Virginia Commonwealth University

NIH Research Projects · FY 2025 · 2021-08

Project Summary Streptococcus sanguinis is a commensal oral bacterium and a pioneer colonizer of human teeth. It is associated with infective endocarditis and can enter the bloodstream through oral lesions, invasive dental procedures or routine oral activities such as chewing, flossing and brushing teeth. Recent developments in genomic technologies have created an opportunity to study streptococcal fitness genes as a whole by systems biology. This application proposes in Aim 1 to identify genes required for S. sanguinis fitness in an infective endocarditis model by screening a unique, comprehensive library of S. sanguinis genome-wide mutants. In Aim 2, a comparable analysis will be performed in saliva, serum and blood using ORF-seq analysis to clarify the findings of Aim 1 and to identify genes required for fitness, but not growth in saliva. The fitness genes in saliva and blood will be compared. Fitness genes that are located at critical hubs will be identified in network analysis and those that are conserved among oral streptococci will be identified by bioinformatics. In aim 3, the gene regulation of fitness genes will be studied in depth. This systematic analysis will provide an unprecedented understanding of streptococcal fitness genes at the molecular level and set the stage for identification of specific targets for new therapeutics or prophylactics.

NIH Research Projects · FY 2024 · 2021-08

Generic orally inhaled drug products (OIDPs) are expected to reduce cost and thereby improve compliance with prescribed dosage regimens, leading to improved control of multiple lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). Despite these advantages, relatively few generic OIDPs have received US Food and Drug Administration (FDA) approval and entered the marketplace due to challenges associated with establishing bioequivalence of inhaled medications, largely related to difficulties in determining regional lung dose. The objective of this study is to develop and validate new open-source computational fluid dynamics (CFD) methods for a solution-based metered dose inhaler (MDI) product that can accurately predict regional drug deposition throughout the airways, and then implement the model to establish in-vitro-in-vivo-correlations (IVIVCs) between US FDA recommended in vitro test metrics and in vivo regional lung deposition. Innovations in this project include first translating our existing methods and techniques to open-source CFD software OpenFOAM. We will improve our existing MDI simulation routines to better capture the physics of MDI spray plume formation and the evaporation of multicomponent droplets for a small-particle solution-based product containing ethanol as a co-solvent. Concurrent in-house experiments will be used to broadly characterize the MDI aerosol and will provide in vitro deposition data in realistic airway geometries to benchmark CFD predictions. Our complete-airway simulation approach will be significantly expanded to improve model realism and enable simulation of deposition during exhalation. Finally, the expanded open-source complete-airway model will be compared with well-documented 2D and 3D validation data of the same MDI product evaluated in human subjects with mild asthma. The developed and validated complete-airway model will then be implemented to develop IVIVCs between the in vitro test metric of aerosol size distribution and regional lung deposition across multiple subject sizes. To accomplish the project objective, the following aims are proposed: Aim 1. Develop enhanced CFD open-source methods for predicting solution-based MDI aerosol formation, transport and upper airway deposition and validate model predictions with existing and new in vitro data. Aim 2. Develop enhanced CFD open-source methods for predicting solution-based MDI transport and deposition throughout the lungs and validate model predictions with 2D and 3D in vivo data. Aim 3. Implement the validated open-source complete-airway MDI model to develop IVIVC relationships between FDA recommended in vitro test metrics and predicted regional lung deposition. Outcomes. Project outcomes are directed toward an ultimate goal of increasing the number of generic inhaled medications in the US marketplace and worldwide, which is expected to reduce consumer cost, improve compliance with prescribed inhaled drug regimens and thereby improve quality of life and the control of multiple lung diseases.

NIH Research Projects · FY 2025 · 2021-08

Project Summary The rising incidence of allergic disease is a public health challenge needing novel interventions. While mast cell activation by IgE has a known role in disease pathology, fundamental aspects of mast cell biology remain unclear. There is a particular need to understand sex-specific effects. Mast cells from female mice have stronger IgE-induced responses, an observation complementing the greater incidence and acuity of allergic asthma among women. In an effort to repurpose drugs, we found that fluoxetine (Prozac) potently suppresses mast cell activation by IgE. These data were consistent in vitro, in vivo, and with human mast cells. However, fluoxetine effects were strikingly female-restricted. Our results indicate fluoxetine has an off-target effect on P2X3, an ATP-activated cationic channel most often associated with pain signaling. We find that mast cells rapidly release ATP in response to IgE signaling, suggesting P2X3 is triggered in an autocrine loop. P2X3 was readily detectable on female but not male mast cells and may explain why female mast cells have stronger IgE responses. Like fluoxetine, P2X3-selective inhibitors greatly suppressed mast cell responses to IgE or ATP. Inhibitors of other P2X proteins had no effect. Our study will test the hypothesis that IgE signaling elicits ATP release that activates P2X3 selectively in females, amplifying allergic inflammation. This pathway could offer an explanation and a clinical target for sexual dimorphism in allergic disease. We have three Specific Aims: Aim 1: We will test the hypothesis that P2X3 enhances mast cell function in females. Aim 2: We will test the hypothesis that fluoxetine acts by suppressing P2X3 function and expression. Aim 3: We will test the hypothesis that P2X3 is a fundamental part of allergic airway inflammation that can be targeted in females.

NIH Research Projects · FY 2025 · 2021-08

Astrocytes are extremely diverse across different brain regions and perform specialized function. Diversity of these cells is generated by initial patterning and then promoted by region- specific communication with neurons to fine-tune astrocytes to the local requirements. While important insights have been gained into the diversity of astrocytes using novel approaches, molecular mechanisms controlling their diversity remain mostly elusive. In the cerebellum, astrocytes differentiate into highly specialized Bergmann glia of the molecular layer, velate astrocytes of the granular cell layer, and fibrous astrocytes of the white matter. This proposal aims at understanding mechanisms that regulate diverse astrocyte subpopulations in the cerebellum. Our results strongly suggest that a transcription factor, Yin Yang 1 (YY1), is essential for sustaining the distinct functions of astrocyte subpopulations in both the developing and the adult cerebellum. Deletion of YY1 in cerebellar astrocytes manifests in contrasting effects in the molecular layer versus the granular cell layer and white matter by post-natal day 20. We found astrogliosis in the molecular layer associated with GFAP+ astrocyte hypertrophy and loss of typical morphology whereas the numbers of GFAP+ astrocytes in the granular cell layer and white matter were drastically diminished. Furthermore, we found that YY1 differentially alters gene expression at the later stages of astrocyte development in a region-specific manner and is continuously needed in mature astrocytes. To test the hypothesis that YY1-dependent chromatin architecture is critical to execute and sustain programs that affect functions of diverse astrocyte subpopulations in the cerebellum, we will: 1. Establish the effects of YY1 on the major functions of subpopulations of cerebellar astrocytes, and 2. Obtain insights into the molecular mechanisms by which YY1 regulates subpopulations of cerebellar astrocytes.

NIH Research Projects · FY 2024 · 2021-08

Modified Project Summary/Abstract Section Many intracellular targets involve intracellular protein-protein interactions that are “undruggable” because the binding surfaces are too large and featureless to be blocked by a standard rule-of-5 compliant small molecule. Recently, there have been attempts to catalog molecules that are orally bioavailable but lie beyond the rule of five (bRo5) to access these targets. Macrocyclic peptides can inhabit this bRo5 space, and a key advantage to using peptides as bRo5 molecules is that there are many mature techniques for finding peptide binders from vast libraries. Arguably, the most powerful of these techniques is mRNA display, which allows creation of peptide libraries containing over 10 trillion variants, 6-7 orders of magnitude larger than a standard peptide library prepared on beads. The extreme diversity of these libraries has enabled many successes in inhibitor development. Yet these successes are disconnected from real drug discovery, because the peptides uncovered are much too large to be bRo5 compliant. Libraries that are short in sequence and bRo5 compliant can be created by mRNA display, but these libraries lack the diversity needed to uncover potent inhibitors because standard mRNA display is limited by the genetic code to ~20 variants at each position. In this proposal, strategies to enhance this positional diversity will be pursued. This will first involve breaking the degeneracy of the standard genetic code through isolation of fully modified tRNA isoacceptors. Based on codon reading rules it is predicted that this will allow the addition of 10 non-canonical amino acids (ncAAs) to the code. The second aim focuses on combining these newly defined codon reading rules and previously described tRNAs with unnatural base pairs to allow expansion of the genetic code to the use of 40 monomers at each position. This aim will also focus on developing and testing a curated group of aminoacyl-tRNAs for the future creation of bRo5 compliant libraries containing billions of variants for use in drug discovery.

NIH Research Projects · FY 2025 · 2021-07

Hypertension contributes to high rates of morbidity and mortality of other chronic conditions, including cardiovascular disease, obesity, diabetes, and end stage renal disease. African Americans (AAs) are more likely to develop high blood pressure and at a younger age compared to other racial/ethnic groups, and despite higher treatment rates, are less likely to have blood pressure under control. Racial disparities persist by sex—AA women have higher incidence of hypertension and earlier onset compared to their white counterparts. Experiences of stress and stress-related coping are thought to be at the root of these disparities. African American women demonstrate greater stress as evidenced by higher allostatic load over the life course relative to male and white counterparts. Higher incidence and earlier onset of hypertension make younger AA women a prime target for prevention; however, the development of effective prevention-focused interventions is inhibited by limited understanding of underlying mechanisms in this subgroup. To fill these critical gaps, this proposal will use a sequential mixed methods approach, including the following: 1) quantifying stress responses in vivo using a 14-day ecological momentary assessment (EMA) protocol with 24-hour physiological monitoring; and 2) developing and testing the feasibility, acceptability and preliminary efficacy of a stress management intervention that may mitigate the effects of the chronic stress on blood pressure levels in young AA women. The candidate for this mentored Career Development Award, Dr. Anika L. Hines, is an Assistant Professor in Health Behavior and Policy at the Virginia Commonwealth University School of Medicine. Her long-term career goal is to: 1) become an independent investigator who explicates the complex and cumulative effects of stress on lived experiences within the context of health disparities; and 2) design and implement innovative, evidence-based interventions and policies to address these stressors using an interdisciplinary, socioecological approach. During this award, Dr. Hines will undergo rigorous didactic and research training, including didactic courses, experiential lab training, and practical research experience, that will substantially build her skills in intervention development and the conduct of randomized controlled trials for behavioral interventions. These career development activities will be conducted within the rich training environment of Virginia’s largest academic medical center with direct guidance from mentors, advisors, and collaborators with expertise in multi-level and behavioral interventions, evidence-based behavioral medicine approaches, stress, qualitative methods, physiological pathways linking stress and cardiovascular risks, advanced statistical analyses, health disparities, and grant development. These activities will provide Dr. Hines with a solid foundation to ensure her successful transition to an independent, interdisciplinary investigator who is well equipped to conduct behavioral intervention trials in service of eliminating health disparities.

NIH Research Projects · FY 2025 · 2021-06

Human papillomaviruses (HPV) are causative agents in ano-genital and head and neck cancers. Our long-term goal is to identify, and develop, novel approaches for targeting these viral diseases; to do this we must enhance our understanding of the viral life cycle and how it interacts with the host. HPV activates the DNA damage response (DDR) during the viral life cycle and uses homologous recombination (HR) to replicate its genome. The Morgan lab has identified several HR factors involved in HPV replication including TopBP1, BRD4, SIRT1 and WRN. We have demonstrated that these factors regulate the levels and fidelity of HPV16 E1-E2 DNA replication in C33a cells. Following WRN depletion, E1-E2 replication switches from high fidelity mechanisms to break induced replication (BIR). BIR is highly mutagenic and occurs when a paused fork is unable to be resolved by WRN. In the absence of WRN there is excess recruitment of MUS81 to E1-E2 replicating DNA, an endonuclease that causes DNA double strand breaks at paused forks in the absence of WRN. This resolves the fork and allows BIR to continue replication, albeit with low fidelity. We present data demonstrating that WRN is a restriction factor for the HPV16 life cycle; in the absence of WRN there is increased cell proliferation, DNA damage and viral replication in organotypic raft cultures. We observed a similar phenotype following SAMHD1 depletion, another HR factor. WRN and SAMHD1 are in the same cellular complex along with other DDR factors involved in HPV life cycles. Our first objective is to determine the roles of the WRN-SAMHD1 complex in controlling HPV life cycles. The central hypothesis is that this complex controls high fidelity replication during the viral life cycle, and that targeting components of this complex along with MUS81 may block HPV replication. Using a novel HPV16 genome, we will investigate the levels and the fidelity of viral replication during the viral life cycle. Our second objective is to determine whether there are altered host DNA replication forks in HPV16 positive cells. The central hypothesis is that differences can be exploited for differential targeting of HPV positive cells. This will be done in association with Pietro Pichierri, a WRN and DNA replication and repair expert; Morgan and Pichierri have already published together. Our third objective is to investigate the activation of the innate immune response (IIR) in HPV16 cells depleted for WRN and SAMHD1. The central hypothesis is that depletion of WRN or SAMHD1 in the presence of the active DDR in HPV16 cells results in excess cytoplasmic DNA fragments that activate the IIR. The outcomes are crucial for achieving our long-term goals. For example, targeting of WRN enzyme activities could boost the IIR in HPV16 positive cells, assisting the adaptive immune response (and immunotherapy) to eliminate HPV positive cells. If there are different factors replicating host DNA in the presence of HPV, these could be targeted to preferentially kill HPV positive cells. Double targeting of WRN and MUS81 in HPV positive cells may block viral replication resulting in cell death following E6/E7 depletion and reactivation of p53/pRb pathways.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT: Conduct disorder (CD) is one of the most prevalent and debilitating psychiatric disorders impacting our youth. Unfortunately, the effects of CD are not limited to these early years. Youth with CD are more likely to develop lifelong mental and physical health problems, which is why CD is responsible for 5.75 million years of healthy life lost. There is mixed evidence supporting the effectiveness of treatment for CD, and this may be because youth with CD have differing etiological mechanisms. Research has shown that there is heterogeneity among youth with CD, with a subgroup of CD youth displaying more severe behavioral and personality symptoms, called callous-unemotional (CU) traits. CU traits is a specifier for CD that designates a high-risk group of youth who engage in chronic violence and criminal behavior, placing a significant burden on families and society. The key distinction is that these youth show distinct personality symptoms that current CD interventions are not equipped to treat, such as callousness towards others, lack of empathy and guilt for their harmful behaviors, and shallow/diminished affect. Understanding the mechanisms behind CU traits is essential for equipping interventions with the knowledge needed to employ a targeted approach for treating CU traits. Longstanding theories suggest that fearlessness is a key mechanism in the development of CU traits. However, biological evidence supporting the association between CU traits and fearlessness is deficient, and the current state of this examination has been restricted because of ecological validity. We will examine the association between fear and CU traits in youth (13-17 years) with CD by measuring sympathetic (pre-ejection period) and parasympathetic nervous system reactivity (respiratory sinus arrhythmia) during immersion in cutting edge virtual reality (VR) fear induction. We will concurrently apply facial electromyography to assess levels of positive and negative valence. We will also determine sex differences in these associations, as well as provide evidence of how biological fear profiles contribute to the stability of CU traits over 12 months during the adolescent period. The potential benefits of the proposal are high. Based on our pilot data, we will test our hypothesis that youth with CU traits will display a unique biological profile to fear that cannot be explained by fearlessness, and these associations are sex-specific. Confirming this finding in youth with CD may lead to reshaping prior developmental theories of CU traits that could lead to improved screening and more favorable gender-responsive treatment strategies for our youth.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT Background: Nearly 20% of women in the United States experience clinically significant depressive symptoms during pregnancy or the postpartum period. Although treatments exist for depressive symptoms such as antidepressants and psychotherapy, many women remain under- or un-treated due to concerns about stigma, side effects, and costs, particularly marginalized women (minority, low socioeconomic status). Further, the standard depression treatments do not address social connectedness, which is a potentially modifiable factor involved in depressive symptoms. A focus on adequate symptom management through safe, non- pharmacologic, accessible therapies that address social connectedness during pregnancy in marginalized women is an urgent clinical and research priority. Preliminary Data: Preliminary evidence from our research team shows a promising pathway for addressing social connectedness in the context of perinatal depression: we have piloted the “Mindful Moms” intervention in a NICHD-funded study (R15HD086835, Kinser); the 12- week intervention involves a brief motivating discussion about symptom self-management plus group-based mindful physical activity (prenatal yoga) sessions. Study findings provide support of protocol feasibility and intervention acceptability, with qualitative findings leading us to hypothesize that social connectedness plays as a key role in symptom management. Also, we have identified epigenetic patterns uniquely related to postpartum depression; we will evaluate the similarity of these DNA methylation patterns related to social connectedness and postpartum depression, as an exploration of mechanisms of social connectedness. Study Design: Using a conceptual framework based upon Individual and Family Self-Management Theory, the overall goal of this two-arm longitudinal randomized controlled trial is to evaluate the effects and mechanisms of this self-management approach in marginalized women with depressive symptoms (n=200), compared to an active control. The first Specific Aim is to evaluate effects by group on depressive symptom severity, anxiety, and perceived stress over time. The second Specific Aim is to understand the role of social connectedness as a moderator of the effects of group assignment on depressive symptoms. The third Specific Aim is to identify genome-wide DNA methylation patterns associated with levels of perceived social connectedness. Potential Impact: The successful completion of our aims will provide important insights into social connectedness as a mechanism to decrease depressive symptoms in a largely understudied, historically marginalized population of women. Further, if proven efficacious, “Mindful Moms” may be a low-cost, sustainable, and translatable option for intervention in perinatal depressive symptoms.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Virginia Commonwealth University (VCU) seeks to renew the Health Educational Research Opportunities for Teachers (HERO-T) program, a two -year research and professional development opportunity for science teachers, and expand the Health Educational Research Opportunity and Exploration for Students (HEROES) enrichment program for secondary students in the Richmond, Virginia Metropolitan Statistical Area. These programs, grounded in theory, are designed based upon a combination of data derived from current literature related to the impact of research experiences for teachers and literature on student engagement in science. The wealth of experience of the investigators, steering committee, and advisory board members will enable the successful implementation of this program. The goals of the HERO-T and HEROES programs are to 1. cultivate in teachers the skills needed to conduct cutting edge biomedical research and translate that research experience into curricula for their students and 2. increase student awareness of and exposure to research in the biomedical sciences and foster in them scientific investigation skills. These goals will be accomplished by: 1. Selecting three new secondary science teachers annually (15 teachers total), HERO-T fellows, to participate in a two year experience consisting of two eight week summer research experiences and a two year professional learning community (PLC) during the school year. 2. Providing teachers with research training, professional development, and coaching. 3. Engaging twenty secondary students annually in the HEROES Academy (up to 100 students total), a monthly enrichment program to build their awareness of careers in biomedicine/ biomedical research and cultivate their scientific investigation skills. 4. Selecting ten talented students annually (50 students total), HEROES scholars, to participate in the HEROES Externship, a one week summer research program, and the Center on Health Disparities Summer Research Symposium. In the first four years of the grant, 12 teachers were trained, 83% from high-need schools and 67% from underrepresented groups. The HERO-T fellows have developed more than 12 lesson plans from their research to be used in an average of 2 classes per fellow per year impacting over 150 students in the first year to now over 1,200 secondary students annually. More than $10,000 has been used to purchase laboratory supplies for the teachers’ classes thus amplifying the long lasting impact of their research experience. Thirty-nine students have participated in the HEROES program. Approximately 75% of the students have come from high need schools and 80% are from underrepresented groups. This grant request is for another five years of support.

NIH Research Projects · FY 2025 · 2021-04

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects ~1% of the population. Progress has been made in elucidating the genetics of ASD through large-scale genome-wide association studies (GWAS) and whole-exome sequencing (WES) studies that have identified several loci associated with ASD. However, a substantial fraction of ASD status cannot be explained by genetic sequence variation. There are multiple reasons to expect that DNA methylation (DNAm) may account for part of this unexplained variation. First, part of the ASD-related genes identified via DNA sequence variation include genes involved in chromatin modification and DNAm. Second, ASD likely originates during prenatal development, a period of dynamically regulated changes in DNAm in the brain. As this remodeling may result in epimutations that can dysregulate brain function, disruption of the DNAm regulation in utero represents a plausible ASD risk mechanism. Third, ASD is associated with several neonatal and other environmental risk factors. Because DNAm can be modified by environmental factors, it may mediate the effect of these risk factors on ASD. The overall aims of this project is to enhance our understanding of DNAm in ASD etiology, and use DNAm marks at for early detection of individuals at risk for ASD. For this purpose we will generate methylome-wide data using samples from ASD cases age 18-25 years and matched controls from an existing Swedish case- control study called Population-based Autism Genetics and Environment Study. In addition, we will use stored neonatal blood samples to generate a second methylation profile for these same individuals at birth. Thus, we will have methylome-wide data from blood for two time-points from all participants, accompanied by longitudinal phenotype information spanning birth to current date obtained from the Swedish registers. We will use a sequencing-based approach to assay the DNAm status of nearly all 28 million common CpG sites in the human genome and will perform a battery of novel statistical analyses including methylome-wide association studies (MWAS) of whole blood and individual cell-types in blood; analyses integrating DNAm information with neonatal risk factors and already existing GWAS and WES data; and studies exploring the role of DNAm in the ASD sex-bias. Significant findings will be replicated in four existing and independent blood sample collections, and studied in the newly generated methylation/expression data from ASD brain samples. Finally, we propose to use neonatal DNAm markers to create multi-marker methylation risk scores (MRSs) for predicting ASD risk.

NIH Research Projects · FY 2025 · 2021-04

Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer death in men over the age of fifty. Bone is the primary site of metastasis in patients whose CaP progresses beyond organ confinement. The absence of curative therapies for metastatic CaP emphasizes the imperative to develop innovative technologies for target-specific delivery of therapeutic agents as well as novel treatment strategies that are efficacious with minimal toxicity. Our investigative team seeks to address different aspects of CaP bone metastasis through a highly integrated and focused research effort that will enhance our comprehension of the mechanisms underlying CaP progression and improve therapeutic strategies to eradicate bone metastases and prevent relapse. Our early work using a subtraction hybridization screen identified two unique genes, i.e., melanoma differentiation associated gene-9 (mda-9) and mda-7/IL-24 from terminally differentiating human melanoma cells. Subsequent research established MDA-9 as a key promoter of cancer invasion and metastasis, whereas MDA-7/IL-24 was recognized as a broad-spectrum anti-cancer therapeutic. Using a newly developed syngeneic pre-clinical model of CaP bone metastasis, we will investigate the interplay between CaP bone metastases and the bone niche orchestrated by MDA-9 and evaluate therapeutic activity of ‘first-in-class’ small molecule inhibitor of MDA-9 (i.e., PDZ1i) for targeting both metastatic CaP cells and the bone niche. By exploiting the exquisite ability and high efficiency of T cells to locate and destroy disseminated cancer cells, especially those in normally inaccessible sites, i.e., bone, we will engineer CaP-reactive T cells to produce MDA-7/IL-24, a unique cancer-selective apoptosis-inducing cytokine, for improved capacity to attack potentially antigenically heterogenous bone metastases. Last, based on the ability of PDZ1i to reprogram the immune niche in the tumor microenvironment, we will combine engineered T cells producing next-generation MDA-7/IL-24 (“Superkine MDA-7/IL-24“, “S7M”), having enhanced secretion and stability, with MDA-9-targeted therapy for synergistic elimination of CaP bone lesions. We anticipate that the insights garnered from these studies will enable a more precise molecular understanding of bone metastasis development for target discovery, rational design of improved cellular immunotherapy, and combinatorial treatment modalities optimized to achieve a maximum therapeutic potential. Successful completion of this multidisciplinary, synergistic research program will provide a rapid path to translate these technologies and strategies into the clinic to safely and effectively manage this most common skeletal complication of CaP.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Opioid overdose is a leading cause of pregnancy-associated mortality with most of these events occurring through the 12 months after delivery rather than the 9 months before delivery. Historically, the bulk of research on opioid use disorder (OUD) in this population has focused on pregnancy rather than postpartum. The ‘Fourth Trimester’ carries significant unique stressors, and women with OUD are especially vulnerable given the addi- tional stressors they face specific to addiction. Comprehensive treatment during pregnancy includes medica- tion for OUD (MOUD) with wrap-around services such as behavioral health and prenatal care. However, post- partum OUD treatment continuation rates are poor, opioid-related deaths increase, and even for women on MOUD within the month of delivery, postpartum overdose risk is not diminished. We urgently need evidence to guide how to improve not only postpartum OUD treatment continuation but also its effectiveness at strengthen- ing recovery, operationalized as abstinence or decreased substance use with improved quality of life. A novel approach to address this critical knowledge gap is to assess a woman’s multidimensional profile (‘phenotype’) at the pregnancy to postpartum transition then use that information to tailor her treatment regimen (e.g., dosing of buprenorphine, addition of other medications, targeted behavioral therapies) going forward after delivery. Multidimensional (‘phenotypic’) variables that determine one’s profile include biological, neurobehavioral and psychosocial factors. This strategy is in line with our current understanding of addiction having a neurobiologi- cal basis modified by one’s psychosocial context that can vary over the lifecourse. The proposed research has three aims: (1) Obtain qualitative data from women on MOUD and providers in perinatal addiction on chal- lenges and promoters of recovery specific to the postpartum period, (2) Use this data to systematically revise a battery developed by NIDA in collaboration with the VCU Institute for Drug and Alcohol Studies to tailor it for women in the postpartum transition and to be feasible in a busy clinical environment, (3) Assess correlations between multidimensional variables, treatment and recovery outcomes through 12 months after delivery. The data from this innovative study will inform a clinical trial of an individualized treatment regimen for postpartum women on MOUD. This project focuses on two high priority areas to NIDA –personalization of addiction treat- ments and OUD through the perinatal period. The PI, Dr. Martin, is an Early-Career Investigator, obstetrician- gynecologist and addiction medicine physician. This career development award will fill gaps in her training nec- essary for her transition to independence in addictions research (in line with Notice of Special Interest NOT- DA-20-037). Specifically, Dr. Martin will advance her knowledge and skills in clinical trials, mixed methods re- search, addiction therapeutics, neurobehavioral science, advanced statistical analyses, professional develop- ment as well as the responsible and ethical conduct of research. She will work closely with a mentorship team and consultants with expertise across these specialties to carry out her career objectives and research aims.

NIH Research Projects · FY 2025 · 2021-03

Abstract/Summary Chemotherapy and radiation can induce breast tumors to enter a prolonged state of growth arrest with characteristics of senescence. A senescent-like state is also characteristic of residual tumor cells that survive after chemotherapy and/or radiation have eliminated the bulk of a tumor cell population. Tumor cells surviving therapy-induced senescence have the capacity to recover proliferative capacity subsequent to their prolonged growth arrest. Recovery and re-emergence of tumor cells from this growth-arrested state could contribute to disease recurrence months or years after the patient has apparently been cured of the primary disease. Several agents have recently been identified as having senolytic properties [e.g. ABT-263 (navitoclax)] which can selectively kill senescent cells. Given that disease recurrence and consequent cancer mortality is frequently associated with the re-emergence of proliferative tumor cells either at the primary disease site or metastatic sites, a primary goal of this project will be to test the hypothesis that senolytic agents can eliminate therapy induced senescent breast tumor cells to prevent, or at least significantly suppress, cancer recurrence. Aim 1 will examine the hypothesis that the senolytic agent, ABT-263, can efficiently eliminate cells induced into senescence by chemotherapy or radiation when used sequentially (and potentially, repeatedly) after the initial therapy. Sensitivity to senolytics will be assessed in senescence-sorted breast tumor cells, prior to senescence and during the course of recovery, and in residual populations surviving after the bulk of the tumor population has been eliminated by therapeutic agents. The effectiveness of a sequential treatment schedule will also be assessed in vivo using xenograft and PDX tumor models. Aim 2 will interrogate the mechanisms whereby the senolytic ABT-263 promotes cell death in senescent breast tumor cells, with a focus on the contributions of pro- and anti-apoptotic BCL-2 family proteins. Aim 3 will examine the immune response to therapy-induced senescent cells alone and after treatment with senolytics. A phenotypic assessment of tumor infiltrating immune cells and studies with selective immune cell depletion in mouse models will define the nature (adaptive and innate) of the antitumor immune response. Ex vivo studies will further determine NK- and T cell-mediated lysis of senescent cells in the absence or presence of ABT-263. The ability of senolytics to induce immunogenic cell death will be a focus. Toxicity to the host will be compared for exposure to senolytics concomitant with chemotherapy (i.e. prior to entry into senescence) and subsequent to induction of senescence.

NIH Research Projects · FY 2025 · 2021-02

The overarching goal of this application is to investigate the role of sialidase and sialic acid in the keystone pathogen Porphyromonas gingivalis (Pg) and their contributions to the pathogenesis of periodontitis. Sialic acid (SA), a group of structurally related nine-carbon sugar acids, plays critical roles in host-pathogen interactions. On the host side, mammalian mucosal surfaces and secretions of the mouth, airway, gut, and vagina are especially sialoglycan-rich, which have a variety of biological, biophysical, antimicrobial, and immunological functions. In addition, a number of cell surface receptors (e.g., chemokine-, immunoglobulin-, and toll-like- receptors) are either sialylated or recognize sialylated ligands, which play critical roles in immune recognition and activation. On the pathogen side, many bacterial pathogens have evolved different mechanisms to target host SA for adherence, invasion, immune modulation and nutrient acquisition, thereby promoting their fitness and pathogenesis. Specifically, bacterial pathogens often use sialidases to hydrolyze host sialoglycans, compromise host immune defenses, and promote their survival in the mucosal niche. Salivary and gingival crevicular fluids contain a high concentration of SA bound to sialoglycans in various proteins such as mucins. Clinical studies indicate that sialidase activity in the oral cavity is positively associated with the severity of periodontitis; thus, it is recommended as a biomarker for periodontitis diagnosis. Accordingly, sialidases have been found in numerous oral bacteria including the keystone pathogen Porphyromonas gingivalis (Pg). Pg lacks genes to synthesize SA. Instead, it encodes a sialidase (PG0352), which is highly conserved among all genome sequenced Pg isolates. Previous studies from our group and others have shown that PG0352 plays a crucial role in Pg capsule synthesis, biofilms, stress response, innate immune responses, and virulence. However, the molecular mechanism underlying these phenotypes remains elusive. In this application, we hypothesize that Pg employs a sialidase and a SA specific transporter to scavenge host SA, which is in turn used to modify Pg cell surface molecules such as capsule and lipopolysaccharide (LPS), thereby intercepting host innate immune defenses, such as complement killing and phagocytosis of neutrophils and macrophage. To test this hypothesis, the following studies are specifically designed and will be implemented: Aim 1: To delineate the biochemical and structural features of PG0352 by using an approach of genetics, biochemistry, and crystallography; Aim 2: To investigate how Pg imports and utilizes SA by using a multidisciplinary approach of genetics, biochemistry, glycobiology, immunology, and biofilm assays; and Aim 3: To elucidate the role of PG0352 in the pathogenicity of Pg by using various in vitro and in vivo approaches. Completion of this project will not only provide mechanistic insights into understanding the role of SA and sialidase in the pathophysiology of Pg and perhaps other oral pathogens as well, but also will pave a way for future development of specific sialidase inhibitors against oral pathogens, which can provide alternatives to mitigate periodontal diseases.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY Cardiovascular disease is the primary cause of death for patients with Duchenne muscular dystrophy (DMD). Arrhythmia and cardiac fibrosis leading to dilated cardiomyopathy are the primary mechanisms of cardiac mortality. Pannexins (Pxs), which are large conductance ion and small molecule channels, have been implicated in other fibroproliferative diseases and are thought to be arrhythmogenic in other model of cardiac disease. Loss of dystrophin, the primary defect in DMD, leads to elevated intracellular calcium (Ca2+) which is also a primary effector of Pxs. The goal of this project is to investigate the mechanisms by which Px1 modulates the development of cardiac fibrosis and arrhythmogenesis in models of DMD cardiomyopathy. Our preliminary data demonstrate a novel role for Px1 in the development of cardiac fibrosis and inducible arrhythmia seen in the D2-mdx model of DMD. Genetic ablation of Px1 in the D2-mdx model (mdxPx1-/-) rescues the cardiac phenotype, including normalization of cardiac fibrosis.as assessed by histopathology and significant reduction in isoproterenol-induced ventricular ectopy. Based on these data, we hypothesize that pathologically elevated intracellular Ca2+, a hallmark of this disease, leads to Px1 activation and results in signaling cascades that activate apoptotic, oxidative, and inflammatory pathways that ultimately lead to fibroblast activation and the development of cardiac fibrosis. We also hypothesize that Px1 channels represent an independent mechanism for ventricular arrhythmia via generation of delayed after-depolarizations (DADs). We with test these hypotheses using the 3 specific aims outlined in this proposal. In Aim 1, we will use transgenic mice with global Px1 deletion in addition to pharmacological Px inhibition to determine if Px1 activation results in triggered arrhythmia. In Aim 2, we will identify the mechanism by which Px1 contributes to cardiac fibrosis in DMD cardiomyopathy using pharmacological and genetic strategies. As Pxs are expressed in both cardiomyocytes and cardiac fibroblasts, Aim 3 will test if fibroblast migration is dependent on Px1 activation in cardiomyocytes and/or fibroblasts using co-culture techniques for human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts. The completion of these studies will help to improve our understanding of the mechanisms of cardiovascular disease in DMD and will provide the basis for further investigation of a novel therapeutic target that has the potential to delay or prevent cardiac mortality in DMD patients. Additionally, this proposal will allow a promising young physician scientist to gain important skill in basic and translational studies in cardiac electrophysiology, cell signaling, and inflammation/fibrosis biology under the expert guidance of a highly accomplished and dedicated mentorship committee. These new skills will provide the foundation for a successful transition from junior investigator to an independently-funded academic physician scientist.

NIH Research Projects · FY 2026 · 2021-01

Project Summary This R35 application proposes to capitalize on previous work produced by the PI over the past 11 years and to further serve as a strong foundation for interrogating the role of sterile cardiac inflammation (inflammasomes) in promoting the progression of heart failure caused by chemotherapy-induced cardiotoxicity and complicated with myocardial ischemia. The overarching goal of this program is to better understand how inflammasome formation/activation in the heart secondary to different stress signals perpetuates myocardial injury and also dissect the contributions of different cardiac cell types during this pathophysiologic process. 1) Previous studies from the PI’s lab have characterized the role of the inflammasome in mediating adverse cardiac remodeling following acute myocardial infarction in preclinical models. 2) Other studies from the PI’s lab also demonstrated that NLRP3 inflammasome inhibition reduced interstitial fibrosis and preserved systolic cardiac function in mice exposed to doxorubicin. Evidence from the literature also supports a pathophysiologic role of NLRP3 in mediating doxorubicin-induced cardiotoxicity. Moreover, studies from the PI’s lab and others have shown that endogenous production of hydrogen sulfide (H2S) is essential for survival during cellular stresses, including ischemia, and that administration of H2S donors further promotes survival. The orally-active H2S- donor SG1002 was shown in a recent Phase I clinical trial to be safe and tolerable in heart failure patients and also to increase blood H2S levels as well as circulating nitric oxide while attenuating BNP. We recently demonstrated that H2S treatment attenuates ischemic and inflammatory (NLRP3 inflammasome) injury following myocardial infarction. Accordingly, modulation of the inflammasome with H2S may represent an important mechanism to limit inflammatory cell death and mitigate cardiomyopathy. Preliminary data demonstrate that increases in cofilin-2 expression and its potential for phospohorylation and oxidation under oxidative stress rises during ischemic injury, which is attenuated with H2S donors. Thus, this proposal provides the opportunity to perform in-depth investigations on role of the cardiac inflammasome and structural proteins, such as cofilin2, in heart failure due to chemotoxicity and also when complicated with myocardial infarction, therefore extending our knowledge on the potential mechanism of cardiotoxicity and facilitating the design and development of novel preventive/therapeutic modalities in the emerging field of cardio-oncology.

NIH Research Projects · FY 2024 · 2020-08

Corticosteroids provide an important drug class for treatment of numerous forms of heart failure and chronic inflammation, together affecting over 432 million people. In Duchenne muscular dystrophy (DMD), drugs that target corticosteroid receptors are used to treat heart and skeletal muscle. Our lab developed a first-in-class dissociative steroid that shows efficacy as both a heart-protective and anti-inflammatory drug. Our work in the mdx mouse model of DMD was critical for moving this drug, vamorolone, into DMD clinical trials (now in Phase 2b). In early cell and animal studies, vamorolone showed anti-inflammatory efficacy while avoiding key side effects. Subsequently, we discovered new readouts of drug activity and important impacts of corticosteroids on dystrophic hearts. Cardiomyopathy is a feature of both the severe muscle disease DMD (caused by loss of dystrophin) and the milder muscle disease Becker muscular dystrophy (dystrophin in-frame deletions). As promising new therapies are being developed that seek to convert severe DMD genotypes into milder Becker- like phenotypes, importance of treating dystrophic hearts should grow because cardiomyopathy is the leading cause of Becker mortality. Moving forward, it will be important to address knowledge gaps regarding the mechanisms of selective steroids, roles of specific steroid receptors in the heart, and impacts of steroids on Becker-like hearts. This knowledge is important because cardiomyopathy is a leading cause of DMD mortality and current corticosteroids have problematic safety profiles. Our long-term goal is to dissect mechanisms of steroid signaling that can be selectively targeted to improve treatment of chronic pediatric diseases. This can greatly improve outcomes for DMD in a way that also impacts much larger groups of heart, muscle and inflammatory diseases. The objective of this grant is to dissect mechanisms of corticosteroid receptors that impact dystrophic cardiomyopathy. The advanced expertise and tools developed by our lab place us in a unique position to accomplish this using a combination of cell culture, receptor mutation, tissue-specific knockout, micro-dystrophin gene therapy, and animal model systems. We propose the central hypothesis that 11β-hydroxysteroid agonists activate receptor transactivation to drive progression of dystrophic heart pathology. Our rationale is that identifying corticosteroid mechanisms which can be selectively targeted will provide a basis for the improved treatment of DMD and other diseases with heart failure or chronic inflammation.

NIH Research Projects · FY 2024 · 2020-07

Project Summary Ischemic cardiomyopathy is the leading cause of death in the world and affects approximately 1% to 2% of the general population. Sphingolipids, a lipid class bearing signaling properties, have been implicated in numerous cardiac pathologies. Sphingolipids are formed by serine palmitoyltransferase, a heterodimeric enzyme comprised of the subunits Sptlc1 and Spltc2. This heterodimer combines serine and palmitoyl-CoA to generate dihydrosphingosine, which serves as a scaffold for generation of all downstream sphingolipids (e.g. ceramides, sphingomyelins, glycosphingolipids, sphingosine-1-phosphate, etc.). Despite their implication in pathology, sphingolipids are required by all eukaryotic cells; depletion of Sptlc2 in cardiomyocytes led to cardiac dysfunction (Lee, SY et al. 2012 J. Biol. Chem). However, previously identified a novel pool of myocardial sphingolipids These lipids arise from a dimerization of Sptlc1 with a novel SPT subunit, Sptlc3. We previously published work showing that Sptlc3 is strongly induced in diabetic cardiomyopathy. Here we show that Sptlc3 is also induced in human ischemic HF and in mouse models of both acute and chronic ischemia. The products of the Sptlc1/3 complex, which we showed are pro-apoptotic, also increase in human ischemic heart and mouse models. Therefore, we propose that the canonical sphingolipids derived from Sptlc1/2 heterodimer are homeostatic, but in some cardiac insults (lipotoxicity, ischemia) Sptlc3 is induced, changing the intracellular sphingolipidome and leading to deleterious outcomes. This would present the opportunity for therapeutic intervention directed toward atypical, Sptlc3-derived sphingolipids, leaving the homeostatic sphingolipid pool intact. The scientific premise behind our hypothesis is that sphingolipid metabolism could be targeted to prevent ischemic injury. Our hypothesis is that ischemia induces these atypical sphingolipids, or a subset thereof, which promote apoptosis and are thereby toxic to cardiomyocytes, and that blocking their production will attenuate ischemic injury. This will be tested in 3 aims: 1-to test whether cardiomyocyte-specific depletion of Sptlc3 will attenuate ischemic injury and/or heart failure in acute or chronic ischemia in mice, 2-to determine the mechanism(s) of Sptlc3 upregulation in acute vs. chronic ischemia and identify the downstream metabolic pathways and resulting subset of atypical lipids that are produced; and 3-to determine the mechanism(s) by which the atypical lipids induce apoptosis. This proposal will establish the role of non- canonical sphingolipids in ischemic cardiomyopathy and will lay the foundation for further research on potential targeting of the pathway as an innovative therapeutic option to prevent ischemic injury and heart failure and improve patient outcome.

NIH Research Projects · FY 2024 · 2020-07

HER2 is an oncogenic receptor tyrosine kinase (RTK). It is overexpressed in about 20% breast cancer (BC) due to gene amplification, known as HER2-positive BC (HER2+ BC). Several HER2 inhibitors are available clinically and have significantly improved disease outcome. However, primary and acquired drug resistance is common. Most patients with advanced disease show disease progression after some time on treatment. Drug resistance is a major unresolved problem in HER2+ BC, and our long-term goal is to find a solution to this problem. In this project, we propose to investigate a recombinant human protein, i.e., PEPDG278D, for overcoming drug resistance in HER2+ BC. PEPDG278D is an enzymatically inactive mutant of peptidase D (also known as prolidase). Exogenously-administered PEPDG278D binds to HER2 and its family member EGFR, and in cancer cells overexpressing the RTKs, PEPDG278D disrupts their signaling units, directs them for lysosomal degradation, and inhibits the growth of the cells in vitro and in vivo. PEPDG278D inhibits HER2+ BC cells that are resistant to current HER2 inhibitors in vitro and in vivo. Yet, PEPDG278D is well tolerated in mouse studies and shows little effect on HER2 and EGFR in normal tissues where expression of the RTKs is very low. Cancer cells lacking HER2 and EGFR are insensitive to PEPDG278D as well. The objectives of this proposal are: 1) to determine the therapeutic activity and mechanism of action of PEPDG278D in HER2+ BC, and 2) to assess PEPDG278D safety and to understand how PEPDG278D spares HER2 and EGFR in normal cells. The central hypothesis is that PEPDG278D targets HER2 and EGFR specifically and its unique binding mode enables it to target overexpressed HER2 and EGFR strongly and selectively, thereby inhibiting drug-resistant HER2+ BC without causing toxicity. The rationale for the proposal is that completion of the research may propel PEPDG278D into clinical evaluation. We propose three specific aims to test the hypothesis: 1) to elucidate the target specificity of PEPDG278D, 2) to assess its therapeutic activity and mechanism of action, and 3) to determine its target selectivity and how it spares HER2 and EGFR in normal cells. An innovative combination of experimental methods will be used, including but not limited to isogenic cells, cells and tumors carrying clinically verified molecular changes that confer resistance to current HER2 inhibitors, primary normal human cells and humanized mice. The proposed research is significant, because it addresses a major problem in HER2+ BC, i.e., drug resistance. Expected outcome of this work includes: 1) showing that HER2 and EGFR are the sole therapeutic targets of PEPDG278D; 2) showing that PEPDG278D inhibits HER2+ BC resistant to current HER2 inhibitors and the underlying mechanisms; 3) showing that HER2 remains a critical therapeutic target in drug-resistant HER2+ BC; and 4) showing that PEPDG278D is non-toxic to normal cells and tissues and understanding the molecular basis. Our findings will have an important positive impact, because they will generate strong enthusiasm for clinical study of PEPDG278D.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Evidence-based programs (EBPs) delivered in school settings show great promise in reducing risk for the development of emotional and behavioral disorders (EBDs) among elementary-aged children. However, efforts to sustain EBPs in school settings face a number of barriers. Improving EBP sustainment in schools thus represents a public health priority, but very little research exists to inform the development of sustainment strategies. In order to address this gap, and build a foundation for sustainment strategy development, the specific factors that predict sustainment need to be identified. To achieve this goal, the proposed project will accomplish three primary aims: (1) Determine if malleable teacher (self-efficacy, burnout, attributions), intervention (usability), and school (climate, classroom atmosphere, classroom-level adversity) factors predict EBP treatment fidelity (adherence, competence) and modifications to EBPs (fidelity-consistent, fidelity- inconsistent) during implementation or sustainment; (2) Assess the impact of EBP treatment fidelity and EBP modifications on child outcomes (disruptive behavior problems, social skills) during implementation and sustainment; and (3) Explore the mechanisms through which teacher, intervention, and school factors influence sustainment outcomes. This project builds on a 4-year, federally-funded randomized controlled trial evaluating BEST in CLASS, an effective, teacher-delivered intervention for K-3rd grade children at risk for EBDs. The sample will include 96 teachers, 384 children, and 12 elementary schools in diverse communities located in VA and FL. During the implementation phase, the teachers randomized to BEST in CLASS will receive a one-day training followed by 14 weekly sessions of practice-based coaching. To achieve study aims, a multilevel, interrupted time series design will be used to examine the relation between baseline factors, treatment fidelity (adherence and competence to the core BEST in CLASS practices), modifications (fidelity- consistent, fidelity-inconsistent), and child outcomes (disruptive behavior, social skills), followed by a mixed method approach to elucidate the mechanisms that influence sustainment outcomes. Independent observers will use a gold-standard, observer-rated measure with strong psychometric properties to rate treatment fidelity and modifications to BEST in CLASS at 8 timepoints during implementation (baseline, 4 weeks, 8 weeks, post- intervention [14 weeks]) and sustainment (baseline, 4 weeks, 8 weeks, 14 weeks). Our long-term goal, consistent with NIMH Strategic Priority 4 to strengthen the public health impact of funded research, is to engage in research to maximize the sustainment of EBPs in school settings and thus improve outcomes for children. This R01 is a critical building block in our effort to realize the promise of EBPs and will help us create the blueprint for a strategy designed to improve EBP sustainment in schools that we can develop with a NIMH R34 proposal and subsequently evaluate with a R01 proposal.

NIH Research Projects · FY 2024 · 2020-07

The uncontrolled growth of breast cancer cells in vital organs is attributable to nearly all the ~40,000 deaths that happen each year in the United States. The long-term goal of this project is to identify new therapeutic strategies to overcome chemotherapy resistance in triple-negative breast cancers (TNBC). The objective of this proposal is to identify biological pathways that can be targeted in addition to the epidermal growth factor receptor (EGFR) to promote apoptosis of disseminated cancer cells. The central hypothesis is that EGFR inhibition leads to activation of cellular stress responses that can be targeted with pro-apoptotic agents. The goal at the completion of this project is for the results to immediately translate to the clinical setting and provide options for current patients with metastatic disease. The proposed work will also develop a unique dataset that can be used for further research towards identifying and developing targeted inhibitors that bypass carboplatin-resistance. The central hypothesis will be tested by pursuing two specific aims: 1) Delineate targetable pathways that are synergistic with Afatinib for cytotoxicity of carboplatin-resistant TNBCs; 2) Determine the efficacy of Afatinib- synergsitic combinations in multiple diverse in vivo models of TNBC. These aims will be pursued using an innovative set of patient-derived xenograft models (PDX) which consist of isogenic pairs that are carboplatin- sensitive or those that have been generated to be carboplatin-resistant. This research proposal is significant because it will identify new therapeutic targets that can be used to treat patients with advanced disease, and then compare the effectiveness of various targeted combinations on surgically unresectable metastases. The expected outcome of these efforts is that we will identify subsets of TNBC PDXs that will favorably respond to Afatinib-based combination therapies, and subsequently that we will identify genomic and/ or proteomic predictors of anti-EGFR response that will make a positive impact on disease management by identifying patients that may benefit from this treatment approach.

NIH Research Projects · FY 2024 · 2020-07

Mutations of the p53 gene are found in the majority of lung cancers and most of these mutations are single amino acid changes instilling gain-of-function (GOF) oncogenic phenotypes, making the GOF p53 oncoprotein an excellent cancer therapeutic target. We propose a radically different approach to current GOF p53 targeting therapeutic concepts in which instead of inhibiting the protein, we weaponize GOF p53 in promoting lung cancer cell death, either by suicide, viral lysis, or both, while leaving normal cells unscathed. This innovative strategy is possible based on our discovery of a unique transactivation mechanism for GOF p53, and from that, our creation of a GOF p53 inducible promoter. Our GOF p53 inducible promoter directs expression of any gene cloned downstream only if the cell has a GOF p53 mutation, with wild-type (WT) p53 having no effect on the promoter and cells with WT p53 or p53 null mutations showing no expression. For our first major goal, we propose using an exciting new oncolytic virus that only replicates, propagates, and kills cancer cells with GOF p53 while having no effect on normal cells. We have placed two adenoviral early genes, E1A and E1B, the genes needed for adenoviral replication, under the control of the GOF p53 inducible promoter within an adenoviral vector. Initial studies show that this virus has remarkable oncolytic ability and specificity for lung cancer cells with GOF p53, with no effect or viral growth whatsoever in cells with WT p53. The killing effects in xenograft tumors with GOF p53 appear as though there is sustained accelerated tumor killing after a short delay of when the virus is injected. We propose to enhance the oncolytic virus by adding additional lysis abilities and by combining the suicide strategy with the oncolytic strategy. Preliminary results look very promising for this combination. For our second goal, we propose devising a means of specifically killing lung cancer cells with GOF p53 mutations by cloning a suicide gene downstream of our GOF p53 inducible promoter. This construct will be introduced into an adenoviral vector so that when the virus infects cells, only cells with a GOF p53 mutation (cancer cells) will die from prodrug treatment. We have created such a virus using the Herpes Thymidine Kinase suicide gene and show striking killing effects and specificity for lung cancer cells with GOF p53 both in culture and in xenograft tumors. We aim to further discover how this strategy works and ways to improve it. We propose the use of the bacterial Cytosine Deaminase suicide gene (bCD) to enhance the bystander effect of our GOF p53 specific suicide virus. In addition, we propose to improve the inducibility of our GOF p53 inducible promoter to further enhance the suicide and oncolytic viruses. The potential impact of this work is far-reaching since these strategies should be applicable for any cancer with GOF p53 mutations, which constitutes over half of all cancers.

NIH Research Projects · FY 2026 · 2020-04

Abstract Neuroimaging has expanded our understanding of brain development from childhood into early adulthood. Adolescent substance use trends have shifted over time, but use of cannabis, alcohol, and tobacco remain prevalent, typically starting during teenage years, when serious mental health conditions also tend to emerge. Although physical health is at its lifetime peak, emerging concerns for teens include increasing rates of depression, anxiety, social isolation, suicidal ideation, and excessive use of screen media. The extent to which early substance use and other environmental exposures may place youth at risk for altered neurodevelopment and adverse outcomes remains poorly understood. A diverse sample of 11,878 9-10 year olds was enrolled from 21 sites across the ABCD Study consortium, and 554 were enrolled at Virginia Commonwealth University (VCU), under RFA-DA-15-015. All participants underwent a comprehensive baseline assessment, including state-of-the-art brain imaging, comprehensive neuropsychological testing, bioassays, careful assessment of substance use, mental health, physical health, culture and environment, and mobile monitoring every 2 years. Interim in-person annual interviews and biannual telephone or mobile app assessments provide refined temporal resolution of behaviors, development, and life events with minimal participant burden. Intensive efforts are made to retain the vast majority of participants through adolescence and beyond and retention rates thus far are very high. Data, securely and privately shared with the scientific community, will enable investigators to: (1) describe individual developmental trajectories in terms of neural, cognitive, emotional, and academic functioning, and influencing factors; (2) develop national standards of healthy brain development; (3) investigate the roles and interaction of genes and the environment on development; (4) examine how physical activity, sleep, screen time, sports injuries (including traumatic brain injuries), and other experiences affect brain development; (5) determine and replicate factors that influence the onset, course, and severity of mental illnesses; (6) characterize the relationship between mental health and substance use; and (7) specify how use of different substances (e.g., cannabis, alcohol, tobacco, caffeine) affects developmental outcomes, and how neural, cognitive, emotional, and environmental factors influence substance use risk.

NIH Research Projects · FY 2024 · 2020-04

Abstract Localized prostate cancer is often categorized as either indolent or aggressive based largely on clinical and pathological features. Despite our understanding of genetic alterations that are associated with different stages of prostate cancer (PCa), there is no clear molecular classification system, which can predict the risk for developing aggressive PCa. As a result, it is currently difficult to discriminate the aggressive and indolent PCa in their early stages, and develop appropriate therapy for the patients with aggressive cancer. Surgery, radiation and less often androgen deprivation therapies are the available treatment options for localized tumor, although cancers of 30-35% of patients recur and some of them evolve into metastatic disease. There are very limited treatment options for advanced stage PCa. It is therefore important to identify the molecular mediators that promote the advancement of PCa. A comprehensive knowledge on the function of these mediators will not only help us to determine the molecular factors that can distinguish the aggressive and indolent PCa but also to establish effective treatment modalities for those patients who are at high risk to develop metastatic cancer. Our preliminary results indicated that Neuropilin-2 (NRP2) could be a mediator of aggressive PCa by regulating the global transcription of genes required for cancer promotion. Mechanistically, NRP2 can translocate from ER to nuclear membrane through retrograde transport and stabilize the transcription machineries necessary for the expression of cancer promoting genes. Based on these novel observations, we hypothesized that nuclear NRP2 is critical for the transcription of genes required for the advancement of PCa. Hence, NRP2 is not only a predictor for aggressive PCa but also a target for effective treatment strategy. Two specific aims have been proposed. In aim 1, we will study the underlying mechanisms of how nuclear membrane-bound NRP2 interacts with the transcription factors in PCa cells and facilitates their activity. We will also determine using a cohort of human PCa tissues whether nuclear NRP2 can be a prognostic factor, which can discriminate between indolent and aggressive PCa. Aim 2 will focus on the molecular mechanism of how NRP2 migrates to nuclear membrane and determine whether inhibition of this translocation can block the prostate tumor growth. Altogether, our proposal will determine how nuclear NRP2 promotes PCa and thus can be an effective predictor for aggressive PCa. Moreover, it will identify whether targeting NRP2 axis such as blocking its nuclear transport is an effective therapeutic approach to treat aggressive PCa.