Fred Hutchinson Cancer Center

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT Recent advances in targeted therapies have prolonged the survival of non-small cell lung cancer patients. However, lung cancer still remains the leading cause of cancer deaths in the U.S. and worldwide, and the 5-year survival rate for non-small cell lung cancer is a dismal 24%. For many tumors, drug-targetable mutations are not identified, limiting therapeutic options to chemotherapy or immune checkpoint inhibitors. For those that do have identified druggable biomarkers, such as EGFR mutations, acquired resistance is near-universal problem. To make progress in extending lung cancer patient survival, it is necessary to identify new targets beyond those that are currently known. Genome-wide CRISPR knockout screens are increasingly used to identify new cancer drug targets. However, these assays still suffer from a major limitation, as single-gene knockouts generally do not reveal the functions of gene families that have redundant functions, such as paralogs. Remarkably, it is now clear that paralogous genes constitute two-thirds of the human genome, so this blind spot appears to be cover much of the genomic landscape. To fully interrogate the function of all human genes, we are developing and deploying new methods for multiplexed gene knockouts. In addition, the same duplications that make paralogs difficult to study could also provide new, potentially customizable, approaches to cancer therapy, since the deranged genomes typical of cancer cells commonly harbor deletions and inactivating mutations in one or more paralogs. We hypothesize that targeting the actively expressed paralog in cancers that have lost or suppressed its paralogous pair could create a synthetic lethal non-oncogene dependency and a therapeutic window for killing cancer cells. Here, we propose to overcome the major barriers impeding the development and application of new synthetic lethal therapies. First, we will build on the success of our paralog work since the first submission by performing rapid and efficient systematic knockout of synthetic lethal paralogs across multiple cell types. Second, we will apply our innovative paralog Perturb-seq approach to determine what paralogs undergo transcriptional adaptation. Last, we will develop reproducible software to perform analysis of dual gRNA CRISPR screens, enabling the broad adaption of our methods for genetic interaction mapping which may go beyond paralogs. Ultimately, our findings will deliver new cancer targets that can be used to develop and deploy new therapies. The impact of this project will be the identification of dozens to hundreds of new paralog genetic interactions and demonstration that some of these can be harnessed to suppress tumor growth and drug resistance. Due to the low anticipated off-target effects of synthetic lethal therapies, these could be applied in combination with existing targeted-, immuno-, or chemo-therapies to suppress drug resistance and improve patient survival.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY/ABSTRACT Herpesviruses such as Herpes simplex virus (HSV) 1 and 2 are ubiquitous DNA viruses that establish lifelong infections. After primary infection, they enter latency and occasionally reactivate, causing recurrent disease. HSV 1 and 2 establish latency in neurons, and reactivation causes lesions of the facial or genital area. HSV-1 and 2 lack vaccines or curative treatments and new therapeutic strategies against chronic HSV diseases are critically needed. During an infection, cells are frequently co-infected by multiple virions, and therapeutic approaches that rely on viral co-infection have great potential. We recently invented a CRISPR-based “viral gene drive” that relies on viral co-infection to replace wild-type viruses with an engineered version. Our strategy was inspired by similar methods developed in insects and uses CRISPR-Cas9 and homologous recombination to efficiently propagate a genetic modification in the viral population, ultimately reducing viral levels. Our discovery uncovered a new way to engineer herpesviruses for therapeutic and research purposes. Our long-term goal is to design gene drives that could be used as curative therapies for HSV infection. Patients suffering from chronic HSV disease could be treated with an engineered virus that recombines with wild-type viruses in the latent reservoir and prevents viral outbreaks. In this innovative application, we propose to determine if a viral gene drive can spread to the latent reservoir and inactivate wild-type viruses in mice latently infected with HSV-1. In specific aim 1, we will use mouse models of HSV-1 infection to test if a gene drive can suppress viral shedding in mice persistently infected with wild-type HSV-1. This will establish the potential of gene drives to cure chronic HSV-1 infection. In specific aim 2, we will use fluorescently-labeled viruses and mathematical modeling to quantify co-infection frequency during gene drive propagation in vivo. The rationale is that gene drives rely on co-infection, and that a better understanding of the biology of co-infection is needed for the development of our innovative therapeutic strategy. We will determine how often a gene drive reaches latently infected neurons and establish if a gene drive can efficiently inactivate the latent reservoir. Altogether, this project will test the therapeutic potential of a breakthrough technology and may lead to novel therapies that significantly improve human health.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT This research proposal aims to develop new immunogens for an HIV-1 vaccine by utilizing advanced protein design techniques and deep learning methods. Conventional structure-guided approaches have limitations in achieving desired structural characteristics. Therefore, this study proposes using RFdiffusion, ProteinMPNN, and AlphaFold2, to generate new germline-targeting gp120 cores based on the pre-fusion native-like structure of the HIV-1 strain 426c. The designed immunogens prioritize maintaining the structural integrity of the pre-fusion gp120 while removing the bridging sheet and re-designing specific regions to maintain the pre-fusion native-like backbone structure. In addition, particular attention is directed towards masking off-target epitopes. In vitro characterization will be performed to evaluate the binding characteristics of these immunogens with germline, broadly neutralizing, and non-neutralizing antibodies. Subsequently, transgenic mice expressing human germline VRC01-class BCRs will be utilized to assess the immunogenicity of selected immunogens presented on nanoparticles and analyze their impact on germinal center B cell responses and memory B cell repertoire. Monoclonal antibodies (mAbs) obtained from immunized animals will be structurally characterized, shedding light on antibody maturation pathways influenced by the immunogen structure. Additionally, this research plan aims to expand the immunogen repertoire by designing gp120 cores based from diverse HIV-1 clades/strains. Furthermore, to enhance immunogenicity, membrane-bound and nanoparticles delivery through self-amplifying mRNA will be explored. The ultimate objective of this research is to gain valuable insights into novel antigen design techniques and their application in HIV-1 vaccine strategies for broadly neutralizing antibodies development. The findings from this study will contribute to the development of immunogens that closely resemble the pre-fusion gp120 state, potentially leading to enhanced B-cell responses capable of generating broadly neutralizing antibody lineages. By addressing the complex features of the HIV-1 Env protein and advancing immunogen design strategies, this research aims to make significant contributions towards the development of an effective HIV-1 vaccine.

NIH Research Projects · FY 2026 · 2024-02

Multi-cancer detection assays offer new opportunities to screen for many different cancers that currently have limited options for early detection. The Cancer Screening Research Network (CSRN) will conduct definitive clinical trials and studies to evaluate these assays. Design and implementation of screening trials involve many challenges. Cancer incidence and death are rare events in an average risk population.Trial designs must balance screening frequency, trial duration, and sample size to achieve trial objectives in a cost-efficient manner. Implementation must anticipate issues such as non-adherence, non-compliance, and contamination. Analysis must accommodate the design while adapting to the circumstances of implementation. The Statistics and Data Management Center (SDMC) of the CSRN will offer a rigorous system for CSRN trial design, management and analysis so that the information generated by CSRN trials forms a sound basis for national cancer screening policy. Our team brings together statistical leaders and clinical trial management experts with experience in major clinical trials networks, including the Women’s Health Initiative Clinical Coordinating Center, the SWOG Statistics and Data Management Center and the Early Detection Research Network Data Management and Coordinating Center. We have expertly designed, implemented, analyzed and reported cancer screening, prevention, and treatment trials. This work has required developing procedures and processes for expert execution of trials and production of reliable results. It has necessitated creating close collaborations with clinicians, scientists, patient advocates, and subject matter experts. And, particularly in the case of screening trials, it has inspired development of novel statistical methods that have become established in the field. Our goal is to promote excellence in all aspects of statistical and data management for the CSRN. To address the critical questions regarding potential benefits and risks of new cancer screening methodologies, the CSRN clinical trials must be designed and implemented with great integrity and efficiency to produce the most knowledge possible within realistic constraints of time and resources. To attain these goals, the SDMC for the CSRN has four specific aims: (1) Integrate the SDMC with the other CSRN components. (2) Provide rigorous and high-quality designs and analysis for CSRN studies. (3) Build a state-of-the-art data system to ensure data quality and integrity for CSRN trials. (4) Develop statistical and data management approaches to a large CSRN screening clinical trial that would build upon the Vanguard study. MCD testing represents a potential paradigm shift for cancer early detection. Successful execution of these aims will ensure that the CSRN produces valid and reliable quantitative results concerning the efficacy and benefit-harm tradeoffs of candidate products to support evidence-based policies concerning their use.

NIH Research Projects · FY 2026 · 2024-02

The promise of early detection to reduce the burden of cancer is an easily understood and highly desired intervention point in the cancer continuum. Recent developments of multicancer detection (MCD) technologies have fueled already strong interest in their use, well in advance of compelling data describing the impact of MCD-based screening on cancer mortality. In addition to limited data available on sensitivity and lead time to assess these MCD tests, the complexities of implementing them in a real-world clinical environment require careful evaluation to assess the frequency of achieving resolution of a positive MCD test (i.e., a specific cancer diagnosis), the nature, timeline and cost of those procedures, and the potential to increase cancer worry and differences in health outcomes. To help to address these critical gaps in data, the Cancer Screening Research Network (CSRN) will be a cooperative group structure with the expertise, infrastructure and access to appropriate patient populations needed to provide high quality evidence on the value of MCD tests in real-world settings. The goal for the Vanguard phase is to design and conduct a randomized controlled feasibility trial of two promising MCD tests. The CSRN Communication and Coordinating Center (CCC) in conjunction with the CSRN Statistics and Data Management Center (SDMC) and the NCI will lead and support this critical national effort. Our specific objectives are to: (1) Provide exceptional scientific and clinical expertise in the design and implementation of multicenter cancer screening trials, and particularly randomized trials evaluating MCD assays. (2) Develop the organizational structure and administrative relationships to assure CSRN success. (3) Develop and maintain effective communication channels and materials to promote CSRN success (4) Support CSRN ACCESS sites in their recruitment and retention efforts. By these efforts, the CSRN will assess the feasibility of and establish the core infrastructure needed to conduct future full-scale trials of MCD tests and other cancer screening studies. Through the efforts of the Vanguard phase, the CSRN will create the infrastructure, data and learnings needed to support full-scale trials and additional observational studies of the most promising MCD tests and other cancer screening approaches.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT Infection with Herpes simplex virus 2 (HSV-2) for which there is no vaccine is a significant cause of human morbidity both in the United States and worldwide. Current understanding of humoral immunity to HSV-2 is based on bulk metrics of antibodies circulating in bloodstream, however everyday battle between virus and host occurs in microscopic tissue microenvironment which represent a unique immunological niche. Our premise is that study of local humoral responses will reveal critical, currently unidentified mediators and mechanisms for immune control of HSV-2. After primary infection, HSV-2 establishes latency in sensory neurons. Clearance and subsequent control of constantly reactivating HSV-2 in tissue are believed to depend on T cells, but clinical observations and mathematical models present evidence consistent with essential role for local humoral responses required to achieve efficient virus control, Our preliminary data has shown that antibody secreting cells (ASCs) are actively present in genital skin during HSV-2 reactivation and long after virus outbreak is contained. Using the power of having sequential samples in individual tissue biopsies over time combined with recent advancements in single-cell RNA sequencing we demonstrated that these clonally expanded cells commit to the long-lived plasma cell lineage implying possible tissue residency, Isolation of antibodies from these unique cells has shown they recognize HSV-2 suggesting they may be directed to as yet unidentified HSV-2 antigens and/or provide functions important for virus control in the tissue. Therefore, the goal of this project is to thoroughly define the immunoprotective role of skin antibody-secreting cells (ASCs) against reactivating HSV-2. Underlying our proposed experiments is the idea that tissue resident ASCs have the ability to contain the virus over long timeframes via secreting HSV-2 specific antibodies that are essential for virus control due to their specificity and/or effector functions. Therefore, we focus on critical features of ASCs such as antigenic specificity, gene expression profile, clonal structure and functional activity of cognate antibodies during symptomatic HSV-2 reactivation and at multiple time points following clearance of a genital skin virus within microscopic sites of infection. We will also evaluate frequency of essential clones of skin ASCs among circulating B cells that have direct implication for rational vaccine design allowing targeted vaccination strategies. Ultimately, our goal is to use the gained insights to develop strategies that will allow for novel approaches to design successful herpes vaccine and development of immunotherapeutic approaches to treat chronic HSV-2 infection.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY Overview: 1.8 million people die of lung cancer each year across the globe. Immunotherapy, particularly using immune checkpoint inhibitors (ICIs), has revolutionized lung cancer treatments over the last years projecting a significant improvement in cancer prognosis; however, response rates of ICIs are only 15-25%, which is in part due to the lower levels of functional immune cells and insufficient infiltration of immune cells into the tumor. Furthermore, about 70% of patients with NSCLC receiving ICIs experience immunotherapy-related adverse events (irAEs) (e.g., metabolic dysregulation, myalgia, and cardiopulmonary disease). An effective intervention that can enhance treatment responses as well as ameliorate irAEs would be a significant advance in clinical care for patients with NSCLC on ICIs. Exercise has been emerging as a novel approach that can enhance response to immunotherapy and reduce irAEs. Exercise can increase the infiltration of neutrophils in the target cells and circulating levels of T cells and NK cells as well as induce tumor angiogenesis in preclinical settings, which has the potential to induce a better response to ICIs and cancer suppression. Furthermore, exercise is proven to be feasible in patients with lung cancer and to increase cardiopulmonary/muscular fitness and metabolic regulation, which also suggests the potential benefits of exercise to reduce irAEs. However, no exercise clinical studies, to date, have been conducted in patients with lung cancer receiving ICIs. Also, it is unclear which exercise prescription, particularly regarding exercise intensity, would yield better immunological and treatment-related outcomes. Therefore, I propose multi-level clinical research where a pilot randomized clinical trial (K99) will explore the preliminary efficacy and mechanisms of different exercise training on immune function, which will inform a larger Phase II trial (R00) to examine the effects of exercise on immunotherapy response rate and irAEs. Research Plan: The K99 study is a three-arm pilot randomized clinical trial that will compare high-intensity interval training (HIIT) vs. moderate-intensity continuous training (MICT) vs. usual care (UC) to explore the preliminary efficacy of exercise on immune function (Aim 1) and provide a proof-of-concept of the mechanisms by analyzing biomarkers of immune function, exercise-induced cytokines, and cancer progression (Aim 2). These findings will inform the R00 study, which is a two-arm phase II randomized clinical trial to examine the effects of exercise (most efficacious training from K99) on immune function (Aim 3a), tumor biomarkers (Aim 3b), and immunotherapy response and irAEs (Aim 3c). Career Development Plan: The proposed comprehensive and transdisciplinary training, involving coursework, mentorship, research involvement, and seminars/meetings, will enable me to (1) obtain the fundamental expertise necessary to comprehend the roles of exercise in lung cancer immunotherapy and (2) become an independent researcher who will lead a research group to pioneer the field of ‘Exercise Immuno-Oncology’.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT Despite treatment advances over the past decade, metastatic prostate cancer (PC) remains a fatal disease. As such, there is an acute need for additional treatment options. The development of therapeutics that specifically target cell surface antigens represent an attractive approach. PSMA is a cell surface glycoprotein that is highly expressed on PC and because PSMA is relatively restricted to prostatic tissues, it represents an ideal theranostic target (i.e. useful for both ‘therapy’ and ‘diagnostics’). 177Lu-PSMA-617, a PSMA-directed radioligand, was recently approved by the FDA. While this is clearly a welcome treatment option, the median PFS has been reported to be <9 months and objective responses only occur in ~50% of patients. As such, strategies to augment the efficacy of PSMA-directed therapies are needed. Given that pre-clinical and clinical data has shown that high, uniform expression of PSMA is associated with optimal outcomes, one approach to augment the activity of 177Lu-PSMA-617 would be to administer a drug shown to increase PSMA expression. Our preliminary data from the University of Washington Rapid Autopsy (UW-RA) program has shown that a substantial proportion of men who die from metastatic castration-resistant PC (mCRPC) harbor tumors that have minimal to no PSMA expression. Mechanistic studies exploring the mediators of PSMA expression have demonstrated that epigenetic modifications represent key regulators of PSMA expression, with DNA hypermethylation of the FOLH1 (PSMA gene) and closed chromatin states associated with PSMA negative tumors. Importantly, we have demonstrated that the epigenetic changes resulting in PSMA repression are reversible and treatment with a variety of HDAC inhibitors results in robust increases in PSMA expression in vitro and in vivo. In this proposal we will conduct a first in field clinical trial testing a novel epigenetic ‘priming’ strategy designed to increase PSMA expression. We will use innovative technologies and unique biospecimens to refine our priming strategy and develop iterative approaches to maximize clinical benefit to PSMA-directed therapies. In Aim 1 we will evaluate the effect of the HDAC inhibitor vorinostat on PSMA expression as determined by PSMA PET imaging. We will also conduct detailed correlative work to assess imaging, blood and tissue-based predictive biomarkers. In Aim 2 we will refine our epigenetic ‘priming’ strategy and conduct preclinical studies that will inform iterative clinical trials. Finally, in Aim 3 we will utilize the UW-RA program to collect metastatic tissue from men who died from mCRPC and received 177Lu-PSMA-617. This will enable us to determine other regulators of PSMA expression and determine mechanisms of resistance to 177Lu-PSMA-617. Importantly, because these patients will also have baseline PSMA and FDG-PET imaging, we will be able to evaluate the dependency of PET imaging features on molecular phenotype. Collectively, these studies will be important to guide the clinical development of PSMA theranostics and will provide novel approaches to overcome primary and secondary resistance to PSMA-directed therapies.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT Relative to the general population, HIV-infected individuals have a 60–200-fold higher relative risk to develop Non-Hodgkin's Lymphomas, including plasmablastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma and diffuse large B-cell lymphoma. They also have an 8–10-fold higher relative risk to develop Hodgkin's Lymphoma compared to uninfected individuals. Many of these malignancies are driven by Epstein-Barr virus (EBV) and result from poor immune control of the virus driven by HIV- dependent immune dysfunction. Thus, a safe and effective vaccine that prevents EBV infection and eliminates the EBV-associated component of risk could have a significant clinical benefit in resource-poor areas where HIV-1 is endemic. The correlate of protection for most successful viral vaccines is neutralizing antibodies. Previously, our lab isolated the first anti-EBV mAb that could potently neutralize infection of both B cells and epithelial cells. We further demonstrated that passive transfer of AMMO1 protected both humanized mice and rhesus macaques from experimental EBV infection. These results strongly suggest that antibodies are important for, if not sufficient to protect against EBV infection, and that a prophylactic vaccine should seek to elicit high titers of AMMO1-like antibodies. To this end, we developed and tested several gH/gL vaccines. All gH/gL vaccines were strongly immunogenic, but they only showed partial protection against experimental EBV infection in humanized mice and macaques. Epitope mapping studies revealed that vaccine-elicited gH/gL antibodies targeting the AMMO1 epitope were subdominant and rare, while antibodies targeting other non- neutralizing epitopes on gH/gL were immunodominant. The goal of this proposal is to obtain a high-resolution antigenic landscape of the gH/gL glycoprotein to define the most critical sites of vulnerability. We will then use this this information to employ structure guided EBV vaccine design intended to immunofocus the antibody response onto protective epitopes like the one targeted by AMMO1, and evaluate their ability to protect against EBV challenge in humanized mice and macaques.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY The thymus, which is the primary site of T cell generation, is extremely sensitive to injury; but also has a remarkable capacity for endogenous repair. However, even though there is continual thymic involution and regeneration in response to everyday insults like stress and infection, profound thymic damage caused by common cancer therapies and the conditioning regimes for hematopoietic cell transplantation (HSCT) lead to prolonged T cell lymphopenia. Furthermore, in the context of allogeneic HCT, the thymus is an extremely sensitive target to alloreactive T cells during graft versus host disease (GVHD). Consequently, identification of therapies that can boost T cell reconstitution in recipients of HSCT is a clinical priority. We have previously identified two distinct pathways of endogenous thymic regeneration, centered on the production of the regeneration factors IL-22 by innate lymphoid cells (ILCs), and BMP4 by endothelial cells (ECs); both of which mediate their regenerative effects by targeting thymic epithelial cells (TECs). More recently we have found that the trigger for these distinct regenerative pathways hinge on the balance between forms of cell death, with immunologically silent apoptosis (which is abundant in thymocytes during steady-state) suppressive to the regenerative program. On the other hand, after thymic damage caused by radiation injury, we found a switch toward immunogenic cell death, with the resulting release of damage-associated molecular patterns (DAMPs) sufficient to promote regeneration. Specifically, we identified that intracellular Zn was released after radiation injury, where it could signal through the G-protein coupled receptor 39 (GPR39) to stimulate production of BMP4 and IL-23, a key upstream regulator of IL-22 production. Separately, we also found that the release of the prototypical DAMP, ATP, was able to signal directly on thymic epithelial cells through purinergic (P2) receptors and promote their expression of Foxn1, key microenvironmental drivers of T cell development. Importantly, our preliminary data also suggests that each of these pathways can be therapeutically targeted to improve thymic recovery in mouse models of HCT. Our preliminary data has also identified putative TEC precursors that are important for regenerating the epithelial compartment and thus promoting regeneration, as well as the emergence with age of aberrant epithelial cells that limit thymic function, including in its reparative capacity after acute damage. Our research program is thus exploring several key questions: What are the triggers and upstream regulators of endogenous tissue regeneration in the thymus? Which cells mediate tissue regeneration in the thymus after acute injury? Are there limitations to endogenous regeneration after HCT across sex and lifespan? Can we exploit these mechanisms of endogenous regeneration to develop therapeutic strategies to boost T cell reconstitution in HCT recipients? The studies outlined in this proposal not only have the potential to define important pathways underlying tissue regeneration but could also result in innovative clinical approaches to enhance thymic function.

NIH Research Projects · FY 2025 · 2024-01

ABSTRACT Blood cancers account for approximately 10% of all malignancies and allogeneic bone marrow transplantation (BMT) is a preferred curative therapy for these serious conditions. While the therapeutic potential of this procedure lies in graft-versus-leukemia (GVL) effects in which donor T and NK cells eradicate host malignancy, BMT outcomes are limited by transplant-related complications, mainly graft-versus-host disease (GVHD) in which donor T cells attack host normal tissues and opportunistic infections. Indeed, 15-20% of BMT patients develop severe GVHD that is fatal, particularly when involving the gastrointestinal tract, however, relapse of the primary malignancy is still high and eventually responsible for the majority (40-60%) of transplant failures and death. Current prevention and treatment of GVHD which rely on the broad suppression of T cells abate GVL effects. Thus, the immunological intervention to minimize GVHD with promoting GVL is unmet need in BMT. The initiation and maintenance of donor T cell responses are dependent on alloantigen presentation by antigen- presenting cells (APC). Although recently the types of APC that induce and enhance GVHD and the immunological mechanisms therein have been elucidated, their role in GVL and the presence of GVL-specific APC remain unknown. In this proposal, we focus on GVL-requiring APC to enhance GVL and precent leukemia relapse. In particular, we will build on our preliminary data to test the hypothesis that donor T cells primed at extramedullary sites migrate into the BM where their anti-leukemic capacity is maintained via alloantigen presentation by recipient stromal APC. We will utilize cutting-edge mechanistic preclinical murine studies, using advanced flow cytometry, multiplexed immunofluorescence microscopy, RNASeq, and REAP/CITE-seq, to understand antigen presentation required for effective GVL and relapse prevention. Finally, we will test a novel attenuated synthetic IL-18 resistant to the IL-18 binding protein (decoy resistant (DR-18)) that has been fused to CXCR4 nanobody or CD117 antibody to selectively enhance IFNg and antigen presentation locally in the bone marrow. The proposal will lead to a new, clinically tractable approach to selectively promote GVL and prevent relapse after allogeneic BMT.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract The long-term objectives of this proposal include the following: 1) to generate cell-type- specific chromatin profiles of the four cell types of the intestine, 2) to identify the effect crippled silencing in intestinal stem cells has on progenitor cells, and 3) to characterize the transcriptional changes associated with an age-related increase in the Polycomb Repressive Complex-2 mark, H3K27me2, in the intestine. Cell-type-specific chromatin profiles aid the specific gene expression patterns necessary for the function and identity of the cell. Complex tissues are comprised of both stem and differentiated cells with different phenotypes, transcriptional profiles, and roles within the tissue. The intestine contains a population of stem cells responsible for replenishing damaged cells from the multiple stimuli exerted on the intestine throughout the organism’s life span. However, the determining factors, especially at the chromatin level, that make each cell type unique are unknown. Investigation into the chromatin modifications of each cell type will provide insight into the unique roles each cell plays in the function of the tissue. To generate gut cell-specific chromatin profiles, I will use CUT&Tag, a robust and sensitive chromatin profiling technique that can be used with low cell numbers. I will generate landscapes for chromatin modifications for all cell types of the gut to comprehensively profile the epigenetic state of this tissue. Because of the importance of silencing modifications for cellular identity and previous observations of loss of chromatin silencing affecting the gut cells, I will investigate the effect of the loss of different chromatin silencing pathways in intestinal stem cells on progenitor cells. Since the intestinal stem cells are the only mitotically active cells in the intestine and the source of all other cells in the gut, their chromatin profiles likely affect the progenitor cells. Additionally, observations of age-related increases in H3K27me2 in the Drosophila melanogaster gut may indicate the protective role H3K27me2 has in preventing pervasive transcription as tissue ages. The insights gained from this project will help us understand the cell-type-specific chromatin profiles of cells of complex tissue and how chromatin modification changes in stem cells affect their progenitors. My proposed project fits the mission of NIGMS to fund research in fundamental biology that increases understanding of the principles that underlie biological processes.

NIH Research Projects · FY 2026 · 2024-01

Project Summary / Abstract: During development, the central nervous system establishes precise connections with the body to coordinate organ function. A crucial component of this communication between the brain and body is the vagus nerve (cranial nerve X), which innervates multiple organ systems including the heart, lungs, and digestive tract to regulate blood pressure, heart rate, respiration, and digestion. Despite this important role, the molecular mechanisms guiding the vagus nerve to these organ targets remain completely unknown. We have developed the zebrafish embryo as a powerful model for interrogating vagus nerve development, taking advantage of its optical clarity and genetic accessibility. The vagus is comprised of both ascending sensory fibers that transmit organ state to the brain, and descending motor projections that deliver reciprocal motor commands to the organs. The vagus nerve also targets pharyngeal arch-derived muscles in the head, and the Moens lab has previously described a topographic relationship between the positions of motor neurons in the brain and their targets in the head, and has discovered a spatio-temporal mechanisms for the development of this map. The preliminary data I present here demonstrates that vagal motor projections to the organs are also organized topographically, where vagal motor neurons innervating different organs (heart, stomach, intestines) are spatially segregated within the hindbrain vagus nucleus. I also observe vagal motor projections reaching the viscera much earlier than their sensory counterparts, leading me to hypothesize that correct motor innervation of the viscera is required for subsequent sensory innervation. Here, I propose to address these hypotheses through the following aims. In Aim 1, I will use genetic tools along with live imaging and single-cell RNA sequencing to determine the molecular mechanisms guiding subsets of vagus motor neurons to the heart and gut. I will identify candidate molecules (transcription factors and cell-surface proteins) determining the topographic organization of somatic innervation and test the role of these candidates using reverse genetics. In Aim 2, I will determine the mechanisms guiding vagal sensory neurons to the appropriate organ targets and test the dependence of sensory innervation on the correct establishment of vagal motor innervation. This work will reveal how a major pathway of communication between the brain and organs is established during development.

NIH Research Projects · FY 2026 · 2023-12

SUMMARY Small cell lung cancer (SCLC) is a lethal tumor type characterized by exquisite response to chemotherapy followed by rapid emergence of chemoresistance. Addition of PDL-1 inhibition to platinum/etoposide chemotherapy leads to improved clinical responses but only a small subset of SCLC patients benefit. Distinct subsets of SCLC have been identified including high-neuroendocrine and low-neuroendocrine (NE) subtypes. High-NE SCLC, expressing high levels of ASCL1 (SCLC-A) or NEUROD1 (SCLC-N) is best understood, with genetically engineered mouse models available. However, our understanding of low-NE subtypes is poor, owing in part to a lack of mouse models. A low-NE immune “inflamed” subset of SCLC (SCLC-I) exhibited increased response to immune checkpoint blockade and high expression of RE-1 Silencing Element (REST), a suppressor of neuronal and neuroendocrine gene expression. Aim 1 tests hypotheses that REST overexpression in a mouse model of SCLC will result in low-NE SCLC and that REST increases immunogenicity. We will use gain and loss of function studies to dissect the contribution of REST to SCLC and to model low-NE SCLC. RNA-seq analyses will use data from mouse GEM models and isogenic cell lines with REST perturbation to identify genes consistently regulated by REST while CUT&RUN will identify direct targets of REST. We will perform gain and loss of function studies to assess pathways through which REST alters the biology of SCLC, with an initial focus on how REST controls the expression of MHC-I. Aim 2 builds from our preliminary data that implicate REST expression in driving chemotherapy resistance. We performed an in vivo functional CRISPR activation (CRISPRa) screen to identify genes that switch chemosensitive patient derived xenograft (PDX) models to become chemoresistant when overexpressed and among the top screen hits was REST. We will study the impact of REST perturbation on chemotherapy response and will test our hypothesis that REST expression will cause a switch to chemoresistance. We will perform immunohistochemistry and molecular analyses to characterize the response of REST-perturbed PDX models to chemotherapy in vivo. With the increased appreciation of low-NE SCLC but poor understanding of the underlying biology, it is critical that this important subset of SCLC be modelled and understood. This proposal provides new in vivo GEM and PDX models of low- NE SCLC, which are essential reagents to link biologically distinct SCLC subsets to the most promising therapeutic approaches.

NIH Research Projects · FY 2025 · 2023-12

ABSTRACT (from parental grant) The recent advent of highly potent inhibitors of the androgen receptor and androgen biosynthesis has had the unfortunate iatrogenic effect of fueling new lethal prostate cancer phenotypes in patients. In particular, non- neuroendocrine androgen receptor-low castration resistant prostate cancer (CRPC), an aggressive form of this disease, is increasing in occurrence amongst patients and is uniformly fatal. The main barriers against therapeutic advances are a paucity of relevant disease models and a very poor understanding of the mechanisms that give rise to this phenotype. The process of protein synthesis has long been considered subordinate to alterations at the levels of DNA and RNA in cancer etiology. However, work from our laboratory and others have revealed that protein synthesis control is a dynamic process that coordinates not only bulk mRNA translation, but also the specialized translation of distinct mRNAs important for cancer phenotypes. Recently, our laboratory has developed and characterized a new in vitro and in vivo toolkit of both human and murine androgen receptor-low CRPC. We have used these models to discover a critical link between androgen receptor signaling and the process of mRNA translation initiation, which is critical for androgen receptor-low CRPC growth. We hypothesize that androgen receptor-low CRPC is driven by the specific translation of distinct mRNA networks, thereby leading to persistent tumor growth, which may represent a therapeutic vulnerability. Our long-term objective is to utilize state-of-the-art mouse models, ribosome profiling, and patient derived xenografts to definitively investigate the fundamental link between the androgen receptor and protein synthesis control in a highly relevant and newly emerging disease course for prostate cancer patients. To do so, we will address the following aims: 1) determine the mechanism by which the androgen receptor communicates with the translation apparatus, 2) delineate how aberrant protein synthesis drives the translation of distinct oncogenic mRNAs, and 3) elucidate the therapeutic efficacy of targeting translation initiation in androgen receptor-low CRPC. Ultimately, these studies are poised to uncover a new paradigm for gene regulation in androgen receptor-low prostate cancer and provide the preclinical basis for targeting the protein synthesis apparatus in an increasingly common highly aggressive disease.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Newly synthesized DNA represents an ideal system to understand the molecule mechanisms governing chromatin structure and gene expression. Chromatin structure controls transcription by modulating DNA accessibility. During DNA replication, chromatin structure must be disassembled to allow for passage of the replication fork, which generates an intermediate chromatin structure distinct from mature chromatin. Failure to reestablish proper chromatin structure leads to spurious gene expression and is associated with a variety of human diseases. Transcription factors (TFs) are a critical component of chromatin structure that regulate transcription by binding to targeted sequences on DNA and altering chromatin structure. Following replication fork passage, TF binding sites become occluded by nucleosomes. As nucleosomes are refractory to TF binding, it is unclear when or how TFs rebind to targeted DNA sequences to reestablish mature chromatin structure. Furthermore, in-spite of the established role for TFs in regulating gene expression, it is unclear what effect TF binding has on chromatin structure. To address these gaps in knowledge, I will use novel techniques termed Nascent CUT&Tag and Nascent Fiber-seq to profile TF binding on nascent DNA and during the subsequent steps of chromatin maturation. In Aim 1, I will use Nascent CUT&Tag to determine the kinetics and mechanisms of TF binding to nascent chromatin. This aim will test whether TFs can associate with nucleosomal DNA during the process of rebinding, or whether accessible DNA is necessary for TFs to bind following replication fork passage. In Aim 2, I will visualize TF binding and nascent chromatin maturation on the single molecule level. By simultaneously visualizing TF binding and surrounding chromatin structure, I will be able to assess the direct impacts of TF binding on critical chromatin features such as nucleosome positioning and RNA polymerase II occupancy. In Aim 3, I will characterize the impacts of nucleosome turnover on TF binding and chromatin maturation. Using the anti-cancer drug aclarubicin, I will drive elevated nucleosome turnover and observe the effects on TF binding and nascent chromatin maturation. These studies will provide critical insights into the features regulating TF binding and chromatin assembly, which will inform our understanding of the underlying mechanisms regulating gene expression and cell fate specification. The training outlined in this proposal will provide a strong foundation to develop as an independent investigator studying the critical relationship between chromatin structure and gene expression.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Differences in care across patient groups can be difficult to identify and address. Colorectal cancer (CRC) care provides a useful case study for examining the long-term outcomes from differences in care. There is ample evidence of differences in CRC screening and treatment by race and ethnicity at multiple steps in the diagnostic and treatment pathway. Most research has focused on differences between Black and White populations. Compared to White people, Black people are: less likely to be screened for CRC; less likely to receive endoscopic tests at high-quality facilities; more likely to be diagnosed at a later stage; less likely to receive curative treatment – even after accounting for stage at diagnosis; and have shorter stage-specific survival. These differences in care have both health and economic consequences. In addition to worse outcomes, Black people have higher CRC-attributable treatment costs. We propose to extend and apply CRC-SPIN, an established microsimulation model for CRC, to synthesize the available evidence related to CRC risk, CRC care, costs of care, and economic outcomes. We will extend CRC-SPIN, which currently simulates the overall US population by gender, to simulate the natural history of CRC among specific racial and ethnic groups represented in SEER data (Asian, Black, Latino, and White populations). Extension of CRC-SPIN to include race/ethnicity will focus on incorporating available information about differences in CRC risk. We will also extend CRC-SPIN to simulate insurance status, which mediates the relationship between race/ethnicity and CRC care. We will use the resulting model to simulate the overall impact of disparate care on health and financial outcomes, and to identify elements of the care process that have the largest impact on outcomes to better guide health policy. Health outcomes will include life years lost, disease-free life years lost, excess CRC, and excess late-stage CRC. Financial outcomes will assess individual , provider- and societal-level costs, including screening costs, treatment costs and lost income. We will use Robust Decision Making approaches to address uncertainty in the differential risk of CRC and differential care. These analyses will assist in identifying interventions that are expected to have the greatest impact on health outcomes, and the costs of these interventions.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT The development of drug resistance is a major cause of cancer treatment failure and mortality. Although much is known about the mechanisms by which tumor cells can become resistant to a given drug, translating this into effective therapeutic solutions remains an unmet clinical need. Here we propose to pioneer the use of patient derived tumor organoids (PDTOs) as a platform to identify and validate novel targets and effective drugs to overcome drug resistance in ovarian cancer, pancreatic cancer, and other tumor types. In our preliminary studies, we show that PDTOs genetically and phenotypically match the tumor from which they were derived and can be used to study the phenotypic consequences of tumor heterogeneity, tumor evolution, and drug resistance. Using a Clinical Laboratory Improvement Amendments (CLIA) approved high complexity assay, we show that PDTO drug sensitivities are highly concordant with known genetic biomarkers, retrospective treatment history and prospective patient responses. Tumor organoids derived from patients who developed in situ drug resistance demonstrate ex vivo resistance to those same drugs but also demonstrate sensitivity to other alternative oncology drugs. Additional preliminary studies show stable disease or tumor regression in patients treated with drugs identified from organoid drug screens. Here, we propose to combine drug screening and molecular profiling of PDTOs derived from a given patient from different anatomic tumor sites and before and after therapy to elucidate the mechanistic basis for drug sensitivity or resistance and to identify novel targets and effective drugs to treat metastatic, drug resistant cancers. Accompanying computational prediction models that integrate large public datasets as well as innovative methods of mechanistic target validation including CRISPR, targeted protein degradation technologies, and epigenetic profiling, will be used to prioritize and advance targets and associated biomarkers with greatest clinical potential. The rationale behind our approach is that identifying targets and effective drugs directly in patient derived samples with known clinical history and outcomes will significantly enhance translation of our findings. This proposal is significant because it will demonstrate the utility of PDTOs as both a research tool for target discovery and validation but also as a clinically useful platform to guide functional precision medicine. The findings and methods developed can be readily applied to other cancer types and clinical challenges, will accelerate preclinical drug and drug target development, and will translate to clinical studies. The models, approaches, and expected outcomes of this proposal are highly responsive to the requirements of PAR-21-274.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Accurate cell division is essential for the development of all organisms. During each cell cycle, chromosomes must be precisely partitioned to daughter cells. Defects in chromosome partitioning generate aneuploidy, the state where entire chromosomes are gained or loss. Aneuploidy is a the most common chromosomal abnormality in cancer cells and is thought to be a major factor in the evolution of cancer. It is also the leading cause of miscarriages and hereditary birth defects in humans. The proposed work will lead to an understanding of the mechanisms that ensure accurate chromosome partitioning. This work is important for maintaining genomic stability and preventing human disease. Chromosome partitioning occurs when spindle microtubules move chromosomes by interacting with kinetochores, the machines that assemble onto the chromosome at a locus called the centromere. Kinetochores carry out a number of functions, such as maintaining load-bearing attachments to the ends of microtubules that are continually growing and shrinking. They also control the cell cycle when there is a defect in kinetochore attachments to microtubules. Our lab will address two fundamental questions about chromosome segregation using in vitro assays: 1) How is kinetochore assembly regulated? 2) How do kinetochore proteins contribute to force-dependent kinetochore-microtubule attachments? We will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, our work will lead toward an understanding of the fundamental mechanisms of chromosome partitioning in all eukaryotes.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Liquid biopsy is a clinical test that involves the analysis of various biomarkers present in the blood or other types of body fluids. It enables the detection of cancer at an earlier stage than traditional imaging or tissue biopsy methods, and thus can lead to reduction of cancer mobility and mortality. As one of the most promising solutions for cancer early detection, liquid biopsy has the potential to revolutionize cancer diagnosis, providing patients with more effective and less invasive care. The liquid biopsy analytes include circulating tumor cells (CTCs), circulating tumor DNA, which is part of cell-free DNA (cfDNA), extracellular vesicles, as well other tumor associated RNAs, proteins and metabolites. These analytes typically have very low levels in blood or other types of body fluid of early-stage cancer patients, leading to difficulties in obtaining reproducible results and low sensitivity and specificity for cancer early detection. While many new assays and techniques have been developed to tackle these challenges, validation of these assays/techniques in different populations and/or cancer types poses many challenges, and these are the main areas of research supported by the liquid biopsy consortium (LBC). As Data Management and Coordinating Unit (DMCU) of LBC, we will support state- of-the-art statistical methods for study design and data analysis, the coordination, implementation, and conduct of collaborative studies across the consortium and work closely with LBC investigators on data analysis by statistical methods, machine Learning modeling and Artificial Intelligence. We will also support consortium coordination and data management, including identification and adoption of standard data elements for liquid biopsy studies. We seek to establish the DMCU as a leading center for innovation and resources in liquid biopsy research, and to enhance, improve and maintain LBC network integration and coordination.

NIH Research Projects · FY 2025 · 2023-09

Project Summary The phase 1 of the Molecular Phenotypes of Null Alleles in Cells (MorPhiC) consortium will produce a catalog of molecular and cellular phenotypes for null alleles of ~1000 human genes using in vitro cellular systems. These rich resources will allow us to study the gene functions in several multicellular systems that often model early human development. The impact of a gene knockout on complex human phenotypes can be highly dependent on the corresponding cell type, cell stage, and tissue microenvironment. Therefore, to generalize the insights from MorPhiC studies to in vivo settings, we need to harmonize MorPhiC resources and the molecular/cellular phenotypes of appropriate cell types or tissues, by a flexible and robust computational framework. We aim to achieve this goal by two complementary approaches. First, we will develop a dynamic gene regulatory network named moDAG: multi-omic Directed Acyclic Graph. MoDAG combines multi-omic data from MorPhiC and other studies and the state-of-the-art statistical methods to estimate a gene-regulatory network. MoDAG models cell types characterized by genome-wide epigenetic or gene expression data. It also accounts for signals from tissue microenvironment by modeling a set of signaling proteins. MoDAG can be used to predict the effect of gene knock out in the in vitro cellular systems, and thus help prioritize the genes to be targeted in future MorPhiC studies. Second, we propose a biologically informed deep learning method named as SDAN: Supervised Deep learning with gene Annotation. SDAN combines molecular phenotype of gene knockout with gene annotation to identify gene sets associated with gene knock out. Gene sets provide more robust characterization of gene knockout than individual genes and thus are more generalizable to different cell types or tissues. The gene annotation used by SDAN is gene-gene interaction network that can be modified according to relevant cell types or tissues. Finally, we apply these two methods to predict the phenotypic outcomes of gene knockouts and assess the association between gene knockouts and human phenotypes. Our computational framework bridges MorPhiC’s resource with accumulating omic data in various human cell types and tissues and provide effective solutions to generate new insights or hypothesis for future studies.

NIH Research Projects · FY 2025 · 2023-09

Since the early 1980s, American Indians and Alaska Natives (AIANs) have maintained the highest rates of commercial cigarette smoking. Currently, 27% of AIANs smoke cigarettes. They have one of the highest rates of developing smoking-related cancers. AIANs have low rates of quitting smoking. The result is that commercial cigarette smoking accounts for half of all deaths among AIANs nationwide. A major cause of these high rates of smoking and low rates of quitting is the lack of access to efficacious smoking cessation interventions among AIANs. Compounding the barrier of lack of access to smoking cessation interventions is the barrier of lack of research on the efficacy of smoking cessation interventions for AIANs. Despite having a high smoking prevalence for over 40 years, a mere 0.3% of all full-scale smoking cessation randomized controlled trials (RCTs) have focused on AIANs. Regarding accessibility, a smartphone application (“app”) has the potential to deliver a low-cost smoking cessation intervention with wide geographic reach to AIANs and in regions of the US with smoking rates as high as 57% in this group (i.e., Northern Plains). Regarding efficacy, our preliminary data provides promising evidence for our Acceptance and Commitment Therapy (ACT)-based smartphone app, called iCanQuit, to help AIANs quit smoking. We compared the iCanQuit app with the NCI’s QuitGuide app among the AIAN subsample (N = 165 recruited from 32 US states) enrolled in our full-scale RCT. This secondary analysis of AIANs showed descriptively higher rates of smoking cessation at the 12-month follow-up (30% for iCanQuit vs. 18% for QuitGuide; OR = 1.96; 95% CI = 0.90, 4.26, p = .089). While encouraging, analyses were exploratory, non-significant, and not a substitute for a full-scale efficacy test. To address weaknesses of prior research, a fully-powered comparative efficacy RCT of iCanQuit vs. QuitGuide focusing nationwide on AIANs who smoke is now needed. Thus, the goal of this project is to conduct a nationally recruited and fully-powered two-arm RCT comparing iCanQuit (n = 388) to QuitGuide (n = 388), in order to determine: (1) the efficacy of iCanQuit relative to the QuitGuide app for biochemically verified 30-day point prevalence abstinence (PPA) at 12 months post-randomization and (2) whether iCanQuit’s (but not QuitGuide’s) 12-month smoking cessation outcomes are significantly mediated by improvements in core ACT-based processes. This study will be the first full-scale RCT of a digital intervention for helping AIANs nationwide stop smoking. Qualitative interviews with (1) a subsample of iCanQuit participants to thematize testimonials of their experience with iCanQuit and (2) AIAN members from our study Community Advisory Board (CAB) will guide our plan for broadly disseminating iCanQuit to AIAN adults nationwide. Positive results would provide a highly accessible and efficacious intervention with potential for sustainability and broad dissemination for AIANs nationwide.

NIH Research Projects · FY 2023 · 2023-09

ABSTRACT Endometriosis is a debilitating gynecologic disease presenting with severe pelvic pain and is characterized by the growth of endometrial-like tissue outside of the uterus impacting 10% of reproductive aged women or an estimated 200 million women and adolescents worldwide. Compared to women without endometriosis, women with endometriosis, especially adolescents and young adults with endometriosis, are at an increased risk of chronic opioid use, dependence, and overdose. Therefore, optimal pain management in endometriosis patients, especially starting in adolescence, is critical and will have significant positive impact on resolving the opioid health crisis. Currently, primary treatment options for endometriosis focus on hormonal suppression and/or excision of the endometriotic lesions, although response to these conventional treatments is variable and as a result, many of those with endometriosis are plagued with persistent pelvic pain. Emerging evidence suggests that endometriosis patients who develop persistent pelvic pain have developed centralized pain, and therefore surgical removal of endometriotic tissue does not fully improve their pain. Since many women with diagnosed endometriosis report their symptoms started during adolescence, this transition from acute to chronic pain is likely happening during adolescence and young adulthood. Thus, studying adolescents and young adults with endometriosis, who are in the early stages of their disease trajectory, is critical to fully understanding who is at higher risk of developing chronic pain. However, data on longitudinal changes in biomarkers and endometriosis- associated pain in adolescents is lacking, resulting in lost opportunity for early interventions. The overarching goal of this innovative application is to improve and optimize pain management for endometriosis through identifying plasma protein biomarkers of chronic pain development in adolescents and young adults with endometriosis. Specifically, we propose to conduct a longitudinal analysis of endometriosis cases diagnosed in adolescence with follow-up data and paired blood samples collected 10 years apart from adolescence to adulthood and apply a state of the art 7000-plex proteomics assay to identify plasma protein biomarkers of centralized, chronic pain development. In addition, we will examine change in plasma proteomic biomarkers in paired blood samples drawn 10 years apart (i.e. at adolescence and adulthood), and together these unique resources will allow prospective investigation of predictors and biological factors related to transitioning from acute to chronic pain or chronification of pain. Results from this study will generate important novel data identifying adolescents and young women with endometriosis who are at greater risk of developing chronic pain despite receiving current standard of care, leading to development of novel pain interventions targeted to a younger population to prevent chronification of pain, which will be a critical step forward to resolving the ongoing opioid crisis.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Prostate cancer is the second most common cause of cancer mortality among men. The majority of these deaths are due to resistance to androgen deprivation therapy and progression to lethal castration-resistant prostate cancer (CRPC). New generation androgen receptor signaling inhibitors (ARSI) that target the AR signaling axis have been used in the CRPC setting; however, the majority of patients still develop resistance. Recently, prostate-specific membrane antigen (PSMA) has become a promising target for positron-emission tomography imaging (PSMA-PET) and targeted therapies, such as the recently FDA-approved radioligand (PSMA-RL) for CRPC patients who progressed on ARSI treatment. Despite a survival benefit for PSMA-RL therapy, the improved outcome is modest and only half the patients show favorable responses. The emergence of resistance to ARSI and PSMA-RL may arise through changes in tumor phenotype, such as trans-differentiation from prostate adenocarcinoma (ARPC) into treatment-related small-cell neuroendocrine prostate cancer (NEPC) and other phenotypes with loss of AR activity. Current methods require a biopsy to diagnose tumor histology, which can be challenging due to invasive procedures accompanied by morbidity and some tumors are not accessible or have poor sample quality. Furthermore, tumor heterogeneity is a major contributor to therapy resistance and is particularly challenging to identify using a biopsy of a single metastatic site. These challenges exemplify major limitations of current treatment strategies and precision medicine for men with CRPC. Circulating tumor DNA (ctDNA) released from tumor cells into the blood as cell-free DNA (cfDNA) is a non- invasive “liquid biopsy” solution for addressing challenges in tissue accessibility. Current research and clinical efforts have focused on the detection of genetic mutations from ctDNA sequencing as potential biomarkers; however, these do not fully explain why treatments fail. The objective of this proposal is to develop and evaluate innovative methods for classifying aggressive CRPC genotypes and phenotypes from ctDNA, overcoming challenges of tumor heterogeneity. The investigators hypothesize that ctDNA can be used to classify tumor subtypes in CRPC and that this can be used to predict treatment outcomes. In Aim 1, they will study tumor heterogeneity in men who have undergone rapid autopsy to evaluate the ctDNA classifiers for predicting heterogeneous phenotypes from post-mortem plasma. In Aim 2, they will determine the utility of ctDNA for predicting prostate cancer treatment outcomes in a prospective cohort of patients treated with ARSI and a subset of patients screened by PSMA-PET and treated with PSMA-RL therapy. They will evaluate the ctDNA classifiers as biomarker tools to aid in the initial allocation of PSMA-RL therapy and inform early indications of treatment resistance. In Aim 3, they will develop extensions to ctDNA methods that infer gene expression and tumor aggressiveness in prostate cancer phenotypes using preclinical mouse PDX models, including in vivo engineering of phenotype mixtures.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Prostate cancer is the second most common cancer and the second leading cause of cancer death in US men, with African American men having the highest incidence and mortality rates. Prostate cancer is strongly influenced by genetic factors, and polygenic risk scores (PRS) of common genetic variants are highly predictive of prostate cancer risk in men from European, African, East Asian, and Hispanic populations. Rare pathogenic variants also contribute to overall and aggressive prostate cancer risk, with 15% of metastatic cases carrying such variation. However, due to high sequencing costs, our knowledge of the contribution of rare genetic variation to prostate cancer risk is largely based on candidate gene studies, with of the few whole-exome or whole-genome studies conducted to date having sample sizes that are large enough for the discovery of novel rare variants/genes. Further, the majority of men included in common and rare genetic variant investigations of prostate cancer risk have been from European ancestry populations, limiting our knowledge on genetic risk of prostate cancer in other populations. The objective of this research is to elucidate genetic factors that contribute to risk of overall and aggressive prostate cancer across diverse populations and how such factors can be combined with lifestyle, environmental, and socioeconomic factors to more accurately characterize risk of prostate cancer. In Aim 1, we will investigate the contribution of rare and common genetic variants to prostate cancer risk across diverse populations, with the goal of validating known genetic risk factors and discovering novel genetic risk regions. In Aim 2, we will investigate whether genetic risk of prostate cancer, as measured by a PRS of known common genetic risk variants, can be modified by rare pathogenic variant carrier status, lifestyle factors, and socioeconomic factors across diverse populations. This research will be conducted in the All of Us Research Program, combining results from Aim 1 with other large-scale investigations to improve our ability to identify novel genetic risk regions. Findings from this investigation are expected to identify novel mechanisms novel mechanisms to target for preventative measures and improve our understanding of the complex interplay of genetic risk and modifiable risk factors of prostate cancer. Further, this investigation is anticipated to improve our ability to identify men at increased risk of overall and aggressive prostate cancer, which could have important screening and healthcare implications for prostate cancer prevention.