Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2018-01

Primary brain tumors in adults represent a heterogeneous and often fatal group of tumors. Current treatment of primary brain tumors heavily relies on surgery, radiation, and chemotherapy and is associated with cognitive impairment and other toxicities. Unlike in many other human cancers, inhibitors of oncogenic kinases have shown inconsistent clinical activity in brain tumor patients and it remains unclear which genetic alterations are critical for tumor maintenance in specific brain tumor types. The goal of this research program is to establish therapeutic strategies that exploit the most common genetic alterations is three specific tumor types, namely mutant isocitrate dehydrogenase (IDH) in low grade glioma, Bruton's Tyrosine Kinase (BTK) in Primary CNS Lymphoma (PCNSL), and EGFR in glioblastoma (GBM). Our research program incorporates the evaluation of tumor biopsies from patients being treated with inhibitors of these pathways, genetic and pharmacological studies in newly-derived experimental brain tumor models, and the development of state-of-the art approaches to quantify intratumoral heterogeneity in cancer signaling and tumor evolution. We believe that our research program will narrow the current knowledge gap regarding oncogene “addiction” in primary brain tumors and provide a framework for mechanism-based combination therapies targeting these signaling nodes and other signaling pathways.

NIH Research Projects · FY 2026 · 2017-12

The rapid accumulation of “big data” has initiated a new model for conducting cancer research and has resulted in a pressing need for a workforce with quantitative skills. To meet this need we used our first NCI R25 funding period to establish the Quantitative Sciences Undergraduate Research Experience (QSURE) at Memorial Sloan Kettering Cancer Center (MSK) in Summer 2018. QSURE provides experiential learning over a 10-week period for up to 10 students in quantitative fields powered by the application of real-world data, including biostatistics, epidemiology, health outcomes, and computational oncology. In only 5 years, QSURE has demonstrated substantial demand for quantitative training and expanded the volume of students who successfully engage in hands-on research in our department by nearly 4-fold. We have achieved the aims of the first funding period and reported high post-program student and mentor satisfaction as well as improved student competencies in all years. Most QSURE alumni (71%) have gone on to pursue a PhD or Master’s in a quantitative field. We will build on these successes and use the next funding period to continue to cultivate a cadre of responsible, rigorous quantitative scientists through an intensive multidimensional curriculum that reinforces successful programmatic components, including hands-on research carried out under the guidance of a faculty mentor, discussion series in the ethical and responsible conduct of research, educational seminars illustrating wide applications of data, training workshops in quantitative methodology, training and opportunities for written/oral scientific communication, and career and professional development. We will integrate new components that address contemporary viewpoints in cancer research. These will include patient led panels and “fireside chats” that highlight the role of research in the patient experience, collaborative activities and moderated discussions that provide broader health perspectives through exchanges with other NCI-funded training programs, and student-led discussions of various works that reinforce the foundations of conducting responsible and rigorous cancer research. We will recruit cohorts of motivated undergraduate interns with a passion for applying quantitative science to reduce cancer morbidity and mortality and evaluate short- and long-term program success, using these outcomes to inform and disseminate best practices. Since initial funding QSURE has demonstrated success and resilience in preparing and facilitating the next generation of research scientists for graduate studies and careers in quantitative fields. During the next funding period, we will reinforce the successes of our first funding period by designing an enhanced curriculum to help students contextualize and broaden their understanding of the potential of quantitative science to advance and support public health. QSURE will continue to prepare and facilitate interns for graduate studies and careers in quantitative fields. This is vital to fulfilling our long-term vision of training strong quantitative scientists dedicated to advancing cancer research.

NIH Research Projects · FY 2026 · 2017-09

Project Summary Alzheimer’s disease (AD) represents an increasing medical and societal problem affecting > 6 million patients in the US, with numbers continuing to grow due to the increase in average age and lifespan. Despite the urgent need, there are still only few therapeutic options available to date, and many fundamental questions about AD pathology and mechanisms remain unresolved. Human pluripotent stem cells (hPSCs) offer a novel strategy to tackle AD, as they allow the study of AD directly in human neurons and glia, at scale, and in a patient-specific manner. Furthermore, there has been considerable progress in generating AD patient-specific or genetically engineered stem cells, in directing their differentiation into neuronal and glial lineages and in devising strategies to study many hPSC lines in parallel, including hPSCs from annotated patient cohorts to match in vitro behavior, human genetics and longitudinal clinical data. However, an unresolved issue is that hPSC derived cells resemble a fetal rather than adult-like or aged state, which represents an age mismatch for the neurons used for AD modeling versus the neurons affected in the brain of patients suffering from AD. Over the last 5 years, supported by R01AG054720, we made considerable progress in devising strategies to measure and manipulate neuronal age in vitro, and we have developed culture systems that can model neuroinflammatory interactions present in the AD brain. Most recently, we developed two novel paradigms to identify age-modifying factors : i) based on a genome wide CRISPR loss-of-function screen we identified genetic disease modifiers in hPSC-derived APPSwe cortical neurons that trigger late-onset phenotypes in AD but not isogenic control neurons, including inhibitors of neddylation s ii) We developed RNAge as a tool to measure cellular age in hPSC-derived cortical neurons, and we performed an in-silico screen using RNAge as a probe set in L1000 perturbation data. Validated hits from the study can trigger neuronal age signatures and late-onset neurodegenerative phenotypes in AD neurons. Here, we will extend in Aim 1, our ability to measure and score cellular age at the single cell level with a particular focus on glial aging and on tracking dynamic experimental systems that either reset, retain, or rejuvenate the age of neurons. In Aim 2, we will assess and compare the performance of novel aging paradigms and ask to what extent aging trajectories and endpoints are shared or distinct. We will further address the role of glial cells and glial aging in an effort to capture both cell autonomous and non-autonomous components of the aging process. Finally, in Aim 3, we will apply the most promising induced aging paradigms to modeling AD. These studies will include neuronal, astrocyte, microglial and combined tri-culture systems; and introduction of AD-related mutations that capture neuronal vs glial AD vulnerabilities. The ultimate goal is to devise aging paradigms that mimic physiological age and AD progression and to establish a new class of disease models to capture a decade long process in a defined in vitro culture system.

NIH Research Projects · FY 2024 · 2017-09

PROJECT SUMMARY Lipid droplets (LDs) are ubiquitous monolayer-bound organelles that function in cellular lipid storage (for metabolic energy or membrane synthesis). LDs form from the ER, but how LDs are formed remains unknown and is a central question for the field. The current model indicates that neutral lipids, such as triacylglycerols (TG), are synthesized in the ER and released into the bilayer. At a critical concentration, TGs de-mix from the phospholipid bilayer in a phase transition that forms nascent LDs that bud toward the cytosol. We hypothesize that proteins are essential to ensure this process occurs in a defined manner and to prevent the formation of “ectopic” and potentially dysfunctional LDs, disrupting ER and cell function. Specifically, two ER proteins – seipin and lipid droplet assembly factor 1 (LDAF1) – operate in the lipid droplet assembly complex (LDACs) in the ER to form LDs. Both proteins form an oligomeric assembly with seipin forming a ring of 10-12 subunits and an equal number of LDAF1 occupying the middle of the ring. While we have identified components of the LD formation machinery and gained some insight into their structures, how these proteins function to facilitate LD formation remains mostly a mystery. Here we propose to utilize the latest tools and approaches, including biochemistry, structural biology, molecular simulations, and cell biology, to address the following questions: How and where is TG made relative to LDACs? What are the molecular structures of the seipin/LDAF1 LDACs? How do these oligomeric complexes assemble/disassemble? Where do LDACs localize in cells? How do they function to organize LD formation? We will address these questions by completing four specific aims. Aim 1 will address the mechanism of TG synthesis in the ER by the DGAT1 enzyme. We will expand on our recent elucidation of the molecular structure of human DGAT1, combining molecular dynamics and biochemical experiments to elucidate the precise mechanism of TG generation and determine how TG is released into the ER membrane for LD formation. Aim 2 will determine how and where LD assembly complexes assemble in cells to form LDs. We will determine the relationship of TG synthesis to LDACs, whether seipin/LDAF1 LDACs localize to ER tubules and how they assemble. Aim 3 will focus on elucidating the molecular structure of the seipin/LDAF1 LDAC in vitro and in cells. We will utilize cell and structural biology approaches, including cryo-EM and cryo-ET to test the hypothesis that seipin and LDAF1 form a ring structure with LDAF1 in center and that these LDACs form at areas of membrane curvature (tubules) where the structure may adopt dynamic conformations and activate of the complex. Aim 4 will determine the molecular function of the seipin/LDAF1 LDAC in vitro and in molecular dynamics simulations. We will reconstitute LD formation to test the hypothesis that the seipin/LDAF1 LDAC catalyzes phase transition of TG in the membrane, ensuring LDs form at these designated formation sites. Successful completion of these aims will advance the molecular understanding of a fundamental process central to energy metabolism and provide information on the mechanistic underpinning of many metabolic diseases, such as obesity, atherosclerosis, and fatty liver disease.

NIH Research Projects · FY 2025 · 2017-08

Project Summary/Abstract This cancer education training project is designed to train oncology care providers to implement tobacco use assessment and treatment (TUAT) in their cancer care settings. Persistent smoking is associated with cancer- specific and all-cause mortality, increased likelihood for second primary cancer, increased risk for disease recurrence, poor response to treatment and treatment-related toxicity. Leading oncology organizations have strongly endorsed tobacco use assessment and treatment as an important metric for high quality cancer care and evidence-based, clinical guidelines exist for assessment and treatment of tobacco use and dependence among cancer patients. Unfortunately, barriers for implementation are many, and adoption of TUAT into real world oncology practice settings remains slow and inconsistent. Although recent surveys demonstrate that oncology providers agree that advising tobacco cessation is an important aspect of cancer treatment planning and that some progress has been made at offering tobacco treatment services as NCI-Designated Cancer Centers, most cancer care settings have not yet established tobacco cessation treatment as standard care, and there exists a lack of training and implementation support needed to achieve TUAT innovation in cancer research and care. To address this continued research-to-practice gap, renewal of this cancer education grant will support refinement and enactment of our current well-received TUAT skills development course and collaborative training program intended to accelerate oncology care providers’ efforts to implement TUAT for tobacco-dependent patients treated in their respective cancer care facilities. Our proposed skills development effort will be enacted during a 6-month period of active training engagement beginning with a 2-day onsite or virtual Workshop followed by six monthly videoconferences (Collaboratory) co-facilitated by Program Faculty with extensive TUAT expertise in cancer. This proposed renewal of this skills development course will enable us to enroll 15 additional cohorts (20 participants/cohort) enabling us to train an additional 300 multidisciplinary participants from diverse cancer practice settings. To date, there has been the strong demand for TTT-O from a large number of oncology care providers from multiple disciplines and a wide variety of cancer care settings. We have exceeded our initial enrollment projects and successfully trained 286 oncology care providers. Participant feedback regarding course content, faculty and format have been outstanding. The findings from our multi-pronged evaluation plan demonstrate statistically significant improvements in TUAT knowledge, attitudes, self-efficacy, skills, and behavior. Ultimately, this cancer education program will improve the capacity of oncology care providers to implement clinical practice guidelines for tobacco use assessment and treatment and reduce tobacco-related morbidity and mortality among cancer patients and survivors.

NIH Research Projects · FY 2025 · 2017-03

Project Summary The microbiota provides many key signals that support development and functioning of the immune system. Interactions between host and microbiota allows for proper induction of immune responses against pathogens. These interactions are also necessary to limit inflammatory immune responses against the microbiota which, if left unchecked, will result in inflammatory conditions including inflammatory bowel disease. Many intestinal cell types including immune cells and the intestinal epithelium recognize and respond to the microbiota. It is unclear how such signals are integrated to support homeostasis. In humans as well as in animal models of disease, compositional changes in the microbiota, also known as dysbiosis, correlate with increased susceptibility to inflammatory disease. Understanding the role of individual microbes within the community would allow for the development of novel mechanistic approaches to restore homeostasis in the case of inflammatory disease. We identified a subset of mucosa associated E. coli that induce intestinal macrophage production of IL-1b. This activates innate protection of the intestinal barrier but also drives proinflammatory T cell responses against these E. coli. Specific characteristics of these E. coli as well as how they interact with the host immune system likely drives these responses. In Aim 1 of the proposed work, we will use in vitro and in vivo models to determine how these E. coli activate IL-1b production. In Aim 2 we will define the regulation and consequence of T cell responses against these E. coli. Together, these studies will identify molecular crosstalk between intestinal microbes and immune system that underlie pro-inflammatory responses against the microbiota. Understanding these signals will allow us to identify mechanisms for regulating these pathways and reducing unnecessary intestinal inflammation.

NIH Research Projects · FY 2026 · 2017-02

Title: Development of the resident macrophage lineage in mouse and human Summary/abstract Our long-range goal in our previous funded RO1 application was to elucidate mechanisms that underlie the development and specification of embryo-derived tissue-resident macrophages in mice. Our program of work aimed to understand how the identities and functions of resident macrophages are specified and to provide tools and concepts to identify pathophysiological mechanisms underlying their roles in developmental, inflammatory, degenerative and tumoral diseases. This work allowed us to identify key novel functions of resident macrophages and mechanisms that underly these functions and to develop new genetically tractable models which pave the way for our current project. Yet, a comprehensive understanding of the developmental origin and functions of mammalian resident macrophages, which has essential implications for genetic and pharmacological investigations of their specialized functions, still faces several important challenges, which are identified and addressed in this renewal proposal. 1) The development of resident macrophage in mice cannot be directly extrapolated to humans because the earlier steps of extraembryonic hematopoiesis and macrophage development present with differences among vertebrates, for example between Zebrafish and mice, and are overall poorly understood. Notably, 2) the early hemato-endothelial progenitor(s) that give rise to EMPs and HSCs are still elusive, which impairs our ability to fully characterize resident macrophages. In addition, 3) it is important to elucidate the roles of individual LDTFs and their subset-specific combinations for the control of tissue-specific functions of macrophages, because, in addition to identify molecular mechanisms that underly macrophage functions, these studies have the potential to unveil therapeutic strategies for leveraging macrophage functions in human. We propose to take advantage of novel genetic tools to identify the hemato- endothelial progenitors that generate the macrophage lineage in mice (AIM1), to identify conserved macrophages progenitors in human using a genetically tractable model for hematopoietic differentiation in human induced pluripotent stem cell (hiPSC)-derived embryoid bodies, and to take advantage of this versatile in vitro model to characterize the role of macrophage LDTFs in the control of macrophage tissue-specific functions (AIM2). We expect that our results will provide a robust experimental basis to transform and improve our understanding of the cellular, genetic, and molecular determinants of resident-macrophage development in mice and, importantly, in human, and provide important genetic tools for the molecular understanding and manipulation of their functions in physiology and diseases.

NIH Research Projects · FY 2025 · 2017-02

Ferroptosis, Cellular Metabolism, and Cancer Abstract Ferroptosis is a form of non-apoptotic cell death driven by cellular metabolism and iron-dependent lipid peroxidation. Although the physiological role of ferroptosis remains elusive, mounting evidence has established that ferroptosis impacts various pathological processes, including cancer. This competitive renewal proposal is built upon what we have achieved during the previous funding cycle and aims to further elucidate the molecular basis of ferroptosis, its interplay with metabolism, and its role in cancer. In the previous funding period, we found that multiple cellular metabolic pathways, such as autophagy, glutaminolysis, and strikingly, the normal metabolic activity of mitochondria, contribute to ferroptotic death. We also found that the CDH1-NF2-Hippo-YAP and PI3K- AKT-mTOR-SREBP signaling pathways, both highly relevant to cancer, regulate ferroptosis through modulating cellular iron homeostasis and lipid metabolism. Moreover, via a whole genome CRISPR/cas9-activation screen, we identified several lipid modifying enzymes as novel ferroptosis suppressors, further underscoring the intimate relationship between lipid metabolism and ferroptosis. Importantly, our TCGA analysis indicates overexpression of one of these enzymes, MBOAT2, predicts poor prognosis in multiple cancer types, including liver cancer, bladder cancer, and pancreatic ductal adenocarcinoma (PDAC). Based on these preliminary results, the central hypothesis of the grant is that lipid modification regulates cancer cell metabolism, invasiveness, and ferroptosis, through modulating cellular lipid storage and membrane composition; and targeting MBOAT2 in combination with ferroptosis induction holds cancer therapeutic potential. To investigate this hypothesis and to define the underlying mechanisms, we will tackle following questions. First, what is the mechanism by which these lipid modifiers protect cells fromferroptosis,do they dictate lipid peroxidation viaaltering specific phospholipidspecies, and do they communicate with SREBP, a master transcriptional regulator of lipogenesis and a potent ferroptosis suppressor (Aim-1)? Second, do these lipid modifiers modulate cellular properties such as cellular storage of lipids as energy source and plasma membrane plasticity? As these cellular properties impact cancer cell invasive/metastatic capability, metabolism, and likely redox homeostasis, is there a functional interplay between ferroptosis and these cancer-relevant cellular processes (Aim-2)? Third and directly relevant to cancer treatment (Aim-3), by using patient-derived tumor organoids, xenograft mouse models, and genetically engineered mouse models (GEMM), we will investigate how our newly-identified ferroptosis suppressors modulate tumorigenesis, metastasis and the responsiveness of cancer cells to ferroptosis induction, and assess whether the combination of MBOAT2 inhibition with ferroptosis induction can be an effective therapy for the treatment of cancer in which MBOAT2 overexpression predicts poor prognosis (we will focus on PDAC in this proposal). Success of the proposed study will lead to an in-depth mechanistic understanding of ferroptosis and its interplay with cellular metabolism, and provide insights into the development of novel, mechanism-based cancer therapies.

NIH Research Projects · FY 2025 · 2017-01

PROJECT ABSTRACT We propose to use single cell studies of clinical isolates to explicate how mutations and differentiation status coordinately regulate cell state, clonal expansion and malignant transformation. We will model sequential activation/inactivation of somatic mutations in defined cell compartments and perform functional studies to uncover critical gene networks and therapeutic dependencies. This will include single cell studies in primary patient samples, use of innovative models which allow for sequential mutational activation/inactivation, and functional genomic approaches to delineate mechanisms of transformation and novel therapeutic dependencies. Moreover, we will delineate crosstalk between different cell types in normal and malignant hematopoiesis and how these interactions impact therapeutic response. These tools will be distributed widely to enable studies of tissue development/homeostasis, cell state changes, transformation, and therapeutic dependencies.

NIH Research Projects · FY 2025 · 2016-08

Project Summary Despite intense efforts, the long-term cure rates of patients with acute myeloid leukemia are inadequate. Resistance to chemotherapy is prevalent, and targets for molecular therapies are only beginning to be defined. For example, mutation of genes encoding transcription factors and epigenetic regulators cause most subtypes of AML, but their molecular pathophysiology and pharmacologic accessibility remain poorly defined. We have now found that leukemogenic gene expression in AML requires distinct molecular interactions between the pioneer transcription factor MYB and its coactivator CBP. Remarkably, peptidomimetic inhibitors of MYB transcriptional coactivation exhibit potent anti-leukemia activity in most molecular subtypes of AML while sparing healthy cells. The central hypothesis of this proposal is that defining and blocking the molecular mechanisms of aberrant transcriptional coactivation in AML will lead directly to improved therapies for patients. Aim 1 will define the molecular mechanisms of aberrant activation of leukemic MYB transcription factor complexes that control oncogenic gene expression. Aim 2 will pursue the preliminary evidence that peptidomimetic and targeted small molecule inhibitors can be used to dismantle leukemic transcriptional complexes in vivo and develop effective therapeutic strategies using accurate genetic and patient-derived preclinical mouse models. Successful completion of this project is expected to yield essential molecular mechanisms and effective therapies of aberrant transcription factors and gene control in AML, thus providing essential insights into a fundamental problem that remains poorly understood. This should have broad and lasting significance for understanding and treating refractory leukemias in particular and human cancer generally.

NIH Research Projects · FY 2025 · 2016-06

RNA decay. The balance between RNA transcription and degradation contributes to regulation of RNA lifetime, quality and abundance. Two principle RNA decay pathways exist in eukaryotes, one degrades RNA 5’ to 3’ while the other degrades RNA 3’ to 5’. The 3’ to 5’ decay pathway requires activities of the RNA exosome, a multi-subunit protein complex that contains a nine-subunit non-catalytic core and two additional subunits that catalyze processive and distributive 3’ to 5’ RNA exoribonuclease activities. Ten of the eleven genes are essential for life in budding yeast, suggesting the importance of the RNA exosome and its activities in cellular function. In addition, reports over the last several years suggest that humans harboring mutations in select components of the 3’ to 5’ decay pathway suffer from diseases ranging from motor neuronopathies to cancer. Fundamental aspects of eukaryotic exosome structure and function have been illuminated; however, many questions remain with respect to how upstream factors target substrates for degradation. As RNA decay pathways play a fundamental role in eukaryotic nucleic acid metabolism and disease, our studies are directly relevant to human health and the NIH mission as misregulation of RNA decay is associated with cancer, inflammation and neurodegeneration. This renewal will address central issues of RNA exosome biology by reconstituting or purifying RNA exosomes and upstream factors for characterization through biochemical, genetic and structural studies to establish functions for 3’ to 5’ decay in vitro and in vivo. Ubiquitin-like proteins. Signal transduction can rely on reversible chemical modifications to relay information. Protein substrates can be covalently modified by ubiquitin and ubiquitin-like proteins such as SUMO (small ubiquitin-like modifier) to regulate processes such as nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcription. Post-translational modification by ubiquitin (Ub) and ubiquitin- like (Ubl) proteins requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 protein ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Ubiquitin and SUMO conjugation play integral roles in eukaryotic nuclear metabolism and cell cycle control and our studies are of direct relevance to human health, cancer, and the mission of the NIH. This renewal seeks to address the functional significance for components of the ubiquitin and SUMO conjugation pathways through structural, biochemical and genetic studies that will establish a basis for Ub/Ubl activation, conjugation by E2 and E3 enzymes, and signaling through characterization receptors that recognize Ub/Ubl-conjugated substrates. The enzymes, mechanisms and cofactors utilized for ubiquitin and SUMO protein conjugation pathways are conserved so our studies are broadly relevant to other Ub/Ubl-related pathways.

NIH Research Projects · FY 2025 · 2016-05

Homologous recombination in meiosis is essential for genome integrity during sexual reproduction, but is also a powerful determinant of genome evolution and puts cells at risk for mutation and chromosome rearrangements. Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. Cells ensure that DSBs are made at the right times, places, and numbers to maximize repair efficiency and minimize risks of deleterious outcomes. This research program aims to understand the molecular mechanisms of meiotic DSB formation and of the processes that regulate DSBs and recombination. Mouse and the yeast S. cerevisiae will be used to explore these critical aspects of chromosome biology. Specific areas of inquiry include the following: · A complex network of pathways controls the number, timing, and distribution of DSBs. In one pathway, the DNA damage-response kinase ATM inhibits formation of additional breaks. A second pathway suppresses DSB formation in places where homologous chromosomes have successfully engaged one another. An important challenge is to understand the mechanisms underlying these pathways. · DSB locations are nonrandom, and this DSB “landscape” has important consequences for heritability and genome stability. The factors shaping the DSB landscape remain poorly understood, but recent advances inform mechanistic hypotheses about the roles of both chromosome-intrinsic and trans-acting factors. These hypothe- ses will be tested using powerful methods for mapping DSB distributions genome-wide at nucleotide resolution. · An essential step in the repair of DSBs by recombination is the exonucleolytic processing of DNA ends, but little is known about the mechanism. An innovative new whole-genome assay for DSB resection will be exploited to define how resection is carried out, how it is regulated, and how it overcomes the barrier to nucleases posed by chromatin. · Erroneous, non-allelic recombination between repetitive DNA sequences yields chromosome rearrange- ments that can be passed on to offspring. Recent work identified genomic locations in mice that are prone to such errors and developed tools to characterize and quantify rearrangements. Important challenges now are to understand the mechanisms of this mutagenic recombination and to understand how cells avoid these errors. · In male mammals, segregation of the sex chromosomes is especially challenging because the X and Y chro- mosomes share only a small region of homology (the pseudoautosomal region, or PAR) within which recombi- nation must occur. Defects in PAR recombination cause sterility or sex chromosome missegregation. Recent work has revealed that the PAR develops complex, dynamic structures during meiosis. Cis- and trans-acting factors critical for this behavior have also been uncovered. Key questions will be addressed concerning the mechanisms that ensure sex chromosome recombination and segregation.

NIH Research Projects · FY 2025 · 2016-02

ABSTRACT Upon encountering antigens, mature B cells express activation induced cytidine deaminase (AID) and undergo immunoglobulin heavy chain (Igh) class switch recombination (CSR) and somatic hypermutation (SHM). CSR proceeds through the obligate generation of DNA double strand breaks (DSBs), which constitute one of the most toxic lesions that can occur in a cell. A single unrepaired DSB can cause cell death or potentiate chromosomal translocations that are hallmarks of many types of cancer, including lymphomas. Thus, mechanisms that promote generation of DSBs and facilitate DSB repair are intergral to both immunity and preservation of genomic integrity. In this proposal we test the notion that non-canonical DNA structures such as G-quadruplexes target the DNA deaminase AID to the chromatin during CSR (aim 1) and that AID can regulate expression of non-Ig genes to influence B cell responses (aim 2). Successful completion of the experiments will have far reaching implications in our understanding of both B cell immunity and B cell lymphomas.

NIH Research Projects · FY 2026 · 2015-09

Intestinal injury is a major complication of allogeneic hematopoietic transplantation, limiting its wider use. The immune system plays a pivotal role in causing intestinal pathology in graft vs. host disease (GVHD) occurring post-transplant, but the immune system can also provide critical contributions to epithelial regeneration and in- testinal repair. We have found that immune-mediated interactions with the intestinal stem cell (ISC) compartment are important contributors to both the damage and regeneration occurring in the intestines in acute GVHD, and the previous funding period resulted in several impactful contributions to understanding immunologic effects on the ISC compartment after bone marrow transplantation (BMT), including that 1) the crypt base ISC compartment is the initial site of donor T cell infiltration within the intestines, where their overproduction of Interferon-g (IFNg) can directly induce Bax/Bak-dependent ISC apoptosis at high concentrations; and 2) Interleukin-(IL-)22 can di- rectly signal to ISCs, promoting their survival and regeneration after damage, and it can be administered thera- peutically post-BMT to promote thymic and intestinal recovery. IL-22 administration can also counteract toxicity from corticosteroids, which reduce ISC proliferation and epithelial regeneration. The experimental work from this project resulted in a multicenter clinical trial treating newly diagnosed gastrointestinal acute GVHD with recom- binant human IL-22. In this renewal, we propose to build upon this progress and conduct a mechanistic evalua- tion of immune-mediated and niche-driven epithelial regeneration in the context of allogeneic BMT. Our new preliminary data indicate profound transcriptomic changes in the ISC compartment in GVHD, identifying a novel pathway of IFNg/STAT1-mediated regulation of ISC c-MYC expression and epithelial regeneration. We believe this pathway induces ISC proliferation at low concentrations of IFNg, whereas high concentrations induce apoptosis. Our new data also indicate that Paneth cell loss may not be driving intestinal pathophysiology in GVHD, and other secretory epithelial cells may also produce growth factors for the niche. Utilizing our experience in ex vivo organoid modeling and ISC immunology, 3-D confocal imaging, and cytokine biology developed in the initial funding period, as well as newly developed approaches for epithelial transcriptomics, we propose to ex- plore these preliminary findings further by 1) investigating the importance of epithelial Myc expression post-BMT using a newly developed model of conditional Myc deletion, 2) dissecting IL-22 and IFNg’s distinct and overlap- ping JAK/STAT responses regulating the ISC compartment, and 3) ablating Paneth cells and other secretory lineage cells to determine their importance for ISC maintenance and epithelial regeneration after transplant. We will then build upon these insights to develop novel approaches manipulating epithelial JAK/STAT signaling and niche function to promote intestinal recovery. This renewal will thus pursue basic mechanisms of transplant biology, mucosal immunology, and stem cell research and extend them to translational investigations with the potential to improve clinical outcomes for transplant patients and others suffering from intestinal pathology.

NIH Research Projects · FY 2025 · 2015-09

PROJECT SUMMARY/ABSTRACT This is a renewal K12 application for the Paul Calabresi Career Development Award for Clinical Oncology to continue a training program for clinical translational research at Memorial Sloan Kettering Cancer Center (MSK). Over the first and second funding periods of our K12 program we have established and executed a uniquely universal training program which extends to all MSK departments engaged in cancer research. The program solicits applications from senior fellows moving into junior faculty positions and junior faculty within the first 3 years of their appointments at MSK. This program provides 3 years funding to selected MD, DO, MD/PhD and DO/PhD scholars, and aims to help scholars transition into becoming independent clinical and translational investigators. In the first two funding periods, MSK has trained 30 K12 scholars with an additional 4 set to onboard on September 1, 2024. Applications for this program will be reviewed by experts in the field both from within and outside of MSK. Reviewed applications are further evaluated by the K12 internal Advisory Committee (AC) which will select the 3-4 best applicants annually. Additional matching institutional funding has allowed and will continue to allow the program to support a maximum of 4 new scholars annually with up to 12 scholars funded at any given time during the funding period. Selected applicants will subsequently be evaluated by an established K12 Educational Committee (EC) who will track the progress of each selected candidate through the program and curriculum. This curriculum will include a pre-established core curriculum built around the Master of Science in Clinical and Translational Cancer Research with additional elective courses derived from available courses offered by the Louis V. Gerstner Graduate School of Biomedical Science. Through written questionnaires K12 scholars will be asked to review the program and provide a progress report annually. To further assess the progress and function of the K12 program, scholars will provide written evaluations of their mentors as will mentors provide written evaluations of the scholar’s progression on an annual basis. Collectively these written evaluations will be utilized by the K12 program to assess the scholar’s progress, the mentor’s fitness, and the overall performance of the program. The program and scholar progress will be evaluated annually by the K12 AC as well as the External Advisory Committee (EAC), which will include one EAC visit during the annual K12 research symposium.

NIH Research Projects · FY 2024 · 2015-09

Project Summary/Abstract The overall objective of this project, entitled “Meaning-Centered Psychotherapy Training for Cancer Care Providers” (MCPT), is to continue facilitate the implementation and dissemination of Meaning-Centered Psychotherapy (MCP), a Research-Tested Intervention Program (RTIP) through a multi-modal training program for cancer care clinicians. There is extensive evidence demonstrating a need for interventions targeting depression, hopelessness, loss of meaning and spiritual and existential distress in patients coping with the challenges of advanced cancer. To address this need, Breitbart and colleagues developed MCP as an innovative and novel intervention to enhance meaning and reduce despair in advanced cancer patients living in the face of death. Since our National Institute of Health (NIH) R25 grant was funded, we have successfully developed MCPT and trained 297 clinicians from a wide variety of clinical institutions and settings. As proposed, we utilized the RE-AIM framework, comprised of five distinct factors: 1) Reach, 2) Efficacy, 3) Adoption, 4) Implementation, and 5) Maintenance, to evaluate the impact of the MCPT program on the translation of MCP into clinical practice. Preliminary results indicate that trainees are extremely satisfied with the MCPT program, have become proficient in the delivery of MCP, have successfully implemented MCP in their own clinical settings, and are maintaining or improving their MCP skills post-training. We are applying for a 5-year renewal of this R25 grant to capitalize on overwhelming interest expressed by cancer care clinicians in our innovative, immersive training and to further disseminate MCP to advanced cancer patients in need of evidence-based psychosocial care. We will offer this training to an additional 336 clinicians during years 6-10 and enhance the follow-up components of the training to further improve MCP skill maintenance, adoption, and implementation in clinical practice. This will also enable us to continue to collect data on the implementation of MCP with patients in the “real world” across diverse clinical settings. The long-term goal of this project is to disseminate MCP to a wide variety of cancer and palliative care treatment settings through the training of a large, diverse cadre of clinicians. Thus, the specific aims of this study are to: Aim 1: Provide and further develop a training program in MCP for cancer care clinicians from multiple disciplines who provide psycho-oncology and psychosocial palliative care services for cancer patients; Aim 2: Evaluate trainees’ MCP skill acquisition through facilitators’ ratings of MCPT participants and participants’ satisfaction with the program and adoption, implementation, and maintenance of skills; and Aim 3: Evaluate the impact of enhanced follow-up training and engagement in MCP community activities on MCP implementation and skill maintenance.

NIH Research Projects · FY 2025 · 2015-04

ABSTRACT ETS family protein alterations (primarily ERG translocations) are present in >50% of human prostate cancers in Western men. We were the first to develop a robust GEM model of ERG-driven prostate cancer, revealing enhanced luminal differentiation and an expanded androgen receptor (AR) cistrome in tumors, suggesting a novel mechanism for ERG oncogenicity via chromatin programming (Chen, 2013). We and others subsequently established that ERG activates a luminal differentiation program in prostate organoids, human prostate cell lines and, by computational analysis of prostate cancer genomes, in clinical samples (Blee et al., 2018; Kron et al., 2017; Li et al., 2020b). Other ETS gain-of-function alterations e.g. ETV4 translocations (Li et al., 2020a) and ETS loss-of-function alterations e.g. ERF repressor mutations/deletions (Bose et al., 2017) also show this phenotype, as does FOXA1 (also amplified or mutated in prostate cancer) but with a contracted AR cistrome (Adams et al., 2019). Having demonstrated that luminal differentiation is a primary feature of multiple oncogenic ETS proteins (and FOXA1), our major goal during the next funding cycle is to understand how ERG activates this differentiation program and how this program results in an oncogenic phenotype. We will pursue three parallel lines of investigation. First, from our biochemical studies using purified full-length proteins and various DNA templates, we find that that ERG (and other ETS factors) cooperatively enhance AR DNA binding through allosteric effects via direct protein-protein interaction; biologically, this broadens the AR cistrome to include novel AR binding sites (Wasmuth et al., 2020). Aim 1 will expand this analysis to assess the role of FOXA1 on AR/ERG interactions. Second, we have built genetically defined prostate organoid models that recapitulate the luminal differentiation effect of ERG within a precisely defined time course. Using this system, we identified epigenetic changes that silence transcription of the basal epithelial master regulator p63, likely explaining the reduction in basal cells. Aim 2 will use lineage tracing, single cell analysis, and CRISPR screening to further elucidate how ERG expands the number of luminal cells and initiates oncogenic transformation. Third, we showed that another oncogenic ETS protein (ETV4) also drives luminal differentiation and is sufficient, alone, to initiate prostatic intraepithelial neoplasia (PIN). Remarkably, these luminal epithelial cells acquire exquisite dependence on AR for survival (in contrast to normal luminal epithelial cells) and consequently display enhanced sensitivity to androgen deprivation therapy (ADT). Aim 3 will explore mechanisms underlying this shift to cell-intrinsic AR dependence by examining changes in the AR cistrome and transcriptome following luminal-specific AR ablation (by genetic deletion) versus systemic androgen deprivation through surgical castration (impairs AR function in prostate stroma and epithelium). We will complement these experiments with single cell analysis of ETS-positive patient samples before and after ADT.

NIH Research Projects · FY 2025 · 2015-04

PROJECT SUMMARY/ABSTRACT An evolutionary conserved developmental program is carefully maintained in hematopoietic stem cells (HSCs). Genetic alterations and epigenetic mechanisms can alter the balance of normal blood development resulting in hematological malignancies. Our laboratory and others have found that the MUSASHI2 (MSI2) RNA binding proteins is highly expressed in the most aggressive cancers and predicts a poor clinical outcome in acute myeloid leukemia (AML) patients. Genetic models have found that MSI2 is required for leukemia stem cell function. Utilizing a new way to identify mRNA targets of RNA binding proteins, we have found that MSI2 activity is increased in leukemia stem cells compared to normal stem and progenitor cells. This surprising finding suggests that RNA binding protein function can be dysregulated beyond just expression differences. We hypothesize that the MSI family of RNA binding protein have differential activity in AML compared to normal cells and that MSI enhances the dysregulated epigenome in AML. We propose two possible mechanisms for this intriguing finding 1) MSI2 associated RBPs compete for MSI2-binding sites and 2) Post- translation modifications can modulate MSI2 activity. Our preliminary data has uncovered that MSI2 can mediate resistance to PRMT5 and that PRMT1 and PRMT5 can directly methylate MSI2. PRMT5 inhibitors are being investigated as therapeutic targets and our proposal suggests a novel link to this pathway and may explain cell context MSI2 activity. Our proposal will utilize new genetic models to characterize MSI2 targets in specific cellular contexts and explore the MSI2 associated program to identify new therapeutic strategies in AML.

NIH Research Projects · FY 2026 · 2014-09

PROJECT SUMMARY/ABSTRACT Hematopoiesis is a tightly regulated process where hematopoietic stem cells (HSCs) generate diverse blood and immune cell lineages through symmetric and asymmetric cell divisions. During aging, certain aberrant hematopoietic clones emerge, creating a premalignant condition known as clonal hematopoiesis (CH), which increases the risk of chronic diseases, such as coronary heart disease, and progression to hematologic malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While HSCs require precise control over symmetric renewal and asymmetric differentiation to maintain blood homeostasis, the regulatory mechanisms of these cell fate choices—and how they contribute to early CH and MDS clonal dynamics—are not well understood. Recent discoveries point to the MSI2-associated RNA binding protein (RBP) network as a critical regulator of these fate decisions in HSCs. Our preliminary data suggest that MSI2 and its RBP network are pivotal for maintaining asymmetric cell division in hematopoiesis, and that a resilience variant in an HSC-specific MSI2 enhancer confers protection against CH and blood cancers. This proposal aims to elucidate how the MSI2-RBP network regulates HSC cell fate decisions, with a focus on understanding the molecular basis of resilience against clonal hematopoiesis. We hypothesize that MSI2 and its associated RBPs drive HSC renewal and differentiation choices and that genetic variation influencing MSI2 expression confers resistance to CH and hematologic malignancies. To test this hypothesis, we propose three specific aims: (1) To define the role of MSI2 in regulating symmetric and asymmetric fate choices in HSPCs; (2) To investigate how a CH resilience variant within an MSI2 enhancer modulates human HSC function; and (3) To examine MSI2's cooperation with genetic drivers of CH, including ASXL1, to understand how MSI2 modulates CH-associated clonal dynamics. Our interdisciplinary approach integrates advanced RNA profiling, CRISPR/Cas9-based genome editing, and single-cell sequencing technologies with new mouse models and human HSC assays. Together, this project will reveal how the MSI2-RBP network controls HSC cell fate, uncovering potential therapeutic targets to mitigate CH progression and prevent hematologic malignancies.

NIH Research Projects · FY 2025 · 2014-09

PROJECT SUMMARY/ABSTRACT Our long-term goal is to significantly impact the fundamental knowledge of muscle biology and provide new approaches for disease treatment. Striated muscle fibers are large multinucleated cells and possess a highly organized cytoarchitecture containing organelles positioned for optimal muscle function. This positioning is particularly evident in the placement of myonuclei, which reside above the sarcomere at the periphery of the myofiber and are positioned to maximize their internuclear distance. Our objective is the identification of mechanisms responsible for myonuclear movement and positioning. Centrally located myonuclei have been used for decades as a hallmark of muscle disease. However, much remains to be learned about the mechanisms that control myonuclear movement normally and the contribution of aberrant myonuclear position to the etiology and/or progression of muscle disease. Building on our published results over funding period (e.g. Metzger et al., 2012; Folker et al., 2012, 2014; Schulman et al., 2013, 2014; Azevedo et al., 2016; Manhart et al., 2018, 2020; Rosen et al., 2019), our specific aims are to first address mechanistically how tendon and motoneurons signal to the myofiber to fine-tune myonuclear positioning. We identified two signaling pathways at the myotendinous junction that regulate nuclear positioning. Likewise, we will dissect the contribution of the motoneuron to nuclear placement. Secondly, we will examine why muscles fail to function optimally when myonuclei are mispositioned. Muscle physiology will be assayed through testing mitochondrial function via quantification of ATP and ROS levels and by testing neuromuscular communication via electrophysiological approaches. We will also investigate the input to muscle function of specific metabolic and signaling proteins that we identified are misregulated as a result of mispositioned myonuclei. Thirdly, we will push forward our dissection of the myonuclear positioning mechanisms from fly to the human system. We will employ human 3D muscle cultures that are co-cultured with motoneurons to define and then perturb myonuclear movement and positioning as the myofibers develop. Our methodologies take advantage of cutting edge, in vivo time lapse imaging approaches that we have developed in Drosophila and will also apply to human 3D cultures to follow myonuclear movement and cytoskeletal dynamics. We will employ the genetic resources available in Drosophila and human cultures to manipulate genes, processes, and cell types for our analyses. These genetic experiments will be supported by biochemical and cell biological approaches. Together the work outlined in this proposal will shed new light on this little understood, but important area of muscle biology. The results of this research will permit us to highlight genes and mechanisms that are candidates for changes associated with different human muscle diseases.

NIH Research Projects · FY 2025 · 2011-06

PROJECT SUMMARY Invasive aspergillosis is devastating fungal infection and the most common form of mold pneumonia worldwide, with an estimated 200,000 cases annually. Aspergillus fumigatus, the most common etiologic agent of invasive aspergillosis, forms ubiquitous airborne spores that humans inhaled on daily basis. In immune competent individuals the respiratory innate immune system prevents the formation of tissue-invasive hyphae, a critical immunologic checkpoint. In patients with hematologic malignancies, in bone marrow and lung transplant recipients, and recently, in intensive care unit patients with COVID-19, numeric or functional defects in innate immune function lead to invasive disease. Despite contemporary antifungal drugs, mortality rates remain at 20- 40% in high risk groups, underscoring the need for improved understanding of the molecular and cellular basis of sterilizing immunity to advance immune-based adjunctive approaches. In the second funding period, we harnessed a fungal bioreporter that reports the mode of cell death to discover that neutrophils and monocyte-derived dendritic cells induce a regulated cell death in engulfed fungal cells. The concept that a higher eukaryote can exploit a regulated cell death machinery in a lower eukaryote is novel and, in the case of A. fumigatus, depends on host NADPH oxidase activity. NADPH oxidase-dependent fungal killing is modulated by two novel, essential intercellular crosstalk circuits that involves the early production of GM-CSF (GM-CSF circuit) and plasmacytoid dendritic cells (pDC circuit). During the next project period, we propose to gain a deeper understanding of the GM-CSF and pDC circuits. In Aim 1, we identify the essential cellular source of GM-CSF and, based on preliminary data, focus on pulmonary endothelial and epithelial cells as regulators of neutrophil-dependent fungal killing. In Aim 2, we define the pDC circuit and candidate transmitters and test models of direct or indirect activation by fungal cells or the lung inflammatory milieu. In Aim 3, we define the mechanisms by which the pDC circuit regulates neutrophils, and test its role in NAPDH oxidase assembly, activation, and neutrophil metabolism via the pentose phosphate pathway and its cooperativity with the GM-CSF circuit to mediate sterilizing immunity. The proposed studies are significant and innovative because they integrate innate immune crosstalk between the pulmonary endothelial, epithelial, and pDC compartments and infected myeloid phagocytes into a comprehensive model of respiratory immunity against mold pathogens. Understanding the induction, regulation, and participants of innate immune crosstalk addresses a critical knowledge gap that will inform immune-enhancing strategies in vulnerable patient groups.

NIH Research Projects · FY 2025 · 2010-02

Summary Immune cells communicate through dynamic cell-cell junctions known as immune synapses. Although the biochemical properties of these synapses have been studied extensively, we know little about their mechanical activities and how these activities contribute to immune function. We use cytotoxic lymphocytes as a model system to investigate the origins and purposes of synaptic force. Cytotoxic lymphocytes fight pathogens and cancer by forming an immune synapse with an infected or transformed target cell and then secreting toxic granzymes and the pore forming protein perforin into the intercellular space. Work from our lab and others suggests critical roles for mechanical forces both in triggering lymphocyte activation and in enhancing the efficiency of killing responses. Our proposed studies, which are divided into two Specific Aims, will address the functional relevance and molecular bases of synaptic force in these contexts. Aim 1 builds on preliminary data indicating that cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells detect the physical stiffening of target cells and use this mechanosensing capacity to identify and destroy cancer cells invading the metastatic niche. To determine if and how this mechanical form of immunosurveillance, which we call mechanosurveillance, shapes anti-tumor immunity in vivo, we will apply multiple murine metastasis models, atomic force microscopy, and analysis of clinical immunotherapy trials. Aim 2 is premised on prior work indicating that CTLs use mechanical force to potentiate the pore forming activity of secreted perforin. This sort of physicochemical synergy demands a high degree of coordination between mechanical and secretory output within the synapse, but how lymphocytes achieve this coupling remains unknown. We have developed an imaging-based biophysical approach to address this problem, which will enable us to establish the mechanochemical choreography of cytotoxicity with unprecedented precision. Our proposed studies will employ technically innovative methods, including super-resolution imaging of lymphocyte force exertion against micron-scale biophysical probes. They will also introduce a number of innovative concepts, including the idea that cellular rigidity can trigger immunosurveillance and the idea that lymphocyte subsets employ distinct mechanical signatures to specify their effector responses. Our work will also address a simple but technically vexing issue that has constrained the field for some time, namely whether mechanobiological principles actually influence immunity in vivo. Understanding the biophysical dimensions of immune synapse function could potentially reveal new strategies for the modulation and assessment of lymphocyte activity in the clinic. As such, the studies described herein are highly relevant to the NIH mission in that they will contribute to the advancement of knowledge that could improve human health.

NIH Research Projects · FY 2026 · 2009-08

Project Summary Reproducible assembly of protein molecules from free amino acids requires external information stored in DNA/RNA (genome). In contrast, assembly of higher-order structures from individual protein molecules is thought to occur de novo, relying on information intrinsic to each protein component. How higher-order complexity emerges efficiently without error using only the scattered information in each building block remains a question hindering core understandings of life. Here we address this question using the biogenesis of the centriole organelle as the example. We found that a proteinaceous structure called the cartwheel is grown inside existing centrioles, which is then split, shed and used as structural templates (or guidance) for reproducing a copy of the centriole with the same size/shape. Uncontrolled cartwheel shedding was further found to underly a human disease called Alström syndrome. By erasing the old guidance, we showed that new/naive centrioles varying widely in size and shape arise, which can propagate reproducibly thereafter in cells otherwise genetically identical. The simplest model explaining our results is that one or more steps of centriole biogenesis are template-based, instructed by information stored and transmitted outside of the genome. This model on which the current proposal is based on will shed light on how cellular complexity emerges reproducibly from components randomly distributed in the cell, redefining the scope of biological information as we currently understood.

NIH Research Projects · FY 2025 · 2009-07

ABSTRACT Upon encountering antigens, mature B cells express activation induced cytidine deaminase (AID) and undergo immunoglobulin heavy chain (Igh) class switch recombination (CSR) and somatic hypermutation (SHM). CSR proceeds through the obligate generation of DNA double strand breaks (DSBs), which constitute one of the most toxic lesions that can occur in a cell. A single unrepaired DSB can cause cell death or potentiate chromosomal translocations that are hallmarks of many types of cancer, including lymphomas. Thus, mechanisms that promote generation of DSBs and facilitate DSB repair are intergral to both immunity and preservation of genomic integrity. In this proposal we test the notion that single proteins can coordinate both DSB formation and mediate end-joining to efficiently generate and repair DSBs. We test the hypothesis that the nucleosomal remodeling protein CHD4 co-ordinates generation and repair of Igh DSBs (aim 1) and the C- terminus of AID mediates efficient DNA repair of Igh DSBs (aim 2). Successful completion of the experiments will have far reaching implications in our understanding of both B cell immunity and B cell lymphomas.

NIH Research Projects · FY 2025 · 2008-09

The CCNY-MSKCC Partnership has successfully created a mutually beneficial, cross-institutional collaboration that has emphasized research across the translational T0-T4 continuum, the creation of an education pipeline for attracting students to careers in cancer research, and the establishment of community networks and resources for conducting community engaged outreach and research for the next five years. The Partnership focus will be Translational Cancer Research. Consistent with this emphasis, our research projects will focus on: 1) understanding the biological and social factors associated with immune checkpoint inhibitors and evaluating an intervention designed to reduce side effect severity and improve treatment attendance; 2) an examination of small molecule regulation of cancer-driving transcription factors using a interdisciplinary strategy of protein chemistry, chemical, and cellular approaches that could lead to targeted treatments relevant to cancers; 3) the impact of AI integrated, UN-style medical interpreting engineering solutions on cancer care; 4) identification of germline and other factors influencing the tumor genome of lung cancers.