Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2026 · 2020-08

Our overarching goals are: i. to define the mechanism(s) of ATM activation by the Mre11 complex; ii. to define the role(s) of the Mre11 complex in DNA replication; iii. To define the role(s) of the Mre11 complex and RTEL1 in response to DNA replication stress. As noted, our focus includes RTEL1 which acts to mitigate replication stress at the telomere. A combination of yeast and mouse genetics, biochemistry, and structural biology are employed to address these issues. Our previous work on these topics led to substantial scientific progress and insight regarding processes relevant to human health. Specific areas of inquiry are: · Genetic screens in yeast and mining of cancer genomic data revealed separation of function Rad50 mutations that affect ATM dependent DNA damage signaling without affecting the DNA repair functions of the Mre11 complex. These mutations constitute a resource for deciphering the mechanism by which the Mre11 complex activates the ATM kinase to initiate DNA damage signaling. · We have obtained a cryoEM structure of the S. cerevisiae MR complex. Ongoing structural analyses of rad50S mutants offer a unique opportunity to address the mechanism of Tel1/ATM activation by the Mre11 complex. · We have identified factors that function at the DNA replication fork in a manner that depends on the Mre11 complex. Those data reveal a link between innate immune signaling and genome integrity. An important focus of our laboratory is to understand the functional role(s) of those factors. Defects in factors that promote accurate DNA replication are highly correlated with human disease, and so understanding this fundamental process is an important priority in our work. · We developed a mammary organoid system examine the role of the Mre11 complex in suppressing breast cancer. Those studies provided compelling evidence that genomic instability activates a chronic interferon stimulated gene transcriptional program is driven by a nuclear innate immune DNA sensor. The attendant chromatin alterations dramatically increase the risk of oncogene induce cancer. This forges a novel link between innate immune signaling and tumorigenesis. · Whereas the Mre11 complex functions at the replication fork, RTEL1 is a helicase that promotes accurate replication of telomeric DNA. We have discovered that RTEL1 influences the abundance and disposition of a long non coding RNA, called TERRA, that is transcribed from the subtelomeric regions of all eukaryotes. We also have preliminary data supporting the view that RTEL1 is a ubiquitin E3 ligase. Our goal in this aspect of our work is to understand the role of RTEL1 in maintaining telomere stability and mitigating telomeric DNA replication stress.

NIH Research Projects · FY 2024 · 2020-07

SUMMARY Genes encoding RNA splicing factors are the most common class of mutations in patients with myelodysplastic syndromes (MDS) and are also common across all other forms of myeloid malignancies. These leukemia- associated “spliceosomal mutations” primarily occur in four genes: SF3B1, SRSF2, U2AF1, and ZRSR2. In three of these four genes (SF3B1, SRSF2, and U2AF1), the mutations occur at specific amino acid residues in a heterozygous manner (so-called “mutational hotspots”) and cause gain/alteration of function. In contrast, mutations in ZRSR2 occur throughout the open reading frame and appear to confer loss of function. Moreover, ZRSR2's normal function makes it unique amongst the commonly mutated RNA splicing factors in leukemias: ZRSR2 is the only frequently mutated factor that primarily functions in the recognition of a rare class of introns known as “minor introns.” Thus, ZRSR2 mutations are significantly enriched in leukemia and exhibit a unique genetic spectrum and function amongst recurrent spliceosomal mutations, yet they are comparatively poorly studied and understood compared to mutations in SF3B1, SRSF2, and U2AF1. Here, we propose to determine the mechanistic, functional, and therapeutic consequences of ZRSR2 mutations in leukemia. Our interdisciplinary team consists of a physician-scientist with expertise in leukemia biology and patient care (Abdel-Wahab) and a basic scientist with expertise in RNA splicing and functional genomics (Bradley). As minor introns are far more conserved than are most other introns, we hypothesize that a cross-species comparisons of the effects of ZRSR2 loss will be particularly useful for understanding how molecular alterations in splicing drive malignant transformation. In addition, we hypothesize that aberrant splicing induced by ZRSR2 loss will enable novel therapeutic approaches. In preliminary experiments, we generated a Zrsr2 conditional knockout (cKO) mouse, assembled a relevant patient cohort, characterized the transcriptomes of our Zrsr2 cKO mouse and ZRSR2-mutant MDS, and performed a functional genomic screen to model and prioritize ZRSR2-regulated splicing events. These studies revealed that ZRSR2 mutations cause mis-splicing of a compact set of genes, that Zrsr2 loss promotes aberrant and increased hematopoietic stem cell self-renewal, that simultaneous ZRSR2 and TET2 collaborate to drive malignancy, and that mis-splicing of specific downstream targets of ZRSR2 promotes clonality. We propose to build on these preliminary studies as follows: Aim 1, Determine how ZRSR2 mutations dysregulate the transcriptome and proteome in leukemia; Aim 2, Determine how disruption of ZRSR2-regulated splicing events drives clonal advantage; Aim 3, Identify the functional basis for the frequent co-occurrence of ZRSR2 and TET2 mutations in leukemia. The significance of these studies is that they will elucidate mechanistic and functional connections between ZRSR2 mutations, RNA mis-splicing, and the initiation of myeloid neoplasms. The health relatedness is that the proposed work may reveal new therapies for MDS and leukemia that specifically kill ZRSR2-mutant cells.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY / ABSTRACT Lung cancer is the leading cause of cancer-related mortality nationwide. In patients with metastatic non-small cell lung cancer, recent randomized trials have demonstrated superior efficacy of combined chemotherapy and T cell checkpoint blockade over conventional treatment. These studies have made combination chemotherapy and T cell checkpoint blockade the new standard of care for patients with lung cancer. However, the mechanisms contributing to the combinatorial efficacy of chemo-immunotherapy remain unknown. Therefore, drug combinations are determined based on historical regimens for lung cancer rather than scientific rationale. Our laboratory has a strong interest in understanding the mechanisms underlying treatment responses as a means of discovering new cancer treatments. The proposal described here builds on our previous work, which identified a combination of targeted therapies that potently induces cellular senescence. In addition to demonstrating durable growth arrest, these senescent cells secrete an array of cytokines that facilitate immune surveillance and tumor cell clearance. Interestingly, our preliminary data suggest that a similar senescent state can be induced by the standard chemotherapy for lung cancer. We hypothesize that this senescence may contribute to the clinically observed combinatorial efficacy between chemotherapy and T cell checkpoint blockade. We propose to characterize chemotherapy-induced senescence, with a particular emphasis on secreted immunomodulatory cytokines. We will leverage orthogonal in vitro and in vivo systems to explore the relevance of senescence to adaptive immunosurveillance, with the goal of determining whether senescence indeed contributes to cytotoxicity in the context of T cell checkpoint inhibition. In addition, we will interrogate tumor specimens from patients treated with chemotherapy, immune checkpoint blockade, or the combination to document the relevance of chemotherapy-induced senescence in patients with NSCLC. The data generated by this proposal will have direct relevance to the current state of NSCLC treatment, and they will facilitate the development of additional, potentially novel, drug combinations. These studies will be led by Dr. Matthew Bott, a junior faculty member on the thoracic surgical service at Memorial Sloan Kettering Cancer Center (MSK) with an interest in lung cancer and immunotherapy. The research will be carried out under the combined mentorship of Dr. Scott Lowe, an international leader in cancer biology, and Dr. Jedd Wolchok, a highly accomplished expert in translational immuno-oncology. MSK offers an outstanding environment for a career in basic and translational research. To achieve his goal of becoming an independent researcher, Dr. Bott has developed a structured curriculum of activities aimed at broadening his knowledge base, expanding his technical skills, and sharpening his methods for scientific inquiry.

NIH Research Projects · FY 2025 · 2020-07

Metastatic prostate cancer is the second largest cause of cancer-related death in American men. A major obstacle to their treatment and to developing new drugs for this disease is the lack of reliable quantitative treatment monitoring methods. Fluid-based tumor monitoring using circulating tumor cells (CTC) has recently proven valuable for prognosis and predicting response to treatment. Current imaging methods for treatment monitoring in prostate cancer involve lesion-counting of new bone metastases or selected index lesions of soft tissue disease; both focus on progression only and cannot assess the full disease burden. This proposal deploys two validated methods, automated bone scan index (aBSI) and lymph node segmentation, to quantify standard imaging results so that a measure of total disease burden on imaging can be integrated with CTC data in a predictive model. The hypothesis that this model will outperform current methods for monitoring response to therapy will be tested via these specific aims: 1) Correlate quantitative post-treatment changes in tumor burden as assessed by imaging and CTC’s; 2) Determine whether a combination of fluid-based tumor monitoring (liquid biopsy) and an imaging assay is more powerful than either assay alone in prognosticating for survival; 3) Identify the optimal combination of liquid biopsy and imaging for different therapies. Models will be built to predict overall survival using imaging, CTC, and prognostic factor data from phase III clinical trials of first-line treatments, docetaxel, docetaxel + radium-223, and abiraterone + prednisone, for an advanced stage of disease, metastatic castration- resistant prostate cancer (mCRPC). Clinical trials including this data are available for analysis due to the recommendations for serial imaging (CT and bone scintigraphy) and CTC assessment as endpoints in the Prostate Cancer Working Group (PCWG) 3 guidelines, co-authored by the study team. The results of this study will create the first reliable, multiparametric, and fully quantitative biomarker that captures clinically meaningful indices of both response and progression in mCRPC; generate evidence to support its inclusion in future PCWG guidelines; and develop methods applicable to new imaging modalities.

NIH Research Projects · FY 2025 · 2020-05

PROJECT SUMMARY/ABSTRACT Aberrant accumulation of senescent cells is a major contributor to age-dependent tissue degeneration and its associated pathologies, including fibrosis, neurodegenerative diseases, atherosclerosis, arthritis, and metabolic disorder. Cellular senescence, a stress response program, is characterized by stable cell cycle arrest and a proinflammatory secretory program that mediates tissue damage and regeneration. Senescence has beneficial effects in tumor suppression and wound healing; however, in aging, senescent cell accumulation contributes to tissue decline, and, if not cleared, eventually causes organ dysfunction. Indeed, in model organisms, ‘senolytic’ approaches, i.e. those that ablate senescent cells, ameliorate these disorders and lengthen healthspan. Our goal is to develop senolytics suitable for clinical development to treat age-associated disorders. Rather than pursuing small-molecule senolytics, which to date have had considerable toxicities, we focus on chimeric antigen receptor T (CAR T) cells capable of targeting cell-surface proteins highly expressed on senescent cells. This renewal proposal builds upon our prior success with senolytic CAR T cells that target a senescence-associated protein called uPAR. These uPAR CAR T cells efficiently ablate senescent cells and improve fibrosis and metabolic dysfunction in old mice. We propose further preclinical development of uPAR CAR T cells. This will include testing for safety and for efficacy in multiple disease contexts, including organ function in old mice, as well as extension of these studies to our recently developed human uPAR CAR T cells. We also propose to develop novel senolytic CAR T cells, first identifying candidate targets by comprehensively characterizing cell- surface proteins that are selectively expressed on senescent cells in multiple contexts of age-associated diseases. This work will have the added benefit of yielding novel markers for the challenging tasks of identifying senescent cells in vivo and delineating their roles in disease. For the top candidate targets, we will develop new senolytic CAR T cells. Finally, to improve the specificity and efficacy of the senolytic CAR T cells, we will apply recent technical advances, namely AND-gated CARs and engineered CD3 signaling domains, to develop second-generation senolytic CAR T cells. Our team combines the extensive expertise of the Lowe laboratory in senescence mechanisms and biology with the expertise of the Sadelain Laboratory in CAR T cell innovations. The feasibility of the proposed work is supported by strong preliminary data. We expect our studies to better define senescent states, generate new insights into senescence biology, and produce the next generation of cell-based therapies for treating pathologies associated with senescence and aging.

NIH Research Projects · FY 2026 · 2020-03

PROJECT ABSTRACT The central theme of this proposal is that a personalized de-escalated chemoradiation (CRT) will maintain efficacy while reducing toxicity versus the standard one-size-fits-all CRT to 70Gy . We completed 4 sequential de-escalation trials for early-stage (T0-2/N1-2c) HPV+ OPC where a hypoxia-specific biomarker, 18F- fluoromisonidazole (FMISO) imaged by positron emission tomography (PET), directed radiation to either 30Gy (FMISO negative scan)[“low-risk”] or to the standard 70Gy (FMISO positive scan). The excellent results led to a phase III FDA approved registration trial for early-stage HPV+ OPC randomizing patients to receive either FMISO PET-directed CRT or standard 70Gy CRT. The results of this trial will provide data for an application for FDA approval of FMISO PET as an imaging biomarker to guide RT for this disease. Given the success in early- stage patients, we postulate that FMISO PET-guided CRT can also personalize treatment for advanced-stage (T3-4/N3/M0) patients. If patients respond to induction chemotherapy with tumors down staged to <T2 & <N3 disease and they have a negative week 2 FMISO PET during CRT, de-escalation to 30Gy can occur without tumor control compromise. Indeed, a completed pilot study demonstrates its feasibility and provides the rationale to initiate a larger phase II validation trial. [Aim 1]. Although FMISO PET has enabled CRT de-escalation, not all centers have access to it. Intra-treatment tumor & microenvironmental changes that occur in response to CRT can be detected through longitudinal imaging with more widely accessible techniques. Therefore, we hypothesize that quantitative imaging biomarkers (QIBs) derived from dynamic contrast-enhanced & diffusion- weighted magnetic resonance imaging, and18F-fluorodeoxyglucose PET can predict a negative week 2 FMISO PET. We propose 30Gy CRT de-escalation by synergizing these QIBs that predict for a negative week 2 FMISO PET with week 2 circulating tumor deoxyribonucleic acid, another useful biomarker that monitors response. [Aim 2] In our trials, early-stage HPV+ OPC patients not eligible for 30Gy CRT (FMISO positive, ~30% of all patients) received 70Gy CRT. Current evidence suggests only 10% of all early-stage HPV+ OPC patients or 1/3 of the FMISO positive cohort require 70Gy (“high risk”). Therefore, de-escalation for the remaining 2/3 of the FMISO positive patients or 20% of all early-stage HPV+ OPC patients (“intermediate risk”) can further reduce toxicity. We will use our novel neural network-based clustered random forest advanced machine learning model that synthesizes all clinical/imaging information to select appropriate intermediate risk patients from the FMISO positive cohort with the goal of further de-escalation. Although FDG PET response, surgery, or induction chemotherapy can select HPV+ OPC patients to receive 45-54Gy, 50-63% of the patients complain of ≥grade 2 mucositis, even with 45Gy when 1.5Gy BID was used. Therefore, once we identified a group of intermediate risk patients, we propose to treat them with 46Gy CRT in standard 2Gy/fraction in a future clinical trial. The goal is to further reduce toxicity without compromise in tumor control compared to the standard 70Gy. [Aim 3]

NIH Research Projects · FY 2025 · 2020-03

The transformation of a normal melanocyte into a melanoma requires factors above and beyond genetic mutations. We have identified lipids from subcutaneous adipocytes as one such contributing factor. When nascent melanoma cells come into contact with these adipocytes, they induce lipolysis and release of fatty acids into the extracellular space. These lipids are then directly taken up into the melanoma cell through FATP1 (Fatty Acid Transport Protein 1). Using a zebrafish model of melanoma coupled with validation in human tissues, we show that genetic and pharmacologic manipulation of FAPT1 interrupts the crosstalk between adipocytes and melanoma. Once inside the melanoma cell, these fatty acids undergo β-oxidation and can fuel tumor proliferation and invasion programs. One end product of this metabolism is the production of acetyl-CoA, which we find can be used to modify histone acetylation within the melanoma cell and lead to widespread changes in gene expression. Despite the importance of the interaction between adipocytes and melanoma cells, it is unknown what signals mediate this cross-talk, or how these lipids are used by the melanoma cell to drive progression. In this proposal, we will take advantage of the complementary strengths of the zebrafish model and human cell culture models to elucidate these mechanisms. In Aim 1, we will test whether catecholamines secreted from melanoma cells, which occurs as a byproduct of melanin synthesis can induce lipolytic programs in the adipocytes. In Aim 2, we will use the rapid transgenic capabilities of the zebrafish to test whether adipocyte-specific knockout of the lipolytic enzyme ATGL abrogates melanoma growth and progression. This will be complemented using an ATGL knockout mouse melanoma model. Finally, in Aim 3 we will determine the mechanisms by which fatty acid derived acetyl-CoA modulates histone acetylation and melanoma cell behavior. These studies will highlight the way in which factors such as lipids from the microenvironment can reprogram tumor cells to enable malignant transformation. Identifying these mechanisms will provide new opportunities for therapeutic targeting of this cross-talk.

NIH Research Projects · FY 2026 · 2019-09

An important public challenge is how we address the multi-layer complexities associated with individuals who have a cancer diagnosis and cognitive impairment and/or functional deficits. Many organizations including the American Society of Clinical Oncology and the National Academy of Medicine (formerly Institute of Medicine) have recognized the need for more research and education about the diagnosis and management of geriatric syndromes in patients with cancer as many healthcare providers (HCPs) are unprepared. The Geriatric Oncology Cognition and Communication (Geri-Onc CC) training program has been designed to train oncologists, primary care providers, and other HCPs (nurses, nurse practitioners, physician assistants, social workers, physical therapists, occupational therapists, etc.) to screen for and conduct an initial assessment to identify cognitive impairment and/or functional decline in the older cancer patient, to learn about factors to consider when treating older cancer patients, and to improve communication with the patient and his/her caregiver. The first 5 years of Geri-Onc CC have been highly successful in terms of the ability to recruit HCP’s from a variety of clinical settings and participants’ ratings of the program, their increased knowledge and self-efficacy, and demonstrated improvements in their communication skills. In this renewal, we plan to build on this success and continue to train the workforce in how to work with older adult cancer patients. Two hundred eighty-eight HCPs working with older oncology patients will participate in the interactive, multi-modal two-day program with ongoing professional development over a 12-month period. Training will occur in 12 cohorts over the 5-year project. We will examine knowledge, attitudes, and use of skills before and these variables as well as uptake and use of communication skills and transfer of learning and skills to the HCPs’ clinical setting up to 12 months after the training. The overarching goal of this cancer educational program is to prepare the workforce to care for this growing, vulnerable, aging population.

NIH Research Projects · FY 2026 · 2019-09

Abstract Our understanding of DNA replication and its relationship with chromatin structure, nuclear organization and gene transcription has advanced significantly through the development of genome-wide assays. Most genome-wide tools to study DNA replication utilize large populations of cells and report on the average behavior of the population; as such, many infrequent or stochastic events that occur as the replication fork progresses through chromatin are missed. Recently, we have developed novel methods to map DNA replication with single-molecule and single-nucleotide precision. In this proposal we will apply this new technology to understand how DNA synthesis can be completed when the replication machinery encounters obstacles. We will specifically test how RNA/DNA hybrids and G-quadruplex DNA influence the progression of DNA replication and whether DNA synthesis can be efficiently restarted downstream of an impediment. In addition to understanding the progression of replication through chromatin, we will also investigate how replication is competed during the process of termination. Using high-resolution assays, we have uncovered a critical role for the DNA helicase Rrm3 in replication termination. We will further define the role played by Rrm3 and test a novel hypothesis that Rrm3 may prevent catastrophic re- replication of the genome.

NIH Research Projects · FY 2025 · 2019-09

Abstract: Dr. Ouathek Ouerfelli directs the Organic Synthesis Core Facility (OSCF) at Memorial Sloan Kettering Cancer Center (MSK) since 2004. He is committed to continue to provide chemistry services for the MSK research community and the development of new tools and therapeutic agents for cancer detection, prevention, and treatment. His core services have been extended to Weill Cornell Medical College in 2019 through an inter-institutional agreement. He possesses more than 30 years of chemistry collaborations and service of MSK investigators, about half of whom are NCI grantees. Along these lines, he contributed to major endeavors by eminent colleagues who are NCI grantees such as Dr. Charles Sawyers (R01 CA155169) which led to an FDA-approved drug, Dr. Howard Scher (P50 CA092629) with projects that are showing promise, as well as Dr. Larry Norton (P01 CA094060) for the development of a topical treatment to chemo- induced alopecia among others. Dr Ouerfelli maintains a state-of-the art facility with expert professional personnel in chemical synthesis. OSCF has now the capability to support medicinal chemistry efforts to evolve agents in support of MSK investigators. Under his leadership, the core has greatly facilitated preclinical studies at the Center.

NIH Research Projects · FY 2025 · 2019-09

ABSTRACT Molecular imaging (MI) originated in the need to better understand the fundamental molecular pathways inside organisms in a noninvasive manner. Over the past two decades, two factors have acted in concert to fuel the ascent of molecular imaging in both the laboratory and the clinic: (1) an increased understanding of the molecular mechanisms of disease and (2) the continued development of in vivo imaging technologies, ranging from improved detectors to novel labeling methodologies. We have established a vibrant and state-of-the-art laboratory-based translational research program. The Lewis lab portfolio is situated at the intersection of various disciplines – radiochemistry, cancer biology, chemistry, pharmacology and engineering. Our program has already demonstrated that our work is not just to generate an “image” but also to non-invasively and quantitatively measure target biology within a cancer. Even with the extensive preclinical advances in cancer imaging that we have accomplished and the unparalleled visualization of cancer biology we have achieved, our ability to translate our findings cannot be understated. Our program has excelled in the clinical translation of new imaging agents, providing new insights into cancer biology in humans. In the realms of this R35 we plan to focus on three main areas of discovery: (1) Can our successful imaging agents be transformed into theranostic agents with the ability to quantify the target through non-invasive imaging while providing concomitant lethality? (2) How can our theranostic agents be optimally deployed to quantitatively and non-invasively interrogate and treat tumor heterogeneity? (3) Following conventional and/or novel targeted therapies, can we image cancer-specific pathways to provide immediate and real-time predictors of response? We will exploit recent findings and novel methods to answer these questions, using an integrated set of imaging, chemical, genomic and cancer biology approaches. As such, the questions posed above will be of more general relevance and will allow us to address concepts related to the interactions between imaging, therapy, and response.

NIH Research Projects · FY 2024 · 2019-09

SUMMARY/ABSTRACT Prostate cancer is the most commonly diagnosed cancer among men in the United States, with an anticipated 164,690 men being diagnosed in 2018. It is also one of the leading causes of cancer death, with approximately 29,430 deaths anticipated in 2018, usually as a result of metastatic castration-resistant prostate cancer (mCRPC). Pathogenic variants in DNA damage repair (DDR) pathway genes are prevalent in a substantial subset of men who develop mCRPC. These germline or somatic genetic abnormalities, primarily insertions and deletions resulting in protein truncations that interfere with DDR, occur in 20-25% of men with mCRPC. While several studies are underway to leverage these findings for men at the latest stages of prostate cancer, genetic variation in the DDR pathway has not yet been fully characterized for men with localized prostate cancer. While there is increasing evidence that some DDR gene aberrations may be associated with aggressive prostate cancer, this also has not been fully characterized. In the United States, where prostate cancer screening is common, over 90% of patients present initially with localized disease. It is at this point in the natural history of the disease when intervention can have the most profound impact. Thus, a major focus of this proposal is understanding the spectrum of DDR gene aberrations that promote aggressive cancers, particularly in men with high-risk localized and oligometastatic disease. Retrospective series demonstrate that DDR variants occur with low frequency in men with low-risk prostate cancer and with higher frequency in men with high-risk localized prostate cancer. This has wide-ranging clinical implications. For instance, mutational status could be used to identify those at highest risk of developing lethal prostate cancer, and therapy could be optimized based on tumor or germline findings. In addition, targeted screening could be implemented to identify those at highest risk of aggressive disease and provide an opportunity for early intervention. The overarching goal of this program is to increase our understanding of the spectrum of DDR gene aberrations that are associated with adverse outcomes in high-risk localized and oligometastatic prostate cancer. This will allow us to optimize the therapeutic approach to patients who have DDR aberrations, to detect and treat lethal disease early, and to improve outcomes for patients and their relatives who carry germline aberrations. In order to achieve our goal, we have assembled a multi-institutional and multidisciplinary group of investigators, including clinical investigators, epidemiologists, statisticians, pathologists, clinical geneticists, computational biologists, bioinformaticians, and basic scientists. Our specific aims are to determine the association between long-term clinical outcome and pathogenic germline and somatic variants in DDR genes across different ethnic groups, to develop treatment strategies for patients with germline or somatic alterations in DDR pathways, and to evaluate the functional significance of different alterations in DDR genes.

NIH Research Projects · FY 2025 · 2019-07

R25 MSK Computational Biology Summer Program: Project Summary / Abstract The Memorial Sloan Kettering Cancer Center (MSK) Computational Biology Summer Program (CBSP) is a cancer education research experience that will enable 15 computer science and applied math undergraduates per year to apply their computer science skills to cancer-based laboratory and clinical research under the mentorship of MSK faculty. Program participants will work with mentors in laboratories centrally involved in basic and clinical research, with a computational focus to their work. CBSP trainees will also attend educational lectures, journal clubs and a wide range of seminars, including several focused on professional development. They will learn how to blend these lessons to present their work in a capstone showcase of their work at the conclusion of the 10-week internship. We anticipate that for some of the CSBP participants, this summer experience will be their first exposure to cancer biology, biomedical concepts, and laboratory research. By providing them with a concentrated and intense exposure to cancer biology, we will ensure that they will have the proper understanding to apply their computational skills to help develop novel methods and mine data for insights toward mechanistic and therapeutic advances. A key goal of this program is to increase the students’ awareness of and interest in careers as computational biologists. Through strategic recruitment of computer science and computational biology undergraduates and exposure to carefully selected mentors and research topics, the CBSP will provide a path toward development of computational biologists capable of informing the vanguard of cancer biology. The CBSP will enhance the students’ knowledge about cancer, genomics, and academic biomedical research, and will foster interest in careers in oncologic fields by pursuing the following Specific Aims: 1. Engage computationally fluent undergraduate students with little previous exposure to biology in innovative scientific research projects that address cause, diagnosis, prevention, and treatment of cancer 2. Provide CBSP students with additional mentorship to promote their professional development within computational oncology and biology in order to foster the trainees’ interest and engagement in academic computational cancer research Through the exposure that the CBSP will enable, we expect to shape the undergraduates’ perception of computational biology applied to cancer research such that many will pursue graduate studies in this field.

NIH Research Projects · FY 2026 · 2019-06

Project Summary/Abstract: Epigenetic biology associates with inherited cellular cues to define diverse cell fates with the same genome. Among essential epigenetic events is protein methylation deposited by more than 60 human protein methyltransferases (PMTs) and recognized by hundreds of effectors to render downstream functions. Because many methylation events are invisible in cellular contexts for conventional methods, our understanding of epigenetic roles of protein methylation is very limited. In the past five years, our laboratory has focused on developing novel chemical biology technologies and implementing them to annotate novel methylation events and their downstream outcomes. We plan to continue this research theme for technological, functional, and conceptual advances. Regarding the technological advance, we envision developing a collection of complementary chemical biology tools to annotate each PMT-target-effector axis in the context of the complex methylome. For potential functional and conceptual advances as supported by our preliminary data, we will characterize nonhistone and noncanonical histone methylation events associated with transcriptional regulation and protein homeostasis. The completion of this proposal is expected to reveal the molecular mechanisms of multiple protein methylation events in their biologically relevant contexts.

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY The objectives of this research program are to understand the structural, functional, and molecular mechanisms of two classes of integral membrane proteins in eukaryotes: ion channels and enzymes that catalyze chemical reactions within lipid membranes. For ion channels, we aim to discover the mechanisms by which the channels conduct ions across cellular membranes, achieve ion selectivity, and are gated. Regarding membrane enzymes, the salient questions we will address include how both water-soluble and lipophilic substrates access membrane-embedded active sites, what underlies chemical mechanisms of catalysis, what conformational changes occur during the reaction cycles, and what constraints the lipid membrane places on these processes. We combine approaches to determine three-dimensional structures (X-ray crystallography and cryo-electron microscopy) with functional analyses (e.g. electrophysiology, enzymology, and biochemistry) to pursue holistic mechanistic understandings of these complex molecular machines. The ion channels under study include the mitochondrial calcium uniporter, the bestrophin (BEST) family of calcium-activated chloride channels, and two-pore domain potassium (K2P) channels. The mitochondrial calcium uniporter is a highly regulated multi-subunit ion channel complex. It is the primary conduit for mitochondrial calcium entry and thereby regulates ATP synthesis and other processes. Our efforts are aimed to discover the channel’s modes of ion selectivity and gating, and to investigate functions of its regulatory subunits. BEST channels form anion-selective pores that are regulated by intracellular calcium, phosphorylation, and changes in cell volume. Mutations in BEST channels cause retinal degenerative diseases. Our efforts are geared to understand the gating and selectivity properties of the channels, with particular attention to: differences among human BEST1-4 channels, interactions with binding partners (e.g. lipids and other proteins), and the possibility that the channels conduct neurotransmitters. K2P channels establish the resting potential of cells and thereby regulate immune responses and neuronal firing. We aim to determine the structures and mechanisms of the channels, with emphasis on regulation by cellular binding partners. Regarding our studies of integral membrane enzymes, current focuses are the enzymes ICMT and RCE1, which catalyze posttranslational modifications of RAS and other CAAX proteins, and the enzymes HHAT and GOAT, which attach acyl groups onto the signaling molecules Hedgehog and ghrelin. We aim to determine atomic structures of these enzymes with substrates, substrate analogs, and products that represent snapshots of their reaction coordinates – and to combine these efforts with experiments that address function. The studies will reveal principles of ion channel and enzyme function, thereby making substantial contributions to the understandings of the physiological processes that these membrane proteins control.

NIH Research Projects · FY 2026 · 2019-03

PROJECT SUMMARY/ABSTRACT This application is Memorial Sloan Kettering Cancer Center’s (MSK’s) proposal to renew grant UG1CA233290 to maintain its status as a Network Lead Academic Participating Site for the NCI National Clinical Trials Network (NCTN). The long-term objectives and specific aims of this project are for MSK to continue to provide scientific leadership in the development of NCTN trials and the activities of the NCTN and NCI Scientific Steering Committees; to contribute to patient accrual on NCTN trials; and to provide mentorship to early-stage and new investigators in clinical trial research. MSK has the proven resources to both bring strong scientifically driven studies to the network and to participate in studies brought forth by other network participants. MSK has a multidisciplinary team of investigators committed to translational research, a large patient population amenable to participation in clinical trials, and superb infrastructure to support such trials. MSK is a Main Member of the Alliance for Clinical Trials in Oncology and NRG Oncology, as well as a Scientific Main Member of ECOG-ACRIN. The multiple Principal Investigators in this Network Lead Academic Site UG1 are experienced leaders in the Network Groups, with a track record of meeting these scientific, accrual, and mentoring aims. As a large center focused solely on cancer, we have a particular expertise and commitment to the NCTN mission of evaluating rare tumors.

NIH Research Projects · FY 2025 · 2018-09

PROJECT SUMMARY/ABSTRACT Small GTPases regulate diverse cellular functions and their aberrant activation plays a key role in disease. Perhaps most significant is their association with cancer, a disease where KRAS is mutated in ~1/3 of patients. With this in mind, the mechanism by which cancer hotspot mutations activate KRAS is a central concept in cancer biology. Under physiologic conditions, KRAS cycles between an active (GTP-bound) and an inactive (GDP-bound) conformation. Its slow intrinsic GTP hydrolysis is catalyzed by GTPase-activating proteins (GAPs). Common mutations in KRAS prevent the stabilization of the hydrolysis transition-state leading to oncoproteins that are thought to be deficient in hydrolysis, insensitive to GAPs, and constitutively active in cancer cells (i.e., `locked' in their active or GTP-bound, state). Emerging therapies are challenging the conventional model of KRAS oncoprotein activation. Perhaps the strongest evidence is provided by inhibitors selectively targeting KRAS G12C, the most common KRAS mutation in lung cancer. G12C inhibitors bind only to the GDP-bound (or hydrolyzed) conformation and trap the oncoprotein in an inactive state by preventing the exchange of GDP for GTP. To be effective, these inactive state selective drugs require intact GTP hydrolysis by mutant KRAS. In a similar fashion, inhibition of nucleotide-exchange (as achieved by targeting factors upstream of KRAS) has been reported to suppress mutant KRAS activation and/or tumor growth. This and other emerging therapeutic effects presented in the preliminary data of this application could not be possible if mutant KRAS GTPases were `locked' in their active state. Our proof-of-principle experiments suggest the presence of cellular proteins that enhance the GTPase activity of mutant KRAS and that KRAS oncoproteins are broadly susceptible to inactive state selective inhibition. We now propose (i) to isolate enhancers of mutant KRAS GTPase activity in cancer cells, (ii) to determine the tertiary structure of common KRAS mutants in complex with their enhancer and (iii) to characterize the effects of novel inactive state selective drugs that suppress common KRAS mutants found in cancer. This work will explain the mechanistic basis responsible for the physiologic inactivation of mutant KRAS and refine the conceptual model explaining how mutations activate KRAS in cancer. The proposed study will pave the way for key advances in cancer biology with a large potential for therapeutic and translational impact in patients.

NIH Research Projects · FY 2025 · 2018-09

ABSTRACT The long-term goal of the SPORE in Soft Tissue Sarcoma is to reduce the morbidity and mortality from soft tissue sarcoma by developing therapies targeted to specific molecular, genetic, epigenetic, and signaling pathway alterations or specific sarcoma type and subtype. To pursue this, we will focus our efforts on 4 broad translational research objectives: 1. Define shared and type-specific molecular mechanisms of sarcomagenesis to identify new rational therapeutic targets; 2. Define mechanisms of resistance to targeted and immune therapies; 3. Clinically validate new therapeutic targets and treatments in soft tissue sarcoma patients and facilitate the development, recruitment, and application of clinical trials that serve both the adult and pediatric populations; 4. Discover specific molecular alterations and new biomarkers that predict outcome and response to targeted and immune therapy. To achieve these goals, we have marshaled an integrated, multidisciplinary group of basic and clinical investigators, all armed with a unique resource, a clinicopathologic and outcomes database prospectively collected over a 41-year period. This database now contains data for over 14,990 patients treated for soft tissue sarcoma at MSK. The database is linked to an extensive sarcoma tissue and blood bank, which in turn is linked to an extensive multi-platform molecular genetic and epigenetic dataset and a collection of primary sarcoma cell lines and patient-derived xenograft (PDX) models of human sarcoma. The SPORE is structured around 3 research projects, 4 cores, and career enhancement and developmental research programs. Each research project focuses on two or more of the 4 broad translational research goals listed above. RP1 (GIST Pathogenesis) aims to elucidate the molecular mechanisms and role of MAX/MGA/MYC genetic perturbations in driving GIST pathogenesis and to develop novel biomarkers and predictive models to improve patient risk assessment and selection for adjuvant therapy. RP2 (Targeting Hippo Dependence) seeks to characterize the role of the Hippo pathway and the eIF4FA complex in genetically complex sarcomas, test the efficacy and toxicity of a new eIF4A inhibitor, TDI-7663, and develop biomarkers of innate and acquired resistance to eIF4A inhibition. RP3 (Synovial Sarcoma Vulnerabilities) seeks to identify novel epigenetic vulnerabilities and immuno-oncologic strategies in synovial sarcoma and potential synergies between them by discovering H3K36 methylation-related dependencies on specific epigenetic regulators, preclinical development of T cells genetically engineered to express a T cell receptor against the SS18::SSX public neoantigen, and a conceptually innovative clinical trial of a multivalent mRNA vaccine encoding the SS18::SSX(1/2) junction sequence and the major cancer-testis antigens in synovial sarcoma.

NIH Research Projects · FY 2025 · 2018-08

Interactions between the immune system and the intestinal stem cell (ISC) compartment are poorly understood, as are the mechanisms by which ISCs respond to immune-mediated insults. In the previous R01 cycle, we made several major discoveries: 1) T cells infiltrate and directly target the ISC compartment in vivo. 2) T cells mediate Interferon-γ-dependent ISC killing in models of graft vs. host disease (GVHD) and autoimmunity. 3) Lymphocyte- derived cytokines also target ISCs to promote JAK/STAT-dependent epithelial regeneration, and this immunobiology can be translated clinically as demonstrated by our recently completed trial of Interleukin (IL)-22 treatment for patients with GVHD. 4) We have identified a new mechanism of epithelial regeneration whereby ISC-derived IL-33 induces the stem cell niche to augment epidermal growth factor (EGF) production after damage. Additionally, new unpublished data uncover the surprising finding that another EGF receptor (EGFR) ligand, Amphiregulin (Areg), which is thought to promote epithelial regeneration, can signal directly to T cells, drive their proliferation, and promote immune-mediated epithelial injury. Preliminary findings show that CD4 T cells upregulate Areg and EGFR upon activation, increasing their expansion, tissue infiltration, ISC loss, and intestinal pathology. The newly discovered IL-33-dependent epithelial regeneration pathway and our new data identifying a pathologic role for the T cell Areg/EGFR axis in tissue damage present a critical translational conflict: the IL-33/EGF regenerative circuit supports the therapeutic potential of EGF administration, but administering EGFR ligands also has the potential to drive further immune-mediated damage by stimulating T cell EGFR. The goals of this project are to investigate 1) the function of the EGFR pathway in activated T cells, 2) EGFR function within the ISC compartment in models of immune-mediated tissue damage, and 3) how these pathways can be targeted to reduce immune-mediated damage and enhance regeneration. Utilizing a combination of in vivo and ex vivo T cell activation models, as well as ex vivo modeling of tissue responses to T-cell-mediated damage using murine and human organoid culture systems, we will test the hypothesis that Areg produced by activated CD4 T cells drives autocrine EGFR signaling and T cell proliferation, leading to tissue infiltration and ISC compartment damage in the allogeneic setting. This represents a potential paradigm shift in the understanding of Areg and EGFR. We will also test the hypothesis that ISCs respond to this damage by enhancing the function of their own niche, identifying ISCs as not just the mediators but the regulators of regeneration. This study will thus mechanistically uncouple pathologic T cell EGFR signaling from regenerative epithelial EGFR signaling to enable rational design of novel therapies for protecting tissues from immune- mediated damage and promoting epithelial regeneration. In collaboration with a team of experts in Areg/EGFR biology, organoid culture systems, autoimmunity, and mucosal immunology, this project will yield a mechanistic understanding of fundamental properties of EGFR function within the immune system and the ISC compartment.

NIH Research Projects · FY 2024 · 2018-08

Project Summary/Abstract There is palpable excitement in the oncology community that we are on the cusp of a major advance in how we treat bladder (urothelial) cancer. Recent efforts to comprehensively define the landscape of genetic alterations in urothelial cancer and to understand their impact on drug sensitivity, as well as the exciting early results with immune targeting strategies suggest that prospective molecular profiling of blood and tumor tissue could improve the outcomes of urothelial cancer patients by personalizing care. This MSK SPORE in Bladder Cancer seeks to leverage recently initiated multicenter efforts to explore the molecular basis of inherited genetic susceptibility, exploit prospective molecular characterization to guide treatment, and to test the efficacy of immunotherapy-based combination approaches. The overall translational aims of the MSK SPORE in Bladder Cancer are to 1) develop predictive biomarkers of response and resistance to immunotherapy, chemotherapy, and investigational treatments; 2) identify germline genetic alterations that confer increased risk for the development of urothelial cancer; and 3) identify mechanisms of immunotherapy resistance and develop combinatorial strategies to enhance immunotherapy response in patients with urothelial cancer. To pursue these aims, we have assembled a multidisciplinary team with complementary expertise in the clinical management of urothelial cancer, inheritable risk, mycobacterial and cancer biology, cancer genetics, molecular pathology, biostatistics, computational biology, and multiplatform data integration. The translational aims of this SPORE will be pursued through four projects, each of which addresses a different clinical state in the evolution of the disease. Project 1 will use prospective molecular characterization to determine, in the context of a cooperative group trial, whether transurethral resection and chemotherapy, without the need for cystectomy, is curative in patients with DNA damage response gene alterations and to identify novel biomarkers of chemotherapy sensitivity. Project 2 will identify and functionally characterize novel germline variants that confer increased inherited susceptibility. Project 3 will seek to identify and validate tumor- and blood-based predictive biomarkers of response to systemic immune checkpoint blockade in patients with metastatic urothelial cancer in the context of a randomized, multicenter trial. Project 4 will seek to identify predictive biomarkers of Bacillus Calmette-Guerin (BCG) response and BCG strains with greater activity as a prelude to future clinical trials. Each of these projects will be supported by the Biospecimen Repository and the Biostatistics and Bioinformatics Core, which will assist with the preparation and analysis of human tissues and genomic, immune, and clinical data, and an Administrative Core will ensure project integration. Finally, developmental research projects and career mentorship are fully integrated into the SPORE to ensure that a future generation of researchers is prepared to further advance our long-term objectives of enhancing therapy, reducing the morbidity of treatments, and ultimately eliminating this disease as a cause of premature death.

NIH Research Projects · FY 2024 · 2018-08

Project Abstract PDA is the most common neoplasm of the pancreas, and is soon to be the second most common cause of cancer deaths in the United States. Surgical resection in Stage I/II patients provides the only opportunity for cure, yet >80% of patients will recur and die of their disease within 2-3 years2. The statistics for Stage III and Stage IV PDA are more dismal, having 12 and 6 month median overall survival times, respectively. Outside of BRCA2 mutations that confer sensitivity to platinum salts or PARP inhibition, or immune checkpoint inhibitors in patients with mismatch repair deficiency, there are few actionable targets in the PDA genome. Thus, it is essential that novel strategies are developed to extend survival. One innovative way to do so is to “treat evolution with evolution”. However, it is first imperative that we develop a deep understanding of PDA evolutionary biology. Our efforts will be focused on three questions with clear mechanistic and translational relevance to this ultimate goal. First, what are the features of clinically relevant intratumoral heterogeneity at the genetic and transcriptional level? Second, how do cell autonomous and non-cell autonomous factors influence the evolutionary dynamics of PDA? Third, how do PDA therapies influence evolutionary trajectories, and can they be more effectively used within the evolutionary context of a tumor? We will rely on whole exome or whole genome sequenced samples of primary and metastatic pancreatic cancer tissues, single cell technologies for copy number alterations or RNA expression, long-term evolution experiments, mouse models and computational models to address these questions. We aim to use the information gained over the period of this work to develop metrics of heterogeneity that will inform clinical management, including identification of the optimal agents and timing of administration based on the evolutionary context of the patients' PDA. Such questions are of broad interest in cancer biology in general and have a strong likelihood to impact upon other tumor types as well.

NIH Research Projects · FY 2025 · 2018-07

PROJECT SUMMARY/ABSTRACT Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near-uniformly lethal. While the most frequent genomic alterations in PDAC are not presently druggable and conventional therapies are often ineffective in this disease, immune-modulatory therapies hold promise to meaningfully improve outcomes for PDAC patients. Development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment, including frequent exclusion of antineoplastic T cells and abundance of immune-suppressive myeloid cells. We recently found that cancer cell-intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we found that GOT2 functions beyond its established role in the malate-aspartate shuttle and promotes the transcriptional activity of nuclear receptor PPARd, facilitated by direct binding to PPARd ligand arachidonic acid. While GOT2 in PDAC cells is dispensable for cancer cell proliferation in vivo, GOT2 loss results in T cell-dependent suppression of tumor growth, and genetic or pharmacologic PPARd activation restores PDAC progression in the GOT2-null context. This cancer cell-intrinsic GOT2-PPARd axis promotes spatial restriction of both CD4 and CD8 T cells from the tumor microenvironment, and fosters the immune-suppressive phenotype of tumor-infiltrating myeloid cells. Our results to date demonstrate a non-canonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, and here we propose to exploit this novel GOT2-PPARd axis to promote a productive antitumor immune response with the following specific aims. Aim 1: Assess the therapeutic potential of targeting the GOT2-PPARd axis in established PDAC. We will perform preclinical evaluation of GOT2/PPARd pathway inhibition together with therapeutic approaches aimed to increase antitumor T cell activity in diverse mouse models, validate our findings in patient specimens with known clinical outcomes and mutational status, and assess heterogeneity across the patient population with respect to the association between GOT2 signaling and T cell spatial regulation. Aim 2: Analyze the immune evasion mechanisms driven by cancer cell-intrinsic GOT2. We will apply in vitro co-culture systems, in vivo assays testing a suite of GOT2 mutants with varying fatty acid signaling capacity, and unbiased transcriptional analyses to understand the stepwise mechanisms mediating paracrine regulation of immune evasion. Aim 3: Interrogate fatty acid-mediated gene regulation by GOT2 and PPARd. We will analyze the genome-wide binding patterns of PPARd and additional immune-modulatory transcription factors putatively regulated by GOT2, and characterize chromatin states in the context of fatty acid signaling perturbations that prevent or permit antitumor immune responses. This work will leverage our recent identification of a novel immune suppression mechanism in PDAC, with the potential to inform on new therapeutic combinations poised for clinical translation.

NIH Research Projects · FY 2026 · 2018-05

PROJECT SUMMARY: The goals of the research proposed for the MIRA renewal are: (i) to understand the mechanisms and structures of enzymes that perform nucleic acid synthesis, modification, and repair; and (ii) to elucidate factors that regulate these events. The project integrates diverse experimental approaches (microbiology, biochemistry, structural biology, genetics) and applies them to model systems ranging from viruses to bacteria to fungi. The principal themes are: (1) The structures, mechanisms, and distinctive specificities of fungal tRNA splicing enzymes Trl1 (tRNA ligase) and Tpt1 (tRNA 2'-phosphotransferase) – as paradigms of an RNA repair system essential for normal cell physiology and as promising targets for anti-fungal drug discovery. We will determine structures of Trl1 and Tpt1 in complexes with nucleic acid and nucleotide substrates and cofactors, and endeavor to capture structural snapshots of intermediates and transition-states along the reaction pathways. (2) The structural basis for RNA recognition and strand joining by ATP-dependent 5'-PO4/3'-OH RNA ligase T4 Rnl1. Rnl1 is a tRNA repair enzyme that the T4 bacteriophage uses to evade a tRNA-damaging host response to virus infection. (3) The unique catalytic mechanism, end-specificity, and regulation of GTP-dependent 3'-PO4/5'-OH RNA ligase RtcB. The RtcB-family ligases are found in all phylogenetic domains. They are agents of diverse RNA transactions, including tRNA splicing (in metazoa and archaea), RNA repair (in bacteria), nonspliceosomal mRNA splicing (in the metazoan unfolded protein response), and the formation of chimeric RNAs in human cells that can undergo retrotransposition into the human genome. (4) Tandem transcriptional interference as a controlling factor in fission yeast phosphate homeostasis. The three S. pombe PHO regulon genes are repressed in phosphate-replete cells by transcription in cis of 5’- flanking lncRNAs that interferes with the PHO mRNA promoters. The lncRNA-mediated interference that underlies the repression of pho1 has afforded us a sensitive read-out of genetic influences on 3'- processing/termination and a powerful tool for discovery of agents and regulators of this step of the Pol2 transcription cycle. These influences include: (i) the Pol2 CTD code; (ii) numerous components of the 3'- processing/termination machinery; and (iii) metabolite control by inositol pyrophosphate 1,5-IP8, an intracellular signaling molecule. We propose to investigate the fission yeast Asp1 kinase/pyrophosphatase enzyme that determines IP8 dynamics and the cellular proteins and pathways that connect IP8 to gene expression.

NIH Research Projects · FY 2026 · 2018-04

Summary Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes [TC(s)], unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a component of cellular stress responses. Stalled forks are foci for genomic instability that causes genetic alterations and can give rise to cancer. Stalled forks must be protected/remodeled/repaired and replication restarted/continued in order to maintain genomic stability. We propose to continue our analyses of replication fork stalling brought about by such factors. We ask: (i) Are replisome collisions with protein-bound R-loops more prone to stall forks than collisions with unbound R- loops? which are only transient obstacles to progression. (ii) Does replication fork reversal preserve replication potential during replication-transcription conflicts? (iii) Does the accumulation of positive superhelicity between replisomes and TCs approaching each other head-on lead to replication fork stalling and/or collapse? And (iv), how do replisomes overcome collisions with RNA polymerases that are themselves stalled by DNA template damage? We have used the MIRA mechanism to begin to transit our focus on bacterial replication systems to replication with human proteins. We will investigate the DNA sequence requirements for loading of double hexamers of the MCM proteins to DNA, as well as the effect of chromatinization of the DNA substrate on the loading reaction. We ask: (i) what are the requirements for various amino acid sequence motifs in ORC1? (ii) What role is played by ORC6 (we currently do not require this protein for loading)? And (iii) what are the effects of histone modifications in the loading reaction? We are also proceeding to reconstitute the complete replication reaction with purified human replication proteins. Such a system will afford unprecedented insight into insults to replication fork progression and cellular stress responses. Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. We propose to continue our analyses of the mechanisms by which the bacterial condensin MukBEF and the cellular decatenase topoisomerase IV cooperate to promote proper chromosome compaction and segregation. We ask: (i) Does the MukBEF complex either translocate on or extrude loops of DNA? And (ii) how does replication proceed through topological domains generated by MukB and Topo IV?

NIH Research Projects · FY 2025 · 2018-02

PROJECT SUMMARY Several human pattern-recognition receptors detect intracellular danger-associated signals, oligomerize into multiprotein complexes called inflammasomes, and trigger a lytic form of cell death called pyroptosis. Inflammasomes are involved in mounting immune responses to pathogens and in maintaining organismal homeostasis, but their hyperactivation can cause cancer, autoimmune disorders, and metabolic dysfunction. As such, it is critically important to characterize the molecular mechanisms that regulate inflammasome activation. NLRP1 and CARD8 are related pattern-recognition receptors that form inflammasomes, but the danger signals that they sense have not been fully established. Notably, ligands that bind to the serine dipeptidyl peptidases 8 and 9 (DPP8/9), including endogenous peptides with Xaa-Pro (where Xaa is any amino acid) N-termini, have been reported to activate these inflammasomes. However, why the innate immune system monitors Xaa-Pro peptide levels is unknown and constitutes a major knowledge gap. Recently, reductive stress, or a profound lack of reactive oxygen species (ROS), was also reported to activate the NLPR1 and CARD8 inflammasomes. The central hypothesis of this application is that reductive stress and Xaa-Pro peptide accumulation are intimately related danger signals that together comprise an overall “danger state” that causes rapid and full NLRP1 and CARD8 inflammasome activation. Specifically, it is proposed that the disordered regions of many cytosolic proteins, including the autoinhibitory N-terminal region of CARD8, are stabilized by intramolecular disulfide bonds; reductive stress abolishes these bonds, destabilizing these sequences and triggering their degradation into peptides by the proteasome. Proline is the most abundant amino acid in disordered protein regions, and therefore reductive stress likely generates many Xaa-Pro peptides. In this way, Xaa-Pro peptide accumulation can serve to confirm that reductive stress is occurring. This central hypothesis has been formulated based on preliminary data produced in the applicant’s laboratory and described in this application. The objective of this project is to determine the relationship between reductive stress, disordered protein degradation, and Xaa-Pro peptide accumulation. This project consists of three Specific Aims: 1) to determine how reductive stress induces the proteasome-mediated degradation of CARD8, 2) to characterize the relationship between reductive stress and Xaa-Pro peptide accumulation, and 3) to determine the relationship between cell metabolism and inflammasome activation. The successful completion of this work will not only clarify the primordial function of these enigmatic inflammasomes, but will also reveal a previously unknown a connection between intracellular redox state and protein stability. Moreover, this work will provide the foundation for future efforts to therapeutically control these inflammasomes for the treatment of human disease.