Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2022-09

Maintaining genome stability requires the intricate coordination of DNA replication, DNA repair, and the DNA damage response. We have developed multi-disciplinary approaches to investigate some of the complex DNA transactions and signaling in these processes that are poorly understood. Our areas of inquiry include the regulation of the replisome and replication forks, the control of homologous recombination intermediates, and dampening of the DNA damage response. Our findings have led to novel hypotheses and testing them will deepen our understanding of critical genome regulation strategies. DNA replication must cope with many types of template barriers. The coping mechanisms entail close collaboration between the replisome and many regulators. One of our long-term goals is to elucidate how various regulators dynamically modify replisome functions. We will apply novel strategies to identify replisome changes and determine how the highly conserved multi-functional Smc5/6 complex promotes replisome function. Another goal of our studies is to determine the control of replication forks stalled at programmed barriers within the ribosomal DNA. These sites suffer topological stress that can drive fork instability. We will investigate how cells maintain the stalled replication forks in the face of this challenge to complete replication. When replication forks stalled by barriers fail to recover, collapsed forks and unreplicated DNA gaps can be repaired by homologous recombination, generating repair intermediates such as Holliday junctions. Resolving such joint DNA structures by specialized cleavage enzymes completes the repair process and prevents DNA entanglement. These enzymes collaborate with a range of regulators to engender efficient repair; however, the molecular roles of many regulators remain unclear. It is our goal to elucidate the mechanisms underlying the roles of these regulators, including the functionally coupled Smc5/6 and Esc2. In addition. we will study Smc5/6, which ties together DNA replication and recombinational control, in molecular detail. Genomic stress caused by DNA replication and repair failure activates the DNA damage checkpoint. While activating this checkpoint is beneficial, its persistence is detrimental to growth. Dampening the DNA damage checkpoint is thus essential to counter such harmful effects, but its mechanisms are understudied. One of our research goals is to identify checkpoint dampening pathways and their licensing mechanisms. This line of study will provide insights into the dynamic control of the DNA damage checkpoint. Outcomes of our proposed studies will expand our view of interconnected genome replication, repair, and stress response processes and inform studies of diseases that are linked to the malfunction of these pathways.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Colorectal cancer (CRC) is now the leading cause of cancer deaths in individuals under 50, yet the causes behind this rapid rise in early onset CRC (EOCRC) remain largely unknown. The microbiome has emerged as potential environmental driver, altering the colon microenvironment through inflammatory/tumorigenic signals, or genotoxins that accelerate carcinogenesis, but why these would disproportionately affect younger people remains elusive. Escherichia coli (E.coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin and have been linked to CRC pathogenesis. While pks+ E.coli are found as commensal bacteria, they can become pathogenic in dysbiosis and inflammation, and are enriched in IBD, polyposis, and CRC patients. Colibactin directly alkylates DNA, leading to mutational A-T rich signatures (SBS-pks) detectable by whole genome sequencing (WGS). Since WGS is not routinely used clinically, we developed an approach to identify SBS-pks signatures in a targeted exon capture assay, MSK-IMPACT. We identify in our institutional pan- cancer cohort of 78,905 tumors 149/1845 samples with MSS CRC with >10% mutations attributable to SBS-pks (Gerstberger et al., in submission). We find that SBS-pks+ CRCs are associated with EOCRC, consistent with a recent WGS study (Diaz-Gay et al., Nature, 2025). To mechanistically investigate why pks+ E. coli preferentially induces CRC in the young, we developed a novel patient-derived organoid (PDO)-microbe co-culture model that leverages reversal of epithelial polarity. Three-month co-culture with human CRC PDOs and NC101 pks+ E.coli or its isogenic ∆pks strain selectively induces SBS-pks signatures in pks+ E.coli co-cultured PDOs. We find that pks+ E.coli activate DNA replication and DNA damage repair (DDR) pathways coupled with cell cycle delay in S phase, indicative of replication stress, and induce a phenotypic switch into more proliferative and regenerative intestinal stem cell (ISC) states that are poised for tumorigenesis. Ageing is associated with delayed cell cycle progression, reduced DNA repair proficiency, proliferation, and stem cell fitness. Using young/old PDO co-culture and mouse CRC models with pks+ and ∆pks E. coli, we will test the hypothesis that (1) the young epithelium has greater DDR and ISC plasticity, enabling error prone repair and dynamic colibactin induced entry into ISC states, while in aged epithelia, colibactin injury induces synthetic lethality due to deficient DNA repair and decreased plasticity. Alternatively, (2) young intestinal epithelia may harbor an epigenetic memory of inflammation from emerging environmental stressors, rendering them poised to tumorigenesis upon colibactin injury, which we will test through epigenomic and inflammatory stimulus-response assays of our existing biobank of young/old normal colon/CRC organoids from patients with/without CRC. Outcomes of this research will inform our understanding of EOCRC mechanisms and have potential implications for exposures and preventative screening.

NIH Research Projects · FY 2025 · 2022-09

The Center for Tumor-Immune Systems Biology at MSKCC SUMMARY The advent of cancer immunotherapies based on immune checkpoint blockade (ICB) has revolutionized clinical care in multiple solid tumor types and demonstrated the power of the immune system to target and eliminate cancer cells. Despite these breakthroughs, the efficacy of ICB-based immunotherapy is limited to a subset of cancers, and even in tumors where ICB is now the standard of care, only a fraction of patients achieve durable complete responses. Addressing these limitations requires (1) improving our fundamental understanding of tumor-immune interactions in immunological contexts where current immunotherapies fail and (2) developing novel strategies for enhancing responses in contexts where they have only partial success. The Center for Tumor-Immune Systems Biology at MSKCC has assembled a multi-disciplinary team of leading investigators in computational biology, immunology, and cancer biology to tackle these challenges. The Center is organized around three Research Projects that integrate computational and experimental studies in mouse models and molecular analyses in patient tumors in both immunotherapy-resistant and -responsive contexts, exploiting novel machine learning modeling of single-cell multiome, highly multiplexed optical imaging using confocal immunofluorescence, and spatial transcriptomic data sets. We will investigate distinct immune microenvironments where cancers are refractory to ICB: metastatic colonization of the brain, an immune- privileged organ where the interplay of cancer cells, astrocytes, and different states of disease-associated microglia dictate modes of invasion (Project I); and mismatch-repair proficient primary colon cancer, where tumors reside in a tolerizing microenvironment and interact with complex cellular circuits of regulatory and conventional T cells, together with metastases to the lymph node and liver (Project II). We will also carry out a systems biology interrogation of cancer cell death mechanisms in models of ICB-responsive melanoma and renal cell carcinoma, based on findings that engineering mitochondrial damage-dependent but caspase- independent cell death elicits anti-cancer immunity and protection against tumor rechallenge (Project III). A Shared Resource Core will interact with all three Research Projects to develop computational methods and establish technologies for spatial analyses of the tumor-immune microenvironment. These studies will advance our fundamental understanding of tumor-immune ecosystems in ICB-refractory microenvironments and of immune responses to therapeutically induced immunogenic cancer cell death in ICB-responsive settings, ultimately leading to novel immunotherapeutic targets and combination strategies. Our team will build on the successes of our previous CSBC U54 Center award for the Center for Cancer Systems Immunology at MSKCC, which produced numerous high-impact studies at the forefront of systems biology and cancer immunology. Our Research Center will also carry out innovative outreach and training activities to disseminate research findings in tumor-immune systems biology and to train young scientists in this critical field.

NIH Research Projects · FY 2025 · 2022-09

Project Summary: Malignant peripheral nerve sheath tumor (MPNST), accounting for 4% of all soft tissue sarcomas (STS), represents an aggressive subtype of STS with poor prognosis. MPNSTs occur in distinct clinical settings: type I neurofibromatosis (NF1)-associated (45%), sporadic de novo (45%), or radiation (RT)-associated (10%). Molecularly, MPNSTs share highly recurrent and biallelic genetic inactivation of three tumor suppressor pathways: NF1, CDKN2A, and Polycomb repressive complex 2 (PRC2) core components, EED or SUZ12. PRC2 loss occurs in more than 80% of all high-grade MPNSTs, and results in global loss of H3K27me2/3 and aberrant transcriptional activation of developmentally silenced master regulators, leading to enhanced cellular plasticity. PRC2 loss in MPNST also leads to aberrant activation of multiple signaling pathways (e.g. WNT signaling), an “immune desert” tumor microenvironment, and primary resistance to immune checkpoint blockade. Currently, there are no effective systemic therapies that provide durable clinical benefit for MPNST. Using a custom RNAi library specifically targeting epigenetic regulators and a pooled negative screen, we identified and validated DNMT1 as the top synthetic lethal candidate with PRC2 loss. We further observed that compared to PRC2-wild-type (wt), treatment with a pan-DNMT inhibitor (decitabine) or a selective DNMT1 inhibitor (GSK862) resulted in significantly enhanced toxicity in various in vitro and in vivo MPNST models with PRC2 inactivation. In contrast to previous studies of DNMT inhibitors in solid tumors that typically demonstrated minimal antitumor effects, DNMT inhibitors imposed significant antitumor effect in the PRC2-loss context through programmed cell death. We hypothesize that PRC2 loss creates a critical dependence on the DNA methylation- mediated ERV regulation in tumor cells and DNMT inhibition may represent a novel therapeutic strategy in PRC2- loss MPNSTs. Here, we propose a proof-of-concept investigator-initiated phase II trial to evaluate the efficacy of ASTX727 (combination of decitabine and cedazuridine) in patients with PRC2-loss MPNSTs. The phase II trial is rich in pre-treatment, on-treatment, and at disease progression research biopsies, as well as exploratory correlative studies using cutting-edge genetic, epigenetic, and transcriptome analyses at single cell level with the goals to evaluate for biomarkers and mechanisms of therapeutic sensitivity and resistance. We believe that this clinical study will generate the pivotal biomarker-driven clinical and translational information for a definitive trial of ASTX727 in PRC2-loss MPNST with the potential to delineate therapeutic strategies in other cancer types with genetic or functional inactivation of PRC2.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Rare monogenic immune disorders have illuminated key aspects of inflammation, but many of the underlying mechanisms remain poorly understood. For example, autoimmune lymphoproliferative syndrome (ALPS), a disorder in which T cells fail to undergo apoptosis, is most often caused by genetic defects in the death receptor FAS or its ligand FASL. However, mutations in caspase-8 or its adaptor FADD – which mediate cell death downstream of FAS – cause a combination of ALPS plus severe immunodeficiency. Since immunodeficiency is not generally observed in patients with FAS or FASL mutations, I hypothesized that FADD-caspase-8 must have an apoptosis-independent function downstream of an immune receptor other than FAS. Indeed, I recently discovered that activation of multiple immune receptors elicits the caspase-8-mediated cleavage of Nedd4- binding protein 1 (N4BP1), a novel cytokine suppressor. This represents a critical point of regulation during inflammation. Notably, deletion of N4BP1 does not ordinarily affect the TRIF-dependent subset of toll-like receptors (TLRs) that activate caspase-8 (e.g., TLR3 and TLR4). However, the impaired cytokine production of caspase-8-deficient macrophages stimulated with a TLR4 agonist is restored to normal by co-deletion of N4BP1. In contrast, N4BP1 deletion leads to exorbitant cytokine responses by the TRIF-independent TLRs (e.g., TLR1/2, TLR7 and TLR9) that do not directly activate caspase-8. Thus, N4BP1 cleavage by caspase-8 inactivates the anti-inflammatory activity of intact, un-cleaved N4BP1. These findings offer a novel mechanistic explanation for immunodeficiency caused by FADD-caspase-8 mutations, whereby the inability to cleave N4BP1 results in its aberrant persistence and constriction of cytokine responses. Like TLR3 and TLR4 agonists, tumor necrosis factor (TNF) also leads to caspase-8 cleavage of N4BP1, endowing TNF with the ability to inactivate N4BP1 and thereby license cytokine production by the TRIF-independent TLRs. This latter finding highlights a key point of molecular crosstalk between the TNF and TLR systems that converges on caspase-8 cleavage of N4BP1. In the current proposal, I have linked the mechanism by which N4BP1 suppresses cytokine production to a series of proteins with both previously recognized and heretofore unknown roles in inflammation. In Aim 1, I will attempt to decipher the mechanism by which N4BP1 controls the activity of this novel kinase-dependent pathway that suppresses inflammation. In Aim 2, I will dissect how N4BP1 suppresses late phase inflammatory gene expression using genome-scale technologies. In Aim 3, I will explore the mechanisms and in vivo consequences of signal integration by the TNF-caspase-8-N4BP1 axis. Together, these aims will provide novel mechanistic insights explaining a key regulatory circuit underlying inflammation. They also will serve to launch my independent research career.

NIH Research Projects · FY 2025 · 2022-09

Abstract Cells respond to stress by upregulating adaptive mechanisms that promote survival or by undergoing cell death when the stress is too severe. Cancer cells take advantage of stress responses in order to survive within harsh cancer microenvironments, and understanding which adaptive mechanisms are utilized to avoid cell death is critical to gaining new knowledge that may be exploited for cancer therapy. It has also become clear that there is not one, but in fact many different forms of cell death that can occur in response to stress, and our studies have contributed significantly in this area. We have shown that some mechanisms have unique effects on the dynamics of cell populations, and that some promote, while others may hinder, therapeutic responses. Our proposed research program will focus on two major areas of discovery: (1) How is cell death regulated in response to stress, and how do particular mechanisms contribute to controlling population dynamics? (2) How do cells respond to nutrient starvation through adaptive mechanisms that involve lysosomes? We will exploit recent findings and methods we have developed to study these overarching questions through an integrated set of cell biological approaches with a focus on imaging-based studies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Genomic sequencing of tumor samples is now a routine component of cancer care, providing unprecedented insight into cancer initiation, progression, and treatment effects. Additionally, novel molecular profiling and imaging techniques are gaining traction, generating ever more data. Ensuring that these data sets are easily accessible and interpretable to scientists and clinicians is of vital importance. Towards this end, we seek to evolve and expand the capabilities of the cBioPortal for Cancer Genomics, a unique platform that enables interactive exploratory analysis of large-scale cancer genomic data. The cBioPortal is the most widely used and most highly cited tool within the cancer genomics community. The public site, with data from 325 cancer studies, is accessed by >34,000 unique users each month. The cBioPortal instance that supports AACR Project GENIE, a multi-institutional data sharing initiative, now hosts genomic profiles from >120,000 tumors. Since the software is available under an open source license, >65 cancer centers and pharmaceutical companies have institutional installations of cBioPortal to analyze their own data. Multiple institutions are making contributions to the software, including the five that are part of this application (Memorial Sloan Kettering Cancer Center, Dana-Farber Cancer Institute, Harvard Medical School, Princess Margaret Cancer Centre, and Children’s Hospital of Philadelphia). To ensure that this vital resource continues to aid the cancer research community and to keep pace with the rapidly advancing fields of cancer genomics and precision cancer medicine, including the continuing increase in the number of profiled tumor samples, we propose to actively sustain and evolve the cBioPortal platform. Specifically, we plan to make improvements across the entire cBioPortal software architecture (Aim 1); this includes significant changes to address key performance bottlenecks, a new API capable of supporting federated queries, a new App Store, and improvements to our cloud infrastructure and data pipelines. We will also support several new molecular data types, add two entirely new cBioPortal views, develop new features for precision oncology, and improve general usability (Aim 2). We propose to continue funding a group of core developers across five institutions, expand the base of code contributors, and continue to collaborate with The Hyve to support biotech and pharmaceutical companies (Aim 3). Finally, to maximize use in the scientific community, we plan to continue to improve community outreach, user support, and training (Aim 4). These improvements will be necessary to ensure that cBioPortal continues to provide an essential service for cancer research and development of new biomarkers and drugs, especially as more cancer centers are using the cBioPortal as part of their precision medicine programs, and as pharmaceutical companies are using it for internal research. We expect that, over the next few years, the cBioPortal will continue to have a strong impact on all areas of cancer research and patient care.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Immunotherapies that boost anti-tumor T cells are landmark breakthroughs in oncology. Yet, current immunotherapies directly activate T cells and are therefore ineffective in ~80% of tumors with few T cells (“cold” tumors). Thus, a primary challenge in oncology is to develop effective immunotherapies for cold tumors. Pancreatic ductal adenocarcinoma (PDAC) is a prime example – ~91% of tumors have few T cells, and thus PDAC rarely responds to current immunotherapies (<2% response rate). Yet, immunotherapy is the most promising option in PDAC, as all other therapies have failed, and only the rare (9%) patients with immunogenic “hot” tumors (high density of intratumoral T cells) survive long-term. Thus, new immunotherapies are urgently needed for PDAC, and the principles can be applied to other cold tumors. To discover new targets that active immunity in PDAC, we contrasted immune cells in hot tumors from rare long- term PDAC survivors to those in more typical cold tumors from short-term survivors. Unexpectedly, we found that hot tumors have ~3-fold higher densities of group 2 innate lymphoid cells (ILC2s) (Moral et al., Nature, 2020). ILC2s are lymphocytes that amplify CD4+ Th2 cells in infection but paradoxically can activate CD8+ T cells in tumors. Using mouse models, we found that ILC2s recruit CD103+ dendritic cells to activate CD8+ T cells and suppress primary PDACs. Through further studies, we have now found that unlike currently presumed, ILC2s can also migrate to suppress metastatic PDAC tumors, express lymphotoxin (LT), a protein that induces tertiary lymphoid structures in tumors, and express the immune checkpoint PD-1 that regulates their anti-tumor function. As we are the first group to report that ILC2s can activate immunity in PDAC, the mechanisms by which ILC2s suppress PDACs, which can thus inform rational strategies to harness them in immunotherapies, are unknown. Thus, we now propose to study how ILC2s migrate to tumors, activate CD8+ T cells, and are functionally regulated. Through integrated, multi-disciplinary study of ILC2 phenotype and function in human PDAC patients, patient-derived organoids, and functional studies in mouse models, we will: 1) define the cytokines that mobilize anti-tumor ILC2s; 2) investigate how anti-tumor ILC2s utilize LT to activate CD8+ T cells; and 3) demonstrate how PD-1 blockade enhances anti-tumor ILC2 function. To ensure a cross-disciplinary approach, we will use an experienced team of investigators with complementary skills in PDAC biology, ILC2 immunology, immunotherapy, organoid models, and computational oncology. We expect our proposal will lay the scientific framework to understand ILC2 cancer biology and guide efforts to harness ILC2s in new immunotherapies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Candidate: The PI, a Medical Oncology Fellow at Memorial Sloan Kettering Cancer Center (MSK), has developed a 5-year career development plan that builds upon his scientific background in immunology and clinical training in medical oncology. He will conduct the proposed research under the mentorship of Dr. Jeffrey Ravetch, an internationally recognized expert in Fc receptors (FcRs). He will also develop new skills in antibody and tumor biology that are critical for his future career focused on understanding the mechanisms that mediate effective responses of cancer immunotherapies. The PI has planned to address the necessary training and mentoring required for his successful transition to independence through select coursework and a robust mentoring plan. The institutional environment of MSK, The Rockefeller University, and an Advisory Committee composed of leaders in the field will not only ensure that the PI’s research project progresses as planned, but also the PI’s transition to independence as a physician-scientist with his own laboratory and grant funding. This research project is also sufficiently different from his mentor’s to avoid competition or overlap. Research Plan: Antibodies targeting immune checkpoints lead to long-lasting clinical responses in a variety of malignancies. However, many patients fail to respond to these therapies and new targets that enhance antitumor immunity are under active investigation. CD47 is a “don’t eat me” signal overexpressed on several types of cancer and is associated with poor prognosis. Its expression protects tumor cells from phagocytosis by interacting with SIRP-alpha (SIRPα), a cell surface receptor expressed on myeloid cells (i.e., macrophages and dendritic cells). Antibodies blocking the CD47/SIRPα pathway enable myeloid-mediated phagocytosis and tumor cell elimination, leading to effective antitumor effects in preclinical studies and early clinical trials. The PI studies have focused on understanding the mechanism of action of antibodies targeting the CD47/SIRPα axis, in particular the contribution of the antibody Fc domain and its binding to FcRs to induce effective antitumor responses. Preliminary data presented in this proposal show that a) Engagement of FcRs by the Fc domain modulates the activity of anti-CD47 and anti-SIRPα antibodies, and b) Fc-optimized anti-CD47 antibodies increases antitumor immunity and infiltration and of tumor-associated macrophages, dendritic cells, and other immune cells. The goal of this proposal is to understand the role of specific FcγRs in mediating effective antitumor responses using a novel humanized mouse model for CD47, SIRPα and FcRs. In this model, Fc-engineered humanized anti-CD47/SIRPα antibodies will be tested alone or in combination with other immunotherapies to determine their effects on infiltrating immune cells. Elucidating the role of FcRs as modulators of response to anti-CD47/SIRPα antibodies may establish a new avenue to maximize their therapeutic actions and provide critical information on the role of innate immune cells in promoting effective antitumor responses.

NIH Research Projects · FY 2025 · 2022-09

T cell receptor mimic (TCRm) mAbs. TCRm mAbs represent a new class of mAbs, structurally identical to traditional mAbs. However, while traditional mAbs recognize 3D conformational structure of a surface protein, TCRm mAbs recognize peptides (9-10 amino acids) derived from intracellular proteins, displayed on cell surface by MHC class I molecules, the complexes traditionally recognized by TCR. This allows an antibody to have access to vast majority, truly tumor-specific antigens, most of them are intracellular proteins. The advantages of mAb therapy are well known that include their high target specificity, high efficacy, limited side effects, prolonged half-life, availability, low cost, and infrequent dosing. In addition to the immune effector functions of a mAb, mAb can also serve as antigen-specific vehicles that can deliver more potent cytotoxic agents such as toxins, drugs, or radiation. Finally, mAbs can be engineered into chimeric antigen receptors or bi-specific antibodies in order to enhance the specificity and the potency of T cell therapy. This project aimed at developing novel TCRm mAbs specific for two important, validated and tumor-specific antigens, as described below. Combining the best inherent features of both TCR recognition and the flexibility and potency of the mAbs as drugs, the new TCRm mAbs could offer a potent, controllable and widely applicable therapy. 1. Developing TCRm mAb to HPV-E7-derived epitope in the context of HLA-A*02:01. Human papilloma virus (HPV) causes hundreds of thousands of cancers worldwide. Most cervical carcinoma cells constitutively express HPV type 16 E6 and E7 oncoproteins, which provide an ideal and specific target for immunotherapies. We selected an immunogenic epitope derived from E7 protein (E7 p11-19) presented by HLA-A*02:01 as the target for the TCRm mAb. This epitope has been reproducibly detected in a majority of cervical cancer biopsies and cell lines in the context of HLA-A*02:01 molecule by mass spectrometry. 2. Developing TCRm mAb to pIRS2 in the context of HLA-A*02:01. Dysregulated protein phosphorylation is a hallmark of malignant transformation. Phosphorylation of serine and threonine residues is retained on peptides during MHC class I and class II antigen processing and presentation on the cell surface. Therefore, phosphopeptides derived from inappropriate phosphorylation of various proteins in malignant cells represent an extraordinary class of tumor specific neoantigens, that are also widely expressed and not patient-specific. We hypothesize that phosphopeptides could be used as shared tumor specific neoantigens. We selected a phosphopeptide of insulin receptor substrate2 (pIRS21097-1105) in the context of HLA-A*02:01 as the target for the TCRm development. This epitope has been identified in various cancer cells by mass spectrometry. We have a set of experimental tools and methods to select, characterize specific mAbs and to test their therapeutic efficacy both in vitro and in vivo.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Mutations arise as a result of exogenous and endogenous processes that leave characteristic imprints or signatures upon the genome. Systematic analysis of these mutational signatures led to the identification of >50 distinct types of single base substitutions (SBS) in human cancer genomes. Revealing the origins of individual signatures is critical for understanding cancer etiology, with potential implications for cancer prevention and therapy. Two of the most prevalent mutational signatures in cancer, termed SBS2 and SBS13, are present in >78% of cancer types and 56% of all cancer genomes, with a particular prominence in breast, bladder, and lung cancers. SBS2 and SBS13 are proposed to be caused by the endogenous APOBEC3 (A3) enzymes, which target ssDNA and RNA of viruses and retroelements as part of the innate immune defense. Correlations between A3 expression, driver gene mutations in A3-preferred contexts, and clinical outcomes suggest that A3 mutagenesis may play important roles in cancer etiology and evolution. Thus, there is strong rationale to understand the mechanisms of A3 activity. However, reliance on engineered model systems and correlative data have caused links between A3 enzymes, mutations in cancer, and cancer etiology to be poorly understood. We have identified human cancer cell lines with endogenous A3 mutagenesis and developed a workflow that enables us to quantify contributions of individual A3 members to mutations. Here, we propose to leverage this workflow to accomplish the following goals: 1) Identify A3 mutator enzymes in cancer types where A3 mutagenesis is prevalent and find biomarkers of their activity; 2) Investigate mechanisms modulating A3 mutagenesis; 3) Determine the functional relevance of A3 mutagenesis in therapy resistance and metastasis. Aim 1 will expand upon our characterization of human cancer cells with active A3 mutagenesis to identify A3 mutators in breast, bladder, and lung cancers. In parallel, we will directly assess the unknown specificity and sensitivity of assays to measure activities of individual A3 enzymes. These experiments may further confirm the speculative A3-etiology of a large number of cancer mutations and quantify contributions of individual A3 enzymes, thus nominating them as putative targets for therapeutic pursuit. Aim 2 builds on our preliminary data to investigate proposed modulators of A3 mutagenesis. These experiments have the potential to broaden the scope of therapeutic opportunities focused on cancer cell evolution. Aim 3 will assess the links between A3 enzymes, therapy resistance and metastasis in breast, bladder, and lung cancer cell lines. These experiments will test predictions from multi-dimensional associations that A3-mutagenesis is a disease-modifying process that can be therapeutically exploited at various stages of cancer evolution. Taken together, these studies will define the etiologies of highly prevalent mutational processes and identify strategies to elicit more durable clinical benefits to targeted therapies and curb metastasis.

NIH Research Projects · FY 2025 · 2022-09

Smoking is the leading risk factor for bladder cancer and is estimated to account for half of the 80,000 new diagnoses each year in the United States. Patients who quit after diagnosis have improved quality-of-life, lower risk of recurrence, and a 3-fold lower chance of dying from bladder cancer compared to those who continue to smoke. Yet only one in 5 urologists delivers smoking cessation treatment to patients, and considerable gaps in patient awareness and guideline-concordant screening and referral exist in the urology setting. These factors contribute to adults with bladder cancer being the least likely to quit smoking after diagnosis, compared to all other cancer survivors. Increased use of evidence-based smoking cessation treatment will improve outcomes for the estimated 100,000 patients with bladder cancer who continue to smoke. The proposed research project aims to investigate and optimize the delivery of evidence-based smoking cessation treatment for patients with bladder cancer at Memorial Sloan Kettering Cancer Center (MSK). Specific Aim 1 will evaluate how smoking cessation treatment is given to patients with bladder cancer by quantifying practice patterns and exploring factors associated with sub-optimal care delivery. Specific Aim 2 will identify determinants of smoking cessation treatment using qualitative methods that will help elucidate multi-level determinants of evidence-based practices. Specific Aim 3 will adapt our current tobacco treatment strategy to the urology context using theory-based implementation science methods informed by our mixed-methods explanatory study (Aims 1 and 2) and will pilot promising strategies. The proposed research represents a significant step towards understanding and improving the delivery of evidence-based smoking cessation treatment for patients with bladder cancer. Our findings will establish a theory-based paradigm to facilitate smoking cessation treatment within diverse surgical oncology contexts at MSK while improving patient outcomes and advancing the field of implementation science. The proposed research project is accompanied by a training plan that will provide the candidate with critical expertise in advanced biostatistics and data science, qualitative research methods, and implementation science. This will be accomplished through a detailed plan that involves coursework, collaboration, and experiential learning corresponding to each Specific Aim of the study. This career development and training plan will give the candidate a set of skills unique among urologists and will be guided by a world-class group of mentors and collaborators at a premier academic medical center and partner hospitals. The members of this multidisciplinary team will each contribute unique expertise to the candidate’s novel and innovative career development plan and research proposal. This career development award will position him to make significant contributions in the future as an independent investigator in health services research and urologic oncology.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Our mechanistic understanding of small (<150 nt) self-cleaving nucleolytic ribozymes that primarily use general- acid base catalysis involving attack of the 2’-OH on the adjacent scissile phosphate to site-specifically cleave the intervening phosphodiester backbone has initially focused on hammerhead, hairpin, glmS, hepatitis delta virus and Varkud Satellite ribozymes, and more recently on twister (Twr), twister-sister (TSr), pistol (Psr) and hatchet (Htr) ribozymes. Our overall goal as reflected from our structure-function studies of the Twr, Tsr, Psr and Htr ribozymes is to expand on our current understanding of the catalytic versatility of RNA with the emphasis on the contributions of active site organization, geometric constraints, activation of the 2’-OH nucleophile, the role of transition state stabilization, protonation of the 5’-oxygen leaving group and the potential of Mg2+ cations in mediating catalysis. One of the challenges in the field relates to whether self-cleaving ribozymes use a common or diverse set of mechanisms, and the extent to which hydrated divalent cations catalyze cleavage chemistry. We have an ongoing collaboration with the Ronald Micura lab (Innsbruck) to study catalytic mechanisms of self- cleaving ribozymes by solving crystal structures of precatalytic, transition and product states in our lab, followed by systematic multi-faceted studies of structure-guided selective catalytic mutants and analogs, as well as pKa measurements of catalytic residues, and pH and temperature-dependence of catalytic rates by the Micura lab. Aim 1: A recent biochemical genome-wide screen resulted in the identification of the naturally occurring self- cleaving hovlinc ribozyme in humans. The sequence of the 168-nt hovlinc ribozyme and its 83-nt minimal functional counterpart contained two pseudoknots with one of them embedded in the cleavage site. The cleavage rate was shown to increase with pH and its inverse correlation with the pKa of divalent cations suggested the catalytic participation of a hydrated divalent cation in cleavage chemistry. We propose to crystallize and determine the structures of the precatalytic, vanadate transition-state mimic and product states of the minimal functional hovlinc ribozyme and follow up with systematic functional studies with the Micura lab of structure- guided modifications and rate measurements towards elucidation of its catalytic cleavage mechanism. Aim 2. This Aim revisits structure-activity relationships of the Twr and TSr ribozymes to resolve discrepancies in the published precatalytic structures and resulting mechanistic insights reported in the literature. The splayed- apart orientation of bases at the cleavage site in a four-way junctional TSr ribozyme by our group contrasts with the stacked bases at the cleavage site in a three-way junctional TSr from the David Lilley lab. We propose to characterize vanadate transition-state mimics of the Twr and TSr ribozymes to resolve the existing discrepancies and mechanistically evaluate the proposed role of catalytic bases and hydrated divalent cations in mediating cleavage chemistry. These studies should elucidate whether Twr and TSr ribozymes, that contain common but topologically distinct conserved sequence elements, use similar or distinct mechanisms for cleavage chemistry.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Skin cancer is the most common type of cancer in the United States. It is critical to detect it early as skin cancers, especially melanoma, can be cured by surgery alone if detected early. As digital technology improves, skin cancer detection, and especially automated skin cancer detection, is increasingly being performed over images either in person or remotely via teledermatology. While artificial intelligence (AI) for skin cancer detection exceeds human performance on static images, algorithm performance on representative, multimodal data is still underdeveloped due to data collected piecemeal with different devices, without consistent image acquisition standards or automated registration. A well-curated dataset of annotated skin images helps meet a unique need beyond machine learning, as primary care clinicians also require expertly annotated images for education and training. We will overcome the lack of imaging standards and disparate data sources problematic in dermatology imaging by developing automated ingestion, organization, registration, and curation pipeline to improve AI for skin cancer detection. The International Skin Imaging Collaboration (ISIC) Archive includes over 2,500 citations, 156,000 images, 100 daily users, and 5 AI grand challenges with over 3,500 participants. The ISIC archive is built upon the open- source, NCI- supported, open-source web-based data management platform, Girder. The Girder platform is highly flexible, and has been extended to multiple applications (e.g., pathology, radiology). The flexibility of the Girder platform will enable us to address four major barriers that prevent our ability to efficiently ingest, host and serve large amounts of multidimensional data at the scale of non-medical image repositories (e.g. ImageNet): (1) need for laborious expert data curation and quality assurance review for protected health information, imaging artifacts, and incorrect labels (SA1.1); (2) limited metadata without content-based features creating cumbersome image retrieval (SA1.2); (3) lack of multimodal viewing capabilities (SA2); and (4) inadequate integration to existing AI and annotation software, preventing flexible, hypothesis-driven experimentation (SA3). The proposed informatics project aimed at data ingestion, multimodal visualization, and organization through ML and computer vision-based automation build on the initial success of the International Skin Imaging Collaboration (ISIC) Archive and the Girder platform upon which it is built. They will enable scaling of the Archive to millions of images, enabling multimodal experimentation with registered reflectance confocal microscopy images, and nimbly facilitate AI and translational experimentation for improved skin cancer detection.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY RESEARCH: Lung cancer remains one of the deadliest cancers in the United States, in part due to tumor plasticity that drives intratumoral heterogeneity and leads to therapy resistance. In order to understand how plasticity impacts tumors, we profiled single cell transcriptomes from genetically engineered mouse lung tumors at various stages. We observed a set of reproducible transcriptional states whose diversity increased over time. Interestingly, we identified and transcriptionally defined a high plasticity cell state that arose in every tumor. We profiled this cell state and identified a robust potential for phenotypic switching, an increased potential for spheroid formation in tumor sphere cultures, an increased proliferative potential and tumor forming ability in allotransplant models, and an enrichment of this plastic cell state after chemotherapeutic stress in vivo. We identified a similar plastic cell state in both primary human lung adenocarcinoma tumors and patient-derived xenograft models, and we found the cell state to be associated with worse survival for patients. Thus, our work suggests that the high plasticity cell state drives tumor progression and resistance to therapy in lung adenocarcinoma. To better understand the functional role of the high plasticity cell state, I propose to i) interrogate the function of the high plasticity cell state in lung adenocarcinoma progression and treatment resistance and ii) define the transcriptional drivers controlling the high plasticity cell state. This work will provide a functional and molecular definition of the high plasticity cell state, which will provide new therapies for lung adenocarcinoma. CANDIDATE & ENVIRONMENT: Dr. Jason E. Chan is an Instructor in the Department of Medicine at Memorial Sloan Kettering Cancer Center (MSKCC). His goal is to become an independent tenure-track physician-scientist investigating tumor plasticity and tumor evolution. He has delineated a 5-year career plan that builds upon his background in bioinformatics and systems biology, genetics, mouse models, molecular biology, and clinical training in medical oncology. This project will provide the ideal training for Dr. Chan to use state of the art genomics and molecular biology techniques, mouse models, and patient-derived xenografts to dissect the role of the high plasticity cell state during carcinogenesis. Dr. Chan will be co-mentored by Dr. Tuomas Tammela and Dr. Scott Lowe of the Cancer Biology and Genetics Program at MSKCC. The candidate’s career development plan includes coursework, workshops, mentoring from an interdisciplinary advisory committee comprising of distinguished basic scientists and medical oncologists, and research experience in the supportive academic institutional environment of MSKCC, a center of excellence in translational cancer research. Successful completion of the project will lead to new approaches for treating patients and will provide a foundation for Dr. Chan to become an independent investigator with his own R01 funded laboratory.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Therapeutic modulation of dysregulated metabolism has emerged as a successful therapeutic strategy for acute myeloid leukemia (AML) harboring oncogenic isocitrate dehydrogenase (IDH) mutations. Inhibition of IDH results in terminal myeloid differentiation of leukemic blasts and led to FDA-approval of IDH1 and IDH2 inhibitors in AML. However, there are currently no metabolism-directed therapies for IDH wild-type AML, which represents the majority of AML patients. Preliminary data presented in this proposal describe the identification of 2- oxoglutarate dehydrogenase (OGDH), a tricarboxylic acid (TCA) cycle enzyme which catalyzes the conversion of alpha-ketoglutarate (aKG) to succinyl CoA, as a previously unknown metabolic vulnerability in AML. Inhibition of this enzyme is sufficient to upregulate cellular aKG and drive myeloid differentiation in AML cells lacking IDH mutations. Currently however, the molecular mechanisms facilitating the change in cell fate with OGDH inhibition remain unknown, as do the genotypic contexts where exploiting aKG-dependent metabolism is most efficacious. The studies proposed seek to rigorously test the hypotheses that, 1) the treatment-refractory TP53- mutant/complex karyotype (CK) AML subset may be particularly sensitive to aKG perturbation, and 2) that the TET family of aKG-dependent dioxygenases which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and impact gene expression, among other chromatin modifying enzymes, serve as effectors of aKG- dependent differentiation. The research plan will utilize in vitro systems to characterize the aKG-dependent epigenetic program, in vivo mouse models to examine OGDH as a putative target in TP53-mutant/CK AML, and patient samples/patient-derived xenografts to determine if aberrant aKG-dependent metabolism sustains human leukemia. The proposed investigations will expand our biological understanding of metabolite use in leukemia and advance a differentiation-based strategy to treat chemotherapy-refractory leukemias that lack conventionally targetable oncogenes. The applicant, Dr. Scott Millman, an Instructor on the Leukemia Service at the Memorial Sloan Kettering Cancer Center (MSKCC), has devised a 5-year career development plan that builds upon his background in molecular biology and biochemistry, and his clinical training in medical oncology. Dr. Millman will conduct the proposed research under the mentorship of Dr. Scott Lowe, an internationally renowned expert in cancer genetics with a proven track record of training successful independent investigators, to develop new skills in functional genomics and leukemia modeling that are essential for his career goal of developing new therapeutic approaches for hematologic malignancies. This mentorship, combined with the ideal training environment provided at MSKCC, will allow Dr. Millman to carry out the proposed research program and transition to an R01- funded independent, physician-scientist position in an academic setting.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT The core mission of the MorPhiC program is to define the function of every human gene through the creation of a comprehensive catalog of null phenotypes using multicellular systems. The impact of gene loss on complex phenotypes is strongly influenced by the cellular context and the genetic background. Therefore, it is essential to develop scalable knockout methods in diverse genetic backgrounds followed by robust phenotyping assays in multicellular systems that are informative of human biology. Our Production Center will leverage our collective expertise in human pluripotent stem cell (hPSC) guided differentiation, organoid engineering, gene editing, and our extensive experience combining large-scale CRISPR-Cas9 knockout phenotyping with hPSC differentiation. We plan to conduct extensive curation and quality control to select a panel of ~100 hPSC lines, including mostly induced pluripotent stem cell (iPSC) lines and some embryonic stem cell (ESC) lines, from diverse ancestral populations, and from males and females to generate an hPSC repository for distribution. We will further prioritize genes affected in neurodevelopmental and metabolic disorders (e.g., autism and diabetes) for conducting knockouts in these diverse hPSC lines for sharing with the scientific community. For investigation of knockout phenotypes, we will optimize three distinct multicellular systems, a micropattern-based gastruloid model for early tri-germ-layer differentiation, a defined neuro-glial tri- culture system, and a 3D pancreatic islet-like organoid culture. Using these multicellular systems with different levels of complexity, we will then conduct extensive phenotyping assays in a multitiered system to allow scaled analysis both in terms of the genes analyzed and the hPSC line background (reflective of the human genetic background). Primary human islets will be included for several phenotyping assays to test the generalizability beyond the hPSC systems. We expect to work with consortium partners to prioritize the target genes for Phase 1 of the MorPhiC project, develop standards for data and resource sharing, and optimize methods for joint analyses. Our Production Center is expected to deliver a rich resource of knockout human pluripotent stem cell lines from diverse genetic backgrounds, extensive knockout phenotyping datasets in multicellular contexts that are informative of diverse human biology, robust and scalable knockout and phenotyping pipelines along with associated transferable methods, and establish strong use cases for the MorPhiC catalog. The optimized mutagenesis and phenotyping pipelines along with the scalable methods will pave the way for a full-scale MorPhiC catalog production effort in Phase 2.

NIH Research Projects · FY 2025 · 2022-08

Few viable treatments exist for patients with locally advanced or metastatic well-differentiated or dedifferentiated liposarcoma (WD/DDLS), which are rare and often neglected orphan cancers. There are approximately 1000 patients per year in the US diagnosed with WD/DDLS, and these cancers generally do not respond to chemotherapy. Recent trials of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, motivated by the finding that the CDK4 gene is amplified in >90% of WD/DDLS, show these agents stabilize disease that had been growing prior to treatment and demonstrated clinically meaningful median progression-free survival (PFS) of 66% at 12 weeks but responses were uncommon. To enhance response rates and improve PFS benefit, we propose to combine the CDK4/6 inhibitor palbociclib with an antibody against the immune checkpoint PD1. Checkpoint inhibitors have some activity in DDLS, leading to partial responses in about 8% of patients. CDK4/6 inhibitors have been shown to increase the efficacy of checkpoint inhibitors and to promote antitumor immunity in breast cancer patients and animal models. In responsive tumors, CDK4/6 inhibitors trigger senescence, causing cells to secrete proinflammatory cytokines and growth factors (termed the senescence-associated secretory phenotype, or SASP), suggesting that these agents may enhance antitumor immunity. To identify patients likely to benefit from this combination therapy, we will investigate mechanisms of response and resistance using pre- and on-treatment biopsy specimens from patients on the proposed phase 2 trial of palbociclib combined with the anti-PD1 antibody INCMGA0012. These studies will focus on the cellular markers previously determined to be required for CDK4/6 inhibitor-induced senescence in WD/DDLS (MDM2 turnover, cadherin 18 expression); markers of terminal senescence (Angplt4), antitumor immune responses; and gene expression. Thus, our Specific Aims are to: (1) Assess the safety and efficacy of CDK4 inhibition using palbociclib in combination with PD1 blockade using INCMGA0012 in 30 patients with WD/DDLS (outcomes: overall response rate, PFS, and overall survival); (2) Examine the roles of senescence, terminal senescence and resultant SASP in response to the combination therapy and, their relationship with immune response; and (3) Characterize the immune microenvironment (specifically CD8+ T cells and PDL1+ tumor cells) and tumor gene expression (assessed with immunohistochemistry and both bulk and single-cell RNA sequencing) prior to and during combined treatment of palbociclib and INCMGA0012, and examine their association with clinical response and outcome. Successful completion of this trial may lead to the introduction of a novel combination immunotherapy strategy for WD/DDLS and identify predictive biomarkers for selection of patients most likely to benefit in both sarcoma and other malignancies.

NIH Research Projects · FY 2025 · 2022-08

A substantial number of people at hereditary risk for cancer could benefit from novel genetic counseling (GC) approaches that enhance education, engagement, and outreach to relatives at greater genetic risk (ARR). When probands carrying pathogenic/likely pathogenic variants (PV) are asked to share medically actionable genetic results with their ARR, less than 30% of ARR complete predictive “cascade” testing, endangering lives. Provider-facilitated outreach to ARR leads to improved cascade testing uptake. Yet, rigorous experimental study designs have not been used to demonstrate comparative effectiveness of this approach for sustainably expanding ARR use of cancer GC and testing, or to investigate whether digital technology may enhance provider-facilitated outreach. Patients with a variant of uncertain significance (VUS) may also benefit from enhanced GC engagement; current standard of care leaves serious risks for misinterpretation by patients and non-genetics providers, and consequent medical mismanagement. Patients may experience negative responses to VUS, particularly when encountering discordant interpretations or recommendations between providers and confusion about how they will receive variant updates. In addition, best practices for follow-up and reassessment of a VUS would benefit from technology to support continuity of patient care with local primary care providers (PCPs). The proposed study addresses these needs by assessing the impact of a new GC model that leverages the increasing digitization of healthcare on psychosocial, behavioral, and implementation outcomes for probands with PV and their ARR, and for patients with a VUS result. We will first enhance an existing digital tool to include features that increase patient use, education, and sustained engagement, creating an. enhanced Digital Genetics Platform (eDGP). Next, we will conduct two RCTs involving either probands with a PV (n=350) and their ARR (n≈3150), or patients with a VUS (n=280). Probands and their ARR will be randomized to the standard of care arm, wherein probands outreach to ARR to encourage testing, or the intervention arm, wherein the GC team is given permission to outreach to ARR with support from the eDGP to expand service use across the U.S. Patients with a VUS will be randomized to obtain follow-up care through the standard of care arm, wherein they are recommended to re-contact the GC care team in 1-2 years, or the intervention arm, wherein they use the eDGP to remain engaged with the GC care team, and receive education and reminders for update appointments. We will also directly assess PCPs’ uptake of digital VUS education. Participants will complete surveys to assess uptake, psychosocial and behavioral outcomes, and intervention implementation readiness and cost. This research has the potential to improve care for patients with a PV and their families, and patients with a VUS, and will ultimately be applicable to the practice of GC and genomic medicine across diseases and clinical settings.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Developing infants are colonized with trillions of bacteria within the intestine. Failure to establish tolerance within a narrow early life window leads to increased risk of immune mediated diseases in later life, including chronic inflammation and cancer. Central to the generation of intestinal tolerance is the peripheral conversion of naïve T cells into regulatory T (pTreg) cells that suppress immune responses to commensal microbes. pTreg cells arise in the intestine at the time of weaning; however, the cell types that instruct pTreg cell fate are not known, limiting our ability to modulate pTreg cells for therapeutic benefit. We recently discovered a fascinating population of antigen presenting cells (APC), enriched within the intestinal lymph nodes during early life. These cells, dubbed Thetis cells (TCs), express the autoimmune regulator Aire, known for its critical role in immune tolerance. Here we set forth the tantalizing possibility that TCs represent a dedicated lineage of tolerogenic APCs. We aim to uncover their role in instructing pTreg cell fate in neonates, and susceptibility to inflammatory disease in later life. Our proposed genetic models allow lineage-specific manipulation of TCs, including deletion of Aire. Through these studies we aim to develop a deep mechanistic understanding of TC function. In our efforts to define the biology of TCs, we seek to understand the ontogeny and development of these cells. Using state-of-the-art lineage tracing approaches and genetic models that allow us to perturb the intestinal micro-environment in a tissue- and developmental-stage-specific manner, we will dissect the cross-talk between stromal and immune cells that drives tissue-specific early life immune development. The overarching goal of this proposal is to establish a roadmap for intestinal immune tolerance, delineating the critical antigen presenting cells that direct tolerance to commensal antigens, and the environmental cues that drive their differentiation. These studies will i) provide an unprecedented view of early life immune development, ii) establish a new framework for peripheral immune tolerance, and iii) reveal potential therapeutic targets for inflammatory and immune mediated diseases.

NIH Research Projects · FY 2026 · 2022-08

Human reproductive success and the development of healthy offspring depend on accurate transmission of genetic material from parent to child. Homologous recombination during meiosis plays a central role in this genetic transmission by ensuring accurate chromosome segregation. Errors in recombination can lead to aneuploidy or mutations in gametes that in turn cause miscarriage or developmental defects in children. Understanding the mechanism and regulation of recombination is thus critical for understanding how meiotic errors affect human fertility and child development, but the molecular principles of recombination remain incompletely understood because of a paucity of biochemical and structural information. Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by the Spo11 protein in collaboration with a suite of accessory factors. We recently overcame longstanding barriers to progress by purifying for the first time recombinant complexes of DSB-promoting proteins. Building on this advance, the Keeney and Patel labs propose to extend their ongoing collaboration to combine biochemical, structural, and single molecule biophysical approaches in vitro with functional experiments in vivo to illuminate the molecular principles that govern how DSB formation by Spo11 occurs. By conducting these studies in parallel on proteins from mouse and Saccharomyces cerevisiae, we will dive deeply into the mechanisms of evolutionarily conserved processes while retaining the ability to explore mammal-specific aspects. Aim 1 will focus on a “core complex” of Spo11 with its direct binding partners TOP6BL (mammals) and Rec102–Rec104–Ski8 (yeast). We will apply cryo-EM, x-ray crystallography, and computational modeling along with biochemical studies to define the structure of Spo11 core complexes and their critical protein-protein and protein-DNA interfaces. We will also test the physiological relevance of our structural and biochemical findings in vivo. To this end, we will use molecular genetic, genomic, and cytological studies in yeast and will employ a novel approach to parallelized genetic screening in mouse by competitively transplanting pools of genetically modified spermatogonial stem cells into testes of germ cell-depleted mice. Aim 2 will focus on the conserved accessory proteins Rec114, Mei4, and Mer2, which are important as a nexus for regulating DSB timing, number and location. We will use NMR spectroscopy, x-ray crystallography, cryo-EM, and computational modeling to define the structures and protein- protein interfaces of heterotrimeric Rec114–Mei4 complexes and of homotetrameric Mer2 complexes. We will use bulk biochemical and single molecule biophysical approaches to define the mechanism and dynamics behind the cooperative assembly of these proteins to form nucleoprotein condensates on DNA, which we hypothesize to be a central feature of their ability to support Spo11 activity. We will also apply a battery of in vivo assays to test functional predictions arising from the structural and biochemical findings.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Metazoan genomes achieve complex gene control by uncoupling regulatory DNA elements from target promoters and allowing regulation at a distance. Thus, a gene can be differentially expressed in different cell types and under different environmental signals or developmental cues. How distal regulatory elements (enhancers) target specific gene promoters, how the search process is shaped by the topology of the genome in the nucleus and how enhancer-promoter interactions are facilitated by regulatory complexes that relay signals to the RNA Polymerase II and control transcription activity remains a mystery. Our goal is to understand molecular and biophysical mechanisms that enable enhancer-promoter communication in human and other mammalian cells. Towards these goals and during the period of this award we will accomplish the following: (i) visualize the dynamic communication of enhancers and target promoters simultaneously with the association of regulatory complexes and gene activity, using novel single-molecule and super-resolution approaches for non-invasive 4D imaging of structure and function of the genome in single live cells; (ii) determine mechanisms by which different classes of architectural proteins shape genome folding, enhancer- promoter communication and transcription kinetics; (iii) dissect the function and interdependencies of individual constituent enhancer elements within complex regulatory landscapes controlling cell identity genes. Our results will establish quantitative frameworks for understanding the biochemistry of transcription regulation in the crowded environment of the nucleus and for interpreting gene regulation and genome organization using soft- matter/polymer physics and related biophysical concepts. These conceptual leaps are needed to ultimately understand physical chromatin organization at sub-Mb scales, the scale most relevant for regulatory genome interactions. Our integrated structure-function approach will provide functional validation and critical tests for gene “regulation-at-a-distance” models. The proposed studies will not only provide substantial new knowledge on the mechanisms of promoter-enhancer communication but will also set the stage for further studies of the interplay of genome topology/organization and gene expression regulation.

NIH Research Projects · FY 2025 · 2022-08

The MATCHES (Making Telehealth Delivery of Cancer Care at Home Effective and Safe) Telehealth Research Center aims to build the evidence base necessary to establish best practices for telehealth-enabled cancer care. The overarching goal of the Center is to create a research hub that generates evidence, trains investigators, and develops the research methods required to ignite the field of precision cancer care delivery. Prior work demonstrates that oncology-focused telehealth can achieve favorable outcomes, but large-scale trials have been limited to specific contexts like palliative care or survivorship. Adoption has been constrained by restricted reimbursement. The MATCHES Center will help remediate this evidence gap by executing prospective trials, conducting observational analyses, and training transdisciplinary researchers. Our research theme focuses on the integration of multi-layered data from telehealth platforms, patient portals, mobile tracking devices, and the electronic medical record to develop analytic methods that support personalized care. We aim to develop a new paradigm in oncology—precision delivery—with the ultimate goal of matching individual patients with the most beneficial combination of clinic-based or telehealth-supported home-setting care at the appropriate time—all based on the totality of dynamically available data. We will accomplish this goal by applying data science methods—including nimble trial designs and machine learning—that have had limited application to telehealth to date. By establishing a research hub that nurtures investigators across disciplines and provides training, tools, data, analytic methods, and venues for sharing knowledge and building partnerships, we expect to accelerate progress in the science of care delivery. Our specific aims are 1) to conduct impactful pragmatic trials of telehealth in oncology, 2) to analyze a large existing cache of multidimensional observational data characterizing telehealth utilization and outcomes, 3) to train investigators and equip them with the skills necessary to innovate within an evidence-based framework, and 4) to integrate telehealth with other data streams and create and apply analytic methods to transform the field of precision care delivery. An Administrative Core will coordinate activities and engage feedback from internal and external stakeholders—including patients and the oncology workforce. Our Clinical Practice Network serves as an innovation laboratory and comprises 7 outpatient clinics in NY and NJ with a shared informatics ecosystem including telehealth capacity, digital monitoring, and a highly trafficked patient portal. This will enable us to launch and execute a large pragmatic trial comparing in-person care to telehealth, with resources designed to support home-based care using a cluster-randomized design. The Research & Methods Core will support the Center by applying data science methods to extract and synthesize insights from telehealth and other data streams to develop methods relevant to advancing precision cancer care delivery and the goals of availability, efficacy, and efficiency to create an optimal experience for people being treated for cancer.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Personal payments from the pharmaceutical industry to US physicians are common and influence physician prescribing. Approximately 80% of medical oncologists receive personal payments from industry each year, with a mean of over $5,500 per physician. These payments may result in differences in the quality of cancer care delivery and potentially affect patient outcomes. Because the selection of different cancer treatments directly impacts patient outcomes such as drug toxicity, financial toxicity, and survival, these outcomes may be affected when industry payments cause changes in physicians’ treatment selection. Despite concerns about the potential influence of industry payments on medical practice, no study has investigated whether the changes in treatment patterns resulting from industry payments have consequences for patients. The short- term project goal is to evaluate the association between industry payments and cancer care quality, patient outcomes, and costs. The long-term research goal is to understand the importance of physician-industry financial relationships within the US healthcare system. Our overall hypothesis is that industry payments affect care delivery and, ultimately, patient outcomes. To test this hypothesis, we will link CMS Open Payments data to Medicare fee-for-service claims. The exposure of interest will be receipt of payments by the treating physician from a manufacturer of a drug relevant to each patient’s treatment decision. Aim 1: Assess the association between industry payments and cancer care quality. We will assess whether receipt of industry payments by physicians is associated with the likelihood that they deliver the guideline-preferred treatment for each patient (1A) and established forms of low-value care (1B). We hypothesize that physicians who received payments are less likely to use guideline-preferred treatments (1A) and are more likely to use low-value interventions (1B). Aim 2: Assess the association between industry payments and patient outcomes and costs of care. We will assess whether patients treated by physicians who received industry payments have differences in patient-focused outcomes such as unplanned acute care utilization, survival time, and quality of end-of-life care (2A) and whether they have differences in Medicare spending (2B). We hypothesize that patients treated by physicians who received payments are more likely to have acute care utilization, shorter survival time, and lower quality end-of-life care (2A) and to have increased Medicare spending (2B). This study will be the first to assess the relationship between industry payments and patient- focused outcomes, substantially increasing our understanding of the health system impact of these payments. These results may inform future policy and practice decisions regarding physician-industry interactions, potentially contributing to improvements in cancer care quality, improved patient outcomes, and lower costs.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Although there are existing epidemiological studies of disparities in cancer incidence and outcomes, there are very few studies of disparities in cancer genomes, which requires both germline, somatic, and clinical data from the same patients. However, studying ancestry-specific genomic alterations is one of the most effective ways to understand the underpinning mechanisms in cancer disparity, and develop potential prevention and therapeutics strategies. Dr. Carrot-Zhang and others evaluated genetic ancestry effects on somatic alterations among 10,678 patients across 33 cancer types from The Cancer Genome Atlas, and highlighted novel ancestry-specific evolutionary trajectories from pan-cancer and tissue-specific analyses. We also suggested that ancestry associations were profoundly tissue specific, and therefore, more samples from diverse ancestries are required for tissue-specific analyses. To that extent, this proposal is based on large sequencing data sets composed of 1,153 lung cancer patients from Mexico and Colombia, and 60,085 lung cancer samples from Foundation Medicine. Our overall goal is to understand the well-known, but mysterious, population-specific genomic differences in lung adenocarcinoma. Our first aim is to systematically characterize the landscape of ancestry effects on genomic features of lung adenocarcinoma, as we are well powered to detect new associations. Then in the second aim, Dr. Carrot-Zhang will develop a novel statistical method leveraging the local ancestry (ancestry of a genomic region) from ancestry-admixed populations to infer the heritability of ancestry-associated somatic features. We will also explore the potential mechanisms underlying genomic differences related to ancestry. Our third aim is to elucidate the influence of ancestry on clinical outcome, in order to improve prognostics and precision medicine for the minority populations. Dr. Carrot-Zhang’s long-term career goal is to improve cancer prevention, early detection and treatment by integrating computational biology, germline genetics, and somatic genomics approaches to understand the mechanisms underlying cancer initiation and progression. The K99 award will further prepare her for a successful independent research career. Dr. Carrot-Zhang’s training will be carried out under the extraordinary mentorship of Dr. Matthew Meyerson (cancer genomics), and an advisory committee consisting of Drs. Alexander (Sasha) Gusev (population genetics and statistical genetics), Rameen Beroukhim (cancer biology), Heng Li (computational method development), and David Kwiatkowski (clinical oncology). The proposed research plan will be facilitated by the outstanding institutional environment of the Dana-Farber Cancer Institute and the Broad Institute. The scientific collaboration with Foundation Medicine will provide exceptional resources for cancer genomics and ancestry-related analyses. The proposed professional development plan will enhance Dr. Carrot- Zhang’s career advancement in laboratory management, grant-writing and leadership.