Univ Of North Carolina Chapel Hill

NIH Research Projects · FY 2025 · 2023-09

Abstract/Project Summary While the effects of alcohol use disorder (AUD) on glutamatergic signaling have been widely reported, the role of delta glutamate receptors (GluD1 and GluD2) in AUD remains unknown. Although they are not involved in traditional excitatory synaptic responses to glutamate, the GluD family is known to mediate many subtler aspects of excitatory signaling which may contribute to the dysfunctions of neuronal activity observed in models of AUD. GluD1 has also been implicated in AUD by several genome-wide association studies. However, no research has been conducted directly evaluating the effect of alcohol exposure on GluD1 function. The bed nucleus of the stria terminalis (BNST) is a forebrain nucleus that has been heavily implicated as a critical hub for neuromodulation following alcohol exposure. Previous work has shown that alcohol exposure regulates synaptic glutamate in the BNST, but the exact mechanisms by which this happen remain unclear. Synaptic glutamatergic transmission is in part mediated by AMPA receptors, ~30% of which can pass calcium (calcium- permeable AMPARs, CP-AMPARs). Our preliminary data suggests that acute withdrawal from chronic intermittent ethanol vapor exposure (CIE) decreases both CP-AMPAR synaptic current and reduces a GluD1 mediated tonic excitatory current in the BNST. Although it has been observed before that GluD1 is expressed in the BNST, these are the first data to that they may be involved in regulating BNST signaling. These data also implicate GluD1 modulation as a possible novel target for neuromodulation following alcohol exposure. This proposal aims to further examine the role of CP-AMPARs and GluD1 in the BNST, how they may interact, and how they are affected by alcohol exposure using a combination of fluorescent in situ hybridization, patch-clamp electrophysiology, and ex vivo calcium imaging. Aim 1 will use GluD1 knockout (GluD1 KO) mice and wildtype (WT) littermates to establish a functional profile of GluD1 in the BNST, as well as its overlap with CP-AMPAR expressing BNST cells. Aim 2 will then evaluate the impact of CIE on BNST CP-AMPAR and GluD1 expression and signaling, neuronal excitability, and spontaneous calcium dynamics in these mouse models. Finally, Aim 3 will use these approaches to examine the translational relevance of BNST CP-AMPAR and GluD1 signaling across different models of AUD.

NIH Research Projects · FY 2024 · 2023-09

Project Abstract Breast cancer remains the second-leading cause of cancer-related death in the US. Obesity is an established risk factor for several aggressive breast cancer subtypes, and is also associated with increased breast cancer metastasis and mortality. On a mechanistic level, diet-induced obesity drives dysregulation of lipid metabolism that results in greater extracellular fatty acid availability, providing a rich energy source for tumor proliferation and migration. While this has been shown to increase the severity of breast cancer progression and metastasis, the therapeutic potential of this metabolic relationship has not yet been investigated. Dependent upon fatty acid availability and linked to metastatic progression of several cancer types, ferroptosis, a mechanism of cell death caused by lipid peroxidation, serves as a targetable mediator between obesity, fatty acid metabolism, and breast cancer progression. Our preliminary findings support the ferroptosis-metastasis link and suggest obesity-induced dysregulation of fatty acid metabolism may exacerbate this relationship. This proposal will use murine models of obesity and metastatic mammary cancer in concert with several advanced mechanistic approaches, including lipidomic quantification of tumor and plasma as well as high-throughput RNA sequencing for assessing transcriptional regulation of lipid metabolism in the primary and metastatic tumor microenvironment, to rigorously test the hypothesis that dysregulation of fatty acid metabolism associated with obesity promotes sensitivity to ferroptosis in murine models of TNBC. This hypothesis will be tested with two integrated specific aims: Aim 1. Quantify the impact of obesity and ferroptosis induction on murine mammary tumor growth, metastasis, and fatty acid metabolism in vivo. To isolate the effect of fatty acid availability from other components of the tumor microenvironment, in vitro exposure to serum from control or DIO mice will be used to assess changes in sensitivity to inhibition of xCT and GPX4 in TNBC. Aim 2. Determine whether the enzyme pyruvate carboxylase (PC), uniquely involved in both fatty acid and glutamate metabolism, protects against ferroptosis in obesity by assessing if suppression of PC promotes sensitivity to ferroptosis in both primary and metastatic tumors of DIO mice. Transcriptional dysregulation of lipid and glutamate metabolism in obese and control shPC TNBC tumors, comparing primary and metastatic tumors, will be quantified via RNAseq. This proposal aims to define molecular characteristics of fatty acid metabolism enriched in breast cancer of patients with obesity to identify therapeutic targets that can decrease breast cancer mortality by limiting tumor progression and metastasis. Combined with the exceptional training environment at UNC, comprehensive mentoring from Dr. Hursting, and a focused training plan, this fellowship will provide a critical foundation for developing my future career as an independently-funded scientist in the fields of nutrition and cancer metabolism.

NIH Research Projects · FY 2025 · 2023-09

TITLE Encoding social arousal within prepronociceptin circuits in the extended amygdala PROJECT SUMMARY/ABSTRACT As a social species, humans thrive on the ability to form social connections for individual well-being, survival, and societal success. Physiological arousal responses regulate this process by encoding information from social stimuli that guides subsequent behavioral actions. Dysregulation of the neural circuitry responsible for encoding arousal responses is thought to contribute to disturbed motivated behavior, a characteristic in many neuropsychiatric disorders. We can track the rapid changes in arousal responses by recording certain physiological metrics such as pupil size, frequency of heartbeats, and respiratory cycles, however, little is known about the neural circuits that regulate these rapid arousal responses and how they influence ongoing social behavior. We recently found that neurons that express the prepronociceptin gene in the BNST (BNSTPnoc neurons) encode the arousal responses that occur rapidly upon exposure to motivationally salient stimuli, such as predator and food odors. Interestingly, BNSTPnoc neurons project predominantly to the medial amygdala (MeA) and the medial preoptic area (mPOA), which are brain regions that regulate aspects of social motivation. Here, we will study how BNSTPnoc neurons that project to either MeA or mPOA regulate social arousal responses and modulate social behaviors. Our global hypothesis is that unique populations of BNSTPnoc neurons will selectively encode arousal responses to social stimuli in a stimulus-dependent manner and independent of encoding behavior. To accomplish this, we will precisely map afferent and efferent circuit connections of BNSTPnoc neurons (Aim 1), test the hypothesis that BNSTPnoc neurons encode social arousal responses (Aim 2), and test the hypothesis that neurons that encode arousal responses influence social behaviors (Aim 3). Identifying the function and natural dynamics of BNSTPnoc neurons is important because we currently do not understand the role of arousal in the generation of behavior and behavioral disorders, nor do we understand the circuitry underlying the contributions of arousal on social behavior. This gap in knowledge prevents us from developing therapeutic strategies that target this potentially critical biological substrate to treat disorders defined by maladaptive social behaviors.

NIH Research Projects · FY 2023 · 2023-09

Project Summary The physical world contains signals encompassing the entire electromagnetic spectrum, and yet human perception of the world is limited to our five senses. To augment our senses, it is essential to first develop methods that can effectively transmit high-bandwidth information to the brain. Researchers working on brain- machine interfaces have successfully extracted movement signals from the brain to control external devices. Yet, methods that augment, restore, or modulate sensory perception are currently limited. Lack of real-time sensory feedback from a brain-machine interface or neuroprosthetic device prevents optimal motor control and thus limits sensorimotor rehabilitation. Loss of sensation due to life-altering injuries and disorders affects the quality of life of millions of Americans. Thus, methods that mimic sensory signals and interface them directly with the brain are an unmet clinical need. This project proposes a novel spinal sensory neuroprosthetic interface using sensory spinal cord stimulation (SSCS) with the ability to augment, restore, and modulate sensory perceptions. This radically innovative approach has the potential to impact a wide array of neurological conditions by addressing sensory restoration and allows for the exploration of the limits of human sensory perception. Pre- clinical experiments in rodents and rhesus monkeys demonstrated that animals learn to detect and discriminate artificial sensations induced by SSCS. To achieve clinical translation of SSCS technology, this proposal involves a feasibility study, conducted in patients undergoing spinal cord stimulator implantation for the treatment of chronic pain. First, the relationship between SSCS-induced sensory perceptual thresholds, just-noticeable differences, and stimulation parameters will be established (Goal 1). Second, human subjects will be trained to detect and discriminate variations in signal intensity and orientation of non-native signals such as infrared, UV light, magnetic fields, etc. using SSCS-induced perceptual sense, and ultimately subjects will learn to use these novel perceptual abilities to navigate a spatial environment with non-native signal cues (Goal 2). Third, lower limb amputee subjects will learn to intuitively perceive movement and location of their prosthetic limbs during locomotion via real-time closed loop sensory feedback using SSCS (Goal 3). This project is innovative because it uses FDA-approved technology (spinal cord stimulation) in a new context, all without changing the patient’s standard-of-care. The ability to augment, restore, and modulate perceptions will be an unprecedented development in the field of sensory neuroprosthetics. Successful execution of proposed goals will not only launch a new line of augmentation research, but it will also showcase that SSCS can be widely applicable in the rehabilitation of patients suffering from sensory deficits due to neurological disorders and injuries.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Women living in low- and middle-income countries (LMICs) face a disproportionate burden of incidence and mortality from cervical cancer, accounting for 85% of incident cases and 90% of mortality globally. Women living with HIV (WLWH), the majority of whom live in LMICs, bear the greatest burden with up to 6 times increased risk of cervical cancer due to higher incidence and persistence of human papillomavirus (HPV) infection. The World Health Organization (WHO) recommends the use of ablation or excisional treatment for suspected or confirmed cervical precancer in LMICs, in a 'screen & treat' strategy to limit loss to follow-up. However, among WLWH, both treatment methods experience high failure rates, with a 30% recurrence of cervical intraepithelial neoplasia (CIN2/3) at two years following thermal ablation. Treatment failure rates are driven partly by high rates of persistent HPV infection following both ablation and excisional treatment in WLWH. High treatment failures are a significant limitation of the current cervical cancer secondary prevention strategy among WLWH and call for studies on feasible, innovative, yet accessible therapies to improve HPV clearance and reduce precancer treatment failure in WLWH. I will build on ongoing U.S-based trials that demonstrate safety and possible efficacy of artesunate, a semi-synthetic derivative of artemisinin for treatment of HPV-associated anogenital lesions. My proposed study will generate preliminary data on the feasibility of using self-administered artesunate vaginal inserts as adjuvant therapy following thermal ablation to improve treatment outcomes among HPV+ WLWH in LMICs. I will leverage my decade-plus collaboration with Ministry of Health-supported HIV clinics in Western Kenya, the Kenya Medical Research Institute (KEMRI), and new and established collaborators to conduct this study successfully. In this planning grant, I propose a pilot, randomized, placebo-controlled trial to investigate the following specific aims: 1) evaluate uptake and acceptability of self-administered artesunate vaginal inserts as adjuvant treatment following thermal ablation in HPV+ WLWH in an LMIC, 2) test and evaluate three methods of assessing adherence to self-administered artesunate to inform a future study protocol, and 3) perform a preliminary comparison of type-specific hrHPV clearance at six months following randomization in the artesunate and matched placebo arms as a statistical planning aim to obtain effect sizes to power a future trial. This study will be the first to evaluate the feasibility of artesunate vaginal inserts for improving the current ‘screen & treat’ cervical cancer prevention strategy among WLWH in LMICs. If found to be feasible and effective, artesunate, which is included in the WHO’s Essential Medications List, could be repurposed in this highly scalable approach to impact a significant global public health problem.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Dr. Benyam Muluneh’s long-term career goal is to become a leading clinician-researcher in oncology by leveraging health behavior theories and implementation science methods to promote adherence, access, and equitable cancer care. This K08 Mentored Research Career Development award (PAR-21-299) is an initial step toward developing his research program as an NIH-funded independent investigator; with this award, he will follow a structured plan, receive expert mentorship, and conduct research in an ideal environment. Oral anticancer (OAC) agents revolutionized treatment of once-fatal malignancies by extending survival and delaying progression; however, success often requires a medication adherence rate >90%. Medication adherence drops from >80-90% in clinical trials (where patients receive adherence support, proactive symptom management, and the study drug is provided free of charge) to ~40% in clinical practice, compromising clinical outcomes. The National Cancer Institute (NCI) recommends cancer centers design and implement health promotion programs (e.g. adherence interventions) guided by health behavior theories, models, and frameworks. Most published adherence interventions lack NCI’s theory-guided approach: they address known barriers (eg. patient education and symptom management) without addressing critical moderators of health behavior (eg. social support, self- efficacy), compromising effectiveness and long-term sustainability. Additionally, many of the existing interventions were piloted in well-resourced academic medical centers and were not adapted to rural and underserved settings. Our team piloted an adherence coaching intervention—consisting of tailored education and barrier mitigation—which increased adherence rates from 48% to 60% in chronic leukemia patients; however, similar to other adherence programs designed by clinicians, our intervention was not optimally effective (adherence <90%) or sustainable beyond the pilot phase. The objective of this proposed study is to enhance the effectiveness and sustainability of our adherence coaching intervention in both urban/academic and rural setting, by integrating social cognitive theory (SCT)—a proven behavioral theory that conceptualizes health behavior through cognitive, environmental, and behavioral influences —with a well-tested planning framework called intervention mapping (IM). We will identify cognitive, behavioral, and environmental determinants of adherence to OACs in both academic/urban and rural settings (aim 1); conduct focus group discussions to adapt our adherence coaching intervention for local contexts (aim 2); and test the refined adherence coaching intervention in a Type I Hybrid Effectiveness-Implementation study (aim 3). Through the following training objectives, Dr. Muluneh will acquire the skills to complete the research aims: gain training and experience in intervention design using health behavior theories (objective 1); gain training and experience designing effectiveness-implementation studies (objective 2); prepare and submit an R01 grant based on findings from the proposed K08 award (objective 3); and gain professional development skills necessary for a successful faculty career (objective 4). This foundational knowledge is necessary to implement and widely disseminate interventions to improve medication adherence in oncology clinical settings.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Alternative splicing wields extraordinary power in controlling protein function, cell development, and tissue identity. Alternative splicing is a co- and post-transcriptional RNA processing mechanism that enables a single gene to generate more than one transcript, thereby expanding the diversity of a cell’s proteome. This ubiquitous mechanism of gene regulation occurs in more than 95% of multiexonic genes in humans. The heart exhibits one of the most tissue specific and highly conserved alternative splicing programs, and the reprogramming of these splicing patterns is a hallmark of cardiovascular diseases. In the heart, genes encoding membrane trafficking proteins are alternatively spliced in a tissue-specific manner; however, how these events are regulated and the functional roles of these isoforms within cardiomyocytes remains elusive. This proposal will investigate one specific membrane trafficking splicing event that takes place in the gene encoding the clathrin heavy chain (CLTC) protein. CLTC classically functions during clathrin-mediated endocytosis but plays unconventional structural roles as well. Cltc exon 31 alternative splicing occurs in the heart, and we have previously observed that deletion of Cltc exon 31 using CRISPR editing in mice impedes the progression of hypertrophy and heart failure after pressure overload. I hypothesize that the regulation of Cltc exon 31 splicing in the heart modulates the endocytic and structural functions of clathrin, which impacts cardiomyocyte cell growth. I will address this hypothesis with two aims. In Aim 1, I will determine the physiological consequences of Cltc splicing in the heart by assessing the impact of CLTC isoform expression on cardiomyocyte cell signaling, cytoskeleton architecture, and protein-protein interactions (immunoblotting, confocal microscopy, and proteomics). In Aim 2, I will identify the regulatory mechanism governing Cltc exon 31 splicing in the heart. I will demonstrate that the quaking (QKI) and polypyrimidine tract binding protein 1 (PTBP1) RNA-binding proteins control Cltc exon 31 splicing and that these RBPs bind with high affinity to consensus motifs located within the Cltc pre-mRNA transcript (protein knockdowns, in vitro reconstitutions, and minigene reporter expression paired with motif deletions). My long-term goal is to have a research-intensive career and become an independent leader in cardiovascular biology. The training that I will receive during this fellowship period will facilitate my scientific, professional, and personal growth by providing opportunities to expand my experimental toolkit, network with esteemed researchers, and increase my confidence as a strong scientist. Importantly, the University of North Carolina at Chapel Hill offers a rich research community of RNA, cardiovascular, genetic, and molecular cell biologists, and I have recruited mentors and collaborators with expertise in each of these fields. All of them are committed to educating and supporting me throughout my fellowship, which will set me up for success with both my research proposal and my future scientific career.

NIH Research Projects · FY 2025 · 2023-09

Today’s incipient and early career trainees in Kidney, Urologic, Hematologic (KUH) fields require training within environments richly populated by accomplished team-science researchers and adept, committed mentors. Trainees must learn sophisticated research tools while simultaneously completing didactic coursework and developing professional development skills. Coordinating these efforts will enable scale-up of high-impact practices to facilitate advanced skills among KUH researchers, and the direct gains of this training program will be realized most by the KUH patient populations we serve. Training the next generation of KUH researchers requires institutions and mentors to synergistically engage in research and training, in settings that support both trainees and mentors. We have brought together KUH researchers at three leading North Carolina (NC) biomedical research universities: University of North Carolina-Chapel Hill, Wake Forest University, and Duke University, as well as from three smaller NC universities with strong science, technology, engineering and mathematics (STEM) degree programs, NC Agricultural & Technical State University, Winston Salem State University and NC Central University. Together, this forms the NC KUH TRIO (Training, Research, Innovation, Outreach, or TRIO) U2C/TL1 Research Training Program. The TRIO Training Core has 3 objectives: 1) provide didactics and hands-on experience in rigorous basic/translational and clinical sciences research; 2) conduct research that includes adoption of emerging technologies and builds on the cross-disciplinary team science across the KUH mission; and 3) establishes and nourishes a community of scholars that expands and sustains KUH research. Multidisciplinary and team research skills will be developed under the Professional Development Core in conjunction with Clinical and Translational Science Award (CTSA) programs. A connected community of TRIO scholars, that spans from high school to early faculty, will be built and supported by the Networking Core. Qualified and motivated TRIO mentors will train both pre- and post-doctoral trainees across broad, highly relevant KUH scientific specialties that span basic/translational, clinical, and technology arenas. Emerging mentors will be guided towards their maturation into well-trained leaders, toward supporting the longevity of the program. TRIO will exploit existing university training infrastructures and programs in biotechnology, public health, translational science, and clinical research. A robust admissions process with targeted recruitment will be used to select the strongest trainees, and rigorous program evaluation will allow real-time improvement in serving the needs of our KUH trainees. The TRIO program will leverage collaborative synergies and provide cohesive programming to train the next generation of KUH researchers and leaders.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Unreliable access to safe and sufficient water for all household uses (i.e., water insecurity) is a common but understudied public health issue. Although water is itself a critical nutrient that is essential for agricultural production, food preparation, and hygienic practices that reduce exposure to enteric pathogens, few studies have examined the role of water insecurity in nutrition; most consider water quality only. A more holistic conceptualization of water insecurity could reveal important pathways by which water accessibility, reliability, and adequacy impact diet and health. Water insecurity presents substantial barriers to cooking nutrient-dense foods, which often require considerable water to be made palatable and safe for consumption. Qualitative studies have reported that water insecurity limits breastfeeding duration and undermines the ability of caregivers to prepare preferred complementary foods. Individuals experiencing water insecurity are also more likely to substitute water with sugar-sweetened beverages, plausibly contributing to weight gain and increased risk of overweight and obesity. The proposed project seeks to expand upon this formative work by examining the extent to which differential exposure to water insecurity impacts diet quality and body composition throughout the life course. A mixed-methods approach is required to comprehensively understand how water insecurity manifests at the household level and subsequently impacts nutritional well-being. The proposed project will therefore use qualitative data to complement quantitative data from 2 existing cohort studies that comprehensively measured water insecurity and diet among infants, children, adolescents, and adults on San Cristóbal Island, Ecuador and in Cebu, Philippines. Specifically, the project aims to qualitatively describe how issues with water availability, accessibility, and use impact food production, purchase, and preparation (Aim 1). Multi-level modeling will be used to assess whether household water insecurity undermines child diet quality (Aim 2) and longitudinal structural equation modeling will be applied to evaluate how changes in household water insecurity throughout early development impact adiposity (Aim 3). The project will address a recognized knowledge gap within the field by using advanced statistical procedures to model water insecurity as a multidimensional, time-varying exposure. Findings from this work have the potential to reveal new targets for addressing the growing burden of malnutrition. Additionally, the proposed rigorous curriculum in nutrition and epidemiology, as well as mentorship from thought leaders in the water and nutrition sectors, will prepare the applicant for an academic career in global nutrition.

NIH Research Projects · FY 2025 · 2023-09

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The NC KUH TRIO Networking Core is designed to build a pipeline of talented trainees who will contribute to KUH related fields across NC institutions of higher learning. We aim to provide support to this pipeline as part of a dynamic and cohesive cohort along their training pathway towards successful and rewarding careers in research. By connecting programmatic and established partners across 3 leading North Carolina (NC) biomedical research universities: University of North Carolina-Chapel Hill, Wake Forest University, and Duke University, and from three smaller NC Universities with strong science, technology, engineering and mathematics (STEM) degree programs, NC Agricultural & Technical State University, Winston-Salem State University and NC Central University, the TRIO Networking Core is poised to reach a broad spectrum of trainees interested in science and research. Networking Core activities will connect early-stage STEM trainees to peers, near-peer trainees, and senior mentors, which will be enhanced through the integration of social media and virtual learning platforms in addition to traditional in-person group learning opportunities, conference meetings, and community outreach events. Together, these activities will support the TRIO Networking Core’s three objectives to: 1) organize a pipeline with a leadership structure built to recruit and support development of the workforce needed to serve the communities impacted by KUH-related diseases, leveraging existing, and creating new KUH-specific programs especially for undergraduate college learners; 2) support the talent of our trainees as emerging interdisciplinary leaders across all levels of KUH-related research by equipping them with the skills required to conduct scientific, collaborative research ; and 3) enhance retention of trainees across the KUH pipeline through community building trainee activities. Close collaboration with the TRIO Professional Development, Training (TL1), and Administrative Cores will provide resources and infuse the Networking Core with the mentors and tools to build the bridges for undergraduate trainees and maintain an open gateway of support for the next generation of researchers. These efforts support the overall goal of nurturing fully prepared researchers who will advance KUH research and advance our goal of developing a strong workforce to advance research and improve outcomes for the broad spectrum of KUH diseases and conditions.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY During DNA replication, a cell must replicate not only its DNA, but also the nucleosomes that package its genome. To meet this extremely high demand for rapid histone protein biosynthesis, all eukaryotes coordinate high expression of replication-dependent (RD) histone gene clusters during S-phase of the cell division cycle. Regulating the level of expression of RD-histone genes during S-phase is important for maintaining genomic integrity and normal cell cycle progression, as too many or too few histones lead to toxic effects such as enhanced DNA damage sensitivity or cell cycle arrest. While the transcriptional activation of RD-histone genes has been characterized, little is known about the negative regulation that occurs as cells exit S-phase or the modulation of transcription that occurs during S-phase. My preliminary research has shown the Drosophila protein Mute (a homologue of human Yarp/Gon4L) is a prime candidate for a negative transcriptional regulator of RD-histone genes. I have found that loss of Mute uncouples expression of RD-histone genes from S-phase in Drosophila embryos. I hypothesize Mute is both restricting RD-histone expression to S-phase as well as regulating levels of expression during S- phase. This proposal seeks to uncover the mechanisms through which Mute represses RD- histone genes and how this repression is connected to the cell cycle. Using an interdisciplinary approach, the mechanisms of Mute’s repression at both the cellular and molecular levels will be explored using a combination of genomic, fluorescent imaging, genetic, and proteomic techniques in the Drosophila model system. This project will enhance our understanding of the role of histone gene regulation in metazoans and provide insight into the mechanisms that govern the regulation of this highly conserved cellular process.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY A “good death” is often desired but seldom delivered for patients with cancer. Because oncologists often do not recognize when a patient is near the end of life (EOL), many patients receive aggressive care until death. To remedy this, prognostic tools are needed to identify patients nearing death. How to incorporate these tools into routine cancer care and use them to prompt best practices, such as serious illness conversations and hospice referrals, is not well understood. The long-term goal is to leverage evidence-based tools to improve EOL care for patients with metastatic breast cancer (MBC). The main objective is to integrate a validated breast cancer prognostic tool into routine practice using implementation science. This research study will pursue three aims: 1) Define the baseline frequency of documented serious illness conversations and hospice referrals among patients with metastatic breast cancer and their relationship to prognostic risk scores; 2) Co-design an implementation protocol with patient, caregiver, and care team partners for use of a metastatic breast cancer prognostic tool in an oncology clinic; and 3) Pilot implementation of a metastatic breast cancer prognostic tool in an oncology clinic and assess implementation outcomes. For the first aim, a retrospective study using EHR data from patients with MBC will quantify baseline EOL best practices and define their relationship to prognostic scores. For the second aim, interviews and user-centered research with oncologists, patients, and caregivers will evaluate factors that impede or enable implementation of the tool in practice and identify desired features of the prognostic tool implementation. These key partners will co-design an implementation protocol. For the third aim, the tool will be piloted in an oncology clinic according to the protocol and evaluated for implementation outcomes using surveys, interviews, and secondary data in a mixed method design. This project seeks to innovate by shifting the paradigm from sole reliance on clinician judgement towards use of evidence-based tools to estimate prognosis. The proposed study is significant for its potential to improve EOL quality for patients with MBC through timely identification of high-risk patients and earlier initiation of EOL best practices. This proposal is submitted by Emily Ray, MD, MPH, a breast medical oncologist and health services researcher at the University of North Carolina in the Cancer Outcomes Research Program. The proposed career development plan will equip her with skills in implementation science, mixed methods, cancer care quality, and multi-site trials to accelerate her research from its evidence basis in large datasets towards interventions aimed at improving cancer care quality. The academic environment at the University of North Carolina, with leading programs in breast oncology, cancer outcomes research, and public health, and her outstanding mentoring team including Dr. Jennifer Leeman, a well-funded, national expert in implementation science, positions Dr. Ray to make an impact through designing, testing, and implementing evidence-based interventions to deliver high-quality, patient-centered end-of-life cancer care.

NIH Research Projects · FY 2024 · 2023-09

Glioblastoma (GBM) is the most common primary malignant brain tumor affecting adults, with a median survival time of 12-15 months. The standard of care for treating GBMs is maximum tumor resection followed by concomitant radiation and temozolomide therapy. However, tumor cells remain in the brain after resection, posing the threat of disease recurrence. Systemically administered treatments like radiation and chemotherapies are not targeted to the microscopic tumor lesions present in the brain post-surgery and are thus ineffective at preventing recurrence for 90% of GBM patients. Our group and others have demonstrated the promise of therapeutic neural stem cells (tNSCs) as a drug delivery platform for treating post-operative GBM due to an innate property known as tumor tropism. tNSCs interact with cytokines secreted by GBM cells, initiating a signaling cascade which results in tNSC migration in the direction of the tumor. This directional migration can be leveraged as a targeting mechanism for the delivery of drugs secreted by genetically engineered tNSCs. However, the platform's durability is limited by rapid clearance of tNSCs implanted directly into the GBM resection cavity. Encapsulation of tNSCs in biomaterials when delivered into the cavity could prevent this rapid clearance and lengthen the duration of therapeutic efficacy. Our group has demonstrated that biocompatible materials such as commercially available hemostats are able to support long-term in vivo tNSC viability. However, these matrices can pose a barrier to tNSC migration, resulting in insignificant tumor killing compared to tNSCs injected in PBS alone. Thus, we discovered that a balance between enhanced tNSC viability and unimpaired cell migration must be reached to optimize tNSCs for long-term GBM therapy. To do so, we will develop a novel adaptation of the 3D printing technology, continuous liquid interface production (CLIP), in which tNSCs are 3D printed into hydrogels in a process known as bioprinting. This results in cell-embedded 3D hydrogels which could be implanted into the GBM resection cavity without any intermediate cell seeding steps. We have shown that bioprinted cell-laden hydrogels exhibit higher seeding consistency than cells seeded externally onto hydrogel surfaces. However, cell behavior and function has not been characterized or optimized inside bioprinted hydrogels. Moreover, the most biocompatible hydrogels which support the longest cell viability exhibit the lowest printing resolution. Thus, we propose to optimize this novel bioprinting strategy by developing a biocompatible and printable resin that can support cell viability for at least one month. Furthermore, we will characterize cell health and functionality pre- and post-bioprinting to ensure that toxic resin monomers and UV light have not compromised the efficacy or safety of the embedded cells. Finally, we will characterize the efficacy of cell-laden bioprinted hydrogels in a post-resection GBM mouse model with unencapsulated cells serving as a comparator. We hypothesize that the optimized bioprinting strategy will result in higher consistency during manufacturing, easier clinical handling, and a longer duration of tumor suppression, leading to improved patient outcomes.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Current therapies for Alzheimer’s disease (AD) do not reverse, or even slow, progression of the disease. This situation is dire and exacerbated by the failure of antibodies directed toward two of the more promising targets—phosphorylated tau and beta-amyloids—to treat the disease. Clearly, new treatments are urgently needed. In 2017, a large genome-wide study associated a naturally-occurring variant (P522R) of PLCG2, the gene encoding PLC-2, with protection from late onset AD. In follow-up studies, this genetic association has remained strong and highly reproducible. Even more encouraging, in clinical studies of patients with mild cognitive impairment, people that carried PLCG2 (P522R) had slower rates of cognitive decline compared to non-carriers. Protection was observed even for patients homozygous for ApoE4, a biomarker strongly linked to AD. In the brain, PLC-2 is primarily expressed in microglial cells where it controls phagocytic and neuroinflammatory processes. It is more highly expressed in pathological areas of patients with AD. In microglia, PLC-2 is activated downstream of both TREM2 (which uses ApoE4 as a ligand) and CSF1R, two transmembrane receptors that are strongly linked to AD. Similarly, PLC-2 activates PKC, which is also linked to AD. Thus, genetic and cellular data strongly support PLC-2 as a novel therapeutic target for treatment of AD. The phospholipase activity of PLC-2 (P522R) is modestly elevated relative to its wild-type counterpart and it is this increased activity in microglia that is generally accepted to protect against AD. We propose to identify and optimize small molecules that selectively activate PLC-2 to reproduce the neuroprotective effects of PLC- 2 (P522R) and treat AD. The research plan relies on complementary high-throughput assays enabled by two fluorogenic substrates for eukaryotic PLCs that we invented explicitly for this research. Consequently, we will pursue three Aims. In Aim 1, in-house collections totaling ~300,000 compounds will be screened for activators of PLC-2 and primary hits verified for activity, selectivity, composition, and purity; cheminformatics will be used to structurally classify hits. In Aim 2, a high-quality model of full-length PLC-2 coupled with molecular dynamics simulations will be used for computational screens of tens of millions of compounds. In Aim 3, a suite of biochemical, biophysical, and cell biological studies will be used to prioritize allosteric activators of PLC-2 with favorable chemical and pharmacological properties. These novel small molecules will be invaluable tools to further understand how PLC-2 (P522R) reduces the risk of AD. The small molecules will also be used as leads for the development of novel therapeutics to treat AD.

NIH Research Projects · FY 2025 · 2023-09

Project Abstract Component A: BD-STEPS Core at the NC Center for Birth Defects Research and Prevention Birth defects are a leading cause of infant mortality and childhood morbidity, yet the etiology of most cases is unknown. Established in 2002 with funding from the CDC, the North Carolina Center for Birth Defects Research and Prevention (NCCBDRP) has been a major contributor to the National Birth Defects Prevention Study (NBDPS) and Birth Defects Study To Evaluate Pregnancy exposureS (BD-STEPS). The next phase of BD-STEPS will continue to build on these important multi-site, population-based case-control studies to identify novel epidemiologic and genetic risk factors for major structural birth defects that can be translated into prevention strategies. The NCCBDRP's Specific Aims are to (1) Implement the BD-STEPS Core protocol in North Carolina including: (a) Utilize the existing state-wide, population-based birth defects surveillance system in NC to actively ascertain at least 150 cases per year among livebirths, stillbirths and elective abortions with a study-eligible birth defect in the demographically diverse 33-county study area; (b) Utilize birth certificates to identify an eligible population-based sample of approximately 100 control infants per year born in the same study area; (c) Conduct clinical case review and classification by clinical geneticist and pediatric cardiologist using abstracted medical records; (d) Partner with CDC's central interviewing contractor to facilitate NC participation in the core interview and online occupational survey; (e) Conduct supplemental data collection about reportable infections before and during pregnancy; (f) Conduct supplemental biospecimen collection of residual newborn blood spots; (2) Establish a unique data resource for investigating prescription medication use during pregnancy and birth defects by linking BD-STEPS and NBDPS participants in NC with health insurance claims records from NC Medicaid and Blue Cross Blue Shield NC; (3) Establish a unique biospecimen resource for investigating prenatal environmental exposures and birth defects by collecting shed deciduous teeth from BD-STEPS and NBDPS participants in NC; (4) Leverage the NCCBDRP's well- established group of investigators and collaborators to develop innovative, high-impact studies with BD-STEPS and NBDPS data on genetics, epigenetics, and gene-exposure interactions associated with birth defects, and epidemiologic risk factors for birth defects including medication use during pregnancy, maternal chronic and infectious disease, and key social determinants of health such as individual- and neighborhood-level socioeconomic factors, acculturation, employment and workplace conditions, and environmental factors; (5) Produce at least seven NC-led manuscripts for publication in high-impact journals using pooled data from BD- STEPS and/or NBDPS during the 3.5-year award period; and (6) Continue our long-standing, successful training program in birth defects epidemiology.

NIH Research Projects · FY 2025 · 2023-09

Approximately one in seven adults in the United States lives with chronic kidney disease (CKD). CKD typically worsens with time and, in its final stage, can result in kidney failure. Contextual factors in rural, eastern North Carolina communities impede optimal management of CKD multimorbidity. In these communities, geographical barriers to medical care, dwindling resources, and underdeveloped health infrastructure have worsened CKD outcomes. CommunityRx-CKD (CRx-CKD) is an evidence-based, low-intensity, health information technology-driven intervention designed to support CKD management in rural eastern North Carolina. CRx-CKD integrates medical (e.g., blood pressure and glucose monitoring, eye and foot care), social (food, housing, transportation), and self-care (weight and stress management, exercise) resources. CRx-CKD comprises three components: brief education on integrated CKD needs, a CKD care plan that includes integrated care referrals, and clinic navigator-led, longitudinal support (12 months) for CKD patients in our trial. Our multidisciplinary, community- engaged research team will test the effects of CRx-CKD through three related aims. In Aim 1, we will assess contextual factors that influence the implementation of an integrated care intervention. We will employ a participatory science approach, known as group model building, to engage patients, caregivers, providers, and other stakeholders and elicit their perspectives on how (a) community factors affect CKD progression in rural North Carolina and (b) how integrated care interventions like CRx-CKD can address CKD multimorbidity in rural North Carolina. In Aim 2, we will develop an implementation blueprint based on the findings of Aim 1 and an assessment of organizational readiness to implement change. We will engage providers and clinic navigators, train them on the CRx-CKD intervention, and test the CRx-CKD integrated clinical workflow using quality improvement strategies. In Aim 3, we will conduct a pragmatic individual-randomized, two- arm (usual care + CRx-CKD (treatment) and usual care (control)) single-blind trial in 25 rural primary care clinics in 12 rural eastern North Carolina counties (N=634 adults with CKD) to assess the effect of CRx-CKD on acute healthcare utilization (primary outcome), self-efficacy for finding resources, knowledge and sharing of integrated care resources, resource use, number of unmet needs over time, ambulatory care utilization, and health-related quality of life. We hypothesize that 12-month acute healthcare utilization will differ between participants receiving CRx-CKD and those receiving usual care.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY At the heart of Alzheimer’s (AD) pathogenesis, tau pathology is linked to neurodegeneration and cognitive decline. We have spent the last decade addressing how tau exerts its toxicity in AD. Among the many tau post- translational modifications (PTMs) that are now known to co-occur and target tau, acetylation of tau’s lysines, which have emerged from recent cryo-EM and mass spectrometry studies, is a particularly relevant and attractive target. Coupled with the fact that acetylation accelerates tau aggregation, prevents normal microtubule binding, and induces prominent AD-like deficits including synaptic dysfunction and cognitive decline, these properties would appear to set this particular PTM apart from many of the other tau PTMs that are known to regulate tau. While acetylated tau is common to virtually all sporadic AD brains, there remain few reliable models to unravel the signaling events and enzymes that converge on this idea. In our view, this gap needs to be overcome if we hope to unravel mechanisms that drive tau pathogenesis. We identified a new signaling pathway in which the Parkinson’s disease (PD)-relevant kinase LRRK2 acts upstream to regulate HDACs and therefore indirectly controls tau acetylation. We hypothesize that LRRK2 and other related “HDAC kinases” act as master regulators of HDAC function, with their end goal of preventing the accumulation of acetylated tau and thereby protecting against tau toxicity. In Aim-1, we develop a new model based on an engineered cytoplasmic CBP acetyltransferase to precisely target cytoplasmic tau and then assess the extent of tau pathology, synaptic dysfunction, tau seeding, and tau propagation. Having narrowed in on HDACs 3/6 as the only tau-associated HDACs among all human HDACs, we will deliver them to neurons and determine whether synergism among HDACs converges onto tau to suppress its toxicity. In Aim-2, we focus on the upstream kinases that coordinate HDAC activity. We explore LRRK2 as a very attractive hit identified in a mini screen that modulates HDAC3/6 function. We will manipulate LRRK2 function in mouse neurons, mice, and human iPSC neurons to evaluate downstream consequences on HDACs and tau. Our proposal will shed light, not only on AD-relevant HDACs, but also open up new therapeutic avenues (e.g., the targeting of upstream kinases) to suppress toxic tau species in the brain. This proposal is therefore both innovative and significant since upstream HDAC regulatory kinases including LRRK2 and PKC hold promise as unanticipated regulators of HDAC activity. This will expand our repertoire of targetable pathways in tauopathies that include AD and even PD as well.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Directed evolution, which adopts principles of natural evolution to the laboratory, is singular in its impact on molecular engineering. As one example, it is responsible for the generation of the majority of approved therapeutic antibodies and those still in clinical development. Despite its progress, a valuable therapeutic niche remains outside its scope: the mammalian cell. While in vitro systems, phage, bacteria, and yeast have lent themselves to laboratory manipulation, mammalian cells have proven less tractable. Consequently, the power of evolution remains inaccessible to drug development pipelines that seek to modulate mammalian cell signaling. Further, many directed evolution campaigns result in biomolecules that fail in critical ways when transplanted to human cells. To address these limitations and advance methods in drug discovery, I will explore and mine viral diversity to create a novel system for molecular evolution in mammalian cells. Next, focusing on the 5-HT2A serotonin receptor, I will create extracellular nanobodies to template a drug discovery campaign via structural determination and in silico docking. The insights gained by these studies will be applied toward the directed evolution of state-specific nanobodies against dark GPCRs. This work will result in a general method for directed evolution in mammalian cells, chemical matter against HTR2A, and new paths forward for the deorphanization of GPCRs.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT The rapid emergence of innovative technologies, therapeutics, and other FDA-regulated products have outpaced the ability to effectively appropriate them for public benefit. Doing so hinges on the capability to expeditiously evaluate these products and effectively monitor them once on the market. However, methodologies for pre- market evaluation through laboratory testing and clinical trials for drugs, devices, and biologics and for post- marketing surveillance of all FDA-regulated products have not kept pace, and key gaps in knowledge exist that would inform regulatory decision making in many areas. In the face of these pressing needs, the Research Triangle Center of Excellence in Regulatory Science and Innovation (Triangle CERSI) will provide a one-stop- shop accelerator to meet FDA’s current and evolving needs in regulatory science and a generative community for regulators, academia, industry, and other stakeholders. A collaboration of the University of North Carolina at Chapel Hill, Duke University, North Carolina State University, and North Carolina Central University, all in close proximity, the Triangle CERSI represents a broad network of investigators and national and international collaborators that bring unique and diverse expertise and resources for regulatory science, including but not limited to novel approaches in statistical methodologies, machine learning and artificial intelligence, imaging, in silico trials, pediatric pharmacology, patient reported outcomes (PRO), population science, and safety assessment across the lifespan, and other areas. The Triangle CERSI includes two Schools of Medicine, a School of Pharmacy, two Schools of Nursing, a School of Public Health, a College of Veterinary Medicine, a leading historically Black university, a national Center for Virtual Imaging Trials, and the Duke Clinical Research Institute, and leverages relationships with nearby companies and organizations in the Research Triangle Park. The Triangle CERSI has three specific aims: 1. Conduct regulatory science projects in collaboration with FDA to deliver major advances in regulatory science and rapidly address a broad range of major, specific, and emerging challenges in response to FDA needs. 2. Establish robust CERSI Core infrastructure to develop, propose, support, enable, and monitor execution of CERSI research projects to facilitate rapid achievement of project deliverables; partner Oak Ridge National Laboratory provides support for data access, computation, and resource sharing. 3. Expand support of the Triangle CERSI and extend its impact through regulatory science information sharing activities, including efforts to diversify the regulatory science workforce. The Triangle CERSI will actively share results and newly developed tools and resources with the FDA, research community, stakeholders, and our institutions’ students and trainees. Together, our overarching goal is to provide an abundance of essential new information, infrastructure, and tools to shorten the drug and device development process, to advance public health, and to inform FDA activities in ways that complement other CERSIs and contribute to achieving the goals of the national CERSI program.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Chronic wounds are a growing public health threat, with over 6.5 million people affected in the US alone, costing upwards of $30 billion annually. The single-most-important cause of delayed wound healing is bacterial infection, often in the form of biofilms that impede antibiotic penetration and force the bacteria into a “dormant” state where they are more tolerant to antibiotics. Antibiotics work so poorly against biofilm-infected wounds due to 1) poor drug penetration and 2) the presence of drug-tolerant persister cells within biofilms. These factors contribute to a 70% infection recurrence rate. Failure to completely eradicate biofilms during antibiotic therapy can result in significant quality-of-life reduction, hospitalization, sepsis, amputation and death. Comorbidities such as diabetes and cardiovascular disease further complicate therapeutic strategies. Every day in the US, 230 patients suffer an amputation due to a chronic wound infection, the majority due to diabetic foot infections. There is an urgent need for improved wound care therapies but with the void in the drug- development platform, innovation is mostly centered around wound closure rather than improving antibiotic efficacy. Recently, the technology of acoustically active cavitation agents, microbubbles and phase-change contrast agents (PCCA), for ultrasound-mediated drug delivery has made several substantial advances and is currently in clinical trials for other applications. In this proposal, we will develop a non-invasive theranostic ultrasound platform to improve delivery of anti-persister drugs into biofilm-infected wounds. Our encouraging preliminary data in an in vivo diabetic mouse model observed improved traditional therapeutic clearance of MRSA biofilms by 94% in chronic wounds using a topical-only approach. Importantly, we achieved complete eradication (below limit of detection) in 3 out of 8 animals. In this project, we propose to optimize the efficacy of our acoustically responsive biocompatible particles and therapeutic ultrasound parameters to potentiate various antibiotics against biofilms of the most common pathogens in chronic wound infections (Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis) in vitro, in conjunction with investigating mechanisms of action by quantifying therapeutic penetration and cavitation activity in vitro (specific aim 1). We will then evaluate this approach in a polymicrobial diabetic chronic wound infection model (specific aim 2). For this, we will focus on Pseudomonas aeruginosa and Staphylococcus aureus, the two organisms that most commonly co-infect chronic wounds. Measures of wound healing, wound closure and reduction in bacterial burden will be quantified and benchmarked against current standard of care systemic antibiotic administration, where cavitation activity will also be evaluated for its ability to predict early response to therapy. Our proposed approach has the potential to have a substantial impact on the treatment of polymicrobial biofilms in chronic wounds.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Pertussis (aka whooping cough) is a serious, re-emerging public health concern despite available vaccines. The acellular vaccine against Bordetella pertussis, used in the U.S. since the 1990s, prevents serious disease, but not colonization or transmission, resulting in a larger reservoir from which infants, who are most vulnerable, can be infected. New vaccines that protect against both colonization and disease are needed. The BvgAS and PlrSR two-component systems control Bordetella virulence. The BvgAS two-component regulatory system (TCS) has long been considered the master regulator of Bordetella virulence. It controls production of all know protein virulence factors, including those in the acellular vaccine. We discovered another TCS, called PlrSR, that is essential for Bordetella viability and for BvgAS activity in the lower respiratory tract (LRT). We found that PlrSR is required for LRT infection even when BvgAS is constitutively active, indicating that PlrSR controls expression of unidentified but critical virulence functions independently of BvgAS. These currently unknown virulence factors could serve as therapeutic targets or new vaccine components, and hence their identification is critical for controlling pertussis in the future. Drs. Cotter and Julio are experts in Bordetella pathogenesis and molecular biology, and Dr. Bourret is an expert in TCS biochemistry. Together, we have characterized PlrS and PlrR proteins biochemically and have discovered a way to bypass the apparent essentiality of plrS in vitro so that we can construct strains to collect gene expression data without knowing the stimuli sensed by PlrS. In Aim 1, we will identify genes regulated by PlrSR using RNA-Seq to reveal positive or negative regulation, and ChIP-Seq to reveal direct or indirect regulation. In Aim 2, we will investigate PlrSR signaling by making reporter fusions to key PlrSR-regulated genes and assessing responses to physiologically relevant stimuli, as well as the consequences of using PlrS lacking PDC or PAS sensory domains. Identification of the PlrSR regulon is low risk/high reward and will lay the foundation for a R01 project. The proposed methods are well-established, suggesting a high probability of achieving our Aims. Identifying the PlrSR regulon will be transformative in understanding Bordetella pathogenesis and enable a future R01 project in which we can determine (i) why PlrR is essential for viability, (ii) the roles of PlrSR regulated gene products in LRT infection by Bordetella, (iii) how PlrSR regulates gene expression, (iv) connections between the PlrSR and BvgAS TCSs, and (v) perhaps gain insight into the stimuli sensed by PlrS.

NIH Research Projects · FY 2025 · 2023-08

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The University of North Carolina at Chapel Hill (UNC) Program in Translational Medicine (PiTM) was first established in 2006 with funding support from the Howard Hughes Medical Institute and later supported by a T32 from the National Institutes of Health. The UNC PiTM is a unique program that fills unmet needs among biomedical graduate students at UNC. Unlike department- and disease-focused programs, the PiTM is cross-department, -diseases, and -disciplines, providing our trainees with a rich exposure to diverse topics in translational medicine. The primary objective of the PiTM is to train a cadre of PhD researchers with the knowledge and skills necessary to recognize, appreciate, and address clinically-relevant biological problems related to human disease from the perspective of basic science. To accomplish this goal, the PiTM trains basic science PhD students to work in multidisciplinary teams composed of scientists, physician-scientists, and clinicians, using state-of-the-art experimental approaches and patient-derived resources. The trainees have a dual (scientist/clinical) mentored experience, a rigorous clinical exposure, and blended core coursework that enhances training in translational medicine. Trainees also receive additional training to build communication and leadership skills essential for future team science and community outreach endeavors. The PiTM structure includes both funded and unfunded trainees (~10-15 matriculants each year), each with access to the same rigorous training opportunities and experiences throughout their graduate tenure. This structure allows us to attract students from different schools, including Medicine, Pharmacy, and Public Health.With this current application, we request support for 6 1-year slots for outstanding PiTM trainees. The PiTM has over a decade of experience in best practices training over 150 basic science PhD students, to perform translational research. The proposed training leverages this experience to create a unique training model for preparing the next generation of PhD researchers to effectively lead and contribute to multidisciplinary teams of translational researchers.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT: Despite the landmark approval of anti-PD1 therapy for non-small cell lung cancer (NSCLC), the majority of patients still progress. Compensatory activation of lymphocyte activation gene 3 (LAG3), to suppress T cell activation is an established mechanism of resistance to anti-PD-1 therapy in cancer. Our recent work suggests that IL-6 is an upstream inducer of LAG3 in patients with NSCLC. We have demonstrated an association between IL-6 and STAT3 activation and decreased response in patients with NSCLC receiving anti-PD-1 therapy. However, we do not know (1) the mechanism of IL-6-induced LAG3 expression which leads to anti- PD-1 resistance, or (2) if IL-6 blockade prevents T cell dysfunction in vivo. This proposal hypothesizes that (1) increased plasma IL-6 induces LAG3 expression via STAT3 activation in peripheral CD8+ T cells leading to T cell dysfunction and decreased response to anti-PD-1 therapy in patients; and (2) LAG3-mediated T cell dysfunction can be rescued with IL-6 blockade in vivo. This proposal will significantly impact future studies regarding immunotherapy resistance in cancer. Specifically, this project will inform our understanding of a novel IL-6-induced LAG3 mediated mechanism of anti-PD-1 resistance and will serve as the pre-clinical rationale for investigating the combination of IL6/STAT3 inhibition and PD-1 blockade in an early phase clinical trial for patients with advanced NSCLC. This proposal will provide Dr. Somasundaram with the opportunity to continue work with (i) animal models (design and oversee murine models), (ii) quantify changes in tumor size, volume and number in mice, and (iii) evaluate the immune landscape in murine T cells by immunohistochemistry (IHC) and flow cytometry, and RNA sequencing (RNAseq). He will continue his current work with patient samples also. These skills will be reinforced by a team of mentors, advisors, collaborators, and core resources available at the University of North Carolina and the University of Pittsburgh. The primary mentor, Dr. Jonathan Serody, is an internationally recognized, NIH R01-funded scientist with 28 years of experience in tumor immunology, and co-mentor, Dr. Dario Vignali, is an NIH R01-funded tumor immunologist with decades of experience in murine research. Also, an advisory committee of accomplished investigators with expertise in tumor immunology, IL-6, LAG3, PD-1, and NSCLC will monitor Dr. Somasundaram’s progress with quarterly meetings ensuring the completion of Dr. Somasundaram’s short-term goal of scientific independence and long-term goal of becoming a tenure-track physician-scientist with expertise in systemic inflammation, tumor immunology, checkpoint blockade resistance, and NSCLC. The results from this proposal will form the basis for an R01 studying the translational role of IL6 induced anti-PD-1 resistance in NSCLC.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Despite the unprecedented clinical success of chimeric antigen receptor (CAR) T cell therapy, its widespread application is limited by lengthy and labor-intensive ex vivo manufacturing procedures that result in: (i) high cost; (ii) delays to infuse CAR cells to patients with rapidly progressing disease; and (iii) CAR cells with heterogeneous composition and terminal differentiation, which limit their engraftment and persistence. There is a clear scientific and medical need for approaches to improve CAR T cell production, including methods to reduce cell processing times, reduce manufacturing costs, and reduce CAR cell differentiation. Recently, our labs collaboratively developed a new technology for CAR cell production called MASTER (Multifunctional Alginate Scaffolds for T cell Engineering and Release). MASTER consists of dry, macroporous alginate materials conjugated to αCD3 and αCD28 antibodies and encapsulating interleukin signaling. CAR generation with MASTER technology involves seeding freshly isolated, non-activated patient PBMCs together with CAR-encoding retroviral vectors and implanting scaffolds back into patients. Once implanted, MASTER mediates every step of the CAR production process, thereby eliminating the current standard procedural steps of αCD3/αCD28 pre-activation, viral transduction with spinoculation and interleukin-mediated CAR expansion. In vitro MASTER-generated CAR cells demonstrate reduced cellular differentiation as compared to CAR cells generated with gold-standard, “conventional” clinical protocols. In vivo MASTER-generated CAR cells demonstrate far superior in vivo cell persistence, enhanced anti-tumor efficacy and far superior prevention of tumor growth after rechallenge. The utility of this system is two-fold: 1) as a transformative therapeutic technology creating enhanced and affordable CAR therapy for cancer care and 2) as a research tool enabling rapid development, prototyping and testing of CAR therapeutic candidates. We have assembled a focused, multidisciplinary team comprised of an expert in biomaterials and drug delivery (Brudno), an expert in viral engineering and protein production (Birnbaum), two specialists in clinical CAR cell production (Chen, Roy) and a clinician focused on CAR cell therapies (Grover). In this proposal we seek to further develop and validate MASTER scaffolds and the associated methods to make them ready for widescale utilization by the research and clinical communities, including researchers in related areas eager to work in the CAR field but deterred by the barriers to test CAR construct in vivo. Leveraging transformative preliminary data that show that the shelf-stable MASTER scaffolds outperform conventional CAR cells in preclinical mouse models of lymphoma, orthotopic pancreatic cancer, and metastatic lung and ovarian tumors this proposal will validate MASTER scaffolds with a wide range of donors and at different scales, with multiple viral vectors and CAR constructs and delineate the phenotype and function resulting from MASTER production of CAR cells. The successful completion of these aims will propel our ultimate vision of low-cost and tunable generation of CAR cells for both liquid and solid tumors and potentially beyond the oncology space.

NIH Research Projects · FY 2025 · 2023-08

Abstract HIV infection of the nervous system results in chronic infection, inflammation, neuropsychiatric problems and cognitive decline in up to 50% of people living with HIV with no effective treatments to-date. Inflammation appears early in the disease process and causes progressive neuronal damage due, in part, to factors released by activated microglia and macrophages. In cultured neurons and mice, expressing the HIV gp120 transgene these factors induce intracellular calcium accumulation, cytoskeletal damage and focal swelling, in a fashion similar to early Alzheimer’s disease (AD) pathology, suggesting a common substrate for disease progression. Age-dependent accumulation of the p75 neurotrophin receptor occurs early in disease and is thought to contribute to pathogenesis by shifting the balance of neurotrophin signaling away from protective, regenerative pathways. Treatment of aging and gp120 transgenic mice with a small non-peptide p75NTR ligand, LM11A-31, suppressed cholinergic degeneration, inflammation and neuronal damage. In cats chronically infected with feline immunodeficiency virus, ten weeks of oral treatment with LM11A-31 prevented degeneration, improved cognitive behaviors, reduced anxiety and CSF viral titers in the absence of any adverse effects on systemic viremia, PBMC FIV burden, or CD4:CD8 T cell ratios. Since p75NTR is normally expressed at very low levels in adult brain but is upregulated in response to injury or disease, it provides a unique target for therapy with minimal potential for off-target effects. The drug is orally bioavailable, crosses the blood brain barrier and has no significant adverse effects in humans at therapeutic concentrations. To explore the potential of LM11A-31 as a disease modifying neuroprotective treatment, the proposed studies will establish the safety and tolerability of LM11A-31 treatment in a small cohort of stable virally-suppressed participants with HIV and mild neurocognitive impairment. Safety measures will be supplemented with exploratory characterization of traditional and novel biomarkers for early detection of inflammation and neurodegeneration in CSF and blood. A novel fMRI Hcorr analysis will be used to provide a sensitive measure of early immune and p75NTR activation with the potential to identify individuals in early stages of neurodegeneration. Serial neuropsychological test results will provide preliminary data and facilitate transition to a subsequent efficacy trial for prevention of cognitive decline. These studies are expected to show that LM11A-31 is safe to use in people living with HIV and to lay the groundwork for a larger efficacy trial designed to demonstration protection from neuronal damage and cognitive decline.