Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2021-09

SUMMARY Aging is the greatest risk factor for cancer development, yet the physiological and molecular mechanisms underlying this relationship remain poorly understood. Undoubtedly, one contributing factor is time – i.e. the years or decades needed for a single cell to acquire sufficient mutations to trigger overt disease. However, other factors certainly play a role, including age-related changes in cell metabolism, DNA damage responses, immune cell function, and the abundance of senescent cells. While understanding how these factors influence tumorigenesis will reveal strategies to improve cancer intervention and treatment, facile models to study cancer in aged animals are lacking. Addressing this critical challenge, we incorporate somatic tissue engineering methods to introduce oncogenic mutations directly into the organs of aged mice, thereby obviating the time and costs of intercrossing and aging cohorts of multi-allelic genetically engineered mouse models (GEMMs). We have developed a range of such “non-germline GEMMs” (nGEMMs) of different target organs and cancer genotypes and shown that the resulting cancers recapitulate molecular and histological features of the corresponding human disease. We have also produced nGEMMs using aged mice and shown that the developing tumors have distinct immune infiltrates with different tumor surveillance capabilities. This proposal combines the unique capabilities of nGEMMs with state-of-the-art tissue analyses to assess the contribution of the aged environment to cancer manifestation in different organ contexts and compares results to settings in which dietary or genetic factors uncouple biological age from chronological age. Implementation of advanced single cell methods will produce a detailed picture of cell type and cell state differences in tumors developing in aged animals and perturbation studies will explore tumor cell intrinsic and extrinsic factors underlying age- related phenotypes. The proposed experiments will benefit from combining our expertise in cancer biology with that of Dr. Laura Niedernhofer, who has extensive experience in studying organismal aging. Our studies will establish broadly portable models for studying cancer in aged mice at a breadth and pace that was previously impossible and produce novel insights into how the epigenetic and physiological processes linked to age contribute to an increase in cancer incidence and/or progression.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY Each day billions of cells die and are cleared rapidly with minimal immunological consequence. This process, clearance of apoptotic cells or efferocytosis, is carried out by phagocytes such as macrophages which are numerically fewer in tissues and are often responsible for clearing multiple dead cells and debris in quick succession. Efferocytosis is thought to be important for preventing autoimmunity and inflammatory disease. However, patients suffering from autoimmunity or inflammatory disease rarely present with symptoms expected from a defect in clearance of apoptotic cells, such as excessive accumulation of apoptotic or necrotic cells. On one hand, you have a process that is occurring in every tissue and organ, yet on the other hand, it remains unclear how this process contributes to or prevents autoimmunity and inflammatory disease. We present a new model that suggests autoimmunity and inflammatory disease arise because of disruptions in how macrophages manage apoptotic cell digestion instead of whether or not macrophages engulf at all. This model suggests that efferocytosis is a dangerous process because the content of engulfed apoptotic cells, such as lipids, amino acids, and nucleic acids, can trigger potentially catastrophic inflammatory responses by macrophages. To prevent this, we hypothesize that macrophages use mechanisms to rapidly detect and respond to the engulfed material, termed rapid response circuits (RRCs). This proposal combines novel experimentation with -omics approaches and informatics to identify and assemble putative efferocytosis RRCs, then uses innovative tools and methods to mechanistically study identified efferocytosis RRCs. Using this approach, we will first examine the common efferocytosis RRCs used by macrophages. Then, we will explore unique efferocytosis RRCs used by tissue- resident macrophages who are exposed to dying cells and debris specific to the tissue of residence. Collectively, this work on RRCs will contribute to a new framework for understanding how failure to appropriately digest apoptotic cells contributes to autoimmunity and inflammatory disease, and possibly reveal novel diagnostics and therapeutics.

NIH Research Projects · FY 2025 · 2021-09

Project Summary: EGFR-mutant lung cancers (LCs) are initially highly responsive to EGFR inhibitors, but cancer adaptation resulting in drug resistance universally occurs. Acquired resistance mediated by lineage plasticity is particularly problematic; EGFR-mutant lung adenocarcinomas (ACs) can transform into either small cell (SC) or squamous cell (SQ) lung cancers. Understanding the molecular determinants of histologic transformation is critical to inform therapeutic strategies to block the emergence of new cell lineage states induced by cancer treatments. We have established that concurrent alterations in TP53 and RB1 are necessary but not sufficient to induce SC transformation in EGFR-mutant LCs; EGFR/TP53/RB1-mutant LCs have a 25% likelihood of transformation over time. In addition, we have assembled a cohort of resected mixed histology tumors (AC/SC and AC/SQ) that serve as a model of transformation where microdissection by histology isolates paired tumors representing pre- and post- transformation states. Using these complementary systems directly derived from patients, we will perform a mechanistic analysis of lineage plasticity utilizing EGFR/TP53/RB1-mutant LCs at high risk for transformation and mixed histology tumors that represent transformation in progress. Our central hypothesis is that while the somatic mutational landscape is critical in establishing conditions permissive of lineage plasticity, actual transformation to an alternative lineage is predominantly epigenetically driven and associated with consistent globally altered patterns of gene expression. Our first aim is to comprehensively molecularly characterize lineage plasticity using parallel whole exome, RNA and bisulfite sequencing focusing on patient samples from before, during (mixed AC/SC and AC/SQ) and after transformation. Resected mixed histology tumors (AC/SQ) will be microdissected and molecularly characterized as paired tumors. The second aim of the proposal is to investigate subclonal dynamics contributing to lineage plasticity using single cell RNA-sequencing. We will interrogate serial samples from our ongoing clinical trial to prevent transformation in patients with EGFR/TP53/RB1-mutant lung ACs and resected mixed histology AC/SC and AC/SQ tumors. Finally, we will utilize our patient-derived xenograft models of transformation to genetically and pharmacologically assess putative drivers of transformation, exploring rational interventional strategies. Our preliminary work has proposed initial targets (Wnt, EZH2, AKT, NOTCH) that will be expanded with findings from this proposal. Novel therapeutic interventions will be proposed based on our findings that can be rapidly translated to the clinical setting for LC and other disease states characterized by lineage plasticity.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT The impact of sex differences in Alzheimer's disease (AD) remains poorly understood, especially in the context of protein-protein interactions within vulnerable regions that drive dysfunction. Despite growing appreciation of the clinical course, presentation, and severity of AD, studies of sex impacting AD development and progression are lacking. Although recent high-throughput and bioinformatics technologies help to understand molecular and genetic basis of sex differences in aging and AD, reliance on static `omics data representing a descriptive inventory of biomolecules measuring changes in their stoichiometry at a given time and condition limits functional insights. Another roadblock is translating these complex datasets into biological insights requires sophisticated computational algorithms, diminishing access and impact to the biomedical community at large. To address these limitations this proposal introduces epichaperomics, an unbiased state-of-the-art `omics approach we invented to generate direct access to interactome perturbations and to the functional outcome of such changes in native biological systems. We posit by applying epichaperomics to well-characterized postmortem human brains that i) capture the disease trajectory, ii) encompass AD vulnerable and less affected regions, and iii) have robust parallel information on patient-specific correlates, will enable rigorous hypothesis- generating analyses on potential impact of stressors and vulnerabilities on disease trajectory, and on interactome as well as connectome dysfunctions as they occur in sex-dependent manner. Through this novel approach we expect to derive mechanistic innovation on specific dysfunctions impacted by sex differences leading to insights into sex-phenotype relationships not available through other `omics platforms. By evaluating, understanding, and anticipating interactome changes through epichaperome formation in relation to sex impact (Aim 1) and subsequent straightforward computational analysis with web-based output (Aim 2), first-of-a-kind proteome-wide insights into the impact of sex differences on interactome networks vulnerabilities and dysfunctions within the hippocampus and regions of the default mode network in relation to the relatively spared cerebellum, both on their nature and trajectory, in vulnerable cells and brain regions will be generated. Information how stressors and vulnerabilities (e.g., genes, environment, hormonal status) interact at cell and brain connectome levels to produce heterogeneous phenotype mapping of disease vulnerability will be produced. We posit a whole new treatment paradigm avenue will open, providing a previously unavailable sex-specific precision medicine approach to AD by understanding and targeting the interactome across the AD spectrum of no cognitive impairment, mild cognitive impairment, and AD dementia through stressor and vulnerability analysis. Raw datasets and data analytics from interactome network studies will be deposited into free-access portals accessible by the scientific community for additional mining and hypothesis testing studies. A web-based user- interface will also be designed facilitating data processing and visualization.

NIH Research Projects · FY 2026 · 2021-09

PROJECT SUMMARY/ABSTRACT Breast cancer outcomes are disproportionately poor in low- and middle-income countries (LMICs) compared to high-income countries. Low breast cancer survival rates in LMICs are primarily attributable to advanced stage presentation and limited diagnostic and treatment capacity. Although national clinical practice guidelines for breast cancer in Tanzania recommend that all breast cancer patients receive hormone receptor (HR) testing, current pathology capacity is unable to meet this need. Pathologic diagnosis, including HR testing, is critical to determining the presence of cancer, extent of the disease, and planning treatment. However, HR testing is available at only two public hospitals in Tanzania. As a result, patients encounter turnaround times of weeks to months, with only a subset of eligible women receiving HR testing and guideline-concordant treatment. To reduce this evidence-to-practice gap, we propose to systematically identify the most effective implementation strategies across all public referral hospitals with pathology services in Tanzania to guide development of a multi-faceted and adaptable intervention (set of implementation strategies). We hypothesize that this intervention will increase the proportion of women who receive HR testing and guideline-concordant endocrine therapy, resulting in improved cancer care, quality of life, and overall short term survival of patients. First, we will conduct a formative evaluation to determine the organizational readiness for change and to identify barriers and facilitators to routine HR testing using mixed methods. Then, we will determine the optimal implementation strategies for the barriers and facilitators identified in the formative evaluation, and develop a multi-component intervention with methods informed by organizational theory. Finally, we will iteratively pilot test the multi-component intervention and will modify the intervention components based on outcomes measures. These studies will be led by Dr. Dianna L. Ng, a junior faculty member in the Department of Pathology at Memorial Sloan Kettering Cancer Center (MSK) with an interest in developing and testing theory-driven, multi-level interventions to increase integration of evidence-based cancer diagnostics in low-resource settings. The research will be carried out under the multidisciplinary mentorship of Dr. T. Peter Kingham (surgical oncologist, global oncology research), Dr. Jamie Ostroff (clinical psychologist, implementation science), Dr. Edda Vuhahula, (pathologist, capacity building in East Africa), and Dr. Britt-Marie Ljung (pathologist, resource- stratified diagnosis). MSK offers an outstanding environment for cancer care delivery and health services research. To achieve her goal of becoming an independent researcher, Dr. Ng has developed a structured career development plan aimed at advancing her knowledge and advanced skills in implementation science in global health.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Nearly all (95%) patients diagnosed with lung cancer report perceiving stigma, defined as a perception and internalization of negative appraisal and devaluation by self and others attributable to a lung cancer diagnosis. Prior research indicates that 48% of patients with lung cancer experience stigma during clinical encounters with their oncology care providers (OCPs), which may be potentially triggered and/or exacerbated by OCPs’ routine assessment of smoking history. Perceived stigma has negative effects on patients’ psychological well- being as well as their medical outcomes. Promoting empathic communication appears to be a potentially effective intervention target to help reduce patients’ perceptions of stigma within clinical encounters; however, no formal trainings exist that focus on teaching empathic communication to OCPs. To address this key need, we developed an Empathic Communication Skills (ECS) training focusing on the communication challenges inherent in OCPs’ discussions of smoking behavior and history with lung cancer patients. Building upon favorable findings from a prior R21 (R21CA202793), our goal is to conduct a national trial of ECS training to facilitate improvements in the medical and psychosocial care of lung cancer patients through de-stigmatizing interactions with OCPs. We will conduct a cluster randomized trial at 16 lung cancer care delivery sites, comparing ECS training (intervention group) with a Waitlist Control Group (WLC) among 160 OCPs (thoracic oncology physicians and advance practice providers) and 960 lung cancer patients (6 patients per clinician). The ECS training will be offered remotely and include all the didactic and experiential training materials that were developed for the pilot trial. To increase the real-world generalizability of our trial, we will leverage two national networks of community oncology practices, the Care Continuum Centers of Excellence coordinated by our lung cancer patient advocacy partner, the Go2 Foundation for Lung Cancer and the Extension for Community Healthcare Outcomes (ECHO) sites for lung cancer care, supported by an American Cancer Society ECHO Hub. The aims of this study are (1) to evaluate the impact of the ECS training on OCP primary outcomes (communication and empathic skill uptake) and secondary outcomes (training appraisal – relevance, novelty, clarity; self-efficacy, attitude towards communication with patients); (2) to evaluate the impact of the ECS training vs. WLC on patients’ reported primary outcomes (lung cancer stigma), and secondary outcomes (perceived clinician empathy, satisfaction with communication, psychological distress, social isolation, and patients’ experience of clinical encounter). Additionally, acceptance of referral to tobacco cessation (for current smokers) and relapse prevention (for former smokers) will be explored; and (3) to examine potential moderators of OCP and patient outcomes. Our central hypothesis is that the ECS training will demonstrate significant improvements in clinicians’ uptake of empathic skills and self-efficacy and will be superior to WLC with regards to patient reported measures of stigma, clinician empathy, satisfaction, and overall experience.

NIH Research Projects · FY 2025 · 2021-09

Abstract The MSK Genomic Data Analysis Center for Tumor Evolution seeks to implement tools, best practices and analytical workflows for studying cancer evolution from cancer genome and transcriptome sequencing data. Over the last 15 years, survey sequencing of patient populations of many cancer types has elucidated novel driver mutations which are mechanistically responsible for disease pathogenesis. The Cancer Genome Atlas (TCGA) and individual laboratory efforts have broadened the understanding of biological processes impacted by somatic mutation and revealed new therapeutic targets that have achieved clinical impact. However, most of this work has been based on bulk DNA sequencing from primary tumors and single biopsies from patients. It is well understood that cancer is an evolutionary process during which clonal expansions within patients generates heterogeneity and phenotypic diversity of cell populations across metastatic sites over time (with or without therapeutic intervention). Indeed, the same targeted therapies developed based on mutation discoveries often select for resistant clones, keeping durable cures out of reach. We will develop analytical methods, tools and software infrastructure to study cancer progression through the lens of evolution, shifting emphasis from analysis of primary tumors to dynamic analyses over clinical trajectories. We expect our program will advance the ability to study clinical trajectories of patients in a more comprehensive approach, including temporal, spatial and single cell analysis to better represent the full clonal repertoires of tumors and to study the determinants of how and why tumors evolve. We use tools, well established in our laboratories, in three key areas: i) variant interpretation from metastatic and post-treatment samples for discovery of therapeutic resistance mutations (Aim 1); ii) multi- sample analysis across anatomic space, and/or time series data from serial biopsy or cell free DNA to track and model clonal dynamics (Aim 2); iii) single cell approaches for clonal decomposition and clone-specific phenotyping within patients (Aim 3). Our team is well positioned to carry out our objectives having developed leading software infrastructures supporting TCGA and clinical sequencing through MSK-IMPACT, development of clinically approved assays for longitudinal monitoring of patients through cell free DNA sequencing (MSK- ACCESS) and through study of clonal evolution at bulk and single cell resolution. We will implement and improve tools to support each of these aims, including Cancer Hotspots, OncoKB, and cBioPortal for Aim 1, PyClone and fitClone for Aim 2 and CloneAlign and CellAssign for Aim 3, tailoring and customizing software to support investigations into the dynamic and evolutionary nature of human cancers. These tools comprise a software infrastructure focused on cancer evolution through variant allele interpretation, multi-sample analysis and single cell investigation. Our infrastructure will enable researchers to automate evolutionary interpretation of disease dynamics to better understand the clinical end points of metastatic progression and therapeutic resistance.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract: Inflammation has long been known to increase risk of tumorigenesis. Epithelial barrier tissues are perpetually exposed to a myriad of environmental and inflammatory insults that necessitates robust regenerative capacity of their tissue-specific epithelial stem cells (EpSCs) to restore barrier integrity. The long term consequences of these inflammatory encounters on the tissue and EpSCs is poorly understood. Here, I seek to understand how exposure to inflammation results in epigenetic and cellular rewiring of EpSCs and their lineages in different barrier tissues and how this rewiring can be maladaptive leading to increased cancer susceptibility. The F99 phase of this proposal is focused on the mechanisms by which inflammatory experience is encoded within the chromatin of skin epithelial stem cells (EpSCs) and how inflammation-experienced skin accelerates tumor formation. I have begun to uncover the molecular mechanisms of how EpSCs acquire and maintain chromatin accessibility at key domains associated with stress response genes that contribute to an inflammatory response. My studies suggest that this phenomenon occurs through the complex and dynamic interplay between transcription factors (TFs) that are naturally present in steady state EpSCs but cannot gain access to stress response enhancers without inflammation-induced TFs. As I unearth the molecular mechanisms involved, I will interrogate how this inflammatory rewiring of skin EpSCs epigenome accelerates tumor formation as EpSCs acquire oncogenic mutations that lead to squamous cell carcinomas (SCCs), a life-threatening, metastatic cancer for which there are few effective therapies. At the completion of the F99 phase, l will have gained strong experience in in vivo high-throughput epigenetics, mouse genetics and epithelial stem cell biology, and transition to a postdoc to gain advanced expertise and training in human cancer and immunology. For the K00 phase, I will shift my focus to how inflammatory experience can reshape colonic epithelium composition in the gut and how this reshaping, along with EpSC epigenetic rewiring, results in colorectal cancer (CRC). Interestingly, colitis can result in colitis-induced CRC that follows a different molecular driven pathogenesis than traditional CRC. Thus, to further understand the mechanisms that drive colitis-induced CRC, I plan to expand my technical expertise to include colitis-induced CRC modeling, single-cell epigenomic and transcriptomic techniques, genetic screening and human organoid modeling. These new approaches coupled with my already strong background in molecular biology and high-throughput genomic analyses, will allow me to address the most pressing and challenging issues in inflammation experience and cancer biology today. With the aid of this award, I intend to continue my research contribution and gain the necessary experience to become an Assistant Professor at a major academic institution. There I will head my own lab and guide my students in epithelial cancer research with the ultimate goal of finding new targets to treat these aggressive cancers.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ ABSTRACT Ionizing radiation (IR), a highly effective cancer therapy, is known to induce cellular senescence, a cell cycle arrest program triggered by the DNA damage response. Cellular senescence results in upregulation of mRNAs encoding secreted factors (including inflammatory cytokines and chemokines), a program referred to as the senescence-associated secretory phenotype (SASP). The SASP modulates the tumor microenvironment, and it has pleiotropic immune-modulatory effects. Although IR sensitivity is known to depend upon host immunity, the contribution of senescence and the resultant SASP is unknown. Our preliminary data suggest that the SASP contributes to both localized and systemic anti-tumor effects of IR in immunocompetent, but not immunodeficient, mouse models. Thus, we hypothesize that the anti-tumor effects of IR, both local and systemic, are mediated in part by the SASP through an immune effector. This project will use a combination of mouse and human models to test this hypothesis and to define the mechanisms through which the radiation- induced SASP alters both local and distant tumor microenvironments. In addition, it will use patient tumor samples to establish clinical relevance in patients with rectal cancer treated with radiation therapy or with radiation combined with immune checkpoint blockade. While this proposal focuses on rectal cancer, it has broad clinical implications, as radiation therapy is a widely used treatment modality for cancer. Dr. Paul Romesser has outlined a 5-year career plan that builds upon his clinical training in radiation oncology and on his research background in cancer biology, radiation biology, genetically engineered mouse models, and patient-derived models. Dr. Romesser will be mentored by Dr. Scott Lowe, an internationally renowned expert in senescence, p53 biology, and mouse modeling with a strong track record of training physician-scientists. Dr. Romesser’s career development plan includes research experience, course work, workshops, and mentoring from an interdisciplinary advisory committee comprising distinguished basic scientists, physician-scientists, and radiation oncologists. He will have the support and infrastructure of Memorial Sloan Kettering Cancer Center, a center of excellence in basic, translational, and clinical cancer research. Successful completion of the research project will lead to new approaches for treating patients with radiation therapy and will provide the foundation for Dr. Romesser to transition to a position as an independent investigator with his own laboratory and R01 funding.

NIH Research Projects · FY 2025 · 2021-08

OVERALL ABSTRACT Despite recent advances in the treatment of acute myeloid leukemia (AML), the majority of AML patients relapse following treatment and the overall five-year survival rate for adults with AML remains 25-29%. Thus, an urgent need to improve therapy for AML patients remains. The MSK SPORE in Leukemia will leverage collective efforts to develop effective targeted therapies and immunotherapeutic approaches for several recurrent molecular subtypes of AML, including some which lack therapeutic options entirely. The overall translational aims of the MSK SPORE in Leukemia are to 1) interrogate genetic and molecular pathways required for AML initiation and maintenance; 2) develop novel targeted therapies and immunotherapeutic approaches for AML based on recurrent genomic alterations and leukemia stem-cell (LSC) specific markers; and 3) identify and validate the mechanism of action, therapeutic efficacy, and predictors of response/resistance of mechanism-based therapies for AML patients. To pursue these aims, we have assembled a multidisciplinary team with complementary expertise in the clinical management of AML, cancer genetics, cancer epigenetics, functional genomics, molecular pathology, biostatistics, computational biology, and multiplatform data integration. We will pursue these aims through four projects, each addressing a different unmet need in the clinical management of AML. Project 1 will elucidate genetic and epigenetic mechanisms of IDH inhibitor therapeutic resistance and perform a clinical trial exploring the efficacy and safety of combining the FLT3 inhibitor gilteritinib with mutant selective IDH1/2 inhibitors for FLT3/IDH-mutant AML. Project 2 will characterize the clinical, molecular, and biological features of complex karyotype (CK) AML, for which there is no treatment, and validate a novel approach to targeting CK AML via inhibition of the metabolic enzyme oxoglutarate dehydrogenase (OGDH). Project 3 will evaluate a novel therapeutic approach for targeting common, poor prognosis spliceosomal-mutant AML subtypes via inhibition of protein arginine methyltransferases in preclinical models and a phase I/II clinical trial. Project 4 will determine the safety and efficacy of a chimeric antigen receptor (CAR) T cell approach targeting a leukemia stem cell-specific antigen while sparing normal hematopoietic stem cells, specifically, a fully humanized CD371 targeting CAR T cell platform bolstered by constitutive IL-18 secretion. All projects will be supported by the Biospecimen, Biostatistics, Genomics, and Bioinformatics Shared Resource Cores, which will assist with the preparation and analysis of human tissues and genomic, immune, and clinical data, and an Administrative Core to ensure project integration. Finally, pilot projects in the Developmental Research Program and career mentorship via the Career Enhancement Program are fully integrated into the SPORE to ensure that a future generation of researchers is prepared to further advance our long-term objectives of enhancing therapy, reducing the morbidity of treatments, and ultimately eliminating this disease as a cause of premature death

NIH Research Projects · FY 2025 · 2021-08

Abstract Natural genetic variation impacts most human diseases, yet predicting how regulatory variants control gene expression and ultimately disease phenotypes poses considerable challenges. First, the polygenic inheritance influencing most conditions requires consideration of a vast number of genes and regulatory elements. This task is challenged by the complexity of gene regulation, where 3D regulatory interactions can link enhancers and genes over large genomic distances. Second, multiple interacting cell types are often dysregulated in disease pathology. This necessitates an understanding of how the collective variants associating with a disease affect each cell type involved in the disease process and subsequently how these dysregulated cellular phenotypes crossregulate and drive subsequent cellular states. In this IGVF project, we will use rheumatoid arthritis (RA), a human autoimmune inflammatory disease, as a case study to develop robust machine learning models of gene regulation to decipher the impact of genomic variation on multiple cellular drivers of pathology—namely, inflammatory T cell and fibroblast subsets found in affected joint tissue. The choice of RA is motivated by its public health importance, specified target tissue, access to clinical samples, considerable knowledge of disease-associated gene loci, and our team’s complementary expertise in machine learning, RA pathophysiology, immunology and inflammation, and single-cell functional genomics. We will develop an advanced machine learning framework to model the effects of allelic variation on gene regulatory networks based on the analysis of epigenomes, transcriptomes, and connectomes of mouse activated T cells and synovial fibroblasts and extend these models to RA patient joint tissue and primary cells. We will train allele-specific gene regulatory models (GRMs) that account for long-range regulatory interactions by integrating single-cell transcriptome and epigenome (sc-multiome) data with bulk 3D interactome analyses. A notable feature of our approach is that we leverage the genetic diversity of evolutionarily distant F1 hybrid mice to provide robust training data for these models, and then apply these advances to the human context through transfer learning. Highly parallelized Perturb-seq experiments in primary synovial fibroblasts from RA patients with single-cell multiomic readouts will then be used to evaluate and refine regulatory models and to train network models that connect gene expression programs to phenotype. Finally, we will combine spatial and single-cell transcriptomics conducted on samples from RA inflamed joints to model the organization and interactions between T cells and sedentary tissue-organizing fibroblasts within local cellular communities. The predictive GRMs that will be generated from our study along with the experimental systems for human disease will be readily transferrable to other polygenic disorders which must consider complex regulatory genomic networks for various interacting cell types in affected tissues.

NIH Research Projects · FY 2025 · 2021-08

PROJECT ABSTRACT The central mission of the T32 “Molecular Imaging in Cancer Biology” program (T32 MICB) will be to develop novel molecular imaging methods, technologies, and platforms that will accelerate the understanding of human cancer biology as a basis for designing curative cancer therapeutic regimens as well as cutting-edge diagnostic, prognostic, and therapeutic tools. This overall goal will be achieved under the auspices of three key themes: (1) “Imaging Fundamental Biology,” (2) “Imaging New Model Systems,” and (3) “Imaging Technology Development.” This T32 MICB research portfolio is situated at the intersection of various disciplines—from basic science including chemistry and physics, structural biology, drug development, cell biology, and developmental biology to cancer biology, invasion and metastasis, and applied disciplines, including pharmacology, nanotechnology, radiochemistry, engineering, and medicine. Fundamental knowledge about the biology of cancer has burgeoned, but the translation of basic science discoveries to clinical advancements can be slow and inefficient; molecular imaging can significantly accelerate this process and a well-trained population of basic scientists well-versed in all aspects of molecular imaging is crucial to the success of this endeavor. The faculty preceptors chosen for this T32 MICB reflect the broad range of expertise and are leaders in their respective imaging fields. They are well-funded and have independent R01 (cancer-related) or R01-like research support (e.g., R35 or HHMI). To ensure that the trainees (five post-doctoral slots and one pre-doctoral slot per year) have a full well-rounded training experience (including resilience and well-being) in preparation for independent careers, our initial design is based on a three-year training program for both the postdoctoral fellows and the predoctoral graduate student trainees. Prior to entry into the T32 MICB program and during the program, the trainees will be informed about the mission of the T32 MICB program, which is to produce scientists with specialized knowledge in advances in cancer biology and imaging technology, within a culture of respect and engagement. Furthermore, trainees will be informed about expected achievements during the program and that their progress will be continuously evaluated and benchmarked.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Predicting the impact of genomic variation requires quantitative modeling to deconstruct the interplay between multiple individual variants and to determine their combined effects on gene regulatory networks (GRNs) that control cell state and cell function. We focus on the GRNs that control early human development as a paradigm. Arguably the most important lineage decision during mammalian development is the decision of epiblast cells to exit the pluripotent state (a state when the cells have the potential to give rise to all somatic cells and germ cells), and differentiate into one of the three primary germ layers, the endoderm, mesoderm, and ectoderm. This pluripotent state and the trilineage differentiation can be captured using cultured human embryonic stem cells (hESCs). Much attention has focused on the GRNs underlying the maintenance of the self-renewing pluripotent state, but the GRNs governing hESC trilineage differentiation remain largely unexplored. We previously conducted genome-scale CRISPR/Cas screens to discover protein-coding genes that regulate the transition of hESCs to definitive endoderm. Based on the genomic and genetic data and machine learning (gkm-SVM sequence analysis), we expanded our initial simple two transcription factor (TF) model to a multiple TF cooperative model. Here we propose an integrative approach examining the hESC transition to definitive endoderm, mesoderm and neuroectoderm germ layer identities to improve the generalizability of GRN models. We will perform quantitative genomic and proteomic measurements with high temporal and single-cell resolution. These quantitative measurements will be combined with perturbation of key GRN elements, core TFs and their target enhancers, to inform the generation of dynamic GRN models. To further improve the precision of our new GRN models, we will map cell trajectories during state transitions through lineage tracing combined with scRNA-seq. Beyond hESC guided differentiation, the physiological relevance of enhancers will be further interrogated in human and mouse organoids (gastruloids) and mouse embryos. We will then apply innovative new computational and algorithmic methods to our multimodal experimental data to generate GRN models, aiming to learn generalizable principles underlying the contribution of genomic variants to cellular and ultimately organismal phenotypes. Developing GRN models for the exit of pluripotency and the acquisition of germ layer identities involves dynamic modeling of the cell state transition, which will not only inform our understanding of early human development, but can also serve as the basis for construction of generalizable GRN models for biological transitions during embryonic development, adult tissue homeostasis and regeneration as well as inappropriate cell fate transitions that occur in pathological conditions such as cancer.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Pancreatic cancer has the highest mortality rate of all cancers, with a 5-year survival rate of only 9%. Surgery still represents the only curative treatment option, though less than 20% of patients are candidates for resection. Approximately 30-40% of patients present with locally advanced unresectable tumors with no significant chance of long-term survival through standard treatments. The use of ablative radiation doses (biologically equivalent doses of 100Gy) produces results that are comparable to surgical resection in patients with inferior prognostic features. However, organ motion, due to respiratory motion, must be managed to minimize toxicity in the gastrointestinal tract. In this project, we will develop novel real-time volumetric MRI technology that can guide radiotherapy to enable the use of ablative doses with minimal risk. Our technique, called MR SIGnature Matching (MRSIGMA), pre-learns 3D motion states and assigns unique motion signatures during an offline learning phase and performs fast signature acquisition and matching during an online matching phase. We have demonstrated real-time tracking of liver tumors with an imaging latency (acquisition plus reconstruction) of about 250 ms using MRSIGMA. We will collaborate with Elekta to implement MRSIGMA on the Unity MR-Linac system and to link the output of MRSIGMA with the multileaf collimator (MLC) system to enable the radiation beam to track the 3D position and shape of the moving tumor in real-time. Specific Aims are as follows: 1. Develop deep learning reconstruction of undersampled dynamic MRI data for rapid motion database generation during offline learning and adaptation during online matching a. Develop a convolutional neural network for rapid reconstruction of motion-resolved data (< 10 seconds) b. Detect anatomical changes, such as motion baseline drifts, and adapt the motion database accordingly c. Perform initial validation on a dynamic MRI phantom and ten volunteers 2. Validate the potential of MRSIGMA for real-time volumetric tumor motion imaging on fifty patients with locally advanced unresectable pancreatic cancer a. Accuracy hypothesis: real-time MRSIGMA is noninferior to a non-real-time XDGRASP reference b. Reproducibility hypothesis: two MRSIGMA scans present equivalent real-time imaging performance 3. Develop and validate on dynamic phantoms the proposed MRSIGMA-guided MLC tracking in collaboration with Elekta a. Develop software to control the MLC with the output of MRSIGMA b. Evaluate tracking latency, geometric error, reproducibility and dosimetric accuracy

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY / ABSTRACT Small cell lung cancer (SCLC) is characterized by rapid growth, early dissemination, and exceptionally poor prognosis. The Rudin laboratory has focused on the study of SCLC for over 2 decades using a fully integrated platform of basic discovery and clinical translational research. Our laboratory has driven fundamental advances in the understanding and characterization of SCLC. We have excelled in successfully translated many discoveries made by our group into clinical testing, including many active trials currently being conducted by our clinical team. Closing the circle, we are also deeply engaged in the molecular characterization of biospecimens from patients receiving these novel therapies, to better inform new directions of laboratory research while maintaining direct disease relevance. In this R35 we will focus primarily on three main areas of future focus for our group. (1) Recent extensive single cell profiling data from our laboratory has defined the exceptional intra- and inter-tumoral heterogeneity of primary human SCLC. We have identified a key subpopulation with stem-like capacity, present in tumors of all SCLC subtypes, and also identified and characterized a novel tumor-infiltrating macrophage subtype exclusively associated with SCLC. The stem-like cell population expands progressively in nodal and distant metastases, and high fraction of this subpopulation confers a strikingly poor clinical prognosis. Defining, characterizing, and targeting these novel cell types could have a transformative impact on patients with SCLC. (2) We have a long-standing interest in lineage plasticity, including histologic transformation from lung adenocarcinoma to SCLC, and tumor evolution between SCLC subtypes. We now have multiple relevant tools in hand to dissect the biology of lineage plasticity in lung cancer, including patient-derived xenograft (PDX) models of lung adenocarcinoma that under different conditions transition to different subtypes of SCLC. We will deeply analyze the drivers of SCLC transformation as a mechanism of tumor escape and acquired resistance in lung cancer. These data will inform approaches to prevent or restrict lineage plasticity as a driver of therapeutic resistance. (3) We have developed new technology allowing controlled in vivo CRISPR/Cas9 gene editing in PDX. We will adapt this system to conduct focused SCLC genetic dependency screens in vivo using a guide RNA library covering the druggable genome. Applied across all subtypes of SCLC, this approach will define novel therapeutic targets for this recalcitrant malignancy.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Melanocytes are a key cell lineage in the skin and are required for skin pigmentation. Defects in melanocyte development and function can lead to pigmentation disorders causing hypo or hyperpigmentation as well as one of the most deadly forms of skin cancer, melanoma. Many of the transcriptional and signaling events which cause these diseases have been explored, however, there has been little investigation into the role of the metabolic environment and intracellular metabolic state of the melanocyte during development. Recent work from our lab has demonstrated that during melanoma progression, adipocytes in the tumor microenvironment can transfer fatty acids to the melanoma cells, promoting invasion. This raises the question of whether fatty acid uptake plays a role in normal melanocytes. Our preliminary data suggests that melanocytes require extrinsic uptake of fatty acids through Fatty Acid Transport Proteins (FATPs) for proper differentiation. In Aim 1, we will investigate the relative importance of fatty acid uptake and de novo fatty acid synthesis in the melanocyte. We will compare multiple mechanisms of fatty acid uptake as well as fatty acid synthesis to determine which play the most important role in melanocyte development and pattern formation. We will also dissect in a stage specific manner when these pathways are required by the melanocyte during differentiation using genetic perturbations. We hypothesize that the importance of fatty acids in the melanocyte reflects certain lineage specific needs, including high levels of fatty acids to support phospholipid synthesis and dendrite formation. In Aim 2 we will examine the importance of fatty acids as building blocks for phospholipid synthesis relative to a requirement for fatty acid breakdown through b-oxidation. There is evidence for the importance of phospholipid synthesis in other dendritic cell types. Because melanocytes also form extensive dendrites which are critical for proper pigmentation we will dissect the importance major phospholipid synthesis pathways to the melanocyte in a stage specific manner. Conversely, melanocytes might break down fatty acids as a source of ATP to support cell proliferation and migration. We will also determine the role b-oxidation in melanocyte development by targeting key proteins in this pathway in a stage specific manner. We will assess the effects of these genetic perturbations on energy production and melanocyte development. To perform these studies we will primarily rely on zebrafish as an in vivo model of melanocyte development. We will use stage specific promoters for the neural crest, melanoblast, and melanocyte to generate stable germline transgenics. Using a combination of genetic methods and image analysis we will investigate the role of fatty acid and lipid metabolism in melanocyte development. When appropriate, we will also apply an in vitro model of melanocyte development, based on the differentiation of melanocytes from human pluripotent stem cells These combined experiments will lead to novel discoveries regarding the role of metabolism in melanocyte development, which could increase our understanding and treatment of pigmentation diseases.

NIH Research Projects · FY 2025 · 2021-07

SUMMARY. The problem: Hormone therapy remains the standard treatment for prostate cancer (PC). While most patients initially respond to therapy, they will ultimately progress to lethal castration-resistant PC (CRPC) within 6 to 12 months. During progression, canonical sources of androgens are replaced with mechanisms that trigger PC growth, even in the absence of hormones, making therapy at this stage very difficult. Proposed solution: We will utilize a newly discovered biological function of prostate-specific membrane antigen (PSMA) to develop an entirely new personalized therapeutic strategy for PC that is significantly different from existing therapies. High levels of PSMA are seen in most aggressive forms of PC and are a predictor for progression. However, the biological role of PSMA remains unknown. Our proposal is based on our recent paradigm-shifting discovery that PSMA provides a so far unknown oncogenic signaling function, where its enzymatic activity triggers an intricate intracellular signaling repertoire, promoting cancer growth through activation of the Pi3K/AKT/mTORC-1 as well as the mTORC-2 signaling cascades. Having charted the interface of PSMA with the main biological signaling cascades in PC, we provide here a novel therapy that disrupts these major signaling pathways. Importantly, inhibition of PSMA led to a survival benefit in mice. We developed in parallel a companion imaging assay to diagnose and monitor PC with a cheap and facile ex vivo bioluminescence-based assay from readily available samples. In this assay glutamated luciferein (GluLuc) is cleaved specifically by PSMA to release luciferin that can be detected by luciferase in urine/prostatic secretions with a bench top assay system. We have already evaluated this assay in mice and patients and have shown that it is superior to the gold standard of PSA levels. Here, Aim 1 will focus on our companion imaging assay. We will measure luciferin released by PSMA in expressed prostatic secretions urine (EPS/U) or urine and correlate the bioluminescence signal with local and metastatic tumor burden and growth. Aim 2 will explore inhibition of PSMA as therapy for PC as monotherapy or in combination with androgen inhibition. Since inhibition of PSMA activity will lead to reduced release of luciferin, we will correlate the reduction of signal with the tumor response. We will repurpose PSMA inhibitors, with a proven safety profile for CNS disease that were abandoned due to low CNS penetration. In Aim 3, we will explore if PSMA inhibition can be used as therapy of CRPC and to prevent metastasis formation. We will also test if it can delay onset of castration resistance. As in Aim 2, we will monitor therapy with the benchtop assay. Tumor growth will be interrogated with MR and PET imaging Our work is significant, as it charts an entirely new path for PC therapy based on the specific biological function of PSMA. The underlying biology of PSMA is highly innovative, as it has never been explored in spite of its significant biological consequences. Furthermore, we also demonstrate an innovative, facile, and inexpensive ex vivo benchtop approach to diagnose, monitor PC and monitor therapy. Ultimately, this will benefit all patients with PC, particularly those with CRPC.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a lethal blood cancer characterized by a clonal expansion of precursor blood- forming cells. Intensive chemotherapy has been the mainstay of AML therapy for decades. Unfortunately, not all patients are fit enough to receive it and mortality due to relapse despite intensive treatment is common. Recently, the FDA approved a lower intensity regimen combining a hypomethylating agent, such as azacitidine (aza), with the BCL2 inhibitor venetoclax (ven), based on phase 3 randomized data showing an overall survival benefit and high response rates across AML prognostic subtypes. The success of aza/ven highlights the apoptotic pathway as an exciting therapeutic target. Venetoclax induces apoptosis by antagonizing the anti-apoptotic function of BCL2, one of many mitochondrial BH3-domain proteins that regulate the threshold at which an AML blast dies. This apoptotic threshold, or priming, in viable leukemic blasts can be measured via a functional cell death assay, called BH3 profiling. I have recently demonstrated that the cell of origin of leukemic transformation influences apoptotic priming and resultant therapeutic sensitivity via alterations in p53 activity. I am interested in understanding how AML cell state, whether established by AML genotype or apoptotic priming, can influence drug sensitivity and clinical outcomes in the context of attenuated p53 function. I hypothesize that BH3 profiling of AML patient samples can serve as a biomarker to predict treatment response to aza/ven. I also hypothesize that complex cytogenetic changes – ensuing from mutant TP53-induced genomic instability – promote AML progression and therapeutic resistance to aza/ven independent of mutant TP53. I believe that this work will address important biological questions with therapeutic implications: 1. Can BH3 profiling assays predict treatment response to aza/ven in xenograft mouse models? 2. Which transcriptional and epigenetic pathways are engaged in AML cells with low apoptotic priming and blunted responsiveness to aza/ven? 3. Is complex karyotype AML in the setting of TP53 loss of function a bystander phenomenon, or does it enhance leukemogenicity and/or resistance to therapies such as aza/ven and chemotherapy? Dr. Sheng Cai, an Assistant Attending at MSKCC, will conduct this study as part of his career development plan, dedicating 85% of his time to research. Dr. Cai is mentored by Dr. Ross Levine, a world expert in hematologic malignancies. He is also advised by Drs. Anthony Letai (who developed the BH3 profiling assay), Scott Lowe, Michael Kharas, Richard Koche, and Andriy Derkach. Dr. Cai's training will include gaining knowledge in biomarker validation and expertise in bioinformatics and genetic mouse models, with the long term goal of developing a research program as an independent investigator in hematologic malignancies developing functionalized biomarker assays for precision oncology.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT With recent enactment of the Research to Accelerate Cures and Equity (RACE) for Children Act, there is a growing impetus to identify pediatric indications for molecularly targeted drugs. Accordingly, there is a pressing need for clinically relevant preclinical studies that can help prioritize pediatric indications for clinical application of nearly the entire universe of cancer drugs currently in development. To meet this need, we established a preclinical testing program that has created >300 genomically-characterized pediatric solid tumor patient- derived xenograft (PDX) models between the pediatric oncology programs at Memorial Sloan Kettering Cancer Center and University of California San Francisco. We propose to leverage this large portfolio of models across a diversity of diseases, along with the deep expertise of the team, to establish a NCI Pediatric In Vivo Testing Program (Ped-In Vivo-TP) Research Team focused on pediatric bone and soft tissue sarcomas, renal tumors, desmoplastic small round cell tumor (DSRCT) and other rare pediatric solid tumors. The Aims of this Research Team are: Aim 1. Integrate with the Coordinating Center and other Research Teams to prioritize agents for preclinical evaluation. We will leverage our team's translational expertise as well as existing connections to disease committees within cooperative groups to inform and facilitate new agent selection and preclinical evaluation in appropriate models to prioritize agents to advance into pediatric oncology clinical trials. Aim 2. Utilize PDX portfolios representative of disease heterogeneity to assess therapeutic agents. We will utilize the >300 PDX models from MSKCC and UCSF, supplemented with models from the PROXC consortium where necessary, to assess 8-10 therapeutic agents/year using study designs matched to the therapeutic question. All preclinical drug testing will be conducted at MSKCC. Aim 3. Align central and local data analyses to ensure rigor in results reporting. For the purposes of prioritization, we will maintain equipoise in validating (“go”) or invalidating (“no go”) therapeutic hypotheses, and will complement central data analyses with advanced local biostatistical expertise. Aim 4. Translational biomarker discovery. We will leverage the combined expertise of our Research Team to identify clinically translatable biomarkers predictive of enhanced response or drug resistance. In some cases, predictive biomarkers may not be genetic, but instead will depend on transcriptional or protein-based assays of target activity. In other cases, genomically identified biomarkers have to be translated to clinically utilizable assays. Together, the expertise and capabilities of this Research Team will support the rigorous evaluation of novel therapeutic hypotheses in clinically-representative models to enable prioritization and translation of the most promising emerging agents into biomarker-informed clinical trials for children with high risk solid tumors.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Inflammasomes detect intracellular danger-associated signals and trigger an inflammatory form of cell death called pyroptosis. The danger signals that the related NLRP1 and CARD8 inflammasomes sense are unknown and represent a major knowledge gap. Interestingly, small-molecule inhibitors of the serine proteases DPP8 and DPP9 (DPP8/9) were recently discovered to induce a danger signal that activates the NLRP1 and CARD8 inflammasomes. However, DPP8/9 inhibitors, in contrast to other inflammasome activators, induce pyroptosis in only a fraction of sensitive cells over relatively long time periods. Thus, it is possible that the co-occurrence of a second danger signal with DPP8/9 inhibition is required for full and rapid NLRP1 and CARD8 activation. The central hypothesis of this application is that a lack of reactive oxygen species, or reductive stress, is the second danger signal required to fully activate these inflammasomes. This hypothesis has been formulated on the basis of preliminary data produced in the applicant’s laboratory and described in the application. The long- term goal of this project is to understand why reductive stress is a danger signal that is closely monitored by the innate immune system. The immediate objective of this application is to determine the molecular mechanism by which reductive stress activates the NLRP1 and CARD8 inflammasomes. This project consists of three specific aims: 1) to characterize the impact of oxidants and antioxidants on NLRP1 and CARD8 activation; 2) to determine the mechanism of GPX1-mediated NLRP1 and CARD8 inactivation; and 3) to determine how TRX1 modulates NLRP1 activation. Successful completion of this project will fill a critical knowledge gap by showing that reductive stress is a key danger signal that activates the NLRP1 and CARD8 inflammasomes. Overall, this work holds tremendous promise to reveal a fundamental new connection between metabolic stress and innate immunity, and to eventually enable these complex inflammasomes to be harnessed for therapeutic benefit.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY The incorporation of Immune checkpoint blockade into anticancer armamentarium has revolutionized cancer therapy in recent years, with FDA approved agents in multiple cancer types. Nevertheless, there is an urgent need to identify additional therapeutic strategies to increase durable response rates. Induction of immunogenic cell death is one of such strategies. The BCL-2 family proteins are central regulators of mitochondrial apoptosis and consist of (1) multidomain antiapoptotic BCL-2, BCL-XL, and MCL-1, (2) multidomain proapoptotic BAX and BAK, and (3) proapoptotic BH3-only molecules (BH3s). BAX and BAK are the essential effectors of MOMP whereas BCL-2, BCL-XL, and MCL-1 preserve mitochondrial integrity. BH3s are death sentinels that relay upstream apoptotic signals to initiate apoptosis by either activating BAX/BAK or inactivating BCL-2/BCL- XL/MCL-1. Through an interconnected hierarchical network of interactions, the BCL-2 family proteins integrate developmental and environmental cues to dictate the survival versus death decision of cells. The research on the BCL-2-regulated apoptotic pathway has not only revealed its importance in both normal physiological and disease processes, but has also resulted in the first anti-cancer drug targeting protein-protein interactions. Recent paradigm-shifting discoveries have shown that BAX/BAK activation in the absence of caspases can trigger the release of mitochondrial DNA to the cytosol through a process called “mitochondrial inner membrane permeabilization” (MIMP), which in turn activates the cGAS/STING pathway and type I interferon response. Hence, the BCL-2 family plays a crucial role not only in the decision of cells to live or commit suicide but also in the decision to die in an immunologically silent or inflammatory manner. The discovery of MIMP and its role in activating immunogenic cell death opens up exciting new avenues for cancer cell death research. However, the molecular and biochemical basis of MIMP remains uncharacterized. Furthermore, it is unclear whether induction of MIMP in tumors will affect the tumor-immune crosstalk and immunotherapy response. In this grant application, we have formulated a comprehensive plan to interrogate the biochemical and molecular basis of MIMP and exploit MIMP as a therapeutic strategy to improve and enhance immunotherapy. Our studies will not only provide novel mechanistic insights into the BCL-2-regulated cell death program but also lay the foundation for targeting the BCL-2 family to induce immunogenic cell death and thereby enhance cancer immunotherapy.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract The mission of the Baylies lab is to deliver basic research findings that will support better therapies across a range of muscle diseases. Our goals are the identification of genes and mechanisms that are essential for the formation and healthy functioning of skeletal muscle, and where these mechanisms go awry in disease states such as muscular disorders (nemaline and centronuclear myopathies), muscle wasting (cachexia, aging), and soft tissue cancer (rhabdomyosarcoma). Specifically, the lab aims to understand key processes that lead to skeletal muscle: cell fate commitment, cell-cell fusion, movement and positioning of organelles such as the nucleus, and muscle fiber growth and maturation. That research is conducted by developing and combining novel genetic, cell biological, imaging, molecular and mathematical approaches, using Drosophila and mammalian muscle cells. Our current investigations focus on a fundamental question: what determines muscle cell size? The mechanisms that control cell size are poorly understood. This is particularly true for a skeletal muscle cell, which may have hundreds of nuclei and is among the largest cells in the human body. Skeletal muscle cells have a remarkable capacity to increase their size in response to exercise (hypertrophy), and to decrease in size upon inactivity, aging, or disease (atrophy). Our work in Drosophila has revealed critical nuclear parameters (number, DNA content, size, activity) that can each be adjusted and coordinated by the muscle cell to generate a particular size. We have also found that the many nuclei in a muscle cell vary in number and activity along the length of a muscle fiber. Key questions we are pursuing over the next five years include: How does a muscle cell generates these regional differences yet globally coordinate the nuclei within a single cell? Are such differences apparent in other organelles? Similarly, what are the specific signals and mechanisms that establish and maintain nuclear identity along the muscle cell; what are the contributions of each nucleus to their local cytoplasmic domain and to the entire muscle cell? How does each nucleus set up its cytoplasmic area and are there regional differences? Finally, under conditions of hypertrophy or atrophy, how are nuclear and cytoplasmic identities and the compensation/communication mechanisms impacted? Altogether, our work will identify defining parameters of muscle cell size under normal, hypertrophic and atrophic conditions, and their physiological range required for muscle function. Our studies will reveal general principles of cell size regulation, provide insight to how improper regulation of these processes results in disease, and inform regenerative medicine aimed at muscle.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract The proposed research will characterize the prevalence and management strategies of gastric intestinal metaplasia (GIM), a lesion of the digestive tract that affects about 12.1 million adults in the United States (US) and is a precursor to gastric adenocarcinoma, or gastric cancer (GC). While mass surveillance of GIM is unlikely to be effective in regions of low-prevalence for GC such as the US, we hypothesize that risk- stratification for targeted screening and surveillance will be cost-effective and can improve outcomes. Extensive GIM has been identified by gastrointestinal society guidelines as a major risk factor for progression to GC, but there is currently a dearth of evidence in understanding the prevalence of GIM overall and by subtype (limited and extensive) in the US population, or how it should be managed. Furthermore, the recently proposed guidelines from the American Gastroenterological Association suggest surveillance of GIM should be considered in racial/ethnic minorities, foreign-born individuals, or those with a history of Helicobacter pylori (H. pylori) infection; however, evidence on the threshold to initiate such interventions and the intervals at which they should be continued is lacking. The research proposed in this K08 application will accomplish three interrelated Specific Aims. In Aim 1, we will utilize data from across the Veterans Affairs health care system to characterize the prevalence of subtypes of GIM and risk factors for progression of GIM to dysplasia or malignancy in the US context. These data will provide a platform for Aim 2, in which we will build a simulation model of the natural history of progression from precancerous gastric lesions to GC to assess which individual- level risk factor profiles could benefit most from screening and surveillance of GIM. The model will utilize 64 different phenotypic profiles, stratified by: gender (male/female), race/ethnicity (non-Hispanic white, Hispanic, black, and Asian), extent of GIM (limited vs. extensive), foreign-born status (immigrant vs. US-born), and H. pylori infection status (previous/current infection vs. no infection). Outcomes will be reported based on number of cancers prevented, survival, and incremental cost-effectiveness ratios (ICERs). Once an optimal profile and screening regimen is identified on the patient level, in Aim 3 we will assess the population-level impact of implementing such a strategy on health care outcomes and costs in the US. The long window of progression from GIM to malignancy and the low incidence of GC make sufficiently powered randomized controlled trials difficult. Simulation modeling allows for the integration of available knowledge to test multiple surveillance regimens of GIM across a broad range of risk factor combinations, which would not be feasible in clinical studies. By award period end, the proposed research will utilize national pathology data coupled with simulation modeling to identify a specific subgroup of high-risk patients that may benefit most from surveillance in the US. The proposal, mentorship and advisory committee, career development plans and institutional support will lay the basis for an independent NIH-funded career.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Rapid detection and response to injury is essential for the survival of all organisms. In animals, wounded tissues must quickly heal and locally regenerate. Failure in wound detection causes acute and chronic conditions ranging from poorly healing wounds and infections to chronically inflamed skins, fibrosis and cancer. Although the exe- cution mechanisms of wound healing (involving cytokines, growth factors, etc.) have been extensively studied, its initiation mechanisms remain little understood. My vision is to develop a genetically and physically plau- sible model of wound detection. There is a fundamental gap in understanding of how wounds are initially detected, and how the first wound signals rapidly transmit information on injury over tissue-scale dis- tances to faraway leukocytes, epithelial, and other cells that participate in healing. I study wound detection in live zebrafish whose wound responses and immune system resemble those of mam- mals yet are better amenable to high-resolution, real-time imaging at high animal throughputs. To this end, my lab combines quantitative intravital imaging with unbiased computational image analysis and various interdisci- plinary approaches ranging from biophysics to mathematical modeling. Over a decade, I have identified three chemical and one physical wound signals: hydrogen peroxide (H2O2), extracellular ATP (eATP), arachidonic acid (AA), and nuclear membrane tension. These discoveries triggered new activity in an old field. Yet, critical mech- anistic gaps remain: How is eATP sensed to mediate rapid wound closure, and how does it instruct faraway cells although it is rapidly broken down in the tissue and cannot diffuse far from a wound? How are H2O2 and AA signals integrated to mediate rapid inflammatory responses to wounds? Wound signals cause inflammation- do they also resolve it? How is wound mechanotransduction regulated on the molecular and cell biological level? These questions are of high basic biological interest, and the pathways they concern are major disease regula- tors. Answering them over the next five years can pave way for novel therapeutic approaches. My work on wound signaling has opened the door to other areas of biology where analogous mechanisms may drive medically important processes, such as infection responses, cancer and bone regeneration/remodeling. Although the primary focus of my group will remain on early wound signaling, I plan to explore some of these new areas, taking advantage of the R35’s flexible funding scheme.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Human cytomegalovirus (HCMV) infects all populations with a penetrance of 50-100% and is kept latent by innate and adaptive surveillance. However, it is a significant cause of morbidity and mortality in conditions of immune reconstitution and suppression, such as in neonates and recipients of solid organ or hematopoietic cell transplants. The T cell response to HCMV through classical HCMV peptide-specific αβ cytotoxic T lymphocytes has been well-studied, and the development of NKG2C+ natural killer cells in response to HCMV infection and reactivation is under active investigation. In addition to these lymphocytes, however, large populations of αβ- TCR CD8 T cells that express NKG2C and other NK-associated receptors have also been observed in HCMV- seropositive healthy donors and patients. These innate-like NKG2C+ CD8 T cells appear to have broad activity against AML and HCMV-infected cells, no activity against uninfected allogeneic fibroblasts, and reduced expression of PD-1 in response to CD3 stimulation. RNAseq analysis has revealed that NKG2C+ CD8 T cells have reduced expression of the transcription factor Bcl11b, critical for cutting off alternative innate fates during the early thymic development of T cells. The central hypothesis of this proposal is that HCMV exposure induces an NKG2C+ CD8 T cell population by diverting clonotypic T cells toward an innate fate through the downregulation of Bcl11b, which alters TCR signaling and promotes alternative recognition pathways beneficial to leukemia patients. The first aim of the proposal is to evaluate the T cell identity of members of the NKG2C+ CD8 T cell population (clonality, TCR specificity and signaling) and how their transcriptional and epigenetic programs are altered from other CD8 T cells by Bcl11b loss. The second aim will assess the function of the NK- associated activating and inhibitory receptors on the NKG2C+ CD8 T cells, with the goal of identifying the mechanism behind their anti-tumor and anti-HCMV activity. Finally, in a collaboration with the Center for International Blood and Marrow Transplantation, an extensive hematopoietic cell transplantation patient sample bank and clinical database will be utilized to determine whether the post-transplantation emergence of an NKG2C+ CD8 T cell population impacts the risk of leukemia relapse and overall survival. Together, the results of these studies will elucidate not only the therapeutic potentials of this innate-like T cell population but also how adaptive and innate fates can be bridged.