Johns Hopkins University

NIH Research Projects · FY 2025 · 2022-06

Project Summary/ Abstract This Mentored Patient-Oriented Research Career Development Award is designed to provide the applicant with the advanced training necessary to establish an independent program of research in the epidemiology of overdose mortality and suicide. A comprehensive training program is proposed, combining formal coursework, mentoring, and hands-on training experiences designed to develop expertise in data linkage/ harmonization, latent class modeling (LCA), qualitative psychological autopsy (PA) and mixed methods research. As overdose deaths increase, they continue to be treated as accidents resulting from changes in opioid use. However, epidemiologic research suggests that many of these deaths are likely suicides, This has important implications for appropriately targeting interventions. We will measure the magnitude of this misclassification of manner of death (MOD; intentionality) and identify factors that may guide medical examiners to more accurately classify suicide decedents. We hypothesize that approximately one third of opioid related deaths of undetermined manner are truly suicides, and that LCA can distinguish subgroups of decedents with greater likelihood of suicidal intent. We will use PA in a subset of previously undetermined intent decedents to test predictive ability of empirically-derived classes and characterize the diverse paths to overdose. We propose to analyze all opioid overdose deaths in Maryland from 2006-2019 (n=13,861) using demographic, social, and clinical data which we will link from the Maryland Suicide Data Warehouse to mortality data from the Office of the Chief Medical Examiner (OCME). First, taking one third of this sample, we will compare cases classified as suicidal (n=115) from accidental (n=756) and undetermined intent (n=3,748) using a three-way multinomial logistic regression. Next, variables found to be most salient comparators will be used in LCA of the remaining cases, agnostic of OCME MOD class (n=9,286). Comparing the empirically derived classes with the OCME designations, we will assess the proportion of designated suicides and accidents in each class. Finally, from each of these latent classes, we will select 40 decedents designated by OCME as ‘undetermined manner’ to be further examined by multiple collateral interview PA, to corroborate these latent classes. By generalizing findings from LCA and PA, more accurate suicide rate estimates can be made. Findings would impact future MOD designation and potentially, how prevention interventions target accidental overdoses and suicides. Training and mentorship plans will leave the candidate well positioned to become an independent physician- epidemiologist, able to utilize both qualitative and quantitative methods for the validation of large linked data sets describing the interrelated suicide and overdose crises. His long term career goals include the elucidation of mechanisms of self injury mortality, the use of mixed methods for the generation and investigation of novel hypotheses regarding pathways to suicide, and the ability to translate these findings into suicide prevention.

NIH Research Projects · FY 2026 · 2022-06

Project Summary Messenger RNA transmits genetic information from DNA to protein. The regulation of mRNA levels is a fine balance between transcription rate and degradation rate. Transcriptional control is well documented and studied. Although the major pathways in mRNA turnover have been identified, accounting for disparate half-lives has been elusive. My lab has shown that codon optimality is a general feature that contributes greatly to mRNA stability in eukaryotes. Codon optimality reflects the disproportionate rate by which the ribosome deciphers each of the 61 codons. The randomness of tRNA selection during the mRNA decoding process manifests in codon optimality wherein tRNA concentrations/functionality dramatically influence rate. Accordingly, codon optimality is ultimately gauged by the relative prevalence of cognate tRNAs, wherein a codon is deemed `optimal' when tRNAs are in excess and conversely `non-optimal' when tRNAs are more limiting. Codon optimality is also determined by the thermodynamic stability of codon/anticodon pairing. Our major advance has been to show that the mRNA degradation machinery monitors ribosome speed and responds to degrade message when ribosome movement is relatively slow. In this proposal, we investigate how the mRNA degradation complex senses ribosome translocation rate as a function of codon optimality. We will determine the precise molecular events that occur in response to ribosome hesitations. Moreover, we focus on biological context where codon optimality is regulated both through mRNA chemical modification and tRNA regulated expression. Lastly the influence of codon optimality is now seen to be the major determinant of mRNA stability in yeast and in early development. Thus work through this project has uncovered a central and critical principle in biology that contributes broadly to gene expression regulation.

NIH Research Projects · FY 2025 · 2022-06

Summary During the last 10 years of both CPTAC2 and CPTAC3, the JHU Proteome Characterization Center (JHU/PCC) has repeatedly demonstrated superior technological innovation and robust data generation that have played critical roles in the success of CPTAC's core mission of accelerating the understanding of the molecular basis of cancer through the application of large-scale proteome and genome analysis technologies to different cancer types. As one of the 3 current CPTAC3 PCCs, we established robust and standardized proteomic analysis protocols and technologies and applied them to 7 human cancer cohorts and an additional 200 patient-derived xenografts from the Patient-Derived Models Repository. Together with the Proteogenomic Data Analysis Centers (PGDACs), we published articles on integrated proteogenomic studies in four cancer types. For our CPTAC4 PCC application, the overarching goal of our PCC is to generate accurate and reproducible data using sensitive, quantitative, and standardized technologies. We will leverage our established Center's infrastructure and capitalize on our success in clinical cancer proteogenomic discoveries to characterize proteins, protein modifications, and protein complexes associated with genomic alterations of cancer from additional human tumor types and pre-clinical models. We will identify unique features that are inherent to proteins such as post-translational modifications covering acetylation and ubiquitination, as well as protein- protein interactions in addition to glycosylation and phosphorylation that have been included in our current PCC. We propose a three-step strategy to characterize defined sets of genomics-characterized samples using technology platforms validated during CPTAC3: (1) Discovery of target proteins from both clinical specimens and preclinical models using quantitative proteomics by tandem mass tags and data dependent acquisition mass spectrometry; (2) Verification of findings using orthogonal data-independent acquisition mass spectrometry; and (3) Confirmation of the verified targets using high-throughput, CPTAC Tier 2 analytically-validated targeted Multiple Reaction Monitoring Mass Spectrometry (MRM-MS) assays. We further propose pilot studies for technology improvement. While this PCC application is focused on the proteomic characterization of clinical specimens and preclinical models, we believe that the understanding of and the expertise in proteogenomic data analysis and translation will be critical for the success of the PCC and the overall CPTAC network. We have assembled a team of outstanding investigators with complementary expertise and years of experience in the CPTAC program (CPTAC2 and CPTAC3), and evidence of successful collaborations with investigators/PIs from the CPTAC PGDACs and Proteogenomic Translational Research Centers (PTRCs). We believe that our PCC offers the best opportunity for the successful characterization of biological and clinical specimens to discover and confirm cancer targets to advance precision cancer medicine.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract Chronic obstructive pulmonary disease (COPD) occurs in over 15 million adults in the US. As many as 35% of those patients develop pulmonary vascular disease (PVD), but the recommendations for assessment and management of PVD in COPD have been limited by key knowledge gaps. Firstly, assessment of the pulmonary vasculature has relied upon invasive right heart catheterization as the gold standard, limiting studies to select populations with severe PVD. Noninvasive techniques, though widely available, have also been primarily validated in populations with severe PVD applying markers that are likely to reflect end-stage PVD, such as right ventricular failure. As a result, another key knowledge gap is the unknown contribution of PVD to COPD morbidity across its observed spectrum of severity. While the concomitant presence of severe PVD is observed to portend increased morbidity and mortality compared to those with COPD alone, the contribution of less severe PVD, at potentially more intervenable points in the disease course is less well-understood. This proposal seeks to address these knowledge gaps in three aims by (SA1) testing the diagnostic accuracy of three noninvasive markers that are selected for plausible sensitivity to early PVD, (SA2) and applying these noninvasive markers to understand the contribution of PVD to respiratory morbidity in cross-sectional and (SA3) longitudinal study designs. This application will add noninvasive markers to a general COPD cohort unselected for PVD and will further assemble a prospective longitudinal cohort of COPD patients enriched for PVD with the same noninvasive testing. The results of this proposal will identify novel noninvasive approaches for assessing PVD, define their clinical relevance across the spectrum of PVD, and establish opportunities to study the use of noninvasive markers as candidate outcomes in clinical trials targeting the pulmonary vasculature to manage COPD. Dr. Balasubramanian’s proposal is a strong training vehicle for her career development, offering her skills in hands-on cohort study design and execution, advanced biostatistical and epidemiologic methodology, and noninvasive and invasive assessment of PVD in COPD through numerous modalities including right heart catheterization, magnetic resonance imaging, speckle-tracking echocardiography, and physiologic measurements of diffusing capacity of the lung. Dr. Balasubramanian will be supported by a strong multi- disciplinary mentorship team with expertise in COPD, pulmonary hypertension, cardiology, radiology, and biostatistics. She further will leverage an exceptional institutional environment with its collaborative and supportive culture, extensive intellectual and physical resources, and strong history of supporting junior investigators. This award will establish Dr. Balasubramanian as an independent investigator with a unique research niche, distinct from her mentorship team, and establish a foundation for future studies evaluating and managing PVD in COPD.

NIH Research Projects · FY 2025 · 2022-06

Project Summary The concept of synthetic lethality has been validated clinically with the use of PARP inhibitors in treating breast cancers with loss-of-function mutations in BRCA1/2. Nevertheless, only 5-10% of breast tumors are caused by inherited mutations in BRCA1/2, highlighting a need to identify new synthetic lethal interactions that can be exploited clinically. In this proposal, we capitalize on our recent discovery of a new synthetic lethal interaction that is exposed by a cancer-specific genetic alteration. Centrosomes are microtubule-organizing centers that catalyze the assembly of the mitotic spindle during cell division. Centrosome duplication is tightly coupled to cell cycle progression and controlled by the master regulatory kinase (Polo-like kinase 4) PLK4. Chemical inhibition of PLK4 activity leads to cell division in the absence of centrosome duplication, producing centrosome-less cells that exhibit delayed mitotic spindle assembly. Although most cancer cells can proliferate in the absence of centrosomes, we recently discovered that inhibition of PLK4 leads to centrosome depletion that selectively triggers mitotic catastrophe in cancer cells overexpressing TRIM37. This has therapeutic relevance as the TRIM37 chromosomal locus is amplified in 50- 60% of neuroblastomas and ~10% of breast cancers. In addition, amplification of this region is associated with aggressive cancers with highly rearranged and unstable genomes. In this application, we will define how TRIM37 overexpression promotes tumorigenesis and increases the vulnerability to PLK4 inhibitors. We will also test the effectiveness of PLK4 inhibition in achieving selective killing of human cancer organoids with TRIM37 amplification. Aim 1 will determine how TRIM37 overexpression increases the sensitivity to PLK4 inhibitors and test if TRIM37-driven centrosome dysfunction contributes to tumorigenesis by promoting mitotic errors. The 17q23 amplicon contains ~30 genes, including TRIM37 and the TP53-antagonizing phosphatase PPM1D. The oncogenic properties of PPM1D overexpression have been validated in several models. Selective PPM1D inhibitors have been developed, but how PPM1D cooperates with other genes encoded in the 17q23 amplicon remains unknown. In Aim 2, we determine if PPM1D overexpression promotes TRIM37-mediated genomic instability and test if PPM1D inhibition can potentiate the action of PLK4 inhibitors in these tumors. Finally, Aim 3 will examine if TRIM37 overexpression confers increased sensitivity to PLK4 inhibition in breast and neuroblastoma tumor organoids. Understanding the role of TRIM37 in tumorigenesis will help define how its overexpression drives the development of aggressive cancers and provide a rationale for the use of PLK4i in the treatment of cancers with genomic amplification of TRIM37.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY It is now clear that the mammalian retina has at least one other photoreceptor class besides rods and cones, consisting of a subpopulation of retinal ganglion cells (RGCs) that express the pigment melanopsin (OPN4) and are intrinsically photosensitive (ipRGCs). These cells project predominantly to non-image-vision centers in the brain, serving functions such as circadian photoentrainment and pupillary light reflex. At the same time, ipRGCs project moderately to the brain's image-vision centers, presumably serving subtle image- forming visual functions such as possibly providing information about absolute light intensity in the visual scene. IpRGCs are known so far to comprise 6 subtypes, M1-M6, which differ in the level of melanopsin content, photosensitivity, somatic and dendritic-field size, exact locations of their dendritic arborizations in the retina's inner plexiform layer, and the detailed locations of their axonal projections in the respective brain targets. To fully understand the ipRGC system, it is important to know their light-response properties and the underlying mechanisms. In recent years, we have made great progress in understanding M1-ipRGCs, including the molecular identities of several key components in phototransduction such as Gαq-subfamily members, the PLCβ4 enzyme and the TRPC6,7 channels. These components closely resemble those found in fly-eye phototransduction. Most recently, we have made the exciting discovery of yet another phototransduction pathway, found in M2- and M4-ipRGCs. This pathway involves a light-induced elevation of cyclic nucleotide, which opens HCN channels (channels gated by membrane hyperpolarization and cyclic nucleotide) to produce membrane depolarization and action potentials. This application constitutes a continuation of these successful investigations. Aim 1 is to seek some details of the HCN-phototransduction pathway still outstanding, including establishing (i) whether cGMP or cAMP is the second messenger, (ii) the identity of the effector enzyme that controls the second messenger, and (iii) the identity of the G protein upstream of the HCN-pathway. Aim 2 is to characterize the phototransduction mechanism(s) still unknown in M3-, M5- and M6-ipRGCs. Our speculation is that the same two pathways are involved, but this requires verification. Aim 3 is to quantify the relative importance of the two signaling pathways in different cell subtypes. We shall study sensitivity, intensity-response relation, single- photon response, and response kinetics of each pathway, hopefully eventually to derive correlations with the respective subtype's macroscopic functions.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY Frailty is a syndromic state of vulnerability that puts adults aged ≥65 years at heightened risk of adverse health outcomes. An estimated 50% of older Americans are prefrail—a pre-clinical stage of frailty that might be more amenable to intervention efforts than frailty. Increasing physical activity is a promising intervention to better manage/help reverse the multisystem dysregulation that drives frailty and sequalae. However, initiating and maintaining habitual physical activity is difficult for sedentary older adults, particularly those encumbered by health challenges. The 2018 US Physical Activity Guidelines recommends that all adults perform ≥150 minutes/week of physical activity and reduce sedentary behaviors. Yet, traditional approaches to increase physical activity do little to address sedentary behavior reduction, especially for older adults. Lower sedentary behavior is associated with improved biological and psychosocial health—independent of meeting physical activity guidelines. Thus, there remains a critical need to implement and evaluate a structured way to reduce sedentary behavior as a potential pathway for habitual physical activity engagement. Using novel objectively measured physical activity metrics, our research group has shown that daily sedentary time, either in total or accrued in a prolonged manner, is associated with frailty. Our observation evidence shows that: 1) daily, non- exercise physical activity declines and becomes more fragmented with age (less continuous activity with longer sedentary bouts), 2) higher daily sedentary time and activity fragmentation are both associated with higher frailty incidence, and 3) sedentary time is positively associated with frailty-related markers of inflammation. We propose a pilot study in which we randomize 60 prefrail community-dwelling older adults to receive one of two interventions, each designed to gradually reduce sedentary time: 1) continuously to form a 30-minute walking bout, or 2) in a bouted manner to form three 10-minute walking bouts. Project goals are to: a) explore the effectiveness within and between interventions to decrease objectively measured sedentary time over 2 months; b) assess decreased sedentary time’s association with i) patient-reported outcomes and ii) frailty- related inflammatory markers. The primary outcome is accelerometer-determined sedentary time. Secondary outcomes include activity fragmentation, patient-reported outcomes, and inflammatory markers. With a transdisciplinary mentoring panel, my career development plan builds on my expertise in aging and physical activity epidemiology to gain proficiency in: 1) developing and implementing clinical trials for older adults, 2) designing interventions to improve health behaviors, 3) conducting frailty and inflammation related research and 4) gaining competencies to become an effective PI and leader. This project utilizes the infrastructure of the Johns Hopkins Institute for Clinical and Translational Research (ICTR) and Beacham Center for Geriatric Medicine which have strong records of supporting early-stage faculty. This award will facilitate my transition to an independent investigator and will also provide informative data for R21 and R01 applications.

NIH Research Projects · FY 2025 · 2022-06

Summary Transcription is the first step to read out the genetic information stored in the chromosome. In eukaryotes, three multi-subunit RNA polymerases (Pols), Pol I, II, and III transcribe the 25S ribosomal RNA (rRNA), protein-coding, and short non-coding RNAs such as transfer RNA (tRNA) and 25S rRNA genes, respectively. A wealth of genetic and biochemical data accumulated over 50 years have uncovered most of the molecular players involved and details of the intricate regulatory circuits. The recruitment and assembly of the initiation complex at the promoter represent one of the key regulatory steps during gene expression. The Pol II initiation machinery includes the activator-bound Mediator along with a series of general transcription factors (GTFs) (TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) that assemble into a ~4-megadalton (MDa) pre-initiation complex (PIC) on core promoter DNA. On the other hand, the Pol III enzyme is recruited onto its promoter with the help of corresponding GTFs (TFIIIC, TBP, BRF1, and B''). This proposal aims to investigate the mechanism of transcription regulation by directly visualizing these transcriptional competent initiation complexes using single-particle cryo-EM. Comparable pictures of these different but evolutionarily related systems at this critical step of gene regulation will provide an unprecedented, comprehensive view of this key process in the central dogma of molecular biology.

NIH Research Projects · FY 2025 · 2022-06

Summary The NEI AMD Pathobiology group concluded that identifying all of the pathogenic signals, prioritizing their contribution relative to one another, and establishing when these signals are pathogenic will lead to effective treatment for each AMD stage. Because preventing or curing early AMD will eliminate the burdens of advanced AMD and reduce its financial burden, this proposal will focus on early AMD. Cellular heterogeneity is a well- recognized phenomenon. While ordered heterogeneity is protective, stress causes disordered heterogeneity that induces disease by aggressive subclones, even if comprised of a limited number of cells. Retinal pigment epithelium (RPE) heterogeneity is well recognized in early AMD, but its pathogenic role is unexplored. The epigenome mechanistically links environmental exposures with the transcriptome to influence biological processes, and epigenetic changes vary across individual cells to cause pathogenic cellular heterogeneity. Smoking, the highest environmental risk factor for AMD, is a powerful epigenetic inducer in part, due to the altered expression of chromatin modification enzymes that can reprogram the transcriptome to disrupt multiple cytoprotective pathways, a fundamental characteristic of a complex disease like AMD. Using ATAC-seq and RNA-seq, we previously linked reduced chromatin accessibility in promoter regions of genes with the RPE transcriptome in early AMD eyes, implicating the RPE as a driver of early AMD. Furthermore, iPSC-RPE cells treated with smoke had chromatin accessibility profiles similar to the RPE from early AMD eyes. While valuable, this global approach does not define RPE heterogeneity or the impact of heterogeneity on RPE functions related to AMD pathobiology. The objective is to define RPE heterogeneity in early AMD due to reduced chromatin accessibility from smoking by addressing the hypothesis that pathologic RPE subsets contribute to early AMD through transcriptome changes induced by epigenetic or genetic alterations. SA1. Define pathogenic RPE heterogeneity by regional location in aging and early AMD by chromatin accessibility and transcriptome modifications. ScRNA-seq and scATAC-seq will be used on the RPE from the macula and periphery from aging and early AMD genotyped globes, and chromatin accessibility and pathologic pathways from cellular subsets will be visualized by ATAC-see and immunohistochemistry. SA2. Determine the extent that RPE heterogeneity i) develops due to chromatin accessibility and transcriptional heterogeneity with aging or smoking, and ii) impacts RPE function in a model of early AMD. ScATAC-seq and scRNA-seq will be used to assess chromatin accessibility and transcriptional RPE heterogeneity in wild-type (littermates), HDAC11-/-, and WT mice treated with HDAC11 inhibitor Mocetinostat exposed to chronic cigarette smoke. The spatial distribution of RPE heterogeneity will be identified using immunofluorescence of biomarkers of cellular subsets and ATAC-see and impact on RPE function will be assessed.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT The overarching research goal of this K23 Mentored Career Development Award is to improve outcomes assessment for microbial keratitis (MK), or infectious corneal ulceration. MK affects 2 million people per year and causes significant harm to vision and quality of life. Meaningful measurement of MK for clinical and research purposes remains challenging. Traditional clinical assessments of MK severity and response to treatment are subjective and imprecise, making it difficult to compare efficacy of treatments in clinical research studies due to low reproducibility and increased sample size requirements. Prior MK clinical trials have used outcome metrics such as best spectacle-corrected visual acuity which are not disease-specific and may fail to capture key information about corneal damage caused by MK. These measurement limitations reduce the quality and impact of MK research. To date, most large-scale prospective randomized trials in MK have failed to demonstrate clinically meaningful or statistically significant differences in treatment response between groups. Single-center MK studies can also lack applicability to other healthcare settings due to differences in patient characteristics and microbial distributions across populations. Developing more objective, reproducible, clinically relevant, and generalizable outcome metrics and predictors for MK would enhance the clinical relevance and statistical power of future multicenter MK studies. New imaging modalities such as Scheimpflug tomography and anterior segment optical coherence tomography (ASOCT) can provide objective, precise, reproducible, and clinically relevant assessments of corneal structure, but these modalities have not been critically evaluated for MK clinical care or research using prospective studies. This application proposes to conduct a prospective cohort study of MK patients at Johns Hopkins. We will collect detailed clinical and microbiologic data and perform serial multimodal imaging using slit lamp photography, Scheimpflug tomography, and ASOCT over 6 months. Aim 1 will compare the reproducibility and concordance of ultrasound pachymetry, Scheimpflug tomography, and ASOCT for objective quantification of corneal thinning in MK. Aim 2 will evaluate Scheimpflug densitometry as a means of objectively quantifying longitudinal changes in corneal scar density in MK. Aim 3 will assess whether certain early anatomic or clinical features can improve prediction of subsequent visual outcomes in MK and whether these predictors are applicable across different MK populations and infection subgroups, indicating the suitability of these novel outcome metrics for use in collaborative multicenter clinical trials. This proposal will provide the candidate with the advanced training and research experience needed to become an expert in clinical trials methodology and an independently funded clinician-scientist in the field of cornea and external diseases. The candidate proposes a comprehensive training plan combining rigorous formal coursework, seminars, and workshops in the intellectually rich environment at Johns Hopkins University; world-class mentorship under Drs. Douglas Jabs, Thomas Lietman, Elizabeth Sugar, and Albert Jun; immersive experience with ongoing prospective multicenter clinical trials; and applied research experience. Specific training areas include: (1) advanced clinical trial methodology; (2) practical clinical trial implementation; (3) advanced biostatistics; (4) clinical and research expertise in microbial keratitis; and (5) career development as a clinician-scientist. Results from the candidate’s research will be used to develop a proposal for a R01 or UG1 funded clinical trial that will improve evidence-based treatment for cornea and external diseases.

NIH Research Projects · FY 2025 · 2022-05

Title: Impact of Genetic and Pharmacological Kynurenine Pathway Suppression on Healthspan, Lifespan and Cellular Changes Associated With Aging in Mice PROJECT SUMMARY/ASTRACT (30 LINES OF TEXT) Through findings from translational studies on both aged and chronically inflamed mice, as well as on aged and frail older adults, we have identified metabolites of the kynurenine pathway (KP) as potential mediators of systemic damage caused by chronic inflammation. We recently identified that KP metabolites including kynurenine, kynurenic acid, 3-hydroxykynurenine and quinolinic acid were significantly elevated in the serum of older mice and robust and frail older adults, and that this was linked to functional decline and neurodegeneration. The family of molecules known as `kynurenines' are derived from the amino acid tryptophan and are precursors for the important electron carrier and coenzyme molecule NAD+. Kynurenines possess unique bioactive properties and some have pathological potential. For example quinolinic acid (QA) and 3-hydroxykynurenine (3-HK) are neuro- and cytotoxic and induce oxidative stress while kynurenine (KYN) and kynurenic acid (KA) are ligands for the aryl hydrocarbon receptor (AhR), whose signaling activity is linked to immunosuppression, senescence and impaired autophagy. Conversely, genetically inhibiting the KP extends lifespan in C. elegans and Drosophila, and pharmacological KP blockade increases lifespan in Drosophila. Reduced dietary tryptophan extends lifespan in rodents, but it is unknown if genetic or pharmacological KP blockade improves healthspan or extends lifespan in mice. In this study, we aim to evaluate the hypothesis that genetically and pharmacologically suppressing levels of KP metabolites can delay functional decline, pathophysiological metabolic changes, mortality and cellular changes associated with aging in mice. To understand the effects of KP suppression on aging, we will determine the effect of suppressing the oxidative stress inducing kynurenines, 3-HK and QA, using kynurenine 3-monooxygenase knock out mice (KMO -/-, Aim 1). We will also determine the effect of suppressing both oxidative stress inducing kynurenines, 3-HK and QA, as well as AhR agonist kynurenines, KYN and KA using the indolamine 2,3 dioxygenase knockout mouse (Ido -/-, Aim 2). We will then determine if pharmacological suppression of toxic kynurenines and AhR ligands can delay aging in mice using 1-methyltryptophan (Aim 3). Additionally, we will determine if pairing all of these KP suppression strategies with NAD+ supplementation will synergistically benefit healthspan, lifespan and characteristics of aging in mice. These studies will inform on the role of the KP in functional decline and aging and the therapeutic potential of KP suppression as an anti-aging intervention.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Cancer sequencing projects have identified a very large number of DNA mutations whose importance in cancer is not yet understood. To better understand the impact of these mutations, our team has produced a software tool for computational analysis of cancer mutations that can analyze millions of mutations at one time. This tool works as a funnel to help researchers to find the small number of mutations that are most likely to be informative from the very large number of mutations discovered in a sequencing project. The software allows users to design ways to combine multiple mutation evaluation metrics, and generate a prioritized list of mutations that are more likely to be biologically important. These evaluation metrics include the molecular consequence, bioinformatic scores to identify pathogenic and driver mutations, frequency of the mutation in human populations, previous occurrence in tumor tissue types, pointers to literature, and visualization of annotated protein structures and networks. A web-based version of the pipeline - Cancer Related Analysis of Variants Toolkit (CRAVAT) has been widely adopted (3000+ jobs submitted/month on average in 2020). We have attracted a user community that spans both basic and clinical cancer researchers, all of whom rely on high-throughput tumor sequencing in their work. In 2019, we introduced OpenCRAVAT, which is distinguished by an open source codebase and an open app store of tools and resources that can be used to better understand the importance and impact of mutations. The app store is driven by the user community; new apps are prioritized based upon user requests and the app store includes many apps that were contributed directly by outside tool developers. The app store currently aggregates tools from over 70 organizations, and these tools can be combined to identify mutations whose molecular impact contributes to tumorigenesis, prognosis and treatment selection. Initial adoption of our OpenCRAVAT tool is encouraging, with over 10,000 local package downloads in the first two years. We expect that OpenCRAVAT will be adopted by a much larger community, given the increasing importance of DNA sequencing data in cancer research. We will continue to ensure that our tools are interoperable with other informatics tools and services, and can be run in different computational environments such as cloud computing and local installation to maintain data privacy.

NIH Research Projects · FY 2026 · 2022-05

Project Summary In the U.S. over 150,000 patients die annually from multidrug-resistant infections, and resistant infections are associated with over $20 billion in healthcare costs. Healthcare-associated infections (HAI) highlight the impact of antibiotic resistance, as more than 40% of HAIs are due to antibiotic-resistant organisms. Children requiring mechanical ventilation are at risk for developing ventilator-associated infections (VAI). VAIs are responsible for 23-53% of all HAIs among children. Up to 87% of pediatric intensive care unit (PICU) patients are treated with antibiotics and treatment for suspected VAIs accounts for 50% of antibiotic use in the PICU. In an effort to inform the diagnosis of VAIs, clinicians often obtain respiratory cultures from mechanically ventilated patients. However, these cultures are obtained from a non-sterile site and more than 50% of endotracheal cultures will grow a potentially pathogenic organism within 3 days of intubation regardless of clinical symptoms. Respiratory cultures cannot distinguish between bacterial colonization and infection, and despite the low specificity to indicate infection, positive cultures prompt clinicians to treat with antibiotics. Safely reducing testing, referred to as diagnostic stewardship, is an emerging strategy to reduce testing overuse and potentially antibiotic overuse. Recently, a novel clinical practice guideline designed at the Johns Hopkins Children’s Center to standardize approach to respiratory cultures in critically ill children safely reduced respiratory culture use by 41%. The long- term objective of this proposal is to develop a customizable diagnostic stewardship program that can improve antibiotic use and prevent antibiotic resistance among vulnerable children. The specific aims are Aim 1) evaluate whether diagnostic stewardship of respiratory cultures among mechanically ventilated children decreases respiratory culture use and antibiotic use without leading to unintended patient harm, Aim 2) identify barriers and facilitators to implementation of diagnostic stewardship quality improvement programs to reduce respiratory culture use among mechanically ventilated patients in 14 pediatric intensive care units, and Aim 3) use a Delphi method, including a panel with nationwide representation, to develop consensus recommendations informing when to obtain respiratory cultures in mechanically ventilated children. Fourteen hospitals participating in a multicenter quality improvement initiative, the Bright STAR Collaborative, are implementing local quality improvement programs to improve respiratory culture use. This proposal will evaluate the implementation of local diagnostic stewardship programs and determine whether these programs represent an effective and safe strategy to reduce antibiotic use in a vulnerable population. The proposed aims will assemble the tools and evidence to broadly disseminate diagnostic stewardship as a strategy to reduce antibiotic use and deliver high value care, and catalyze similar work in non-ICU pediatric and adult populations.

NIH Research Projects · FY 2025 · 2022-05

Poor cardiovascular health (CVH) contributes to high levels of morbidity and mortality in the United States, with profound health disparities by race/ethnicity, socioeconomic status, and geography. Sustained improvement in CVH requires 2-generation strategies in settings where those at elevated risk for poor CVH already receive care. Evidence-based home visiting (HV) provides an ideal setting to reduce health disparities, reduce maternal morbidity, and promote CVH in infants and children in ways that can be continued across the life course. Prior studies of CVH intervention effectiveness have not tested interventions across HV models, incorporated emerging technologies such as mHealth and telehealth, combined HV with services in other settings where mothers and children receive care, acknowledged mediators and moderators of effectiveness, nor used individual and composite maternal and child CVH metrics to assess outcomes. The Early Intervention to Promote Cardiovascular Health of Mothers and Children (ENRICH) program aims to address these limitations. It will test a common implementation-ready intervention to promote maternal and child CVH and reduce CVH disparities in the context of multiple evidence-based HV models. The success of ENRICH requires a Resource and Coordinating Center (RCC) with sophisticated content and methods expertise and resources to support study design and implementation of a common intervention and research protocol attuned to heterogeneity in individual, family, and community context. The Johns Hopkins RCC multidisciplinary team combines the expertise of the Home Visiting Applied Research Collaborative (HARC), the Johns Hopkins Center for Clinical Trials and Evidence Synthesis (CCTES) and the Johns Hopkins Center for Health Equity. This RCC team offers an unparalleled combination of a national HV platform, expertise in health equity, and clinical trials infrastructure to create new management strategies, organization concepts and impactful analyses to assure that ENRICH achieves its potential. Our RCC includes: (1) stakeholder-built HV Precision Paradigm; (2) content expertise and leadership in HV and CVH; (3) state of the art methods to design and implement a common protocol, analyze data, disseminate results, and translate for policy and practice (4) advanced methods to assess “What works best, for whom, in which contexts, why and how?” and assess ideal CVH metrics for both mothers and children; (5) tight linkage with HV and CVH stakeholders; and (6) expertise in engaging and training Early Stage Investigators and HV staff professionals. The RCC will apply the strengths of three JHU divisions (Public Health, Medicine and Nursing) to ENRICH to generate impactful evidence on effectiveness of the ENRICH intervention and powerful data on generalizability to enhance translation. The RCC will assure that ENRICH achieves its full potential and generates rigorous, innovative, timely and actionable new knowledge to reduce maternal morbidity, promote CVH and reduce CVH disparities in mothers and young children.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY The malaria parasite P. falciparum continues to cause significant morbidity and mortality, with 228 million clinical cases and 405,000 deaths in 2019. With progress towards controlling malaria stalling in many high burden countries and the continuing spread of drug resistant parasites, an effective malaria vaccine is urgently needed. RTS,S, a vaccine currently under implementation, has modest efficacy (~30%) and immunity waning rapidly. Importantly, this vaccine does not target the disease-causing forms of the parasite. An efficacious vaccine targeting Plasmodium blood-forms is required to reduce parasite burden, clinical disease and sequelae to severe disease. Our proposal aims to address this gap by developing a multi-modal P. falciparum vaccine targeting the blood-stage parasite and evaluating its efficacy using a relevant primate model of human malaria. We will build this multi-modal vaccine by incorporating two vaccine candidates with distinct effector mechanisms. The individual components have been tested rigorously and reproducibly in pre-clinical efficacy models. The first is a sub-unit vaccine candidate (AMA1-RON2L complex), designed to enhance antibody quality by increasing the proportion of neutralizing antibodies targeting AMA1. Using a structure-based approach we have now designed this subunit vaccine to cover AMA1 polymorphisms and generate strain-transcending, neutralizing antibodies. The second is a whole blood-stage parasite vaccine that induces a strain-transcending, anti-parasite response through direct cell-mediated killing. This multi-modal vaccine will be formulated with a novel cationic liposomal adjuvant that potently activates both the humoral and cell-mediated arms of the immune system, thereby providing a human-compatible adjuvant platform. We will perform dosing studies to optimize the P. falciparum multi-component vaccine in mice and evaluate impact of biological sex on the vaccine-induced immune response. We will also assess memory B cell and T cell responses induced by the multi-modal P. falciparum vaccine (Aim 1). Next, we will assess immunogenicity and protective efficacy of this multi-modal vaccine against homologous and heterologous P. falciparum in Aotus nancymaae and evaluate the persistence of the immune responses using a delayed re-challenge model (Aim2). Lastly, we will use validated immunological assays and apply cutting-edge technology (single-cell RNASeq) to help inform our understanding of immune correlates of protection. The major deliverable of this project will be a novel, pre-clinically validated, multi-modal P. falciparum blood-stage vaccine in a human-compatible liposomal adjuvant that can be progressed towards clinical trials.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Neuroprotective actions of cystathionine γ-lyase through gasotransmitter hydrogen sulfide signaling. Hydrogen sulfide (H2S) is a gaseous signaling molecule or gasotransmitter which serves key roles in the central nervous system. However, specific targets and mechanisms of H2S action in the brain are obscure. Herein, we propose to elucidate the signaling pathways modulated by H2S in the brain that are neuroprotective to arrive at therapeutics targeting Alzheimer's disease (AD). H2S is generated from the amino acid cysteine which, in turn, is synthesized by cystathionine γ-lyase (CSE) via the transsulfuration pathway in the brain. H2S is also synthesized by two other enzymes, cystathionine β-synthase (CBS) and 3- mercaptopyruvate sulfurtransferase (3-MST), in the brain. We have demonstrated previously, that H2S and cysteine metabolism are dysregulated in AD. One of the modes by which H2S signals is via a posttranslational modification termed sulfhydration or persulfidation, wherein the reactive –SH group of cysteine residues on target proteins is converted to an –SSH group in a fashion analogous to nitrosylation by nitric oxide (NO), where the –SH groups are converted to –SNO groups. Although sulfhydration and nitrosylation modulate diverse physiological processes ranging from response to inflammation to neuroprotection, the molecular mechanisms by which cysteine and H2S/NO axes of gasotransmitter signaling affect neuronal function are yet to be deciphered. In Aim 1 of this project, we will monitor expression and activity of the three H2S biosynthetic enzymes, CSE, CBS and 3-MST in the mouse brain at various ages. Sulfhydration status in normal as well as mice lacking CSE will be assessed. The specific proteins modified by sulfhydration will be identified, and sites of sulfhydration mapped on them. The interplay of sulfhydration with nitrosylation in neuronal function will be monitored. In Aim 2, we will analyze the involvement of H2S in stress responses and behavior. In Aim 3, we will identify differentially sulfhydrated proteins in the 3xTg-AD mouse model of AD. By studying the function of CSE and H2S in the brain, we set a goal to better understand signaling mediated by cysteine, H2S and protein sulfhydration in the context of neuronal signaling in AD. Understanding the regulation of H2S signaling in the brain helps to determine the basic physiological pathways involved in neuroprotection and pins down the nodes for precision therapeutics and development of biomarkers for AD and other age-related neurodegenerative diseases involving dysregulated H2S signaling. 1

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT The goal of this K08 Mentored Clinical Scientist Research Career Development Award application is to provide the candidate with advanced skills needed to establish an independent research program investigating the pathogenesis and therapeutics of monogenic high myopia (HM) and inherited retinal dystrophies (IRDs). Our overall hypothesis is that increased TGFβ signaling drives HM progression and structural changes in the posterior segment indicative of pathological myopia (PM) in Marfan syndrome (MFS) and Rbp3-mediated retinitis pigmentosa (RP). To test this hypothesis, the specific aims are to: 1) Determine the extent that Smad2/3 activation contributes to myopia progression, PM changes and retinal degeneration in MFS and Rbp3-/- mice; 2) Define a role for TGFβ-dependent MAPK activation in myopia progression, PM changes and retinal degeneration in MFS and Rbp3-/- mice. This is based on an extensive review of the literature and our high-quality preliminary data demonstrating that: 1) MFS mice have significantly greater axial length (AL) and -9D myopic shift compared to WT littermates; 2) MFS mouse eyes show increased Smad2/3 and MAPK (Erk1/2, Jnk1/2, p38) activation in the ciliary body and retina; 3) AL and myopic shift are reduced in MFS mice after Erk inhibition; 4) Smad3-/- mice display shorter AL and a prominent hyperopic shift compared to WT littermates. These data indicate that TGFβ downstream pathways represent novel therapeutic targets for myopia and/or PM in MFS. Prior studies illustrated that loss-of-function mutations in RBP3 cause autosomal recessive RP with HM (-12 to -17D) in humans, while Rbp3-/- mice show markedly increased AL and myopic shift, as well as in-vivo and ex-vivo evidence of progressive retinal degeneration. This offers an opportunity to evaluate a role for TGFβ signaling in this etiologically-distinct form of monogenic myopia, with the goal of identifying and targeting common drivers of disease progression. The candidate is proposing a comprehensive training plan, combining formal coursework, meetings, seminars and workshops overseen by his diverse, experienced mentorship team. His specific training goals include to: 1) Refine his knowledge of in-vivo phenotyping of myopia and PM in mice, encompassing mouse eye biometry, autorefraction, fundus photography and optical coherence tomography (OCT); 2) Develop skills in in- vivo phenotyping of IRDs and their complications in mice, including ERG, OCT, and optomotor response (OMR); 3) Enhance his skills in ex-vivo histopathological and biochemical analysis of mouse myopia and IRD models, including PM changes (e.g. retinal detachment, retinoschisis, posterior staphyloma) and retinal degeneration (e.g. retinal thinning, photoreceptor loss); 4) Acquire management skills to build a successful independent laboratory; 5) Develop leadership skills in collaborative research; 6) Refine his communication and writing skills to successfully apply for R01 funding; 7) Continue training in responsible conduct of research. His training plan will be executed in coordination with the research activities described above. Results from this proposal will be used to develop a future R01 research plan that will facilitate the candidate’s transition to independent research.

NIH Research Projects · FY 2026 · 2022-05

Project Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal form of liver cancer that primarily affects children and young adults without cirrhosis. There are no approved systemic therapies for FLC, and it is usually refractory to treatment approaches developed for other forms of liver cancer. A chimeric transcript between DNAJB1 and PRKACA was identified as a signature genomic event in FLC and leads to constitutive activation of PKAc, but pharmacological inhibition of PKAc for FLC with traditional small molecule inhibitors has been infeasible due to on-target toxicity. Prior work from our group and others has demonstrated that neoantigens derived from gene fusions, including the DNAJB1-PRKACA fusion in FLC, can stimulate strong T cell responses. Furthermore, all patients with FLC share an identical amino acid sequence at the fusion junction, allowing a single “off the shelf” neoantigen-specific vaccine to be utilized nearly universally for this cancer. Neoantigen- specific vaccines are most effective in combination with other immunomodulatory agents including ICIs to prevent T cell exhaustion. Our overall hypothesis is that a neoantigen-specific vaccine targeting the DNAJB1- PRKACA chimera will synergize with ICIs to elicit a specific antitumor immune response in FLC. We will conduct a clinical trial of a vaccine targeting the DNAJB1-PRKACA chimera (FLC-Vac), in combination with nivolumab and ipilimumab, in patients with unresectable FLC. We will further determine if FLC-Vac combined with ICIs increases the number of neoepitope-specific T cells with specificity for the DNAJB1-PRKACA chimera in the peripheral blood that traffic to the tumor. Multiplex immunohistochemistry (IHC) on paired pretreatment and on- treatment biopsies will further define the mechanisms of immune response and resistance to immunotherapy in FLC. We will use these samples to identify T cell receptors (TCRs) specific for the FLC fusion protein in the context of the patient's HLA. Using TCRs from our trial and from endogenous responses identified in untreated specimens, we will use humanized mouse orthotopic models to determine the relative efficiency of processing and presenting specific epitopes from the DNAJB1-PRKACA fusion in diverse MHC contexts. These models will involve the endogenous presentation of the FLC fusion within tumor lines that we will treat with primary cells transduced with our identified TCRs, allowing us to compare the targeting efficiency of TCRs specific for the corresponding fusion epitopes. This work may advance a novel treatment paradigm for FLC, a tumor for which there is no standard or effective systemic therapy, and will have important implications for targeting recurrent ”undruggable” driver genes in other immune-resistant tumor types. Identifying optimal peptide-HLA-TCR combinations for targeting the DNAJB1-PRKACA fusion will also lay the groundwork for the next generation clinical trials for FLC, including adoptive cell therapies with specificity for the DNAJB1-PRKACA fusion.

NIH Research Projects · FY 2025 · 2022-04

Project Summary The COVID-19 pandemic disproportionately impacts Latinos in the US. Failing to provide equitable access to vaccines will exacerbate the profound disparities in COVID-19 and other health conditions among Latinos communities. Our team has established a community coalition and identified effective interventions to reduce disparities among low-income limited English proficiency (LEP) Latino communities using qualitative, survey, and implementation science methodologies. We will test the efficacy of a combination intervention REDES (“Networks”) - a social network and mobile health (mHealth) enhanced community health worker (CHW) intervention - to address COVID-19 vaccine hesitancy and uptake among a cohort of Latinos and their networks in Maryland. We will recruit 300 index participants who have taken the COVID-19 vaccine. Half of the index participants will be randomized to the experimental group (n=150). CHWs will train them to be peer mentors to conduct peer outreach in-person and by text messages and to promote vaccine acceptance and uptake with their networks. Index participants in the control group (n=150) will receive an equal attention intervention. In addition, we will recruit unvaccinated primary and secondary network members of indexes for study participation and COVID-19 vaccination. We will enroll an estimated total of 1,590 primary and secondary network members. All participants will be followed prospectively at 3, 6, 12, and 18 months after baseline intake. The specific aims are: 1) Evaluate the efficacy of a combination intervention REDES to promote COVID-19 vaccine uptake among Latinos; 2) Examine ongoing barriers and facilitators of vaccine uptake among Latinos and their networks to tailor our intervention and address new challenges, and 3) Evaluate the implementation determinants and outcomes of REDES to inform future broad-scale implementation. Our multi-disciplinary research team brings together Latino health, social network, mHealth interventions, vaccine hesitancy expertise; a long-term history of community engagement with partner organizations in Maryland; and existing bilingual/bicultural CHW capacity and a rich local infrastructure for COVID-19 response. If this combination intervention demonstrates the efficacy, we will develop an implementation strategy toolkit, both in English and Spanish, for community partners and health departments interested in replicating the approach. This proposal primarily focuses on COVID-19 vaccination, but knowledge gained will be relevant to future vaccine equity initiatives, such as COVID-19 boosters (if needed) and seasonal influenza vaccination.

NIH Research Projects · FY 2026 · 2022-04

Project Summary Antibody-mediated targeted immunotherapies are highly effective in killing cancer cells. T cell leukemias and lymphomas, collectively known as T cell cancers, affect ~100,000 patients worldwide each year. Relapsed T cell cancers respond poorly to aggressive chemotherapy with a 5-year survival between 7% to 38%. Thus, T cell cancers particularly warrant antibody-mediated targeted immunotherapy to improve patient outcomes. However, developing a T cell cancer targeting immunotherapy is challenging as the immunotherapy will have to preserve enough healthy T cells to maintain a functioning immune system. T cells express the T cell receptor (TCR) on the cell-surface. Although all T cells express TCR, they can be distinguished based on the TCR beta chain constant region (TRBC) which is derived from one of two gene segments, TRBC1 or TRBC2. I led a team that demonstrated that in healthy T cell populations, about 45% of cells express TRBC1 while the other 55% express TRBC2. However, clonal T cell cancers express either TRBC1 or TRBC2 (Paul et al., Sci. Transl. Med. 2021). Thus, I hypothesize that antibody-mediated specific TRBC1 or TRBC2 targeting will eradicate the clonal T cell cancers while preserving half of the healthy polyclonal T cell population. I also developed a TRBC1- targeting bispecific antibody (αTRBC1) that selectively kills TRBC1+ T cell cancers (and TRBC1+ healthy T cells) while preserving the healthy TRBC2+ T cells in vitro. These in vitro observations will require confirmation in animal models before initiation of future human clinical trials. For Aim 1, I will determine the in vivo activity of the αTRBC1 bispecific antibody. I will test the ability of αTRBC1 antibodies to induce tumor regression in multiple mouse models of T cell cancers. I will then examine if the remaining healthy TRBC2+ T cells retain all of the immune cell subsets required for a functioning immune system. I will also test if therapeutic pressure from the αTRBC1 bispecific antibody will give rise to a low TCR expressing T cell population that will be resistant to therapy. For Aim 2, I will test feasibility of TRBC2-targeting on T cell cancers. As a TRBC2-targeting antibody is currently unavailable, I will use phage display to develop TRBC2-specific antibodies. I will then test the cytotoxicity of TRBC2 targeting antibodies in vitro and in multiple mouse models of T cell cancers. Our TRBC- directed antibodies will fill an unmet need for the treatment of T cell cancer patients. The proposed in vivo and mechanistic studies will provide the pre-clinical validation required for the initiation of an early-phase human clinical trials that will test the safety and efficacy of TRBC1- and TRBC2-targeting antibodies. My long-term goal is to become an independent laboratory-based physician-scientist focusing on developing novel therapeutic approaches for T cell cancers. Through this proposal, I will also acquire the research skills and career experience needed to reach this goal. These skills will be critical to the development of a relevant, impactful translational research program in human T cell leukemias and lymphomas.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract The interpretation of thousands to millions of variants identified in whole exome and genome sequencing (WES/WGS), respectively, is a challenging task and requires variant frequency together with phenotype information that are not available in most public databases. The development of tools that enable data sharing of genomic data together with phenotypic features is essential for rare disease gene discovery. In 2013 we created GeneMatcher (10,574 users in 93 countries, cited in >380 publications), the first and most used web- based tool to connect individuals (researchers, clinicians, patients, etc) around the globe with interest in the same genes, variants or phenotypes. In 2019, we developed VariantMatcher (625 users in 43 countries, 428 queried variants, 61 matched variants), to share variant-level and phenotypic data from WES/WGS. To enable variant prioritization for investigators around the world, we developed PhenoDB (> 143 unique downloads of source code, 1,828 users from 65 countries). This freely available, web-based tool allows users naïve to bioinformatics to store, share, analyze and interpret patient phenotypes and sequence variants from WES/WGS. Users currently store in one instance of PhenoDB extensive phenotypic data (described with 3,646 terms mapped to HPO) from 37,712 individuals in 9,711 families with an average of 6.8 features/affected individual, describing > 2,282 diseases. Annotated WES/WGS variants are available for 7,724 individuals from 4,446 families. PhenoDB facilitates data sharing and analysis access world-wide. As of 1 December 2020, 6,151 sequenced individuals in PhenoDB were consented and submitted for phenotypic and genotypic matching through VariantMatcher. However, the ongoing development of new sequencing methods, analysis pipelines and data sharing platforms requires development of novel functions in our highly utilized resources and integration with other newly created platforms to ensure that thousands of investigators can perform state of the art analysis of genomic and phenotypic data to better interpret candidate variants. There is a critical need to maintain and improve tools that allow genomic and phenotypic data integration and sharing. To fill this need, we will leverage input from users of PhenoDB, GeneMatcher and VariantMatcher, collaborations with creators of tools and platforms with complementary functions, and our evolving expertise in sequencing analysis and gene discovery to improve our community resources. To this end, we will: 1. Implement new functions in GeneMatcher, VariantMatcher and PhenoDB to enable additional types of queries, variant filtering and prioritization pipelines. 2. Connect GeneMatcher and VariantMatcher with other, community-wide data sharing tools and integrate PhenoDB with other workflow platforms. 3. Expand our PhenoDB Genomic Education program.

NIH Research Projects · FY 2025 · 2022-04

Abstract AKT is one of the most important protein kinases in insulin signaling. In response to insulin, AKT becomes active and phosphorylates critical metabolic effectors, including TBC1D4, GSK3, TSC2, and FOXO. These proteins regulate glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane, glycogen synthesis, lipid and protein synthesis, and glucose production in adipose tissues, skeletal muscles, and livers. Abnormalities in AKT activation have been linked to insulin resistance in type 2 diabetes. AKT is activated by two other protein kinases, mTORC2 and PDK1. mTORC2 phosphorylates the hydrophobic motif of AKT and opens the catalytic domain. PDK1 then phosphorylates AKT to activate its enzymatic activity. The activation step by PDK1 is controlled by the recruitment of AKT and PDK1 to the plasma membrane. However, understanding of how mTORC2 is regulated to phosphorylate AKT is limited. To fill this critical knowledge gap, this grant application tests the hypothesis that KRAS4B, RHOA, and mTORC2 form a supercomplex (termed KARATE) to direct the enzymatic activity of mTORC2 toward AKT in insulin signaling. Toward this goal, we will identify the mechanism, localization, and regulation of the KARATE assembly. We will also determine the physiological function of KARATE in glucose homeostasis. We will employ multiple innovative tools, including: 1) our recently developed total biochemical reconstitution system for KARATE- mediated AKT phosphorylation; 2) a Dictyostelium bioreactor that enables the purification to functional human proteins to high homogeneity with critical post-translational modification; 3) our novel KARATE peptide inhibitor for in vitro and cellular studies; 4) our CRISPR-generated knockout cell lines for RHOA, KRAS and mTORC2 subunits; and 5) tissue-specific RHOA-knockout mice and phospho-defective RHOA mice. We anticipate that the successful completion of the work will significantly advance our understanding of insulin signaling and establish a solid foundation for future studies. Ultimately, this will help translate the fundamental biology of AKT signaling into medical treatments focused on KARATE for metabolic syndrome.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Orofacial clefts (OFCs) represent the most common group of craniofacial malformations in humans affecting approximately one per 1,000 live births worldwide. OFCs include cleft lip (CL), cleft palate (CP) and cleft lip with cleft palate (CLP), which can occur as isolated malformations, with another malformation or as part of a recognized malformation syndrome (often Mendelian with incomplete penetrance). OFCs are commonly categorized into two anatomically and embryologically distinct entities based on embryologic and epidemiologic patterns: cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Among all infants born with an OFC, 70 percent of CL/P cases and 50 percent of CP cases occur as isolated, non-syndromic malformations. Non-syndromic CL/P occurs more frequently in males than females (ratio 2:1) whereas non-syndromic CP occurs more often in females (ratio approximately 1:1.14). Substantial variation in birth prevalence rates of non- syndromic CL/P has been reported across populations, with Asian populations having higher birth prevalence rates compared to European populations, and African populations having the lowest birth prevalence rates. Risk to OFC shows strong evidence of genetic control with estimated heritability up to 90%. Recent genome- wide association studies have clearly shown multiple genes play a role in the etiology of OFCs, but with substantial heterogeneity among families and across populations. To date, approximately 50 different genes have been identified as significant in such genome-wide studies of OFCs, with about two dozen having substantial replication and/or functional studies. However, despite a long history of scientific research into the genetic control of OFC, much of the heritability remains unexplained (which may reflect the genetic heterogeneity influencing risk to OFC, where a number of different genes with both rare and common variants control risk), and it remains difficult to clearly identify underlying causal genes. Moreover, sex differences in risk to OFC and parent-of-origin effects traditionally have not been the focus of genetic studies, and X chromosome variants have largely been ignored. In this application, we are using existing genomic data from family-based studies in different ethnic groups to specifically study the underlying mechanisms for differential risk to OFC between the sexes. Specifically, we will (i) use case-parent trios to detect different genetic OFC risk effect sizes and parent of origin effects, (ii) use a novel method to characterize sex differences in the genetic architecture of OFCs accounting for potential cleft type differences and similarities, and (iii) conduct association tests for variants on the X chromosome. In addition, we will use genomic data from extended multiplex pedigrees to identify highly penetrant genomic X-linked variants. The family-based designs allow us to study common and rare variants, parent-of origin effects, and allow us to assess the impact of de novo variants. In all aims, we will attempt to use functional data from external data bases to conduct an “in silico” validation of our findings.

NIH Research Projects · FY 2026 · 2022-04

Project Summary Immune checkpoint inhibition emerged as promising cancer treatment strategies. However, only a subset of patients respond to these therapies at present. Significant clinical evidence indicates that abundant tumor infiltration of effector T cells and B cells is a prerequisite for the success of the immune checkpoint inhibition therapies. However, these lymphocytes are largely excluded from many patients' tumors, which makes the tumor unresponsive to the immunotherapies. High endothelial venules (HEV) are specialized venules that serve as gateways for naïve lymphocyte recruitment, and these blood vessels can develop ectopically in tumors. High HEV density in tumors correlate with favorable clinical outcomes, suggesting that the promotion of intratumoral HEV formation would offer a novel opportunity to improve immunotherapies. The small GTPase R-Ras balances angiogenic sprouting and vessel maturation, and normalizes tumor blood vessels. Several lines of evidence indicate the importance of R-Ras for the formation of intratumoral HEVs. We hypothesize that intratumoral HEVs shape the tumor immune landscape through efficient recruitment of lymphocytes, thereby creating the immunostimulatory microenvironment favorable for immune checkpoint inhibition therapies. We propose a critical role of R-Ras in this process by facilitating HEV formation in tumors. Using novel genetic models of loss- and gain-of-function of R-Ras in endothelial cells, Aim 1 will demonstrate that R-Ras facilitates the formation of HEVs within the tumor vasculature and determine how these HEVs affect the tumor infiltration of T cells, B cells, dendritic cells, and other immune cell types in immunogenic mouse mammary tumor and melanoma. The influence of intratumoral HEVs on cytokine environment will also be determined. The analyses will be conducted by immunofluorescence, ELISA, FACS, and RNAseq. Clinical cancer specimens will be examined to corroborate the findings from the mouse studies. Aim 2 will functionally characterize tumor HEVs and analyze intratumoral priming of HEV-recruited T cells to a specific antigen using adoptive transfer of naïve OT-1 T cells to OVA-expressing tumors. Aim 3 will determine how intratumoral HEVs impact the immune destruction of tumors and the responsiveness to PD-1/PD-L1 inhibition therapies. As the gateways for T cell/B cell recruitment to tumors, intratumoral HEVs are potential new targets to reprogram the tumor immune landscape and to improve patients' response to immune checkpoint inhibitors. The expected outcome of this study will provide the proof-of-concept for such ideas.

NIH Research Projects · FY 2026 · 2022-04

Modified Project Summary/Abstract Section Limited data exists on adults at higher risk of poor mental health and suicidality across the life course in rural areas of the United States, though where data are available, they suggest higher rates of mental disorders for rural adults affected by social stressors. Technology-delivered mental health interventions are being increasingly explored among the general population and hold promise for use among rural communities, but they must take into account the experiences of this population to be effective. This research team from the Department of Mental Health and Key Populations Program at Johns Hopkins University and Emory University will use online approaches to enroll 2000 adults at heightened risk of poor mental health from rural counties and small cities across the US to follow for 12 months with repeated questionnaires to: 1) examine social, geographic, and economic factors associated with increased prevalent depression and suicide ideation or attempt; 2) determine whether these exposures are associated with incident depression or suicidal ideation or attempt; and, 3) compare the relative acceptability of various technology-delivered interventions for depression and suicide prevention tailored to addressing identified exposures. This study will produce a comprehensive investigation of social, economic, and geographic factors related to prevalent mental health outcomes among rural adults, as well as by what pathways they may predict incident depression, suicidal ideation and attempts. The study will also provide substantive evidence-based recommendations for the development or adaptation of technology- delivered interventions to address mental health among rural communities.