Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY This career development award, Acupuncture for Cognitive Health in Older Survivors of Prostate Cancer (ACHIEVE), will train Dr. Liou to become an independent clinician-investigator with expertise in integrative therapies for cognitive health and symptom management in prostate cancer (PC) survivors. BACKGROUND: By 2030, PC survivors are expected to become the largest group of cancer survivors in the United States. Cancer-related cognitive dysfunction (CRCD) is a significant concern in this growing population, but evidence- based treatments are lacking. Most CRCD studies in PC have focused on the neuro-toxicities of androgen deprivation therapy (ADT). However, the literature on the association between ADT and CRCD remains mixed, suggesting other factors may play a role in CRCD. Growing research from other populations demonstrates that co-morbid symptoms (anxiety, depression, fatigue, insomnia, nocturia) may contribute to cognitive dysfunction. These symptoms are prevalent among PC survivors, but their role in CRCD remains poorly understood in the PC population. Expanding CRCD research beyond the non-modifiable factors (prior ADT exposure) and focusing on modifiable factors (co-morbid symptoms) has the potential to inform novel treatments approaches for CRCD. Based on Dr. Liou’s preliminary studies, acupuncture improves objective CRCD directly, but it may improve subjective CRCD indirectly through co-morbid symptoms. Informed by this prior work, Dr. Liou will test a novel acupuncture intervention that directly targets CRCD with the optionality to jointly address symptoms co-occurring with CRCD. RESEARCH: Dr. Liou will evaluate the association between co-morbid symptoms and CRCD in a cross-sectional study of PC survivors (Aim 1). Dr. Liou will then evaluate the feasibility and preliminary effects of acupuncture for CRCD and co-morbid symptoms in a pilot randomized controlled trial of PC survivors (Aim 2). CAREER DEVELOPMENT: Dr. Liou will acquire competence in CRCD evaluation, PC symptom science, clinical trial design, biostatistics, and grant-writing. His training will be supported by mentorship, coursework, independent study, workshops, conferences, and preparation of manuscripts and grants. MENTORS: Dr. Liou will be mentored by a team of internationally-recognized researchers with complementary expertise in CRCD, neuropsychological assessment, PC symptom science, integrative medicine, oncology acupuncture, clinical trial design, biostatistics, and PC medicine. ENVIRONMENT: Memorial Sloan Kettering Cancer Center will provide the institutional support and resources for the research. IMPACT: The proposed studies have potential to advance multi-factorial understanding of CRCD and expand non-pharmacological treatment options for prevalent symptoms affecting the aging PC population. Dr. Liou will acquire the preliminary data to support a competitive R01 grant application to test the efficacy of acupuncture for CRCD and comorbid symptoms in PC survivors. This award will establish the foundation for an independent research program with sustained impact in PC survivorship care, cognitive health, and integrative oncology.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT RUNT-related transcription factor 1 (RUNX1) is a master regulator of hematopoiesis and leukemogenesis. RUNX1 mutations are identified in 10-20% of patients with acute myeloid leukemias (AML). RUNX1-mutant AML is characterized by chemoresistance and poor prognosis. Lineage infidelity is prevalent in RUNX1-mutant AML and has been proposed as a potential mechanism of therapeutic resistance. However, the mechanisms by which RUNX1 mutations confer lineage infidelity in AML and the specific contribution of lineage infidelity to the pathogenesis of RUNX1-mutant AML remain poorly understood. Leukemogenic RUNX1 mutations may possess potential mutant-specific functionalities in leukemogenesis and lineage specification. NRAS mutations are the most common co-mutated genes in RUNX1-mutant AML exhibiting lineage infidelity, suggesting that NRAS mutations cooperate with pathogenic RUNX1 mutations to promote leukemogenesis and lineage infidelity. Current preclinical models including inducible Runx1 null mice and germline Runx1 R174Q mutations are not ideally suited to test this hypothesis. In this proposal, we will utilize a novel inducible, reversible Runx1R174Q allele, alone or together with cooperating Nras disease alleles. This will allow us to characterize the mutant-specific functionalities of RUNX1, the impact of comutations on leukemic transformation and lineage infidelity, and address the requirement for RUNX1 mutations in leukemia initiation and maintenance. The specific aims of this project are: 1) Characterize lineage infidelity, genetic heterogeneity and their prognostic relevance in RUNX1-mutant AML. 2) Determine the mechanisms by which Runx1R174Q and Runx1R174Q + NrasG12D induce leukemogenesis and lineage infidelity. 3) Investigate the necessity of Runx1R174Q mutations in disease initiation and maintenance. These studies will lead to better understanding of disease mechanisms and new modes of therapy, which will also shape the focus of my future independent lab. Wenbin Xiao, MD, PhD, an Assistant Member at MSKCC, will conduct this project as part of a 4-year career development plan, dedicating 75% of his time to research with remainder spent on clinical work. Wenbin is mentored by Dr. Ross Levine, a world expert in hematologic malignancies. He is also advised by Drs. Omar Abdel-Wahab, Kristian Helin and Richard Koche at MSKCC, and Dr. Ulrich Steidl at Albert Einstein College of Medicine. He will collaborate with Dr. Andriy DerKach and Dr. Elli Papaemmanuil both at Department of Bio- Statistics of MSKCC. Wenbin’s training will include gaining technical laboratory skills, knowledge in the novel leukemia mouse model with dual recombinases, knowledge in the epigenetic regulation, and formal training in bioinformatics. In the short term, the project goal is to publish two papers on the findings from this research. In the long term, the goal is for developing a research program and obtaining R01 funding to become an independent laboratory investigator in hematologic malignancies.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY The development of novel therapies is at the core of improving cancer outcomes worldwide. However, currently used metrics to capture clinical benefit of these novel therapies may not always capture treatment success or failure. The overarching goal of the proposed research is to increase precision by leveraging metrics that integrate imaging with other measures of tumor response such as changes in plasma circulating tumor DNA (ctDNA) in patients undergoing novel therapies. The proposed research builds on clinical proof-of-principle by the investigative team using next-generation sequencing (NGS) of ctDNA that decreases in variant allele frequency (VAF) of selected alterations can be observed prior to conventional radiologic response, and that increases in VAF often occur several weeks to months before radiologic progression. Building on the foundation of NGS, MSK-ACCESS, a highly sensitive deep sequencing liquid biopsy assay, was recently developed by the investigative team based on its FDA-authorized counterpart MSK-IMPACT performed on tumor tissue, which enables the identification of actionable genetic alterations that can be targeted with drugs. Additionally, the research builds upon extensive experience of the investigative team with “basket trials” evaluating the activity of precision genome-driven and immunomodulatory therapies, whereby the enrolling criterion is a putative biomarker regardless of cancer type, which has put a premium on serially collecting co-clinical trial ctDNA samples along with MSK-IMPACT testing on the tumor tissue, providing a baseline genomic profile to guide ctDNA-based disease monitoring. The diverse and extensive collection of prospectively collected ctDNA samples within these trials provides timepoints that can be compared to regulatory grade pre-treatment, on-treatment, and post-progression imaging assessments via computed tomography (CT) and/or magnetic resonance imaging (MRI), and positron emission tomography (PET). Specific Aim 1: To evaluate the correlation between early changes in ctDNA variant allele frequencies (VAF) with best response to therapy via conventional and advanced imaging assessments in early-phase targeted or immunomodulatory clinical trials. Specific Aim 2: To identify if plasma ctDNA trends can more precisely predict longitudinal clinical benefit (measured by progression-free survival) in patients who fall within the broad response category of stable disease via RECIST in early-phase clinical trials. Specific Aim 3: To identify the median time prior to which rising ctDNA levels presages eventual radiologic progression in patients who initially benefit from targeted or immunomodulatory therapy (i.e., complete/partial response or stable disease as best overall response) in early-phase clinical trials. Impact: The insights from this study will lay the groundwork for integrating advanced imaging and ctDNA-based biomarkers in the future that may be used by regulatory agencies around the globe for the purpose of assessing and approving novel precision therapies and ultimately allow the possibility for personalized precision medicine.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT The overarching goal of the NCI Small Cell Lung Cancer (SCLC) Coordinating Center has been to “break the logjam” of fundamental knowledge gaps resulting in a 30+ year lack of progress on this lethal malignancy that was designated a “recalcitrant cancer” by the US Congress and NCI. Our U24 Coordinating Center has done this by promoting and facilitating information sharing, interaction, and collaborative research among NCI- supported investigators studying SCLC. Over our initial five years of support, this Coordinating Center has significantly increased research progress within the SCLC research community through efforts including, but not limited to, the creation and ongoing expansion of comprehensive preclinical and clinical -omics databases, centralized annotation and cataloging of available SCLC models, distribution and sharing of such models, centralized data analytics, a centralized Consortium website, initiation and coordination of an interactive work- in-progress (“WIPs”) forum as well as planning and oversight of annual meetings. Of all of this, the role of the Coordinating Center in facilitating interactive WIPs (usually with ~100 attendees from dozens of institutions including patient advocates) with unprecedented information and resource sharing, has made possible tremendous progress in overcoming knowledge gaps, all of which are documented in a large number of peer reviewed publications. This renewal application will continue the exceptional research momentum the SCLC Consortium has generated, and build on this initial success through support of new initiatives. These include centralized support for databasing and comparative analysis of genetically-engineered mouse models (GEMMs), and multi-parameter characterization of patient-derived xenograft models developed across the Consortium. These additional resources will be of direct utility to the SCLC research community. As part of the U24 renewal, given the remarkable growth in scope and diversity of SCLC research, we have developed a new multi-PI structure including Charles Rudin (MSK), John Minna (UTSW), with addition of two exciting young SCLC investigators who have contributed important new insights into SCLC molecular and cellular biology - Trudy Oliver (University of Utah), and Lauren Byers (MDACC). All four have been personally very active both in the U24 activities and in their own individual SCLC U01 and R01 research. The complementary strengths of this leadership team cover all of the important areas of SCLC basic, translational, and clinical trials research. Dr. Rudin will continue as contact PI, providing administrative leadership and coordination. We emphasize that the products of the Consortium will ultimately benefit human health through fostering development of novel strategies for disease prevention, early intervention, immune surveillance, metastasis suppression, and therapeutics.

NIH Research Projects · FY 2025 · 2022-06

ABSTRACT – not changed Discovery of human radiotracers that serve as companion diagnostics and/or aid in understanding abnormal biological processes that underlie cognitive disorders, such as Alzheimer’s disease (AD) and other brain disorders is an area of high translational priority towards key milestones tied to the implementation of the National Plan to Address Alzheimer’s and Related Dementias and a specific requirement of PAR-20-038. Our study proposes the discovery and evaluation of a radiopharmaceutical agent for the positron emission tomography (PET) imaging of epichaperomes, emerging targets in AD. Epichaperomes, long-lived oligomeric protein scaffolding platforms, are among the earliest mediators of AD pathogenesis. They negatively impact the interactions of proteins important for neuronal function, such as synaptic plasticity, cell-to-cell communication, protein translation, cell cycle re-entry, axon guidance, metabolic processes and inflammation, leading to proteome-wide defects in protein-protein interaction networks, and in turn cell- and brain-network dysfunction and cognitive decline. We discovered both epichaperome drugs (eg. PU-AD) and companion diagnostics (eg. [124I]-PU-AD PET) and translated them to clinic. To image epichaperomes, we discovered [124I]-PU-AD, a [124I]- labeled epichaperome probe. In a pilot feasibility clinical study, [124I]-PU-AD provided proof-of-principle that epichaperomes are imageable and quantifiable in patients by PET. In preclinical models, it demonstrated that epichaperomes form in AD in a disease-relevant region- and age-dependent manner. The next step is to make epichaperome imaging probes practical for widespread clinical use. We posit replacing the 124I label with 18F will significantly improve sensitivity, spatial and temporal image quality, reduce radiation burden and imaging times, improve production costs and availability, thus increasing the clinical applicability of the probe. We here propose a plan for the discovery of the 18F epichaperome PET imaging agent with emphasis on steps such as synthesis, identification of lead candidates, tracer characterization, safety, dosing, preclinical validation and IND-enabling studies for a proposed future Exploratory Investigational New Drug Application. We assemble a multidisciplinary team with a history of successful collaborations (>40 papers, >20 PET tracers in clinic) and designed 3 Specific Aims to accomplish our goal: Aim 1. Identify F-containing epichaperome probes with favorable affinity, selectivity and BBB permeability; Aim 2. Investigate the probe’s specificity and sensitivity in detecting epichaperome-mediated dysfunction in AD mouse models; and Aim 3. Perform IND-enabling studies for a proposed Exploratory Investigational New Drug Application. Outcomes of this work are novel PET probes for use as precision medicine tools to image in vivo early molecular dysfunction in the brain and as companion diagnostics for epichaperome targeted therapies, both research areas of high translational priority.

NIH Research Projects · FY 2025 · 2022-06

SUMMARY Lung cancer is the leading cause of cancer-related deaths in the U.S. Curative radiotherapy + chemotherapy is the standard of care for patients with inoperable or unresectable disease that has spread beyond the primary tumor to the lymph nodes. Unfortunately, this treatment approach has a high recurrence of 15%-40% and advanced treatments including immunotherapy combined with radiation increase toxicity to organs. Spillover radiation to normal organs at risk (OAR) results from treatment margins to account for uncertainty in localizing tumors and OARs. Despite being part of standard equipment, information from in-treatment room cone-beam computed tomography scans (CBCTs) is currently used only in limited ways for patient positioning during treatment, without simultaneous online localization of the tumor and each OAR. This proposal will use innovative artificial intelligence (AI) methods, that have been trained from both CT and magnetic resonance imaging (MRI) studies, to create auto-segmentation tools that can accurately localize the tumor and key OARs online at treatment setup. The proposed novel AI methodology is called “Cross-Modality Educed Learning” or CMEDL (‘c-medal’). The key advantage of CMEDL is that MRI datasets, even from different patients, can be used, to guide the CT/CBCT network and “learn” to extract features that emphasize the difference between tissue types and produces accurate segmentations even in areas with little inherent contrast such as the mediastinum. For the first time, the clinical utility of what could be called AI-Guided Radiotherapy (AIGRT) segmentation tools will be systematically studied in relation to their potential impact on treatment margin reduction and normal tissue toxicity modeling for longitudinally segmented tumor and healthy tissues on CBCTs. Proposed AIGRT tools would provide increased geometric confidence as well as provide a better basis for an after-delivery estimate of delivered dose, and treatment toxicity, enabling better risk-benefit assessments for potential treatment adaptations. Aim 1: Apply CMEDL methodology to develop lung tumor and OAR segmentations on planning CTs. Aim 2: Extend the CMEDL methodology to longitudinally segment tumors and OARs on weekly CBCTs, incorporating patient-specific anatomic and shape priors from planning CTs. Aim 3: Determine whether CMEDL can enable improved (safer) lung cancer radiotherapy dose characteristics by performing automated planning and delivery simulations, using in-house planning system. Project goal: To develop and rigorously test AIGRT tools for lung cancer radiotherapy treatments. Potential impact: If successful, these innovative AI tools could be deployed routinely, enabling (1) smaller margins and less radiotherapy toxicity for patients, including those with very difficult-to-treat centrally located tumors and (2) providing tools for monitoring the need for plan changes. These AIGRT tools could potentially be deployed to other disease sites, and once established be made widely available as a pragmatic, generalizable technology for geometry guidance throughout the radiation treatment.

NIH Research Projects · FY 2026 · 2022-06

Modified Project Summary/Abstract Section ABSTRACT Taxi ROADmAP (Realizing Optimization Around Diet And Physical activity) is an effectiveness-implementation hybrid type 1 design using the Multiphase Optimization Strategy (MOST) to address the overweight and obesity crisis in a growing essential worker population: taxi and for-hire vehicle (FHV) drivers (Lyft, Uber, etc.). MOST, an innovative framework, involves highly efficient randomized experimentation to assess the effects of individual treatment components to guide assembly of an optimized treatment package that achieves target outcomes with the lowest resource consumption. Hybrid trials, which blend effectiveness and implementation studies, can lead to more rapid translational uptake and more effective implementation. There are over 750,000 licensed taxi and FHV drivers in in the U.S. and over 185,000 in New York City (NYC). They have higher rates of overweight/obese range body mass index (BMI) than New Yorkers in general (77% vs 56%) and have high rates of elevated waist circumference, sedentary behavior, and poor diets. Obesity contributes to cardiovascular disease and cancer. Modifiable factors, such as physical inactivity, compound drivers’ likelihood of obesity and related diseases. While there is much evidence on effective multicomponent lifestyle interventions, such as the Diabetes Prevention Program (DPP) and Look AHEAD, focused on weight loss in some populations, these programs were not designed to be translatable to community settings and are considered too expensive to be widely disseminated. There is a paucity of data on how to optimize such approaches for all people, to reduce costs but still lead to meaningful weight loss. Even less literature addresses implementation potential and strategies for such interventions. ROADmAP builds on our unique preliminary work and uses a hybrid type 1 design and MOST, to address these gaps. ROADmAP will test 4 evidence- and theory-based (Social Cognitive Theory [SCT]) behavior change intervention components, which we developed and piloted, and which include DPP and Look AHEAD features. We will use MOST to identify which of the 4 components contribute most significantly and cost-effectively to weight loss among NYC drivers recruited at workplace health fairs (HFs) and virtually. Objectives are to apply MOST to design an optimized version of a scalable lifestyle intervention for taxi/FHV drivers, and then to conduct a mixed methods multistakeholder process evaluation to facilitate widespread intervention implementation.

NIH Research Projects · FY 2026 · 2022-06

Project Summary/Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, with a 5-year survival rate of 15%. Only a small proportion (16%) of lung cancer cases are diagnosed at an early stage, when it is more likely to be curable, thus we need improved strategies to identify high-risk individuals for intensive surveillance. Although cigarette smoking and other environmental factors are risks for lung cancer, it is estimated that 10-25% of lung cancers occur in never-smokers, highlighting the potential role of inherited genetic factors in lung cancer. Familial lung cancer as well as genome wide association studies have identified few lung cancer predisposing genes, which only explains 14% of all inherited risk for lung cancer. Hence, most of the genetic risk for lung cancer remains unexplained. With the adven t of the next-generation sequencing technologies, emerging evidence suggests the contribution of pathogenic germline mutations in DNA damage repair (DDR) genes in lung cancer susceptibility and etiology. Our preliminary data shows that about 6.8% of lung can cer patients harbored pathogenic mutations in DDR genes including high and moderate penetrant mutations in ATM, BRCA2, CHEK2, ERCC2, NBN and TP53. We hypothesize that the genetic alternations in DDR genes may modify the intrinsic and extrinsic (tobacco smoking or environmental) risk factors of lung cancer. The objectives of our proposed study are to determine the clinical significance of inherited mutations in DDR genes in lung cancer, study the interplay between germline-somatic mutational architecture and functionally characterize them to understand the mechanism of lung cancer susceptibility. Our findings will inform clinical and preventive management by elucidating genotype-phenotype correlations, penetrance (risk) modification, and clinical outcomes in genetically defined cohorts. For the proposed study, we will leverage the ongoing MSK-IMPACT initiative, an institution-wide effort to perform genomic testing using paired tumor–normal tissue samples in 10,000 patients with lung cancers as well as whole exome sequencing for a selected 400 lung cancer patients who had either family history of any cancer, or early diagnosis (age at diagnosis <50 years) or personal history of multiple primary tumors. In aim 1, we will discover germline mutations in DDR genes using novel analytical framework by integrating germline-somatic data for variant interpretation. We will replicate our findings in a case-control cohort in collaboration with the International Lung Cancer Consortium and England Genomics UK and determine risk associated with lung cancer in a case-control cohort and the pattern of inheritance in families (Aim 2). We will establish the clinical and functional significance of the germline mutations using the CRISPR gene editing approach and generate patient- derived xenograph (PDX) models from patients carrying germline mutations in DDR genes to test the therapeutic options; this will open the new paradigm of research and treatment options for lung cancer guidelines for lung cancer patients and their family members (cascade testing) for early detection.

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY/ABSTRACT - Overall SARS-CoV-2 continues to cause severe morbidity and mortality in the ongoing pandemic. Future RNA virus epidemics and pandemics are inevitable. New clinical-trial-ready antivirals are urgently needed RNA viruses of pandemic potential. COVID-19 has further underscored the need for early, global access to clinic-ready compounds. Beyond coronaviruses; flaviviruses and picornaviruses also cause frequent and ongoing epidemics worldwide and have no effective therapeutics. Maintaining a portfolio of novel, clinic-ready therapeutics are critical for our future pandemic preparedness. The AI-driven Structure-enabled Antiviral Platform (ASAP) AViDD Center will develop novel chemical assets that have antiviral activity against three target viral families. ASAP will leverage state-of-the-art structure-enabled technologies capable of leveraging recent advances in AI/ML and computational chemistry in identifying, enabling, and prosecuting discovery campaigns against novel viral targets. ASAP is built on principles of open science and rapid dissemination (enabled by a dedicated Data Infrastructure Core). ASAP builds on the successful COVID Moonshot, an open science collaboration that recently secured $11 million from the Wellcome Trust via the WHO Access to COVID Tools Accelerator (ACT-A) to fund preclinical development of a novel oral noncovalent SARS-CoV-2 antiviral acting against the main protease (MPro). Beginning with a high-throughput X-ray fragment screen, the discovery team spent just 18 months and $1M to reach the preclinical phase. ASAP will mirror this rapid, cost-efficient approach: automated structural biology at Diamond Light Source (Frank von Delft); AI/ML synthesis models from PostEra (Alpha Lee); nanoscale chemistry and covalent fragment libraries from Nir London; massively distributed free energy calculations on Folding@home (John Chodera); an industrial medicinal chemistry team led by MedChemica (Ed Griffen); and antiviral assays and virology expertise at Mount Sinai (Kris White; Adolfo García-Sastre). ASAP augments this seasoned antiviral discovery team with new approaches to resistance-robust targeting (Karla Kirkegaard and Matt Bogyo, Stanford) and deep mutational scanning (Jesse Bloom, Fred Hutch). ASAP is supported by the Drugs for Neglected Diseases Initiative (DNDi) (PI Ben Perry), and Letters of Support from Takeda, Pfizer, Novartis, and Grupo Insud. ASAP Impact: ASAP will become the nexus of a robust global antiviral discovery community. Our open science approach focuses on ensuring global, equitable access to therapeutics to combat future pandemics. We aim to produce a robust antiviral pipeline consisting of 3 new Phase I ready candidates, 6 lead optimization campaigns, 9 fragment-to-lead campaigns, and 10 structure-enabled resistance-robust viral targets. Our associated data packages will accelerate follow-on development and investment.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Breast cancer is the leading cause of cancer-related death in women. Triple negative breast cancer (TNBC) has the poorest prognosis of all major subtypes of breast cancer. The anticipated success of immunotherapy has not been seen in TNBC. The role of T-cells in TNBC and immunotherapy has been well-studied but the role of tumor- infiltrating B-cells (TIL-B) is less well-known. Present in up to 60% of breast cancers, TIL-B are associated with improved prognosis and response to chemotherapy. Evidence suggests that TIL-B respond directly to tumor antigens in TNBC. Utilizing single-cell sequencing of tumor-infiltrating lymphocytes in TNBC, we have identified high expression of a low diversity of paired heavy and light chain B-cell receptor segments which is definitive evidence of clonal selection, suggesting B-cells have undergone affinity maturation and can recognize antigens. In order to clone antibodies and identify their targets in TNBC, we have successfully sequenced the B-cell receptors of the most abundant B-cell clones we have identified thus far. Pre-clinical data also suggests an important role for B-cell antibody production in immunotherapy efficacy. Based on these data, we hypothesize that B-cells in TNBC recognize tumor-associated antigens and produce biologically relevant tumor-specific antibodies. To test our hypothesis, we propose the following aims: 1) Discover the role of clonally selected B- cells/plasmablasts in human TNBC, 2) Characterize the endogenous antibody response against TNBC, and 3) Evaluate the impact of immune checkpoint inhibition on B-cell receptor diversity and phenotype in human TNBC. Successful execution of these aims will support a role for B-cells in the anti-tumor immune response and lay the foundation for development of novel B-cell based therapies and/or biomarkers in TNBC. In addition to advancing scientific knowledge, this proposal provides training to a physician-scientist. Dr. Downs-Canner is a practicing breast-surgical oncologist with a background in cellular immunology. Her long-term goal is to combine her clinical and research expertise to develop an independently-funded research program focused on immunotherapy in breast cancer. She benefits from two well-established mentors with track records of training clinician-scientists and an extremely supportive research and practice environment. In order to achieve her long-term goals, the candidate's proposal includes a structured career development plan and training in: 1) bioinformatics 2) antibody cloning techniques and development of expertise in: 1) B-cell biology and 2) increasingly complex hypothesis driven experimental design, execution, and analysis. The proposed plan includes mentored experiential learning, course work and conference participation, frequent mentor meetings and a gradual increase in research independence. At the completion of the award period, the candidate will be prepared to apply for independent funding for her research program.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Randomized trials demonstrate aerobic training (AT) attenuates treatment-induced impairments in physiological and psychosocial outcomes in a broad number of cancer patient populations. However, whether AT specifically impacts the tolerability of cancer treatment is largely unknown. To address this fundamental knowledge gap in exercise-oncology research, the objective of this study is to evaluate the dose-response of AT on treatment tolerability and related outcomes in patients with locally advanced rectal cancer (LARC) initiating total neoadjuvant therapy (TNT). LARC is an ideal model in which to conduct a definitive trial of AT on treatment tolerability for several reasons: (1) high rate of LARC diagnoses annually in the U.S. (>40,000), (2) poor tolerability of TNT (<60% of patients complete the recommended regimen), and (3) strong biological rationale (TNT-induced impairment in hematological function is the major cause of poor tolerability, and AT is demonstrated to enhance hematological function in preclinical and clinical studies). Therefore, in this phase 2 randomized trial, a total of 225 inactive (<60 mins of moderate-intensity exercise/wk) patients with LARC scheduled to initiate TNT will be stratified by sex (male vs. females) and age (<55 years vs. >55 years) and randomly allocated (1:1:1 ratio) to receive: 90 mins/week, 150 mins/week, or 300 mins/week from pre-treatment to pre-surgery (~32 weeks). All AT dose regimens will be prescribed according to standard AT principles and implemented using our digital AT platform permitting all sessions to be performed in patients’ homes with remote real-time monitoring. We will address 3 specific aims: AIM 1: Determine dose-response of AT on TNT treatment tolerability. AIM 2: Evaluate AT dose-response on hematological function. AIM 3: Explore AT dose-response on tumor clinical outcomes. The proposed study directly addresses an unmet clinical need by testing, for the first time, the dose-response effects of AT on multiple treatment-related outcomes in patients with LARC receiving TNT. The proposed study will improve behavioral intervention protocols for patients undergoing cancer treatment by using our digital exercise approach that expands access to AT for patients not residing within close proximity of a research site. Receiving cancer treatment is not a qualifying condition for exercise therapy and, as such, exercise is not currently considered a standard aspect of cancer management. Therefore, if successful, findings from this investigation will also shift clinical paradigms regarding exercise therapy in cancer by adding to a growing body of evidence supporting integration of AT into standard clinical cancer care.

NIH Research Projects · FY 2026 · 2022-05

PROJECT ABSTRACT Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which emerge intrinsically within genomes. If left unchecked, these can be disastrous to the individual and/or the population. Thus, such conflicts induce strong pressures to innovate opposing mechanisms of repression. This is patently manifest in paradoxical meiotic drive systems, in which the wildtype activities of selfish genes can have deleterious consequences to bias the sex ratio of progeny (typically eliminating males), or to induce frank sterility. However, relatively little is known about the molecular mechanisms of meiotic drive genes and their control. Our recent work provides first evidence that related, recently-evolved, sex-biasing and sterility-inducing systems in the non-model fruitfly D. simulans are suppressed by endogenous siRNA loci. This provides a unique foundation to study how meiotic drive loci are tamed at the post-transcriptional level. Although little is generally known about the biochemical function of selfish meiotic drive loci, our data support testable models in which these D. simulans distorter gene products may interfere with chromatin packing in sperm. Finally, we will examine the broader evolutionary implications for how endogenous siRNAs may unlock maps of intragenomic conflicts in multiple Drosophila species, and examine a rationale for analogous molecular battles during mammalian meiosis. Our multidisciplinary investigations will elucidate mechanisms that link tissue-specific de novo gene activity and small RNAs with rapid genome dynamics that may impose the reproductive isolation of individuals, potentially early steps on the path towards speciation.

NIH Research Projects · FY 2026 · 2022-05

ABSTRACT Metastatic castration-resistant prostate cancer (mCRPC) is a lethal, incurable disease that will kill ~33,500 patients in the US in 2020. Responding to the urgent need for novel treatments that are safe and efficacious, and leveraging the high expression of prostate specific membrane antigen (PSMA) in mCRPC lesions, several small-molecule-based targeted radionuclide therapies (TRTs) have been developed. Among them, targeted alpha therapy agent (TAT) with [225Ac]-PSMA-617 in particular has demonstrated striking responses in the treatment of refractory patients — even achieving complete and durable responses in a subset of patients. However, many responding patients have discontinued treatment due to non-target toxicity. Salivary gland toxicity (irreversible xerostomia) and potential renal toxicity place hard limits on patient eligibility, maximum dose and maximum number of doses, severely restricting the use of [225Ac]-PSMA-617. As such, there is an urgent and unmet need to develop strategies that can reduce the unwanted side effects of these treatments without compromising treatment efficacy. We in turn are proposing a simple method to reduce salivary gland and kidney toxicity by reducing the effective specific activity (ESA) of [225Ac]-PSMA-617 by addition of PSMA-11. In our preliminary studies, reducing the ESA of [68Ga]-PSMA-11 and [177Lu]-PSMA-617 with PSMA-11 led to significantly reduced salivary gland and kidney uptake without compromising tumor uptake in mouse models of prostate cancer. We have assembled a highly qualified and collaborative team of researchers — including radiochemists, medical physicists, nuclear medicine physicians, genitourinary oncologists, veterinary pathologists and toxicologists — to unequivocally demonstrate the efficacy of our methodology for reducing the salivary gland and renal radiation dose of [225Ac]-PSMA-617 or other PSMA-TRT agents in clinically relevant mouse and rat models. As part of our proposal, we will determine the range of ESAs that will reduce salivary gland and kidney dose of [225Ac]-PSMA-617 by > 75% without compromising tumor radiation dose in mice and rats; demonstrate that salivary gland and renal function are maintained long-term(~2 years post-treatment) while eliminating tumor burden; demonstrate the methodology’s applicability to other PSMA-TRT agents; conduct a GLP toxicology study of PSMA-11 at required doses (5–10 mgs/patient) to establish its safety; and make the data available to all researchers in order to facilitate clinical trials. The experiments are being conducted as IND- enabling studies for near-term clinical translation. Once established, our simple but innovative approach will refine treatment with [225Ac]-PSMA-617 and other PSMA-TRT agents by reducing toxicity to salivary glands and kidneys without compromising treatment efficacy and help to extend the lives of mCRPC patients while maintaining their quality of life.

NIH Research Projects · FY 2025 · 2022-04

Project Summary/Abstract Chemical Exchange Saturation Transfer (CEST) MRI uses selective radio-frequency (RF) pulses to saturate the magnetization of exchangeable protons on a variety of molecules and macromolecules, including proteins, which, due to fast chemical exchange with bulk water, results in a decreased water MRI signal. The CEST contrast depends on the chemical exchange rate (kex), which is pH sensitive, and the volume fraction of the exchangeable proton pool (fs) that is sensitive to protein and metabolite concentrations. The sensitivity of CEST MRI to pH and protein/metabolite concentrations has proven to be a powerful tool for imaging a wide range of disease pathologies. For example, the amide proton CEST contrast from endogenous proteins has been used to distinguish pseudo-progression from true progression in malignant gliomas, differentiate between radiation necrosis and tumor progression, and image the tumor's extracellular pH. However, clinical translation of these CEST-MRI methods has been hindered by the qualitative nature of the image contrast, long image acquisition times, and the complex data processing required. Efficient methods for quantification of kex and fs are needed to produce high-quality pH and volume fraction maps required to move many of these studies forward into the clinic. In this proposal a CEST magnetic resonance fingerprinting (MRF) method that enables accurate quantification of both proton exchange rates and volume fractions in a fraction of the time required by conventional pulse sequences will be developed and optimized. These novel techniques exploit deep learning methods to enable the simultaneous quantification of multiple tissue maps from a single measurement. The improved CEST-MRF method will enable the acquisition of accurate pH, water T1 and T2, and protein/metabolite concentration maps in acquisition times of less than 5 minutes. The sequence will be adapted to a clinical scanner, and a novel multi- slice method will be implemented to obtain whole brain coverage (Aim 1). Next the CEST-MRF acquisition schedule will be optimized to maximize the parameter map discrimination and accuracy using a deep learning approach for the parameter map reconstruction. The parameter map reconstructions in normal human subjects will be validated with conventional CEST and test-retest studies (Aim 2). Lastly, the optimized CEST-MRF method will be used to evaluate the change in the quantitative parameter maps before and after radiation therapy to assess the potential role of CEST-MRF maps as predictive imaging biomarkers for brain metastases (Aim 3).

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY As tumors progress, cancer cells acquire characteristics that allow them to adapt to various stresses. In fact, one of the best predictors of patient outcome is disease stage at the time of diagnosis, as advanced tumors are more aggressive and difficult to treat. However, the underlying mechanisms that potentiate increased cell plasticity throughout cancer progression remain poorly understood. The ability of cancer cells to adapt has posed a particular problem for the use of targeted therapies, which are frequently rendered ineffective by the emergence of acquired resistance. The goal of this work is to elucidate molecular mechanisms that regulate the cell cycle and cell fate decisions to influence cancer progression and resistance to targeted therapy. In the F99 phase, I aim to identify novel factors that regulate the retinoblastoma (RB) pathway and influence the cellular response to inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6). CDK4/6, in complex with Cyclin D, phosphorylate and inactivate the tumor suppressor RB to drive cell cycle progression. Recently developed CDK4/6 inhibitors have shown some promise in the clinic, but every patient given these inhibitors eventually progresses, creating an urgent need to identify mechanisms of resistance. Using an in vitro genome-wide CRISPR/Cas9 screen, I recently identified loss of the E3 ligase adaptor AMBRA1 as a potential mechanism of resistance to CDK4/6 inhibition. Further, AMBRA1 loss increased Cyclin D protein stability. I hypothesize that AMBRA1, with its accompanying E3 ligase complex, targets Cyclin D for degradation, and that AMBRA1 loss could be a mechanism of resistance to CDK4/6 inhibitors in vivo. I will use molecular and biochemical assays to identify the E3 ligase that cooperates with AMBRA1 to target Cyclin D. In addition, I will combine tumor barcoding with multiplexed CRISPR/Cas9-mediated gene targeting in mouse models of non-small cell lung cancer to determine whether loss of AMBRA1 leads to CDK4/6 inhibitor resistance in vivo. In the K00 phase, I aim to elucidate the molecular mechanisms regulating cell identity in lung adenocarcinoma (LUAD). Treatment of LUAD with small molecule inhibitors targeting mutant receptor tyrosine kinases can lead to relapsed tumors that have transdifferentiated into small cell lung cancer, an aggressive neuroendocrine cancer with limited treatment options. However, the mechanism of transdifferentiation is largely unknown. I propose to develop cell line and mouse models of this transdifferentiation process in order to identify factors that regulate LUAD cell identity and ultimately identify means to prevent or reverse transdifferentiation. Together, this body of work will elucidate fundamental principles of acquired resistance and disease progression in lung cancer, which may also be applicable to other cancer types.

NIH Research Projects · FY 2026 · 2022-03

This D43 supports the Nigerian Cancer Research Training (NCAT) Program, a collaboration between Memorial Sloan Kettering Cancer Center (MSKCC) USA and institutional partners in Nigeria. The program aims to generate Nigerian research partners capable of collaborating with American investigators on cancer questions requiring diverse populations or environmental conditions unavailable in the US. Research conducted in Nigeria is cost-effective and directly supports questions relevant to US patients. MSKCC is currently sponsoring an immunotherapy trial in Nigeria, for example, for colorectal cancer (CRC) patients to determine whether impressive US results are reproducible in a different population, yielding insights into the tumor immune environment that will advance American cancer care. To perform additional trials, we need well trained Nigerian cancer researchers as partners. This program meets that. Aim 1 strengthens individual capacity by supporting an MSKCC epidemiologist and mixed-methods scientist to deliver virtual and in-country training in Nigeria, in addition to global health research training for US surgery residents. This will benefit the American scientific community by building expertise for global trials at MSKCC. We will also train a multi-disciplinary group of Nigerian researchers with an online program from Harvard University. This will provide Nigerian researchers with the foundation to collaborate on trials important to MSKCC and American cancer patients and ensure training in producing research that can be replicated and reproduced. Aim 2 builds institutional capacity through a cohort of 46 Nigerian Clinical Research Scholars and 7 NCAT postdoctoral fellows receiving team science, scientific rigor, and grant-writing training. These Nigerian faculty are the collaborators US researchers currently need for research projects focused on cancer questions of importance to US cancer patients. A pilot study, for example, that is now evaluating a urine metabolite point-of-care test for CRC diagnosis is cost-prohibitive in the US. This illustrates the program's value. Mentorship is provided to Offices of Clinical Research at Nigerian hubs and a program evaluation uses the Kirkpatrick Model. Aim 3 is focused on building a sustainable cancer research program to perform research studies with American centers by creating broader systemic relationships at the national, regional, and global levels through training in scientific leadership, advocacy, rigor, reproducibility, communication, grant writing, and health systems, with mentors drawn from oncology, epidemiology, statistics, and nursing research. These are all required skills for successful research collaborations. Ongoing funded projects provide hands-on training, and the NCAT program creates durable links between Nigerian and US faculty to answer cancer research questions of importance to American clinicians and patients.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Acute myeloid leukemia (AML) is the most common form of leukemia in adults. New therapeutic strategies are urgently needed for AML, as the overall five-year survival rate remains at less than 25 percent. Agents that activate the CARD8 inflammasome were recently discovered to trigger a non-inflammatory form of lytic cell death in hematopoietic cells, including AML cancer cells, and thus have potential to become new anti-AML drugs. Currently, the only pharmacological agents known to activate the CARD8 inflammasome are small molecule inhibitors of serine proteases DPP8 and DPP9 (DPP8/9). Unfortunately, DPP8/9 inhibitors also activate the related NLRP1 inflammasome, which, unlike the CARD8 inflammasome, triggers a highly inflammatory form of cell death in other cell types and thereby limits the therapeutic window of DPP8/9 inhibitors for the treatment of AML. The central hypothesis of this application is that the differences between NLRP1 and CARD8 can be exploited to develop selective CARD8 inflammasome activators. The preliminary data produced in the applicant’s laboratory and described in this application show that inhibitors of the enzyme PEPD selectively activate the CARD8 inflammasome and kill AML cancer cells without simultaneously activating the NLRP1 inflammasome. The objective of this application is to develop PEPD inhibitors as new therapeutic agents for the treatment of AML. This project consists of three specific aims: 1) to optimize potent and selective PEPD inhibitors; 2) to determine the mechanism of action of PEPD inhibitors for selective activation of CARD8 in AML cells; and 3) to explore the therapeutic potential of PEPD inhibitors in mouse models of AML. Successful completion of these aims will identify and characterize the first agents that selectively activate the CARD8 inflammasome, and obtain preclinical proof of concept for the utility of such agents in treating AML. Overall, this work has high potential to not only reveal fundamental mechanisms that regulate inflammasome activation, but also to harness inflammasome activation for therapeutic benefit against cancer.

NIH Research Projects · FY 2026 · 2022-03

Metastasis causes >90% of cancer death. The persistence and lethality of metastasis is driven by cells capable of self-renewal, slow cell-cycling, tumor re-initiation, and therapy resistance, termed metastasis stem cells (MetSCs). Development of effective strategies for eliminating metastasis requires a better understanding of the mechanisms that MetSCs exploit for survival. We recently demonstrated that (1) disseminating colorectal cancers (CRC) undergo a dynamic phenotypic switch from an LGR5+ tumor-initiating cancer stem cell (CSC) state to a distinct LGR5lowL1CAM+ state required for metastasis. (2) This phenotypic plasticity of MetSCs is retained in ex vivo patient derived organoids, which can be used to dissect mechanisms of plasticity. (3) L1CAM+ MetSCs are functionally distinct from intestinal tumor-initiating LGR5+ CSCs: L1CAM is required for organoid formation, the regeneration of intestinal epithelium after colitis, and tumor formation after metastatic dissemination. But unlike LGR5, it is dispensable for epithelial homeostasis or intestinal tumor initiation. In contrast to tumor initiation, where homeostatic stem cells undergo oncogene-driven hyper proliferation in intact tissues, metastasis subverts a regenerative mechanism to survive and regrow outside an intact epithelial niche. (4) We have shown that the principal driver of L1CAM expression is loss of epithelial integrity itself, acting via loss of E-cadherin intercellular adherens junctions to transiently displace the transcriptional silencer REST/NSRF from chromatin in quiescent MetSCs, in turn derepressing expression of L1CAM and other genes required for tissue regeneration1. Proliferation, restoration of epithelial structures, and macrometastatic outgrowth, on the other hand, require high REST levels. Our evidence suggests that MetSCs cells are regenerative stem cells that emerge directly in response to loss of epithelial integrity to drive repair, a phenotype of physiological wound healing that is redeployed in MetSCs. In this proposal, we will define the molecular mechanism by which REST chromatin binding is dynamically regulated in MetSCs, and how this in turn enables cell fate plasticity from stemness to proliferation. Our preliminary data implicates the mRNA binding protein ZFP36L1/2 in REST- mediated metastatic plasticity. Project hypothesis: The ZFP36L1/2REST axis is a master regulator of cell fate plasticity in intestinal epithelial progenitors. Aim 1: Define the function of the ZFP36L1/2-REST axis in normal and neoplastic intestinal stem cell self-renewal, differentiation, and proliferation. Aim 2: Dissect the molecular mechanism of ZFP36L1/2-mediated cell fate plasticity. Aim 3: Determine the functional consequences of ZFP36L1/2-REST dynamic regulation in metastatic seeding and colonization. Results will define mechanisms of cancer progression and identify putative therapeutic targets to limit regenerative plasticity, with potential to impact clinical outcomes.

NIH Research Projects · FY 2026 · 2022-02

Regulation of protein multi-functionality by 3′UTRs SUMMARY Many protein functions are mediated by protein complexes whose formation is often regulated by abundance as higher levels increase the chance to encounter an interaction partner. mRNAs contain a coding region that is translated into protein, but they also contain a 3′ untranslated region (3′UTR). In addition to regulation by abundance, my lab discovered that protein function can be regulated by 3′UTRs as during protein synthesis 3′UTRs mediate protein-protein interactions. 3′UTR-dependent protein complex assembly is mediated by the local translation environment. Each mRNA generates its own translation environment that consists of the proteins bound by the mRNA together with the recruited proteins. As a result, mRNA isoforms with alternative 3′UTRs – that often differ substantially in length – provide drastically different translation environments, and thus encode different protein functions. Currently, thousands of 3′UTR-dependent functions are unknown because they cannot be inferred from canonical protein functions. We have developed a method to systematically identify protein functions mediated by long 3′UTR isoforms of multi-UTR genes using a CRISPR-based approach. We will identify 3′UTRs that mediate so far unknown protein functions involved in the evasion of cell death, in the regulation of migration, and differentiation. We currently know of two ways to achieve 3′UTR-dependent functions. As described above, an mRNA that contains a long 3′UTR can generate its own translation environment. Moreover, mRNAs can use elements in their 3′UTRs to localize to pre-existing translation environments that are formed by phase-separated cytosolic compartments. Within these large cytosolic membraneless organelles the environment is generated by many mRNAs together with their recruited proteins. We discovered such a compartment called TIS granule network. We determined hundreds of enriched mRNAs and observed that usually only half of transcripts with the same 3′UTR localize to TIS granules. This implies that proteins can have alternative functions depending on whether they are translated in the cytosol or in TIS granules. Our goal is to investigate how proteins change their function when translated within TIS granules. To study TIS granule-dependent protein functions, we have engineered cells that are unable to assemble TIS granules. For candidates whose mRNAs are strongly enriched in TIS granules, we are investigating if translation in TIS granules controls the addition of post- translational modifications, the establishment of specific protein complexes, or if it suppresses protein aggregation. If successful, our research will reveal a widespread role of mRNA in the compartmentalization and physical scaffolding during translation. It will show how elements in 3′UTRs contribute to the diversification of protein function. In the long-term, it will facilitate the development of mRNA therapeutics where inclusion of specific 3′UTR elements allows mRNAs to encode proteins with more robust or alternative functions.

NIH Research Projects · FY 2026 · 2022-02

Ferroptosis and Cancer Cell Signaling Summary Programmed cell death (PCD) plays important role in normal biology, and its deregulation contributes to the development of various diseases. Ferroptosis is a PCD modality driven by cellular metabolism and iron- dependent cellular lipid peroxidation. Mounting evidence indicates that ferroptosis is involved in multiple pathological conditions, including cancer. Therefore, understanding the mechanisms of ferroptosis is important for both fundamental biology and disease treatment. While most mechanistic investigation of ferroptosis focuses on intracellular molecular events, our recent studies revealed a conceptually novel mechanism for ferroptosis regulation that is non-cell autonomous: in epithelial cells, E-cadherin-mediated intercellular interaction suppresses ferroptosis through intracellular Merlin/NF2-Hippo signaling; antagonizing this signaling axis unleashes the activity of the proto-oncogenic transcriptional co-activator YAP to promote ferroptosis through regulating multiple ferroptosis modulators. As E- cadherin and Hippo-YAP signaling are key regulators of epithelial mesenchymal transition (EMT), our work provides mechanistic insights into the recently published observation that mesenchymal and metastatic properties of cancer cells are highly correlated with the sensitivity of cancer cells to ferroptosis induction. Our preliminary studies furthersuggest that malignant mutation of E-cadherin and multiple components in the Merlin- Hippo-YAP signaling pathway can be used as biomarkers predicting cancer cell responsiveness to future ferroptosis-inducing therapies. Considering that loss of function mutations of tumor suppressors E-cadherin, NF2, and Lat1/2 (components of Hippo signaling), as well as super-activation of YAP oncoprotein, are all malignant events that make cancer cells more resistant to common therapies and to apoptotic cell death, our finding that these same mutations instead render cancer cells more sensitive to ferroptosis induction is unexpected and highly important both conceptually and clinically. Based on these preliminary results, in this proposal, (1) intercellularly, we will investigate the molecular basis underlying the role of E-cadherin in transducing signals into the intracellular machinery, thus functioning as both a tumor suppressor and counter-intuitively, an inhibitor of ferroptosis; to further expand this concept, we will determine if other cell adhesion molecules can also regulate ferroptosis via similar mechanism; (2) intracellularly, we will determine how YAP dictates ferroptosis sensitivity via its transcription co-regulating activity; and (3) relevant to cancer, as E-cadherin mutation is highly frequent in gastric cancer, a fatal disease currently without effective treatment, we will investigate the role of E-cadherin tumor suppressor in determining gastric cancer cell sensitivity to ferroptosis, and the potential role of E-cadherin-regulated ferroptosis in gastric cancer metastasis. Genetically engineered mouse models for gastric cancer, as well as gastric cancer patient- derived tumor organoids and xenograft mouse models, will be used for this preclinical investigation. Taken together, success of the proposed research will unveil in-depth mechanisms of ferroptosis, as well as its functional communication with various cancer-relevant intercellular and intracellular molecular events. The proposed research will also lead to the identification of biomarkers that predict cancer responsiveness to future ferroptosis-inducing cancer therapy.

NIH Research Projects · FY 2026 · 2022-01

Project summary The human flu virus undergoes fast antigenic evolution driven by the challenge of the host immune system. Circulating viruses are a moving quasi-species of high genetic and antigenic diversity, which is structured in distinct clades representing niches with separate avenues of escape from human herd immunity. Specifically, viral-immune co-evolution follows a Red Queen’s dynamical pattern of competing lineages, which is further modulated by global transmission patterns, host population structure, and herd-immunity acquired by previous infections and vaccination. This process poses a challenge for vaccine strain selection: to determine a single strain from each of the four seasonal lineages to provide the best protection from the viruses that are expected to dominate almost a year in advance. Here we posit that shape and dynamics of the global viral population can be understood and predicted from the underlying human population immunity. In this project, we will develop a comprehensive and objective computational approach to provide a mechanistic understanding of the influenza virus-host immune interaction on the host level, to quantify the selection imposed on the virus on the global evolutionary scales. Specifically, we will build biophysical models, both for the B-cell and T-cell driven immune recognition of epitopes in a host, to accurately characterize the immune structure of the human population to best represent the fitness effects acting on the virus on the global scale. For the model of B-cell immune recognition (Aim 1), we will leverage diverse antigenic human serology assays to prepare a detailed map of antigenic effects of mutations and epistatic interactions. The data will be cross-mapped on the dataset of sequences of globally circulating viruses and our detailed phylogenies for each of the four seasonal lineages. The T-cell immune recognition (Aim 2) will be based on computational machine learning predictions of epitopes, combined with novel biophysically motivated models for prediction of immunodominant antigens. Host population geographically diverse HLA diversity will be accounted for estimating selective pressures imposed on the population of the virus. These components, together with a component describing the selective pressure due to previous vaccinations, will be used to optimize a joint fitness model (Aim 3). Information theoretic approaches will be used to optimize and evaluate the predictive power of the combined model. We will objectively quantify the significance of each of the components and validate the predictions on the historical sequence and epidemiological data. With the resulting fitness model we will define principled criteria for vaccine strain selection, to optimize the coverage and efficacy of the vaccine in the future populations of the of seasonal human influenza viruses.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Chromosomal instability (CIN) is a hallmark of cancer characterized by high rates of chromosome mis-segre- gation during cell division. CIN can generate nuclear aberrations termed micronuclei when a chromosome or chromosome fragment lags during anaphase and fails to join the main chromatin mass that will form the prima- ry nucleus. Micronuclei recruit nuclear envelopes but defects in construction lead to frequent rupturing, loss of compartmentalization, and an unregulated exchange of proteins and small vesicles with the cytoplasm. Mi- cronuclear envelope rupturing causes broad dysfunction and is associated with extensive DNA damage and genomic rearrangements, including clustered mutational phenomena such as chromothripsis and kataegis, which are commonly observed in cancer genomes. Ruptured micronuclei can also activate the pro-inflammato- ry cGAS-STING pathway, which plays essential roles in anti-tumor immunity. These observations suggest that micronuclei may represent key platforms for genome evolution and immune activation in cancer. The mecha- nisms driving DNA damage and immune activation at micronuclei are poorly understood. The laboratory dis- covered that the endoplasmic reticulum (ER)-associated exonuclease TREX1, which is mutated in a variety of human immune diseases including Aicardi-Goutières Syndrome, accumulates at micronuclei upon micronu- clear envelope rupture where it resects micronuclear DNA and limits cGAS-STING activation. Therefore, TREX1 occupies central positions in key pathways with diverse roles in human health and disease. Conse- quently, there is strong rationale to understand mechanisms of TREX1 activity and engagement with cytosolic DNA. The long-term goals of the laboratory are to determine mechanisms of DNA damage, clustered mutage- nesis, and immune activation at sites of nuclear envelope rupture. The specific Aims of this proposal are to 1) Elucidate mechanisms of TREX1 structure and function, 2) Determine how TREX1 is recruited to micronuclei, and 3) Dissect pathways of micronuclear DNA damage. Each objective is supported by extensive preliminary data. Aim 1 will focus on a previously uncharacterized region in TREX1, which is essential for its ability to de- grade cytosolic DNA and inhibit cGAS activation. Aim 2 will build on results showing that TREX1 DNA binding function is dispensable for its localization to micronuclei, while its association with the ER is essential. Aim 3 will use a new method to purify micronuclei to dissect sources of micronuclear DNA damage. Taken together, these data will provide fundamental insights into cancer genome evolution, explain how previously uncharac- terized TREX1 mutations cause Aicardi-Goutières syndrome, and may identify new strategies to improve anti- tumor immunity.

NIH Research Projects · FY 2025 · 2021-12

Barrett’s esophagus (BE) is the only known precursor for esophageal adenocarcinoma (EAC), a highly lethal cancer with rising incidence and median survival <1 year. Substantial health-care resources are devoted to BE screening, surveillance, and treatment. Gastroesophageal reflux-induced injury of the lower esophagus and chronic inflammation are key drivers of BE development, but molecular pathways underlying risk are not well defined. Recent genome-wide association studies (GWAS) led by members of our team identified >20 novel genetic susceptibility loci for BE/EAC, providing new insights into the inherited genetic component of risk. Nevertheless, little progress has been made in bridging associations to biology. Consistent with GWAS of other complex diseases, all BE index variants map to non-coding regions, lack obvious biologic function, and are in linkage disequlibrium with many other SNPs, any of which may be causal. The vast majority of functional variants underlying GWAS signals are believed to map to and alter activity of regulatory elements including enhancers, in an allele-specific manner, and in turn modulate expression of downstream genes involved in risk. Importantly, such regulatory effects may be tissue- and condition-specific. To begin prioritizing candidate functional variants for experimental interrogation, we developed a customized informatics scoring pipeline using comprehensive in-silico annotations from multiple public resources. We selected four high-scoring BE risk loci for evaluation using luciferase reporter assays in esophageal cell lines, and found that two of four regions exhibited allele-specific enhancer activity. CRISPR-mediated deletion of the enhancer region at both loci correlated with downregulation of several candidate risk genes. Motivated by these successes, we seek to expand our integrative framework for elucidating functional consequences of BE-related genetic variation. We hypothesize that such variation is biologically expressed through alterations in transcriptional regulation and downstream gene expression. Our goal is to identify functional variants, risk enhancers, and target genes underlying BE risk, leveraging unique resources and complementary statistical/experimental approaches. In Aim 1, we will define candidate causal variants via Bayesian fine-mapping, using the largest BE GWAS world- wide, and further prioritize leading candidates via functional-potential scores. In Aim 2, we will perform new transcriptome profiling of reflux-exposed gastroesophageal junction tissues and constituent cells, and identify candidate BE risk genes and pathways via eQTL colocalization and network-based analysis. In Aim 3, we will validate candidate functional variants using luciferase reporter enhancer activity assays; identify target genes of risk enhancers via CRISPR-mediated enhancer deletion and RNA-Seq; and interrogate pathways influenced by prioritized target genes in Aims 2 & 3 via CRISPR-mediated gene knockdown/overexpression and RNA- Seq. This study will advance noncoding GWAS signals into functional biological signatures and support future efforts to develop novel preventive/interventional strategies for BE/EAC.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY/ABSTRACT Research: In lung adenocarcinoma (LUAD), neuroendocrine (NE) transformation to small cell lung cancer (SCLC) is associated with metastasis and resistance to targeted therapies. This lineage plasticity often leads to LUAD and SCLC admixed in the same tumor. We demonstrated that laser-microdissected LUAD and SCLC intratumoral components share truncal mutations, confirming NE transformation. SCLC itself is classified as classical, variant, and non-NE subtypes. Preclinical studies demonstrate that variant and non-NE subtypes have increased risk for metastasis and chemoresistance. It is poorly understood what gene regulatory mechanism drives SCLC transformation and SCLC subtype switching. Single-cell RNA and ATAC sequencing (scRNA-seq, snATAC-seq) in samples of combined LUAD/SCLC histology present an ideal platform to characterize the intratumoral heterogeneity of NE plasticity. As a control reference, we completed scRNA-seq in a cohort of de novo SCLC (Chan, et al. bioRxiv, under review at Cancer Cell). We performed scRNA-seq in an initial cohort of combined LUAD/SCLC and found significantly increased intratumoral subtype diversity in transformed SCLC (T- SCLC). We found Notch suppression in T-SCLC and reactivation with subsequent SCLC subtype diversification. We observed overexpressed SOX2 and ELF3 in pre-transformed vs classical LUAD, and PHOX2B and ELF3 in T-SCLC vs de novo SCLC. We hypothesize that under RB1 and TP53 loss, key transcription factors (SOX2, PHOX2B, ELF3), epigenetic regulators, and modulation of Notch signaling all contribute to NE transformation and subtype diversification. We will leverage scRNA-seq and snATAC-seq in samples of combined LUAD/SCLC histology to 1) identify molecular markers of subclonal populations, 2) reconstruct the regulatory network, and 3) validate transcriptomic and epigenetic drivers of NE plasticity in preclinical in vitro and in vivo models, including an EGFR+ LUAD patient-derived xenograft undergoing NE transformation after osimertinib treatment. Candidate: Dr. Joseph Chan, MD, PhD is a Medical Oncology Fellow at MSKCC. He aims to become an independent, tenure-track physician-scientist investigating lineage plasticity in metastasis and treatment resistance in cancer. His mentors Drs. Charles Rudin and Dana Pe’er are leading experts in lung cancer and single-cell sequencing, respectively. Dr. Chan proposes a five-year period of mentored training to acquire wet lab and advanced computational skills. His wet lab training will include 1) single-cell library preparation and 2) genetic manipulation of preclinical models for functional validation. His computational training will include 1) snATAC-seq analysis and 2) advanced machine learning. His advisory committee—Drs. Charles Sawyers, Helena Yu, Ronan Chaligné, and Christina Leslie—will guide his training and research. Environment: MSKCC is a cancer center renowned for patient care, innovative research, and training for junior faculty seeking careers as independent physician-scientists. MSKCC houses the Single Cell Research Initiative that advances single-cell sequencing, which will support this proposal for research and career development.

NIH Research Projects · FY 2025 · 2021-12

PROJECT SUMMARY Lysosomes are small, membrane-bound organelles that participate in numerous critical processes including macromolecular degradation, secretion, membrane repair, signaling, nutrient sensing and cellular metabolism. Central to lysosomal function is its acidic lumen, which can reach pH values as low as 4.5. The low pH activates degradative enzymes that break down proteins, damaged organelles and other macromolecules into building blocks that can be recycled for cellular use. In neurons, defects in lysosomal function can lead to accumulation of potentially cytotoxic macromolecules such as ab, a-synuclein, tau and others. Consequently, lysosomal dysregulation is associated with numerous human diseases, including many neurodegenerative diseases. In this application, we propose experiments that will elucidate the molecular mechanisms of two lysosomal ion transport proteins, TMEM175 and CLC-7, whose mutation can lead to defects in lysosomal homeostasis and are associated with disease in humans and mice. TMEM175 is a lysosomal K+ channel that was identified in as a highly potent risk-factor for the development of Parkinson’s Disease. In cells, TMEM175 establishes a membrane potential between the lysosomal lumen and the cytosol and is critical for lysosomal and cellular homeostasis. Loss of TMEM175 leads to dysregulation of lysosomal pH, deficiencies in autophagy and mitophagy and an increased susceptibility to cytotoxic stress. Despite its importance in Parkinson’s Disease and cellular homeostasis, both the molecular details of TMEM175 function and its physiological roles are only starting to be understood. We recently determined cryo-EM structures of TMEM175 in open and closed states that demonstrated that TMEM175 is structurally unrelated to other K+ channels. Moreover, the structure confirmed that its gating, permeation and selectivity mechanisms are distinct from those characterized for other K+ channels. Through a combination of biophysical, biochemical and structural analyses, we will determine the permeation, gating and selectivity mechanisms underlying TMEM175 function and gain insights into how its mutation can lead to lysosome dysfunction and disease. CLC-7 is a member of the CLC family of Cl- channels and Cl-/H+ exchangers that requires a b-subunit, OSTM1, to function in lysosomes and the ruffled border of osteoclasts. CLC-7 is a lysosomal Cl-/H+ exchanger whose mutation can lead osteopetrosis, lysosomal storage disease and developmental delay. Notably, several of the mutations associated with associated with disease in humans result in changes in gating. While studies of prokaryotic and eukaryotic CLC proteins have established a framework for the transport cycles, the gating of CLCs is poorly understood at the molecular level. Using structural and electrophysiological approaches, we will elucidate mechanisms by which pH and ligands regulate the gating of CLC-7. These studies will serve as a foundation for better understanding the role of CLC-7/OSTM1 in lysosomal physiology.