University Of Tx Md Anderson Can Ctr

NIH Research Projects · FY 2025 · 2024-09

Advances in single-cell technologies have enabled three-dimensional (3D) genome structure profiling and simultaneous capture of the transcriptome and epigenome within a cell. Quantitative tools are, however, still unable to fully leverage the unprecedented resolution of single-cell high-throughput chromatin conformation (scHi-C) data and integrate it with other single-cell modalities. To address this challenge, I propose to (1) Develop a single-cell gene-body associating domain (scGAD) scoring system to explore single-cell 3D genomics data in units of genes. (2) Construct machine learning-based models to impute histone modification and 3D chromatin interaction for simultaneously profiling of each cell's epigenomic features and 3D chromatin architectures. Subsequently, I will develop an epigenomic regulatory score (ERS) model to infer the cell-type-specific promoter-enhancer regulation programs at the highest singlecell and single-gene resolution. (3) Validate and extend scGAD and ERS pipeline to CAR-T immunotherapy study to gain insights into the impact of distal gene regulation variations on patient responses. In Aim 1, preliminary analysis on human and mouse brain tissues demonstrated that scGAD extracts gene features agreeing well with the scRNA-seq data from the same system. As a result, scGAD facilitates the projection of cells from 3D genomics data onto reference panels constructed by scRNA-seq embeddings with known cell-type annotations. Hence, scGAD provides an unprecedentedly accessible and accurate cell type annotation method based on 3D chromatin architectures. Furthermore, the successful integration of cells from different modalities into the same network facilitates information sharing across 3D chromatin structures, the transcriptome, and the epigenome. Aim 2 leverages such multi-modal networks to build an ERS model. ERS jointly models the histone profiles at the promoter and distal neighborhoods of the target gene and the 3D spatial proximity between them. Therefore, the ERS scores quantify the regulatory effects of distal elements on a per gene and cell basis. Aim 3 will extend the integration framework in Aim 1 and 2 using scRNA-seq as a multi-modality bridge to CITE-seq data for a deeper annotation, especially for the Peripheral Blood Mononuclear Cells. This enables the in-depth investigation of the apheresis samples from the Acute Lymphoma Leukemia patients to gain insight into the roles of distal regulatory elements on gene expression and their impact on the CAR-T cell response.

NIH Research Projects · FY 2025 · 2024-09

Adolescent and young adult cancer survivors (AYACSs) face multiple challenges, including impaired psychosocial health, isolation, limited access to psychological services, and unmet needs for psychosocial support. Despite these challenges, there is a lack of interventions to mitigate these challenges and improve the quality of life (QOL) of AYACSs. Furthermore, previous studies among AYACSs have often overlooked the range of developmental stages and preferences concerning psychosocial care. While expressive writing interventions have demonstrated positive effects on various health outcomes among older adult cancer survivors and healthy young adults, they have primarily been implemented as individual-based approaches. One innovative idea to adapt such interventions for AYACSs is to combine individual-based writing with social support from a group of peers experiencing similar challenges. Thus, this proposed study aims to develop and evaluate a virtual, group-based expressive writing intervention, grounded in a conceptual framework tailored to the unique developmental needs of AYACSs and incorporating their communication preferences. The overarching goal is to enhance the QOL and reduce stress of AYACSs. Aim 1(K99) is to develop a virtual, group-based expressive writing intervention and assess the feasibility and acceptability. To promote group cohesion, group composition preferences (i.e., age-similar or general groups) will be assessed before the intervention. Aim 2 (K99) is to refine and optimize the intervention based on quantitative and qualitative data obtained from Aim 1, in collaboration with stakeholders such as the MD Anderson Young Adult Advisory Council, AYA clinic providers, and AYA organizations, as well as the PI’s mentorship team. Finally, Aim 3 (R00) is to assess the effects of the intervention on QOL, perceived stress, and stress biomarkers compared to a control group. If successful, this intervention will be ready for widespread expansion. The PI’s long-term career and research goal is to become an independent oncology nurse scientist in the areas of cancer prevention and control, developing and evaluating novel psychosocial interventions to improve QOL among AYACSs. To achieve this goal, the PI seeks specialized training in various areas, such as psychosocial and behavioral interventions, understanding the developmental needs of AYACSs, proficiency in quantitative, qualitative, mixed-methods research, and stress biomarkers, and professional development. The proposed training areas will be obtained through hands-on training with mentors, taking formal courses, and active participation in conferences and institutional trainings. In summary, the proposed research plan and training will enable the PI to transition to independence by providing the necessary skills and preliminary data to successfully compete for future R01-level grants.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Esophageal squamous cell carcinoma (ESCC) is a lethal cancer with a 10%-25% 5-year survival rate. Given the importance of early detection, the initiation process of ESCC must be better defined. To study this process, we focused on preneoplastic cells, which have the potential to progress to neoplastic cells. We comprehensively analyzed single-cell RNA-sequencing (scRNA-seq) datasets from human ESCC and healthy donors, as well as normal adjacent to cancer datasets, to reveal the spatial characteristics of preneoplastic cells. We also analyzed scRNA-seq data from a mouse model of carcinogen-induced ESCC to identify features of preneoplastic cells in a temporal context. Strikingly, molecular analysis revealed that preneoplastic cells, though histologically normal, exhibited elevated copy number variations and reduced TP53 and CDKN2A signaling compared to healthy normal samples in both spatial and temporal datasets. The CELF2 gene emerged as a robust marker of preneoplastic cells across both datasets, validated by an increased presence of CELF2+ cells in Trp53 and Cdkn2a double knockout (PC) organoids. These CELF2+ cells showed increased plasticity, acquiring cancer stem cell features and undergoing epithelial-mesenchymal plasticity (EMP). Aim 1 is to determine the cellular reprogramming in CELF2+ cells when TP53 and CDKN2A are ablated. The reprogrammed stemness will be validated by comparing the tumorigenicity of CELF2+ and CELF2- cells of PC organoids. Additionally, tumorigenicity of a genetically engineered mouse model with Trp53 and Cdkn2a deletion will be tested in the presence or absence of CELF2+ cells. Cellular reprogramming will also be evaluated by comparing the exact cell lineage of wild-type and PC organoids and PC-derived tumors. Employing cutting-edge genetic barcoding, we will trace the entire cellular trajectory. Cell lineage established in Aim 1 will be the most reliable reference for preneoplastic cells and normal esophageal cells. Furthermore, cell lineage and population analyses of PC-derived tumors will elucidate the mechanism of clonal selection during tumorigenesis. Aim 2 will address the role of EMP-undergone CELF2+ cells in immune evasion. Although EMP is conventionally known for its function in invasion and metastasis, its role in immune evasion is less studied. Our preliminary experiment identified mesenchymal-CELF2+ cells in the tumor niche release chemokines and cytokines. Aim 2 will disclose the efficiency of tumorigenicity when mesenchymal-CELF2+ cells are co-cultured with immune-rejected cells. In addition, the candidate chemokine and cytokine genes will be manipulated in preneoplastic cells to test our working model. Collectively, the proposed study will elucidate the cellular and genetic mechanisms of ESCC initiation. Under the guidance of Dr. Jae-Il Park, a mentor, Dr. Ko's research skillset, such as mouse genetics, organoids, and cancer signaling, as well as the skills of writing, teaching, and mentoring, will be expanded during the K99 phase. Completing the research and training will significantly facilitate Dr. Ko’s transition to an independent investigator.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Poorly defined fitness features enable a subset of cancer cells to survive therapy, ultimately giving rise to treatment-refractory tumors. The expanded use of sequencing tools have led to the recognition that refractory tumors often lack a genetic mechanism of resistance, and instead co-opt a regenerative program characterized by activation of developmental, inflammatory, mesenchymal and stem cell features. Importantly, these regenerative states are conserved across diverse cancers of epithelial origin, and are potentially reversible. Our preliminary data indicate that therapy resistance in colorectal cancer (CRC) is fueled by preexisting cells via a YAP-driven regenerative program that is strikingly similar to the consensus molecular subtype 4 of CRC, a poor risk phenotype often observed in treatment-refractory tumors. Our central hypothesis is that a preexisting population drives regenerative reprogramming and tumor escape through epigenetic adaptations that can be targeted therapeutically. First we propose characterizing the preexisting (pre-resistant) state using barcode lineage tracing and single-cell technologies. Using patient-derived organoids (PDOs) to isolate the founding clones of resistance, we will study the contribution of preexisting versus actively gained fitness features through whole-exome (WES) and RNA sequencing (RNA-seq). Using pre-clinical models of resistance to KRAS-inhibition (KRASi), we will define the role of genetic and non-genetic drivers of tumor escape. To develop approaches that abrogate regenerative reprogramming, we will perform a focused shRNA screen targeting 50 chromatin regulators. To expedite clinical translation, we will test readily available compounds targeting the top hits of the screen, in combination with standard therapies. We believe that our innovative tools will maximize our opportunity to develop treatment approaches with immediate clinical impact. During the award period, the candidate will conduct research at Weill Cornell Medicine (WCM) and Memorial Sloan Kettering (MSK) under the mentorship of Dr. Lukas Dow and an advisory committee. The candidate will commit at least 9 person-months of his professional effort to the research and career development activities outlined here. With his mentor and advisory committee, the candidate has designed a 5-year plan aimed at expanding his knowledge and expertise in cancer research, including single cell technologies, computational biology, genetic engineering, and functional screens. The goal of the career plan is to launch an independent career as a laboratory investigator focused on therapy resistance and biomarkers of response in CRC.

NIH Research Projects · FY 2024 · 2024-09

Project Summary: Broad Impact: Diabetic peripheral neuropathy is a condition that affects millions of people worldwide. Here we propose a set of experiments that will determine the role of insulin dysregulation and tau phosphorylation by GSK3β in T1DM and diabetic peripheral neuropathy. We believe these experiments will lead to a breakthrough in our understanding of diabetic neuropathy that will impact development of new therapeutics for years to come. Specific aims: Based on previous publications and our preliminary data, we hypothesize that insulin deficiency in the STZ model will upregulate the activity of GSK3β and drive up the level of phosphorylated tau within the neurons of the sciatic nerve resulting in neural dysfunction, IENF loss and mechanical and thermal hypersensitivity. To test this hypothesis, we will first identify increases in tau phosphorylation and dysregulation of the insulin signaling pathway using high content microscopy and western blot techniques. Then, we will confirm the role of GSK3β in tau phosphorylation with a GSK3β specific inhibitor and TRPV1Cre x GSK3βFL mice. Last we will develop new in vivo and in vitro modeling systems using AAV vectors that can be used to examine the effects of GSK3β activation in the absence of the insulin dysregulation that accompany T1DM. Career development and goals: In order to provide further growth of Dr. James Nichols’ career, the K99 phase of this award will be focused on 1) additional training in specific research techniques (High Content Microscopy, AAV vector delivery and Cre/lox systems, etc.); 2) professional development through mentorship, scientific writing, grant writing and attendance of scientific meetings; and 3) education in the field of peripheral neuropathies through quarterly meetings between Dr. Nichols and his mentorship team, daily mentoring by Dr. Andrew Shepherd, and collaborative opportunities with other PIs in the Neuroimmunology Lab. Career goals: Dr. Nichols’ long term career goal is to become a professor at a college of veterinary medicine or a college of medicine, so he can continue his research in the field of neuropathic pain, continue to encourage medical students who wish to follow a career in research, and participate in the education of the next generations of medical students. Environment: The Laboratories of Neuroimmunology within the Department of Symptom Research at MD Anderson Cancer Center is comprised of over 40 scientists at various levels of their career ranging from Professors to student volunteers. This environment is perfect for collaborations with other labs and provides access to a plethora of core facilities to which the candidate has ready access. During the R00 phase of this award Dr. Nichols will seek a similarly equipped environment to continue his studies in neuropathic pain, and the development of therapies for DPN.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY This application aims to address the urgent need for precision in intercepting hereditary diffuse gastric cancer (HDGC) associated with pathogenic germline CDH1 mutations, elevating lifetime cancer risk up to 80% for developing advanced diffuse gastric cancer (AGC), particularly at a young age. HDGC patients pose unique challenges in cancer interception and risk reduction due to the aggressive nature of AGC, which is associated with a poor prognosis. Consequently, those with pathogenic CDH1 mutation are advised to undergo prophylactic total gastrectomies (PTG), However, the incidence of AGC in CDH1 carries varies significantly, with only 25-45% of mutation carries develop AGC while many patients remain negative during surveillance (normal-appearing mucosa, N) or stable at the T1a stage (intramucosal stage which is considered as the “precancerous” lesion of AGC in the context of HDGC) without progression to AGC in years or even lifetime. These facts indicate that PTG, a major operation associated with chronic physical and psychosocial disabilities, may not be necessary for all CDH1 mutation carries, however, previous attempts to stratify risk based on specific variants or family cancer history have not been successful and tools to identify patients at high risk of AGC development, potentially requiring PTG, are currently lacking. This is largely due to our limited understanding of the earliest events that drive T1a lesion development and progression. To fill this void, we assembled the Center for Gastric Pre-Cancer Atlas of Multidimensional Evolution in 3D (GAME3D) that is composed of scientists who work closely together bridging multiple disciplines, have longstanding expertise in pathobiology of HDGC, and who developed innovative workflows for multimodal characterization and modeling of the N-T1a-AGC pathologic continuum in CDH1 germline mutation carriers. The overall objective of our GAME3D Research Center is to develop a multimodal, single-cell, dynamic, and 3D spatial atlas of normal-appearing mucosa, T1a lesion, and AGC through longitudinal samples from CDH1 mutation carriers that will inform of transition of at-risk mucosa to T1a lesions and then to AGC. Aim 1 will build a multimodal, dynamic, 2D spatial atlas that maps the transition of T1a lesion from at-risk mucosa and its subsequent evolution to AGC. Aim 2 will employ revolutionary technical and computational approaches and build a spatiotemporal three-dimensional (3D) atlas at single-cell resolution that captures the N-T1a-AGC pathologic continuum. Aim 3 will leverage complete use cases and 2D and 3D atlases to build computational models of tumor initiation in normal-appearing gastric mucosa, pathogenesis and progression of T1a lesions, and, ultimately, the N-T1a-AGC pathologic continuum. Our atlas is of high-value to the current HTAN repertoire as it will provide an exceptional view into the single-cell spatial landscape of the N- T1a-AGC pathologic continuum, thereby generating an invaluable resource for HTAN and the field to understand the earliest phases in human AGC development in high-risk CDH1 mutation carriers and thus identify targets that are ideal to intercept this trajectory.

NIH Research Projects · FY 2025 · 2024-09

PROJECT ABSTRACT Pancreatic adenocarcinoma (PDAC) is an almost uniformly fatal disease most often diagnosed after the development of metastases. The tumor suppressor gene, TP53, is the most commonly altered gene in human cancer and has been recognized as a genetic driver of PDAC in up to 75% of patients. While the functions of TP53 mutations within cancer cells continue to be studied, knowledge of its non-cell autonomous roles in the primary tumor microenvironment remain limited. The ultimate goal of our work is to exploit targets that arise in PDAC tumor cells or in the tumor microenvironment (TME) as a consequence of p53 mutation for translation into patient therapies. Recently, our laboratory discovered a cooperative signaling node between the top genetic PDAC drivers, oncogenic KRAS and mutant p53, engaged through interactive binding between mutant p53 and CREB1. This mutant p53/CREB1 complex subsequently activates multiple pro-metastatic transcriptional networks within tumor cells. Our preliminary data indicate that mutant p53/CREB1 GOF also drives non-cell autonomous functions through the activation of WNT/β-catenin signaling elements that direct cancer associated fibroblasts (CAFs) to curate the primary TME into pro-metastatic landscapes. We hypothesize that targeted disruption of the mutant p53/CREB1 complex is a viable opportunity to reverse cell- and non-cell autonomous functions of mutant p53 for therapeutic translation in PDAC. Accordingly, the objective of this grant proposal is to gain additional mechanistic insight into how p53 mutations in tumor cells drive pro-metastatic interactions with cancer associated fibroblasts and to test if disruption of the mutant p53/CREB1 complex subverts vitals steps in the metastatic cascade. Using genetically engineered mice and patient derived model systems, in aim 1 we will confirm essential elements of the mutant p53/CREB1-WNT/β-catenin signaling axis that consort with cancer associated fibroblasts to enhance migratory and invasive phenotypes. Moreover, we will test the efficacy of combined targeted approaches to disrupt multiple levels of the mutant p53/CREB1-WNT11 signaling axis to mitigate metastasis. In aim 2, we will clarify the role of the mutant p53/CREB1-WNT/β-catenin signaling axis in the curation of the fibrotic TME as it relates to chemoresistance and metastasis while testing its reversibility using genetic and pharmacologic strategies. In Aim 3, we will use human PDAC biospecimens and derived organoids to correlate our findings from Aims 1 and 2. The proposed research is significant because p53 mutations are present in 70 percent of PDAC patients and remain untargetable. As such, our approach to disrupt non-cell autonomous mutant p53 gain of function could expose new therapeutic vulnerabilities or inform rational combinatorial treatment strategies. The proposed research is innovative because we will study and modulate a novel mutant p53/CREB1-WNT/β-catenin signaling axis to undermine multiple checkpoints in the metastatic cascade. Given the prevalence of mutant p53 in human cancers, knowledge gained from this proposal also has broad application to other malignant disease sites and entirely aligns with the overarching mission of the NIH.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Our goal is to broadly understand the biological roles of arginine methylation, a very common post-translational modification (PTM). This PTM is deposited by at least nine protein arginine methyltransferases (PRMTs) in mammals. Like lysine methylation, arginine methylation of a substrate often recruits an effector molecule to the newly created methyl-motifs. Unlike lysine methylation, relatively few effects have been identified for methylarginine marks. This paucity of identified methylarginine (Rme) mark effectors and the signaling roles of the known effectors define a knowledge gap that will be addressed here. By performing proteomic screens, we have identified a novel reader for Rme marks, called SART3. This effector does not harbor a Tudor domain which is usually found in “traditional” readers of Rme marks, but rather it carries a series of HAT (Half-a-TPR) repeats that are rich in aromatic amino acids. Here, we plan to characterize this interaction and investigate the possibility that other TPR repeat-containing proteins may be involved in sensing arginine methylation. Also, we are investigating the functions of two known effects of Rme marks, called SND1 and TDRD3. Through a combination of mouse work and protein array studies, we have determined that SND1 can likely directly activate the kinase activity of S6K2, and that TDRD3 can play a role in the DNA damage response.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Within a patient’s tumor, malignant cells are surround by a complex and dynamic collection of cell types, which includes the intratumoral microbiota. However, there is a fundamental lack of understanding on how microbes within the tumor microenvironment (TME) interact with human components of the tumor, or how they might contribute to disease progression; presenting a significant barrier to progress. Comparative microbiome analysis has revealed an enrichment of distinct invasive bacterial communities in tumor tissue compared to adjacent normal tissue. Large-scale epidemiological studies have demonstrated a significant correlation between high intratumoral load of specific oncomicrobes, such as Fusobacterium nucleatum in colorectal cancer (CRC), with disease relapse and poorer survival. Yet, while pan-cancer tumor-tissue microbiome studies have exposed the pervasive presence of intratumoral bacterial communities within most cancer types, most of these studies to date have centered on bulk tissue molecular analysis, which obscures the spatial distribution and localized impact of the microbiota within tumors. My team recently discovered that viable intratumoral bacteria have a heterogenous distribution within human oral and CRC tumors, where they form bacterial-colonized microniches (BCMs). These BCMs are metabolically active, dominated by the anaerobic bacterium Fusobacterium nucleatum, and characterized by myeloid cell infiltration, upregulation of immune checkpoint proteins PD1, CTLA4 and Lag3, and reduced T-cell infiltration. Intratumoral heterogeneity (ITH) and associated phenotypic diversification of cellular subpopulations within the TME remain major factors for cancer progression and therapy resistance. In order to understand how the TME contributes to cancer progression and treatment resistance it is imperative that we assess interactions of all components of the tumor ecosystem, both mammalian and prokaryotic. The central hypothesis of this proposal is that BCMs within tumors are contributing to ITH by modulating cancer cell functions, as well as the influx and spatial distribution of immune cell types in colonized tumor regions. In Aim 1, to understand the focal tumor distribution of BCMs, their stroma-immune-tumor cell spatial interactions, we propose to functionally map cellular and metabolic features of BCMs within patient tumor tissues in-situ. In Aim 2 we will delineate the human cell types within patient tumors that harbor intracellular bacteria, the identity of those intracellular bacteria, and their impact on host transcriptional programs at the single cell level. In Aim 3, using animal model systems, we seek to examine the role of the oncomicrobe Fusobacterium nucleatum in remodeling cellular components of the TME. My team has the breadth of experience to accomplish this project as it seeks to apply multi-omic approaches to delineate microbial and host interactions within the TME. Our goal is to advance mechanistic understanding of host-microbiota interactions within the TME with the potential to reveal novel approaches for cancer prevent and treatment.

NIH Research Projects · FY 2025 · 2024-09

Research Summary: High-grade serous ovarian cancer (HGSOC) is the most common histologic subtype of ovarian cancer. Despite ongoing efforts to develop effective surgical and treatment regimens for advanced HGSOC, the overall survival rate is only 30%, in part due to late diagnosis. HGSOC is among the most chemo-sensitive of all epithelial malignancies at diagnosis. However, a subset of advanced stage patients fails to respond to initial therapy and has a dismal prognosis. Therefore, understanding the mechanisms through which HGSOC metastasizes, and develops intrinsic chemoresistance is crucial to improve the efficacy of treatments and survival of patients. The proposed Ovarian Cancer Atlas Research Center (OCARC) will use and integrate multiple state-of-the-art molecular and cellular profiling platforms to construct a three-dimensional (3D) ovarian cancer atlas characterizing HGSOC metastasis, and the development of intrinsic therapeutic resistance using a diverse patient cohort diagnosed with advanced HGSOC. The Center is composed of an Administrative Core and three connected units: a Biospecimen Unit, a Characterization Unit, and a Data Processing Unit. The multi-PI team from MD Anderson Cancer Center, and the University of Arkansas, brings together internationally recognized experts in ovarian cancer biology and treatment, mass spectrometry, bioinformatics, and imaging. This team will have the complementary multidisciplinary scientific expertise required for the integration of the multidimensional, multiparametric data needed to construct a 3D ovarian cancer atlas and address the key research problems proposed. The Administrative Core will provide the support necessary for the proposed OCARC infrastructure. The Biospecimen Unit will acquire specimens, collect clinicopathologic information, and prepare tissue sections. The Characterization Unit will perform spatially resolved subcellular transcriptome profiling on FFPE tissue sections using the novel STOmics platform; 3D metabolomic and peptidomic and glycomic profiling using high-resolution mass spectrometry imaging; and 3D stromal and tumor cell profiling using the COMET multiplex immunofluorecence platform. The Data Processing Unit will build a data warehouse hosting all the data generated by the Characterization Unit and the Biospecimen Unit. It will be responsible for integrating all available data to identify the key signaling circuits that underlie the 3D spatial changes of phenotypic profiles. The final atlas will identify differences in the cellular and molecular content, and cell-cell crosstalk signaling networks in HGSOCs from (1) primary and metastatic sites; and (2) intrinsic chemosensitive and chemoresistant HGSOCs. It will bring deeper insight into the multidimensional HGSOC tumor ecosystems associated with intrinsic chemoresistance. We expect that our results will be used by academic researchers, clinical researchers, and industry partners to improve our understanding of the molecular basis of ovarian cancer and better stratify disease management and improve patient outcome. Our results will have the potential to lead to the development of novel agents against chemoresistant HGSOC.

NIH Research Projects · FY 2025 · 2024-09

Abstract Cardiovascular disease (CVD), including coronary heart disease (CHD) and stroke, is the leading cause of mortality and morbidity in both men and women in the US. Well-established risk factors for CVD include modifiable lifestyle and non-modifiable host/biological variables, such as smoking, diet, BMI, age, sex, and race/ethnicity, as well as quantitative cardiovascular traits, such as total cholesterol, triglyceride, high- density/low-density lipoprotein cholesterol, blood pressure, and fasting glucose. However, it is not yet clear through which molecular mechanisms these lifestyle/host risk factors influence the cardiovascular traits and CVD risk. Large-scale multiplatform omics data, including whole-genome sequencing, DNA methylation, and gene expression data, have been recently generated in tens of thousands of well-phenotyped individuals in the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. These large-scale multi-omics and phenotype data resources provide an unprecedented opportunity to investigate the relationships among environmental/lifestyle risk factors and cardiovascular traits, and how molecular phenotypes work as intermediate factors between them. However, it remains a significant analytical challenge to delineate those complex relationships in the presence of multiple types of high-dimensional omics data. The objective of this research is to capitalize on the multi-omics and phenotype data in the TOPMed program and our recently developed statistical methods for high-dimensional mediation analysis to quantify the extent to which DNA methylation and gene expression mediate the effects of environmental/lifestyle risk factors on (Aim 1) quantitative cardiovascular traits and (Aim 2) the risk of incident CVD. We will identify mediating markers based on high-dimensional variable selection methods and estimate the total mediation effects of DNA methylation and gene expression, each alone and jointly, using 6,000 individuals in the Framingham Heart Study (FHS) study as the discovery cohort and over 2,000 and 1,000 individuals, respectively, in the Women’s Health Initiative (WHI) and Multi-Ethnic Study of Atherosclerosis (MESA) as validation cohorts. This study will provide deep insights into molecular phenotypes and biological pathways that mediate the effect of lifestyle/host risk factors on cardiovascular traits and CVD risk. Our findings may contribute to the discovery of novel biomarkers and therapeutic targets for CVD, and biomarker-based precision prevention for this devastating disease. The multi-omics-based mediation analysis pipeline established and refined through this proposed research will also be applicable to other heart, lung and blood diseases in the TOPMed program.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT: OVERALL Oropharyngeal cancer (OPC) is rising in epidemic proportions, largely owing to rise in highly curable oncogenic human papillomavirus (HPV) attributable disease. HPV-associated OPC is often diagnosed in young individuals presenting with near normal functional status and high quality of life (QOL) at diagnosis. Standard therapy offers excellent two- and five-year survival probability of 95% and 79%, respectively, yet at a high cost in QOL lost. Long-term survivors risk a host of debilitating delayed adverse sequelae of therapy. Randomized trials seeking to mitigate toxicity by reducing the intensity of systemic therapy or decrease radiotherapy dose in HPV- associated OPC surprisingly resulted in inferior survival (e.g., RTOG-1016, HN005). Other noteworthy efforts to optimize the therapeutic ratio (e.g., transoral robotic surgery [TORS] and highly conformal radiotherapy) still yield significant early toxicities published extensively by the investigators, as well as numerous late symptoms that persist or develop many years into survivorship as independent drivers of unemployment in young survivors (under age 60) and decisional regret about cancer therapy long after cure (median 7 years survival). The investigators have further published extensively on three distinct, but inter-related highly morbid delayed adverse sequelae that threaten QOL and health: 1) mandibular osteoradionecrosis (ORN), 2) late radiation- associated dysphagia (late-RAD), and 3) late lower cranial neuropathy (LCNP). To advance precision health of OPC survivors, we must integrate these individual lines of inquiry to better describe the intersection of actionable phenotypic presentations, trajectories, and clusters of adverse events and novel therapies to mitigate chronic disability as adverse events evolve from early to late survivorship. The absence of well curated long-term survivor cohorts was the stimulus for the development of the MD Anderson Oropharynx (MDA-OPC) cohort, an active, single institution, prospective longitudinal cancer cohort. Since 2015, 1,750 OPC patients have enrolled with interdisciplinary characterization of exposures, diagnostic/staging, treatment, disease control, and validated clinician- and patient-reported survivorship outcomes. Survivors are in longitudinal follow-up by the Patient-Reported Outcomes/Function (PROF) Core which has to date acquired 31,370 PRO questionnaires and 3,687 toxicity imaging studies. The MDA-OPC cohort is an unparalleled resource for integrated OPC specific Projects focusing on delayed treatment sequelae in this rapidly growing cancer survivor population. The OPC- SURVIVOR P01 Program Project aims to maintain and enrich MDA-OPC infrastructure, extending outcomes data collection beyond 5-years into long-term survivorship to support three integrated Projects focused on: 1) osteoradionecrosis (OPC-ORN), 2) cranial neuropathy (OPC-NERVE), and 3) late radiation-associated dysphagia (late-RAD). Our central hypothesis is that clinically feasible PRO and objective measures uncover phenotypes and trajectories of delayed cancer therapy induced adverse events for earlier detection and mechanistically targeted mitigation strategies.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Protection of stalled replication forks is crucial for cells to respond to replication stress and maintain genome stability. Recent research has provided strong evidences that genome instability can trigger inflammatory response when chromosomal or short stranded nuclear DNA are released into the cytoplasm and detected by a cytosolic DNA sensing pathway, the cGAS-STING-dependent pathway. Emerging evidences indicate that DNA repair or replication fork processing in the replication stress response is connected with activation of the innate immune response. However, much remains unknown about the link between stalled replication fork protection and innate immune response. In addition, the physiological consequence of replication stress- induced innate immune response in vivo remains to be defined. Our recent study reveals a role of Abro1 and FANCD2 in linking replication fork protection with restriction of innate immune response. We found that stalled replication fork degradation induced by Abro1- or FANCD2-deficiency leads to accumulation of cytosolic single- stranded DNA (ssDNA) and activation of cGAS-STING-dependent innate immune signaling. Increased cytosolic ssDNA including ribosomal DNA (rDNA) binds to cGAS activating cGAS-dependent innate immune signaling and secretion of proinflammatory cytokines/chemokines. In addition, Abro1 and FANCD2 limits replication stress-induced Processing bodies (P-bodies) formation and P-bodies are capable of modulating activation of the innate immune signaling upon replication stress. We propose to determine the underlying mechanism by which replication fork protection limits the induction of innate immune response and define fork degradation-induced innate immune response in vivo. We will carry out the following specific aims: (1) to delineate stalled fork degradation-induced activation of innate immune signaling; (2) to determine the involvement of P-bodies in stalled fork degradation-induced activation of innate immune signaling; (3) to determine the physiological effect of Abro1 deficiency-induced replication fork degradation associated secretory phenotype in vivo in mice. Overall, this project will provide novel insights into the link of replication fork protection and innate immune response, contributing to establishing new possibilities for the treatment of inflammatory disorders and cancer in the future.

NIH Research Projects · FY 2025 · 2024-08

PROJECT ABSTRACT Patients with hematological malignancies, and immunocompromised patients are highly susceptible to Candida infection. Based on the immune status of the host, the infections may be mild to life- threatening. The goal is to find novel treatment approaches to give long-term protection against opportunistic Candida spp. without disturbing normal gut microflora. The rationale underlying this proposal is that the development of CAR-T cell and CAR-Macrophage based cell therapies will target and recruit host immune system to protect against opportunistic Candida infection. Recently chimeric antigen receptors (CAR) T-cell based therapy has revolutionized cancer treatment strategy and showed overall success rate of more than 90% in hematological malignancies. In pre-clinical studies Aspergillus specific CAR T cells has shown significant reduction in fungal burden. Thus, the objective of this proposal is to develop CAR constructs to target systemic Candida infections without disturbing normal gut microflora. We will generate CAR constructs by fusing extracellular domain of the pattern recognition receptors with cytoplasmic domain of the activation receptors (CA-CAR). The central hypothesis will overcome barriers and immune evasion tactics imposed by the Candida spp. The central hypothesis will be tested by pursuing two specific aims.: Aim 1). Generation and functional characterization of T-cells expressing CA-CARs: Aim 2). Generation and functional characterization of macrophages expressing CA-CARs: Aims 1 and 2 can be done simultaneously without being dependent on each other. These include (i) generating CAR constructs by fusing Candida specific targeting domain with cell activating receptors; (ii) expression of CARs in T cells and macrophages using molecular biology and tissue culture techniques; and (iii) performing fungicidal assays by monitoring fungal growth real-time using image scanning microscope. Functional efficacy studies will be done by co-culturing Candida yeast and Candia hyphae. In-vivo efficacy studies will be done in NSG mice bearing systemic candidiasis. The proposed research is significant, because it is the first cell-based therapy designed to target Candida that spares the normal commensals. It is also significant because it will develop a platform that can be extended to study and control other opportunistic infections. To protect patients from unanticipated CAR T cell/ CAR-Macrophage related toxicity, we will include a mutant epidermal growth factor receptor (mEGFR) safety switch to induce apoptosis in CAR T cells. This can be activated by targeting with mEGFR specific antibody. The results of this study will have an important positive impact especially to overcome barriers such as virulence, antifungal resistance and recruit host immune system for clearing pathogens. The expected outcomes are that this proposal will lay the groundwork to develop suitable CARs to control other opportunistic pathogens and provide long-term benefit.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Treatment with immune checkpoint inhibitors (ICI) has been revolutionary in melanoma and other cancers. However, only a subset of patients will have durable responses to first-line therapy and immune-related toxicities remain a significant challenge. Given the critical, unmet need for safe, effective and broadly applicable strategies in this setting, we will conduct a prebiotic food-enriched diet (PreFED) intervention combined with standard-of-care ICI in metastatic melanoma patients. This study seeks to intervene on insights from our own work and others demonstrating that (1) gut microbiome profiles and dietary habits predict response to ICI in melanoma; (2) well-characterized gut commensals consistently implicated in ICI response are highly responsive to prebiotic dietary interventions; and (3) the gut microbiome is a therapeutic target in ICI-induced toxicities. With the overarching goal to test rationally-designed, microbiome-targeted dietary interventions that cancer patients undergoing active therapy can practically implement and sustain throughout treatment, PreFED is a scalable approach focused on providing multiple key microbiota-accessible nutrients through provision of prebiotic foods and nutritional counseling to selectively stimulate beneficial gut microbes that enhance and sustain the overall gut ecosystem. Nutrient and microbiota-derived metabolites from these interactions support the central mechanisms underlying a favorable immune response to ICI (e.g., balancing inflammation and effector T-cell function). Thus to further interrogate mechanism and test whether PreFED- manipulation of the microbial community could be effective as an adjunctive therapy to ICI, we will transplant paired patients’ fecal samples into germ-free mice to examine the impact of post vs. pre-diet microbiome on melanoma growth and ICI response, as well as mucosal and anti-tumor immunity. Through these studies, we will evaluate the effect of the PreFED on the gut microbiome, host metabolome, and the mucosal, systemic and antitumor immune response to ICI. Integrative analyses of both human and mouse studies will provide significant insights on diet-induced changes in the microbiome and microbiota-mediated changes in the metabolome that influence the immune response to ICI – and highlight avenues whereby patients’ diets may influence the success of other microbiome-targeted strategies being developed in this setting. Notably, to progress to future multicenter trials with robust translational and clinical endpoints, we will define the safety and efficacy profile of PreFED as an adjunct to current ICI regimens (anti-PD1 monotherapy, anti-PD1+LAG3, anti- PD1+CTLA4). Though our studies will focus on metastatic melanoma, dietary approaches to enhance the gut microbiome, anti-tumor immunity and ICI response are relevant and applicable to other stages and cancer types. Accessible, appropriately refined and successful dietary interventions offer a cost-effective and safe adjunct that could be implemented across practice settings and geographies, addressing significant healthcare disparities and ultimately offering the potential to improve care for many patients.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY/ABSTRACT Severe hyperthermia (heatstroke) is due to a failure of thermoregulation that can lead to cell death in various tissues resulting in multisystem organ failure. Hyperthermia generally results from overexertion due to severe physical activity and/or prolonged exposure to elevated temperatures. Patients who suffer from anhidrosis or hypohidrosis, due to disease or certain medications, such as anesthetics, diuretics, anticholinergics, and amphetamines, are likewise at greater risk. Apart from the negative effects of hyperthermia, in the clinic a variety of approaches are being utilized to induce localized hyperthermia in the treatment of various cancers, as many tumors exhibit enhanced sensitivity to heat. Despite the general importance of heat shock responses, however, the mechanisms by which heat induces cell death remain unclear. Heat shock reportedly induces cell death through the activation of various canonical pathways involving the initiator caspases-2, -8, and -9; however, we and others find that while these pathways can serve to amplify the cell death signal, they are largely dispensable for heat shock-induced cell death, particularly at higher temperatures or following longer exposures. Instead, in a surprising discovery, we have found that Bim mediates heat shock-induced cell death, independently of its BH3 domain, through an interaction with the LC8 dynein light chain subunit of the dynein motor complex (DMC). In binding to LC8, Bim promotes anterograde trafficking of lysosomes from the perinuclear region to the cell periphery, where they become sensitive to the effects of heat shock. Additional data indicate that peripheral lysosomes undergo direct membrane permeabilization (LMP) or exocytotic release following heat shock, resulting in the release of cathepsins into the cytoplasm or extracellular space, respectively. Moreover, knockout or depletion of cathepsin L (CatL) renders cells highly resistant to cell death. Lastly, while cathepsins released into different cellular compartments play roles in mediating cell death, targeted localization of the cathepsin inhibitor, cystatin B (CSTB), to the nucleus results in profound suppression of heat shock-induced cell death. Thus, our overall hypothesis is that Bim plays a critical noncanonical role in regulating lysosomal number and positioning within cells, and as a result, it determines the susceptibility of peripheral lysosomes to heat shock- induced LMP/exocytosis, cathepsin release, and cell death. In four specific aims, we will determine: (1) the roles of Bim in lysosome number and positioning; (2) the roles of Bim in heat shock-induced LMP and lysosome exocytosis; (3) the roles of nuclear CatL in heat shock-induced cell death; and (4) the roles of Bim in two mouse models of localized and whole-body hyperthermia. Collectively, the proposed studies will provide important insights into an unexpected role for Bim in regulating lysosomal trafficking with far reaching implications for Bim biology in general and heat shock-induced cell death in particular.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT The proposed study seeks to determine if DNA methyltransferases from members of the microbiota contribute to bacterial pathoadaptation in the tumor niche, direct xenogeneic epi-modifications on the human genome, and can be exploited as therapeutic targets in cancer prevention/treatment. Within a patient’s tumor, malignant cells are surrounded by a complex microenvironment encompassing a range of non-transformed cells and also a diverse collection of microorganisms. For example, Fusobacterium nucleatum (Fn) is significantly enriched in colorectal adenocarcinoma compared to adjacent normal tissue. Although cellular and animal models have supported a role for this bacterium in cancer initiation and progression, we still have little information as to how they directly contribute to cancer. In published work, our group has demonstrated that Fn, which is usually part of the oral microbiome and not found in the lower gastrointestinal tract, is an invasive intracellular bacterium that effectively colonizes the colorectal cancer (CRC) niche. In preliminary studies, we isolated and characterized the genomes and epigenomes of >150 Fn strains from CRC tumors and the oral cavity and discovered that within the Fn subspecies Fn subsp. animalis, there are two distinct clades that differ in their enrichment in CRC, which we named Fn oral-clade and Fn CRC-clade. We show that Fna CRC-clade is the only Fn group significantly enriched in human tumors and fecal specimens. Of the many differences between these two clades, perhaps one of the most striking is their distinct DNA methylome signatures, with CRC-clade uniquely harboring Gm6ANTC methyl-modifications. We, therefore, hypothesize that the DNA methyl-modifying enzyme (M.FnI) responsible for m6A methylation at this motif contributes to Fn CRC-clade virulence during carcinogenesis. We propose to test this hypothesis using genetically engineered CRC-clade clinical isolates in cell culture and animal model experiments to determine if M.FnI regulates bacterial gene expression to promote their pathoadaptation (Aim 1). Additionally, we speculate that M.FnI can act as a nucleomodulin to directly interfere with the human host epigenome and gene regulation. We will investigate this possibility through ectopic expression of M.FnI in human CRC cell lines and pre-cancer organoid models to delineate its nucleomodulin potential (Aim 2). Finally, we seek to develop chemical probes specific for M.FnI to aid in authenticating it as a therapeutic target in CRC prevention and progression (Aim 3). While we focus here on a single pathogen and single disease, successful completion of these aims will provide fundamental knowledge on the role of microbial epigenetic systems in tumor colonizing microbes. Further, this work has potential to reveal a hidden paradigm of host microbe-epigenetic crosstalk underlying the oncogenic process in bacterial-colonized, hypermethylated tumors. As such, if our core hypothesis is true, this work could have a far-reaching impact beyond CRC.

NIH Research Projects · FY 2025 · 2024-08

Abstract/ Summary Pancreatic ductal adenocarcinoma (PDAC) represents one of the most aggressive malignancies, with more than 50% of patients with pancreatic cancer presenting with metastatic disease, mainly in the liver, at the time of diagnosis. We have previously described the presence of microbes in pancreatic cancer which could determine outcomes. Furthermore, we revealed that microbial modulation can affect tumor microbial composition, immune cells landscape and tumors growth. We have now preliminary data showing microbial distribution across tumors and cellular compartments. Moreover, we have been able to perform spatial microbial functional assessment at regional and single cell level. We have identified similar microbes in primary tumors and matched liver metastasis, suggesting microbial seeding from primary tumors to liver. More evidence for this is our findings revealing that NETosis, a process of host defense against microbes, is upregulated in liver during the metastatic process. The overall goal of this proposal is to deeply characterize the location, distribution and spatial functionality of microbes within the tumor microenvironment of primary tumors as well as their influence in tumors growth, pre- metastatic niche and liver metastasis formation. To this end, we will use multiple methodologies including state-of-the-art microbial-host imaging, regional and single cell microbial-host sequencing to determine the location of bacteria within the cellular compartments, patterns of distribution, functionality and species characterization together with screening culturomics (Aim 1). We will then use multiple mouse models to fully characterize the role of human PDAC microbes in primary tumor growth, metastatic niche and liver metastasis formation by adding human- pancreatic cancer derived microbes allowing in vivo tracking of microbial-containing cancer cells as well as local and global microbial ablation, in vitro organoid microbial co-cultures and assessment/modulation of NETosis to determine their role in mediating microbial effects (Aim 2). The success of this proposal has high potential for the development of novel microbial modulation strategies that target the tumor microenvironment which could ultimately lead to improved outcomes in patients with pancreatic cancer.

NIH Research Projects · FY 2025 · 2024-08

PROJECT ABSTRACT Lymphedema is a debilitating, life-long iatrogenic sequalae of cancer treatment which is becoming an important survivorship issue due to improved cancer survival. Patients with inflammatory breast cancer (IBC), a highly aggressive form of breast cancer, require systemic therapy, surgery, and radiation therapy for oncological control. These treatments place them at the highest risk of developing lymphedema, with approximately 50% developing it within 12 months following surgery. Therefore, developing strategies to predict and prevent treat lymphedema would significantly impact the well-being of patients with IBC. General strategies for treating lymphedema include physical therapy and the continuous use of compression garments, and surgical treatments have recently been introduced that can provide some improvement in the swelling and symptom burden. Given the incomplete outcomes of these interventions, especially for a high-risk group like IBC patients, focus has recently shifted to risk-reducing surgeries. Immediate lymphatic reconstruction (ILR) of lymphatic vessels at the time of axillary lymphadenectomy (ALND) has resulted in significantly lower rates of lymphedema among breast cancer patients for patients who received ILR. Unfortunately, none of the studies on ILR have focused on IBC patients, or have investigated longitudinal outcome measures, which would enable comprehensive outcome assessment, including identification of potential biomarkers. We propose a single-arm, clinical trial investigating the impact of ILR on the development of lymphedema in IBC patients at a single, high-volume center specializing in treatment of IBC patients. We hypothesize that ILR in patients with IBC is preventative against lymphedema development as defined by objective clinical threshold measurements, and prevents changes in lymphatic architecture and pumping characteristic of lymphedema visualized using near-infrared fluorescence lymphatic imaging (NIRFLI), as well as functional alterations in peripheral blood immune cells associated with lymphedema development. This hypothesis will be addressed in the following Specific Aims: (1) to determine the incidence of clinical lymphedema in IBC patients following ILR; (2) to establish a longitudinal, dynamic, imaging-based profile of IBC patients following ILR using NIRF-LI to provide real-time visualization and objective characterization of changes in lymphatic vessel architecture and function; and (3) to identify a blood-based, inflammation-related signature of lymphedema in patients with IBC through longitudinal analysis of serum samples from our clinical trial cohort, using an inflammatory cytokine/ chemokine array. The proposed trial will allow objective assessment of lymphatic vessel anatomy and functional changes in lymphedema, as well as to define immune characteristics that correlate with lymphedema development. If successful, this work would provide impetus to change the surgical standard-of-care for patients with IBC and help direct highest risk patients to specialized treatment centers for surgical intervention.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT: Novel PET Imaging to Guide Therapy in Hepatocellular Carcinoma Liver cancer is the third leading cause of cancer death worldwide, with hepatocellular carcinoma (HCC) constituting more than 75% of all liver cancers. Locoregional therapies (LRTs), such as transcatheter arterial radioembolization (TARE) with Yttrium-90 (Y90), play a critical role in the care of patients with HCC. For patients undergoing LRT, time is of the essence; the time required to determine how an HCC responds to LRT is a critical determinant in the overall care algorithm and outcome for these patients. Current methods to predict response to Y90 LRT rely on standard-of-care (SOC) anatomical imaging, such as magnetic resonance imaging (MRI) or computed tomography (CT), which remain ambiguous for months following treatment. During this critical waiting, physicians and patients face uncertainty with respect to disease status, with eligibility for future interventions frequently compromised by ambiguities associated with determining treatment outcomes. In this project, we propose to evaluate an innovative molecular imaging approach to predict response to Y90 LRT much earlier than SOC imaging. We have previously evaluated 18F-FSPG PET in liver cancer and found it to be highly suitable for lesion detection and evaluation of response to therapy. Cancer cell accumulation of 18F-FSPG is mediated by xCT (SLC7A11) transporter activity, which is highly correlated with resistance to radiation. Thus, we hypothesize that 18F-FSPG PET represents an ideal biomarker to predict the response of HCC to Y90 LRT at baseline or soon after therapy, to identify residual, Y90-resistant disease and early recurrence. Complementary to imaging, liquid biopsy involves the collection of blood or other body fluids for the detection of tumor-derived molecular specimens, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). In this application, we propose to determine if ctDNA levels are actionable in the setting of patients with HCC undergoing Y90 LRT, triggering future 18F-FSPG PET/CT examinations in these patients. Our overall objective is to evaluate 18F-FSPG PET as a means to predict early response and recurrence following Y90 therapy and to determine whether PET imaging and ctDNA are correlated. Our overarching hypothesis is that 18F-FSPG PET will predict response to Y90 therapy and tumor recurrence sooner than standard-of-care (SOC) imaging and will be highly correlated with ctDNA liquid biopsy. We will test our hypothesis, in two Specific Aims. Aim 1. To evaluate the relationship between 18F-FSPG uptake in HCC lesions, ctDNA in blood, and clinical response to Y90 radioembolization therapy by SOC imaging. Aim 2. To evaluate voxel-wise correlations between post-treatment 18F-FSPG intra-tumoral accumulation and SOC imaging in patients with HCC who have undergone TARE. Clinical Impact: This study holds the potential to improve HCC disease management by providing novel imaging and liquid biopsy approaches that predict response to Y90 LRT much earlier than conventional means.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT Patients with lung cancer, on the most prevalent cancer diagnoses in the United States, tend to experience debilitating physical and psychological sequelae. Common symptoms include reduced lung function, dyspnea, fatigue, sleep disturbances, and depression compromising their physical function and quality of life (QOL). Consequently, patients have a high need for care and support. Patients’ family members are their most important and valued source of support and care; yet, caregiving is physically and emotionally taxing. In fact, family caregivers report high rates of psychological distress, fatigue, and sleep disturbances, which may undermine the quality of care they are able and willing to provide to the patient. Including caregivers in supportive care interventions may not only reduce caregiver burden but may potentially improve patient outcomes beyond the typical patient-oriented programs. Thus, there is a need to establish evidence-based dyadic interventions targeting both patient and caregiver outcomes. To this end, we have systematically built a program of research testing a patient-caregiver dyadic yoga program to address the needs of this vulnerable population. The parent R37 project seeks to examine the efficacy of an instructor-led dyadic yoga program regarding improved objective physical function and QOL outcomes in both patients and caregivers while patients are undergoing standard thoracic radiotherapy. With the goal to facilitate the large-scale implementation of this promising intervention with a flexible, cost-effective delivery strategy, we now propose to deliver the intervention on-demand via a mobile application. Under the proposed R37 extension project, we seek to field test a mobile app prototype and assess the feasibility, usability, and acceptability of the app-based yoga program in 20 patient-caregiver dyads. We will examine social determinants of health variables as correlates of these study outcomes to ensure that the app is acceptable to families from diverse backgrounds. We will use a mixed-methods approach to understand the experience of participants with the intervention and its delivery using qualitative accounts. Participant feedback will inform the need to refine and enhance the yoga app. The proposed innovative work will provide rich pilot data that will inform a subsequent, larger trial seeking to test the effectiveness of the app-based program in the community setting. Thus, this study represents a compelling next step of this program of research to support this vulnerable patient-caregiver population. Together, the results of the parent project—a rigorous, single-blind randomized controlled trial with a stringent comparison group—beautifully dovetail with the knowledge gained from the proposed pilot trial to inform future implementation research and ultimately, the clinical care of this high need population.

NIH Research Projects · FY 2026 · 2024-08

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotes. The mammalian genome undergoes tightly regulated global demethylation early development where faithfully inherited DNA methylations are essential. In adult soma, while DNA methylation is largely stable, its aberrations have been widely observed in aged and malignant tissues. Under these conditions, broad hypomethylated domains have been observed at gene deserts and genomic repeats, a phenomenon linked to excessive proliferations. In the meantime, focal hypermethylation occurs at lineage specific promoters, accompanied by widespread methylation variations at conserved regulatory loci. The molecular basis and functional consequences for these DNA methylation abnormality in adult tissues remain poorly understood. In the current study, we investigate the role of DNA methylation in determining adult stem cell fates under homeostasis and upon stress. In particular, we focus on the regulation of retrotransposons (RTs) by DNA methylation and how their impact adult stem cell function. Differing from tandem repeats in the centromere and telomere that are constitutively heterochromatin, RTs are interspersed genomic repeats that exist in both heterochromatin and euchromatin state. Compared to protein-coding genes comprising 1.5% of the genome, RTs constitute 40% of the genome and exert many regulatory functions, but are understudied due to their sequence repetitiveness and less conserved nature. During pre-implantation, RTs residing in the heterochromatin are transiently derepressed due to global demethylation and play an essential role in embryonic development. In adult somatic tissues, RTs are largely suppressed but become aberrantly induced when heterochromatin is destabilized, such as during aging and cancer. What pathways control RT levels in adult tissues, their impact on lineage gene expressions, and their physiological role under homeostasis and upon stress remain unclear. To address these questions, we use mouse skin as our model that harbors well-characterized, highly accessible, and genetically amenable adult stem cells. Epidermal and hair follicle stem cells in the skin fuel the homeostatic postnatal remodeling, drive wound induced regeneration, and undergo functional decline during aging. By using genetic models lacking what we have found to be a crucial epigenetic repressor of RTs in adult skin, we examine (1) the molecular basis for DNA and histone methylation mediated RT silencing, and (2) the mechanism by which DNA methylation and RT dynamics regulate adult stem cell fate decision in a physiological setting. We hypothesize that DNA methylation at RTs could either locally impact neighbor gene expressions or titrate rate limiting methylation machinery to regulate lineage gene levels at a distance, both of which contributing to adult stem cell fate decisions. Our study will provide insights into the molecular interconnection between euchromatin and heterochromatin in the context of adult stem cell fate decisions and tissue fitness.

NIH Research Projects · FY 2025 · 2024-08

Abstract Adenoid Cystic Carcinoma (ACC) is a common salivary gland cancer for which there is no standard systemic therapy. ACC has a heterogeneous behavior; while most patients have an indolent disease course, approximately 30% have a very aggressive disease. There are currently no standard markers to prognosticate or for selecting treatment. Epigenetic changes are tightly linked to tumorigenesis and cancer progression and numerous studies imply that DNA methylation may serve as a valuable biomarker to identify molecular disease subtypes. However, studies evaluating whole ACC methylome and the evolutionary trajectories of methylation during the initiation and progression of ACC are lacking. We identified methylation intertumor heterogeneity in ACC and differentially methylated gene regions according to ACC transcriptomic subtype. Additionally, we also identified circulating tumor DNA in blood of ACC patients and their levels correlated with disease recurrence, response to therapy or disease progression, suggesting clinical utility of ctDNA dynamics. Our central hypothesis is that the DNA methylation signature in ACC can identify prognostically distinct and therapeutic relevant molecular subtypes and circulating methylation markers may be used as a non-invasive tool for real-time subtyping stratification and treatment monitoring. In Aim 1 we will examine the DNA methylation and transcriptomic profiles in 200 clinically annotated ACC tumors with long term follow-up and assess the value of DNA methylation in refining transcriptomic based ACC subtyping. In Aim 2 we will profile the methylation/transcriptome of paired primary and metastatic tumors to identify metastatic specific changes and develop a metastatic propensity score to predict metastatic site. In Aim 3 we will profile DNA methylation in circulating free DNA from matched tumor and blood samples, collected as part of a prospective study already initiated, and develop a blood-based methylation score for ACC subtyping. Additionally, we will validate the significance of our blood-based methylation score in detecting ACC subtype evolution and to predict treatment response using blood samples collected as part of our clinical trials. Our research will be the first to investigate ACC DNA methylation markers to identify aggressive ACC at initial diagnosis and throughout tumor progression. Accurate diagnosis through a simple blood test will allow clinicians to detect the evolution of the disease in real- time and guide treatment selection.

NIH Research Projects · FY 2025 · 2024-08

Project Summary The three-dimensional (3D) chromatin architecture in mammals represents a remarkable yet not fully understood biological process. The high-throughput chromosome conformation capture (Hi-C) technique, known as Hi-C, is instrumental in identifying diverse chromatin structures. Despite this, the ways in which the 3D chromatin architectures in T cells are influenced by the tumor immune microenvironment, and how this impacts cancer outcomes, remain largely unexplored. Renal medullary carcinoma (RMC) stands out as a particularly aggressive cancer that predominantly affects young individuals of African descent, especially those with sickle cell disease (SCD). Treatment options for RMC are currently limited. The most promising among them are immune checkpoint blockades (ICIs). However, to truly harness the potential of ICI-based therapies, there's an urgent need for a deeper understanding of the RMC immune microenvironment and the underlying mechanisms of RMC. The 3D chromatin architecture in cells related to RMC remains a relatively uncharted territory. Guided by the expertise of cancer biologist Dr. Liuqing Yang, epigeneticist Dr. Wenbo Li, and Dr. Pavlos Msaouel, a clinician specializing in rare renal carcinomas, our team has harnessed the power of Hi-C. We aimed to delineate the distinct chromatin structures in CD8+ T cells from SCD patients in contrast to those from healthy individuals and to understand the ramifications of these changes on the development and prognosis of RMC. Intriguingly, our preliminary findings highlight significant modifications in the 3D genomic structure of CD8+ T cells derived from SCD donors. A notable observation is the diminished chromatin loop stemming from the SLC7A11 locus in the SCD condition. This locus plays a pivotal role in ferroptosis, a specialized form of programmed cell death involving iron. Building on these insights, we postulate that ferroptosis in SCD-induced CD8+ T cells contributes to the immune resistance observed in RMC, with altered chromatin structures being central to this process. Our project is anchored in three specific objectives: 1) Establish the connection between altered chromatin architecture and the molecular pathway linking SCD and RMC tumorigenesis; 2) Delve into the 3D genomic mechanisms driving ferroptosis in CD8+ T cells in SCD; 3) Assess the significance of both ferroptosis and the altered genomic architecture in CD8+ T cells on the overall disease landscape. Our team envisions that within a decade, ICI and CAR T-cell therapy will emerge as frontline treatments for RMC, enhancing cure rates and addressing the current limitations of standard care. By delving into the role of altered 3D chromatin structures in RMC's molecular mechanisms, we hope to equip the scientific community with insights crucial for devising strategies that safely and effectively sensitize RMC to ICI and CAR T-cell therapies. The data generated from our investigations will lay the groundwork for early-phase clinical studies led by Dr. Pavlos Msaouel. These studies will be pivotal in addressing questions about the next generation of ICI and T-cell therapies tailored for RMC.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Neoadjuvant and perioperative chemoimmunotherapy are the standard of care for the treatment of patients with resectable non-small cell lung cancer (NSCLC). Unfortunately, the majority of patients with resectable NSCLC do not benefit from neoadjuvant/perioperative immune checkpoint inhibitor (ICI)-based therapies for reasons that are unclear. Efforts to develop more effective treatment strategies that bypass therapeutic resistance have been hampered by our limited understanding of the mechanisms that govern response to ICIs and that thus could serve as validated biomarkers to guide neoadjuvant/perioperative ICI treatment. Our long-term objectives are to develop rational neoadjuvant/perioperative combination therapies that prevent resistance and increase benefit from ICI-based treatments. Recently, we analyzed NSCLCs from patients treated with chemotherapy (CT) plus dual ICIs in the phase 2 NEOSTAR trial (PI: Cascone) and found those from responders contained enhanced B cell fractions and markers of tertiary lymphoid structures (TLS) compared to nonresponders. These findings were recapitulated when using neoadjuvant combinations of ICI plus novel immunomodulatory agents in our recently reported phase 2 trial, NeoCOAST. To build on these observations from our clinical trials, we performed preliminary spatial-omics analysis of a pilot cohort of NSCLCs from patients treated with neoadjuvant CT+ICI and with varying response to therapy. We found that pathologic response was associated with aggregation of B cells into organized structures (e.g., TLS) with elevated expression of anti-tumor B cell signatures and enhanced cell-cell communication with the tumor immune microenvironment. Preclinically, we found that neoadjuvant ICI-based therapy was the most effective treatment at reducing frequency of metastases and prolonging survival in our human-relevant models of spontaneously metastatic NSCLC and therapeutic efficacy was once again tightly coupled with enhanced B cell infiltration. Consequently, we hypothesize that B cell transcriptional, immunogenomic, and spatial landscapes in neoadjuvant CT+ICI-treated NSCLC patients differ by MPR, and that B cell-mediated cytotoxic antitumor responses, including their interaction with T cells, augment the efficacy of neoadjuvant ICI-based therapy in early-stage NSCLC. We will test our hypothesis by determining the transcriptional, immunogenomic, and spatial landscapes of B lineage cells in human NSCLC treated with neoadjuvant ICI-based therapy (Aim 1); interrogating the impact of B cells on T cell-mediated responses to neoadjuvant ICI-based therapy in murine models of NSCLC (Aim 2); and investigating the utility of B cell-centric phenotypes in predicting benefit of neoadjuvant ICI-based therapy in resectable NSCLC from unique clinical trials (Aim 3). This proposal will leverage a platform of unique, global clinical trial cohorts of NSCLC, novel human-relevant mouse models of NSCLC, as well as state-of-the-art single-cell and spatial multi- omics technologies to define the role of B cell lineages in antitumor responses to neoadjuvant ICIs and provide a framework for the development of B cell-associated therapies aiming to cure a greater number of patients.