Rockefeller University

NIH Research Projects · FY 2026 · 2020-02

Project Summary This proposal addresses the monumental health burden of chronic hepatitis B virus (HBV) infection, for which there is no effective cure. Long-term objectives are to develop and apply new methods to study HBV biology and make discoveries that inspire curative therapies. In this proposal, we build on the HBV RNA launch method and our experience with deep mutational scanning to address key points of interest among HBV advocates. These include understanding HBV genetic diversity and how it influences drug resistance. How mutations that commonly arise under immune pressure in the chronic phase of infection may alter disease pathogenesis. And how HBV organizes its genome into a multilayered masterpiece of economy. One specific goal of this proposal is to study mechanisms of resistance to core protein (Cp) allosteric modulators (CpAMs), a class of compounds that interfere with HBV nucleocapsid assembly or stability. We will develop a new cell culture method to accomplish this and complement and confirm results with in vivo studies in human liver chimeric mice (huFNRG). Our approach uses self-amplifying RNAs to deliver complex libraries of HBV variants to cultured cells, which overcomes certain obstacles with existing methods. These studies will provide a comprehensive, low-biased view of Cp sequence requirements. While the primary goal is to study mechanisms of CpAM resistance, the method is not limited to studying Cp, and comprehensive fitness maps generated in the process also have the potential to uncover basic truths of HBV biology. A second goal is to apply the HBV RNA launch method to study the effects of mutations in the HBV precore mRNA, which arise under immune pressure and reduce the production of the HBV e antigen. We hypothesize that besides reducing e antigen levels, certain clinically relevant mutations may also increase the production of double-strand linear viral DNA and, consequently, the frequency of viral DNA integration into host chromosomes. The dominant view in the literature is that HBV integrants are incapable of producing infectious viruses; however, they provide an alternative source for viral protein expression with important implications for ongoing clinical trials and the definition of a cure. Lastly, a third goal of this proposal is to continue applying the already fruitful RNA-based deep mutational scanning approach we established to further “deconstruct” the information-dense HBV genome and define protein and nucleic acid sequence requirements for its replication. Specifically, we focus on identifying requirements for each of the following steps: packaging the viral pregenomic RNA into nucleocapsids, reverse transcription to make DNA, and conversion into the stable, episomal form that persists in infected hepatocytes. The proposed work is important because chronic HBV infection is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide, and existing therapies are rarely curative. New tools and fundamental insights are needed to support a pipeline for therapeutic development leading to a cure, which we know is possible but is inefficient.

NIH Research Projects · FY 2025 · 2019-09

Project Summary/Abstract This R50 proposal is a companion to our recently renewed R35 (CA210036-08), which is focused on the role of telomeres in cancer. Telomeres are required for the survival and proliferation of human cells and play a critical role in cancer. Excessively long telomeres at birth predispose to a wide variety of cancers, presumably because long telomeres delay the Hayflick limit to a stage in tumorigenesis when incipient cancers have already disabled the cell cycle arrest response to short telomeres. Conversely, in the short telomere syndromes (e.g., dyskeratosis congenita and Coats plus syndrome) loss of telomeric DNA curbs the vitality of stem-cell compartments and instigates multi-organ failure. This proposal focuses on CST, the trimeric ssDNA-binding complex composed of Ctc1, Stn1, and Ten1 and its associated Pola/primase. CST– Pola/primase has a dual role at telomeres: it mediates maintenance of the telomeric C-strand and it regulates telomerase, preventing the excessively long telomeres that lead to cancer predisposition. As described in the preliminary data, we recently discovered a new end-replication problem that is not solved by telomerase. This problem arises from the inability of the replisome to sustain lagging-strand synthesis when it reaches the end of a linear DNA, a phenomenon we demonstrated using in vitro DNA replication in collaboration with Dr. Joseph Yeeles (LMB, Cambridge). I showed that the C-strand of telomeres shortens by ~60 nt during lagging-strand DNA synthesis in vivo and that the CST–Pola-primase complex is required to counteract this shortening. My additional preliminary data showed how CST–Pola- primase is recruited to telomeres. In collaboration with graduate student Sarah Cai, I demonstrated that CST binds to the shelterin subunit POT1, not TPP1, as had been anticipated. We derived the Cryo-EM structure of POT1 bound to CST and found that POT1 must be phosphorylated to recruit the complex to telomeres. These pathbreaking findings form the basis of my current proposal to determine how recruitment of CST– Pola/primase is regulated. My aims are to identify the kinase that phosphorylates POT1 to allow CST binding, study its regulation at telomeres, determine how dephosphorylation of POT1 releases CST–Pola/primase into the active fill-in complex, and how CST controls telomerase. I am a long-term member of the Unit Director’s group and a pivotal contributor to our R35 program. I have developed new technologies that are essential for our research. I am highly skilled, rigorous, creative, collaborative, and completely committed to the Unit Director’s research on the role of telomeres in cancer. My career goal is to continue to excel and support the success of the R35 through innovative research that delivers breakthrough discoveries.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY The research proposal aims to investigate the novel regulatory sequences and elements underlying novel phenotypes, to gain a deeper understanding of the genetic basis of morphological and cellular innovation. The evolution of morphological structures and traits is complex, and the origins and genetic mechanisms that drive the development of new cell types, tissues, and organs are not fully understood. Our long-term goal is to uncover the processes that govern morphological and functional diversity and complexity, which is a crucial step in understanding the evolution of complex life. Despite progress in this field, our current understanding is still limited. The goal of this research is to understand the origins of novel regulatory sequences and elements, how they are integrated into regulatory networks to contribute to genic and phenotypic innovation, and further impact evolution. To accomplish this, the laboratory has two major focuses: the study of how new genes and phenotypes, including expression phenotypes, are regulated, and the deciphering of the principles of sex-biased regulation from an evolutionary biological perspective. The first part of the proposed research aims to investigate the mechanisms governing the gain of expression of evolutionarily young genes in Drosophila. Specifically, the project will focus on the regulatory basis of new genes in Drosophila and using scRNA-seq and scATAC-seq to pinpoint enhancers and promoters for new genes and new expression. Additionally, the project will investigate the mechanism of pre-meiotic dosage compensation and identify putative novel players. The second part of the proposed research will focus on the genetic and epigenetic mechanisms for sex-biased novel expression. This will include using deep learning to reveal the basis of sex-biased chromatin accessibility, investigating the origin of novel enhancers from poised sequences, and studying the role of distant enhancers in gene expression novelties and sex-biased novelties. Overall, this project will use cutting-edge techniques and approaches to gain insight into the mechanisms that drive the evolution of new genes and their expression patterns in Drosophila. This study will provide important insights into the evolution of transcription regulatory networks and their contributions to novel traits, including expression phenotypes. Altogether, our integrative approach will help to elucidate the origination and evolution of novel regulatory circuits and their contributions to phenotypic innovation and evolution.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY/ABSTRACT     Cancer  development  accompanies  with  the  dynamic  evolution  of  immunity,  a  well-­known  process  termed  as  immunoediting.  However,  the  underlying  mechanisms  of  the  transition  between  each  phase,  from  immune  surveillance to final escape, still remain a lot to discover. This proposal aims to study immunoediting during liver  cancer development and progression, with a focus on senescence and metastasis. Senescence is a cell cycle  arrest program that limits the expansion of damaged cells and can trigger anti-­tumor immunity that leads to their  elimination  in  vivo,  serving as a  potent  barrier  to  tumorigenesis.  However,  during  tumor  initiation,  the effective  clearance of senescent cells is compromised, warranting a deeper mechanistic understanding of this process.  My doctoral research aims to identify critical molecular and cellular players driving anti-­tumor immune responses  during senescence surveillance triggered by wildtype p53, which is known to modulates cancer immunity. The  long-­term objective of my thesis project is to define the mechanisms of how senescent cells are susceptible to  immune  surveillance  and  how  these  mechanisms  are  evaded  or  bypassed  during  cancer  development  and  progression. As described in Specific Aims 1.1-­1.3, my thesis work has demonstrated that the p53 restoration  triggers regression of liver cancers in an immunocompetent host. Using different immunodeficient mouse strains  and pharmacological approaches perturbing specific immune compartments, our preliminary data suggests that  adaptive  immunity  plays  a  key  role  in  senescence  surveillance.  RNA-­seq  and  mass  spectrometry  were  conducted  on  both  proliferating  and  senescent  tumor  cells  and  revealed  several  senescence-­enriched  cell  surface factors related to epithelial-­immune cell interactions. In Specific Aims 1.4 and 1.5, we aim to functionally  interrogate  the  role  of  these  senescence-­induced  factors  as  novel  senescence  surveillance  effectors,  with  a  focus on the regulatory network of antigen presentation pathway and, by exploiting multiplexed in vivo genetic  screens established in the Lowe laboratory. My postdoctoral research will continue to study immunoediting with  a slight change of the focus from the epithelial-­tumor angle to a more immunology-­rich perspective, applied to  the problem of metastatic immune escape. The proposal aims to investigate the molecular changes of NK cells,  shown to have control of early metastasis, after having physical interaction with metastatic cells. During different  stages of  metastatic  colonization,  tumor-­engaging  NK  cells are  labeled  via  “SynNotch”  technology and  will  be  subjected to single-­cell RNA-­seq to unveil the NK cell heterogeneity (Specific Aim 2.1) and ATAC-­seq to reveal  potential  epigenetic  mechanisms  of  immune  exhaustion  with  functional  perturbation  of  the  altered  programs  employed (Specific Aim 2.2). The proposed postdoctoral research will increase our mechanistic understanding  of NK biology during the metastasis outbreak, paving new paths to harness innate immunity against cancer. In  all, these two projects will offer distinct insight into immunoediting, of which the elucidated mechanisms could be  exploited for developing novel immunotherapies, jointly with existing ones for more effective cancer control.

NIH Research Projects · FY 2025 · 2019-06

ABSTRACT Licensed influenza vaccines commonly elicit strain-specific immunity, failing to provide protection against antigenically distinct strains with the capacity to cause pandemic outbreaks. Therefore, the development of a universal influenza vaccine with the capacity to elicit lifelong protection against diverse influenza virus strains is critically needed to prevent future influenza pandemics. Influenza hemagglutinin (HA) represents a key vaccine target, as it is the major glycoprotein expressed on the surface of influenza virions and mediates viral entry and fusion. HA comprises two distinct functional domains: (i) the globular head, which is highly variable due to antigenic drift, and (ii) the stalk domain, which is structurally conserved among diverse influenza strains. Influenza infection or vaccination commonly elicits immune responses against immunodominant, strain-specific epitopes on the HA head. By contrast, immune responses against highly conserved epitopes are limited and short-lived, despite conferring broad and heterologous protection. Refocusing the immune response towards conserved, immunosubdominant HA epitopes, while avoiding eliciting strain-specific immunity, represents a promising strategy for the development of a universal influenza vaccine that would confer broad protection against diverse influenza strains. To achieve this, the proposed studies aim to develop and evaluate novel HA immunogens, termed mosaic HAs (mHA), in which the immunodominant, strain-specific epitopes at the head domain have been replaced from those of exotic HAs which humans are naïve, while immunosubdominant, conserved epitopes have been retained. In previous studies, we have dissected the mechanisms by which antibodies, through specific interactions of their Fc domains with activating Type I Fcγ receptors (FcγRs), such as FcγRIIa on dendritic cells and with the Type II FcγR, CD23, on B cells modulate cellular and humoral immunity against influenza, respectively. Given the immunomodulatory consequences of Fc-FcγR interactions, the proposed studies will develop Fc-engineered mHA immunogens that will engage and activate specific FcγR pathways on defined effector cell populations to elicit broad and long-lasting immunity against diverse influenza strains. We anticipate that the proposed studies will lead to the design, selection, and pre-clinical evaluation of innovative immunogens with the capacity to confer durable and heterologous protection against influenza. These studies are expected to have important implications not only for our efforts towards the development of a universal influenza vaccine, but also provide the framework for the design of vaccines with long-lasting and broad protection against other viral pathogens, such as SARS-CoV-2.

NIH Research Projects · FY 2026 · 2019-05

Animals exhibit astonishing diversity in their behavior, yet almost nothing is known about how evolutionary variation in neural circuits gives rise to species-specific behavioral variation. Here I propose to take advantage of recent advances in genome editing and develop an innovative approach to reveal how evolution sculpts brain circuits. Using CRISPR genome editing technology, we are translating neurogenetic tools from D. melanogaster to other Drosophila species, allowing for the first high-resolution anatomic and functional neural circuit mapping across species. By directly comparing the homologous sensory processing pathways in closely related drosophilids, we will precisely pinpoint where adaptive changes have occurred within the nervous system to produce species-specific mate preferences. The rapid evolution of Drosophila courtship allows us to systematically probe how parallel changes in behavior have been independently implemented in different species, shedding light on the types of changes that are permissible and preferable within brain circuits. Mapping the sites of anatomic and functional change within these pathways will further enable us to study their underlying molecular basis, using transcriptional profiling of the relevant neural populations to provide a definitive link between genetic and behavioral variation. Together, the proposed studies will transform our understanding of the molecular, cellular, and circuit-level changes that generate adaptive behavioral variation across species. As the etiology of many brain disorders is aberrant neural circuit wiring, a deeper understanding of the link between genes, neural circuits, and behavior could have profound consequences for mental health.

NIH Research Projects · FY 2026 · 2019-05

Project Summary Chromosome inheritance during cell proliferation is fundamental for living systems. Failures in this process cause diverse diseases, such as cancer. Through the development of various innovative methods and the exploitation of the unique Xenopus egg extract cell free system, our research program studies structure, function, and regulation of the nucleosome - the fundamental structural unit for chromosomal DNA - in chromosome inheritance and integrity. 1) Roles of nucleosomes in mitotic chromosome structure, integrity and function. High resolution 3D structural analysis of nucleoprotein complexes on functional chromosomes has been impossible. We propose to combine an innovative cryo-EM method and nucleosome manipulation method that we developed in Xenopus egg extracts to study how nucleosome dynamics and integrity are regulated on mitotic chromosomes. 2) Centromere-associated repeats and DNA methylation. Mutations in DNMT3B, ZBTB24, CDCA7, and HELLS cause Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome. We have demonstrated that CDCA7 is a critical activator for the nucleosome remodeling by HELLS. Expression of HELLS, CDCA7 and its paralog CDCA7L are linked to various cancers. While multiple roles of HELLS, including DNA methylation, loading of macroH2A, and nonhomologous end joining (NHEJ), have been reported, it is not clear how CDCA7 contributes to these diverse processes and how they are related to immunodeficiency and cancers. We will address this question through dissecting the molecular function of CDCA7 and HELLS. 3) Mitotic regulation of cGAS. cGAS is a critical innate immunity pattern receptor targeting pathogenic DNA. cGAS binds to DNA and becomes activated to synthesize cyclic GMP-AMP (cGAMP). cGAMP activates STING, which then triggers signal transduction pathway to promote inflammation. How does cGAS avoid being activated by the host’s chromosomal DNA? We gave an answer to this question by demonstrating that the nucleosome directly binds cGAS to block its DNA-dependent activation. However, when cGAS binds to chromosomes during mitotic arrest, cGAS slowly becomes reactivated and induces apoptosis. Using Xenopus egg extracts where we can precisely control nucleosome assembly, DNA, and cell cycle stages without being confounded by downstream events, we will dissect the mechanism of mitotic suppression and reactivation of cGAS.

NIH Research Projects · FY 2025 · 2019-04

Obesity is expected to affect nearly 50% of adults in the United States by 2030 and is a major risk factor for type 2 diabetes, cardiovascular disease, and many types of cancer. Obesity is characterized by an accumulation of white adipose tissue, which can become dysfunctional in the setting of chronic overnutrition, contributing to the sequelae of excess adiposity. Mice and humans also possess thermogenic brown and beige adipocytes, which convert chemical energy into heat and have been associated with potent anti-diabetic and cardioprotective benefits. The metabolic effects of thermogenic adipocytes extend beyond energy dissipation, with these cells also serving as a sink for toxic metabolites, suppressing inflammation and fibrosis, and secreting paracrine and endocrine mediators. Murine beige fat shares significant similarities with brown fat described in adult humans, with both displaying highly cold-inducible activity and a common molecular program. Thus, beige adipocytes in mice are an attractive model system for dissecting the mechanisms underlying the broad benefits of thermogenic fat. However, fundamental questions regarding the origin, dynamics, and function of these cells remain unanswered. In studying crosstalk between the sympathetic nervous system and beige fat, we found that beige adipocytes form at postnatal day 10, independent of sympathetic innervation and cold stimulation. Starting at postnatal day 28, these cells suppress their thermogenic properties and become dormant, but can be reactivated upon cold exposure in adults. We also discovered that dormant beige adipocytes possess heretofore undescribed functional properties, protecting against tissue inflammation in obesity. Based on these findings, we propose that postnatal beige adipocytes are developmentally hard-wired cells that become thermogenically dormant in early adulthood and are reactivated to provide the major source of inducible beige adipocytes in adults, while also serving as a central regulator of adipose tissue homeostasis. We will address this hypothesis through two specific aims: (1) We will elucidate the origin and dynamics of beige adipocytes, using lineage tracing models coupled with in vitro characterization and in vivo transplantation experiments to define the committed postnatal beige fat cell progenitor and the tissue niche factors triggering these cells’ development. (2) We will dissect the function of postnatal beige adipocytes in normal physiology and obesity using mouse models with temporally regulated ablation of active or dormant postnatal beige adipocytes, along with single nuclear transcriptomic datasets to define the molecular properties of these cells. We will also employ a new approach to monitor and characterize interactions between active and dormant postnatal beige adipocytes and other adipose tissue cell types. This proposal will provide a new conceptual understanding of beige adipocytes and the first insights into the role of dormant beige adipocytes in adipose tissue and whole-body homeostasis. In addressing key knowledge gaps in the field, findings from these studies could serve as the framework for targeting postnatal beige adipocytes for the treatment of obesity, type 2 diabetes, and associated diseases.

NIH Research Projects · FY 2026 · 2019-02

PROJECT SUMMARY/ABSTRACT The long-term goal of our research is to decipher the essential molecular mechanisms underlying successful cell division. Errors in this process have been linked to developmental defects and diseases such as cancer. We now know essentially every protein needed for cell division. However, analyzing the precise molecular mechanisms needed for error-free division has continued to be very challenging for at least three reasons. First, cell division in human cells is a highly dynamic process that can be completed in <1 hour, with key steps such as chromosome-microtubule attachments and nuclear envelop reformation taking only a few minutes. Second, the microtubule-based structures that dynamically self-assemble and function in dividing cells can be ~1000- times larger than their nanometer-sized protein components. Third, this multi-step process depends on several distinct protein-protein interactions that can be transient and mitosis-specific. To address these challenges and fill gaps in our knowledge we have: (i) Discovered and characterized cell-permeable chemical inhibitors of key mechanoenzymes (e.g. AAA, ATPases associated with diverse cell processes). These chemical probes can be used to rapidly (typically, within minutes) inhibit or activate (through relief from inhibition) protein function in dividing human cells. We combine these fast perturbations with state-of-the-art microscopy (e.g. lattice light- sheet microscopy) and quantitative image analysis to dissect mechanisms underlying cell division dynamics in human cells. To identify target-specific phenotypes we carry out parallel inhibitor dose-dependent analyses in matched cell lines that are either inhibitor-sensitive or -resistant. (ii) Deciphered how micrometer-sized features (e.g. microtubule length or overlap length) can effectively be measured by nanometer-sized proteins to generate proportionate outputs (e.g. tags or force). For these studies we have generated a biochemical ‘toolbox’ comprised of recombinant forms of the augmin complex, isotypically-pure human tubulin, key microtubule- associated motor and non-motor proteins and g-TuRC (g -tubulin ring complex), the major microtubule nucleator in human cells. (iii) Developed and applied chemical proteomics approaches to ‘capture’ and profile direct, transient and context-dependent protein-protein interactions in living cells. The research proposed benefits from our expertise and will combine chemical, structural and cell biology approaches to answer long-standing questions, including: (a) What are the functions of different AAA mechanoenzymes during cell division and how are their activities regulated? (b) What is the structural basis of g-TuRC-dependent microtubule nucleation and how does this complex contribute to centrosome-dependent and -independent microtubule formation during mitosis? Our research should provide new insights into fundamental mechanisms, uncover general principles that inform on other cellular processes (e.g. microtubule organization in neurons), and may provide starting points for developing new therapeutics.

NIH Research Projects · FY 2025 · 2018-09

ABSTRACT Instead of conferring protection, IgG antibodies against dengue virus (DENV) have been correlated with increased susceptibility to symptomatic dengue disease, as patients with pre-existing antibodies against DENV experience at a higher rate the symptomatic form of dengue disease, which often includes life-threatening complications. With over 2.5 billion people being at risk of DENV infection and with >100 million new infections occurring annually, DENV represents a tremendous burden to global human public health, necessitating the development of efficacious therapeutic or vaccination approaches to control DENV infection and disease. Using in vitro cellular assays, several studies have previously suggested that anti-DENV antibodies can mediate enhanced viral infection of Fcγ receptor (FcγR)-expressing myeloid cells; a phenomenon referred to as antibody dependent enhancement (ADE). However, ADE of viral replication alone cannot account for the complex pathophysiological features of symptomatic dengue disease, as well as for the wide spectrum of clinical disease severity observed among symptomatic patients. During the previous funding period, in collaborative studies with the Institute Pasteur in Cambodia, a DENV endemic area, we demonstrated that dengue disease susceptibility, as well as disease severity are associated with the induction of specific glycoforms of IgG antibodies (afucosylated) that exhibit increased affinity for the activating FcγRIII receptor. In parallel mechanistic studies, we developed a novel in vivo model of dengue disease that expresses the full array of human FcγRs and is permissive for DENV infection. Using this model, we demonstrated that a critical step in the in vivo pathogenesis of dengue disease is the engagement of FcγRIIIa on macrophages by afucosylated IgG antibodies, which are enriched in severe dengue cases. FcγRIIIa-afucosylated IgG antibody interactions result in aberrant macrophage activation, inflammatory sequelae, significant morbidity, and mortality. These findings implicate the FcγRIIIa- afucosylated Fc axis as the basis for dengue disease susceptibility and pathogenesis. Using a unique set of biospecimens from DENV-infected patients, as well as our recently developed mouse models of ADE of dengue disease, the proposed studies aim to (i) dissect the mechanisms that drive the induction of IgG Fc afucosylated glycoforms upon DENV infection and (ii) characterize the downstream effector responses that are initiated upon engagement of FcγRIIIa by afucosylated IgG antibodies and contribute to disease pathogenesis. We anticipate that our findings will significantly advance our understanding of the pathways that regulate IgG Fc glycan heterogeneity during DENV infection and drive dengue disease pathogenesis, having a broader impact on the study of immune responses against other viral pathogens that are characterized by aberrant IgG Fc fucosylation.

NIH Research Projects · FY 2026 · 2018-08

PROJECT SUMMARY Generation of high affinity antibodies in germinal centers (GCs) is a fundamental immunological process that provides protection against infection. Antibody affinity maturation follows a prototypical Darwinian evolutionary framework, in which rare GC B cells that acquire affinity-increasing mutations in the antigen-binding portions of their immunoglobulin genes selectively undergo massive proliferation, leading to their expansion within the GC population at the expense of lower-affinity clones. Through iterative rounds of selection and expansion, GC B cells increase their affinity toward a particular pathogen or antigen, while being exported as memory or plasma cells and contributing to the affinity of serum antibody over time. GC B cells are among the fastest dividing mammalian cells and are uniquely equipped with a distinct cell cycle program that allows them to divide every 4-6 hours. At its extreme, this robust expansion program can lead to clonal bursts, in which a single B cell can take over a 2,000-cell GC in just a few days. Interestingly, GC B cells can enter S phase in the apparent absence of mitogen, as if by “inertia,” hence their rapid mode of cell division is termed “inertial cycling”. Notably, many of the key drivers of GC-derived B cell lymphoma, such as cyclin D3, are critical for triggering and sustaining strong cell cycles in GC B cells. Despite its importance in the expansion of high-affinity B cell clones and development of GC-derived lymphomas, the precise cellular dynamics and molecular pathways that underlie inertial cycling of GC B cells remain poorly understood. This gap in our understanding is primarily due to a lack of tools with the necessary spatial and temporal sensitivity to resolve GC B cell cycles in vivo. The main goal of this research proposal is to define the cellular and molecular processes that underly the unique cell cycle programs of GC B cells. To achieve this, we will characterize the spatiotemporal dynamics of inertial cycling at a single cell level with intravital two-photon microscopy and fluorescent activity sensors (Aim 1); and we will determine the principles that coordinate inertial cell cycling and mutability of B cell receptors (Aim 2). Completion of the proposed Aims will provide a comprehensive understanding of the specialized inertial mode of GC B cell cycling and will contribute new insights into the fundamental mechanistic basis of how GCs select for high affinity B cells. In addition, the findings from this proposed Aims will better our understanding of GC- derived lymphomas, where inertial cycling becomes coopted for malignant transformation.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY Alzheimer's disease (AD) is a complex, multifactorial disease that leads to profound neurodegeneration, cognitive decline, and eventually death. There is strong evidence that the Alzheimer's beta-amyloid peptide (Aβ) is an important driver of the disease. However, the mechanisms by which Aβ is toxic are not defined. There is increasing evidence that inflammation and vascular abnormalities contribute to AD pathology. One pathway that links these two processes is the plasma contact system. We have found that this system can be initiated by Aβ and is activated in AD plasma from human patients and mouse models. Furthermore, inhibition of the contact system in AD mice improves their pathology and cognition. We propose to further define how the contact system and its components, specifically high molecular weight kininogen (HK) and coagulation factor XII (FXII), contribute to AD pathophysiology. We have found that Aβ protofibrils are the most effective Aβ species to activate the contact system. Aβ protofibrils are also the main target of lecanemab, the promising new FDA-approved antibody therapy for AD patients. We will investigate how lecanemab might interfere with the contact system to generate its beneficial effects in humans and determine if both lecanemab and a contact system inhibitor could have a synergistic effect on AD pathology. We will also study the structural interaction between Aβ protofibrils, HK, and FXII to determine mechanistically how Aβ causes contact system dysfunction in AD. Finally, we will explore the potential of an anti-HK antibody as a new therapeutic avenue for AD.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY Alzheimer's disease (AD) is a multifactorial disorder with many pathogenic elements. One contributing factor is vascular dysfunction, which can be both a result of the primary AD pathogenesis and a cause of neuronal loss and subsequent cognitive impairment. The molecular mechanisms by which AD and the vascular system intersect and influence each other are still unclear. We have been working for two decades to try to better define this molecular interaction. We have shown that beta amyloid (Aβ), the peptide that is a driver of AD, interacts with fibrinogen and increases blood clot formation. These clots have an altered structure and are resistant to lysis. These abnormal clots can contribute to reduced blood flow and increased inflammation, both of which could contribute to vascular contributions to AD. We have now found that the Aβ/fibrinogen complexes can have toxic effects independent of clotting. In hippocampal slice cultures, Aβ/fibrinogen complexes are more toxic than either Aβ or fibrinogen alone. These results identify a new mechanism by which the interaction of Aβ and fibrinogen can lead to neuronal dysfunction. Mutant and longer forms of Aβ are sometimes much more toxic that the classical Aβ42 and Aβ40 species. We show that some mutant forms of Aβ that are more toxic interact much more strongly with fibrinogen. These Aβ variants provide an entrée for studying the mechanisms by which Aβ/fibrinogen complexes are toxic in AD. Therefore, our main goal is to explore the cellular and molecular mechanisms by which Aβ variants and their fibrinogen complexes negatively affect the brain.

NIH Research Projects · FY 2025 · 2018-01

Our long-term goal is to understand how glia contribute to nervous system development, function, and information processing. Glia constitute a large fraction of cells in the vertebrate nervous system and surround neuronal receptive endings to form isolated compartments. Most excitatory synapses are glia-ensheathed, as are sensory-neuron receptive endings and neuromuscular junctions. Major gaps remain in our understanding of glia. While developmental specification of some glia has been explored, programs governing astrocyte or sensory organ glia differentiation are not clear. How glia form and regulate compartments around synapses and other neuronal receptive endings is also not understood. Glia have been proposed to regulate neuronal activity, yet the effector mechanisms are not fully explored. Finally, neuron structural and functional plasticity may, in part, be under glial control, yet the details are not at hand. Thus, much remains to be learned about glial functions and their underlying molecular programs. In many animals, neurons are born in excess, and the final neuronal complement is determined in part by glial and other secreted cues controlling cell death. Glial manipulation, thus, often leads to neuronal demise. A long-standing goal has been to identify in vivo settings for studying glia-neuron interactions that bypass the neuron-survival problem. We have taken a major step towards this goal by pioneering the nematode C. elegans as a facile and relevant system for studying glia and their nervous system contributions. We showed that C. elegans possess glia, and that these ensheath sensory-neuron receptive endings, highly resembling glial structures found in vertebrate sense organs, as well as envelop the CNS, wrapping around defined synapses. Like vertebrate astrocytes, these latter glia tile, subsuming specific CNS domains, express transcription factors promoting gliogenesis in vertebrates, and express ion and neurotransmitter transporters, channels, and neurotransmitter receptors. The development of these glia bears uncanny similarities to the radial glia-to-astrocyte developmental transition in vertebrate brain development. Importantly, in C. elegans, neuron survival does not require glia, but glia manipulation results in major deficits in neuron shape and function. C. elegans therefore offers a unique in vivo arena to study glia and their effects on the nervous system. Here we aim to investigate three interrelated aspects of glia-neuron biology. (1) We will determine how astrocytic glia develop and regulate synaptic function. (2) We will determine glia guided brain assembly. (3) We will study a new cell death program resembling glia-dependent neurodegeneration. In addressing these questions, we challenge the view that only neurons underlie the phenomena under study, and posit that glia are integral regulators.

NIH Research Projects · FY 2025 · 2017-07

Project Summary Intestinal intraepithelial lymphocytes (IELs) are T cells that form one of the key branches of the mucosal immune system, providing a first line of immune defense against pathogens and possibly against epithelial cancers due to their location at the critical interface between the intestinal lumen and the core of the body. Consistently, dysregulation of IELs leads to loss of mucosal barrier integrity, susceptibility to enteric infections and inflammatory bowel diseases (IBD) and cancer. In recent years some of the mechanisms controlling the development and function of IEL populations against enteric pathogens have been elucidated, including work developed during the first funding cycle of this proposal. In addition to their role in immune surveillance against enteric infections, recent data suggest a γδ T cell-associated gene signature as the most favorable prognostic factor across cancer types, including colorectal cancer (CRC). CRC is the second most deadly cancer in the United States, affecting over 140,000 people each year, killing approximately 50,000 in the US. Up to 20% of IBD patients develop CRC, although the majority of CRCs develop in patients without underlying inflammation. In both the common forms of CRC and IBD-induced CRC tumor-elicited inflammation triggers EC damage resulting in microbial invasion, which sustains inflammation that in turn drives cancer progression. Therefore, IEL surveillance of the mucosal barrier may play dual roles in CRC: (i) prevention of CRC progression and early dissemination by immune cell-mediated killing or additional anti-tumor responses; (ii) promotion of CRC progression and metastasis through inflammatory cytokines or immune-regulatory molecules. Based on existing literature in murine and human CRC, our recent work, and extensive preliminary data presented here, we hypothesize that γδ IEL epithelial surveillance is crucial for the regulation of tumor formation. We show that at steady state, the majority of intestinal γδ IELs express Vγ7 or Vγ1 TCRs and IEL hallmarks including a cytotoxic machinery. However, in both colitis-associated (AOM+DSS) and mutation-associated (CDX2-APC) models, CRC progression was associated with relative reduction of Vγ7 or Vγ1+ and accumulation of γδ IELs expressing Vγ6 or Vγ4 TCRs, which produce IL-17 and express PD-1. In Aim 1, we will address whether tissue-resident Vγ7+ or Vγ1+ γδ IEL subsets play a role in immune surveillance of the epithelium, preventing tumor formation. In Aim 2, we will characterize γδ IEL subsets that accumulate during CRC progression and may facilitate tumor growth. Studies proposed here will characterize γδ IEL behavior during early and late stages of CRC development using a combination of innovative imaging approaches. We will also track interacting ECs and surrounding IELs during CRC using a novel mouse model to identify cellular partners and single cell transcriptomics. Inducible intersectional genetics will be used to target differentiation or function of γδ IELs, while γδ IEL subsets enriched in different stages of CRC will be targeted using novel strains lacking specific V-gamma; these murine lines will be subjected to gnotobiotic and infection models using complementary CRC approaches.

NIH Research Projects · FY 2025 · 2017-05

Project Summary: Bacterial natural products have a long history of serving as lead structures for the development of new therapeutically relevant small molecules. Despite the tremendous early success of bacterial natural product discovery programs, both academic and industrial drug screening efforts have largely deprioritized natural products in recent years due to unacceptably high rediscovery rates, which were often taken as an indication that nature had few novel small molecules left for us to identify. Extensive sequencing of both bacterial genomic and metagenomic DNA indicates that this is, in fact, not the case. Instead, it appears that we have just begun to scratch the surface of the structural and functional diversity of small molecules encoded by the global microbiome. In most environments uncultured bacteria still significantly outnumber their cultured counterparts, and even when it is possible to grow bacteria in the laboratory they usually only express a small subset of their natural product biosynthetic gene clusters. My research group is focused on the development of culture-independent (metagenomic) approaches for identifying novel natural products encoded provides a means of accessing this large fraction of previously inaccessible biosynthetic diversity. In these studies, the need to culture bacteria is circumvented by extracting DNA directly from environmental samples and cloning it into easily cultured bacteria. This proposal is designed to continue our development of culture-independent methods for accessing the natural products encoded in soil metagenomes and to increasingly apply these methods to the discovery and characterization of new bioactive small molecules. In particular, our studies will include the development of new sequence-based and functional metagenomic screening methods as well as the use of heterologous expression and synthetic bioinformatic natural product discovery approaches to generate novel small molecules from soil metagenome derived biosynthetic gene clusters. Method develop studies will be focus on overcoming key technical hurdles to enable the more efficient and higher throughput discovery of structurally and mechanistically different natural products from metagenomes using both bioinformatic and unbiased functional screening approaches. As with traditional natural product discovery programs, our ultimate goal is to identify bioactive small molecules that have the potential to be used as lead structures for the development of novel therapeutics. Although our metagenomic methods can be applied to the identification of compounds that can target almost any disease, we will primarily focus on the discovery of new antibiotics. This choice is driven by the long history of natural products being the most productive source of potent antibiotics with unique modes of actions and the clear biomedical need for new antibiotics that are effective against clinically relevant, multi-drug resistant, Gram-positive and Gram-negative pathogens.

NIH Research Projects · FY 2026 · 2017-01

PROJECT SUMMARY Generating an appropriate antibody response is critical for protection against reinfection and for the effectiveness of vaccination, particularly in the context of viral diseases. In addition to well-studied quantitative parameters such as antibody titer and affinity, other, more qualitative parameters related to the clonal composition of the response also play critical roles in antibody-mediated protection. These include antigen and epitope specificity, which is key to viral neutralization capacity and antibody breadth, and overall clonal diversity, which strongly influences the degree of immunodominance and therefore the ability of viruses to escape immunity by mutation. Despite their importance, such “ecological” aspects of GC biology remain poorly understood and systematically understudied at the mechanistic level. Our long-term goal is to develop a mechanistic understanding of how the competitive waxing and waning of B cell clones at the various stages of the immune response shapes the ultimate composition, specificity, and protective efficacy of serum antibody. In our previous studies, using multicolor “Brainbow”-based B cell fate- mapping models, we focused on the germinal center (GC) and memory phases of the response, revealing how highly diverse early responders are funneled towards oligoclonality, first progressively by GC selection (including in chronic gut-associated GC) and then dramatically by secondary boosting. We now propose to extend our work using these same tools to investigate the clonal dynamics of prolonged selection in long-lived virus-induced GCs (Aim 1) and of the progressive differentiation of plasmablasts and plasma cells from GC precursors (Aim 2). We also propose a new “molecular fate-mapping” system to determine how clonal dynamics impact the ultimate composition of serum antibody (Aim 3). This allows us to investigate the B cell biology of serum-level phenomena such as antigenic imprinting/original antigenic sin, immunodominance, and viral escape. We expect our findings will provide greater mechanistic understanding of how the composition and protective effectiveness of serum antibody is determined by the dynamics of B cell clonal competition, with implications for the design of effective vaccination strategies for influenza, HIV, and SARS-CoV-2.

NIH Research Projects · FY 2026 · 2016-11

Project Summary Chronic mucocutaneous candidiasis (CMC) is characterized by lesions of the nails, skin, and oral and genital mucosae by the fungus Candida albicans. Autosomal recessive (AR) IL-17RA, IL-17RC, and ACT1 deficiencies, and autosomal dominant (AD) IL-17F deficiency underlie ‘isolated CMC’, while AR CARD9, ROR-g/gT, ZNF341, IL-12p40, and IL-12Rβ1 deficiencies, AD STAT3, IL6ST/GP130, and JNK1 deficiencies, and AD STAT1 gain-of- function (GOF) underlie ‘syndromic CMC’. Cells with IL-17RA, IL-17RC, ACT1, or JNK1 deficiency respond poorly to IL-17A and IL-17F. Cells with IL-17RA or ACT1 deficiency also respond poorly to IL-17E (IL-25). Patients with ROR-g/gT, ZNF341, STAT3, IL6ST, or JNK1 deficiency, or STAT1 GOF, display low proportions of IL-17A/IL-17F (IL-17A/F)-producing T cells. Since 2008, we have made major contributions to these discoveries that causally connected inborn errors of IL-17 immunity with CMC. Three outstanding enigmas are (i) the mechanisms by which STAT1 mutations can be GOF and by which they impair the development of IL-17 T cells, (ii) the mechanisms by which inborn errors of STAT3 that impair IL-17 production underlie CMC, while inborn errors of STAT3-activating cytokines IL-6, IL-21, and IL-23 apparently do not, and (iii) the genetic etiology of about half of the patients with isolated or syndromic CMC. We first intend to test the hypotheses that (a) STAT1 GOF mutations impair the dephosphorylation of nuclear STAT1 by disrupting the formation of antiparallel dimers, thereby preventing the accessibility of specific phosphatases, and that (b) excessive responses of T cells to IFN-a, IFN-g, and IL-27 collectively impair the development of Th17 cells. We then intend to analyze the production of IL-17 cytokines by leukocytes from patients with IL-6R, IL-21R, or IL-23R deficiency, in comparison with known etiologies of CMC, testing the hypothesis that the isolated disruption of IL-6, IL-21, or IL-23 only weakly impairs the development of Th17 and related IL-17-producing lymphocytes. We finally intend to discover novel CMC-causing genes using genome-wide (GW) approaches, based on GW linkage (GWL), and whole-exome or whole-genome sequencing (WES/WGS). On the three fronts, we have exciting preliminary results. We found (i) that all STAT1 variants tested are GOF due to impaired dephosphorylation by the tyrosine phosphatases TC-PTP and PTP1B, by disruption of antiparallel dimers formation; and the concomitant addition of STAT1-dependent cytokines IFN-a/b, IFN-g, and IL-27 inhibits the development of IL-17 T cells from naïve CD4+ T cells with STAT1 GOF; (ii) patients with AR IL- 6R or IL-23R deficiency and CMC, implying incomplete penetrance; and (iii) patients with syndromic CMC and mutations of cRel, RelB, MAP3K6 (also known as ASK2), ZNF375 (zDHHC5), or UBASH3B (TULA-2). From a biological standpoint, this research will provide new insights into the mechanisms of mucocutaneous immunity to fungi, while dissecting the molecular and cellular control of human IL-17. From a clinical angle, this work will provide new molecular diagnoses for patients and genetic counseling for families, while paving the way for new cytokine-based approaches in patients with CMC.

NIH Research Projects · FY 2025 · 2016-09

Project Summary This project focuses on the role of telomeres and DSB repair in genome instability in cancer. Numerous recent WGS studies have revealed that most cancer genomes carry a remarkable level of structural changes, affirming the need to understand how this genome instability arises. In this context, our work asks how telomeres affect tumorigenesis with emphasis on the two major contributions of telomeres in cancer: the telomere tumor suppressor pathway and telomere-driven genome instability. During the current funding period, we have provided genetic evidence for the telomere tumor suppressor pathway and showed that the correct telomere length setting at birth prevents cancer in a wide range of tissues. We have dissected the mechanism by which telomere crisis, a stage at which telomere shortening drives genome instability in checkpoint-deficient cancer clones, instigates breakage-fusion-bridge (BFB) cycles, chromothripsis, and kataegis. We have provided the first evidence that telomerase can create new telomeres (neotelomeres) at DSBs and propose that neotelomere formation can mold the cancer genome by increasing the fitness of cells struggling with ongoing BFB cycles. Finally, our lab continued its work on the role of 53BP1 in DSB repair and PARPi treatment of BRCA1-deficient cells, showing that, unlike what was generally believed, 53BP1 does not block resection but recruits the CST-Pola/primase complex to fill-in resected DNA ends. These findings set the stage for our future work, in which we aim to continue our path-breaking research and the mentoring of future cancer researchers. Examples of projects we will pursue are: 1. Using an innovative approach, we will use CRISPRi screens for repressors of neotelomere formation and query hits for gene loss/mutation in cancer. 2. Our proposal that neotelomere formation can terminate BFB cycles and enhance the viability of cells with dicentric chromosomes will be tested in an in vitro model for induction of BFB cycles. 3. To gain deeper insights into the telomere tumor suppressor pathway, we will determine how telomere length is regulated. 4. Following a recent demonstration that cancer cell lines with short telomeres are exceptionally sensitive to loss of the telomeric factors CST and TRF1, we will determine the mechanistic basis of these vulnerabilities in hopes that our insights may point to new treatments. Our aim is to derive deep insights into how cancer genomes are altered with the overarching goal of providing oncologists with information that can inform their decisions on diagnosis, treatment, and prevention. 1

NIH Research Projects · FY 2025 · 2016-07

The overall vision of the Rockefeller University Center for Clinical and Translational Science (CCTS), supported by the CTSA program, is to develop, demonstrate, and disseminate innovative programs so as to create a model clinical research enterprise built on rigor, responsible and transparent reporting, and scientific integrity to empower translation of paradigm-breaking discoveries into better human health. The overarching goal of our KL2 Clinical Scholars Master's degree program is to prepare our trainees to be successful, scientifically independent, translational investigators who can lead translational teams on projects to improve human health. The core of the program is a mentored translational research experience in which the Scholar serves as a translational team science leader, and develops, conducts, analyzes, and disseminates the results of a human participant study under the guidance of a distinguished senior scientific mentor and with the assistance of a cadre of Translational Science Team Experts/Educators. This experience is complemented by a didactic curriculum including tutorials in Clinical and Translational Science, Biostatistics, Bioinformatics, and Epidemiology; a weekly Clinical Research Seminar by outstanding translational investigators; a private weekly meeting with the seminar speaker; team science leadership training; a graduate level scientific course; Humanities and Translational Science special events; and training in the Responsible Conduct of Research. Our program benefits from the scientific strength of the mentors in the program, with 10 of the mentors of Scholars during the current grant period being elected members of the National Academy of Sciences. Thus, our specific aims are: 1. To train physician scientists and doctoral level health professionals as outstanding, scientifically independent translational investigators in a 3 year mentored KL2 Clinical Scholars Master's degree program that includes: a) leadership of a human participant protocol from conception to reporting, b) a didactic curriculum to help the Scholar master the competencies to function as a rigorous independent translational scientist, c) focused instruction and feedback to develop the team science leadership competencies required to successfully lead a translational science team. 2. To introduce new educational programs to maximize Scholars' educational experience and ability to consider a wide variety of career choices, as well as benefit from emerging new technology and resources: a) Team science leadership training, b) Pharmaceutical production and formulation, d) Ontology-driven human phenotyping, e) Searching Electronics Health Records and causal inference, e) From discovery to health-enhancing product, and f) Engaging communities as full partners. 3. Reinforce the importance of achieving the highest possible levels of rigor, reproducibility, and reporting (R3) and assist Scholars in developing best practices early in their research careers so that they can incorporate new methods and policies to constantly improve their adherence to evolving standards.

NIH Research Projects · FY 2025 · 2016-07

The overall vision of the Rockefeller University Center for Clinical and Translational Science (CCTS), supported by the CTSA program, is to develop, demonstrate, and disseminate innovative programs so as to create a model clinical research enterprise built on rigor, responsible and transparent reporting, and scientific integrity to empower translation of paradigm-breaking discoveries into better human health. Our new initiatives are designed to: 1) ensure that all of our studies set the highest standards; 2) ensure that our trainees have the team science leadership skills needed for a career in translational science; and 3) enhance scientific rigor, reproducibility, and reporting. These new areas build on the infrastructure we have developed to integrate mechanistic science into community engagement, reach out to basic scientists, speed the development of health- enhancing products, and create an optimal training environment. Our specific aims are: 1. To provide a robust infrastructure to support the conduct of clinical investigation at the highest levels of participant safety, scientific rigor, bioethics, and regulatory compliance. We will enhance our multidisciplinary Translational Research Navigation (TRN) Program and our collaboration with Clinical Directors Network (CDN), a Practice Based Research Network of Community Health Centers, to ensure that our studies meet the highest standards of design, feasibility, method validity, and transparent reporting, supporting our active participation in the CTSA Trial Innovation Network. 2. To ensure that every discovery at Rockefeller has the best chance of improving human health. We will enhance our robust pipeline of programs to support basic and clinical investigators move their paradigm-breaking discoveries into new therapeutics by leveraging the resources of the Tri-Institutional Therapeutic Development Institute, the Robertson Therapeutic Development Fund, and the University affiliated biotechnology company, Bridge Medicines .3. To educate the entire research workforce team in conducting translational research at the highest levels of participant safety, scientific rigor, bioethics, and regulatory compliance. We will further enhance the multidisciplinary team science emphasis of our educational programs for KL2 Clinical Scholars, Clinical Research Nurses, basic scientists, and community clinicians, with a focus on our new Team Science Leadership training with the CTSA Hubs at Yale, University of Pennsylvania, and Columbia. 4. To study and improve the clinical research enterprise both at Rockefeller and nationally. We will continue to use the CCTS as a laboratory for investigating the clinical research enterprise itself and methods to improve it, including obtaining outcome data from research participants, enhancing rigor, reproducibility and reporting, speeding the evaluation of novel therapeutics via in-house formulation, and creating 'super teams' of CTSA Hubs to disseminate our model of community engagement harnessing basic mechanistic studies to address community-identified health needs. We will disseminate the results throughout the CTSA consortium and scientific community by publications, presentations, and active participation in CLIC and CD2H initiatives.

NIH Research Projects · FY 2025 · 2016-05

Project Summary Transcription is the major control point of gene expression and RNA polymerase (RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. The basic elements of the transcription cycle, initiation, elongation, and termination, were elucidated through study of prokaryotes. A detailed structural and functional understanding of the entire transcription cycle is essential to explain the fundamental control of gene expression and to target RNAP with small-molecule antibiotics. Moreover, a complete understanding of how a complex, molecular machine uses binding and chemical energy to effect conformational changes that drive the cycle, and how regulators modulate the cycle, is of fundamental interest. The transcription cycle is marked by a series of stable complexes (core è holo è RPo è EC) that interconvert through transient intermediates. The transitions between stable states are points of heavy regulation that are poorly understood due to the lack of structural information. Major transitions include: Holoenzyme + promoter DNA è open promoter complex (initiation) Open promoter complex è elongation complex (promoter escape, s dissociation) Elongation complex è core RNAP + DNA + completed RNA transcript (termination) Each of these transitions is characterized by unstable, transient intermediates that are extremely challenging for structural biology. Cryo-electron microscopy (cryo-EM) has emerged as a powerful method to visualize these transient states. We are combining cryo-EM with other approaches to mechanistically and structurally characterize transient intermediates that govern transitions in the bacterial transcription cycle, including promoter melting, the initiation to elongation transition, and transcription termination. These findings will provide insight into the behavior of macromolecular machines throughout biology.

NIH Research Projects · FY 2025 · 2016-04

ABSTRACT Our studies over the past two decades have focused on clarifying the mechanisms by which anti-tumor immunotherapies elicit their therapeutic effects. As a result of our studies, the importance of Fc-FcγR mediated effector pathways for the elimination of tumors has been elucidated, resulting in the optimization of these interactions in second-generation anti-tumor immunotherapeutics with improved clinical activity. One of the therapies developed as part of our previously funded NCI studies is now being tested across three clinical trials with early evidence of promising activity. While strategies improving antibody-based therapeutics through Fc engineering have resulted in more effective anti-tumor antibodies (Abs) with significantly improved survival, the long-term goal of immunotherapy is to develop therapeutic strategies that will elicit memory responses and effectively eliminate recurrences, resulting in improvements in overall survival. This current proposal aims to mechanistically investigate general strategies to accomplish this goal by focusing on 1) inducing tumor vaccination using anti-tumor monoclonal Abs (mAbs), 2) define the mechanisms by which agonistic and antagonistic immunomodulatory mAbs enhance anti-tumor vaccination, and 3) explore how the tumor microenvironment may be manipulated to improve these immunotherapeutic strategies. Our preliminary results have indicated that anti-tumor Abs can elicit long-term cellular memory responses when appropriate Fc-FcγR interactions are integrated into these Abs. Manipulating both the cellular effector responses and the tumor microenvironment through the use of Fc-optimized immunomodulatory Abs can further augment these pathways and result in long-term memory responses.

NIH Research Projects · FY 2025 · 2016-02

Project Summary The yellow fever 17D live-attenuated vaccine has been administered to millions of people and is a highly successful and safe vaccine providing essentially life-long protection. However, the mechanisms of attenuation are poorly understood, and rare individuals suffer from sometimes fatal severe adverse events (SAEs) including yellow fever-like viscerotropic disease, or neurologic disease. Epidemics of this mosquito-borne disease continue to occur in tropical and sub-tropical regions of South America and Africa, so understanding the mechanisms of 17D attenuation and the factors predisposing to SAEs is critical for development of safer vaccines and SAE prevention or treatment strategies. Work of others and of ours in the prior funding period determined that type I interferon (IFN) responses are greater after infection of cells with 17D compared to the virulent Asibi parental strain. New preliminary data demonstrate that the prophylactic IFN-induced antiviral state is also more effective at controlling 17D. Importantly, our work uncovered mutations in genes encoding type I IFN receptor chains (IFNAR1 and IFNAR2) and autoantibodies against type I IFNs as factors causing SAEs after 17D vaccination, thus linking the observed IFN phenotype with 17D attenuation in vivo. We also discovered a differential requirement between 17D and Asibi, mediated by the viral NS4B protein, for the host factor TMEM41B, with increased innate immune responses to 17D in TMEM41B KO cells. Prior studies have been hampered by the disparate specific infectivities of 17D and Asibi, precluding meaningful comparisons in bulk cell-based assays. New preliminary data demonstrates that 17D and Asibi harboring the 17D E protein (Asibi+17D E) have similar specific infectivities, replicate with similar kinetics in the HepG2 hepatocyte cell line, and maintain previously described differences in IFN transcription and secretion. Unexpectedly, levels of phosphorylated STAT1 are similar in 17D- and Asibi+17D E-infected cells. In this project continuation, in Aim 1 17D and Asibi+17D E will be used to examine pattern recognition receptor activation, including kinase activation and downstream transcriptional responses using phosphoproteomics and RNASeq. The mechanism by which Asibi+17D E blocks IFN secretion, the process by which STAT1 phosphorylation occurs and the consequences to infection and role of individual ISGs in controlling 17D and Asibi infection will be elucidated. In Aim 2 interactions of Asibi and 17D with both pro- and anti-viral host factors will be characterized through imaging-based methods allowing single cell resolution. We will examine responses in the infected cell and the influence of individual genes (at genome scale) on innate immunity and infection. Patient-specific mutations associated with SAEs will be functionally interrogated as we have done previously. In Aim 3 the role of NS4B in differential TMEM41B requirements will be elucidated using proteomic and mutational approaches. These studies will uncover critical host-pathogen interactions that can be exploited to develop safer vaccines, SAE treatment or prevention approaches, and new drug targets. The information gained can also be broadly applied to vaccine development for other flaviviruses.

NIH Research Projects · FY 2024 · 2015-09

Project Summary (Overall) The chromatin landscape impacts fundamental cellular processes including gene expression, DNA damage repair, and cell fate and differentiation, all of which are extensively dysregulated in cancer. The collective number of oncogenic mutations in chromatin regulators has led to the emerging view of driver mutations underlying cancer epigenomes. In keeping with theme, over the past several years our current collaborative program members have been critical to the discovery and characterization of ‘classical’ oncohistone mutations in the histone H3.3 N-terminal tail. These mutations globally alter chromatin by inhibiting the activity of chromatin modifying enzymes. While much progress has been made to understand these effects, important questions remain including the nature of the dysregulation of chromatin modifying enzymes by these mutations and how these mutations lead to cancer. In addition, Program members have recently identified an expanded number of cancer-associated somatic histone mutations that occur in as many as 4 % of human cancers and involve both globular and tail domains of all four core histones. These findings generate additional important questions such as if the newly observed histone mutations have functional effects on chromatin and through what mechanisms they rely on. Given that some of the most prevalent mutations are in the globular domains of histones, we hypothesize that these mutations affect nucleosome structure and/or integrity. Lastly, we and others have also identified a novel function of the EZHIP protein, which is overexpressed in posterior fossa A ependymomas, and acts as an oncohistone mimic to directly inhibit the Polycomb Repressive Complex 2 (PRC2) function. The single goal of our Program is to illuminate the molecular mechanisms underlying classical and novel “oncohistone” mutations and oncohistone mimics to advance the diagnosis and exploration of therapeutic avenues for the cancers. Specifically, we will: i) develop and employ novel patient sample-, cell culture- and animal model-based systems to recapitulate oncohistone-associated cancers and investigate the underlying pathogenic mechanisms; ii) evaluate the activity of a comprehensive set of novel cancer-associated histone mutations using a multidisciplinary approach that includes genetics (barcoded oncohistone libraries, mouse models, barcoded-cell lines), epigenetics (ChIP-seq, ATAC-seq, DNA-methylation profiling), transcriptomics (RNA-seq), and chemical biology (“designer chromatin”, small molecule inhibitors); iii) define the mechanisms by which oncohistones, and oncohistone-mimics, dysregulate the Polycomb Repressive Complexes (PRC1 and PRC2) activity to promote gliomas and bone tumors; and iv) identify which of novel histone mutations perturb chromatin states (and by what mechanisms) to subsequently cause cellular phenotype using newly developed high-throughput biochemical and yeast genetic screening tools. These studies will inform future work towards the development of therapeutic strategies designed to ameliorate the pathogenic effects of histone mutations and oncohistone mimics in cancer.