Fred Hutchinson Cancer Center

NIH Research Projects · FY 2025 · 2025-06

eDyNAmiC (extrachromosomal DNA in Cancer) Human genes are arranged on 23 pairs of chromosomes, but in cancer, tumour-promoting genes can free themselves from chromosomes and relocate to circular, extrachromosomal pieces of DNA (ecDNA). These ecDNA do not follow the normal “rules” of chromosomal inheritance, enabling tumours to achieve far higher levels of cancer-causing oncogenes than would otherwise be possible, and licensing cancers with a way to evolve and change their genomes to evade treatments at rates that would be unthinkable for human cells. The altered circular architecture of ecDNAs also changes the way that the cancer-causing genes are regulated and expressed, further contributing to aggressive tumour growth. These unique features make ecDNA-containing cancers especially aggressive and difficult to treat. Cancer patients whose tumours harbour ecDNA have markedly shorter survival. Despite being first seen over fifty years ago, the critical importance of ecDNA has only recently come to light, and the scale of the problem is substantial. ecDNAs are present in nearly half of all human cancer types and potentially up-to a third of all cancer patients. The collective current understanding of how ecDNA form, how they function, how they move around the cell, how they evolve to resist treatment, how they impact the immune system, and how they can be effectively targeted are lacking. We bring together an internationally recognized, pioneering interdisciplinary team of cancer biologists, geneticists, computer scientists, evolutionary biologists, mathematicians, clinicians, and patient advocates to boldly create novel insights and resources and to provide transformative solutions to one of Cancer’s Grand Challenges. A core team of experienced and productive ecDNA investigators will work with new investigators in the ecDNA and cancer fields to bring completely new perspectives and approaches to this daunting challenge. By bridging cutting-edge and diverse approaches and insights from cancer genomics, yeast genetics, epigenomics, artificial genome synthesis, longitudinal patient tracking, combinatorial and machine learning algorithms, mathematical modelling, immunobiology, and innovative chemistry we will develop a new understanding of the role of ecDNA in cancer, and we will find new ways to drug the undruggable. This bold programme, which consists of 7 work packages and a committed international infrastructure, generates new and unusual collaborations that would simply be impossible under any other type of funding mechanism. Our programme endeavours to foster bold innovative solutions to one of the hardest problems in cancer and to one of the greatest challenges facing cancer patients.

NIH Research Projects · FY 2026 · 2025-05

ABSTRACT Immune memory is a critical aspect of adaptive immunity. The presence of antigen-specific memory cells can provide immune control against infection, and for this reason, immunological memory is a cornerstone of vaccine protection. Adaptive immunity has long been thought to be the sole domain of B and T cells, but exciting new studies of natural killer (NK) cells memory responses has challenged this paradigm. In the case of HIV, there is evidence from animal models that rare NK cells can mount long-lasting memory responses to HIV antigens, but there is limited data that define HIV-specific NK memory in humans. NK memory has been most clearly demonstrated for HCMV, where recent studies have demonstrated the persistence of NK cell clones using a novel method for cell lineage tracking called ASAP-seq. ASAP-seq uses mitochondrial DNA mutations as endogenous barcodes for long-lived NK cell clonotypes, much like receptor rearrangements are used to mark B and T cell lineages. In addition, this method captures protein expression that can be used to identify markers associated with long-lived NK cell clones. ASAP-seq also captures epigenetic data that provides insights into the cellular changes that are associated with the NK memory cells of interest. Here we propose to apply this method to samples from a unique 30-year cohort of HIV infected women, where there are banked longitudinal samples starting prior to HIV-infection. Thus, we will examine NK cell populations before and after HIV infection, which will enable detection of new NK memory cell clones that arise/expand after HIV acquisition. In addition, we will perform ASAP-seq on HIV+ cases with and without HCMV co-infection to control for the potential confounding effect of NK memory responses to HCMV. Together, these complementary approaches are designed to identify NK memory cell clones that are specific to HIV. If HIV-specific NK memory is discovered through this innovative work, it will lay the foundation for many future studies, ranging from basic studies to define epitope specificity, and HIV-specific NK cell phenotype and function to more applied work to harness these responses to prevent and cure HIV infections.

NIH Research Projects · FY 2026 · 2025-05

Project Summary: While much is known about factors that stimulate the proliferation and differentiation of stem cells, less is known about the factors that prevent their differentiation and preserve their function. One factor implicated in the maintenance of an undifferentiated stem cell state is the non-canoncial Notch ligand, Delta like homologue 1 (DLK1). DLK1 is widely expressed during embryogenesis where it regulates stem cells to control organ size. In the adult, DLK1 expression is limited to select tissues, including hematopoietic stem cells (HSC), where it has been shown to enhance the generation of primitive murine hematopoietic precursors. Yet, the precise effects of DLK1 on stem cell proliferation and differentiation remain elusive. In preliminary experiments using cord blood CD34+ cells, we have found that DLK1 knockdown decreased CD34+ precursors following in vitro culture and reduced their engraftment in immune deficient mice. In contrast, DLK1 overexpression led to an increased proportion of the least mature CD34+CD90lo precursors in vitro and an increased proportion of CD34+ cells in vivo. Moreover, exposure of CD34+ cells to exogenous DLK1 extracellular domain (ECD) increased longer-term engraftment. Using scRNAseq, we observed that DLK1 knockdown in highly enriched HSC shifted the transcriptional state from a quiescent HSC (qHSC) to an activated HSC (aHSC) during short-term culture. These findings compel further assessment of DLK1 effects on HSC quiescence in EC co-culture, where previous studies have shown expansion of repopulating cells and preliminary studies suggest the expansion of transcriptionally-defined qHSC following cell division in vitro. However, the mechanism by which DLK1 elicits these effects is unclear. DLK1, an intrinsically disordered protein, has been reported to inhibit multiple receptor mediated cell signaling pathways activated by extracellular ligand, including the TGFb, FGF, Notch, and insulin pathways, each relevant to hematopoietic stem cell function. In preliminary studies using the TGFb and Notch pathways as models, we have shown that DLK1 inhibits receptor activation via its ECD. Additional studies suggest that ECD phase separation induces clusters in the plasma membrane, termed condensates, that co-localize with TGFb and Notch receptor components, leading to altered receptor dynamics. Herein, we test our hypothesis that DLK1 regulates HSC quiescence by elucidating: whether DLK1 maintains and/or expands qHSC by inducing aHSC to return to quiescence (Aim 1); how DLK1 interactions with receptor components within condensates alters receptor signaling (Aim 2); and whether inhibition of multiple signaling pathways is required for the effects of DLK1 on HSC (Aim 3). If successful, these findings will suggest that DLK1 maintains qHSC by inhibiting the ability of receptors to respond to multiple environmental signals that induce HSC proliferation and differentiation through the alteration of receptor dynamics within DLK1 condensates. These findings will provide the foundation for novel strategies to enhance stem cell engineering, including the expansion of true repopulating HSC for transplantation.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY/ABSTRACT Patients with genetic blood diseases and disorders are commonly treated with blood (hematopoietic) stem cell (HSC) transplants from healthy, human leukocyte antigen (HLA)-matched sibling donors. Unfortunately, for most patients, there is no HLA-identical sibling donor available, and transplantation of HSCs from alternative donors can lead to life-threatening complications such as graft-vs-host disease (GVHD). To avoid severe side effects, especially from GVHD, correcting the patient's own HSCs—a process called gene therapy—has become a promising alternative treatment approach for genetic diseases and disorders affecting the blood. Despite significant progress in the field of HSC gene therapy, currently available treatment strategies are greatly limited by the requirement of highly sophisticated infrastructure in specialized facilities similar to bone marrow transplantations limiting the accessibility of this treatment option for low- and middle-income countries. The main bottleneck is the current inability to safely and efficiently perform HSC gene therapy directly in the patient (in vivo). The ability to genetically modify HSCs in vivo would 1) improve the feasibility of HSC gene therapy, 2) enable portable gene therapy, and 3) avoid expensive infrastructure. With the aim to refine the target for HSC gene therapy, we recently performed comprehensive studies in our nonhuman primate (NHP) stem cell transplantation and gene therapy model. This study identified a highly HSC-enriched cell population that contained all required cells for rapid short-term recovery and robust long-term multilineage engraftment of gene- modified cells providing the ideal target for in vivo gene therapy. Most importantly, we recently reported highly efficient encapsulation of gene-editing nucleases into polymeric and lipid nanoparticles (NPs). NPs can be surface modified for targeting, lyophilized for enhanced portability, and have been successfully used in vivo. Here, we hypothesize that targeting our novel HSC-enriched phenotype in vivo performing injections of NPs directly into the bone marrow stem cell compartment will allow us to overcome current limitations in HSC gene therapy. In Aim 1, we will optimize our NP formulations that can be loaded with next-generation double-strand break independent gene-editing agents and functionalize the surface for targeting. Untargeted as well as targeted NPs will be tested in vitro on cell lines and primary human HSCs for efficiency and specificity. In Aim 2, we will evaluate different versions of NP in the humanized mouse model to determine the biodistribution after intraosseous injections, on-target specificity, and long-term efficiency in vivo. Finally, in Aim 3 we will focus on the scale-up and clinical translation using our preclinical NHP large animal model. We will perform short-term studies to evaluate the biodistribution and safety profile of intramarrow NP injections, long-term studies to monitor persistence of editing, as well as in vivo selection to increase the frequency of gene editing.

NIH Research Projects · FY 2026 · 2025-04

SUMMARY Kinetochores (KTs) are large protein structures assembled upon centromeres during mitosis that bind to microtubules (MTs) of the mitotic spindle to orchestrate and power chromosome movements. We have recently discovered that a high proportion of human glioblastoma isolates suffer from a lethal form of KT stress, which is triggered by oncogenic mitogen-activated protein kinase signaling [Mapk stressed KTs (MaSKs)]. MaSKs arise when the Ras-Raf-MEK-ERK cascade is inappropriately active in mitosis, resulting in hyperstimulation of a network of KT kinases that, in turn, hyperphosphorylate KTs, decreasing their MT binding capacity and causing excessive KT-MT turnover. The purpose of this grant is to reveal mechanistic details of our MaSK model while revealing MaSK relevance to KT and cancer biology. Our overarching hypothesis is that oncogenic RTK-Ras-Raf-MEK-ERK signaling alters the activity of key proteins involved in KT regulation causing cancer-specific destabilization of KT-MT attachments and requirement for MaSK-associated compensatory mechanisms. Aim 1 will investigate the nature, function, and regulation of MaSKs in glioblastoma isolates. Aim 2 will reveal the occurrence of MaSKs in patient tumors. Aim 3 will elucidate MaSK-induced vulnerabilities in cancer and transformed cells. MaSKs are highly relevant to cancer research. Our MaSK model provides a direct mechanism for chromosome instability induced by oncogenic Ras pathway signaling, which is a key knowledge gap. Moreover, we have only found MaSKs in cancer or transformed cells, where they trigger novel genetic and molecular dependencies not observed in normal cells. MaSK-containing cells differentially rely on two non-essential domains of the mitotic protein BubR1/BUB1B to facilitate recruitment of PP2A phosphatase to KTs to counteract MaSK-induced KT- MT instability. Further, MaSKs themselves can serve as biomarkers for tumors and can be leveraged to discover new MaSK-targeted therapies and to define a patient responder class.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY/ABSTRACT The core aim of this study is to systematically investigate and mitigate the impact of preanalytical variables on the accuracy and reliability of targeted, multiple reaction monitoring mass spectrometry (MRM-MS)-based immune protein measurements in cerebrospinal fluid (CSF) from pediatric brain tumor patients participating in clinical trials. Recently, immune therapies such as immune checkpoint inhibitors or intrathecal (i.e., introduced directly into the cerebrospinal fluid) delivery of chimeric antigen receptor T-cell (CAR-T) therapies have shown promise for treating poor prognosis pediatric brain tumors, yielding improved outcomes for some. However, more research is needed to optimize safety, to identify patients most likely to respond, to monitor response in real time, & to understand immune response timing and mechanisms (pharmacodynamics) to develop strategies to increase efficacy. This study will: (i) identify and characterize sources of CSF biospecimen preanalytical variation affecting MRM-based assay performance for quantifying immunomodulatory protein biomarkers in CSF, (ii) develop, validate, and disseminate a preanalytical quality control standard operating protocol (SOP) to counteract preanalytical variation impact on the performance of MRM assays quantifying immunomodulatory proteins in CSF, and (iii) validate the biospecimen SOP through real-world testing by analyzing prospectively collected clinical trial samples at two CLIA (Clinical Laboratory Improvement Amendments) labs to demonstrate harmonization. Tools established through this work will enhance the interpretation of clinical trial outcomes through data reliability & provide valuable insights into the immunological mechanisms underlying the effects of immune-based treatments in pediatric brain tumor patients enrolled in clinical trials. The work also has broader significance for immune analyses of CSF in primary and metastatic adult brain tumors, as well as in neuroinflammatory disorders where immune activation either causes or contributes to the pathogenesis (e.g., multiple sclerosis, Alzheimer disease, Parkinson disease, Huntington's disease, amyotrophic lateral sclerosis, stroke and traumatic brain injuries myelitis).

NIH Research Projects · FY 2026 · 2025-02

Hispanic adults, the largest and fastest growing population in the United States (US), have higher rates of smoking-related morbidity and mortality. They lack access to standard smoking cessation treatments primarily due to the absence of medical insurance and guidance from healthcare providers to quit. Needed now is rigorous research on methods to enable Hispanic adults to access efficacious evidence-based interventions to quit smoking. Two widely available cessation methods that hold promise for Hispanic adults are: (1) smartphone applications (“apps”) providing behavioral intervention, and (2) nicotine replacement pharmacotherapy (NRT). Apps can be freely accessed and widely available, with 91% of Hispanic adults owning smartphones. iCanQuit, based on Acceptance and Commitment Therapy, is the only app proven efficacious for smoking cessation in a Phase III randomized trial (N = 2415). Among Hispanic participants (N = 220) in the parent iCanQuit trial, results showed significantly higher self-reported 12-month 30-day point prevalence abstinence (PPA) rates for iCanQuit participants compared to QuitGuide participants—an app that follows US Clinical Practice Guidelines (34% iCanQuit vs. 20% QuitGuide; OR = 2.20; 95% CI: 1.10, 4.41, P = .02). Regarding medications, NRT has high reach. While combining behavioral intervention with NRT generally results in higher quit rates than behavioral intervention alone, existing evidence primarily stems from in-person clinical and telephone-based trials focused on non-Hispanic adults. Although some research suggests underutilization of quit smoking medications among Hispanic adults, our research indicates that providing free NRT improves its uptake. Most pertinently, we found that Hispanic iCanQuit participants who used NRT on their own within 3 months post-randomization showed descriptively higher self-reported 12-month 30-day PPA rates compared to non-users (45.5% users vs. 28.8% non-users, OR = 2.24; 95% CI: 0.54, 9.27, P = .26). It is unknown whether providing Hispanic adults with free NRT in combination with iCanQuit cost-effectively yields higher biochemically verified quit rates than iCanQuit alone. Thus, in a fully-powered RCT, this proposal will: (1) determine whether iCanQuit combined with free NRT (n = 427) has significantly higher biochemically verified 30-day PPA than iCanQuit alone (n = 427) at 12 months post-randomization; (2) determine the cost-effectiveness of iCanQuit plus NRT vs. iCanQuit alone, as measured by cost per quitter, cost per life year gained, and cost per quality-adjusted life year (QALY) gained. We will also apply the RE-AIM dissemination framework and conduct qualitative research on the barriers and facilitators to dissemination of each intervention to Hispanic adults nationwide. This rigorous trial will determine whether providing iCanQuit alone is sufficient or whether adding NRT cost-effectively yields higher quit rates for Hispanic adults who smoke.

NIH Research Projects · FY 2026 · 2025-01

Project Summary Inhibitory mechanisms and negative feedback loops are in place soon after a T cell is successfully activated to temporally limit and restrain effector function, which is critical to prevent excessive tissue destruction. Some inhibitory signals are CD8 T cell intrinsic and others are provided by professional antigen-presenting cells (APCs), regulatory T cells (Tregs), cytokines and metabolites. Most studies examining the regulation of T cell responses in tissues have been performed in the mouse model system, while the nature of activating and inhibitory signals for T cells in human tissues is still poorly defined. T cells in human tissues, particularly human barrier tissues, appear to be continuously exposed to an array of activating and inhibitory signals. We report here that pro- and anti-inflammatory signals are present in healthy, sterile human tissue. We propose to test the hypothesis that activating and inhibitory signals in the form of cytokines and metabolites are present even in healthy, sterile human tissue to regulate effector function of CD8 T cells located in the tissue. Importantly, based on our preliminary data we propose that the tissue itself provides at least some of the pro-inflammatory signals at steady state suggesting that even healthy tissues can direct T cell responses. Defining which immunomodulatory signals are present in healthy as well as acutely infected human tissues is relevant to better understand how immune quiescence is achieved while also maintaining immune responsiveness and function. We established a human cohort that will allow us to define how the balance of activating and inhibitory signals signals is altered during an acute tissue infection. Ultimately, this may reveal new strategies to specifically curtail unwanted T cell activation and return to a state of tissue homeostasis.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT Prevention holds the greatest potential for reducing cancer burden in the population. Recent breakthroughs in genomics technologies have propelled advancements in deciphering molecular events and understanding cancer causes, leading to improved risk stratification and precision prevention. The objective of this application is to develop statistical and computational approaches to harness state-of-the-art genomic data and translate these findings into precision prevention through risk-based intervention strategies. Tumors are heterogeneous and unraveling tumor heterogeneity to distinguish indolent versus aggressive tumors will facilitate the understanding of underlying disease process. Emerging spatial omics technology, which simultaneously profiles both molecular features and spatial locations, provides critical information about the tissue microenvironment essential for understanding disease development. There are significant challenges in analyzing such data, including non-comparability of spatial omics images from different samples, high-dimensional data, and limited sample size. The goal of Aim 1 is to develop (a) deep learning-based approaches that combine unsupervised and supervised loss functions for learning common features predictive of poor clinical outcomes and (b) robust and efficient data integration approaches for assessing the association of individual's risk factors with these features, leveraging external existing biomarker summary information to improve efficiency while accounting for data source heterogeneity. Identifying risk factors that are associated with tumor subtypes linked to adverse outcomes can better stratify the population into different risk levels, enabling tailored prevention approaches such as determining when to start screening. This requires careful cost-effectiveness analysis using large-scale observational data. However, analyzing such data is complex due to non-random utilization of intervention and confounding. Further, the timing of screening is continuous and subject to censoring, as some individuals may have died or been diagnosed with the disease before initiating screening. The goal of Aim 2 is to develop statistical methods for assessing the cost-effectiveness analysis of time-varying screening, including (a) a causal- inference-based estimation procedure to quantify the benefit and cost of the intervention; (b) leveraging improved risk prediction from Aim 1 to determine “when to start screening” based on its impact on the benefit and cost in the population. Through our collaboration, we will apply the methods to data from the Genetics and Epidemiology of Colorectal Cancer Consortium to gain insight into carcinogenesis and risk assessment and evaluate their translational impact in the Women's Health Initiative cohort. Since our methods are also applicable to other studies, we will develop R or python-based open-source software packages, along with detailed manuals and data processing pipelines. These resources will be made available in public repositories or on our websites.

NIH Research Projects · FY 2025 · 2024-12

Summary All cases of multiple myeloma (MM) are preceded by precursor state termed as monoclonal gammopathy of undetermined significance (MGUS). Therefore targeting precursor states may be an effective approach to prevent this malignancy. In a recent randomized trial, we have shown that lenalidomide led to reduced risk of progression of smoldering myeloma (SMM) to clinical myeloma. In prior studies, we have shown that the immune system is capable of recognizing myeloma precursor lesions. In recent studies, we have also shown that immune dysfunction is an early feature of myeloma suggesting that earlier application of immune modulation may be needed to mediate immune prevention. In particular, evolution of clinical myeloma is associated with attrition of stem-like memory T cells and the accumulation of terminally differentiated effector T cells. In this proposal, we will test the hypothesis that immune control of myeloma precursor states is determined in part by the cell states and spatial architecture of immune cells in these conditions. We further posit that immune modulation to alter these properties could prevent evolution of clinical myeloma. This will be evaluated with the following specific aims. In Aim 1, we will evaluate whether properties of immune cells at baseline in SMM correlate with the risk of progression to clinical myeloma. Aim 2 will evaluate changes in circulating and bone marrow immune cells in patients undergoing therapy with iberdomide or iberdomide and dexamethasone under a randomized phase II clinical trial. Studies in aim 3 will evaluate the effect of immune modulation on spatial heterogeneity of immune cells in preclinical model systems. Together, these studies will not only provide new insights into improving immune modulation as a strategy to prevent clinical myeloma but may also help develop novel immune based combinations to treat myeloma.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Flow cytometry is a specialized technology that characterizes cells on a single-cell basis and is heavily used in the fields of immunology, infectious diseases and cancer biology. As flow cytometry technology has advanced, the multiplexing capability has markedly expanded allowing for more markers to be examined simultaneously. These advances have increased the complexity of experimental design of and of the expertise required to appropriately analyze and detect potential artifacts hidden in the resulting data. Advanced training is critical to appropriately implement these technologies in resource-limited settings where exposure to scientists and mentors with the requisite expertise is limited. Although complete mastery can take years, the practical as well as the theoretical tools needed to achieve competency can be taught in a relatively short workshop. The African Flow Cytometry Workshop has been held biennially since 2005 in Cape Town, South Africa with the aim of enhancing both the theoretical and technical flow cytometry knowledge of African immunologists so that this cutting-edge technology can be applied to critical studies being conducted on the continent. This technology has unique capabilities to help address scientific questions of particular relevance in Africa given the high prevalence of disease caused by the three major global pathogens, HIV, tuberculosis and malaria. As flow cytometry instruments with greater capabilities have become more available in Africa, the number of scientists needing this advanced knowledge has also increased. Training opportunities that exist for African investigators remain very limited and can be very costly. The African Flow Cytometry Workshop is structured over five full days and consists of a combination of lectures and hands-on tutorials with homework assigned each evening and an initial and final exam. Participant numbers are limited to 20, to ensure appropriate interaction between faculty and students and to facilitate peer-to-peer interactions. We have conducted nine previous workshops and this experience has demonstrated the need for this type of training and has shown successful outcomes for many prior participants (Nemes et al, 2016). As in prior workshops, the selection process is conducted in a thoughtful way in order to choose participants across geographic regions in Africa who are likely to apply their training and train others in their home laboratory or institution. In fact, a survey of prior workshop participants demonstrated that almost 90% of our survey respondents reported having trained peers at their home institution after the workshop, often by adapting teaching materials provided by the workshop. In an effort to perpetuate the training, we have incorporated a strategy to invite and select high-achieving past participants to join the workshop faculty, which has served to deepen their understanding of flow cytometry and provide them with leadership training. The workshop training is therefore maximized through a “train the trainer” model and through the development of a specialized network of African flow cytometry experts in the field of infectious disease.

NIH Research Projects · FY 2026 · 2024-12

SUMMARY Small cell lung cancer (SCLC) is an aggressive cancer type with no targeted therapies available. It is critical that we gain a better understanding of the genes that drive SCLC and that we model key SCLC subsets. Employing genetically engineered mouse (GEM) models is central to this effort. To identify candidate tumor suppressive and biologically important pathways, we applied genome-scale functional screens to a cellular model of early- stage SCLC. We identified a stress activated protein kinase (SAPK) pathway, as tumor suppressive in SCLC cellular model systems. Map2k4, Map2k7 and Jun were particularly strong screen hits. The SAPK/AP-1 pathway is a target of deletions and mutations in human SCLC, with targeted sequencing revealing MAP2K4 deletion or mutation in ~3% of SCLC. In human datasets, AP-1 transcriptional activity is associated with ASCL1 target gene expression, linking this pathway to the SCLC-A subtype. Moreover, our preliminary data confirms potent acceleration of SCLC tumorigenesis with Map2k7 deletion in a sensitized Rb/p53-deleted mouse model. In this proposal we explore the biological roles for this SAPK axis in SCLC tumorigenesis. We will leverage study of cellular models of SCLC, in vivo genetically engineered mouse models, and human tumor datasets to define roles for the SAPK pathway in SCLC tumor suppression. We will generate and characterize GEM models of SCLC with deletion in Rb/p53 along with either Map2k7 or Map2k4 to elucidate effects of SAPK suppression on tumor initiation and progression. We will employ cellular models to interrogate the impact of SAPK perturbation on early stage and late-stage SCLC cells. We will perform RNA-seq analyses to identify transcriptional programs consistently regulated by a MAP2K4/MAP2K7 axis upstream of c-JUN/AP-1. These analyses will use SCLC tumor tissue from mouse models along with isogenic SCLC cell lines with SAPK pathway perturbations. We will also perform CUT&RUN occupancy studies to determine the impact of SAPK perturbation on genomic binding of JUN and FOS and ATAC-seq to examine chromatin accessibility. Integrative analyses will then identify candidate direct transcriptional targets of AP-1 relevant to tumor suppression for functional perturbation studies. There is increasing appreciation that SCLC exhibits different subtypes based on transcriptional features. Our preliminary data support the notion that SCLC differs in the level of SAPK/AP-1 activation, likely via both genetic and via transcriptional suppression of pathway components. Changes in this pathway may also be linked to neuroendocrine features and transcriptional subtype of SCLC. This proposal will elucidate an unappreciated tumor suppressor axis in SCLC and will also help us understand factors that govern SCLC transcriptional state and SCLC subtype.

NIH Research Projects · FY 2026 · 2024-12

Antigen-specific therapies have long been pursued to improve outcomes in acute myeloid leukemia (AML). Most exploited are monoclonal antibodies (mAbs) targeting the membrane-distal V-set domain of CD33, a glycoprotein displayed on leukemic blasts in almost all cases and possibly leukemia stem cells in some. Improved survival of some patients with gemtuzumab ozogamicin validates this approach but many patients with CD33+ AML do not benefit from this antibody-drug conjugate, prompting interest in developing improved CD33-directed therapeutics. Because AML cells are sensitive to radiation, -emitting radionuclides are ideal to arm anti-CD33 mAbs. Unlike -emitters, they deliver a very high amount of radiation over just a few cell diameters to enable precise and efficient target cell kill. Early trials with an anti-CD33 mAb labeled with actinium-225 have been conducted, but important shortcomings include high costs, long half-life, and release of free daughter radionuclides after decay of 225Ac with risk of non-specific toxicity to healthy tissues. We hypothesize astatine-211 (211At), an -emitter we have focused on because of its shorter half-life and because it decays without any long-lived or potentially dangerous daughter isotopes, will provide a novel, superior payload for CD33-directed radioimmunotherapy (RIT). Using humanized mice, we have generated a panel of fully human mAbs recognizing either the V-set domain or the membrane-proximal C2-set domain of CD33. Since the V-set but not C2-set domain is missing in some CD33 variants, C2-set domain-directed mAbs can recognize all naturally occurring variants of CD33 (i.e., are “CD33PAN mAbs”). With these mAbs available, we now plan to optimize CD33-directed RIT and have assembled a multidisciplinary team of investigators with complementary expertise in developing radioimmunoconjugates and other mAb-based therapeutics for AML to accomplish this task. We will focus our efforts on 3 connected areas of research. In the first, we will identify a candidate mAb clone to be used as basis for the clinical therapeutic. In the second, we will examine to what degree internalization and decrease of cell surface display of CD33 upon continued mAb exposure decreases the efficacy of 211At-CD33 RIT and will test the value of CD33 delivery vehicles with reduced internalization properties. In the third, we develop improved conjugation/astatination technologies to overcome the limitations inherent to the conjugation of mAbs with the currently used bifunctional boron cage molecule, isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10-NCS). Expected results will define a new form of CD33-RIT that should be readily translatable into the clinic for patients with AML and other CD33+ neoplasms, for whom current treatment outcomes are unsatisfactory.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY (from parental grant) The root cause of most breast cancer deaths is metastasis. By dissecting the molecular events driving it, the research community can develop new therapeutic approaches to eradicate and prevent metastatic disease. One promising avenue of research involves the cooperative behavior of tumor cells. Conventionally, metastasis is conceptualized as the dissemination of individual tumor cells to distant organs. However, recent studies by the Cheung research group and others have established that clusters of tumor cells metastasize to distant organs more efficiently than single cells in mouse models, and that circulating tumor cell clusters are associated with poor patient outcomes and therapy resistance in humans. The molecular mechanisms responsible for aggression in tumor cell clusters and the optimal therapeutic strategies to eliminate clusters have remained obscure. Recently, the Cheung laboratory has found that clustered tumor cells display heightened levels of apoptosis resistance, cell proliferation, and changes in molecular expression that indicate that the cells are cooperating with one another. These studies reveal that the tyrosine kinase EGFR is activated at cell-cell contacts in clustered tumor cells, and they establish that EGFR and the low-affinity EGFR ligand Epigen are necessary for cluster- dependent proliferation and metastatic colonization. The proposed project will test the hypothesis that tumor cell clusters are highly metastatic because they contain a private signaling environment involving EGFR, Epigen, and the transcription factor Fra-1, and that disrupting this signaling environment will neutralize clusters’ metastatic potential. The Cheung lab has already developed technically innovative organoid and murine models to study cluster-based signaling and its impact on metastasis in vivo. Using these models, the lab will first determine whether cluster-induced metastatic efficiency depends specifically on local activation by Epigen. Second, the lab will determine the impact of Fra-1 transcriptional programs and signaling feedback loops on metastatic processes specific to tumor cell clusters, as well as whether this program depends on the presence of Epigen. Third, the lab will supplement its experimental findings by studying the association between EGFR, Epigen, and long-term recurrence and mortality data from human breast cancer datasets. Through this integrated approach, the Cheung lab will develop an understanding of the cooperative molecular mechanisms that underlie the propensity of tumor cell clusters to metastasize. As described in the proposal, this understanding is likely to reveal molecular vulnerabilities that can be exploited to develop new anti-metastatic therapies. Although the work proposed here focuses on uncovering therapeutic strategies to target tumor cell clusters in breast cancer, the findings will potentially be relevant to a wide range of tumor types.

NSF Awards · FY 2024 · 2024-10

Artificial intelligence (AI) works by learning from patterns in data. Building AI technologies depends on acquiring data for training models. Responsible development of AI as part of public interest technology (PIT) requires building AI that benefits the public interest while safeguarding data used to power AI systems. Safeguarding data require tradeoffs between the level of protection provided and the usefulness of the models created with the data. These tradeoffs create a tension that PIT organizations must resolve. This project engages a multi-disciplinary team across sectors in a combination of ethnographic and computational research to develop novel approaches that can support PIT organizations in deploying data safeguards to build AI. The project uses disclosure limitation techniques to protect the privacy of sensitive information in AI training data. Deploying these techniques, including newer techniques like differential privacy, require making tradeoffs that affect stakeholders in the AI lifecycle. For example, strong privacy protection reduces statistical accuracy, which may ultimately reduce the model usefulness. The project will develop novel methods and best practices for navigating these aspects for PIT organizations. The project will: (1) use ethnographic approaches and qualitative inquiry to identify socio-technical decision points and challenges at PIT organizations; (2) create and evaluate novel approaches to participatory engagement of stakeholders in the deployment process; and (3) build software and communication tools for evaluation and transparency of AI systems that use differential privacy. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-09

Project Summary/Abstract Rapid advancements in genomics have revolutionized healthcare, technology, research, and beyond. The demand for professionals with genomics skills, knowledge, and critical thinking is also increasing. Currently, genomics skills are primarily acquired at major research institutions, leaving instructors at under-resourced institutions unsupported. Community and technical colleges, which educate a diverse student population and entry-level workers, face barriers in developing genomics curricula due to high teaching loads and limited resources. We propose Faceting GEMs (Genomic Education Modules) for the Entry-level Workforce to address this gap at under-resourced community colleges. Our program will (1) provide accessible, research- focused genomics training modules, (2) train and motivate Partner Site faculty, (3) foster faculty community, support, and collaborations, and (4) use scalable online platforms to promote genomics education. We will create active learning modules built around authentic research experiences to deeply engage the capable and driven participants at our Partner Sites. To address the scarcity of professional development opportunities for community college faculty, our program offers an annual in-person "Learn-a-thon" at the Lead Site (Fred Hutch). The Learn-a-thon will engage Partner Site faculty in research, train them to lead the modules, and foster collaboration for module improvements. We will prioritize creating an authentic support network by offering both synchronous and asynchronous forms of communication, while also providing funds for the Partner Site faculty to visit each other and build deeper support systems. Outside of the classroom, Instructor Guides and materials will be provided to encourage adoption of the modules by other genomics educators. We will leverage our experience bringing online courses to millions of learners by scaling and publishing our module materials as free Massive Open Online Courses (MOOCs), benefiting other educators and learners worldwide. All materials will also be freely available on our website to promote broad availability. Finally, we will assess our impact through evaluation rooted in pedagogical practices. The GEMs program leverages the established Genomic Data Science Community Network (GDSCN), a collaborative that has worked to diversify the genomic data science research community by engaging faculty from Historically Black Colleges and Universities (HBCUs), Hispanic Serving Institutions (HSIs), Tribal Colleges and Universities (TCUs), and Community Colleges (CCs) across the United States. Our work proposed here builds upon this trusted collaboration, ensuring continuity and sustained momentum in innovating genomics curriculum.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT The CANOE Partnership: Cancer Awareness, Navigation, Outreach, and Equitable Indigenous Health Outcomes U19 Cooperative Agreement is proposed in response to the need to improve cancer outcomes for American Indian and Alaska Native (AI/AN) communities nationally, with an emphasis on the Washington State (WA) catchment area of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium, a National Cancer Institute (NCI) designated Comprehensive Cancer Center. The proposed work builds on the Consortium’s substantial community engagement with Tribes and Tribal Organizations over the past 22 years, beginning with the Spirit of EAGLES Special Population Network in 2002, up to and including an ongoing R01 “ Digital smoking cessation intervention for nationally-recruited American Indians and Alaska Natives: A full-scale randomized controlled trial ” (R01CA284687; PI: Bricker) Disparities in cancer outcomes for the AI/AN population are due to multiple social determinants. Three major sources of these disparities, amenable to intervention, are: 1) behaviors to reduce cancer risk (e.g., smoking cessation), 2) access to appropriate screening by way of imaging technologies (e.g., appropriate application of chest computed tomography and mammography) and colorectal cancer (CRC) screening procedures such as fecal immunochemical test (FIT), and colonoscopy; and 3) primary prevention. We propose a multidimensional approach to addressing these sources of disparate cancer outcomes in partnership with our Tribal and community collaborators at the South Puget Intertribal Planning Agency (representing the Chehalis, Nisqually, Skokomish, Shoalwater Bay, and Squaxin Island Tribes) and the Black Hills Center for American Indian Health (Rapid City, SD). Together, we will use community based participatory research (CBPR) approaches and the Indigenous Cancer Health Equity Initiative Model to empower and engage Tribes and Tribal organizations through our three research projects. Our Overall Specific Aims are: 1) Improve rates of cessation of commercial tobacco smoking among a nationally recruited sample of AI/AN adults (Research Project 1); 2) Improve rates of lung, colorectal, and breast cancer screenings among our Tribal partner populations in the Consortium’s catchment area (Research Projects 2 & 3); 3)Prepare the next generation of researchers in Indigenous cancer equity and provide them with resources to obtain preliminary data to inform future cancer equity research in Indian Country (Pilot Grant Program); and 4) Develop infrastructure to support equitable engagement of Tribal partners and Indigenous Frameworks in cancer research. (Administrative and Community Engagement Cores). The overall public health impact of the proposed work will be high, given the focus on smoking cessation, cancer screenings and vaccinations that altogether will prevent and control cancers that are highly prevalent and are responsible for a large share of disparate mortality rates among Indigenous populations. The public health impact of this work will be high, given its focus on modifiable behaviors and on future generations of Indigenous Cancer Health Equity researchers.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY The HIV reservoir is a population of latently HIV infected cells that persists in people living with HIV on suppressive antiretroviral therapy (ART) and prevents cure of HIV. Dr. Reeves and colleagues will study the mechanisms that create and sustain the HIV reservoir throughout treated and untreated infection. This team will use a mathematical modeling approach to integrate existing published and unpublished as well as novel experimental data ranging across five stages of infection: early and chronic untreated (Aim 1) and early and long-term treated, and analytical treatment interruption (Aim 2). Data will consist of HIV viral loads and genetic sequences (RNA and DNA), cell counts (CD4+ and CD8+ T cells), and clonality of CD4+ T cells defined by T cell receptor sequencing. We will adopt a novel within-host phylodynamic “wiphy” model to specifically study HIV reservoir biology by specifically interrogating the various mechanisms driving its persistence vs. clearance – including cellular proliferation of infected cells and adaptive immune selection against certain HIV sequences. We will determine which mechanisms predominate in which stages, and provide a comprehensive understanding of HIV reservoir creation, maintenance, and evolution throughout all infection stages. This research will aid in the building of next-generation models of HIV reservoir seeding and persistence dynamics. This work will help the HIV cure field gain a deeper understanding of how the HIV reservoir is created during untreated infection and maintained during treated infection. Results should aid in efforts to design therapeutics towards a functional or sterilizing HIV cure.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS) are a group of age-related marrow failure disorders characterized by cytopenia and anemia. It is estimated that 10,000 new cases of MDS are diagnosed in the United States each year. This is caused by the functional decline of hematopoietic stem cells in the marrow because of acquired genetic mutations that corrupt their ability to make blood cells effectively. Clinical management of MDS remains challenging with limited treatments available, and stem cell transplantation remains the only curative therapy. However, most patients are unable to receive transplantation due to advanced age and co- morbidities. Thus, there is a pressing need to better understand the complexity of the disease which can lead to the development of novel therapies. Somatic mutations in splicing factor genes including SRSF2, U2AF1, SF3B1 and ZRSR2 are found in most patients with MDS. Patients carrying splicing mutation are generally older, have inferior prognoses, and are at a higher risk of transforming to secondary leukemia. Recent studies found that mutations in splicing factor genes trigger an elevated level of cellular R-loops, which consists of a RNA:DNA hybrid structure and a displaced single-stranded DNA. While R-loops are natural by-products of active transcription, if left unresolved, they could pose as a threat to genome integrity due to their propensity to induce double strand breaks. Here, we propose to test the hypothesis that targeting R-loop-induced genomic instability is an attractive therapeutic strategy in MDS cells that carry endogenous mutations in RNA splicing factors. Our preliminary data suggest that cells mutated for splicing factors are exquisitely sensitive to clinically approved PARP inhibitors, such as Olaparib, and a novel PARP1-specific inhibitor, AZD5305. Moreover, we provide evidence that PARP can physically associate with R-loops to dampen R-loop-induced genomic instability, and that PARP1 activity at R-loops could be used as a predictive biomarker for PARP inhibitor response. This proposal has two specific aims. In Aim 1, we plan to dissect the underlying molecular mechanisms of PARP inhibitor sensitivity in the context of splicing mutant MDS, and to test the therapeutic potential of PARP inhibitors in pre-clinical MDS models. In Aim 2, we plan to expand and identify combination therapy strategies with PARP and PARP1-specific inhibitors. We first plan to determine whether that combining PARP and ATR inhibitors can induce synergistic effects in MDS cells with RNA splicing mutations. Next, we plan to identify and test novel sensitizers of PARP1-specific inhibitor to evaluate optimal combination treatment strategy using pre-clinical models. Successful completion of this proposal will lead to the validation of new treatment options that can hopefully improve the outcomes MDS patients.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Current immunotherapies, such as anti-PD-1 immunotherapy, have low objective responses in patients, necessitating the development of novel therapies that boost immune responses to cancer and responses to these treatments. Further, most immunotherapies in the clinic, anti-PD-1 immunotherapy included, predominantly act on T cells. While combinations of T cell-directed therapies can lead to excellent outcomes for some, most patients still have no objective responses to combinations of these immunotherapies. Thus, it is imperative to identify novel therapeutic targets that work synergistically with anti-PD-1 immunotherapy and other T cell-directed therapies. Natural killer (NK) cells are innate lymphocytes that can control tumors through direct cytotoxicity or their immunomodulatory production of cytokines and chemokines. NK cell abundance in the tumor correlates with increased patient survival and patient responses to immunotherapies. We previously found that a key immunomodulatory function of NK cells is their production of the cytokine FLT3LG which regulates type 1 conventional dendritic cell (cDC1) abundance in the tumor microenvironment (TME). cDC1s are an important antigen presenting cell that shapes anti-tumor T cell responses. We found that NK cell production of FLT3LG regulates cDC1 abundance in the TME which leads to increased patient survival and responses to anti-PD-1 immunotherapy. We hypothesize that targeting the immunomodulatory effects of NK cells in the tumor microenvironment will lead to more protective cDC1s, better tumor-directed T cell responses, and increased efficacy of immunotherapies. Thus, it is critically important that we define how NK cells are regulated in the tumor microenvironment. In particular, the cellular and molecular mechanisms regulating NK cell phenotype and function in the tumor are unknown, and the molecular mechanisms controlling NK cell production of the cytokine FLT3LG are unknown. In Aim 1 of this project, we will determine tumor-intrinsic features that regulate NK cell phenotype and function in the tumor. In Aim 2, we will define the mechanisms that regulate NK cell production of Flt3L. In Aim 3, we will elucidate the mechanisms regulating NK cell phenotype and function in human metastatic melanoma. To address these aims we have developed ectopic tumor models and in vitro NK cell stimulation assays that allow us to define the molecular mechanisms regulating NK cell production of Flt3L. We have also developed complimentary human immunology studies of metastatic melanoma samples that allow for the study of NK cells and their immunomodulatory effects in a translational setting. The studies outlined in this proposal will provide answers to longstanding questions about how NK cell phenotypes and functions are regulated in the TME and will be foundational to the development of novel immunotherapies that target the immunomodulatory effects of NK cells to increase a patient’s immune response to cancer.

NIH Research Projects · FY 2025 · 2024-09

Colorectal cancer (CRC) incidence and mortality rates vary significantly across population groups and these differences extend beyond access to screening and health care suggesting underlying contributors to disease etiology, progression, and response to treatment. By studying population groups with different cancer rates, we enhance our ability to identify the molecular characteristics and contributing factors driving these differences. Despite advances in biotechnology, tools for analyzing tumor and host molecular genetics and genomic biology have not been fully utilized to address these differences. Our team has the expertise to use cutting-edge technologies to drive innovative translational cancer research aimed at developing novel prevention, early detection, diagnosis, and treatment approaches. To achieve our goal of reducing differences in CRC incidence and mortality between population groups, we propose the following specific aims: Aim 1: Improve risk stratified screening and early detection of CRC across population groups by developing risk prediction models that perform similarly well across population groups. Aim 2: Reduce differences in CRC-specific mortality across population groups by discovering and validating novel molecular and biological changes related to risk of lethal CRC and response to treatment in CRC patients that can guide surveillance and treatment selection for CRC survivors. Aim 3: Discover novel therapeutic targets for CRC across population groups with varying mortality rates and test potential clinical interventions aimed at reducing CRC mortality by advancing our understanding of differences and similarities in the genetic, molecular, and microbial characteristics of CRC in different populations and testing the effectiveness of novel interventions in CRC clinical trials. During the P20 SPORE phase we assembled a robust biorepository and clinical data from a large CRC patient population and through our long-term leadership in genetic epidemiology we have access to the world’s largest CRC germline genetic data set from population groups with different incidence and mortality rates. Utilizing these resources, our program will conduct four impactful projects supported by three centralized cores - a) leadership and administration, b) biospecimens, pathology and molecular technologies, and c) biostatistics and bioinformatics. Our Career Enhancement and Developmental Research Programs will cultivate new investigators and novel translational research projects. Through this integrated effort we aim to build a lasting translational research program to understand and reduce differences in CRC incidence, mortality and overall disease burden.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Adoptive T cell therapy (ACT) has been effective against certain blood cancers but has had limited success against solid tumors, such as pancreatic cancers, largely because ACT T cells become dysfunctional within the tumor-microenvironment (TME). There is a big knowledge gap on the causes of dysfunction in ACT T cells, mainly because there are so many T-cell-intrinsic regulators and T-cell-extrinsic TME suppressive factors that can all potentially contribute to ACT T cell dysfunction. With the ultimate, goal of overcoming T cell dysfunction and improving therapeutic efficacy, I seek a more comprehensive understanding of how ACT T cells are molecularly programmed by intrinsic epigenetic regulators to become dysfunctional, and how extrinsic TME factors further suppress the function of ACT T cells using ACT-treated mouse models and data from ACT-treated pancreatic cancer patients. Specifically, using single-cell multi-omics (including epigenomics) and spatial omics, along with the computational analysis and functional experiments, I aim to: 1) Identify targetable T cell-intrinsic epigenetic regulator(s) of ACT T cell dysfunction in TME, 2) Identify targetable intra-TME cellular & molecular interactions that suppress ACT T cell function. My previous wet-lab & dry-lab training, and my extensive experience with the single-cell analysis of both cancer and the immune cells, make me well-suited for the project. The project can significantly clarify the fundamental biology of T cell dysfunction and reveal actionable clinical strategies for improving ACT efficacy. The proposed computational frameworks should also be broadly applicable to a wide spectrum of contexts. The benchmark datasets should also serve as a rich, resource to the broad field of cancer immunotherapy for hypothesis generation and testing. My long-term goal is to lead an independent research group at a top research university, using systems biology to study quantitative questions in immuno-oncology, especially ACT against solid tumors. Over the course of this award, I will be supported by primary mentor, Dr. Philip Greenberg, a pioneer of ACT therapy for cancer, with a remarkable track record in science and mentoring. My co-mentors, Drs. Henikoff, Newell, Gottardo, Setty, Chapuis, are all renowned experts of epigenomics, single-cell and spatial omics, computational biology and clinical science, respectively, providing complementary training for my independence transition. They will all assist me in navigating the transition to an independent faculty position. Work will be conducted at Fred Hutch Cancer Center, which offers all the state-of-the art facilities required for the successful of the Aims in addition to a collegial scientific & training environment, and strong institutional support.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Despite advances in cancer therapy, metastatic disease is overwhelmingly fatal and accounts for most cancer- related deaths. Systemic therapies fail to control disseminated disease due to a mix of innate and acquired resistance. The processes that drive drug resistance and metastasis are a combination of tumor cell-intrinsic mechanisms, such as genetic and epigenetic changes, and extrinsic factors, including the tumor immune microenvironment (TIME). I propose a comprehensive research and training plan to investigate both dimensions. F99 Phase: My doctoral work focuses on a cancer cell-intrinsic mechanism, the role of Claudin-1 (CLDN1) expression in promoting chemoresistance and metastasis in colorectal cancer (CRC). My work thus far (Aim 1A) details a novel mechanism in which CLDN1 interacts directly with the receptor tyrosine kinase Ephrin type-A receptor 2 (EPHA2) in CRC and inhibits its degradation. The increase in protein-level EPHA2 enhances AKT signaling and CD44 expression to promote cancer stemness and chemoresistance. Previous work has shown that CLDN1 expression promotes CRC metastasis in mouse models. My future work (Aim 1B) will investigate the molecular details of the CLDN1/EPHA2 interaction; compare patterns of CLDN1 and EPHA2 expression in patient-matched normal, primary tumor, and metastatic tumor tissues; and look specifically at the role of the CLDN1/EPHA2 interaction in promoting metastasis. Aim 1 will provide training in bioinformatics, organoid culture, and orthotopic metastasis models. K00 Phase: In my postdoctoral phase, I will study the extrinsic factors influencing breast cancer (BC) dormancy and metastasis in the liver. Cancer cells spread to distant organs early in tumor development. These disseminated tumor cells (DTCs) can enter a state of dormancy and survive initial therapy, only to reactivate years later, resulting in relapse. It is well-accepted that the factors regulating dormancy and reactivation depend on the microenvironment of the host organ. Even though hepatic BC metastases carry the worst prognosis, the factors regulating dormancy and reactivation in the liver are understudied, particularly in a fully intact TIME. In Aim 2.1, I will use immunocompetent mouse models to study the role of T-cells in regulating BC dormancy in the liver. Evidence suggests that hepatic stellate cell (HSC) activation may be a critical factor in DTC reactivation. Activated HSCs are a hallmark of non-alcoholic fatty liver disease (NAFLD). NAFLD is the most common liver disease in the world, but its impacts on BC metastasis are not well studied. Aim 2.2 will use mouse models of NAFLD to study its effect on BC dormancy and metastasis. Aim 2.3 will use innovative ex vivo whole tissue slice cultures to study the signaling networks regulating BC dormancy and immune evasion. Aim 2 will provide training in cancer immunology, advanced cancer models that accurately model the TIME, and analytical techniques capable of single-cell resolution. The proposed studies will offer insights into the causes and therapeutic vulnerabilities of metastatic disease and will provide a multidimensional training program that will prepare me for a career as an independent cancer researcher.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY The cardiovascular system is an essential organ key for development. This highly efficient network of vasculature structures constantly undergoes expansion and trimming to maintain vessel homeostasis. Angiogenesis – the budding of new vasculature structures from preexisting vessel systems, is one of the primary processes contributing to network expansion. Multiple cellular receptors function together to ensure successful proliferation and motility of endothelial cells for angiogenesis. One of these receptors is Tie2, a receptor tyrosine kinase (RTK) expressed exclusively on the endothelial cell surface. Tie2's primary role, upon binding to angiopoietin-1 (Ang-1), is to initiate and maintain tight cell-cell junctions across the endothelial monolayer. Like other RTKs, Tie2's proposed activation pathway involves the dimerization of the receptors to propagate a signal across the cell membrane to phosphorylate downstream targets. While the role of Tie2 in angiogenesis is clear, little is known about the structural changes that Tie2 undergoes that allow for dimerization. The Ang-1 binding site is distant from the proposed dimerization site. Therefore, information must travel across multiple domains, and there is currently no mechanism for this transduction. In addition, our understanding of cell signaling pathways has been described as most straightforward possible, often rendering them as exclusively linear with a single ligand and receptor. However, within the cellular context, proteins and ligands are constantly engaging with other signaling pathways in addition to their own. Integrins, a membrane receptor that has a role in cellular adhesion and motility, have been observed to engage with Tie2 on the cellular level. But the molecular details remain elusive. Therefore, a structural investigation into Tie2's activation and modulation with angiopoietin and integrin would contribute to a more complete picture of Tie2 biology. To this end, I will combine cutting-edge structural biology techniques with traditional cellular biology to dissect these structural changes in Tie2. My goals are to elucidate the molecular mechanism of Tie2 dimerization and the receptor cross-talk that occurs with integrin α5β1. I have designed experiments with two specific aims to understand how Tie2 is structurally impacted by angiopoietin-1 and integrin α5β1. Aim 1 will examine and describe the impact of Ang-1 binding on the full-length Tie2 receptor using cryogenic electron microscopy (cryoEM) and cell-based assays. The results from this aim will push the limits of our understanding of Tie2's structural architecture and ligand-induced dimerization of RTKs. Aim 2 will determine the structural role of integrin α5β1 in modulating Tie2's signaling axis. I will use a combination of cryoEM and Förster resonance energy transfer to identify the key residues of this receptor-crosstalk. Results from this aim will shed light on the functional importance of receptor cross-talk and how structures of the receptors can influence communication across signaling pathways. By studying Tie2, we can better understand its structure and potentially develop new therapies targeting this receptor.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract An organism’s ability to grow, develop, and reproduce are some of the defining characteristics of life. Central to growth and reproduction of organisms ranging from single-celled baker’s yeast to humans is the ability of a cell to replicate its genome and accurately divide the genome into two daughter cells. Errors in the replication or division of the genome can result in genetic changes that cause disease or are lethal to the cell or organism. Segregation of the genome is accomplished through an intricate series of steps wherein spindle microtubules must successfully bind sister chromatids and pull one copy of each chromosome into each daughter cell. The kinetochore, a conserved megadalton protein complex, mediates microtubule attachment to chromosomes. Although prior work has successfully charted many kinetochore components, as well as key regulatory steps in kinetochore assembly and function, this process is still incompletely defined. The goal of this proposal is to understand how post-translational modifications, specifically ubiquitin, contribute to kinetochore assembly and function in the budding yeast, Saccharomyces cerevisiae. Using a combination of proteomics, yeast genetics, and biochemistry, I will generate a comprehensive map of kinetochore regulation by the Mub1/Ubr2 E3 ubiquitin ligase complex (Aim 1) and investigate how a large family of E3 ubiquitin ligases, the cullin-RING ligases, regulates kinetochore function (Aim 2). Combined, these approaches will allow me to address how ubiquitylation influences kinetochore function and generate new knowledge surrounding kinetochore regulation. Given the highly conserved nature of the kinetochore, this work will likely identify principles of kinetochore regulation that apply to multiple organisms. Understanding these principles could provide insight into the cellular adaptations that occur in response to pathological changes in chromosome number (aneuploidy), a common feature of cancer cells. The training facilitated by this fellowship, along with my previous research experiences, will allow me to develop the skills necessary to become an independent academic investigator, with the long-term goal of establishing a research program that uses yeast and mammalian systems to study mechanisms of kinetochore regulation.