Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2023-08

High rates of incidence and prevalence of oral cancers occur in 15-20 developing low-and-middle income countries (LMICs) in Asia and Africa. Visual clinical examination followed by biopsy is the standard for diag- nosing oral lesions. But the low and variable specificity of 16-100% of visual examinations results in biopsies of an estimated 37-51% indeterminate lesions (1.4-2.1 million lesions in India, alone) and in benign-to-malig- nant biopsy ratios of 2-24. Patient compliance for biopsy and follow-up care is low (35-63%) in LMIC settings due to pain, fear, time and cost. Our novel solution is noninvasive imaging with a low-cost handheld reflec- tance confocal microscopy (RCM) - optical coherence tomography (OCT) device. Diagnosis and grading of oral dysplasia are based on cellular atypia in the epithelium and underlying architectural changes. RCM imag- ing shows cellular morphology in the entire epithelium to depth of 300 µm. OCT imaging shows epithelial lay- ers and underlying lamina propria to deeper depth of 1 mm. Combined RCM-OCT imaging with a single de- vice will enable simultaneous imaging of cellular atypia and architectural changes in co-located fields of view to guide diagnosis, grade dysplasia, monitor progression to malignancy and assess invasion. Stratification, with a quantitative RCM-OCT scoring algorithm, will guide triage of oral lesions into low-grade dysplasia, which can be monitored or immediately treated with non-surgical therapies, versus high-grade, which may be immediately biopsied, versus carcinoma, which will be surgically excised. Diagnosis may be combined with treatment, all integrated in a single patient visit - a “one stop shop” patient care paradigm. We are an academic-industry team at Memorial Sloan Kettering Cancer Center (New York, NY), Physical Sciences Inc. (Andover, MA), Cali- ber Imaging and Diagnostics (Rochester, NY) and our LMIC collaborators at Tata Memorial Hospital (TMH, Mumbai). For FOA PAR-21-166, innovation is defined to be “likelihood of delivering a new capability to end- users.” Innovations will be in delivering an RCM-OCT device with a new probe for intra-oral imaging and in designing a quantitative diagnostic scoring algorithm to guide diagnosis and treatment, in real-time, at the bed- side. The device will be delivered to TMH and will ultimately cost $25,000, when scaled up and locally manu- factured in LMICs, which will support dissemination of RCM-OCT as a new and affordable imaging capability in LMICs. In preliminary studies, RCM-OCT imaging detected oral lesions and cancers with sensitivity of 100% and specificity of 80%. Our specific aims are (1) to design a handheld RCM-OCT device for imaging in the oral cavity; (2) to prospectively test on 4,422 patients for diagnosis, grading of dysplasia and assessment of invasion in oral lesions and cancers in vivo. Testing will be in LMIC settings, at TMH in Mumbai and in their regional clinic in Varanasi. Affordability, delivery of care in LMICs: the return-on-investment on our device can be in 6-9 months at cancer centers, while the cost of care to deserving patients can be as little as zero to 50 cents for the imaging procedure. Our initial success in India will seed global effort in LMICs.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Meiotic recombination is essential for the reductional cell division in mammalian germ cells and thus for the development of haploid gametes, i.e., sperm and eggs. Recombination is initiated by hundreds of DNA double- strand breaks (DSBs) introduced genome-wide that are catalyzed by the SPO11 protein. Faithful transmission of the genome to subsequent generations requires proper repair of these numerous DSBs, primarily through recombination with the homolog. DSB formation is regulated in meiotic cells by the ATM kinase, which is known to be a primary responder to DSBs in mitotic cells, such that in the absence of ATM, meiotic DSBs increase ~10-fold. We recently discovered that meiotic DSBs are at risk for provoking germline rearrangements, in particular deletions and tandem duplications involving nonhomologous end-joining, especially in the absence of ATM. These events are consequential in terms of disrupting the genes in which these hotspots occur as well as the associated PRDM9 binding sites that govern recombination at those loci. Thus, our findings reveal a previously hidden potential for germline mutagenesis that is likely to affect human health and genome evolution. In humans, recent long-range sequencing of Icelanders supports this impact. This proposal pursues aims to understand the mechanisms that give rise to these events, the range of events at meiotic DSBs, and the effect of age. We hypothesize that other rearrangements are possible at meiotic DSBs than what we have previously identified. Thus, in the first aim, we propose to determine the range of mutagenic outcomes that can arise from meiotic DSBs, including long-range deletions and duplications and chromosomal translocations. In the second aim, we examine factors that may impact the formation of deletions. We focus on the effect of DNA end processing at two steps, SPO11 removal and end processing, and recombination. Further, we address whether gaps formed at nearby DSBs are substrates for homologous recombination and the impact of paternal age in the rearrangement events at meiotic DSBs.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Autologous CD19-directed chimeric antigen receptor T-cells (CAR-T) have resulted in extraordinary response rates in relapsing and refractory large B-cell lymphoma (LBCL). However, over 60% of CD19-CAR-T recipients will experience disease recurrence or progression. Most of these patients will die from their disease. Mechanisms of CAR-T treatment failure are partially understood and biomarkers informing patient outcomes and management have limited clinical utility. Our central hypothesis is that orthogonal modalities (e.g., clinical, molecular, genomic, and radiomic [quantitative measures from medical images]) complement one another, together providing information on resistance mechanisms and patient outcomes beyond that accessible through any individual modality. We present results suggesting that machine learning is an effective methodology for synthesizing and modeling multiple sources of data together. Cancer cells harness genomic heterogeneity to evade pressure applied by immunotherapies, such as immune checkpoint inhibitors. Our preliminary data also demonstrate that TP53 genomic alterations strongly determine response to CAR-T. Furthermore, using transcriptomic profiling, we found that cancer cellular pathways required for effective transmission of CAR-T cytotoxic signals are distorted in TP53-altered lymphoma. These early findings provide a proof-of-concept for the utility of genomics to inform disease biology and risk after CAR-T. We hypothesize that tumor genetic aberrations in cellular pathways used by CAR-T cells to exert cytotoxicity drive treatment resistance by rendering cancer cells insensitive to CAR-T stimuli and supporting immune escape. In Aim 1, we will use comprehensive genotypic and phenotypic tumor profiling before and after CAR-T to study the role of a priori determined genes and pathways in mediating inherent and acquired treatment resistance. We also hypothesize that orthogonal modalities for patient and tumor profiling are complementary, and their integration into a unified, multimodal model could accurately predict CAR-T outcomes. In Aim 2, we will synthesize data from multiple modalities and use machine learning algorithms to predict CAR-T response and identify novel biomarkers. To meet our goals, we have compiled one of the largest CAR-T patient and sample biobanks. A group of leading experts in immunology, genetics, pathology, radiology, machine learning, and bioinformatics will guide the candidate in this multidisciplinary work. If successful, we expect our combinatorial approach to uncover genetic features underlying inherent and acquired CAR-T resistance and identify new druggable targets. Furthermore, our machine learning approach will support treatment personalization by establishing decision support systems and identifying biomarkers of high-risk patients. Finally, we will introduce novel methodologies for modeling CAR-T outcomes, which are extendable to other forms of treatment.

NIH Research Projects · FY 2026 · 2023-08

RNA-binding proteins (RBPs) play key roles in RNA splicing, editing, nuclear export, translation, turnover, and subcellular localization. Reflecting their importance, RBPs and their cis-regulatory elements (CREs) have broad implications in human health: mutations in RBPs or CREs have well-established roles in cancer, developmental defects, particularly in neural development, and in neural degenerative diseases. Using a combination of a high-throughput, in-vitro-selection-based RNA binding assay, RNAcompete, and machine learning (ML) models trained to map from an RBP’s protein sequence to its RNA binding preferences, this project will endeavor to assign RNA sequence- and structural-context binding preferences to all human RBPs, all vertebrate RBPs, and the vast majority of metazoan RBPs. These specificities will then be used to detect and assign function to RBPs and cis-regulatory elements (CREs) in human genomes, as well as those of other model organisms. The specificities, machine learning models, and predicted CREs will be distributed widely via publication, open-source software, and user-friendly web tools like cisBP-RNA. This project has the potential to transform cancer and human genetics research supporting the estimation of the functional impact of germline or somatic mutations on post-transcriptional regulation (PTR). By improving the reconstruction of PTR networks, this project will speed research in this emerging field toward a complete understanding of this key process. This project will also permit the study of the evolution of PTR by developing tools to reconstruct PTR networks in other organisms based solely on genomic and transcriptomic data. RNAcompete will be used to assess the RNA sequence-binding preferences of the 511 still-uncharacterized RBPs in humans and D. rerio (zebrafish), thereby establishing a complete catalog of binding preferences for all likely sequence-specific RBPs in these two species. These data will be combined with binding data for >500 other RBPs from a variety of sources and used to train an ML model that reconstructs RNA-binding preferences given RBP protein sequences. These models will also leverage recent advances in de novo prediction of protein structure from sequence. RBPs will be assigned roles in PTR based on (i) the location and conservation, in human transcripts of their predicted target CREs, (ii) the correlation of their expression with the PTR fate of their putative target transcripts, and (iii) other, more powerful regression methods like the Inferelator. CRE predictions will be continuously improved using in vivo data to recalibrate in vitro motif models and to improve in silico predictions of transcript RNA secondary structure. Our predicted CREs and reconstructed PTR networks will be validated by comparisons with in vivo data collected by our team and others.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT CANDIDATE: One of my long-standing interests is to understand the mediators of a successful antitumoral T cell response. In this application, I am proposing a series of studies where I combine my graduate study background in T cell biology with the expertise of the laboratory of my mentor Dr. Scott Lowe in in vivo cancer modeling and tumor suppression. My research in the Lowe lab at Memorial Sloan Kettering Cancer Center (MSKCC) has focused on how senescence, a common consequence of conventional anticancer therapies, affects the function of T cells. Here I am delineating a plan for transitioning to independence that will allow me to: (i) complete a set of experiments that are leading to discoveries I will be able to continue exploring as an independent investigator; (ii) Acquire a set of expertise and further develop a research line that will separate me from my present and past mentors and (iii) develop a series of professional skills needed for leading a lab. RESEARCH: Cellular senescence in cancer cells is a stress-induced program that results in stable cell cycle arrest and in secretion of a plethora of cytokines that can affect the behavior of other cells, including immune cells. Senescence has been shown to have an immunostimulatory or immunosuppressive role in different contexts. Cellular senescence is a common outcome of antitumoral therapies, yet the precise mechanisms by which senescence alters adaptive anti-tumor immune responses remain largely unexplored. This project aims at studying the functional consequences of senescence on T cells, utilizing novel model of cancer I have developed. In Aim 1, I will explore how different drivers of senescence can lead to a T cell-infiltrated or a T cell-excluded tumor microenvironment. In Aim 2 I will explore how an immunostimulatory form of senescence affects the functionality of T cells that are specific for senescent and non-senescent cancer cells. These studies will inform on how senescence can affect an antitumoral T cell response and on how to rationally design better anticancer senescence-inducing approaches. ENVIRONMENT: MSKCC provides an ideal environment for me to accomplish my training and research goals, and successfully transition to an independent faculty position at an academic institution. My mentor Dr. Lowe is a world leader in cancer biology, with a particular expertise on tumor suppressor programs, mouse models, and functional genetics. In addition, I have assembled an advisory committee of three established scientists with relevant and complementary expertise and strong commitment to mentoring (Drs. Pe’er, Rudensky, and Rosen), who will support my transition to independence by providing valuable research and career guidance. Together with the collaborative environment and broad spectrum of resources at MSKCC, this support network creates optimal conditions for the successful completion of the proposed research and career development plans.

NIH Research Projects · FY 2025 · 2023-08

Older adults with cancer (OACs) comprise a large and growing proportion of cancer patients and survivors. Many OACs have unmet behavioral health needs with significant negative implications for their quality of life, physical health, and adherence to and recovery from cancer care. Efficacious interventions to address these needs have been developed but their implementation into cancer care is limited. Dissemination and implementation (D&I) research can address this gap by creating implementable interventions and identifying strategies for integrating these interventions into routine cancer care. Existing D&I research resources are limited by a lack of tailoring to project-specific needs, a focus on trainees and early career faculty, and insufficient resources to meet current demand. The proposed project addresses these limitations by developing a new interdisciplinary research Center, the Center for Implementation Research in Cancer in Later Life (CIRCL) that will infuse D&I research methods into new and existing projects across all stages of behavioral intervention development for OACs. CIRCL will consist of Administrative, Training, and Research Cores. The Administrative Core will provide leadership, support, and oversight to ensure that the mission and aims of CIRCL are accomplished. The Core will consist of an Executive Committee, Administrative Support Team, External Advisory Council, and OAC and Caregiver Council. The Training Core will provide resources and networking opportunities to train investigators to integrate D&I science methods into their research including a webinar series, work-in-progress webinar, mentorship program, and resource library. CIRCL’s Research Core will evaluate the research needs of investigators and provide resources to facilitate research on the D&I of behavioral interventions for OACs. The Core will administer a national research needs assessment to inform the format and content of CIRCL resources and will support a pilot awards program and annual research conference. The overall project objectives will be met across two phases. During the R21 phase (years 1-2), core components of CIRCL will be developed including hiring administrative staff, launching the CIRCL website, establishing the Advisory and OAC and Caregiver Councils, administering the research needs assessment, and developing standard operating procedures for all CIRCL activities. During the R33 phase (years 3-5), CIRCL’s three cores will work to train researchers and support D&I research projects. The resources available through CIRCL will be advertised nationally with outreach to researchers in academic medicine and community hospital settings. Benchmark evaluation during the R21 phase will ensure CIRCL resources are developed in a timely manner in preparation for the R33 phase. Evaluation of the R33 phase will assess utilization of CIRCL resources and the impact of those resources on the advancement of D&I research on behavioral interventions for OACs.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) offer great promise for basic research and for applications in disease modeling. The initial challenge for exploiting this potential was to direct stem cell differentiation towards specific nerve cell or glial fates relevant to disease. Over the last few years, we have developed many such protocols that now enable researchers to generate > 50 distinct human cell types in a dish. However, a major remaining challenge is the fetal rather than adult-like features exhibited by the resulting cells, which limits their usefulness. The reason for their immaturity is unclear but may be linked to a cell-intrinsic, clock-like mechanism that controls the timing of maturation. While it takes 9 months for a human baby to develop, from conception to birth, the same process takes only 20 days in a mouse. Those dramatic timing differences are recapitulated in a dish, where the maturation of human cells may require many months to reach adult-like properties. Interestingly, we observe such timing differences in both 2D and 3D culture including in neural organoids. Even after transplanting human cells into the mouse brain, cells continue to follow a human-specific maturation trajectory, despite being surrounded by an adult host microenvironment. Here we will address this challenge by building assays to measure and quantify neuronal and glial maturation and by developing strategies to override the intrinsic maturation clock. Towards these goals, we have established a unique stem cell-based assay to produce nerve cells at very high precision and in a temporally synchronized manner. The resulting cells then progressively mature from fetal to adult-like stages over a period of several months allowing us to define markers that predict the neuronal maturation state. In Aim 1, we will build on these preliminary data and establish stage-specific “fingerprints” of nerve cell maturation to determine maturation states at unprecedented precision. In addition, we will characterize the maturation of glial cells (astrocytes and microglia) in a novel tri-culture system to test whether the presence of glia can improve neuronal maturation. In Aim 2, we will apply maturation “fingerprints” as a readout for identifying factors that can accelerate maturation timing. In preliminary studies, we have identified chemicals and genes that are strong candidates for driving neuronal maturation. We will further validate those findings in the tri-culture system to determine the combined effect of intrinsic and extrinsic maturation factors. Finally, we will perform mechanistic studies to understand how those factors induce more adult-like features in human cells. In Aim3, we will test our optimized maturation strategies in more complex 3D culture systems to assess whether induced maturation strategies impact other developmental processes such as cell migration and organization. Finally, we will assess whether “induced maturation” strategies can be adopted to achieve accelerated timelines of neuronal maturation upon transplantation into the developing murine brain, a strategy that could enable new human disease models for the study of neurodevelopmental or neuropsychiatric disorders in vivo.

NIH Research Projects · FY 2026 · 2023-08

Genetic instability is a hallmark of cancer. The cell has evolved an intricate set of pathways that sense and repair DNA damage, which is an inevitable consequence of cellular metabolism and the environment. Failure of a repair pathway, due to either overwhelming DNA damage or pathway inactivation by somatic or inherited mutations, can lead to the propagation of genetic errors that confer a selective advantage to the cell and drive the development of cancer. Among the many DNA repair pathways, those that respond to lesions on both strands of the DNA are particularly important, as their failure can lead to chromosomal instability that can accelerate the loss of tumor suppressor genes and the amplification of oncogenes. Such lesions include DNA double strand breaks (DSBs) and stalled replication forks, which are DNA structures arising during the duplication of the genome. The objective of this proposal is to understand how the cell senses DSBs and stalled forks, and how it triggers a response with wide-ranging effects that include arrest of cell growth and initiation of repair programs. We plan to use the method of cryo-electron microscopy (cryo-EM) to determine the 3-dimensional structures of protein assemblies involved in these processes. Structural information – essentially detailed images – will help us better understand how these pathways work, how they fail in cancer, and may ultimately help identify new approaches to intervene therapeutically. Central to the sensing of a stalled replication fork is the ATR protein kinase that signals to other proteins by phosphorylating them. ATR and its partner ATRIP sense persistent single-stranded DNA (ssDNA) and a dsDNA-ssDNA junction – two defining features of a stalled fork. The ssDNA is coated by the replication protein RPA, which recruits ATR- ATRIP. The dsDNA-ssDNA junction is sensed by another protein complex that loads a clamp, termed 9-1-1, onto dsDNA. 9-1-1 then recruits the TopBP1 protein, which binds to ATR-ATRIP and turns on the phosphorylation activity. This is one assembly, reconstituted from purified proteins, that we plan to investigate with cryo-EM. We also plan to investigate a related assembly, where TopBP1 is replaced with the ETAA1 protein, and which senses different features of a stalled fork. Another aspect we plan to investigate is the remodeling of the stalled fork to facilitate its sensing and repair, and its protection during this process. These functions are carried out by 12 FANC proteins mutated in the inherited Fanconi Anemia Cancer predisposition syndrome. FANCM remodels the fork and recruits a 9-protein complex (FA Core complex) that puts a clamp consisting of FANCI and FANCD2 onto the DNA, likely to protect the fork. The sensing of DSBs is mediated by ATM, protein kinase mutated in the cancer syndrome Ataxia-Telangiectasia. DSB ends, together with a 3- protein complex termed MRN, activate ATM and initiate the DSB response. Our third major goal is to understand how this process works at the level of 3-dimensional structure.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Older adults with cancer (OACs) comprise a large and growing proportion of cancer patients and survivors. Many OACs have unmet behavioral health needs with significant negative implications for their quality of life, physical health, and adherence to and recovery from cancer care. Efficacious interventions to address these needs have been developed but their implementation into cancer care is limited. Dissemination and implementation (D&I) research can address this gap by creating implementable interventions and identifying strategies for integrating these interventions into routine cancer care. Available D&I research infrastructure is limited by a lack of tailoring to population and project-specific needs, a focus on trainees and early career faculty, and insufficient resources to meet current demand. The proposed project addresses these limitations by developing new interdisciplinary research infrastructure, the Center for Implementation Research in Cancer in Later Life (CIRCL) that will infuse D&I research methods into new and existing projects across all stages of behavioral intervention development for OACs. CIRCL will consist of Administrative, Training, and Research Cores. The Administrative Core will provide expert leadership, support, and oversight to ensure that the mission and aims of CIRCL are accomplished. The Core will consist of an Executive Committee, administrative Support Team, external Advisory Council, and OAC and Caregiver Council. The Training Core will provide resources and networking opportunities to train investigators to integrate D&I science methods into their research including a webinar series, work-in-progress webinar, mentorship program, and resource library. The purpose of the Research Core is to evaluate the research needs of investigators and provide resources to facilitate research on the D&I of behavioral interventions for OACs. The Core will administer a national research needs assessment to inform the structure and content of CIRCL resources and will support a pilot awards program and annual research conference. The overall project objectives will be met across two phases. During the R21 phase (years 1-2), core components of CIRCL will be developed including hiring administrative staff, launching the CIRCL website, establishing the Advisory and OAC and Caregiver Councils, administering the research needs assessment, and developing standard operating procedures for all CIRCL activities. During the R33 phase (years 3-5), we will utilize this established infrastructure to train researchers and support D&I research projects. The resources available through CIRCL will be advertised nationally with targeted outreach to investigators in underrepresented groups to increase diversity in the research workforce. Benchmark evaluation during the R21 phase will ensure CIRCL resources are developed in a timely manner in preparation for the R33 phase. Evaluation of the R33 phase will assess utilization of CIRCL resources and the impact of those resources on the advancement of D&I research on behavioral interventions for OACs.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States, with 60 000 cases diagnosed annually and five-year survival rate of only 10%. It is projected to become the second- leading cause of cancer-related mortality by 2030. PDAC is also associated with poor quality of life (QOL), including cachexia, in approximately 80% of patients. Cachexia is a contributing cause to 30% of deaths in PDAC and has no approved pharmacologic treatment. PDAC is also strongly associated with hyperglycemia. Half of patients have diabetes at the time of diagnosis and 80% experience hyperglycemia during treatment; the association is likely bi-directional, meaning that there is both evidence that diabetes causes PDAC and that PDAC causes diabetes. As a result, many patients with PDAC must use antidiabetic drugs such as metformin, sulfonylureas, or insulin. Hyperglycemia management during PDAC is an important aspect of supportive care with direct implications for QOL. Comparative effectiveness research on hyperglycemia management in PDAC is almost certain to improve supportive care by identifying which drugs best balance glucose control, side effects, and maintenance of healthy weight. This need for evidence in this specific population is especially pressing because the usual hierarchy of diabetes drugs may be inverted in PDAC patients. For example, prescribers deprecate sulfonylureas in routine diabetes practice in part because they promote weight gain, but that property could make them especially useful in patients with PDAC. Very little research has been done comparing sulfonylureas to metformin (the most widely used antidiabetic drug) or other alternatives as a supportive care intervention in PDAC. This proposal aims to close this evidence gap by, in Aim 1, conducting a retrospective cohort study testing the hypothesis that, compared to metformin, sulfonylureas are associated with better weight maintenance in patients with PDAC, and in Aim 2, enrolling 40 patients with hyperglycemia undergoing systemic treatment for PDAC in a trial assessing the safety of glipizide, a sulfonylurea, with respect to the key safety outcome of rate of hypoglycemia and the key effectiveness outcome of reducing blood glucose levels. This project will lay the groundwork for phase 3 clinical trials to determine whether choice of antidiabetic drug can improve QOL and even overall survival in patients with PDAC.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT There has been a renewed interest in how oncogenic driver mutations and tumor suppressor losses contribute to cancer-associated alterations in cellular metabolism. Much of the effort has been focused on the avidity with which most cancer cells take up glucose only to release most of the glucose carbon as lactate, a process known as aerobic glycolysis or the “Warburg Effect”. This seemingly wasteful metabolism has puzzled cancer biologists for decades. Nevertheless, aerobic glycolysis has been shown to be a sustainable way to support the continuous production of glycolytic intermediates that are utilized in de novo synthesis of proteins, lipids, and nucleic acids. Over a decade ago, the Thompson laboratory embarked on analyzing tumor utilization of glutamine, the second most common nutrient present in extracellular fluid. While glucose is metabolized by cancer cells primarily in the cytosol, we found that glutamine was metabolized primarily in the mitochondria. Similar to glucose, we found that the majority of the carbon taken up as glutamine was secreted as lactate, a process now known as glutaminolysis. Since that time, the study of glutaminolysis has focused on the role of glutamine as an anaplerotic substrate to maintain mitochondrial function as carbon is taken out of the TCA cycle in the form of citrate to fuel fatty acid biosynthesis and as aspartate to support nucleotide biosynthesis. Tumor cell avidity for glutamine in vivo and the ability of glutamine catabolism to maintain oxidative phosphorylation through TCA cycle anaplerosis has been confirmed in vivo. However, the role of glutaminolysis in supporting tumor nitrogen metabolism is less well understood. Although inhibitors of glutamine metabolism have been explored in cancer therapy, their success in the clinic has been limited in part because of our incomplete knowledge of tumor nitrogen metabolism. Understanding the role of nitrogen metabolism in supporting cancer cell survival and growth has become the central focus of the Thompson laboratory. We are currently exploring the hypothesis that glutamine-dependent mitochondrial glutamate accumulation provides the cell with an intracellular reserve of reduced nitrogen that can be directed toward mitochondrial support of de novo polyamine production, amino acid biosynthesis, and glutathione generation. We are also studying how the differential fates of mitochondrial glutamate are regulated by growth factors, as well as by oncogenes and tumor suppressors. While the normal pool of mitochondrial glutamate is fed by extracellular glutamine uptake, we also plan to test whether the combination of lactate and ammonia that accumulates in the tumor microenvironment (TME) under nutrient-poor conditions can be utilized to restore mitochondrial glutamate and cytosolic glutamine to levels that support adaptive translation and cell survival. These results will help clarify how cancer cell avidity for nitrogen is satisfied based on nutrient availability and the presence of specific oncogenic mutations and tumor suppressor losses. The insights gained will help to shape new approaches for the diagnosis and treatment of cancer.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Increased oncogene expression mediated by focal amplifications is a common mechanism for oncogene activation in human cancers. Two major mechanisms leading to oncogene amplification have been described: chromosomal amplification and non-chromosomal amplification. The latter mechanism is characterized by the presence of multiple copies of circular DNAs that are thought to originate following the fragmentation and subsequent circularization of pieces of chromosomes. These “extrachromosomal circular DNAs” (ecDNAs) have long been known as “double minutes” for their appearance in metaphase spreads and by the lack of centromeric sequences. In the past few years renewed interest in this class of cancer-associated chromosomal rearrangements has been fueled by technological advances and by the realization that, due to their random segregation at mitosis, ecDNAs can accelerate tumor evolution, mediate drug resistance, and generally promote a more aggressive phenotype. Despite substantial progress, however, several key questions regarding the biology of ecDNAs, their dynamics during the early stages of tumor formation, and their contribution to tumor initiation and progression, remain unanswered. This is in part due to the lack of effective means to engineer and track ecDNAs in normal cells and in model organisms. Our group has extensive expertise in the generation and characterization of germline and somatic mouse models of human cancers, and we have pioneered the use of somatic genome editing to engineer chromosomal rearrangements in mice. In this grant application, supported by strong preliminary data, we describe a novel general strategy to model ecDNAs in cells and in mice. We have already generated three new genetically engineered mouse strains in which the formation of ecDNAs containing the oncogenes most commonly amplified in human cancers can be induced in a temporally and spatially controlled manner. Using a similar strategy, we have also generated cell lines in which formation of specific ecDNAs can be induced and tracked non-invasively using fluorescent reporters and selectable markers. We propose to use these innovative tools and reagents to address the following key questions: 1) Can oncogenic ecDNAs initiate tumor formation and/or accelerate tumor progression and metastasis in vivo? 2) How do oncogenic ecDNAs respond to changes in intracellular and extracellular environment? 3) Are there mechanisms preventing ecDNA formation and propagation in primary cells? 4) Is the presence of ecDNAs in cancer cells associated with unique therapeutically actionable vulnerabilities? 5) Can ecDNAs be transmitted horizontally between cells?

NIH Research Projects · FY 2025 · 2023-07

Research: The ability to monitor malignant tumor burden below the limit of radiographic detection remains a major unmet need. Liquid biopsy for circulating tumor DNA (ctDNA) offers promise, however, deep targeted sequencing methods – the conventional approach in the field – face a sensitivity plateau in low volume cancer due to the sparsity of ctDNA signal. Whole genome sequencing (WGS) of plasma overcomes this sensitivity barrier by expanding the number of informative sites to the thousands of somatic single nucleotide variants observed across the genome in solid tumors. We showed with our tumor-informed MRDetect framework that WGS of plasma can increase liquid biopsy sensitivity by 1-2 orders of magnitude beyond deep targeted sequencing methods. To expand applicability and overcome MRDetect’s need for matched tumor tissue, I built MRD-EDGE, a plasma-only (de novo) classifier that uses advanced machine learning to increase error suppression and amplify ctDNA signal. My preliminary data shows that MRD-EDGE can quantify ctDNA tumor burden during the nadir of response to immunotherapy in patients with advanced melanoma, and can demonstrate a response to treatment as early as 3 weeks after first infusion. MRD-EDGE therefore enables precise monitoring of malignant disease burden in response to therapy using standard WGS alone. In this proposal, I aim to first radically improve MRD-EDGE sensitivity by including epigenetic features that inform likelihood of cancer mutagenesis, which I hypothesize will allow for unprecedented plasma-only liquid biopsy sensitivity. I will then use the optimized MRD-EDGE platform to define early response or resistance to immunotherapy in metastatic melanoma, which will establish ctDNA as a biomarker that can complement or replace imaging. Finally, I will optimize MRD-EDGE for use in lung cancer and use the platform to monitor response to neoadjuvant immunotherapy and detect postoperative minimal residual disease. I expect that ultra-sensitive monitoring of ctDNA dynamics in the neoadjuvant period can guide precision adjuvant therapy in the postoperative period and thereby provide a transformative impact on patient care. Candidate: I am an Instructor of Medicine at Memorial Sloan Kettering Cancer Center (MSK) and a Visiting Fellow at the New York Genome Center (NYGC). I have outlined a 5-year career development plan to transition to an independent, tenure-track physician-scientist investigating the detection and monitoring of solid tumors through ultrasensitive liquid biopsy. I will conduct the proposed research under the mentorship of Dr. Dan Landau, an internationally recognized expert in liquid biopsy and cancer genomics. I will use the K08 award to further develop skills in next-generation sequencing methods and analysis and advanced machine learning. MSK and the NYGC are ideal environments in which to pursue my scientific and career goals. Both institutions have world class research communities and an outstanding track record of training independent physician-scientists.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The transcription factor p53 is one of the most critical barriers to tumorigenesis. p53 is mutated in over half of all human tumors, and p53-mutant tumors typically carry a worse prognosis. As a tumor suppressor, p53 appears to act at the transition from benign to malignant disease, preventing transformation of cells that have already acquired some pro-tumor features, such as oncogene activation or DNA damage. Decades of research have sought to identify what makes p53 a potent tumor suppressor, as such knowledge could inform effective strategies for cancer prevention and treatment. This research has shown that p53 can be activated by a variety of signals, and in turn activated p53 can regulate a wide variety of cellular processes, including cell survival, senescence, genomic stability, and plasticity. However, the inducers and actions of p53 vary with context, and there is still no consensus on what signals engage p53 during early neoplasia and what biological programs are most important for its tumor-suppressive functions. Furthermore, we still do not know the key events following p53 loss that enable transition to malignancy. Attempts to gain this knowledge have been hampered by a lack of tools to directly study p53 in the specific cells undergoing transformation within endogenous contexts. To address these gaps in knowledge, we will study the events surrounding p53 activation and loss during the initiation of pancreatic ductal carcinoma (PDAC), an aggressive cancer in which p53 loss—which occurs in 70% of PDACs—enables progression from benign precursor lesions to full-blown cancer. We recently developed new mouse models of PDAC that allow us to “see” p53 in action as cells progress through the benign-to-malignant transition. Among the premalignant pancreas cells, we discovered a subpopulation that shares many transcriptional features with established tumor cells, and thus these cells appear to be transitioning from premalignant to malignant. These transitioning cells are also the cells with the strongest p53 activation, and so they provide a unique opportunity to study p53 engagement and tumor-suppressive function at the benign-to-malignant transition. Using single-cell transcriptional and spatial analyses, new computational approaches to infer cell state transitions, and our well-established platform for rapid genetic perturbations in vivo, we will define the cell-intrinsic and cell-extrinsic events that lead to this p53-active transitioning cell state. Furthermore, we will investigate the mechanisms by which activated p53 suppresses neoplastic transformation in the transitioning cells, as well as the events that influence cancer initiation immediately following p53 loss. This project will provide novel insight into the mechanisms that drive PDAC initiation and provide a direct and detailed characterization of p53 in action in endogenous contexts. Given the high prevalence of TP53 mutations in human cancers, we expect the insights into tumorigenesis to be applicable to many cancer types.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Candidate: Maria I. Carlo, MD is an Assistant Attending in Medicine at Memorial Sloan Kettering Cancer Center (MSK) with a dual appointment in the Genitourinary Oncology and Clinical Genetics services. Dr. Carlo's clinical and research interests are in hereditary RCC, a cancer that has a poor prognosis when identified in advanced stages. Under the mentorship of Kenneth Offit, MD, MPH and Ari Hakimi, MD she has begun work to elucidate the role of KEAP1 in the susceptibility to RCC and to define the phenotype of RCCs with KEAP1 mutations. Dr. Carlo's goal is to develop an independent laboratory to do translational work in the genetic predisposition to RCC and its implication for cancer screening and targeted treatment. Career Development Plan: Drs. Carlo, Offit, and Hakimi have developed a plan to ensure that Dr. Carlo has the necessary training, mentorship, and support to effectively transition to an independent researcher who can successfully lead genomic discovery studies in RCC. This plan entails formal courses in genetic epidemiology, bioinformatics, and cancer modeling, informal collaborations with scientists from MSK laboratories, and training with personnel from MSK core facilities. Dr. Carlo has organized an Advisory Committee with expertise relevant to her proposal, and they will guide her in successfully completing the goals of her proposed research. Dr. Offit and the Advisory Committee will also guide Dr. Carlo to ensure progress in the promotion process and garnering independent research funding towards the end of the K08 award period. Research Plan: Despite several known genetic RCC syndromes, the majority of familial RCC remains unexplained. The proposed project will use a large cohort of 928 patients with RCC who have undergone parallel tumor and germline targeted exome sequencing. In a subgroup of patients with RCC of unclassified histology, germline and somatic predicted loss-of-function variants were identified in KEAP1, which encodes a negative regulator of NRF2, the key activator of the antioxidant response pathway. These tumors are histologically similar to Fumarate Hydratase (FH)-deficient RCCs, which arise from germline mutations in the FH gene. Loss of function of FH or KEAP1 can activate the NRF2 pathway. Dr. Carlo hypothesizes that, similar to FH, KEAP1 loss-of-function germline variants increase risk of RCC, and mutations in KEAP1 contribute to the development of RCC in an NRF2-dependent manner. Dr. Carlo aims to (1) characterize KEAP1-mutated RCC using genomic, transcriptomic and metabolic techniques, and (2) delineate the effects of KEAP1 and FH mutations on malignant transformation in RCC model systems. The overarching goal is to elucidate the role and implications of germline and somatic KEAP1 mutations in RCC to direct cancer screening and develop rational targeted therapies.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a genetically complex and heterogeneous set of diseases characterized by a diverse set of mutations. Although many patients initially respond to treatment, many end up relapsing. Over the last decade, an appreciation of the genetic diversity and clonal hierarchy in AML has opened the door to novel therapeutic targets and therapeutic approaches to specific AML subtypes. Moreover, AML has been found to bear unique metabolic features with therapeutic implications. Most importantly, mutations in the enzymes isocitrate dehydrogenase (IDH1/2) have led to clinically approved drugs. However, many patients become resistant to this therapy as well, further underscoring the need for new strategies to target dysregulated metabolism in leukemia. Through the development of novel microcoil platforms to explore leukemia metabolism with HP MR (Jeong et al. Science Advances 2017) we have identified a new metabolic vulnerability in the glycolytic metabolism of leukemia (Jeong et al. Cell Metabolism 2021). This reliance on glycolytic metabolism alters not only glucose flux to lactate, but also one-carbon flux through the serine pathway, which facilitates the metabolism of glutamine. Moreover, we found that genetically targeting or pharmacologically inhibiting the enzyme that mediates flux through this pathway (PHGDH) capitalizes on a new vulnerability in these cells. Importantly, this targeting does not affect normal hematopoietic cell growth. Thus, building upon extensive collaboration between our labs and ample preliminary data, we aim to employ innovative approaches to study metabolism (Keshari Lab), including by developing non-invasive probes to measure changes in glycolysis and oxidative stress with hyperpolarized magnetic resonance imaging. This metabolism will be characterized in well- defined models of AML (Kharas Lab), with both genetic and pharmacological modulation, in order to develop a strategy to assess leukemia-stem-cell-driven AML metabolism and the inhibition of serine metabolism. Altogether, these studies will result in new mechanistic insights and novel cancer therapies.

NIH Research Projects · FY 2026 · 2023-07

SUMMARY We seek to develop a new class of drugs for RAS-MAPK driven cancers by targeting the interfacial binding sites of key regulatory complexes within the cascade. By moving away from conventional active site-based drugs, we have the potential for a unique class of compounds with advantages in terms of selectivity, target engagement, therapeutic index, and combinatorial activity to mitigate the emergence of drug resistance. The MAPK/ERK Kinase (MEK) MEK is a shared effector of KRAS and BRAF, which are among the most frequently mutated oncogenes and protein kinases across all human cancers. As such, MEK has long been pursued as a drug target in oncology, and more recently in immunotherapy and aging. However, many drugs that target MEK are limited due to on-target associated toxicities and drug resistance. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. My laboratory has made initial steps in this direction through the determination of X-ray crystal structures of MEK bound to the RAF paralog Kinase Suppressor of Ras (KSR), and in complex with various MEKi, including the first ever co-crystal structures bound to the clinical drug trametinib (Khan et al., Nature, 2020). Unlike most targeted therapies, trametinib was serendipitously identified through phenotypic screens, and X-ray crystal structures had been lacking. Our novel structural and functional insights have revealed an unexpected mode of binding in which the inhibitor pocket for trametinib is formed through the interface between MEK and KSR, revealing KSR as a direct co-receptor of the drug and trametinib as an ‘interfacial binder’. Moreover, our studies suggest that the unique therapeutic properties of trametinib derive from the ability of the drug to bind at the interface of the complex. Building from these insights, we have developed a tool compound, trametiglue, with enhanced interfacial binding properties and several novel pharmacological features, including unprecedented potency and an ability to overcome a common resistance mechanism to trametinib and other clinical MEKi. This proposal focuses on developing an advanced set of analogs through structure-based design and synthesis. Our targets include advanced trametiglue analogs, including paralog-selective molecular glues to target individual MAPK signaling complexes that have been implicated in RAS-MAPK driven cancers and sensitivity to currently available drugs (Aim 1). In vivo target engagement and optimization of drug-like properties with this expanded set of analogs (Aim 2). Testing in preclinical cancer models, including patient derived organoids and xenografts (Aim 3). There are over 5 million individuals diagnosed globally with RAS-MAPK driven cancers on a yearly basis. Despite recent therapeutic breakthroughs with, for example KRAS-G12C and BRAF-V600E inhibitors, over 90% of RAS-MAPK tumors are unactionable. Advances in this proposal will lead to next generation drugs and a new therapeutic modality for selectively antagonizing RAS-MAPK driven malignancies in patients.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Parkinson's disease (PD) is one of the most common neurodegenerative disorders. It is characterized by the progressive loss of dopamine neurons in the substantia nigra (SN) pars compacta, and their projections onto striatal neurons and the accumulation of α-Synuclein aggregates often into Lewy bodies. The pathogenesis of PD is not fully elucidated but there is vast evidence supporting complex loops of neuroinflammatory cascades, mitochondrial dysfunction, degenerating neurons, sustained microglial activation and other pathophysiological mechanisms that together amplify a relentless progression towards neuronal loss in the nigra and beyond. α- Synuclein (α-S) aggregates are implicated either directly in serving as a template that is transmitted transneuronally and seeding further aggregation, and/or in amplifying the neuroinflammatory loop, leading in both cases to neuronal dysfunction and death. To date, there are no therapeutic options that lead to the regeneration of lost neurons or to the restoration of circuitry. Our group has pioneered the derivation of functional dopamine neurons from human embryonic stem cells (hES) and we have just completed a Phase 1 clinical trial for the bilateral intrastriatal grafting of these cells. There is much excitement about the restorative potential of stem cell derived neurons in PD, but there remain multiple challenges. Here we propose to study the impact of microenvironmental alterations in the brain in the context of 2 different mouse models: the 3K mouse model which expresses a triple mutant form of α-synuclein based on the human E46K mutation, and exhibits histological hallmarks of PD as well as progressive motor and other behavioral abnormalities; the second model consists of the intrastriatal injection of preformed α-synuclein fibrils (PFF) which spread transneuronally through the brain to form pathogenic α-synuclein inclusions, leading to loss of DA neurons and behavioral deterioration. These models are predicated on two different hypotheses and will serve as great tools to study inflammation and its impact on behavior. In addition, we will graft the mice with the same hES cell derived dopamine neurons used in the clinical trial to analyze the impact of the microenvironment on the neurons' survival and phenotype, as well as on their ability to rescue behavior. We will capture grafted cells as well as host microglia and astrocytes at key timepoints during the in vivo lifespan of the grafts to establish dynamic maps of cell lineages, maturation, microglial and astrocytic phenotypes and potentially activation of neurotoxic signals. In the last aim, we will engineer the human ES cells to delete the SNCA gene encoding α-synuclein in an attempt at increasing the resistance of the grafts to neurotoxicity and potentially the transmission of pathogenic α- synuclein. Data obtained in this proposal will serve to further enhance our understanding of neuro-inflammation in different PD microenvironments and could result in enhanced strategies for cell grafting including the use of gene edited cells that are resistant to neuroinflammation.

NIH Research Projects · FY 2026 · 2023-06

SUMMARY This project endeavors to build a nanosensor array platform technology to detect whole disease fingerprints from patient biofluids to facilitate diagnosis, screening, and biomarker discovery efforts. Serum biomarker measure- ments are widely used as diagnostic indicators, but many markers are not sufficient for assessments of disease state. Major factors limiting diagnosis and screening using most biomarkers include their low specificity for dis- eases and the overall dearth of established molecular markers. Innovative approaches are needed to identify new biomarkers and/or improve screening and diagnostic efforts in the absence of validated biomarkers. We believe that the differentiation of diseased from normal biofluids may be achieved by the detection of a “disease fingerprint” through the collection of large data sets of molecular binding interactions to a diverse set of moder- ately-selective sensors, which are used to train machine learning algorithms. We will build a sensor array com- prising organic color centers (OCCs, covalently-modified carbon nanotubes) to transduce subtle differences in physicochemical properties of molecules in biofluids. With sufficient diversity, the sensors can differentiate bi- ofluids by disease status with the aid of machine learning processes. In preliminary experiments, we found that a library of OCC-DNA nanosensors exhibited sensitive and differentiated spectral variation to probe an ensemble of molecular binding events. Via machine learning algorithms, we built a prediction model of nanosensor re- sponses that reliably identified high-grade serous ovarian cancer (HGSC) substantially better than the estab- lished, FDA-approved biomarker, CA125, using an initial set of 264 patient serum samples (Nat Biomed Eng, 2022). Despite advances in the understanding and management of HGSC, survival is currently poor when diag- nosed at later stages, and detection is uncommon at early stages. Surgery is the first-line treatment, and cancer recurs in 70% of patients in remission. Secondary surgery can prolong survival but only if performed early enough to enable complete resection. Improved detection of early-recurrent and early-stage HGSC would therefore markedly increase survival rates. We plan to develop a robust diagnostic sensor platform to improve early de- tection of ovarian cancer and recurrence, and to accelerate biomarker discovery processes. Additionally, quan- titative analysis of proteins bound to the sensors can determine the unique pattern of protein adsorption respon- sible for the disease-specific spectral responses, thereby potentially facilitating biomarker discovery. We propose to investigate: 1) the diversity of molecular sensitivities of OCC-DNA nanosensor elements required to differen- tiate patient samples, 2) machine learning-based classification of disease, focusing on early-recurrence and early-stage HGSC, 3) the molecular mechanism of the sensor response, and 4) the potential of the array to facilitate identification of novel biomarkers. Successful completion of this work will result in a validated platform to enable concomitant identification of disease and acceleration of biomarker discovery processes in HGSC, with applicability to many potential indications.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY T cell–mediated autoimmune diseases result from the breakdown of tolerance mechanisms in self-reactive CD8 T cells. However, many aspects of autoimmune CD8 T cell differentiation remain enigmatic, including where and how autoimmune T cell populations arise and are maintained and what molecular programs define autoimmune T cell states. Type I diabetes (T1D) is a CD8 T cell–mediated autoimmune disease; T1D pathogenesis is complex and involves immune infiltration of the pancreas and destruction of insulin-producing β cells by CD8 T cells. The non-obese diabetic (NOD) mouse model is a clinically relevant model of T1D, which shares many features with human disease. Utilizing the NOD model, we investigate autoimmune β cell-specific CD8 T cells differentiation state dynamics over the course of T1D. We identified a stem-like progenitor CD8 T cell population in the pancreatic lymph node that self-renews and gives rise to differentiated progeny, which migrate to the pancreas and destroy β cells. The goal of this application is to generate a deep mechanistic understanding of the niche- dependent intercellular interactions and signals in the pancreatic lymph node that maintain the autoimmune stem-like progenitor T cell pool and regulate differentiation, and to use this knowledge to develop strategies for therapeutic interventions. We will (i) employ innovative imaging and sequencing approaches to identify the spatial organization of pancreatic lymph node niches that determine diabetogenic T cell responses, (ii) determine the functional roles of key transcription factors controlling autoimmune T cell differentiation and test whether deletion or enforced expression of these transcription factors can alter autoimmune T cell states, and (iii) investigate autoimmune β cell-specific CD8 T cell states in human pancreatic lymph nodes and pancreas from organ donors with T1D. If successful, the proposed studies will provide important insights into autoimmune β cell-specific CD8 T cell programming in mouse and human T1D and could yield promising molecular and cellular targets for the prevention or treatment of T1D and other T cell-mediated autoimmune diseases.

NIH Research Projects · FY 2025 · 2023-06

Two-thirds of oral and oropharyngeal squamous cell carcinomas (OSCCs) occur in low- and middle-income countries (LMICs), with 5-year survival rates of only 10-40%. The poor survival rate in LMICs is due to late diagnosis and treatment. Thus, it is imperative to detect potentially malignant lesions early and expeditiously. To meet the need for oral cancer screening in low resource settings (LRS), we will develop and validate a low- cost mobile phone-based imaging device powered by computer vision and deep learning image classification algorithms to guide patient triage. We are a multi-institutional team comprising of optical imaging and machine learning engineers and oral/head-neck oncologists, at the University of Arizona, Memorial Sloan Kettering Cancer Center and Tata Memorial Hospital (TMH, Mumbai, as the LMIC setting). In preliminary studies, our team has developed and tested the hardware: a dual-mode polarized white light imaging (pWLI) and autofluorescence imaging (AFI) mobile device. Non-expert field healthcare workers read images with (low) sensitivity of 60%. Additionally, a preliminary deep learning classification algorithm, implemented on a cloud- based server computer, demonstrated improved sensitivity of 79% and specificity of 82%. Our proposal is to address the key remaining hurdle – improving the reading skills of non-expert field healthcare workers – locally in LRS in LMICs, which do not have internet and cloud connectivity. We will develop and validate the required software: machine learning (deep learning) image classification algorithm on a mobile phone, to guide field healthcare workers in triage of oral lesions into benign (patients can go home) versus suspicious (patients referred to clinician for follow up care). The innovations will be in design and integration of computer vision (image mosaicking) and deep learning classification algorithms on a mobile phone-based imaging device, to provide high accuracy and consistency for screening. Novel aspects will be in (i) the deep learning approach for dual-mode image contrast: pWLI contrast for color and texture of normal features (increasing specificity) and AFI contrast associated with malignancy (increasing sensitivity) and in (ii) engineering of the algorithm for use on mobile devices, via teacher student learning-based knowledge distillation techniques The clinical innovation will be first-in-humans testing for improvements in sensitivity and specificity relative to that of purely visual interpretation, for routine use by non-expert field healthcare workers in LRS. In the R21 project, we will develop a mobile deep learning-based oral lesion screening and patient triage algorithm and demonstrate feasibility in a cancer care setting (TMH’s main hospital in Mumbai). In the R33 project, we will optimize the algorithm, test and validate in a large study in a field setting at TMH’s regional clinic in Varanasi. Successful completion of this project will deliver urgently needed capabilities to field healthcare workers in LRS, for early detection and triage of oral potentially malignant lesions, improving early oral cancer detection rates, allowing timely referral to specialists, improving treatment outcomes and improving quality of life for patients in LMICs.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT High grade serous ovarian cancer (HGSC) is the most lethal gynecologic malignancy. Patients with increased risk of ovarian cancer due to inherited syndromes–most notably carriers of pathogenic variants of BRCA1/BRCA2–are recommended to undergo prophylactic risk reducing salpingo-oophorectomies (RRSO) because there is no effective ovarian cancer surveillance. Despite undergoing surgical removal of ovaries and fallopian tubes, 10-20% of patients will experience primary peritoneal cancer, an ovarian cancer related malignancy. As such there is a dire need to understand the early events in HGSC development to improve therapeutic decisions for high-risk patients. Importantly, a recent study found that the presence of serous tubal intraepithelial carcinoma (STIC) at RRSO was associated with 10.5% risk of developing primary peritoneal carcinoma (PPC) compared with a 0.3% risk in patients with normal fallopian tube histology at the time of surgery. HGSC is characterized by high rates of genomic instability arising from ongoing chromosome missegregation due to defective mitotic machinery or errors in DNA repair and replication. We hypothesize that genomic instability is a critical event in the transition between STIC and invasive HGSC, endowing cancer cells with karyotypic diversity needed for invasion and immune evasion. We will investigate this hypothesis by 1) Quantifying cGAS, a marker for cytosolic DNA, in fixed patient samples as a biomarker for chromosomal instability. 2) Interrogating disruption of the local immune microenvironment in STIC and HGSC as a function of aneuploidy 3) Quantifying rates of aneuploidy in single cells as a marker for early transformation and 4) Integrating the molecular and imaging markers to identify and validate features associated with invasive STICs. An understanding of the processes that mediate the transition between non-invasive STIC to invasive HGSC will lay the groundwork for discovery of early detection markers. Moreover, as genetic testing increases and identifies high-risk patients for RRSOs and even normal risk patients more often undergo opportunistic salpingectomies, it is likely we will identify patients with STICs. This project will allow us to focus on patients with evidence of genomically unstable STICs who may be at a greater risk to develop subsequent PPC and thus may require closer surveillance and/or adjuvant chemotherapy. Moreover, the FFPE tolerant scWGS technology developed as part of this proposal will allow us to interrogate the timing of genomic instability and clonal relationships between STIC and HGSC. This will fundamentally enhance our understanding of the events in ovarian cancer initiation and lays the foundation to further improve early detection of HGSC in patients.

NIH Research Projects · FY 2025 · 2023-06

Project Summary Metastatic colorectal cancer (mCRC) is the second leading cause of cancer-related mortality in the United States, and annually accounts for nearly 500,000 deaths worldwide. Currently, the small molecule kinase inhibitor (KI) regorafenib is the primary second line therapy for metastatic CRC that is not treatable with immunotherapy or anti-EGFR therapies. However, regorafenib generally provides only modest improvements in survival— typically months—and often at the cost of significant side effects. Proposed targets for regorafenib include kinases that act within tumor cells as well as non-autonomously; however, with over 500 possible targets in the human kinome, the exact mechanism by which this compound operates remains controversial and not fully known. This presents a daunting challenge; without a verifiable target or mechanism, no clear path exists to guide the development of improved therapies for mCRC. Here, we propose an alternative approach to drug development that focuses on kinase networks in the context of the whole animal. Specifically, we will take a multidisciplinary approach to define kinases that are beneficial to inhibit (‘pro-targets’) or avoid (‘anti-targets’) in the context of KRAS-variant CRC. Using Drosophila and mammalian models, we will identify kinases that—when reduced—alter the efficacy of regorafenib and similar compounds. We will also conduct extensive structure-activity relationship analyses, evaluating how modifications in already identified lead compounds impact changes in efficacy and therapeutic index. Finally, we will use computational structural biology to convert our chemical genetic insights into highly optimized and precise polypharmacological leads. In this final step, we generate new analogs to selectively eliminate putative anti-target activity while maintaining or increasing inhibitory activity against other beneficial targets. We have used our chemical genetic platform to identify a promising lead compound, APS5-86-2, that demonstrates significant activity relative to regorafenib in several mCRC models, including human patient derived xenografts (PDX). Comparative analysis suggests that the improved activity of APS5-86-2 relative to regorafenib derives from distinct polypharmacology on several RTKs and critical cancer drivers, including CDK9, AURKA, EGFR, BRAF, and RAF1. In this proposal, we examine the mechanism and importance of these and other putative pro- and anti-target kinases using genetic analysis and in vivo target engagement. The objective is to identify the kinase networks that mediate KRAS-variant mCRC by combining chemical biology with genetics, and to then derive inhibitors that best attack these networks through structure-based drug design. We have been successful previously with a similar approach, but in less complex tumor models (Dar et al., Nature, 2012; Sonoshita et al., Nature Chem. Bio., 2018); here we seek to extend our platform to a more prevalent disease with the goal of directly impacting mCRC by creating new, highly differentiated, and improved drugs.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Abstract Candidate: Asmin Tulpule, MD, PhD, is a pediatric oncologist and physician-scientist whose long-term goal is to lead an independent laboratory-based research program studying the basic biology and therapeutic targeting of fusion oncoproteins in cancer. His recent first-author paper in Cell, as well as contributing authorship on publications in Nature Medicine, Nature Genetics, Cell Reports and first-author papers from his graduate work in Cell Stem Cell and Blood, demonstrate his potential as a cancer researcher. This K08 application is crucial for his ongoing career development by providing him with key mentorship and training in 1) Mouse modeling of human cancer and pre-clinical drug testing, 2) Microscopy techniques to study Biomolecular Condensates, and 3) Training in RTK and RAS/RAF biology. Research: Resistance to standard of care tyrosine kinase inhibitor (TKI) therapy is inevitable for the majority of patients with RTK fusion-driven lung and other advanced cancers and remains the major cause of mortality. Dr. Tulpule recently found that multiple RTK fusion oncoproteins self-assemble into membraneless cytoplasmic RTK protein granules that locally coordinate RAS activation and are critical for oncogenesis. The goal of this proposal is to develop mechanistically informed therapeutics that target the unique oncogenic signaling properties of this cancer-specific biomolecular condensate. The following specific aims are proposed: 1) To test SHP2 inhibitors in xenograft models as a new RTK protein granule-directed therapeutic strategy, and 2) To determine how membraneless RTK protein granules activate RAF kinases. Mentorship and Training: Dr. Tulpule’s training will be accomplished through direct teaching of laboratory techniques, formal coursework, academic conferences, and frequent meetings with his Advisory Committee. Asmin's primary mentor is Trever Bivona, MD, PhD, a thoracic oncologist with expertise in molecularly targeted therapies and RAS biology. Dr. Bivona has mentored four post-doctoral fellows to independence within the past five years and has extensive research support from the NIH (4 NCI-funded R01’s). Dr. Tulpule has assembled a tremendous team of physician-scientists for his Advisory Committee including co-mentor Alejandro Sweet-Cordero, MD (pediatric oncologist and expert in xenograft modeling), Bo Huang, PhD (expert in high-resolution imaging techniques), Kevin Shannon, MD (expert in RAS biology and mouse models), and William Weiss, MD, PhD (expert in cancer signaling). This Advisory committee has a significant track-record of mentoring trainees to independence and will provide the necessary scientific and mentoring support. Environment. Dr. Tulpule's training and research will occur at University of California, San Francisco, a world- renowned center of biomedical research with almost $700 million in support from the NIH (ranking 3rd among all institutions). He has the full support of the Division of Pediatric Oncology and the UCSF Cancer Center, both of which have a long-track record of supporting physician-scientist investigators.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Hypertension (HTN) is the most common cardiovascular (CV) comorbidity among patients with breast cancer and is an important modifiable risk factor for adverse CV events during and after cancer treatment. Work by our group and others has shown that HTN is an important risk factor for cardiotoxicity caused by curative breast cancer treatments including anthracyclines and human epidermal growth factor receptor 2 (HER2) targeted agents, which occurs in up to 20% of patients receiving these therapies and presents with a reduced left ventricular ejection fraction or heart failure. Furthermore, cardiotoxicity is a leading treatment-limiting toxicity that interferes with curative cancer treatment delivery, worsens cancer outcomes, and leads to persistent impairment of cardiorespiratory fitness in long-term survivors of breast cancer. CV disease is now a leading cause of morbidity and mortality among breast cancer survivors who are living longer due to advances in cancer care, therefore strategies to mitigate CV risk in patients with breast cancer are critically needed. No standard treatment option is currently available to prevent cardiotoxicity during cancer treatment, and no guidelines exist to inform the optimal approach to blood pressure control during cancer treatment. Multiple trials have shown that intensive blood pressure control is associated with CV risk reductions, however exclusion of patients with cancer represents an important limitation of these trials. The association between HTN and cardiotoxicity risk provide a strong rationale for optimizing blood pressure control to improve CV health and reduce cardiotoxicity risk in patients with HTN who are most vulnerable, however no previous trial has assessed the role of intensive blood pressure control on the cardiotoxic effects of breast cancer treatment. The objective of this study is therefore to evaluate intensive systolic blood pressure (SBP) control in women with HTN at risk for cardiotoxicity during BC treatment and the effects of intensive SBP control on biomarkers (imaging, functional, and circulating) of cardiotoxicity. Using a randomized controlled trial design, 130 patients with breast cancer at increased risk for cardiotoxicity (defined by baseline SBP ≥130 mm Hg and treatment with anthracyclines with or without HER2- targeted therapy) will be randomly allocated (ratio 1:1) to intensive SBP control (goal SBP <120 mm Hg) versus standard SBP control (goal SBP <140 mm Hg) prior to initiating breast cancer treatment. Aim 1: Evaluate the efficacy of an intensive SBP control intervention during active BC treatment in patients at risk for cardiotoxicity. Aim 2: Evaluate the effects of intensive SBP control on imaging and functional biomarkers of cardiotoxicity. Aim 3: Assess the effect of intensive SBP control on circulating biomarkers of cardiotoxicity. The results from this investigation will: 1) establish critical data to inform clinical implementation of intensive SBP control for patients with breast cancer at risk for cardiotoxicity, 2) provide functional and mechanistic insights into the effects of intensive SBP control on mitigation of cardiotoxicity risk, and 3) guide future cardio-oncology practice recommendations on the role of HTN management to improve CV health in patients with cancer.