Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2024-04

Project Summary Caregivers in emerging and young adulthood account for nearly half of all caregivers in the United States; however, they are significantly underrepresented in cancer caregiving scholarship. Little is known about their unique psychosocial needs, and there is a dearth of cancer caregiving resources to support them. Emerging and young adult caregivers (EYACs, ages 18-35) are a particularly vulnerable caregiving population. They experience higher rates of psychological distress than their older counterparts, as well as lasting impacts to their developmental trajectory as a result of their caregiving experience. Caregiving at this age is particularly challenging when an EYAC must provide care to a parent with cancer, as they must undergo a distressing relational shift = that creates psychological and communication challenges unique to this patient-caregiver dyad. Diagnosed parents are known to withhold information about their cancer from their adult children, which creates further difficulties for EYACs in communicating and coping with their parents. Open family communication during cancer has been linked to better social, psychological, and physical health outcomes for both caregivers and patients. Thus, there is a significant need for a communication skills intervention directed at EYACs of their parents with cancer to help them navigate the interpersonal challenges associated with their caregiving role that can lead to distress. The goal of this project is to identify the complex communication needs unique to this age group and adapt an existing caregiver communication skills training intervention (Healthy Communication Practice, HCP) to meet their age-specific communication needs. The K99 phase of this project will include two aims: 1) identify EYACs’ unmet communication skill needs via online survey, and 2) use the survey findings to adapt the intervention materials and pre-test them in EYAC focus groups. The R00 phase of the project will pilot test the new EYAC-tailored intervention for feasibility and acceptability. The objective of this application will not only create the first communication support intervention specifically designed for EYACs, but it will also equip Dr. Kastrinos with the necessary skills and training to complete the proposed research and transition to research independence. She will advance her training in four key areas: 1) psychosocial intervention development and adaption in cancer caregiving, 2) advanced mixed-method and quantitative design and analysis, 3) designing and conducting RCTs, and 4) professional skills development. She will complete both her training and the proposed research with the full support of her mentors (Drs. Allison Applebaum, Smita Banerjee, Yuelin Li), her collaborators (Drs. Carma L. Bylund and Carla L. Fishers) who are the creators of HCP, and her advisory board (Drs. Kathryn Greene and Youngmee Kim). At the end of her R00 phase, Dr. Kastrinos will submit an R01 application to test the efficacy of the new intervention in a fully powered randomized controlled trial. This K99/R00 plan will enable Dr. Kastrinos to achieve her goal of filling a critical research and resource gap for EYACs and launch a research program addressing their needs.

NIH Research Projects · FY 2026 · 2024-04

PROJECT ABSTRACT The management of metastatic bladder/upper tract (urothelial) cancer has been transformed over the past five years, with FDA approval of anti-PD-1/PD-L1 antibodies, an FGFR-selective kinase inhibitor, and most recently two antibody-drug conjugates (ADCs). Despite these advances, most patients with metastatic urothelial cancer still die from their disease. The HER2 receptor tyrosine kinase (encoded by the ERBB2 gene) is implicated in the pathogenesis of several cancer types, and drugs that target HER2 have an established role in the management of breast, lung, and esophagogastric cancers. While HER2 mutations and/or amplification are present in approximately 20% of metastatic urothelial cancers, older HER2-targeted antibodies and kinase inhibitors had only modest clinical activity in urothelial cancer. Unprecedented clinical activity was recently demonstrated with a new generation of HER2-directed ADCs in breast and lung cancers resistant to older HER2- targeted therapies, including HER2-low breast cancer and ERBB2-mutated lung cancer. The current proposal is based on 1) preliminary data indicating frequent mutational discordance of ERBB2 in paired primary and metastatic tumors collected from individual patients with urothelial cancer; and 2) durable responses to second- generation HER2-directed ADCs in a subset of patients with HER2-expressingurothelial cancer. We hypothesize that pre-existing HER2 expression heterogeneity will be a common mechanism of resistance to HER2-targeted ADCs in urothelial cancer and that molecular imaging can identify those patients most likely to achieve durable responses. To test this hypothesis, we will leverage a largest-of-its-kind prospective molecular characterization effort and a novel molecular imaging platform (89Zr-ss-pertuzumab PET) to define the prevalence of HER2 expression heterogeneity in urothelial cancer, its association with ERBB2 mutational status, and its impact on HER2-targeted ADC response. We will accomplish these translational objectives through three broad approaches: 1) We will perform molecular analyses of paired primary and metastatic tumors collected from patients with urothelial cancer; 2) We will explore the extent of lesion-to-lesion HER2 heterogeneity in metastatic urothelial cancer using a bespoke HER2 PET imaging platform (89Zr-ss-pertuzumab PET); and 3) We will use 89Zr-ss-pertuzumab PET and tumors collected before treatment and at the time of disease progression on HER2- targeted ADCs to study the impact of HER2 heterogeneity on the durability of response to this novel drug class. Mechanisms of HER2 ADC resistance will be functionally explored using patient-derived urothelial cancer organoid and xenograft models. Given the promising clinical activity of HER2-directed ADCs in patients with urothelial cancer, we predict that the studies proposed will directly influence the design of future clinical trials of HER2-targeted therapies for patients and establish the clinical utility of HER2 PET as a predictive biomarker of response in patients with urothelial cancer being considered for HER2-targeted therapy.

NIH Research Projects · FY 2026 · 2024-03

Project Summary During mammalian development, a single cell gives rise to thousands of diverse and functionally distinct cell-types. Understanding how each cell-type is determined during development is one of the central questions in biology with far-reaching consequences for human health and regenerative medicine. While much of our current understanding of how cell-fate decisions are made is based on either temporally-resolved and non-destructive methods (e.g., time-lapse microscopy) or high-throughput but destructive genomic assays (e.g., single-cell RNA-seq), a new method that allows continual observation of each cell throughout the developmental process will fill the major gaps existing in our understanding of cell-fate transitions during mammalian development. Here we propose to develop molecular recording methods that enable the concurrent, non-destructive, high-throughput measurements of past cellular events and the current cell-type. Our recent methods, DNA Typewriter and ENGRAM, use precision genome editing to record cell lineage information and key transcriptional signaling events to the cell’s genome, which are recovered along with the transcriptome at the single-cell level. During the mentored K99 phase, I will further improve our methods by increasing the lineage recording efficiency (Aim 1) and testing it in the synthetic mammalian embryo systems (Aim 2). After I transition to independence in the R00 phase, I will expand the molecular recorder platform to concurrently capture diverse key cellular events (Aim 3). As our preliminary data on DNA Typewriter and ENGRAM demonstrate, we are in a strong position to carry out described molecular recording in model development systems. We anticipate that molecular recording of lineage and key signaling events in the synthetic embryo systems will deepen our model of early mammalian development. Together, our proposal will serve as a strong foundation as I transition into my independence and continue developing a general molecular recording platform.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY/ABSTRACT Current standard tri-modality therapy (chemotherapy, radiation, and surgery) for locally advanced rectal cancer is associated with high morbidity and only cures disease in about 75% of patients. We hypothesize that a biomarker-driven approach that gives matched targeted therapy to patients early in the treatment of locally advanced rectal cancers will lead to high anti-tumor activity and provide the chance to avoid radiation and/or surgery and its associated morbidities. We have already pioneered such an approach for mismatch repair deficient locally advanced rectal cancer, where clinical complete response was seen in all patients who completed 6 months of upfront treatment with immune checkpoint inhibitors (23/23, 100% complete clinical response), allowing for the omission of radiation and/or surgery. In this proposal, we focus on patients with HER2-amplified rectal cancer and hypothesize that induction therapy with HER2-targeted treatment will lead to tumor regression and modify the need for tri-modality therapy. We propose an investigator-initiated phase II study (NCT05672524; MSK IRB# 22-185) that recently opened to enrollment; all patients will receive tucatinib plus trastuzumab induction HER2-targeted therapy for 6 weeks and continue tucatinib plus trastuzumab with addition of standard-of-care chemotherapy for 4 months, followed by disease reassessment before standard chemoradiation and before surgery. Patients with clinical complete response to induction HER2 therapy alone or combined with chemotherapy, or to the combination followed by chemoradiation, will undergo observation with a chance for an organ-sparing management approach. Our central hypothesis is that the primary tumor will be highly sensitive to HER2-targeted therapy alone or in combination with standard chemotherapy, leading to a higher frequency of clinical complete response than with standard total neoadjuvant therapy. Treatment of rectal cancer in patients provides the unique opportunity to obtain on-treatment longitudinal tumor and blood samples to evaluate mechanisms of response and resistance. We hypothesize that levels of HER2 expression and consequent dependence on HER2 homodimers underlie drug response and will determine HER2 expression in biopsy samples and with 89Zr-trastuzumab PET/MRI and correlate with response rate and progression-free survival. Circulating tumor DNA will be tested as a biomarker for response. Established and newly generated patient-derived HER2-amplified colorectal cancer organoids will be used to model and study acquired resistance, taking advantage of our team's expertise in generating these three-dimensional, multi-cellular structures, which both recapitulate the biology of rectal cancer and provide a needed resource that expands the limited HER2- amplified rectal cancer models currently available. We hypothesize that key resistance mechanisms will consist of decreased HER2 expression, secondary alterations that activate ERK, and shifts in tumor transcriptional programs. Together, we will define the efficacy and biological effects of biomarker-directed therapy and aim to transform treatment for patients with HER2-amplified locally advanced rectal cancer.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Small-Molecule Penetration and Efflux in Gram-Negative Bacteria Gram-negative bacterial infections are increasing in incidence and novel antibiotics are urgently needed to combat this growing threat to public health. These pathogens have high intrinsic resistance to antibiotics due to their combination of a two-membrane cell envelope, which presents a permeability barrier to small molecules, and prevalent efflux pumps, which eject molecules that have successfully penetrated the barrier. A major obstacle to the development of novel antibiotics is our poor understanding of the structural features of small molecules that correlate with penetration and efflux across this barrier. As a result, large screening campaigns of existing discovery libraries have mostly failed to provide new antibacterials. Similarly, while potent biochemical inhibitors can often be identified for new targets, converting them into compounds with whole-cell antibacterial activity has proven challenging. To address this critical problem, we have developed a comprehensive experimental and computational platform to evaluate and model Small-molecule Penetration and Efflux in Antibiotic-Resistant Gram-Negative bacteria (SPEAR-GN, “speargun”). We have established all of the key enabling technologies required and strong proof of concept for the effectiveness of this platform. We will now use our platform to assemble the larger datasets required to train robust machine learning models of penetration and efflux that will be of broad utility in antibacterial drug discovery. Herein, we will design and synthesize chemical libraries to map bacterial penetration and efflux space, analyze compound accumulation in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii using isogenic strain sets that decouple outer-membrane penetration from efflux, evaluate the role of the BamA outer membrane protein in facilitated permeation of antibiotics, develop machine learning models based on the assay data to predict compound accumulation, and use the models to design and synthesize new molecules with improved accumulation in Gram-negative bacteria. Success in this project will provide a major advance in the field of antibacterial drug discovery to address this major public health threat. This project will be carried out by an established multidisciplinary team with a proven track record in this field and extensive combined expertise in library synthesis and medicinal chemistry, biochemistry and microbiology, high-throughput assays and mass spectrometry, cheminformatics and machine learning, and antibacterial drug discovery.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Current approaches to small molecule drug discovery are slow, expensive, and prone to failure. The abundance of data for structural biology has led to the emergence of a variety of computer-aided drug discovery methodologies aiming to leverage this structural data to predict compound affinities to prioritize compounds for synthesis in the pursuit of potency. Alchemical free energy methods, which use rigorous statistical mechanics to predict the free energy of binding, have led the way in providing useful predictions for improving or maintaining potency in hit-to-lead and lead optimization phases of structure-enabled programs. Numerous offerings---such as Schrödinger FEP+, Orion NES, CCG AMBER-TI, and the Open Force Field Consortium have emerged that provide engineered solutions with widespread industry adoption for structure-enabled discovery, integrating advances from academia that my lab has been fortunate to contribute to over the last 15 years. In contrast to physical methods such as alchemical free energy calculations, the emergence of machine learning models based on deep learning architectures has provided a complementary tool for computer-aided drug discovery. While physical models can generalize broadly across properties of interest for many target proteins, they lack the ability to easily learn from data generated for a discovery program. Machine learning methods, on the other hand, can readily learn from data, but often lack the ability to predict target-specific properties due to their need for large training sets, generally limiting their utility to properties of relevance to drug discovery that do not depend on the target, like ADMET properties. This proposal builds on our highly successful work in alchemical free energy calculations by proposing a new generation of hybrid physical / machine learning models that overcome the limitations of each method on its own: By endowing alchemical free energy calculations with the ability to learn at multiple scales, we aim to bring these tools into the next decade. Building on our extensive history of innovation in alchemical free energy calculations for drug discovery, we will (1) significantly increase their accuracy via the integration of ML potentials; (2) expand their domain of applicability beyond affinity to encompass conformational and target selectivity, resistance, interactions with structurally-enabled toxicity targets, and physical properties like membrane permeability, lipophilicity, and solubility; (3) eliminate accuracy-limiting challenges associated with current calculations such as sampling of protonation and tautomeric states, structured waters, and ions; (4) introduce learnability into every aspect of alchemical free energy calculations to enable predictions to become systematically more accurate as project data is collected and greatly reduce the cost of evaluating very large virtual synthetic spaces with alchemical-like accuracy; and (5) cast alchemical predictions in a Bayesian framework to enable the propagation of uncertainties in the underlying models into predicted affinities, selectivities, and other properties.

NIH Research Projects · FY 2026 · 2024-03

PROJECT ABSTRACT The success of chimeric antigen receptor (CAR) T-cell therapy in solid tumors requires antigen targets with no on-target, off-tumor toxicity, effective tumor infiltration, cytotoxicity and proliferation in an immunosuppressive environment, and revival of antigen stress-induced exhausted CAR T cells. We translated CD28-costimulated CARs (M28z) that target mesothelin (MSLN), a cancer-associated antigen that we have documented expression in majority of solid tumors; 64 patients have been treated to date, with no on-target, off-tumor toxicity. Having demonstrated that regionally administered CAR T cells avoid pulmonary sequestration and benefit from early antigen-activated CD4 helper CAR T-cell function, we delivered CAR T cells intrapleurally in patients with malignant pleural mesothelioma (MPM), promoting tumor infiltration. To address T-cell exhaustion, we either treated patients with anti-PD1 agent after CAR T cells or employed tumor-specific checkpoint blockade by CAR T-cell intrinsic PD1 dominant negative receptor (PD1DNR); 34 patients have been treated to date, with no CAR- or PD1DNR-related toxicities and with responses by imaging, and increased survival. To promote IFNγ-mediated cytotoxicity shown to be essential for solid tumor killing, we exploited a c-KIT mutation, D816V (KITv), as a costimulatory domain. KITv CAR T cells show antigen- activation induced IFNγ signaling, enhanced cytotoxicity, and when added as signal 3 to CD28 (signal 2), provide a synergistic function, resist TGFβ-mediated suppression, and prolong functional persistence. Clinically available kinase inhibitors provide an on/off, tunable safety switch for KITv CAR T cells. To effectively deliver these next-generation CAR T cells to solid tumors, we developed a translational strategy of non- ablative, tumor-targeted radiation therapy (RT) to generate a chemokine gradient that facilitates systemically administered CAR T-cell chemotaxis, tumor infiltration, proliferation, and persistence. Herein, we seek to translate the M28zKITv-PD1DNR CAR T cells to address key limitations in solid tumor cell therapy. In UG3 phase, we will explore the hypothesis that PD1DNR checkpoint blockade extends beyond tumor cells and counteracts PDL1-expressing M2 macrophages with immune suppressor function (Aim 1). We will define optimal regimen of non-ablative, tumor-targeted RT to promote tumor infiltration of systemically administered CAR T cells, achieving efficacy similar to that with regional delivery. In Aim 3, we will submit an IND application, a process with which we are familiar and have track record of success. In UH3 phase, we will conduct a phase I study to investigate the safety, functional activity and efficacy, and markers of response in patients with MPM. The significance of our approach lies in its effective combination of solid tumor-specific– scFv that is on-target and safe (MSLN), costimulatory domains (CD28, KITv), checkpoint blockade (PD1DNR), and a strategy of promoting solid tumor-infiltration (RT) of CAR T cells. The impact of our proposal extends beyond MPM (>150,000/year pleural cancers in the U.S.); majority of aggressive solid tumors express MSLN.

NIH Research Projects · FY 2026 · 2024-03

Imaging plays an increasingly important role in studying development and complex tissue formation across molecular, cellular and tissue levels. Fluorescence 3D time-lapse imaging allows every cell in a tissue or entire organism to be imaged, tracked and measured over hours and days to analyze lineage differentiation and dynamic cell behaviors. Other modalities, e.g., electron and expansion microscopy reveal fine structural phenomena, while emerging genomics technologies, e.g., spatial transcriptomics, seek to merge with microscopy to further provide systematic molecular information. Effective tools of image analysis are crucial to extract, integrate and interpret the information. We propose to leverage the power of deep learning to develop image analysis tools for systematic in vivo single-cell analysis, and to leverage such analysis to study collective cell behaviors in tissue morphogenesis, with three Aims. First, we will develop deep learning methods for accurate cell tracking, which is the essential first step to trace cell lineages and measure dynamic cell behaviors. We aim to produce a tool that can deliver substantial cell lineages with hundreds to thousands of cells imaged over hours to days in a wide range of model organisms and organoid cultures. Second, we will automate landmark-based image registration, which is crucial for cross-modality data integration. We propose a generalizable approach that uses statistical templates and Neural Networks to address the unique challenge in developmental images, namely heterochrony of developmental processes that creates combinatorial configurations of landmarks and complex systematic co-variance. Third, we study a novel Planar Cell Polarity (PCP) scheme in C. elegans, which we discovered through cell tracking and deep learning of cell movement patterns. By dissecting the compound polarity scheme and context specific regulators, we aim to understand how the conserved core PCP pathway can coordinate with different polarity pathways in order to orchestrate diverse motile behaviors in diverse developmental processes. By integrating technology development and hypothesis driven research, our proposal will further our understanding of embryogenesis and complex tissue formation.

NIH Research Projects · FY 2025 · 2024-03

PROJECT ABSTRACT The success of chimeric antigen receptor (CAR) T-cell therapy in solid tumors requires antigen targets with no on-target, off-tumor toxicity, effective tumor infiltration, cytotoxicity and proliferation in an immunosuppressive environment, and revival of antigen stress-induced exhausted CAR T cells. We translated CD28-costimulated CARs (M28z) that target mesothelin (MSLN), a cancer-associated antigen that we have documented expression in majority of solid tumors; 64 patients have been treated to date, with no on-target, off-tumor toxicity. Having demonstrated that regionally administered CAR T cells avoid pulmonary sequestration and benefit from early antigen-activated CD4 helper CAR T-cell function, we delivered CAR T cells intrapleurally in patients with malignant pleural mesothelioma (MPM), promoting tumor infiltration. To address T-cell exhaustion, we either treated patients with anti-PD1 agent after CAR T cells or employed tumor-specific checkpoint blockade by CAR T-cell intrinsic PD1 dominant negative receptor (PD1DNR); 34 patients have been treated to date, with no CAR- or PD1DNR-related toxicities and with responses by imaging, and increased survival. To promote IFNγ-mediated cytotoxicity shown to be essential for solid tumor killing, we exploited a c-KIT mutation, D816V (KITv), as a costimulatory domain. KITv CAR T cells show antigen- activation induced IFNγ signaling, enhanced cytotoxicity, and when added as signal 3 to CD28 (signal 2), provide a synergistic function, resist TGFβ-mediated suppression, and prolong functional persistence. Clinically available kinase inhibitors provide an on/off, tunable safety switch for KITv CAR T cells. To effectively deliver these next-generation CAR T cells to solid tumors, we developed a translational strategy of non- ablative, tumor-targeted radiation therapy (RT) to generate a chemokine gradient that facilitates systemically administered CAR T-cell chemotaxis, tumor infiltration, proliferation, and persistence. Herein, we seek to translate the M28zKITv-PD1DNR CAR T cells to address key limitations in solid tumor cell therapy. In UG3 phase, we will explore the hypothesis that PD1DNR checkpoint blockade extends beyond tumor cells and counteracts PDL1-expressing M2 macrophages with immune suppressor function (Aim 1). We will define optimal regimen of non-ablative, tumor-targeted RT to promote tumor infiltration of systemically administered CAR T cells, achieving efficacy similar to that with regional delivery. In Aim 3, we will submit an IND application, a process with which we are familiar and have track record of success. In UH3 phase, we will conduct a phase I study to investigate the safety, functional activity and efficacy, and markers of response in patients with MPM. The significance of our approach lies in its effective combination of solid tumor-specific– scFv that is on-target and safe (MSLN), costimulatory domains (CD28, KITv), checkpoint blockade (PD1DNR), and a strategy of promoting solid tumor-infiltration (RT) of CAR T cells. The impact of our proposal extends beyond MPM (>150,000/year pleural cancers in the U.S.); majority of aggressive solid tumors express MSLN.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The maintenance of genome stability across generations is critical for human health and relies on the efficient repair of spontaneous DNA lesions and the faithful duplication of all chromosomal DNA prior to cell division. The highly conserved DNA sliding clamps PCNA and 9-1-1 are critical for these genome maintenance mechanisms in eukaryotic cells by acting as mobile hubs for the assembly of the protein complexes mediating the signaling and repair of DNA damage and conducting the faithful replication of the nuclear chromosomes. The dynamic association of PCNA and 9-1-1 with chromosomal DNA is controlled by a set of four conserved and related ATP-dependent clamp loader complexes that each perform non-redundant genome maintenance functions in the cell. How eukaryotic clamp loaders target their client clamps to sites of DNA replication and repair and load the clamps around DNA has been the subject of intense investigation for several decades. Using advanced biochemical reconstitution approaches and cryogenic electron-microscopy (cryo-EM), we have recently determined the first structures of active eukaryotic clamp loader:clamp:DNA complexes, which revealed the molecular basis for the substrate specificities of the yeast RFC and Rad24-RFC complexes and resulted in a significant revision of current clamp loading models. Building on this work, here we propose to extend and advance the approaches established by us for the yeast RFC:PCNA and Rad24-RFC:9-1-1 clamp loader systems to characterize the molecular mechanisms of the yeast and human orthologues of Ctf18- RFC:PCNA, Elg1ATAD5-RFC:PCNA, and RAD17-RFC:9-1-1. The innovative approach leverages the expertise of Dr. Remus’ laboratory in the biochemical reconstitution of eukaryotic DNA replication and the expertise of Dr. Hite’s laboratory in the characterization of the conformational landscapes of protein complexes by cryo-EM. The proposed studies will determine the mechanistic basis for the functional specialization of CTF18-RFC (Aim 1), uncover the currently unknown mechanism of PCNA unloading by Elg1ATAD5-RFC:PCNA (Aim 2) and visualize the spectrum of conformational states of the human RAD17-RFC:9-1-1 complex to reveal the evolutionary conservation or divergence of the 9-1-1 checkpoint clamp loading mechanism (Aim 3). Collectively, this work will provide novel mechanistic insight into fundamental genome maintenance pathways.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT Mutations in isocitrate dehydrogenase (IDH) enzymes are hallmarks of a variety of deadly cancers, including acute myeloid leukemia (AML), glioma, cholangiocarcinoma, chondrosarcoma, and T cell lymphoma. Mutant IDH enzymes drive cancer through an unusual mechanism – they produce a metabolite called 2- hydroxyglutarate (2HG) that poisons gene expression machinery and locks malignant cells in a stem cell-like state. Drugs that inhibit mutant IDH enzymes induce durable clinical responses in some patients with IDH- mutant cancers, leading to FDA approvals for AML and cholangiocarcinoma. However, despite near universal inhibition of 2HG production, over half of patients do not respond to IDH inhibitors. Even for patients who initially respond to IDH inhibitors, most eventually acquire resistance to the drugs. While the mechanisms of resistance to IDH inhibitors remain incompletely understood, emerging evidence suggests that acquisition of specific co-occurring mutations during tumor evolution results in a loss of dependence on 2HG. Therefore, we need new treatment approaches that target IDH mutations in different ways beyond simple inhibition of the enzyme. We previously identified an unusual pattern of resistance mutations in the dimer interface of mitochondrial IDH2 wherein they occur in trans (on the other allele) relative to the 2HG-producing active-site mutation. Here, we show that in contrast to the in trans mutations that drive drug resistance, forced expression of a dimer-interface mutation in cis with the active-site mutation resulted in mitochondrial dysfunction and impaired growth of leukemia cells. Biochemical and structural studies demonstrated that the in cis dimer- interface mutation enabled IDH2 to aberrantly use NADH as an additional cofactor and dramatically enhanced production of 2HG. Seeking to exploit the toxicity exerted by the in cis dimer-interface mutation, we performed a chemical screen and identified small molecules capable of mimicking this aberrant enzymatic activity with selective toxicity towards IDH2-mutant leukemia cells. Thus, we hypothesize that hyperactivation (rather than inhibition) of mutant IDH offers an unexpected and effective new strategy to target IDH-mutant cancers. This hypothesis will be rigorously tested in three Specific Aims. Aim 1 will use enzyme assays and structural approaches to elucidate the biochemical basis for mutant IDH2 hyperactivation. Aim 2 will employ in vitro and in vivo cancer models to define the mechanisms of toxicity arising from hyperactivation of mutant IDH2. Aim 3 will utilize biochemical and functional approaches to determine if the approach of hyperactivation can applied to cytosolic IDH1 mutations. The proposed studies will reshape our understanding of the oncogenic properties of 2HG and the biochemistry of neomorphic IDH activation. More fundamentally, this work will demonstrate the feasibility of an entirely novel hyperactivation approach as a strategy to harness oncogene-mediated toxicity that could be applied to a wide range of oncogenes and cancer contexts.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY/ABSTRACT: Single cell sequencing technologies have rapidly advanced over the past five years and have become essential tools in a broad array of fields, ranging from developmental biology to human genetics. One major advance was the development of high-throughput barcoding of RNA or DNA within droplets, which allowed for preparation of sequencing libraries as a pool rather than in a laborious 96 well plate format. This advance enabled sequencing analysis of thousands of cells in one batch. However, there has not been the same breakthrough in scale when sequencing both DNA and RNA from the same cell as established techniques involve sorting cells into plates or wells and ultimately separating DNA and RNA before library preparation. The separation of RNA from DNA is necessary because conventional PCR amplification requires a 95oC melting step, which degrades RNA. In this proposal, we will seek to optimize DNA amplification through recombination polymerase amplification (RPA), an isothermal form of PCR which can be performed simultaneously with reverse transcription of mRNA into cDNA. In this way, both RNA and DNA can be captured into barcoded, sequencing libraries within the same droplet. The technology could have impact in many different fields in which genotype-phenotype correlation is needed, such as characterizing genetic polymorphisms or genetic variants in cancer. In SA1, we will demonstrate proof-of-principle of same cell DNA amplicon and RNA transcriptome technology via isothermal RPA and reverse transcription. In SA2, we will determine the quantitative performance of the methodology in two use applications and test capability to genotype heterozygous alleles. If successful, this methodology could be widely adopted by laboratories in diverse fields to answer questions related to genotype-phenotype correlations.

NIH Research Projects · FY 2026 · 2023-11

Project Summary Three-dimensional (3D) brain organoids from human pluripotent stem cells (hPSCs) provide a tremendous opportunity to model brain development and disease. Over the last few years, we have developed many hPSC-based protocols that now enable researchers to routinely generate > 50 distinct human cell types for modeling both Central Nervous System and Peripheral Nervous System disorders in a dish. More recently, our lab and others have translated those 2D approaches into 3D “guided” neural organoid protocols to generate specific regions of the human brain. Further complexity can be achieved by combining individual organoids into multi-region assembloids, or by spatially restricted patterning to induce distinct human brain regions within a single organoid. However, currently available organoid and assembloid models have major limitations, including a poor representation of several progenitor and neuronal cell populations such as outer radial glia (oRG) and several cortical interneuron lineages. We posit that the missing or underrepresented cell types may depend on signals provided by non-neural lineages (vascular and immune cells). In our preliminary work, we have developed an improved hPSC-based cortical organoid model which dramatically enhances the generation of oRG, cortical-derived interneurons, and cortical excitatory neurons with increased levels of maturity. This was achieved by treatment with leukemia inhibitor factor (LIF) resulting in the activation of STAT3 and mTOR signaling pathways. Data from human fetal cortex development identifies vascular pericytes are a major source of LIF. Remarkably, we demonstrate that integrating hPSC-derived brain pericytes can substitute for LIF treatment in cortical organoids. Here, in Aim 1 we will optimize and assess robustness of the LIF organoid protocol and the generation of oRG across multiple human PSC lines. We will further assess whether oRG give rise to intermediate progenitors, cortical interneurons and cortical excitatory neurons using a unique oRG-specific double reporter hPSC line and genetic fate mapping tools. Finally, we will determine how changes in mTOR signaling impact oRG behavior and whether phenotypes observed in models of Tuberous Sclerosis (TSC) are linked to aberrant oRG function. In Aim 2, we will build an organoid system that integrates key non-neural cell types. We will establish a modular platform that combines cortical organoids with 3D spheroids (“microtissues”) comprised of vascular brain pericytes and/or microglia. These novel microphysiological platform will be utilized to determine both LIF-dependent and LIF independent effects on cortical development. Finally, we will assess whether such a more complex microphysiological platform will enable improved modeling of the developmental defects related to mTOR signaling and TSC.

NIH Research Projects · FY 2026 · 2023-09

Project Summary: The most common mitochondrial DNA (mtDNA) abnormality is a deletion of 4977 base pairs called the common deletion (CD), associated with mitochondrial pathologies and widespread in aging. The CD primarily manifests in the brain and muscles when deleted molecules exceed 60% of total copies, known as heteroplasmy. However, the mechanisms that cause harmful deletions and why neuronal and muscle cells are particularly vulnerable to CD remain unclear. Major obstacles to studying the CD is the lack of tools to manipulate mtDNA and the inability to generate the CD in a controlled manner. Here, we developed a series of methodologies to overcome these barriers. Specifically, we generated an inducible quasi-dimeric TALEN that generates the CD in isogenic settings and at defined heteroplasmy states. With this tool, we will establish low, medium, and high levels of CD heteroplasmy in embryonic stem cells that we will then differentiate into muscle, neuronal, and fibroblast cells and elucidate the consequence of this harmful deletion in a cell-type-specific manner and its impact on cellular aging. Furthermore, we will explore the cell-type-specific distribution of CD, Identifying the pathways that sustain mutant mtDNA propagation in post-mitotic cells while promoting its elimination in dividing cells. Combining novel genetic tools with extensive experience in genome stability will resolve the long-standing mystery of preferential mutant mtDNA propagation in post-mitotic cells and have significant implications for numerous mitochondrial pathologies and aging.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Melanoma (MEL) is a model malignancy for studying the mechanisms of cancer immunotherapy. Antibodies that block negative regulators of T cell function, termed immune checkpoint blockade (ICB), have transformed the treatment of MEL and other solid cancers. Although some patients have durable disease control, many fail to respond or progress after initially experiencing tumor regression. Therapeutic resistance is enriched in two molecularly defined MEL subtypes. Twenty eight percent of MELs possess activating mutations (Mut) in the driver oncogene NRAS, the second most common Mut RAS isoform. Beyond MEL, Mut NRAS occurs in other prevalent malignancies, including colorectal cancer (CRC). We and others recently discovered that patients with Mut NRAS MEL and CRC have a significantly shorter time to treatment failure. Separately, ~30% of MELs acquire mutations in beta-2-microglobulin (B2M), an essential component of the human leukocyte antigen class I (HLA-I) complex, following ICB progression. Cancers with Mut B2M are intrinsically resistant to CD8+ T cell killing. Thus, two major gaps in knowledge that limit the potential of immunotherapy in MEL and other common cancers include: (1) identification of immunogenic antigens expressed by Mut NRAS tumors, and (2) therapeutic strategies to overcome genetic loss of HLA-I presentation. We hypothesize that cancers with Mut NRAS or Mut B2M can be therapeutically targeted using T cell receptor (TCR)-based immunotherapies. In support of our hypothesis, we discovered using a mass spectrometry (MS) screen that the three most common NRAS hotspot substitutions generate shared (or “public”) neoantigens (NeoAgs) presented by a prevalent HLA allele. Using a unique collection of biospecimens from patients who express an NRAS public NeoAg, we generated T cells specific for these epitopes, retrieved their TCR gene sequences, and transferred public NeoAg reactivity to polyclonal T cells. These results confirm the immunogenicity of screen-identified NRAS public NeoAgs and enable the development of TCR-based therapies. We further discovered that a significant proportion of MELs undergo direct killing by T cells that express an HLA class II (HLA-II) restricted TCR. Using a genome-scale CRISPR screen, we found that cancer eradication is preserved when B2M and other HLA-I genes are disrupted. Building on these preliminary data, we propose in Aim 1 to develop a novel therapeutic approach for cancers expressing an NRAS public NeoAg using TCR genetic engineering and adoptive cell transfer. In Aim 2, we will study the physical mechanisms underlying NRAS public NeoAg TCR specificity, including the unique capacity of some TCRs to accommodate multiple hotspot substitutions. In Aim 3, we will define the molecular basis for direct cancer cell killing by HLA class II-restricted TCRs and test combinations to enhance the antitumor efficacy of adoptively transferred CD4+ T cells. By completing these aims, we will develop novel, mechanism-based cellular immunotherapies for Mut NRAS and HLA-I deficient cancers.

NIH Research Projects · FY 2025 · 2023-09

The Oncology-focused Postdoctoral Training In Care Delivery and Symptom Science (OPTICS) T32 program will mentor and train physicians and scientists with PhDs in social or quantitative sciences to conduct research focused on innovative cancer are delivery that narrows the gap between outcomes that are possible based on contemporary scientific understanding of cancer prevention and treatment and outcomes that are actually achieved. This persistent gap results in a critical need for translational researchers who are focused on risk reduction, symptom science, and innovative care delivery and have rigorous training in research methods required for care delivery transformation and leadership of health system–level interventions to improve treatment outcomes. The cornerstone of the OPTICS program will be a 2-year mentored research experience in which trainees will conduct research aligned with one or more of four thematic areas: 1) Data Science, 2) Risk Mitigation, 3) Symptom Science, and 4) Care Delivery. OPTICS trainees will receive 1) intensive mentorship and the resources necessary to execute a focused research project in one or more of the thematic areas; 2) training in core methods necessary for impactful research through coursework, seminars, workshops, and reading groups; and 3) training in skills required for career building, including protocol development and execution, management, grant writing, team building, patient engagement, dry-laboratory organization, and the responsible conduct of research. OPTICS will be co-led by 2 PhDs and 2 MDs at Memorial Sloan Kettering Cancer Center (MSK) with long track records of impactful research, mentorship, and successful knowledge translation. This structure will afford participants methods training and the ability to launch interventions in the community and the clinic. OPTICS will train 6 postdoctoral fellows each year (3 new appointees and 3 re-appointees) with a team of 36 core program faculty, 10 emerging mentors, and 6 research supporters with relevant supporting skills, expertise, and resources. Required core program training at the New York City campus of MSK and its partner Weill Cornell Medical College is supplemented by an array of elective learning opportunities customized to each trainee’s needs and learning style. Trainees will obtain the skills necessary to develop, test, and implement new approaches to optimizing patients’ experiences by focusing on risk reduction, symptom control, communication, and new models of cancer care delivery. These skills are crucial to address the challenges brought about by the tremendous growth in the complexity and chronicity of cancer care. OPTICS aims to prepare trainees for impactful careers focused on innovations to optimize cancer care quality and translation of knowledge to ensure that discoveries made in the laboratory and clinic realize their full impact on population health and well-being.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Mechanisms underlying selective vulnerability from cells to networks across the Alzheimer's disease (AD) spectrum remain unknown, limiting our understanding of disease and hampering development of effective therapies. We propose to identify protein-protein interaction (PPI) network dysfunctions in brain cells and regions as a gateway to selective vulnerability mechanisms in AD. To gain systems level insights, we propose to leverage our discoveries in stress biology linking interactome network perturbations to the formation of long-lived oligomeric scaffolds termed epichaperomes, and to employ a novel `omics platform called epichaperomics that provides direct information on PPI network changes. Preliminary studies indicate epichaperomes change how thousands of proteins interact and negatively impact PPI networks important for neuronal function, including synaptic plasticity, cell-to-cell communication, protein translation, cell cycle re-entry, axon guidance, metabolic processes and inflammation, leading to cell and connectome-wide dysfunction and cognitive decline. Parallel studies in transgenic mice and iPSC-derived neurons demonstrate epichaperome formation is a key event that negatively impacts cellular function, from early prodromal disease stages and throughout disease progression. Preliminary results in transgenic mice and postmortem AD brains suggest epichaperome formation occurs principally within vulnerable brain cells and regions. Accordingly, we hypothesize epichaperome formation, and in turn of epichaperome-mediated PPI network imbalances, over decades, not only results in defects within intrinsic neuronal proteins and protein pathways but also intercellularly, where it disrupts intrinsic network connectivity of cells and of brain circuits. We posit vulnerable neurons and brain regions have a higher propensity to accumulate epichaperomes, and epichaperome-mediated dysfunctions. In accordance with NOT-AG-21-040, we propose to uncover mechanisms of PPI dysfunctions within individual brain cells and regions as a portal into selective vulnerability in AD, which remains unknown and a key missing piece. We aim to i) investigate mechanisms that enable (i.e., epichaperomes, Aim 1) and ii) those that execute (i.e., impacted proteins and protein pathways, Aim 2) context-specific dysfunctions in PPI networks. As a key element in linking stressors-to- phenotype, we aim to uncover cell- and region-specific vulnerabilities within PPI networks induced by individual stressors (Aim 3). Results provide first-of-a-kind insights into the spatio-temporal formation and distribution of epichaperomes across the AD spectrum and their relationship to clinical, pathologic, and genetic vulnerabilities. Outcomes are critical proteome-wide insights into interactome vulnerabilities, both on the nature and trajectory within vulnerable brain cells and brain regions. Raw datasets and data analytics will be deposited directly into free access sites for mining and hypothesis testing by members of the scientific community. In addition to defining technically challenging mechanistic insights into selective AD vulnerabilities, innovation includes diagnostics and therapeutics, as epichaperome-mediated dysfunctions are both imageable and targetable.

NIH Research Projects · FY 2025 · 2023-09

In 2014, the state of Maryland, under a federal waiver, enacted an all-payer Global Budget Revenue (GBR) model that prospectively set limits on hospital revenue. It also required the state to limit growth in per-capita spending and mandated reductions in preventable complications and readmissions. GBR implementation was associated with savings to the Medicare Trust Fund and considerations are now underway to expand the program to other regions. However, there is limited understanding of GBR’s impact on the delivery of cancer- related services. It is possible that while GBR may incentivize reduced healthcare expenditures and care improvements on average, it could be associated with unintended effects and poor performance for cancer patients by limiting access to effective cancer treatments. GBR may have deleterious effects on prevailing cancer care by encouraging adverse patient selection across various patient populations due to concerns about higher spending and worse clinical outcomes. Current evaluations of the GBR program have not examined these impacts. We aim to address this evidence gap in this proposal. Our research is important because acute hospital care, the focus of GBR incentives, is a key driver of overall spending and regional variation in spending for patients with cancer. The objective of this proposal is to systematically examine, via a difference-in-differences design, the impact of the GBR model on spending, quality-of-care, and utilization among fee-for-service Medicare beneficiaries and nonelderly Medicaid and commercial insurance beneficiaries with cancer in Maryland compared with similar patients in control states. Our central hypothesis is that the financial incentives in GBR will lower spending, improve care quality, and facilitate a shift in the site of care for chemotherapy administration across our populations of interest. Additionally, we hypothesize that GBR implementation will lead to varied clinical outcomes and spending across various patient populations. We will test our hypotheses and achieve our objectives with the following specific aims: Aim 1: Quantify the impact of GBR on risk-adjusted spending for beneficiaries undergoing chemotherapy. Aim 2: Assess the impact of GBR on the likelihood of chemotherapy receipt and on care quality for beneficiaries undergoing chemotherapy. Aim 3: Assess the impact of GBR on the type of chemotherapy (physician-administered vs. oral) and site of physician-administered chemotherapy (hospital outpatient department vs. physician office setting). Aim 4: Assess the differential effects of GBR implementation on care delivery across various patient populations who are undergoing chemotherapy. Our findings will meaningfully advance our understanding of how to deliver efficient, high-quality cancer care to adult patients. It will also provide timely information to policy makers that would guide updates to GBR and mitigate the risk of unintended consequences in future global budget initiatives.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Clonal hematopoiesis (CH) is a common phenomenon defined as the presence of somatic mutations in hematopoietic stem and progenitor cells (HSPCs) and their expansion in the absence of overt hematological disease. CH-mutant mature, myeloid cells are believed to generate an inflammatory microenvironment promoting the fitness advantage of mutant HSPCs. These, in turn, would expand at higher rates and differentiate into more elevated numbers of myeloid cells, thereby establishing a positive feedback loop between inflammatory signaling and clonal expansion. Yet, whether CH-mutant HSPCs can also trigger cell-autonomous inflammatory signaling to provide a selective clonal advantage for themselves remains unknown. CH increases the risk of hematological malignancy, cardiovascular disease, and mortality from solid tumors. Due to these adverse outcomes and the high prevalence of CH in the elderly, there is an unmet need to develop novel therapies. Targeting inflammation specifically in mutant HSPCs —to avoid disruption of general immune responses— may be a potentially effective strategy. My predoctoral research (Aim 1) aims to identify inflammatory mediators of CH-mutant HSPCs and evaluate their potential as therapeutic targets to restore oligoclonal hematopoiesis. To find novel, cell-autonomous inflammatory pathways in CH, I have designed an sgRNA library to target inflammation-associated genes. Using this library for high-throughput CRISPR/Cas9 screening, I have identified both general and genotype-specific inflammatory dependencies of CH-mutant murine HSPCs. In this proposal, Specific Aim 1.1 seeks to validate the negative selection hits, demonstrate that, when present, the hit genes confer a selective advantage to CH-mutant HSPCs versus wild-type counterparts, and delineate the specific role of credential top hits in clonal expansion. In Specific Aim 1.2, I will use small molecule inhibitors targeting the candidate genes —both ex vivo and in mice— to identify gene expression and cytokine profile changes spanning the hematopoietic cell subsets. I will then assess differences between genetic and chemical approaches concerning their efficiency in achieving adequate target inhibition. My postdoctoral research (Aim 2) will focus on the role of inflammation at the nexus of aging and CH by uncovering the transcriptional and epigenetic mechanisms by which age-related inflammation promotes CH and potential malignant transformation to leukemia. Overall, these two projects, which will use human samples to validate the mouse findings, will lead to developing new therapies targeting inflammation to halt or revert CH and mitigate its clinical sequelae. I, the applicant, will conduct this proposal in the laboratory of Dr. Ross Levine at Memorial Sloan Kettering Cancer Center (MSK), one of the world's leading institutions in cancer treatment and research. MSK's rich environment and abundant resources in conjunction with the support of the Gerstner Sloan Kettering Graduate School, guarantee the successful completion of the proposed research and career development plans.

NIH Research Projects · FY 2026 · 2023-09

Project Summary CRISPR-Cas are prokaryotic adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements, such as phages and plasmids. CRISPR-Cas systems acquire immunological memories during infection by integrating short fragments from the invader’s genome into the CRISPR locus of the host. These fragments, called “spacers”, are later transcribed into CRISPR RNAs that are loaded on Cas nucleases and guide them to recognize and cleave infecting nucleic acids. Depending on their genetic composition, CRISPR- Cas systems are classified into six types (I-VI). While spacer acquisition has been extensively studied in type I and II systems, type III systems are just now starting to be explored. The overall goal of this application is to define the molecular mechanisms that govern spacer acquisition by the prevalent, yet less studied, type III-A CRISPR-Cas system, and understand its implications during CRISPR-Cas defense and tolerance. Preliminary work on the type III-A system of Staphylococcus epidermidis revealed that this system preferentially acquires new spacers by two independent modes. The first mode acquires spacers from some, but not all, highly transcribed genes, and spans their entire transcribed region. The first aim of this proposal is to elucidate how the acquisition machinery recognizes specific genes as substrates for preferential acquisition. This will be achieved by dissecting the DNA sequences that recruit the spacer-integrase complex to specific genes, finding host factors that mediate gene-specific spacer acquisition, and test for the physiological relevance of this process during the CRISPR-Cas immune response. The second mode of acquisition by the type III-A system is similar to the previously studied type I and II systems, where spacers are acquired from free dsDNA ends at the bacterial chromosomal terminus, in a manner that is dependent on the cell’s DNA-repair machinery. Such self-targeting spacers are expected to induce autoimmunity and be negatively selected, however we found them to be stably fixed in the bacterial population, suggesting the existence of unknown mechanisms that inhibit targeting by Cas nucleases at this site, thus preventing CRISPR autoimmunity. The second aim of this proposal will define the genomic context that allows self-targeting spacers to be tolerated, analyze the temporal dynamics of CRISPR- Cas immunity at free DNA ends, and explore the genetic components needed for CRISPR-tolerance and accumulation of self-targeting spacers. This proposed work will not only transform our conceptual understanding of the spacer acquisition process, but also could lead to CRISPR-based technological developments in molecular biology and diagnostics. To achieve these goals, I have assembled a team of experts in the fields of transcription, DNA repair, bioinformatics, biochemistry and biophysics. Their guidance, along with the continued mentorship of Prof. Luciano Marraffini and the scientific environment of the Rockefeller University, will allow me to perform the proposed research, as well as to develop writing, mentorship and communication skills, that will support my successful transition to an independent career.

NIH Research Projects · FY 2025 · 2023-09

SUMMARY Magnetic resonance imaging-guided adaptive radiotherapy (MRgART) allows for safer treatment of otherwise difficult-to-treat soft-tissue cancers in the abdomen, such as inoperable pancreatic cancers that occur close to highly mobile and radiosensitive gastrointestinal (GI) organs. MRgART enables daily replanning to compensate for organ shape variations through improved visualization of the tumor and nearby organs. However, nearby abdominal organs move considerably between and during treatment fractions and, crucially, accurate tracking of the dose distribution accumulated in those tissues is currently unavailable. Consequently, tumor prescription coverage is still often constrained to sub-optimal levels by design to conservatively reduce the risk of radiation toxicity to GI organs. We hypothesize that accurate estimates of doses to the surrounding mobile healthy organs, accumulated over all fractions, would enable a less conservative and more effective treatment of the full extent of the disease. Hence, the key clinical need we will address, to ensure improved local control and to reduce rates of local tumor progression and morbidity, particularly in the tumors adjacent to luminal GI organs, is the development of reliably accurate deformable image registration (DIR) methods to estimate the spatial dose accumulated to the mobile GI luminal organs throughout treatment from previous fractions. This proposal addresses the key need by developing, rigorously validating, and systematically measuring the gain in target coverage with an innovative deep learning DIR dose accumulation utilizing a cohort of virtual digital twins. In Aim 1, We will develop patient-specific virtual digital twin cohorts modeling 21 different temporally varying realistic GI motions encompassing respiratory and digestive motion. The twins will combine analytical modeling with the widely used XCAT digital phantoms. In Aim 2, the virtual digital twins will be used to optimize and rigorously validate our innovative progressive registration-segmentation deep learning network for GI organs. The key technical novelty of this approach is its ability to perform spatio-temporally varying regularization to model large deformations, not possible with most DIR methods. In Aim 3, the potential clinical gain of using AI- DIR dose accumulation compared with the clinical standard with conservative limits to the high dose region will be systematically simulated with a variety of GI tract motion using the VDT datasets. Potential impact: The developed and validated AI-DIR techniques, validated for realistic physiologic GI motions, will be applicable beyond pancreatic tumors and will apply to other GI soft-tissue cancers. Ultimately, the availability of well- validated dose accumulation techniques could enable clinicians to quantitatively determine the accumulated radiation dose distribution to luminal GI organs and appropriately account for the spillover radiation, thus leading to more personalized, safer, and possibly more effective radiation treatments.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Despite significant progress in understanding cognitive change associated with cancer and cancer treatments, little is known regarding longer-term cognitive outcomes over developmentally meaningful intervals in older age (5-20 years post-treatment) and potential association with frailty. Previous work from our lab and others, focusing on the direct effects of cancer and cancer treatment, has moved the field forward for a better understanding of these direct effects but is limited with regard to tracking long-term cognitive trajectories due to 1) focus on cognition before and after treatment and short intervals following treatment completion; 2) longitudinal designs that introduce practice effects and selective attrition that distort true cognitive trajectories; and 3) short intervals that also limit the ability to assess accumulation of deficits, i.e., frailty, to potentially associate with cognitive declines. We will assess cognition in 210 cancer survivors diagnosed between 55 and 60 years of age and 210 non-cancer controls at three cross-sectional age bands: 65-69; 70-74; 75-80. Cognitive assessment will consist of online, remote administration of a validated platform of cognitive-experimental measures for use in cancer survivorship and standard neuropsychological measures, together with collection of cancer and treatment variables. Frailty as indexed by accumulation of deficits, i.e., medical comorbidities, polypharmacy, social detriments of disease (e.g., smoking, obesity), psychological disturbance, and functional limitations / declines in activities of daily living, will be collected in parallel. Aim 1: Examine cognitive differences and trajectories between breast cancer survivors and age and education matched controls controlling for cognitive reserve and APOE. Aim 2: Examine the association of deficit accumulation with cognition and interaction with group status. Aim 3: Explore potential associations between APOE status and deficit accumulation and combined effects on cognition. This research is significant because cancer and cancer treatments have been identified as disease drivers of aging and deficit accumulation and has significant clinical implications in that interventions that reduce deficit accumulation may be effective in maintaining cognitive function. This research is innovative because it utilizes a cross-sectional design that avoids practice effects and cognitive neuroscience measures that allow for assessment of cognitive trajectories and their association with deficit accumulation at developmentally relevant timespans.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY This proposal outlines a five-year career development program for Caleb Lareau, Ph.D. to prepare him for an independent research career in human genomics to study cellular processes underlying complex disease. The candidate will conduct his postdoctoral training at Stanford University, which provides an outstanding environment to complete the proposed research and develop skills in massive-scale computational analyses, genomics technology development, and immunology. Dr. Lareau’s mentors and advisors, including Drs. Satpathy, Kundaje, Greenleaf, Howitt, Curtis, and Anderson, have diverse technical expertise relevant to all aspects of the proposal and track records of guiding trainees to independence. Further, the candidate will utilize world-class resources available through the Stanford School of Medicine, Office of Postdoctoral Affairs, and NHGRI-funded Centers at Stanford to acquire career development skills while interfacing with leaders in genomics. Additionally, the research infrastructure within his mentors’ labs will enable him to efficiently perform the scientific aims, receive training in areas encompassed by this proposal, and transition to independence. The goal of this work is to develop single-cell genomics methods to chart somatic evolution throughout the human body. Evidence from recent bulk sequencing studies has indicated that somatic evolution occurs in almost all tissues, but current approaches lack sensitivity to resolve clonal expansions, associated cell states, or their prevalence throughout the body. A key bottleneck in studying somatic evolution has been limitations of genomics technologies, which if addressed, may lead to insights into the pathogenesis of diseases like cancer. In Aim 1 (K99), the candidate will establish a new single-cell approach for measuring accessible chromatin, protein abundance, and mitochondrial DNA mutations to identify clonal expansions and related cell state changes. In Aim 2 (K99), the candidate will apply multi-omics technologies to identify the origins and expansions of macrophages within human gynecological tissues and tumors. After transitioning to a faculty position for Aim 3 (R00), the candidate will focus his effort on creating massive-scale single-cell whole-genome sequencing methods that are paired with functional measurements, including RNA or protein quantification. All Aims will utilize and build upon cutting-edge single-cell multi-omics technologies to study somatic evolution. Together, the pursuit of this research will result in tools and insights that will directly inform properties of human tissue physiology and aid in the early detection, characterization, and understanding of age-associated diseases, including cancer. All protocols, data, analytical frameworks, and software tools that are produced during the duration of this research will be freely distributed. In total, the proposal will lead to novel insights into the molecular signatures and regulation of somatic evolution and serve as an effective training program for Dr. Lareau to launch his independent career as a tenure-track investigator.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY / ABSTRACT Cancer is the second most frequent cause of death in children under 15 years of age, and primary central nervous system (CNS) tumors are the most frequent cause of cancer-related childhood deaths. Medulloblastoma (MB) is the most frequent malignant childhood brain tumor (incidence of 5.5/million/year). About 40% of cases occur in children <5 years old, which can be sub-divided by biological markers into two groups: low-risk group, biologically defined by either Wingless/Integrated (WNT) or Sonic Hedgehog (SHH) activation TP53-wt, while the high-risk group is defined by non-WNT/non-SHH biology. As WNT-activated MB is extremely rare in early childhood, only young patients (<5 years of age) with low-risk (SHH-activated) MB are eligible and have an excellent prognosis if treated with either of the two randomized arms in this research study. Craniospinal irradiation (CSI) is an integral component in the treatment of MB; however, because of the devastating impact upon the central nervous system (CNS) and neurocognitive outcomes, it must be avoided whenever possible given the significant interference with educational and vocational attainment. Consequently, maintaining or improving neurocognitive and QoL functioning is an essential opportunity for early childhood survivors who can now be cured with treatment that does not include CSI. The Prospective International SIOPE/CONNECT phase-III study to improve neurocognitive outcomes in young children with low-risk medulloblastoma (YCMB-LR) is the first ever randomized study directly comparing two highly effective irradiation-sparing treatment regimens, Head Start 4 and HIT-SKK, which will take place at pediatric oncology centers across Europe and North America and is the first to include neuropsychological and QoL outcome as the primary objective. Aim 1) Compare the overall intelligence and IQ subdomains as measured by the Wechsler Preschool and Primary Scale of Intelligence administered 2.5 years after diagnosis between patients with newly diagnosed, non-metastatic, SHH-activated, TP53-wt MB randomized to the interventional arms A (Head Start 4) or B (HIT- SKK). Aim 2) Compare the trajectory between the two randomized groups at baseline and again at 2.5 years post diagnosis for: a) overall intelligence and IQ subdomains, b) behavioral development and c) QoL, along with analyses at 2.5 years post diagnosis for: d) fine motor dexterity and processing speed, e) visual-motor integration, f) executive functioning, and g) social-emotional functioning. Aim 3) Several quantitative imaging metrics with regard to brain volumes and white matter injury will serve as ancillary noninvasive biomarkers for comparison of the two interventional arms in Aim 1, and will be statistically correlated with the neurocognitive, QoL and behavioral outcomes in Aim 2. Impact: Our work will define the new “gold standard” of treatment in early childhood low-risk MB that is associated with better neurocognitive outcomes with less severe late-effects and ultimately yield a better QoL in survivorship, while simultaneously improving and harmonizing international diagnostic and therapeutic standards not only for MB, but also for other pediatric CNS tumors.

NIH Research Projects · FY 2025 · 2023-09

Abstract: Eukaryotic cells solve the end-replication and end-protection problems through the addition of telomere sequences to the ends of chromosomes. Proper regulation of telomere length is critical for genome integrity, regulation of cellular lifespan, aging, and cancer. Over the years, genetic and biochemical studies have shed an enormous amount of light on how this process is controlled. However, the cell biology of this process in the crowded nucleus remains poorly understood, and the timing, dynamics, and spatial coordination of telomere extension are unknown. To address these gaps in our understanding, we will exploit the MS2 tagging system and Halo-fluorophore to visualize single molecules of endogenous telomerase in live cells. Here, we will decipher discrete and critical steps as hTR traffics from Cajal bodies to telomeres. Contrary to earlier FISH data in fixed cells, our preliminary data using diffraction-limited and super-resolution imaging modalities combined with single-molecule FISH show that hTR is broadly distributed throughout the nucleus. At telomeres, we show that following TPP1-driven recruitment, stable interactions are established between the enzyme and its substrate by RNA:DNA base pairing. Our goal is to apply photoactivation and photobleaching experiments to test the role of the catalytic subunit, hTERT, in the gating of hTR between the Cajal bodies and telomeres. In addition, we will engineer a short telomere to depict telomerase dynamics at critical telomeres that need to be elongated. Lastly, we will perform a proximity-based labeling and purification methodology to investigate the factors that control key steps of telomerase trafficking to short telomeres. All in all, our innovative approach offers a detailed view of the precise mechanics of telomere extension at physiological timescales and opens many future avenues for the study of the link between telomere maintenance and aging as well as cancer.