Johns Hopkins University

NIH Research Projects · FY 2025 · 2023-08

Project Summary Despite the progress made in precision oncology, clinicians typically face a vast volume and variety of next- generation sequencing and molecular data that is frequently intuitively processed to support high-stakes decisions. Overall, currently available resources that assist with next-generation sequence data interpretation are limited by manually performed, complex, time-consuming, and error-prone gene queries and ultimately lack the necessary information for prioritizing emerging therapies in a scalable manner. Importantly, the integration of genomic with clinical data has been severely hampered by the lack of advanced analytical tools that match genomic targets with molecularly-driven therapies. These barriers, together with health disparities, widen the gap between an exponentially increasing drug development field and the actual benefits for patients with cancer. The overarching goal of the proposed research is to link clinical with computational precision oncology and enable clinical decision-making in genomically defined groups. We propose to develop a precision oncology decision support framework for automated, scalable, and precise matching of actionable next-generation sequencing findings with targeted therapies. We will then test its clinical utility and value in the several clinical settings within the Johns Hopkins Molecular Tumor Board, in Johns Hopkins partnering community medical centers as well within two ongoing clinical trials for women with breast cancer. To enhance the generalizability of our analytical toolkit past our local academic environment, we have designed the platform's architecture such that it allows for ingestion and harmonization of next-generation sequence data from multiple sources, implements a common data model to map clinical elements to standardized terminologies and leverages ensemble natural language processing to generate actionable mutation-targeted therapy pairs. These attributes provide the foundation for the toolkit's potential widespread use and implementation in health care settings outside our local academic environment. While significant advances have been made in advanced diagnostics for tumor profiling, a solid backbone that supports the practical implementation within and across health care systems is lacking. The underlying premise of the proposed research is that it will ignite cross-institutional real-world genomic data analysis initiatives and genotype-driven clinical trials that will be beneficial for health systems and patients. Notably, our precision oncology decision support platform will enhance the implementation of precision oncology at institutions that do not readily have access to in-house expertise in clinical genomics. We envision that this streamlined automatic and scalable process will improve care, enhance patient outcomes and define national standards in how treatments are selected and tailored to individual patients.

NIH Research Projects · FY 2025 · 2023-08

Biologically important sounds, such as animal vocalization, speech, and tonal music, contain rich harmonics with spectral energy clustered at integer multiples of the fundamental frequency. Although the exact neural coding mechanisms for harmonic sounds remain unclear, recent experiments show that harmonic sensitivity is widespread in the auditory cortices of the marmoset. Since cortical harmonic sensitivity spans multiple octaves, it is derived presumably by combining subcortical inputs that typically prefer only a single frequency. We propose to study harmonic coding in auditory cortex of the marmoset by simultaneous recording of many individual neurons which are probed automatically by an online adaptive stimulus optimization procedure based on explicit computational models of the underlying neural circuits. Conventional methods are incapable of fully characterizing complex harmonic responses because of the combinatorial explosion of the stimulus space, which is a general obstacle for sensory coding research. We propose to overcome this obstacle using an adaptive online approach to harmonic stimulus design. We will apply two broad types of methods, one is to find the optimal stimulus that best drive a neuron, and the other is model-based stimulus design that can effectively identify each given model and compare competing models by finding the stimuli that best distinguish them. We will develop: (1) automated system to characterize harmonic sensitivity of individual neurons across multiple layers of auditory cortex using Neuropixels recording probes, (2) automated system to characterize harmonic sensitivity in auditory cortex across multiple octaves of frequencies using two-photon imaging, and (3) generative circuit models for efficient coding of harmonic sounds in auditory cortex. By restricting the stimuli to harmonic sounds, which are complex enough but still tractable, we believe our methods are more likely to achieve significant success. We have obtained promising preliminary results in several successful online neurophysiological experiments using single-unit recording. Extending our online methods to Neuropixels recording and two-photon imaging is a logical next step and may potentially benefit many researchers working on related problems. We expect to obtain full stimulus-response landscapes of cortical neurons together with inferred circuit models that may explain how exactly a higher-level cortical representation of harmonics may arise from simpler input components, and all these representations will be examined in the context of efficient coding of natural sounds.

NIH Research Projects · FY 2025 · 2023-08

Kidney transplant (KT) offers a significant survival and morbidity benefit over dialysis, making it the preferred treatment modality for end-stage kidney disease. While late allograft failure has a multifactorial etiology, one of the largest contributors is the development of donor specific HLA antibodies (dnDSA), leading to allograft loss at a median 3-5 years post detection of antibodies. Donor specific HLA antibodies develop against short amino acid sequences within the HLA antigen. Each HLA antigen has multiple epitopes that can interact with the recipient immune system, and antibody-verified epitopes are termed “eplets”. Mismatched epitopes can be identified and enumerated using various molecular mismatch software packages. However, not all epitopes are equally likely to induce an antibody response in the recipient, as specific “high-risk” eplet mismatches were found to be disproportionally associated with dnDSA formation. Avoidance of high-risk mismatches between donor and recipient at the time of organ allocation is one way to improve long-term allograft survival because it would reduce the number of potential dnDSA targets. Variable immunogenicity is an accepted concept however details about which mismatches are high risk has not been well established. I propose to establish a multi-site pediatric kidney transplant (KT) cohort with full HLA genotyping on recipients and donors to perform such an analysis. This will inform the development of an adaptive allocation model, that can better account for the entangled and dynamic nature of allocation systems. The Organ Procurement and Transplant Network (OPTN) has mandated the development of a new allocation model, to develop a composite allocation scoring system that can account for dynamic changes in multiple recipient and donor characteristics. There is insufficient data to inform such a model on how to handle HLA mismatch on an epitope-level. My work with the multi-site cohort will inform how to best inform incorporate molecular mismatch analysis and high-resolution tissue typing data into an adaptive allocation model. My career goal is to become an independent clinical researcher focused on improving outcomes for KT recipients by studying the adaptive and humoral immune response to the allograft and conducting clinical trials to test interventions to reduce the burden of disease. By completion of the proposed research and didactic training at the Johns Hopkins School of Public Health, I will obtain a PhD in Clinical Research Methodology and develop a unique skillset that will allow me to establish an independent research career in transplant immunology. Specifically, I will gain expertise in multi-site study design and execution, large data management and analysis, advanced computational modeling, and application of immunogenetics to clinical practice.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Women who use drugs (WWUD), specifically heroin, shoulder a disproportionate burden of the HIV epidemic in Tanzania. Despite advances in HIV prevention biomedical interventions, including pre-exposure prophylaxis (PrEP), PrEP uptake has been slow in Tanzania. Mental disorders, including depression, anxiety, and post- traumatic stress disorder, are high among WWUD and are known barriers to HIV prevention behaviors, including PrEP engagement. Inattention to the high prevalence of mental health problems in WWUD, is likely to undermine efforts to increase the use of HIV prevention strategies, including PrEP, among this highly vulnerable, yet understudied population. Motivational interviewing (MI) and cognitive-behavioral therapy (CBT) are evidence-based interventions that have been successfully delivered by paraprofessionals, both globally and in Tanzania, for a range of health conditions, including substance use disorders and mental disorders, and to improve uptake and adherence of HIV prevention strategies. The Common Elements Treatment Approach (CETA) is a transdiagnostic, CBT-based intervention developed for use in low- and middle-income countries for delivery by lay counselors with no or limited previous mental health training. The goal of this study is to adapt MI for PrEP engagement (MI-PrEP) and a combined CETA and MI-PrEP interventions (CETA+MI-PrEP) for delivery by paraprofessionals in Dar es Salaam, Tanzania, to optimize PrEP engagement among WWUD. The specific aims of this study are to: (1) identify barriers and motivators to PrEP engagement, and the types of paraprofessionals who could most effectively deliver the MI-PrEP and CETA+MI- PrEP interventions using in-depth interviews and focus groups with WWUD, peer- and non-peer community health workers, lay counselors, and mental health specialists, (2) systematically adapt MI-PrEP and CETA for WWUD with co-occurring mental disorders in Tanzania using the ADAPT-ITT model, and (3) assess the feasibility, fidelity, and preliminary efficacy of the MI-PrEP and CETA+MI-PrEP interventions in a three-arm pilot feasibility trial with 120 WWUD with co-occurring symptoms of mental disorders. A combination of interviews, structured surveys, and supervision will be used to assess for intervention feasibility and fidelity. The preliminary efficacy of the intervention will be estimated for the primary outcomes of PrEP engagement and secondary outcomes of opioid use, symptoms of depressive, anxiety, and post-traumatic stress, and PrEP motivation and barriers. This pilot feasibility study will: (1) advance our understanding of the unique factors that contribute to the PrEP engagement among WWUD, (2) address the HIV prevention needs of an understudied, yet extremely high-risk population, and (3) result in culturally appropriate and locally adapted MI-PrEP and CETA+MI-PrEP interventions that can be formally tested in a Hybrid Type I Effectiveness- Implementation randomized controlled trial.

NIH Research Projects · FY 2025 · 2023-08

7. PROJECT SUMMARY / ABSTRACT People who inject drugs (PWID) face multilevel barriers to accessing HIV-related services, including low demand for services, resulting from stigma, low knowledge/awareness of services, and prioritization of immediate basic needs (e.g., food, housing, addiction) over remote health concerns. In rural areas, these barriers are exacerbated by service scarcity. Syringe service programs (SSPs), a cornerstone of HIV prevention among PWID, are trusted by this population and have the potential to extend the reach of other evidence-based services (pre-exposure prophylaxis [PrEP], medication for opioid use disorder [MOUD], HIV treatment, etc.). Telemedicine provides a sustainable method to deliver services where they are scarce. We successfully piloted telehealth delivery of HCV treatment and MOUD for PWID at rural SSPs. We will adapt and expand on our telemedicine pilot to bring HIV prevention and treatment services to rural SSPs. However, service availability does not equate to uptake. Social network-based interventions have proven effective in disseminating health information and HIV risk reduction among PWID. Peer-based interventions have an unparalleled potential to overcome stigma and trust barriers in marginalized groups, reach “hidden” group members (i.e., those who do not access services at the SSP), and empower individuals to make positive changes. Low rates of HIV testing and diagnosis among PWID remain a barrier to linkage to antiretroviral therapy initiation (for HIV infected) and PrEP (for HIV-uninfected). A key innovation of our intervention will be to combine dissemination of information with distribution of HIV self-testing kits through PWID networks. HIV self-testing strategies are endorsed by the World Health Organization and have been shown to increase testing coverage and new HIV diagnoses in high prevalence regions of sub-Saharan Africa and among MSM, but have been little explored among PWID. We propose to evaluate a model that leverages peer educators to disseminate HIV self-testing kits and information while also providing peer navigation to network members for HIV prevention and treatment services uptake via telemedicine at rural SSPs We plan a hybrid effectiveness/implementation approach. Aim 1a: We will use a community-engaged approach to adapt a preexisting PWID-focused network intervention to a rural context with the goal of increasing HIV self-testing and HIV service uptake. Aim 1b: We will determine the effectiveness of the intervention compared with an equal-attention control condition, using a rigorous randomized trial design. Aim 2a: In contemplation of adoption and sustainability, we will use mixed methods to characterize implementation processes and outcomes. Aim 2b: to explore intervention mechanisms, we will establish the extent to which SSP and HIV service uptake are related to experiences of social influence and stigma.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Ureteral injury represents one of the most serious complications of pelvic surgery, with a majority of these injuries occurring during gynecological procedures. This injury is particularly problematic during hysterectomies because of the proximity between the ureter and nearby blood vessels. One barrier to progress is the absence of clinically available technology to identify relative positions of the ureter, uterine arteries, and tool tips with suf- ficient depth penetration and image contrast. We previously demonstrated that photoacoustic imaging achieves simultaneous detection of critical structures with approximately 25-30 dB contrast at centimeter depths, allowing for complete avoidance of the ureter and better targeting of the uterine arteries. However, to advance this tech- nology into surgical practice, we need to establish the optical, acoustic, and navigation parameters necessary to achieve optimal detection of tool tips, blood vessels, and ureters. Optimizing photoacoustic imaging system designs and providing informative real-time feedback during hysterectomies will enable these surgeries to be performed without the complications that are typically associated with ureteral injuries, including extensive re- peat surgeries, complete kidney failure, sepsis, acute renal insufficiency, and patient death. Our long-term goal is to develop guidance technology to differentiate critical structures in real-time during surgery. The overall objective of this proposal is to establish optimal parameters to advance photoacoustic technol- ogy toward differentiation of ureters, uterine arteries, and tool tips during hysterectomies. Aim 1 of this project will define the light delivery requirements for optimal visibility of laparoscopic surgical tool tips and underlying structures. Aim 2 will integrate and optimize sound reception components and parameters for photoacoustic imaging of the ureter, uterine artery, and tool tips. Aim 3 will pursue in vivo demonstrations of robotic hysterec- tomy navigation with photoacoustic imaging system components. These three aims will be tested independently with a combination of simulation, cadaver, swine, and human patient studies, resulting in multiple possibilities for deploying the proposed technology. Successful completion of the proposed project will establish a series of viable photoacoustic imag- ing system designs to enable ureter avoidance during hysterectomies. This project is innovative because of the novel integration and refinement of photoacoustic approaches and techniques to distinguish the ureter from the uterine artery. The project results are anticipated to have a significant impact on patients undergoing laparoscopic hysterectomies, robotic hysterectomies, and other robotic surgeries (e.g., radical prostatectomies, thoracic surgeries), with possible extensions to additional surgeries wherein critical structures reside in close proximity. The proposed research aligns with NIBIB’s mission to accelerate the application of biomedical tech- nologies by supporting research to advance the development of new tools for visualizing critical structures to target or avoid during minimally invasive surgeries.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Over the last two decades, three highly pathogenic human coronaviruses, including SARS-CoV-2, have emerged, arguing that future deadly pandemics of new or re-emerging coronaviruses and other RNA viruses are almost inevitable. The urgent need to treat these fatal infectious diseases has prioritized the discovery and development of novel effective antivirals. However, despite the unprecedented efforts to address the COVID-19 crisis, highly effective COVID-19 therapeutics are severely limited due to the lack of a mechanistic understanding of coronavirus replication and pathogenesis. Most of the current research and therapeutic development efforts focus on SARS-CoV-2 RNA polymerase (RdRp) or spike protein, but their success can be severely undermined by a unique SARS-CoV-2 proofreading mechanism that excises incorporated RdRp inhibitors or by the rapid emergence of drug-resistant spike protein mutants. Therefore, it is imperative to develop innovative antiviral strategies targeting distinct, and in some cases yet to be identified, essential components of the viral life cycle. The intricate virus-host interplay constitutes a sophisticated regulatory network that dictates the outcome of virus infection, affording promising opportunities to be explored for innovative antiviral therapeutics. However, current knowledge of coronavirus-host interactions is mostly limited to the virus-host arms race in activating or blocking the interferon-dependent signaling pathways or in competing for host translation machinery. By contrast, virus- host interplay in coronavirus gene expression and regulation is largely uncharted territory. This major gap greatly hinders the design of novel antiviral agents targeting these much-overlooked coronavirus-host interactions that are critical for viral survival and host antiviral responses. The central objectives of this proposal are to define novel molecular mechanisms and functional roles of multiple recently discovered SARS-CoV-2-host interactions in modulating the viral replication and transcription processes and to identify new SARS-CoV-2-host interactions linked to novel mechanisms of viral gene regulation. With a combination of structural biology, protein-RNA biochemistry, single-molecule biophysics, cell virology, and computational approaches, we will accomplish these tasks through three tightly interwoven aims. In Aim 1, we will determine the molecular and structural basis of these newfound interactions between host factors and the viral replication-transcription machinery. In Aim 2, we will delineate the biological functions of these crucial host factors in modulating viral replication and transcription. In Aim 3, we will develop an innovative capture-identification-visualization experimental pipeline to discover new host factors and novel viral replication-transcription-regulating mechanisms. Together, these studies will establish an innovative conceptual framework to study coronavirus-host interactions and reveal new targets for the development of novel anti-coronavirus therapeutics.

NIH Research Projects · FY 2025 · 2023-08

People living with HIV infection (PLWH) on ART live longer today but the incidence and prevalence of chronic diseases is significantly higher that it is in those without HIV. As many as 50% of PLWH today have been reported to have left ventricular diastolic dysfunction (DD), which is associated with atrial fibrillation, exercise intolerance, and the progression to heart failure with preserved ejection fraction (HFpEF). The responsible mechanisms for DD and its progression in contemporary PLWH populations are poorly understood but our preliminary studies suggest that impaired cardiac energy metabolism may be a central factor linking previously reported risk factors to DD. ATP is absolutely required for the normal myocellular relaxation and considerable pre-clinical data and our pilot clinical studies using 31P magnetic resonance spectroscopy (MRS) suggest an “energetic myopathy” as a basis for the DD in PLWH. In addition, inflammation is increased despite combined ART and viral suppression in PLWH and is known to impair mitochondrial function. We propose here to examine cardiac high energy phosphate metabolism, its causes, and its relationship to left ventricular diastolic dysfunction (DD) and DD progression in PLWH. The central hypothesis is that cardiac mitochondrial energy metabolism is impaired even in well-treated PLWH, and promotes the development and progression of DD in PLWH as well as the consequences of DD including cardiac remodeling and HFpEF assessed with echocardiography, increased circulating heart failure biomarkers, heart failure symptoms and decreased exercise performance. The specific aims are 1) to define the scope and extent of myocardial energetic abnormalities at rest and exercise using 31P MRS/MRI in PLWH, 2) to probe the factors underlying cardiac muscle mitochondrial and energetic abnormalities in PLWH, including those unique to PLWH (ART history and cumulative viral history) and others more common in PLWH (increased inflammation, immune activation, insulin resistance, cardiac fibrosis, and/or higher cardiac muscle lipids by MRI), and 3) to determine the functional consequences of observed cardiac muscle energetic changes in PLWH, particularly the presence and progression of DD. The studies will leverage the expertise, resources, and established PLWH cohorts at Johns Hopkins and collect novel cardiac energetic, diastolic function, quantitative exercise tolerance and biomarker data. The results of these studies will deliver novel understandings of the type and extent of myocardial energetic-mitochondrial abnormalities in PLWH, the factors prevalent in PLWH that are most closely related to impaired cardiac mitochondrial-energetic metabolism, and the functional consequences, most importantly diastolic dysfunction. These studies, characterizing the presence and functional consequences of what appears to be a “mitochondriopathy” of cardiac and skeletal muscle in PLWH promise new avenues to better understand the pathophysiology of DD in PLWH and suggest the selection and design of metabolic strategies to reduce the personal and societal impact of HIV disease-related functional decline in this important and growing population.

NIH Research Projects · FY 2024 · 2023-08

Over 316 million people are living with chronic hepatitis B and as many as one quarter of people living with HIV have hepatitis B virus (HBV) co-infection. HIV-HBV co-infection increases levels of HBV replication, liver disease development, and the risk of hepatocellular carcinoma. The key to HBV persistence is nucleus- resident, long-lived covalently closed circular DNA (cccDNA) coding for all viral proteins. Nucleos(t)ide analogue therapy (NUC) effectively reduces HBV DNA in the serum by halting reverse transcription of pregenomic RNA (pgRNA). Recently, NUC therapy has been associated with cccDNA transcriptional silencing leading to reduced pgRNA levels in the liver and HBV RNA in the serum, however cccDNA quantities across the liver remain stable. Efforts to develop a cure for HBV focus on the removal or long term control of cccDNA activity, the latter yielding a functional cure. An alternative treatment to NUC is pegylated interferon-α (PEG- IFN), a potent antiviral therapy with comparatively greater rates of functional cure (7%), defined as a durable loss of serum HBV surface antigen (HBsAg). A study following participants before and after PEG-IFN found that treatment non-responders had elevated proportions of spliced HBV (spHBV) DNA from total HBV DNA, supporting a role of spHBV in modulating IFN responsiveness. There are 20 identified spHBV transcripts derived from pgRNA, a subset of which encode noncanonical HBV proteins. A limited number of studies have demonstrated that spHBV expression disrupts IFN response signaling and possibly alters cccDNA transcription. Given their putative role in modulating the host immune response and viral transcription, we propose to compare spHBV in HBV mono-infection and HIV-HBV co-infection. Combining use of a novel multiprobe multiplex droplet digital PCR with direct sequencing, we will characterize spHBV in HIV-HBV co- infection and HBV monoinfection in serum from individuals in the MACS/WIHS Combined Cohort Study. We expect an enrichment of spHBV RNA within total HBV RNA in co-infection compared to mono-infection. Additionally, we will model the decay of spHBV during NUC in HIV-HBV co-infection and compare spHBV expression in the same person’s liver and blood. Additionally, we will perform deep sequencing of hepatocytes with high vs low HBV transcription in an HIV-HBV coinfection ancillary study of the Hepatitis B Research Network, focusing on innate response pathway genes. We will then overexpress candidate spHBV that are likely to affect IFN responses in HepG2-NTCP cells. Conversely, we will use siRNA to knockdown candidate spHBV and measure IFN responses in an HBV infected HepG2-NTCP cells. Using this model in the context of HBV infection, we will measure the interplay between cccDNA transcription, spHBV expression, and innate signaling. This study will be the first characterization of spHBV in HIV-HBV co-infection and has the goal of identifying novel therapeutic targets specifically affecting cccDNA transcription in CHB.

NIH Research Projects · FY 2024 · 2023-08

Coronary artery calcium (CAC) testing provides a direct measure of an individual’s atherosclerotic burden that is robustly associated with cardiovascular disease (CVD) risk. Accordingly, CAC scoring has a IIa recommendation in the 2018 AHA/ACC Cholesterol Treatment and 2019 ACC/AHA Primary Prevention Guidelines and is also recommended by many other national and international guidelines for the allocation of primary prevention medications. The clinical implications of the Agatston score can vary significantly based on the age, sex, and race/ethnicity population percentile score, which is particularly crucial for estimating long- term or lifetime CVD risk, especially for younger persons in whom a low Agatston score may commonly be reflective of premature atherosclerosis and significantly increased lifetime risk. However, current guideline- recommended CAC percentile scores were developed from only one NHLBI cohort comprised of predominantly middle-aged participants and there is no centralized website or resource with all the available tools to facilitate interpretation of the CAC score. Consequently, there is an unmet need to 1) develop CAC percentile data across the lifespan that is more representative of the US population and more diverse across racial/ethnic minorities, particularly South Asians, who have a significantly increased CVD risk yet are not included in current percentile score calculators and 2) create a centralized website and mobile app to aid in the interpretation of CAC at the point of care. Therefore, using standardized data harmonization techniques, we propose to pool data from 7 NHBLI cohorts and 3 real-world clinically-derived cohorts to create the gender- balanced, racially/ethnically diverse CAC Synthetic Cohort Lifetime Pooling Project (CACSC-LPP) of approximately 20,000 NHBLI participants with CAC data across the lifespan (age 30-95 years old) and an additional ~90,000 participants from clinical CAC cohorts. Harmonizing the individual participant level data will pragmatically leverage the enormous NIH investment (monetary and time) in these cohorts to create a new pooled dataset that expands the clinical impact beyond what was originally envisioned. It will also create an ideal foundation to iteratively expand the CACSC-LPP to ensure better representation for all persons living in the US, with an initial focus on South Asians. Additionally, we will build upon our prototype CAC-tools website and companion app so that clinicians and patients can enter a CAC score (plus any other available risk factor data) and retrieve their CAC percentile. This website/app will serve as the definitive source for all CAC related tools and would be well positioned to be endorsed by future national CVD guidelines (see LOS, Drs. Lloyd- Jones & Arnett). Through the creation of the 1) CACSC-LPP, 2) CAC percentile calculator across the lifespan, and 3) centralized CAC website/app this project will improve personalized CVD risk prediction, implementation of ACC/AHA primary prevention recommendations, while also creating the infrastructure for multidisciplinary and cross-institutional collaborations to further improve our understanding of CAC for CVD risk prediction.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY The Ccr4-Not complex is the major regulator of codon optimality-mediated messenger RNA decay, a mechanism that is intimately tied to translation rate. The disruption of co-translational mRNA decay events can have major physiological effects, leading to haploinsufficiency or contributing to cancer. For instance, CNOT3, a subunit of the Ccr4-Not complex, was recently identified as a tumor suppressor that is mutated in 7.9% of adult T-cell acute lymphoblastic leukemias (T-ALLs). Recent work in our lab identified that Not5 (yeast homolog of human CNOT3) directly binds to the E-site of slowly translating ribosomes, leading to the preferential decay of non-optimal mRNA transcripts. This interaction was recently found to be conserved in humans, suggesting a mechanism for how cells use the mRNA decay machinery to alter transcript levels. Though the structure of Not5 interacting with elongating ribosomes has been resolved, nothing is currently known about how Not5 interacts with members of the Ccr4-Not complex to facilitate mRNA decay. Thus, teasing apart how the decay machinery mechanistically regulates the degradation of mRNA will be critical in understanding how cells regulate transcript levels as well as open avenues of therapeutic intervention for a broad range of haploinsufficiency diseases and cancer. To address this gap in knowledge, I will mechanistically dissect how the Ccr4-Not complex assembles onto actively translating ribosomes and assess how the subunits of this complex coordinate the removal of the poly-A tail and 5’-cap of mRNAs using the budding yeast, Saccharomyces cerevisiae as a model system. Preliminary data from our lab shows that Not5 binding to the ribosomal E-site is necessary for the recruitment of the mRNA decay factor, Dhh1, to translating ribosomes, but the details of this recruitment pathway remain opaque. We do not yet understand if Not5 is involved in the recruitment of the remaining mRNA decay factors and how this recruitment might coordinate decay events. Likewise, the loss Dhh1 is known to exhibit decapping defects and recently we found that the loss of this factor also exhibits defects in pol-A tail removal of mRNAs, meaning Dhh1 recruitment may serve to bridge these 3’-5’ decay events. I hypothesize that Not5 recruits the remaining Ccr4-Not subunits to actively translating ribosomes resulting in the timely decay of mRNA transcripts. I will address this hypothesis through the following specific aims: Aim 1) I will systematically determine the assembly of the Ccr4- Not complex subunits onto the translating mRNP structure and determine if this assembly is Not5- dependent. Aim 2) I will characterize the contacts between Dhh1 and the Ccr4-Not complex to determine if their interaction mediates the 3’-5’ communication of mRNA decay. The proposed studies will enhance our mechanistic insight into how mRNA half-lives are regulated and provide the basis for detailed understanding of this role in human disease.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Telomeres are specialized DNA-protein complex that protect the ends of linear chromosomes. Mammalian telomeres are composed of highly conserved tandem repeat sequences of duplexed (TTAGGG)n, ending with a 3’ single stranded (TTAGGG)n sequence that is bound to telomere specific proteins known as the shelterin complex. In healthy somatic cells, progressive loss of telomeres results in cellular senescence. In 85% of cancer cells, however telomeres are elongated by telomerase, while the remaining 15% of cancers use alternative lengthening of telomeres (ALT). ALT pathway leads to overexpression of Telomeric repeat containing RNA (TERRA), a long noncoding RNA that is transcribed from telomeres. TERRA is capable of trans annealing by invading the telomeric duplex to form an R-loop (DNA-RNA hybrid) structure. Consistently, the accumulation of R-loops is one of the hallmarks of ALT cancer. In addition, a recent study showed that R-loop formation is promoted by Rad51 recombinase, but it is antagonized by RNase H1. Despite the mounting evidence of TERRA’s critical role in telomere structure and function, our molecular understanding of TERRA is limited. Here, I propose to elucidate the molecular mechanism underlying TERRA recruitment and invasion into telomeres in the context of shelterin proteins and the extent to which TERRA regulates telomere length in cells. Aim 1 will probe TERRA-induced R-loop formation and the role of shelterin proteins. My preliminary results using single molecule (sm) FRET show that TERRA trans anneals to telomeric DNA, and unexpectedly, the resulting R-loop exhibits dynamic movement that is stabilized by TRF2. I also employed an sm-colocalization assay to measure the efficiency for trans annealing of TERRA to telomeric DNA. My results from this assay show that the presence of non-TERRA sequence, which represents the subtelomeric DNA, as well as the presence of G-quadruplex (G4) telomeric overhang significantly enhances TERRA binding to telomeric duplex. Aim 2 will elucidate the mechanism underlying TERRA recruitment and invasion by Rad51 and shelterin proteins. I present my preliminary results using an sm-colocalization assay which show that RAD51-TERRA complex invades telomere duplex more efficiently than TERRA alone. Aim 3 will measure telomere length as a function of TERRA levels in ALT- and telomerase dependent cancer cells. Additionally, this aim will study the role of TRF2, POT1, Rad51, RNaseH1 in TERRA regulation of telomeres in cells. Completion of these aims will provide a molecular mechanism underpinning TERRA function in telomere protection and length regulation and thereby help develop treatment for ALT positive cancers in which TERRA is upregulated.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Organisms use a variety of molecular mechanisms to adapt to their environments. RNA editing occurs widely across organisms and generates non-synonymous codon changes in mRNAs, thereby altering the amino acid sequence of proteins. In cephalopods and fungi, this ‘recoding’ generates incredible diversity in proteins across most cellular processes. However, the functions of RNA recoding in these organisms are largely unknown. How is RNA recoding used to support physiological needs and facilitate adaptation? The research proposed here investigates how cephalopod and fungal RNA recoding regulates the function of proteins involved in two core cellular processes: microtubule-based transport and DNA replication and repair. This work will illuminate how RNA recoding modulates protein function to support phenotypic plasticity and adaptation and will advance our understanding of the regulation and functions of highly conserved cellular machineries. In Aim 1, Dr. Rangan will investigate how RNA recoding diversifies the function of microtubule motor protein complexes. In the K99 phase, she will evaluate the effects of RNA recoding on dynein and kinesin motor complexes using in vivo cargo transport assays and single-molecule motility assays. She will also investigate how RNA recoding of motor proteins is coordinated at different temperatures in squid to facilitate transport. In Aim 2, Dr. Rangan will investigate how RNA recoding alters the function of DNA replication and repair proteins. In the K99 phase, she will characterize the effects of RNA recoding on DNA polymerases epsilon and zeta using assays for mutation rate, fidelity, and processivity. In the R00 phase, she will evaluate how temperature-dependent recoding of DNA polymerases alters function and extend this characterization to other proteins involved in DNA replication and repair. In Aim 3, Dr. Rangan will explore how RNA recoding of DNA replication machinery influences genomic mutation rate and bias in the filamentous fungus Neurospora crassa. In the K99 phase, she will use RNA-seq to evaluate temperature-dependent changes in RNA editing in Neurospora ascospores. During the R00 phase, she will perform mutation accumulation experiments with recoding site mutants and wild type fungi to elucidate the role of RNA recoding in mutagenesis. Dr. Rangan is committed to developing an independent research program centered around investigating how RNA editing in diverse organisms supports phenotypic plasticity and adaptation. To facilitate her transition to independence, she will attend diverse scientific conferences and participate in UCSD classes on topics of career development and lab management. She will receive guidance and support from her mentoring committee and her primary mentor, Sam Reck-Peterson. This development plan, combined with training in bioinformatics and computational genomics (with Ludmil Alexandrov, UC San Diego) as well as Neurospora biology and genetics (with Katherine Borkovich, UC Riverside) will prepare her for success in an independent career.

NIH Research Projects · FY 2025 · 2023-08

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two age-related neurodegenerative diseases that share genetic and pathological underpinnings. Recently, disruptions to the composition and function nuclear pore complex (NPC) have been identified as a prominent and early pathomechanism underlying neurodegenerative diseases including ALS, AD/FTD, and HD. As the NPC and surrounding nuclear envelope (NE) environment critically control fundamental cellular processes including nucleocytoplasmic transport (NCT), gene expression, and genome organization, NPC and NE homeostasis is essential for neuronal function and survival. Using induced pluripotent stem cell (iPSC) derived neurons (iPSNs) and postmortem human CNS tissues, we have recently identified a significant injury to the NPC characterized by the reduction in specific Nups from the human neuronal NPC that culminates in impaired nuclear protein import and contributes to the dysfunction and mislocalization of the RNA binding protein TDP- 43, a prominent pathological event in neurodegeneration. This NPC injury cascade is initiated by the aberrant nuclear accumulation of CHMP7 in sALS and C9orf72 ALS/FTD neurons. CHMP7 is an ESCRT-III protein that while normally localized predominantly to the cytoplasm, is thought to function primarily in ESCRT-III mediated nuclear surveillance events. The cell biological events and molecular mechanisms underlying CHMP7’s contribution to NPC and NE surveillance and homeostasis are beginning to be understood in yeast and non- neuronal mammalian cells during cell division, NE breakdown, and NPC insertion. However, little is understood regarding CHMP7’s role in maintaining NPC and NE homeostasis in long-lived, non-dividing human neurons where NPC components are infrequently exchanged, and NE breakdown does not routinely occur. Our preliminary data highlight the fact that the molecular events that regulate the nuclear localization and activation of CHMP7 mediated nuclear surveillance in neurons are not perfectly concordant with those recently identified in yeast and dividing mammalian cells suggestive of distinct neuronal mechanisms that give rise to CHMP7 initiated pathogenic cascades in ALS/FTD. In the proposed studies, we will employ iPSNs from a large number of sALS and C9orf72 ALS/FTD patients and appropriate controls and utilize a combination of candidate based siRNA knockdown, biochemical protein interaction techniques, recently developed and optimized live cell and super resolution imaging strategies, and expression of a newly generated CHMP7 variant plasmid library. Collectively, these studies will identify the molecular mechanisms that facilitate the nuclear entry (Aim 1) and nuclear export (Aim 2) of CHMP7 as well as the initiation of CHMP7 mediated NPC injury (Aim 3) to give rise to NPC injury cascades in ALS/FTD. Therefore, these studies will provide an essential understanding of the cell biological and molecular events that facilitate CHMP7 mediated NPC injury in human neurons and provide insights into therapeutic strategies for alleviating this early and significant pathogenic cascade in ALS/FTD.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Adolescents and young adults (AHIV) ages 12-30 years have disproportionately poorer outcomes across the HIV care continuum, including lower rates of adherence to oral ART (oART) and viral suppression (VS) than older adults, correlating with individual risk of poor health and disease progression and public health risk of secondary transmission. AHIV are a priority population in the Ending the HIV Epidemic in the United States Initiative (EHE). Unique multi-layered factors (e.g., cognitive development, psychosocial determinants, system barriers) underlie AHIV's nonadherence and VS; therefore, AHIV-specific interventions are needed to address those factors and ultimately improve VS. Toward this aim, our group has shown that AHIV are more likely to be retained in care and achieve and maintain VS if cared for at clinics with youth-friendly structures and services, more likely to achieve VS with single tablet regimens (STR) vs. multi-tablet oral ART (oART) regimens, and have high interest in ART strategies not taken orally, specifically long-acting injectable ART (LAI-ART). These findings underscore the importance of engaging AHIV in decision-making and providing access to alternative biomedical strategies that obviate daily adherence, like LAI-ART, should they choose. Though uptake and rollout has been slow, LAI-ART (cabotegravir/rilpivirine) administered q4-8 weeks has been approved for HIV treatment in those >12 years-old who have achieved VS—excluding up to 50% of all AHIV due to their decreased likelihood of VS. We have reported provider biases and disparities in ART initiation by patient age and have concern that AHIV may have decreased access to LAI-ART, resulting in a tiered system that precludes LAI-ART access from those who may want and benefit from it most. AHIV need biomedical and care delivery innovations to address their challenges meeting EHE goals by optimizing feasibility of delivering novel biomedical tools for AHIV through minimizing medication and healthcare system barriers. The central hypothesis of the Strategies to AchieVe Viral Suppression for Youth with HIV (SAVVY) Study is that informed choice, counseling on ART options, and facilitating access, will increase rates of achieving and sustaining VS among AHIV. The study proposes to present and solicit informed choice of ART among both AHIV with and without VS, including facilitating LAI-ART (if VS can be achieved). For AHIV who prefer LAI-ART, the SAVVY intervention will support AHIV in successfully meeting or maintaining criteria (VL<50 copies/mL) for LAI-ART consideration, and deploy a focused team that facilitates access to LAI-ART. Study outcomes include VS and health-related quality of life (HRQOL) for AHIV undergoing the SAVVY intervention, identification of implementation barriers utilizing an established implementation science framework, and assessment of the cost-effectiveness of SAVVY using an economic-epidemiologic model. Our interdisciplinary team has expertise to implement the SAVVY study and we anticipate that the results of The SAVVY study will inform the real-world implementation and optimization of LAI- ART AHIV toward reducing disparities in outcomes and ending the epidemic for the key population of AHIV.

NIH Research Projects · FY 2026 · 2023-07

African American prostate cancer patients are known to be diagnosed at an earlier age, present with aggressive disease, and are twice as likely to succumb to prostate cancer than other demographic groups. Despite a major focus on socioeconomic factors, recent findings strongly argue for the existence of biological factors driving cancer disparities. In my efforts to understand biological drivers in prostate cancer, I applied a novel DNA damage detection method, Repair Assisted Damage Detection (RADD), and established that African American tumors have more DNA lesions overall than EA tumors, especially uracil lesions. Our lab has previously demonstrated that the homocysteine-methionine pathway is a metabolic hallmark of African American prostate cancers, which fuels the progression of the folate cycle that is required for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). An upregulation of metabolites in the de novo pyrimidine biosynthesis pathway and altered levels of folate cycle metabolites were identified in African American prostate tumors, suggesting the link between uracil metabolism and uracil lesion accumulation. Uracil lesions are repaired by the base excision repair pathway. We have also shown that expression of XRCC1, a protein involved in coordinating base excision repair function, was lower in African American prostate tumors, indicative of defective base excision repair. These data collectively suggest that African American tumors exhibit the consequences of thymidylate stress, where the ability of thymidylate synthase (TYMS) to convert dUMP to dTMP is obstructed, resulting in dysregulation of base excision through the retention of uracil lesions. The increased amounts of uracil lesions in African American men could be a cumulative result of changes in nucleotide metabolism, which is caused by altered levels of components in the folate cycle and one-carbon metabolism. These components, such Vitamin B12 and folate, are known to be reduced in African American men, which could result from either dietary deficiencies or due to an aberrant homocysteine-methionine cycle, or both. While we understand the molecular consequences of reduced folate as it relates to uracil accumulation, the circumstances surrounding the DNA damage response through the regulation of Vitamin B12 and folate is currently unknown. The hypothesis of this proposal is that altered levels of Vitamin B12 and folate contribute to thymidylate stress, resulting in base excision repair pathway dysfunction and promoting prostate cancer progression in African American men. In this proposal, we will 1) determine the clinical relevance of homocysteine, Vitamin B12, and folate with prostate cancer progression in African American men and 2) elucidate the role of homocysteine, Vitamin B12, and folate regulation on TYMS function and base excision repair in African American prostate cancer patients. Successful completion of these aims will establish a link between metabolism and DNA repair in African American men with prostate cancer and help develop metabolic biomarkers to stratify these patients for DNA repair inhibitor therapies.

NIH Research Projects · FY 2026 · 2023-07

This project will elucidate the biological mechanisms by which obesity alters the myocardium in patients with heart failure and a preserved ejection fraction (HFpEF) to ultimately derive sorely needed precision- guided therapies. HFpEF currently represents more than half of all heart failure worldwide, its prevalence is rising, morbidity and mortality are substantial, and yet we still have very few effective therapies. It is a major unmet medical need and a disease priority for the NIH. One of the factors that has made it difficult to treat is a major transformation over the past two decades such that most patients are now obese, many severely, with diabetes and metabolic syndrome also very common. This obesity-metabolic syndrome (OMS-HFpEF) phenotype has altered disease manifestations and progression and worsened prognosis. Yet our knowledge of the underlying myocardial pathobiology effects from obesity are limited. Johns Hopkins has established a dedicated clinical HFpEF Center that obtains detailed phenotyping of HFpEF patients, the majority being OMS-HFpEF, African American, and female. The phenotyping includes obtaining myocardial biopsies that have already provided novel insights into cellular and molecular features. Our recent studies revealed less fibrosis than predicted, and that fat metabolism and glucose metabolism seem both to be depressed in OMS-HFpEF, pairing abnormalities found in HF with reduced EF and obesity/diabetes respectively, in essence a worst of both worlds that limits fuel flexibility. Yet unlike HFrEF, oxidative phosphorylation seems enhanced particularly in obese patients. We also find a strong inverse correlation between obesity and calcium-stimulated myofilament function – being very depressed in OMS-HFpEF ± diabetes, hypertension, or LV hypertrophy. Obesity is thus a major driver for fundamental changes in HFpEF. This R35 Program dissects metabolic and sarcomeric dysregulation in OMS-HFpEF, starting with analysis of human myocardial tissues, and testing abnormalities in animal models that have both marked OMS and cardiac hemodynamic stress. Models are benchmarked to pair with human molecular/cellular pathobiology, rather than only organ level physiology as historically done. Our metabolic studies will determine the fuel substrates used by OMS-HFpEF heart, where bottlenecks in fuel metabolism occur, how these maybe circumvented and what the impact is, which metabolites are formed that can impact epigenetics (histone modifications) to alter gene programs controlling metabolism and other key cellular functions. Major interest is on Krebs cycle intermediates such as citrate, succinate and fumarate and polyamines, that can impact histone methylation and acetylation. Our myocyte studies will determine how obesity depresses sarcomere function, find the protein(s) and structural changes involved, their causes, and prove causality. Lastly, we will test therapies to improve metabolic flexibility and sarcomere performance in OMS-HFpEF that can lead to precision-guided medicines for this common phenotype.

NIH Research Projects · FY 2025 · 2023-07

Active surveillance (AS) is the preferred management option for low risk prostate cancer (PCa) patients who would benefit from conservative treatment. However, due to the lack of reliable methods in the initial clinical evaluation to identify true low-risk PCa patients for AS enrollment and during AS monitoring to detect a rising risk of progression, patients who could benefit from conservative management through AS are often over-treated, yet at the same time patients initially chosen for AS with a missed high-risk disease are under-treated. The goal of the proposed EDRN Biomarker Characterization Center (BCC) is to develop and validate in vitro diagnostic multivariate index assays (IVDMIA) that combine a panel of biomarkers into a single-valued numerical index with the intended use for the clinical unmet needs for 1) assisting in the preoperative assessment of PCa aggressiveness and decision for enrollment into AS; and 2) detecting a rising risk of progression during AS to triage patients for additional and possibly more invasive procedures for needed disease reclassification. The objective for the IVDMIA development is to improve specificity while maintaining a high negative predictive value in order to safely enroll more patients with true low-risk PCa into AS and reduce the number of unnecessary biopsies and or costly workup procedures for patients in AS. To achieve this goal, we propose an integrated BCC at the JHU consisting of a multi-disciplinary team including PIs from current EDRN BDL (Dr. Hui Zhang) and BRL (Dr. Daniel W. Chan), and a previous CVC (Dr. Alan Partin). The targeted population is JHU AS patients with >20 years of enrollment and clinical follow-up. Our team has many years of experience in biomarker discovery, verification, validation, and translation into clinical diagnostics and the development of IVDMIA, e.g. OVA1, the 1st proteomics IVDMIA cleared by the FDA (2009). We plan to take advantage of the serum biomarkers already discovered for aggressive PCa from our current BDL and BRL and begin the verification and validation in the targeted AS population by our BRL. In parallel, our BDL will focus on the discovery of new candidate serum, urine and tissue biomarkers by applying cutting edge technologies to the AS population, such as mass spectrometry based high throughput proteomics, protein modifications, and single cell analysis of laser- capture-microdissected tissues. We plan to combine these biomarkers into IVDMIAs. Finally, we will work with our industry partners to translate these IVDMIAs into CLIA certified and/or FDA cleared/approved clinical diagnostics. We believe with these innovative, yet, practical approaches, our BCC offers the best opportunity to make significant contributions to the EDRN network and address the critical clinical unmet needs for PCa patients. If the over-treatment, under-treatment, decrease in unnecessary biopsies, and increase in biopsy accuracy can be successfully addressed, the morbidities associated with PCa diagnosis and treatment can be significantly decreased, while enhancing the detection and treatment of clinically significant PCa. In addition, our BRL, a CLIA and CAP certified clinical laboratory at JHU, will serve as a resource center for the EDRN network.

NIH Research Projects · FY 2025 · 2023-07

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with an overall 5-year survival of 11%. Due to its asymptomatic nature and lack of methods for early detection, the majority of PDAC patients (> 85%) present with non-localized tumors. This highlights the need to detect PDAC at an earlier, localized stage. Intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN) offer a unique opportunity to identify premalignant lesions to serve as targets for early detection strategies. Dr. Randall Brand and others proposed a two-step surveillance approach for early detection of PDAC: 1) identification of high-risk populations through clinical evaluation with an elevated PDAC prevalence close to or above 1% and 2) development of serum biomarker(s), for repeated testing at intervals to detect subjects in surveillance with a rising risk of PDAC (prevalence ~ 10%) for additional imaging. The goal of this proposal is to identify serum biomarkers and develop in vitro diagnostic multivariate index assays (IVDMIAs) and incorporate them into an “early detection through surveillance” workflow for the detection of early-stage PDAC and its precursor lesions. The intended use of these IVDMIAs are 1) to assist in the clinical evaluation of high-risk subjects to be included in surveillance for the early detection of PDAC, and 2) to detect rising risk of PDAC or high-risk IPMN in the longitudinal evaluation of subjects in surveillance. The project has five specific aims: 1. To discover and develop serum-based PDAC early detection biomarkers through integrated proteomic analysis of serum/tissue samples from early-stage PDAC, IPMN, and benign and healthy controls using a multimodal and phased approach with corroborative supporting evidence from tissue-based proteomic analysis and immunohistochemical (IHC) verification. 2. To use a by-design approach driven by predefined intended uses that are both clinically meaningful and practically feasible to develop and evaluate serum biomarkers for IVDMIAs. 3. To develop and optimize multiplex analytical assays for selected biomarkers and apply them to generate high-quality biomarker data for IVDMIA development and clinical evaluation. 4. To collect and assemble large clinical specimen sample sets for both IVDMIA algorithm development and independent validation. 5. To participate in collaborative activities with other PCDC-RUs. To be successful, the proposed project requires a multi-disciplinary, systems approach and the support of critical technology, data science, and clinical specimen resources. Our team is a unique ensemble of experts in PDAC and IPMN pathology for early detection, clinical chemistry/assay development, clinical proteogenomics, and statistical/machine learning for IVDMIA development. Most importantly, the team members individually and collectively all have a long-standing history of active research with accomplishments in biomarker development and translation into clinical tests, including the first proteomic IVDMIA test cleared by the FDA. We believe with these innovative yet practical approaches, our RU offers the best opportunity to make significant contributions to the PCDC network and address critical clinical unmet needs.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Dr. Jonathan Webster is an Assistant Professor of Oncology in the Division of Hematologic Malignancies at The Johns Hopkins University School of Medicine. He is a member of the Leukemia Group and has completed the Science of Clinical Investigation curriculum at the Johns Hopkins Bloomberg School of Public Health. His primary mentor, Dr. Richard Jones, is a Professor of Oncology and the Director of the Bone Marrow Transplantation Program. His co-mentor, Dr. Ravi Varadhan, is a Professor of Oncology in the Division of Biostatistics and Bioinformatics. His advisory committee includes Drs. Gojo and Smith, faculty experts in leukemia clinical trials, and Dr. Luznik, a laboratory-based expert in allogeneic blood or marrow transplantation (alloBMT) and immunology. Support from the K08 award will enable Dr. Webster to gain additional research skills, receive mentorship in authoring publications, develop grants, and perform his research project. Dr. Webster's goal is to become an independent investigator and leader in the emerging field of post-alloBMT therapies. The use of nonmyeloablative conditioning (NMAC) coupled with improvements in supportive care and graft-versus-host disease (GVHD) prophylaxis, such as high-dose post-transplantation cyclophosphamide (PTCy), have led disease relapse to overtake transplant-related mortality as the major cause of treatment failure following alloBMT for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Two factors play a particularly important role in post-alloBMT relapse: peri-transplant measurable residual disease (MRD), and the ability of donor T lymphocytes to generate a graft-versus-leukemia (GVL) effect. The prophylactic post-transplant use of targeted therapies reduces relapses in high risk leukemias, but most patients lack targetable mutations. In this application, Dr. Webster proposes to examine a more broadly applicable approach using the bispecific antibodies blinatumomab and flotetuzumab as post-alloBMT maintenance therapy in ALL and AML, respectively. Dr. Webster has significant preliminary data demonstrating the safety of blinatumomab in this setting and the ease with which post-transplant maintenance therapies can be given following PTCy. The overarching goal of this proposal is to decrease relapse following alloBMT in ALL and AML. He will achieve this by: 1. Conducting a clinical trial of blinatumomab as post-transplant maintenance to assess safety and relapse-free survival. 2. Conducting a clinical trial of flotetuzumab in post-transplant patients to assess safety. 3. Assessing the impact of post-transplant maintenance therapies on the activation and expansion of T lymphocytes, T cell receptor (TCR) diversity, T cell gene expression, and the depletion of cells expressing the target antigens (CD19 and CD123). These studies will inform the development of randomized trials of post-transplant maintenance therapies at the cooperative group level. Data regarding the efficacy of post-alloBMT maintenance therapies in patients with peri-transplant MRD will inform future patient selection, while the immunologic correlates may reveal biomarkers of response.

NIH Research Projects · FY 2025 · 2023-07

Modified Project Summary/Abstract Section The failure to engage specific and appropriate metabolic programs can impair or alter T cells, and thus lead to ineffective, or even overexuberant, immune responses. Central to metabolism are mitochondria, which serve as central hubs of energy generation and biosynthetic activity. Work from our group and others has provided mechanistic insights into how mitochondrial metabolism is critical to T cell differentiation and function. However, knowledge about why these organelles change shape to maintain metabolism, biosynthetic capacity, and function, and in what ways mitochondrial remodeling influences T cell activation, differentiation, or effector molecule expression is lacking. Investigating how dynamic changes in mitochondria regulate metabolism to impact CD4+ T cells will enhance our fundamental understanding of immunobiology and of how to manipulate these cells for disease therapy in the context infection, autoimmunity, and inflammatory diseases. In our initial experiments we found that unlike other Th cell subsets, Th17 cells, a cell type necessary for maintaining gut homeostasis and implicated in certain types of autoimmunity and inflammation, had elongated mitochondria in a fused network, as well as tight cristae morphology. These results suggested a differential role for mitochondrial fusion and the protein OPA1, which mediates both membrane fusion and cristae morphology, in these cells. OPA1 deletion had no discernible effect on Th1 and Th2 cell differentiation or cytokine production in vitro, and while Th17 cell differentiation was also unimpaired, IL-17 expression was drastically reduced. Further, mice with a T cell-specific OPA1 deletion were resistant to developing pathology in experimental autoimmune encephalomyelitis (EAE), a Th17 cell-mediated autoimmune disease of the central nervous system. Finally, our data revealed a role for liver kinase B1 (LKB1) in regulating the cellular response to OPA1-deficiency, and in restraining Th17 cell IL-17 expression when mitochondrial fusion is perturbed. In these initial studies we also observed that Tregs had mitochondria that appeared more elongated, making them look more like Th17 cells than other Th subsets. We now propose to investigate the role of mitochondrial dynamics in Treg cells, in addition ot Th17 cells, by using mouse models of colitis and graft-versus-host disease (GVHD). The balance of Treg and Th17 cells is known to be a critical factor in controlling autoimmunity and inflammation. In this proposal we aim to understand how mitochondrial dynamics influence the function of these diverse CD4+ T cell subsets. Our overall goal is to dissect the function of OPA1 in Th17 cell and Treg cell function, and to determine how MM fusion interfaces with LKB1 signaling to modulate cellular metabolism to limit IL-17 expression in settings of mitochondrial disruption or stress in vitro and in vivo, and if and how this may impact Treg suppressive capacity. We propose to 1) explore the role of mitochondrial dynamics in CD4+ T cells, 2) determine the extent to which LKB1 controls the response to OPA1-deficiency in T cells, and 3) investigate how metabolic changes upon disrupting mitochondrial fusion influence Th17 and Treg cell function.

NIH Research Projects · FY 2025 · 2023-07

Infertility effects around 15% of couples that are of reproductive age worldwide. Issues relating to infertility often arise due to errors in the process of meiosis, the specialized cell division where a single diploid cell gives rise to four haploid gametes. The general sequence of events in meiosis includes one round of DNA replication, the separation of homologous chromosomes (meiosis I), and then the separation of sister chromatids (meiosis II). During the process of chromosome segregation, the cell organizes bipolar spindles to evenly divide the genetic material. Like most mitotically dividing cells, spermatocytes rely on the centrosome to be the microtubule organizing center responsible for the formation of bipolar spindles. The centrosome consists of two centrioles and associated proteins known as pericentriolar material (PCM). Centrioles are cylindrical organelles that exist in pairs, one centriole oriented perpendicular to the other. The PCM that surrounds them consists of ordered, dynamic proteins that are necessary for cellular functions such as protein degradation, organelle trafficking, and spindle assembly. The PCM also acts as the spindle organizing center by concentrating the γ-tubulin that serves as nucleation sites for assembling the microtubules necessary for bipolar spindle formation and chromosome segregation. During cellular division, centriole duplication occurs. Polo-like kinase 4 (PLK4) is responsible for this duplication event in mitosis. PLK4 is regulated through autophosphorylation and proteasomal degradation to ensure centriole duplication only occurs once during the cell cycle. However, in meiosis the cell must undergo centriole duplication twice and centriole duplication is no longer coupled with DNA synthesis, but rather takes place during prophase I and interkinesis. Furthermore, PLK4 protein levels remain high during spermatogenesis and its activity is likely regulated by a novel mechanism. Because of this added complexity, the role of PLK4 in relationship to centriole duplication during meiosis is not yet understood. To determine the role of PLK4 in meiosis, conditional knockout (cKO) and overexpression (OE) mouse models, which allow for depletion or upregulation of PLK4 in early prophase spermatocytes, are utilized. In the Plk4 cKO model, due to the lack of functional PLK4, centrioles fail to duplicate, and the spermatocytes enter meiosis I with a single centrosome. In contrast, centrioles are over duplicated in the Plk4 OE model. It is our goal to utilize these models to further characterize the role of PLK4 as well as elucidate other regulatory mechanisms and components required for centrosome biogenesis during mammalian spermatogenesis. As centrosome biogenesis is critical for chromosome segregation during spermatogenesis, and centrioles are required for sperm flagella formation, our studies will contribute to understanding causes of infertility and gamete aneuploidy. We will address the following aims in this proposal: Aim 1: Determine novel aspects of bipolar spindle formation during mammalian spermatogenesis Aim 2: Explore the regulatory mechanisms required for centriole duplication during mammalian spermatogenesis

NIH Research Projects · FY 2025 · 2023-07

Project Summary Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer. Our groups have pioneered liquid biopsy approaches for detection and characterization of cancer. Recently we have developed a genome-wide approach for analysis of cfDNA fragmentation profiles called DELFI, DNA evaluation of fragments for early interception. We demonstrated that fragmentation profiles of healthy individuals from low coverage whole genome sequencing reflect nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. Through the analysis of cell-free DNA fragmentation patterns, we identified patients with localized cancer and this tool of early detection could result in better patient outcomes. Lung cancer is the most lethal cancer in the world, and its incidence continues to increase worldwide. There is an urgent, unmet clinical need for development of noninvasive approaches to improve cancer screening for high-risk individuals and ultimately the general population. A clear understanding of molecular changes along the pathway of lung tumorigenesis is critical for identifying biomarkers related to carcinogenesis and tumor progression. Biomarker development for early detection of lung cancer has broad clinical applications in screening as well as for distinguishing malignant from benign pulmonary nodules. Tools to better predict the fate of early lesions non-invasively would be invaluable for early detection of lung cancer, when curative approaches are more likely to succeed. Unlike targeted deep sequencing approaches that would be cost prohibitive for broad use in a screening population, our approach is affordable, highly scalable, and may lead to more effective strategies for clinical intervention. The recent intersection of cancer genomics with novel noninvasive blood tests could revolutionize cancer screening. The purpose of our proposed research is to study the origins and molecular characteristics of cell-free DNA fragments along the progression of Lung Cancer, profiling these alterations in preneoplastic lung lesions likely to progress to invasive cancer, and in treatable lung tumors, as well as in normal controls and in benign lesions. We aim to implement new features to further optimize our DELFI molecular test in plasma. The proposed plan is to test and validate our approach in both accrued samples and a prospective lung cancer screening population. Ultimately, this approach already shows great promise as a pan-cancer early detection strategy and we intend to expand our research in this direction in collaboration with other EDRN centers. We envision that these analyses will be rapidly translated into the clinical setting, providing new noninvasive approaches for early cancer detection.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Up to 1 million Americans experience acute respiratory failure (ARF) and require mechanical ventilation in an intensive care unit annually. Studies repeatedly revealed incomplete penetration of proven-effective, sometimes life-saving, evidence-based practices (EBP) for these patients, and it is unclear how to select optimal implementation strategies that can bridge the gap between evidence and practice. Common approaches to selection have inherent limitations. For example, concept mapping and implementation mapping rely heavily on stakeholder perspectives, are labor-intensive, and may focus on stakeholder preferences instead of strategies with the greatest potential impact. Quantitative approaches are also challenging because important determinants of practice - such as individual motivation and organizational culture - are difficult to measure at scale. One important goal of implementation is to reduce variability in the uptake of EBPs attributable to clinicians and the environmental setting. While clinical practice should vary in response to patient factors and preferences, implementation programs try to overcome clinician and environmental factors (e.g. insufficient knowledge or resources) that limit EBP uptake. Applying the Consolidated Framework for Implementation Research (CFIR) to this conceptual model, the domains of Individuals and Inner Setting should have minimal influence on adherence to EBPs after a successful critical care implementation program. We hypothesize that variability attributable to the CFIR domains of Individuals and Inner Setting is lower among patients when a treatment is supported by high-quality evidence compared to patients for whom the existing evidence for a treatment is weaker. Our overall objective is to demonstrate 1) how established multilevel modeling techniques can be used to estimate the proportion of variation in the use of EBPs that is attributable to the CFIR domains of Inner Setting and Characteristics of Individuals, and 2) how the resulting information can inform selection of implementation strategies and evaluate their effectiveness. As a proof of concept, we will study two proveneffective interventions - low tidal volume ventilation for acute respiratory distress syndrome and bag mask ventilation during intubation. We will use existing multicenter datasets from the Low Tidal Volume Universal Support: Feasibility of Recruitment for lnterventional Trial (LOTUS-FRUIT) cohort study and from 3 randomized trials that collected data on the use of bag-mask ventilation.

NIH Research Projects · FY 2025 · 2023-07

HIV treatment and prevention are essential to reducing healthcare-related expenditures and promoting and the health of the US population. Although comprising approximately 9.5% of the US population, Latino men account for more than a quarter of all new HIV diagnoses in the United States. People living with HIV (PLWH) are at risk for exposure to interpersonal trauma (IT) and among Latinos living with HIV, male immigrants face increased risk of IT related to their HIV status and immigration journeys. IT has been shown to adversely impact victims’ biopsychosocial and emotional health and has been associated with poor health outcomes such as mental illness and poor engagement in care (EIC) among people living with HIV (PLWH). Latino men are disproportionately impacted by HIV, and EIC-related outcomes such as retention in care among Latinos are well below US and international goals. Latino PLWH also have low engagement in mental health care and are at risk for low emotional support (ES)—a protective resource shown to buffer the negative psychosocial, emotional, and behavioral impacts of IT. Unfortunately, little is known about the impact of IT on EIC among Latino immigrant men living with HIV (LIMWH) and the protective psychosocial resources that influence EIC among LIMWH in the context of IT. To better understand these relationships among LIMWH and inform HIV prevention and treatment, this study will utilize the recent Patient Health Engagement (PHE) model describing psychological and emotional processes involved in EIC. The purpose of this exploratory sequential mixed methods study is to generate a rich understanding of the experiences of LIMWH with EIC and IT and the psychosocial resources that HIV care teams can promote to improve EIC and psychosocial well-being among LIMWH by addressing the following specific aims: Aim 1a: Understand the experiences of LIMWH (n=20) with IT and the psychosocial and emotional influences that impact their EIC. Aim 1b: Understand the perspectives of HIV care team members (n=15) caring for LIMWH regarding the impact of IT and psychosocial and emotional influences on EIC in this population. Aim: 2: Examine the association between exposure to IT on the continuum of EIC among LIMWH (n=100) while controlling for individual and interpersonal covariates. Aim 3: Evaluate the association between ES and the continuum of EIC among LIMWH while controlling for individual and interpersonal covariates. The proposed study will use preliminary results from semi-structured qualitative interviews with LIMWH and HIV care providers from the Baltimore-Washington metro area to inform a quantitative survey assessing the associations between IT, EIC and ES. Reflexive Thematic Analysis (RTA) will be used to analyze qualitative interview data and identify themes and key psychosocial variables to be included in the survey and subsequent analysis using multivariable logistic regression models, model testing, and subgroup analysis. This iterative approach incorporating the perspectives of LIMWH and HIV providers will strengthen the validity of the findings and inform future research and psychosocial interventions among LIMWH to promote optimal EIC, psychosocial wellbeing, and progress towards Ending the HIV Epidemic.