defective pathway coordination. In Project 2, using X-ray crystallography, we will elucidate the features of DNA substrate and ligase interaction that dictate accurate versus mutagenic outcomes during final nick sealing step at atomic resolution. This project will be extended with cryo-EM to define the structural architecture of large BER multi-protein complexes scaffolded by XRCC1 that dictates accurate repair pathway coordination. With these 2 Projects, my laboratory will launch a new and unique aspect of the research conducted by my group which seeks to better understand the mechanism by which a multi-protein repair complex coordinate during BER and answer several key questions regarding how a tight coordination is vital for maintaining the integrity of our genomic DNA, functions normally and how altering these functions stemming from a failure in the repair pathway coordination leads to disease.

NIH Research Projects · FY 2025 · 2022-08

Pancreatic cancer (PC) is the fourth leading cause of cancer death and often goes undiagnosed until it has already advanced and metastasized. Aberrant changes in O-glycans, such as increased expression of truncated carbohydrate antigens (Tn, sialylated Tn/STn), are commonly observed in PC. However, the mechanistic involvement of these truncated O-glycan structures in PC progression and metastasis is under-explored. Hence, our study is focused on investigating the mechanistic role of truncated O-glycans during early metastatic dissemination in PC. The O-glycosyltransferase Core 1 β1,3-Galactosyltransferase (C1GALT1) catalyzes the second step of mucin-type O-glycan biosynthesis by adding galactose to the first sugar N-acetylgalactosamine (Tn) that forms the Core 1 carbohydrate structure. Such structures are usually elongated to mature O-glycans found on normal tissue, but their extension may be truncated at the Tn-glycan stage during cancer due to inactive C1GALT1 activity. Our preliminary data demonstrated the loss of O-glycosyltransferase activity, C1GALT1, in a subset of (poorly differentiated) human PC tissue. Further, CRISPR/Cas9-based C1GALT1 knockout (KO) in PC cells resulted in aberrant O-glycosylation (increased Tn and STn glycans). Along with glycan alterations presented upon oncogenic glycoproteins (mucin glycoproteins and cancer stem cell markers), our studies also indicate increased tumorigenicity and metastasis of C1GALT1 KO PC cells. We have also observed O-glycan truncation present on CD44, a cancer stem cell marker, in C1GALT1 models. Interestingly, knockout of C1galt1 along with KrasG12D and Trp53R172H/+ mutations in mouse models resulted in early-onset (in 3 weeks) and early distant metastasis (in 10 weeks) of PC. Based on these observations, our major goal is to investigate the mechanistic role of truncated O-glycans in PC progression and metastasis. Based on these observations, we hypothesize that "Truncated O-glycans on cancer-associated glycoproteins (mucins and stemness markers) induce the early onset of progression and metastatic dissemination in pancreatic cancer." To test this hypothesis, the following aims are proposed. The first aim will investigate the functional impact of C1GALT1 expression and aberrant glycosylation profile on cancer-associated glycoproteins in pancreatic cancer. The second aim will elucidate how truncated O-glycans (such as Tn and STn) on membrane-bound mucins and stemness markers facilitate pancreatic cancer metastasis. The third aim will determine, in vivo, the impact of truncated O-glycans in the early onset of pancreatic cancer metastasis using C1galt1 knockout KC and KPC mice. The proposed studies will establish the association of aberrant expression of truncated Tn and STn glycans with differential membrane- bound mucin function during PC progression and metastasis. This study will significantly contribute to our knowledge of mucin glycobiology in cancer. Altogether, this proposed study will also pave the way for developing novel therapeutics for modulating membrane-bound mucin function in PC.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY Pancreatic ductal adenocarcinomas (PDA) are unrivaled in their lethality. PDAs have the highest 1- year, 5-year, and 10-year mortalities of any cancer and are expected to become the second-leading cause of cancer-related death by 2030. An invasive PDA represents the coordinated evolution of cell- intrinsic and extrinsic processes and capabilities that subvert and repurpose the dictums of normal tissue composition, architecture, and physiology to foster unbridled growth and colonization. This new organizational entity is constructed largely at the behest of the mutated epithelial cell. The resulting PDA neo-organ contains a minority of tumor epithelial cells amidst a heterogeneous sea of non- epithelial cells; a complex interstitial stew of proteins, proteoglycans and glycosaminoglycans, together with both freely mobile and complexed water; and a paucity of vessels that otherwise resemble a normal vasculature in lacking fenestrae or interendothelial junctions, but that are collapsed under intense interstitial pressures. We have undertaken a systematic exploration of the cell autonomous and non-cell autonomous processes that drive PDA pathogenesis and resistance. We have developed genetically engineered animal models that faithfully recapitulate the clinical syndrome, metastatic behavior, histopathology and molecular features of the human disease as primary platforms to both uncover critical principles of disease biology and to rigorously test strategies to overcome them. Through such investigations we have identified unusually high concentrations of intratumoral hyaluronan (HA) as the primary culprit in the extraordinarily elevated interstitial pressures in PDA that, in turn, cause the vascular collapse and hypoperfusion characteristic of this disease. The stromal barrier to perfusion also serves as a primary mechanism of drug resistance in limiting the penetration of systemically delivered agents. We have additionally identified multiple mechanisms of immune suppression that prevent the development of an endogenous effector T cell response. Collectively, these unique aspects of stromal biology in PDA conspire to create a drug- and immune-privileged sanctuary for unimpeded growth of the pancreas cancer cell. Very recently, we have elaborated strategies to overcome critical aspects of these physical and immunological barriers to therapy revealing a perhaps unexpected degree of vulnerability once the barriers are breached. We describe a series of continuing investigations into this overarching strategy of stromal re-engineering to build upon the significant inroads made – and the important lessons learned – in the hopes of radically transforming the approach and prognosis for this formidable disease.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Replication stress leading to genome instability is an early driver of tumorigenesis and has been associated with overexpression of oncogenes. In normal cells, activation of the DNA damage response (DDR) pathway serves as a barrier to tumorigenesis leading to cell cycle arrest inducing cellular senescence or cell death in response to high burden of genome instability. However, in cancer cells upon oncogene-induced replication stress the protective barrier of DDR, cell death and senescence is bypassed leading to uncontrolled cell proliferation. Therefore, deciphering the mechanisms that bypass oncogene-induced replication stress and senescence will help understand the basic science underlying disease progression and will identify new targets for therapy. Ewing sarcoma (EWS) is driven by a chromosomal translocation and in-frame gene fusion between EWSR1 and ETS family of transcription factors. In majority of the EWS cases, the chromosomal translocation results in the generation of EWS-FLI1 oncogene. EWS-FLI1 functions as an aberrant transcription factor that drives the development and progression of EWS. Expression of EWS-FLI1 oncogene leads to oncogene-induced replication stress and genome instability. However, the molecular mechanism underlying bypass of EWS-FLI1 oncogene-induced replication stress response pathways is largely unknown. Our preliminary data shows that USP1 deubiquitinase is overexpressed in EWS cell lines and tumors. USP1 regulates DDR and is required for genome stability and stem cell maintenance. We find that USP1 expression is regulated by EWS-FLI1 in EWS. Importantly, inhibition of USP1 activity using small molecule USP1 inhibitors resulted in growth arrest of EWS cell lines indicating that USP1 expression and activity is important for EWS cell proliferation and progression. Notably, USP1 depletion led to a decrease in the levels of HELLS chromatin remodeling protein. The function of USP1 or HELLS in EWS pathogenesis has not been investigated. In this study, we will examine the regulation of HELLS by USP1 deubiquitinase (Aim 1), determine the mechanism by which USP1 promotes EWS cell proliferation (Aim 2), and determine the effect of USP1 knockdown on EWS tumor formation in vivo and the efficacy of USP1 inhibition in combination with chemotherapeutic drugs at suppressing EWS cell proliferation (Aim 3). Successful completion of this study will unravel novel mechanistic insights into USP1 mediated bypass of EWS-FLI1 oncogene-induced replication stress and help evaluate USP1 targeted treatment strategies for EWS.

NIH Research Projects · FY 2025 · 2022-08

Abstract Text Many antitubulin agents, such as paclitaxel (Taxol), have been used extensively for treatment of several types of cancer, including ovarian, lung, pancreatic, and breast cancers. Despite their wide use in cancer treatment, however, patient response is highly variable and drug resistance remains a major clinical issue. It is therefore essential to identify prognostic markers to predict the patient response and to enhance drug sensitivity. Protein kinase R (PKR) plays significant roles in innate immune response to viral infection and tumorigenesis. The biological significance of PKR in antitubulin chemotherapeutics and underlying mechanisms have yet to be defined. Through Phos-tag-based kinome-wide biochemical screens, we identified PKR as a critical regulator in antitubulin cytotoxicity. Our preliminary data suggest that inactivation of PKR confers resistance to Taxol in ovarian and breast cancer cells. Enhanced expression of PKR potentiates taxol cytotoxicity in vitro and in vivo. We further identified novel phosphorylation sites on PKR during antitubulin-mitotic arrest and in normal mitosis. Mechanistically, our findings also suggest that PKR controls Taxol chemosensitivity through modulating Bcl2 expression. Our hypothesis is that the PKR-Bcl2 axis functions as a therapeutic target for antitubulin agent-based chemotherapeutics in treatment of drug-resistant and/or recurrent patients. We will test our central hypothesis by three specific aims. Aim 1: Examine the functional significance of PKR in antitubulin chemotherapeutics in vivo; Aim 2: Determine how antitubulin drugs regulate PKR; Aim 3: Elucidate the mechanisms and downstream signaling of PKR in antitubulin chemosensitivity. The identification of new regulators and/or signaling pathways triggered by antitubulin drugs will shed light on the mechanisms underlying chemoresistance. Our study suggests that combining kinase activators (e.g., being characterized in this application) for PKR or Bcl2 inhibitors (FDA-approved) with antitubulin agents will have enhanced efficacy in treatment of drug-resistant and/or recurrent patients. Our findings also suggest that profiling PKR-Bcl2 signaling status of tumors (mRNA/protein levels and activity) can be useful to predict the patient response to antitubulin chemotherapeutics.

NIH Research Projects · FY 2026 · 2022-08

Abstract Superoxide dismutases (SODs) are the major regulators of oxidative stress and therefore the first line of defense to protect organisms against metabolic- and environmentally-induced reactive oxygen species (ROS). Human mitochondrial manganese SOD (MnSOD) expression is modulated to prevent ROS-based damage, promote redox homeostasis, and maintain proper cell signaling. Our research goal is to understand the molecular basis of how MnSOD uses coupled proton-electron transfers to dismute superoxide. For this, the 3D arrangement of all atoms is needed, most importantly the position of protons. Our recent technical advancements with neutron crystallography at Oak Ridge National Laboratory have overcome the limitations of X- ray crystallography – revealing proton positions with high detail while also allowing control of the metal electronic state. In this research project, MnSOD neutron maps will reveal the proton relays to the active site metal and the protonation states of metal- bound ligands. The scientific hypothesis for this study is that MnSOD transfers protons from a small group of water molecules via partially solvent-exposed amino acids to the nearly completely buried manganese for the dismutation of superoxide to hydrogen peroxide and molecular oxygen via cyclic metal redox reactions. The specific aims are to characterize the electron-coupled proton relays of MnSOD by investigating the proton environment of (1) the resting states of the reduced and oxidized manganese active sites, (2) the product inhibited Mn-peroxo complex, and (3) the superoxide bound enzyme. Spectroscopy on crystals will be performed to help design/understand crystallographic experiments, and computational chemistry studies on neutron derived all-atom structures will help tie the results together and test our interpretations about the enzymatic activity. The resulting protocols, methods, and structures will be of specific interest to those in the fields of structural biology, antioxidants, and metallo-enzymology and of interest to biologists in general.

NIH Research Projects · FY 2024 · 2022-07

Abstract: Contemporary estimates suggest that more than 40% of people living with chronic HIV-1 infection (PLWH) have diastolic heart failure (dHF), a harbinger for adverse clinical outcomes including pulmonary abnormalities, frequent hospitalizations, and sudden death. To date, the molecular causes for dHF in PLWH remain poorly understood. This paucity of information and a lack of treatment options have prompted the OAR to list “Strategies to Prevent and Treat HIV-Associated Heart Diseases” as areas of high priority for HIV research. We hypothesize that that “elevation of the cytotoxic glycolysis metabolite, methylglyoxal (MG) is a primary cause for dHF development in PLWH.” This elevation in MG is arising from HIV-1 induce upregulation of glycolysis in infected immunocytes followed by ischemia-induced increase in glycolysis in vascular cells and cardiac myocytes. This multi-PI project brings together the expertise of Drs. Keshore R. Bidasee (M-PI, heart failure) and Santhi Gorantla (M-PI, humanized mice and HIV-1 infection) with assistance from Dr. Prasanta Dash (HIV-1 eradication and cardiovascular complications), to (1) Define pathobiological trajectories of dHF in relation to MG levels in HIV-1 infected Hu-mice with and with ARD treatment; (2) Characterize mechanisms by which MG increases in HIV-I infected immunocytes and in myocytes, macrophages and vascular cells under with and without ARD and hypoxia (3) Show that lowering MG will blunt dHF in HIV-infected Hu-mice with and without ARD. Accomplishments of these aims will not only define a novel link between glycolysis and early-onset dHF in the setting of HIV-1 infection, but the data could pave the way for the development of urgently needed therapeutics to mitigate this disease in PLWH.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Medulloblastoma (MB) is the most common childhood brain tumor arising from the cerebellum. Many factors influence the proliferation, differentiation, and migration of cerebellar granular neuronal precursor (GNP). Among them, MDM2 is a major nexus between tumor suppressor TP53 and hedgehog (Hh) signaling in GNPs and promotes MB tumor growth and metastasis. In addition, PI3K and BRD4 signaling also play key roles in MB cell growth, cancer stem cell (CSC) proliferation, and tumorigenesis. Further, MB treatment is challenging due to the development of chemoresistance, inefficient drug transport across the blood brain barrier (BBB) and drug induced neurotoxicity. Hh inhibitors are effective initially to treat SHH-MB, but their repeated use develops chemoresistance due to mutations in smoothened (SMO) but can be overcome by modulating GLI, which is downstream of SMO. In our preliminary studies, we synthesized a series of potent BRD4/PI3K dual inhibitors by modifying structure of parent compound SF2523. One of the compounds 8-(2,3-dihydrobenzo[b][1,4]dioxin- 6-yl)-2-morpholino-4H-chromen-4one (abbreviated as MDP5) was found highly potent. We then determined X- ray crystal structures of the recombinant BD1 and BD2 domains from BRD2 in complex with MDP5. While MDP5 showed higher potency in DOAY cells compared to SF2523 (12.6 µM), IC50 values for MDP5 and SF2523 were similar potency on HD-MB03 MB (MYC amplified) cells. MDP5 decreased the target downstream proteins like p-AKT, MYCN, Cyclin D1, and increased the degradation of MYCN protein indicated by p-MYCN (ser 54). We also discovered a small molecule JW-475A which is a potent dual MDM2 and XIAP inhibitor. MDP5 and JW- 475A (a dual MDM2 and XIAP inhibitor) effectively inhibited the proliferation of MB cells in a dose dependent manner, with significantly higher cell killing when these drugs were used in combination. Treatment of MB cells with the combination of these two drugs significantly decreased the colony formation capacity compared to individual drugs. We prepared PEG-DSPE based lipid nanoparticles (LNPs) with 4.9±0.1% and 4.8±0.1% loading for MDP5 and JW-475A. BBB penetrating targeted LNPs were prepared by surface decorating with rabies virus glycoprotein (RVG) peptide-peptide. Our hypothesis is that inhibition of BRD4/PI3K and MDM2/XIAP simultaneously using MDP5 and JW-475A represents a promising strategy to inhibit MB tumor in vivo. Further, we will use RVG-PEG-DSPE LNPs to encapsulate MDP5 and JW-475A, which have poor drug transport across the BBB. Our specific aims are to i) Synthesize novel MDP5 derivatives as dual function BRD4/PI3K inhibitors and characterize in vitro activity; ii) Evaluate anti-cancer efficacy of JW-475A in combination with MDP5 in vitro.; iii) Formulate MDP5 and JW-475A into LNPs decorated with RVG peptide and determine their biodistribution, therapeutic efficacy, and systemic/organ toxicity in in SHH and MYC driven cells and PDX-based orthotopic and transgenic SmoA1 MB mouse models. Long-term significance. Successful completion of this project will provide a platform technology for treating brain tumors using this innovative LNP-based combination therapy.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease that can cause a debilitating, flu- like illness in acute cases, or a life-threatening endocarditis in chronically infected patients. Q fever patients present with few distinguishing clinical features, and chronic disease requires a minimum of 18 months of antibiotic treatment, highlighting the need for new therapeutics. An obligate intracellular pathogen, Coxiella survives inside a vacuole within infected cells that has characteristics of a functional phagolysosome, including active proteases and phosphatases and moderately acidic pH, a physicochemical parameter to which C. burnetii is exquisitely adapted. C. burnetii is a strict moderate acidophile capable of efficient nutrient transport, catabolism and replication only within a narrow pH range under both host cell-free and intracellular conditions. The objective of this application is to identify host and pathogen factors that function to maintain both CCV and bacterial cytoplasmic pH. Aim 1 will test the hypothesis that C. burnetii controls bacterial cytoplasmic pH via carbonic anhydrase-dependent metabolism of CO2 and also how the metabolically dormant Small Cell Variant of C. burnetii protects the pathogen against acid stress. Aim 2 will test the hypothesis that C. burnetii regulates CCV pH by manipulating host lysosomal biogenesis. Understanding the molecular mechanisms behind C. burnetii survival within the acidic CCV will allow identification of potential therapeutic targets.

NIH Research Projects · FY 2025 · 2022-07

Project Summary Severe hemorrhage from extremity injuries is a significant cause of battlefield deaths and preventable trauma fatalities in civilian medicine. Tourniquet use is the most effective means of arresting life-threatening limb hemorrhage in the pre-hospital setting and creating bloodless surgical fields in orthopedic and vascular surgeries; however, tourniquet-related ischemia and subsequent reperfusion (IR) can cause serious IR injuries. These IR injuries have led to the limitations of tourniquet use. Exploring the mechanisms and finding effective therapies can resolve the limitations of tourniquet use and improve outcomes and quality of life in post- traumatic patients. The neuromuscular junction is structured to transmit electrical signals from motor nerve terminals to nicotinic acetylcholine receptors (nAChRs) for affecting muscle function. Our pilot data demonstrated that some nAChR clusters are fragmented in mice with 6 weeks of tourniquet-induced IR. Therefore, in Aim 1, we will determine the relationship between the fragmentation of nAChR clusters and muscle contractile dysfunction in long-term tourniquet-induced IR. Additionally, our preliminary studies have targeted a specific signaling pathway that links fragmentation of nAChR clusters in the injured muscle, namely the inflammatory cytokine-cyclin-dependent kinase 5 (Cdk5-catenin-rapsyn signaling pathway. In Aim 2, we will test if the inflammatory cytokine-Cdk5-catenin-rapsyn signaling pathway mediates fragmentation of nAChR clusters in long-term tourniquet-induced IR. In Aim 3, we will investigate the therapeutic effect of a novel anti- inflammatory drug on long-term functional and structural recovery of the neuromuscular junction via inhibition of pro-inflammatory cytokines in mouse models of tourniquet/IR injury. We will design in vitro and in vivo studies to address our overarching hypothesis that anti-inflammation will promote repair of the neuromuscular junction. Overall, proposed studies will unveil cellular and molecular mechanisms responsible for long-term neuromuscular junction disorder in tourniquet-induced IR. These studies will provide further information that the neuromuscular junction could be a potential therapeutic target in tourniquet/IR injuries, especially through the application of a novel anti-inflammatory drug in this proposal. This approach has significant potential to resolve the limitations of clinical tourniquet use, thereby improving outcomes and quality of life in post- traumatic patients.

NIH Research Projects · FY 2025 · 2022-07

Project Summary: Functional analysis of the Clp Protease Systems in Chlamydial Growth and Differentiation Chlamydia is an obligate intracellular bacterial pathogen that causes a range of serious diseases in humans. In developed countries, Chlamydia trachomatis is the primary cause of bacterial sexually transmitted infections (STI). Indeed, recent reports from the Centers for Disease Control highlight the increasing incidence of STIs, with chlamydia infections consistently outpacing all other types. In developing countries, C. trachomatis is not only a significant cause of STI, but it is also responsible for the primary cause of infectious preventable blindness, trachoma. The major concern of chlamydial infections is that they are often asymptomatic and undiagnosed, which can lead to chronic sequelae. These include pelvic inflammatory disease, tubal factor infertility, and reactive arthritis for C. trachomatis. Consequently, chlamydial diseases remain a significant burden on health care systems around the world. In adapting to obligate intracellular growth, Chlamydia has significantly reduced its genome size and eliminated genes from various pathways as it relies on the host cell for its metabolic needs. This pathogen has also adapted to alternate between different functional and morphological forms during its normal growth, also referred to as its developmental cycle. These observations, combined with its obligate intracellular dependence, makes Chlamydia a difficult organism with which to work. However, recent development of genetic tools to study chlamydiae mechanistically have significantly enhanced our understanding of this pathogen. This proposal applies a combination of these new genetic techniques and classical biochemical studies to evaluate the role of conserved protease systems in chlamydial growth and pathogenesis. The hypothesis of the proposed work is that Chlamydia uses two separate protease systems to regulate its growth and transition between developmental forms as well as to respond to stress. Major goals of the proposal include (i) characterizing the function of the different protease systems both in vitro and in vivo and (ii) identifying and validating substrates of these protease systems. Results will advance our understanding of this important pathogen and lead to the design of novel therapeutic agents that are specific for Chlamydia. This in turn will allow for minimal effects on normal flora for patients receiving treatment for this highly prevalent disease.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY DNA-Protein crosslinks (DPCs) are irreversible covalent crosslinks of proteins to DNA that stall replication forks during DNA replication. Unrepaired stalled forks lead to DNA breaks and fork collapse, leading to genome instability, cell death or senescence. SPRTN is a DNA-dependent replication-coupled metalloprotease that catalyzes proteolysis of DPCs during DPC repair. SPRTN also regulates replication fork progression and translesion DNA synthesis. Ruijs-Aalfs (RJALS) syndrome patients with bi-allelic mutations in SPRTN protease domain are prone to genome instability, segmental progeria and early-onset hepatocellular carcinoma. Regulation of SPRTN protease function is critical for accurate DNA replication fork progression and DPC repair. This proposed study is designed to characterize novel regulators of SPRTN and investigate the molecular mechanism underlying SPRTN-mediated replication-coupled DPC repair. Investigating the regulation of SPRTN and SPRTN-mediated DPC repair pathway will further our understanding of the DPC repair pathway, delineate the mechanism of RJALS syndrome, and help develop novel strategies for sensitizing cancer cells to chemotherapy by targeting SPRTN-mediated DPC repair pathway.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract PROJECT SUMMARY Cardiovascular complications are the leading cause of maternal mortality in the United States. The burden of poor cardiovascular health largely falls on communities with lower socioeconomic status and limited access to quality care, thus impacting health outcomes. After school care (ASC) can be intentionally designed to mitigate barriers to health by empowering communities to address modifiable risk factors for poor health. ASC for adolescents has been shown to improve health outcomes and mitigate certain cardiometabolic risk factors, including poor nutrition and exposure to psychosocial stressors. However, the long-term impacts of adolescent participation in ASC on cardiovascular health and its impact on future pregnancies are not known. In this proposal, we will partner with a nationally recognized ASC (Girls Inc., Omaha) to address this gap in the scientific literature. We hypothesize that ASC with nutritional and emotional resiliency components will positively impact adolescent modifiers of adult cardiovascular disease. To test this hypothesis, we will compare nutritional antioxidant status (Specific Aim 1), hair/saliva cortisol levels (Specific Aim 2), and vascular elasticity (Specific Aim 3) in current/former ASC participants versus age-matched controls. Dietary patterns and emotional resiliency will be measured using validated questionnaires. Nutritional antioxidant status (a marker of cardiovascular inflammation) will be measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Cortisol levels (a marker of psychological stress) will be measured in hair and saliva using enzyme-linked immunoassay (ELISA). Vascular reactivity (a marker of latent endothelial disease) will be measured using a VENDYS® test. Our research will inform the design of ASC for adolescents and contribute to the mitigation of cardiovascular complications in young women. In addition to this dissertation research, I have developed a training plan to enrich my scientific and professional development as a physician-scientist trainee at the University of Nebraska Medical Center (UNMC). I will achieve the following training goals: build foundational knowledge and technical skills in pediatric health, develop capacity to independently design and implement research projects, master written and verbal scientific communication skills, and integrate my clinical and scientific skillsets. Throughout my graduate training, UNMC will provide essential coursework, grant writing workshops, seminars, and other skill-building opportunities that enhance my technical expertise and professional development. Altogether, the research and training opportunities outlined in this proposal will empower me to become an adept physician-scientist and an innovative leader in the field of pediatric health

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY Alveolar bone is a critical tissue for tooth and dental implant retention. Increasing alveolar bone mass in patients who lose this tissue as a result of periodontal disease or trauma is crucial for successful dental implant therapy (e.g., loss of bone around a tooth extraction site prior to implant placement). Currently, bone grafts (e.g., iliac or mandibular bone) or artificial bone grafts are commonly used for alveolar bone regeneration therapy. However, most of these therapies require extensive surgical procedures, which present risks of many complications, particularly in aged patients. Therefore, the development of new alveolar bone regeneration techniques that do not require surgical procedures is urgently needed. Herein, in this proposed study, we aim to develop an injectable and biomimetic highly porous nanofiber microsphere-based therapy for healing critical- sized alveolar bone defects. We recently developed an exciting approach for the fabrication of biomimetic nanofiber microspheres consisting of short electrospun nanofiber segments without limitation to certain compositions. Cells can attach and proliferate on the surface of such nanofiber microspheres. Working with Dr. Reinhardt (Co-I), we also demonstrated that mineralized short nanofibers incorporated with E7-BMP-2 peptides showed promise for healing a critical-sized socket defect model created in rat maxillae, following extraction of the first molar teeth. In addition, our most recent study revealed that BMP-2/QK peptides conjugated nanofiber microspheres can significantly enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and tubular network formation of human umbilical vein endothelial cells (HUVECs). Based on these findings, it is hypothesized that the injectable highly porous nanofiber microspheres in combination with biomimetic delivery of signaling molecules and/or incorporation of BMSCs could greatly promote alveolar bone regeneration after minimally invasive administration to critical-sized alveolar bone defects in rats. To test the hypothesis and accomplish the primary objective, our strategy is three-fold: i) Demonstrate the fabrication of porous nanofiber microspheres with controlled composition, structure, and coupling of signaling molecules; ii) Examine the effect of engineered porous nanofiber microspheres with biomimetic delivery of signaling molecules on cellular response; and iii) Determine the bone regenerative capacity of injectable porous nanofiber microspheres in combination with biomimetic delivery of signaling molecules and/or BMSCs for healing alveolar bone defects in rats. We expect to identify the critical factors of biomimetic and injectable highly porous nanofiber microsphere-based therapy that contribute to alveolar bone regeneration. Also, we expect successful completion of these aims to lay the foundation for developing injectable bone grafts that could greatly accelerate healing of alveolar bone defects without invasive surgical procedures.

NIH Research Projects · FY 2025 · 2022-06

Abstract With a critical need to establish a workforce of specialists in neurodegenerative diseases focusing on Alzheimer’s disease, there is an increasing demand for multidisciplinary scientists in the biomedical research community. Despite this significant need, there are few training programs that provide experience in academic and clinical settings. This proposal for an Alzheimer’s Disease and Related-Dementia Drug Discovery training program is critical to meeting that increased demand. The overall goal of this program is to fund five predoctoral students from the Colleges of Pharmacy and Medicine to become experienced leaders in interdisciplinary research with the ability to collaborate effectively on cross-disciplinary teams with broad expertise to address challenges within target-based drug discovery. The program will help the selected students to develop keen scientific rigor and translational skills necessary to compete and excel in the future workplace, including but not limited to academic institutions, government agencies, for-profit businesses, and private foundations. Importantly, the program faculty are drawn from a wide array of disciplines with many active collaborations. Twelve mentors are dedicated to this training program, all either tenured or in tenure track positions, with the experience and funding to undertake their training and mentoring roles. These faculty will provide trainees with a unique program in which to learn techniques and methodologies of collaborative research projects. The proposed training program is composed of formal didactic courses, discussion-based journal clubs and seminars, shadowing of a clinician and mentored research leading to a doctoral thesis and Ph.D. degree within 5.5 years of entering graduate school. This innovative program will produce an outstanding output of trainees with high accomplishments that will in turn catalyze our recruitment of the best students for this interdisciplinary training program at the University of Nebraska Medical Center.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT The internalization and recycling of receptors is a key biological process in all eukaryotic cells. The early/sorting endosome is the initial destination of receptors internalized from the plasma membrane (PM). This endosome serves as a major sorting station from which receptors are shunted to late endosomes and lysosomes for degradation, or are recycled back to the PM through a transitory network of vesicular and tubular recycling endosomes. Whereas a decade ago most researchers thought that active sorting directed proteins to the degradation pathways, targeting to the recycling pathway was thought to be largely a passive process that occurs by default. However, recent evidence supports active sorting to the recycling pathways by specific sorting nexin (SNX) and other proteins that bind to the cytoplasmic tails of receptors and specifically target them for recycling. Although recycling is an essential process for all mammalian cells, its complex regulation is poorly understood including the sorting of receptors on endosomal membranes, the budding and fission of vesicles and tubules from the endosome, and the transport of receptors back to the PM. As such, our knowledge of endosomal function lags substantially behind that of receptor internalization mechanisms. A key group of regulatory proteins that controls sorting and trafficking at the endosome is the retromer complex. Originally identified in the retrieval of biosynthetic cargo from endosomes to the Golgi complex, the retromer has recently been implicated in the regulation of a variety of key cellular pathways both within and beyond the scope of endocytic trafficking including endocytic recycling, mitochondrial homeostasis, the centrosome cycle and ciliogenesis. The retromer complex also interacts with other key endocytic regulatory proteins, including the tubular endosome scaffold MICAL-L1; its interaction partner and endosomal fission modulator, EH domain containing 1 (EHD1); and a host of SNX proteins that mediate endosomal cargo sorting. The retromer also links to the actin cytoskeleton via the WASH complex. Our laboratory has been focusing on an overall understanding of the mechanisms by which endocytic regulatory proteins function both in endocytic pathways and in non-endocytic trafficking. Our primary expertise is in biochemistry and molecular cell biology coupled with advanced light microscopy, but we recognize the need to incorporate in vivo components into our approach and have ongoing collaborations with other groups to examine these processes in whole organisms, including zebrafish and worms. In our studies, we will address significant and as-yet-unresolved biological problems such as: 1) how endosomal fission is regulated and linked to sorting and recycling and 2) how key endocytic proteins mediate the biogenesis of the primary cilium.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY The majority of patients diagnosed with rectal cancer will receive radiation therapy to treat their tumors. Long- term complications from radiation therapy directed to the colorectal region are bowel fibrosis, rectal wall damage, bowel incontinence, rectal and bladder bleeding and pelvic fracture and there are no FDA approved treatments to protect normal rectal and anal tissues from radiation-induced damage. Radiation exposure leads to free radical-mediated oxidative damage to normal tissues leading to fibrosis. Cancer cells have increased metabolic production of reactive oxygen species (ROS), relative to normal cells, which have been shown to drive cancer progression. Thus, suppressing radiation-induced ROS would act as both a radioprotector in normal tissues while inhibiting pro-survival and progression pathways in cancer cells. BMX-001 is a small molecule antioxidant, which scavenges ROS. Preliminary data demonstrate that BMX-001 protects rectal tissues from radiation- induced damage, while enhancing colorectal cancer killing. The overall hypothesis of this proposal is that BMX- 001 will protect normal tissues from radiation-induced damage, while not protecting the cancer cells in patients undergoing radiation therapy of rectal and anal cancers. Specific Aim 1 will determine the mechanism(s) by which BMX-001 protects from radiation-induced epithelial dysfunction and how BMX-001 prevents blood chemo- radiation toxicity. It will be determined whether BMX-001 prevents hematological chemoradiation toxicity through the NRF2/MnSOD signaling and potential biomarkers of oxidative stress and GI damage will be identified in a chronic in vivo model of radiation-induced damage. Specific Aim 2 will determine the safety and efficacy in patients through Phase II clinical trials as a radioprotector of rectal cancers. A randomized phase II clinical trial will determine whether BMX-001 is an efficacious radioprotector of normal tissues in rectal cancer patients undergoing total neoadjuvant chemoradiation therapy followed by surgery. Specific Aim 3 will identify potential biomarkers that can be used to demonstrate radiation damage and radioprotector efficacy of BMX-001 in human clinical specimens obtained from the Phase II trial. Surgically resected tumor and adjacent normal rectal tissues from BMX-001 or placebo controlled patients after chemoradiation will be evaluated for inflammation, normal tissue damage, fibrosis and oxidative stress. In addition, oxidative stress markers will be evaluated in blood and urine samples. The completion of the studies will provide an in depth mechanistic understanding of the mechanisms by which BMX-001 inhibits normal tissue injury and whether BMX-001 can be used in rectal cancer patients as an effective radioprotector and could be universally adapted for treatment of other cancers receiving pelvic irradiation.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY: Chronic pain is a poorly managed health disorder and common treatments (e.g. opioids) have significant issues associated with them: risk of addiction, constipation, cognitive impairment, motor impairment, fatal respiratory depression, and others. Developing new, non-opioid based pain therapies is a major effort of the scientific community; however, the identification of new targets is still a bottleneck in this endeavor. G protein-gated inwardly rectifying potassium (GIRK) channels are members of the inwardly rectifying K+ channel (KIR) family. GIRK channels and known to couple to the μ-opioid (μOR), which is a key target of opioid analgesics, and multiple lines of evidence indicate that the therapeutics utilize GIRK channel-mediated hyperpolarization to produce their analgesic effect in rodents. In addition, variations in the Girk2 gene (KCNJ6) are associated with decreased therapeutic efficacy of opioid analgesics and pain sensitivity in humans. Work from our labs has shown that systemic administration of GIRK1/2 activators, alone or in combination with morphine, can produce analgesia, and can enhance the effects of morphine in preclinical mouse models of acute pain and persistent pain. In addition, we have observed that systemic administration of our GIRK activators does not result in significant motor impairment, nor does it appear to be perceived as rewarding in the conditioned place preference assay (CPP). In order to develop first-in-class, IND-ready GIRK1/2 activators, we will optimize our lead scaffold with the appropriate activity, selectivity and DMPK properties. We will utilize an iterative medicinal chemistry approach coupled with an acute inflammatory (carrageenan) pain model to determine a PK/PD model that can be used to progress compounds into further chronic models of pain. The selective GIRK1/2 activators will offer a unique opportunity to help advance the field toward a first-in-class therapeutic agent.

NIH Research Projects · FY 2026 · 2022-04

Zinc (Zn) deficiency has emerged as a growing public health problem. In fact, an estimated 17% of the global population is deficient. Animal studies have demonstrated that even marginal zinc deprivation leads to significantly impairs physiological functions. This is especially true in the gut where zinc is required to maintain intestinal homeostasis. Zn deficiency-mediated loss of intestinal homeostasis and microbial dysbiosis have recently been proposed as major mechanistic pathways for the development and severity of inflammatory bowel disease (IBD). Specifically, Zn deficiency is common in patients with IBD with a prevalence ranging from 15% to 40%, likely due to diet deficits and increased intestinal loss. In addition, a common genetic variant of the Zn transporter ZIP8 (rs13107325; A391T) has been associated with an increased risk of Crohn’s disease. In the context of gastrointestinal health, it is also notable that zinc is also an essential nutrient for bacteria. As such, commensals must compete for and scavenge zinc from their host, which likely further effects the host’s ability to acquire adequate levels of zinc. Bacteria utilize numerous strategies to acquire metal, such as the secretion of small molecules known as metallophores (i.e., siderophores and zincophores). Overgrowth of pathobionts, which express high levels of metallophores, is hypothesized to be one mechanism by which the microbiota contributes to IBD pathogenesis. In this application, we propose a new paradigm in which bacterial metallophore production is a key mechanistic pathway leading to accelerated IBD disease severity/inflammation. Specifically, we hypothesize that IBD disease status is associated with a unique subset of microbial metallophores and further hypothesize that IBD-associated metallophores exacerbate disease severity. Four key findings support this hypothesis: First, humans with the A391T allele have and increased prevalence of IBD and have significantly altered intestinal microbial communities. Second, Zip8 393T-KI mice have increased susceptibility to chemically induced colitis. Third, microbial metallophores are associated with the development of adherent-invasive Escherichia coli (AIEC)-mediated colitis. Fourth, a novel class of zinc transporters (zincophores) are produced by a wide-range of known gastrointestinal bacterial species many of which are over-represented in IBD dysbiosis. To test our hypothesis that bacterial zincophore production is a key mechanistic pathway leading to increased IBD disease severity, we propose three research Aims: Aim 1 will establish cross-talk between host genetics, gut microbial composition, bacterial metallophores, and dietary Zn levels as a link to IBD severity. Aim 2 will determine and characterize the effects of bacterial metallophores on intestinal epithelial health. Aim 3 will seek to validate the association of bacterial metallophores with IBD disease using a well characterized IBD biobank.

NIH Research Projects · FY 2026 · 2022-03

ABSTRACT The goal of this multi-PI project is to understand how Staphylococcus spp. bind to the skin surface. Although S. aureus has the capability to cause significant disease in humans, most Staphylococcus spp. do not cause disease but instead act as commensals or mutualists and adhere to the skin surface providing beneficial aspects to the host. These benefits include inhibition of pathogen colonization by synthesis of unique antimicrobial compounds in addition to appropriate immune system development. However, we do not know how Staphylococcus spp adhere to skin. This application hypothesizes that species of staphylococci that colonize humans do so in a conserved manner via an Aap (or SasG in S. aureus) orthologue that binds corneocytes. Aap is a rod-like fibrillar protein that functions in initial adherence to corneocytes but also functions to mediate intercellular accumulation. To address this hypothesis, we have proposed the following specific aims: (1) understand the dynamics of Aap-mediated corneocyte adherence; (2) define the ligand specificity of Aap and SasG lectin domains and (3) investigate S. aureus SasG-dependent mechanisms of skin colonization. Overall, the proposed studies will address how staphylococci bind to skin, what ligand is bound, and thus could uncover new layers of crosstalk between pathogen and the host, potentially leading to novel therapeutic modalities for treating staphylococcal infections.

NIH Research Projects · FY 2026 · 2022-03

A craniotomy is performed to access the brain for procedures that include tumor resection, localization and resection of epileptogenic foci, and aneurysm clipping, where the bone flap is replaced intraoperatively. Despite prophylaxis, infectious complications after craniotomy range from 1-3%, with approximately half caused by Staphylococcus aureus (S. aureus), which forms a biofilm on the bone flap that is recalcitrant to antibiotics. We have developed a mouse model of S. aureus craniotomy infection that shares important ultrastructural, MRI, and immune attributes with human disease, which can be exploited to identify mechanisms for infection persistence. Our preliminary results suggest that T cells maintain S. aureus in a biofilm state to minimize the shedding of planktonic bacteria into the brain. Ultimately, this helps protect the CNS parenchyma but does not clear the biofilm. This is supported by the fact that bacterial burden was unchecked in Rag1 KO mice and in WT animals following CD4+ T cell depletion, reflecting increased planktonic bacteria in the brain and galea. Furthermore, T cell loss coincided with an attenuated activation signature in microglia, macrophages, and granulocytes, with significant reductions in several IFN-ɣ-regulated genes, including CXCL10, indicative of T cell-innate immune crosstalk. We aim to understand this regulation in addition to T cell-biofilm crosstalk to devise novel strategies to promote biofilm eradication. This possibility is feasible given our innovative bacterial scRNA-seq data, where activated CD4+ T cells induced the expression of several S. aureus virulence genes, including protein A (spa) that binds the Fc portion of antibody to inhibit opsonophagocytosis. CXCL10 has been reported to induce Spa shedding from the bacterial membrane, which raises the intriguing possibility that CXCL10 induction by T cells promotes Spa release to block S. aureus phagocytosis, which is supported by our preliminary data where craniotomy infection was significantly reduced with a S. aureus spa mutant. This proposal will examine the hypothesis that CD4+ T cells regulate antimicrobial responses in the brain and galea by targeting microglia/macrophages vs. PMNs/G-MDSCs, respectively, to prevent bacterial outgrowth and limit invasion into the brain. In response, S. aureus alters its transcriptiome to augment virulence factor expression to promote biofilm persistence. This represents a host-pathogen triad that dictates infection outcome and the molecular mechanisms responsible for bacterial persistence in the brain and galea will be examined in the following Specific Aims: 1) Identify the critical CD4+ Th subset and antigen specificity in controlling S. aureus outgrowth during craniotomy infection; 2) Determine key mechanisms of T cell-innate immune crosstalk that dictate biofilm growth and CNS invasion; and 3) Identify S. aureus biofilm genes that are critical for subverting T cell effector function during craniotomy infection. An improved understanding of how T cells shape the innate immune landscape and S. aureus virulence during craniotomy infection may be leveraged to enhance antimicrobial activity and biofilm clearance to reduce patient morbidity.

NIH Research Projects · FY 2026 · 2022-03

There is a global aim to reduce the burden of chronic hepatitis B (CHB) infection and prevent the development of HBV-associated end-stage liver disease and cancer. The improvement of existing therapeutics is expected to help achieve this goal. Specifically, the usage of once two-month injectable nucleos(t)ide analogs in combination with immunomodulating antiviral compounds instead of life-long daily pills has the strong potential to help to achieve a functional cure for CHB. To this end, we propose to transform water-soluble antiviral drugs, first-line drugs tenofovir (TFV) and entecavir (ETV), and immunomodulating drug tizoxanide (TIZ) into hydrophobic lipophilic crystalline prodrugs. We will formulate them as nanosuspensions suitable for intramuscular injection. The efficient optimization of physicochemical properties of nanocrystals is expected to improve their pharmacokinetics (PK) and pharmacodynamics (PD) profiles. This optimization will enhance uptake of the prodrug nanocrystals by liver macrophages and hepatocytes to ensure a slow release and sustained therapeutic drug concentrations at the site of hepatitis B viral replication. The treatment with long-acting TFV, ETV, and TIZ is expected to decrease dosing frequency, limit toxicity, and facilitate sustained viral suppression and treatment. A functional cure for HBV is expected to be achieved via multifactorial mechanisms, including inhibition of viral polymerase, prevention of cccDNA formation, and the clearance of HBV micro-chromosomes via stimulation of host innate immunity by TIZ. Thus, the overall objective of this proposal is to develop clinically translatable, long-acting, injectable, antiviral drug nanoformulations to increase adherence and enhance drug delivery to sites of persistent HBV infection, thereby facilitating sustained viral suppression and finite cure. To this end, three specific aims are proposed: Aim 1: Develop long-acting anti-HBV prodrug nanoformulations and evaluate the drug efficacy. Here, we will apply pronucleotide (ProTide) and a modified HepDirect prodrug technology to transform existing drugs into hydrophobic prodrugs suitable for formulation as nanosuspensions to achieve prolonged therapeutic active drug concentrations in hepatocyte. This is expected to improve drug biodistribution to infected hepatocytes without compromising drug potency and safety profile. The prodrug formulations will be screened in vitro in human macrophages as a potential drug depot and in infected hepatocytes as final targets. The anti-HBV activity of prodrug nanocrystals will be examined in vitro and in vivo using HBV-infected humanized mice. Aim 2: To develop long-acting TIZ nanoformulation and evaluate the mechanisms by which TIZ suppresses HBV replication in infected hepatocytes. Aim 3: To evaluate the synergistic efficacy of the selected long-acting TIZ and NUC formulations and evaluate the ability of this combination to eliminate HBV cccDNA from hepatocytes and significantly reduce the concentration of HBsAg.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Pancreatic cancer goes undiagnosed for a number of reasons including, anatomical location, vague and non- localizing symptoms, and a lack of adequately sensitive and specific biomarkers of disease. Combined with lack of blood biomarkers, early detection of pancreatic cancer remains a significant challenge. Unfortunately, this limits therapeutic strategies that are most efficacious. Consequently, approximately 20% of patients with pancreatic cancer are eligible for curative surgery. Of those patients, approximately 75% will have recurrent disease within the next 5 years, even those patients thought to have had negative margins. Despite the abysmal statistics for patients with pancreatic cancer, there are now several pancreatic cancer imaging probes in preclinical and clinical development to try to improve disease removal. Moreover, the increased use of neoadjugant or induction thereapy has resulted in additional patients that become eligible for surgery, if they restage at a lower stage. The opportunity to develop molecularly-targeted probes to pancreatic cancer to improve surgery and the increased numbers of patients that may benefit from surgery are key motivating factors for this project. Mucin-16 (MUC16) is an attractive marker for pancreatic cancer since it is highly overexpressed in malignancies, but not inflamed or healthy pancreas. Recently, we evaluated a murine antiMUC16 antibody, termed AR9.6, with cross-reactivity to human MUC16. In these preclinical studies, we demonstrated durable pancreatic cancer enhancement to at least 6 days postinjectin using IRDy800-labelled AR9.6 compared to an isotype control. Using a pancreatic cancer xenograft, AR9.6-IRDye800 was able to effectively enhance orthotopic and metastatic disease. Metastasis detection is an added benefit because it would prevent patients from unecessary surgery. Murine antibodies, however, are not suitable for clinical translation. Consequently, a humanized form of AR9.6 has now been developed by our collaborative team. An Initial investigation of IRDye800 conjugated to humanized AR9.6 suggests strong binding to MUC16 and in vivo targeting. Therefore, the goal of this project is to perform the preclinical development of NIRF-labelled, humanized AR9.6 to detect PDAC for improved R0 resection and to detect peritoneal metastasis. This goal will be addressed by three specific aims: To (1) develop and evaluate humanized AR9.6-NIRF for targeting of MUC16; (2) ascertain the preclinical contrast-enhancement and safety profile of humanized AR9.6-NIRF conjugates; and (3) demonstrate surgical efficacy of IRDye800-AR9.6 in preclinical models of pancreatic cancer. We hypothesize that the aberrant MUC16 overexpression in pancreatic cancer will allow specific targeting pancreatic cancer with fluorescently labelled huAR9.6. Completion of this research project will result in a fluorescence-guided surgery contrast agent that will be able to (1) improve the rate of R0 pancreatic cancer resections and (2) be able to identify peritoneal metastasis, which will prevent unnecessary surgery and allow optimized treatment strategy.

NIH Research Projects · FY 2025 · 2022-02

ABSTRACT The single-stranded DNA binding protein (SSB) is the founding member of the nineteen-partner SSB interactome. There is a fundamental gap in understanding the mechanism of action of the interactome, the first prokaryotic family of oligosaccharide/oligonucleotide binding fold (OB-fold) genome guardians identified. The continued existence of this gap represents an important problem because, until it is filled, a complete and clear understanding of genome stability will be lacking. This understanding is crucial as defects in OB-fold genome guardian family members have disastrous consequences for genome stability. In higher organisms, mutations in the three OB-folds of BRCA2 ultimately result in cancer, and the proposed studies are therefore directly relevant to human disease. Consequently, the long-term goal is to understand the molecular mechanism of the SSB interactome. The main objective of this proposal is to understand how SSB interacts with, and regulates, several partner proteins and itself, to maintain genome integrity. The central hypothesis is that two regions of SSB known as the linker and the OB-fold, (present in both SSB and interactome partners), are the key elements to all aspects of protein function. The rationale for the proposed research is that once it is known how SSB physically and functionally interacts with both itself and its partners, a clearer understanding of the events required to maintain genome integrity, mediated by this OB-fold family of genome guardians, will be obtained. The central hypothesis will be tested by pursuing two specific aims: 1) determine the role of the linker/OB-fold network in SSB function; and 2) determine the mechanism of action of the SSB interactome. Under the first aim, a combination of in vivo and in vitro binding assays, HDX-MS, protein crystallography, cryo-EM, and single-molecule biochemistry will be used to determine whether the linker/OB-fold interface is the primary means of SSB-partner binding. Under the second aim, enzyme kinetics, genetics, real-time super-resolution microscopy, and single-molecule biochemistry will be used to understand how the linker/OB- fold network mediates SSB interactome function in the dynamic reactions of DNA replication, recombination, and repair. The proposed research is innovative because of the combinatorial strategy taken, the novel single-molecule approaches used, and the care that we will take in elucidating SSB interactome function using full-length proteins. The proposed research is significant because it will allow, for the first time, the development of a clear picture of SSB interactome function in DNA metabolism and the maintenance of genome integrity.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Gemcitabine (GEM), a frontline drug, shows limited efficacy due to its rapid metabolism and inefficient delivery to the desmoplastic pancreatic tumor site. Hedgehog (Hh) signaling activates pancreatic stellate cells (PSCs) and plays a critical role in the formation of desmoplasia and proliferation of cancer stem cells (CSCs). KRAS is predominantly mutated in pancreatic cancer (PC), yet KRAS remains a difficult target. Since inhibition of mTORC1/2 increases ERK phosphorylation, we propose combination therapy of GEM with ONC201, which is an AKT/ERK dual inhibitor to effectively treat PC. ONC201 inhibits cell proliferation and induces TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Further, we have adopted a stroma depletion strategy by sequentially administrating Hh inhibitor MDB5 for reducing physical barrier of drug delivery to the tumor site. While sonic hedgehog (Shh)-deficient tumors have reduced stromal content, such tumors are aggressive with increased vascularity and metastatic potential. Therefore, reduction of desmoplasia by inhibiting Hh pathway will allow efficient delivery of ONC201 and GEM loaded into EGFR targeted NPs to the pancreatic tumor site. We have identified an effective combinatorial treatment strategy using clinically viable inhibitors, which can be applied to PDAC tumors with different KRAS mutations. In our preliminary studies, (i) compared to free GEM, mPEG-co-PCC-g-GEM-g-DC NPs increased GEM accumulation in orthotopic tumor by 2.5-fold. To control GEM release into the tumor, we synthesized mPEG-co-P(Asp)-g-DC-S-S-GEM with GEM payload of 14% w/w. There was 90% GEM release from the polymer upon incubation with L-glutathione (GSH). Combination of GEM with ONC201 showed synergy in killing resistant PC cells in vitro and reduced tumor growth in vivo more effectively than their monotherapies. We also synthesized 2-chloro-N1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis(2- pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5), which inhibited Hh ligands and CSC markers more efficiently than vismodegib. Targeted NPs were prepared and optimized by decorating their surface with EGFR binding peptide GE11 at different ligand density. Systemic administration of MDB5 loaded GE11-NPs into PC tumor bearing mice resulted in higher drug concentration in the tumor at 4h post administration compared to non-targeted NPs. Therefore, we hypothesize that sequential administration of MDB5 loaded NPs will increase GEM and ONC201 delivery to the tumor and result in synergistic inhibition of PC by reversing resistance induced by desmoplasia and CSC proliferation more efficiently. Our specific aims are to i) assess the effects of ONC201 and GEM combination in GEM resistant PC cells in vitro and in vivo, ii) development of targeted redox sensitive nanomedicine of MDB5, ONC201 and GEM, and iii) nanoparticulate delivery of MDB5, ONC201, and GEM combination in orthotopic, PDX and spontaneous KPC mouse models. Long-term impact is to develop novel strategies to reduce desmoplasia-induced chemoresistance in PC using multifunctional nanomedicine of MDB5, GEM and ONC201.