Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Allogeneic hematopoietic cell transplantation (allo-HCT) is often associated with a clinical complication known as graft-versus-host disease (GVHD), a major driver of mortality after allo-HCT. Current approaches to the prophylaxis of GVHD are primarily immunosuppressive therapies that can dampen the intended activity of the transplanted immune cells against the tumor and cause delayed immune reconstitution. Gastrointestinal (GI) microbiota, the highest microbial colonization in the body, has long been understood to contribute to the pathophysiology of GVHD. Indeed, patients undergoing allo-HCT are subject to dramatic immunological and microbiota perturbations, developing pre-HCT and continuing during transplant; GI microbiome diversity before and after transplant are independently associated with clinical outcome. When GVHD occurs, the GI tract is frequently involved, and patients often succumb to GVHD. New approaches to prevent GVHD and other transplant-related complications are urgently needed. We hypothesize that restoring the health of the intestinal microbial community early post-HCT is feasible and associated with improved transplant outcomes and immune reconstitution. In 2 studies of fecal microbiota transplantation (FMT) in allo-HCT, our group reported that an FMT intervention is a safe way to restore microbiota diversity. However, FMT has batch-to-batch variability depending on specific donors and carries the risks of transmission of infectious organisms or antibiotic-resistance genes. Here, we propose an entirely novel approach: a rationally designed oral-ecobiotic capsule that delivers a defined composition of pure bacterial strains. An oral, defined blend of strains offers advantages of predictable and reproducible pharmacology and would be immediately scalable to future studies. On this multi-PI proposal, we will capitalize on samples from an ongoing, investigator-initiated multicenter placebo-controlled phase 1b trial, led by PI Doris Ponce, which is first-in-human evaluating a clonally-derived multi-strain bacterial consortia for the prevention and restoration of the GI microbiome (NCT04995653). Our group has shown in a large multicenter study that early intestinal microbiome dysbiosis occurred universally among centers and had an association with transplant outcomes. In addition, innovative preclinical models of microbiome dysbiosis serve as the basis for the translational study and proposed microbiome analyses. Project objectives: Along with PI Marcel van den Brink, to evaluate the effects of intestinal microbiota restoration in allo-HCT recipients. We will pursue 2 specific aims that will evaluate the effects of microbiome restoration in the GI microbiome, allo-HCT outcomes, and immune reconstitution. Expected outcome: Results will provide new mechanistic insights into interactions between the intestinal microbiome and host immunity. Impact: Findings will inform future research not only for the reduction of transplant-related complications including GVHD, but also for other conditions in which GI injury occurs such as chemotherapy and/or radiation-induced toxicity and non-malignant inflammatory GI conditions. The proposed project outlines an entirely novel framework for targeting the GI microbiome in HCT.

NIH Research Projects · FY 2026 · 2023-05

Project Summary Tumor-reactive cytotoxic T lymphocytes (CTLs) often progress to dysfunction defined as T cell exhaustion. Marked by expression of the programmed cell death protein 1 (PD-1), the exhausted T (Tex) cell lineage is a developmental continuum wherein PD-1low Tex progenitors give rise to terminally dysfunctional PD-1high Tex cells. Notably, the immune checkpoint blockade therapy revives Tex progenitors, but not terminal Tex cells, calling for exploration of their differentiation mechanisms and means of therapeutic intervention. In a murine cancer model, tumor development induces differentiation of tumor-associated macrophages (TAMs) in association with generation of Tex cells. Transcriptome analysis revealed that TAMs exhibit shared characteristics with type 1 dendritic cells (DC1s) including expression of the transcription factor interferon regulatory factor-8 (IRF8). IRF8 promotes TAM presentation of cancer cell antigens to CD8+ T cells similar to DC1, but TAMs differ from DC1s in promoting high PD-1 expression. Importantly, macrophage-specific deletion of IRF8 attenuates Tex cell differentiation, and suppresses tumor growth. Furthermore, human TAMs express IRF8, and a TAM IRF8 gene signature tracks with a Tex cell gene signature in multiple cancer types. Based on these findings, we hypothesize that terminal Tex cell differentiation is driven by IRF8-expressing TAMs with a tolerogenic antigen-presenting cell (APC) function in the tumor tissue, and such a TAM-Tex cell regulation axis can be targeted for novel cancer immunotherapy. To test this hypothesis, we will first determine how IRF8 is induced in TAMs, and how it promotes TAM APC function. By performing chromatin profiling experiments and using genetic mouse models, we will assess whether the TAM-enriched transcription factor Batf2 enables IRF8 autoactivation via the +32kb Irf8 enhancer element. IRF8-deficient TAMs are defective in acquiring cancer cell antigens. Using mouse strains with macrophage- or cancer cell-specific deletion of the B2m gene, we will investigate whether IRF8 promotes TAM acquisition of antigens through cross-presentation or cross-dressing. Secondly, we will define how the tolerogenic function of TAMs is specified, and how it can be reprogrammed for therapy. Compared to DC1s, TAMs express lower levels of interleukin-15 (IL-15), but exhibit heightened transforming growth factor-b (TGF-b) signaling. By generating macrophage-specific gain- or loss-of-function mouse models, we will explore whether blockage of TGF-b signaling reverses the tolerogenic APC function of TAMs in an IL-15-dependent manner, and whether overexpression of IL-15 in macrophages is sufficient to induce T cell-stimulatory TAMs in genetic models and in a cell therapy setting. Compared to DC1s, TAMs have a smaller cell size. We will investigate whether and how activation of the metabolic regulator mammalian target of rapamycin complex 1 (mTORC1) reprogram TAMs to be immunostimulatory APCs. Successful completion of this project will not only generate mechanistic insights into APC control of Tex cell differentiation in cancer, but also guide the targeting of the TAM-Tex cell regulation axis for therapy of a wide range of malignancies.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Mutations in the KRAS GTPase are some of the most frequent gain-of-function alterations in cancer. KRAS mutations are found in 25% of patients with lung cancer and nearly half of these involve a G12C substitution. Lung cancer is the leading cause of cancer related mortality and, considering that more than 250,000 people are diagnosed each year with lung cancer in the US, developing therapeutic strategies that directly targeting KRAS oncoprotein is of paramount importance. The discovery of inhibitors that selectively target KRAS G12C has been one of the most exciting recent developments in precision oncology. We previously showed that the inhibitors trap the oncoprotein in an inactive (GDP-bound) state by blocking its reactivation by nucleotide exchange and that such inhibition requires the breakdown of GTP by mutant KRAS in cancer cells. Inactive state selective KRAS G12C inhibitors (e.g., sotorasib and adagrasib) have a 37-43% objective response rate (ORR) and a 6-8 month median progression free survival (PFS) in patients with lung cancer. Sotorasib was approved by the food and drug administration (FDA) as a second line treatment in patients with KRAS G12C- mutant lung cancer. Despite these early successes more work is needed if we are to dramatically affect the survival of patients with KRAS mutant cancer. The current proposal seeks to identify factors responsible for modulating drug adaptation and resistance in patients and to identify improved KRAS-directed therapeutic approaches. The first part of the proposal builds on our preclinical work showing that isogenic lung cancer cells bypass G12C inhibition in a non-uniform manner. Our preliminary data suggest this occurs because of the conformation-specific nature of available G12Ci and the synthesis of new KRAS G12C in some drug-treated cells. In Aim 1, we will determine the heterogeneity in response and adaptation to treatment with inhibitors targeting either the active or the inactive conformation of mutant KRAS. The studies will be performed in parallel in clinical specimens and in patient derived models to ensure robustness and reproducibility. Aim 2 builds on our recent effort to study acquired resistance in patients treated with sotorasib or adagrasib. Here we will focus on alterations that cause resistance to emerging active state selective drugs, relying initially on patient-derived models and then on clinical specimens, once these drugs enter clinical testing in late 2022. In addition, prospective studies with barcoded populations will investigate the clonal interactions that guide the selection of resistance-causing alterations. In Aim 3 we will study the mechanisms by which the alterations or phenotypes identified in the preliminary data or in Aims 1 and 2 lead to resistance. The early success of KRAS G12C inhibitors has inspired the development of additional KRAS inhibitors that are entering clinical trials and promise to improve the survival and quality of life of patients with KRAS driven cancer. The proposed work will be of vital importance in maximizing the therapeutic potential of these long-awaited therapies.

NIH Research Projects · FY 2026 · 2023-05

SUMMARY: The problem: In molecular medicine multiple parameters are combined for a more inclusive evaluation towards personalized medicine. A thorough characterization of a patient’s tumor upfront provides better outcomes, i.e., better insight affords higher survival rates. In contrast and almost anachronistically, PET imaging (the most sensitive and quantitative imaging method) is “monochromatic” as it can only assess one parameter at the time, lacking depth of information. Suitable imaging tools that allow visualization of more than one target in patients are needed, akin an in vivo cytometry. Optical imaging utilizes multiple parts of the spectrum to visualize several targets simultaneously, but this is not feasible for whole-body clinical imaging due to the limited penetration of light. Single-photon emission computed tomography (SPECT) can distinguish several isotopes based on the energy of their emissions, but spectra often overlap and the required collimation significantly decreases sensitivity. Different tracers could be imaged sequentially with PET but multiple scans increase the dose exposure from the required CT scans. It also requires sufficient decay of one tracer over time to be able to image the remaining one, decreasing convenience for patients. For three or four different isotopes this requires an even more complex coordination. As a solution, we propose the new modality of multicolor PET (mPET), which allows for simultaneous PET acquisitions of up to four different radiotracers at the same time. This new imaging paradigm utilizes one standard (pure) positron emitter together with positron-gamma emitters that produce triple (positron-gamma) coincidences, where a prompt gamma emission immediately follows the positron and identifies the isotope. We already imaged two isotopes in a standard PET scanner with the aid of the additional gamma signal but without energy discrimination. Here, we utilize the energy of the gamma signal as “barcode identifier” for the corresponding isotope while the spatial information is carried with the 511 keV annihilation photons. The prompt gamma requires detection without spatial decoding, which is achieved by an add-on gamma detector with sufficient energy discrimination and temporal resolution that is synchronized with the PET scanner. We established this system already and imaged three isotopes together. Here, we will in Aim 1 optimize the mPET set-up and then employ mPET to address important clinical/biological problems: In Aim 2, we will dissect the tumor microenvironment, interrogating signatures important for prognosis. In Aim 3, we will use mPET to interrogate important players in checkpoint inhibition therapy (CD4+ / CD8+ / PD-L1 / macrophages) simultaneously over time to predict response and will explore cellular therapies by following the injected cells to their target. The overall impact of this study will be significant, as mPET represents a true paradigm shift, allowing imaging of several radiotracers simultaneously. We demonstrate the power of this novel approach with clinically relevant approaches. More tracers asses a tumor better than one tracer alone, will provide a deeper insight into relevant tumor signatures, resulting in improved patient outcome.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY/ABSTRACT This proposal comprises a three-year research and career development program for Rachel Niec, MD, PhD to achieve independence as an investigator at the intersection of immunology and intestinal epithelial biology. Dr. Niec completed her doctoral training in immunology at Sloan Kettering Institute and Internal Medicine Residency and Gastroenterology Fellowship at Weill Cornell Medical College. The research and career development activities will occur at Rockefeller University. Dr. Niec will engage in career development activities including didactics, workshops in grant writing and lab management, acquisition of technical skills and scientific expertise, conference presentations, and a comprehensive mentorship program through her dedicated mentoring team. Dr. Niec has a strong background in cell biology and, over the course of this K08 award at the Rockefeller University, aims to expand her skills in advanced microscopy, transcriptomics and stem cell (SC) biology to study how features of the intestinal SC (ISC) niche interpret diverse cues to direct SC activity in health and in disease. Inflammatory bowel diseases (IBD) are inflammatory conditions focused within the intestinal epithelium and driven by genetic, environmental, and microbial factors. Intestinal epithelial maintenance is dependent on ISC which in turn rely on their niche for local signals to direct their activity. While many niche factors emanate from local sources, intestinal epithelial tissues is subject to systemic changes suggesting vascular features may regulate ISC activity. While lymphatics abnormalities have long been appreciated as a feature of IBD, the vascular features of the intestinal niche that control ISC behavior are unknown. The central hypothesis is vasculature is a key regulator of ISC function, through direct signaling between vasculature, ISC and other niche cells. Two specific aims are proposed: (1) Define the contribution of lymphatic capillaries to the cellular network comprising the intestinal stem cell niche; (2) Determine the impact of lymphatic derived Reln and lymphatic:stem cell interactions on ISC maintenance and function. These aims will be addressed using tissue clearing and advanced 3D imaging, established and newly developed single cell and spatial resolution transcriptomics, and novel in vitro coculture and in vivo molecular genetic approaches. The significance of this proposal lies in its relevance to fundamental mechanism of epithelial maintenance and regeneration and to pathogenesis of IBD. The proposal is innovative in the combination of advanced methods in imaging and transcriptomics and their application to SC niche biology and intestinal inflammation. Long-term, Dr. Niec aims to apply the expertise gained to identify environmental and inflammatory signals that maintain and regulate the intestinal SC niche in homeostasis and disease to improve prevention and treatment of IBD.

NIH Research Projects · FY 2026 · 2023-04

The last decade has seen an exponential increase in multimodal cancer -omics data due to the development of high throughput cutting-edge technologies that capture DNA, RNA, protein and metabolite level data. There is a critical need for training the next generation of data scientists in genomics who can be tasked to translate the complex integration of these high dimensional data to deliver precision oncology using sophisticated statistical and computational methods and tools. Due to growing enticements from industry, there is significant threat of “brain drain” from academia that is especially prevalent among those with data science and high dimensional computational skills. This proposal seeks to develop the Memorial Sloan Kettering Cancer Center’s Genomics Research Experience for Master’s Students (GEMS) Fellowship Program, a structured and specialized program that targets master’s level trainees in biostatistics, statistics, data science, computer science or related quantitative discipline (6 per summer over 5 years). The GEMS program is a hands-on, 12-week immersive and interdisciplinary summer research experience in cancer genomics with several components that make the program unique: access to the world's leading resources of cancer genomics data and tools, a quantitative and scientific dual-mentoring model, pairing with a peer advisor, and a lecture/mini workshop series on cutting-edge genomic technologies and high dimensional data analysis given by program faculty who are world experts. The fellows will gain experience working with whole-genome and whole-transcriptome next-generation sequencing data and obtain a real understanding of high-dimensional data analysis, advanced statistical genomics concepts and modeling techniques, parallel computing and reproducible research paradigms. This combination of large data resources, computational infrastructure, didactic lecture and hands-on workshop series from program faculty creates a unique environment in which the following aims will be pursued: 1) develop a genomics research internship program that annually recruits 6 students to provide them a 12-week immersive hands-on research training experience addressing cutting edge cancer genomics research questions; 2) develop and facilitate a bi-directional evaluation plan to provide timely assessment and feedback for the participants and their mentors; and 3) track participants' career development over time to evaluate the success of the program and to support program alumni to pursue quantitative careers in genomics. GEMS will be co-led by 2 PDs at Memorial Sloan Kettering Cancer Center with long track records of impactful research, mentorship, and successful knowledge translation. The dual team mentoring approach will prepare students for the inter-disciplinary translational science workforce and will learn to become critical thinkers. GEMS will prepare trainees for impactful careers as -omics data scientists and will obtain work-force training in genomics cancer medicine.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Patients with gastrointestinal (GI) metastases in the peritoneal cavity suffer high morbidity, chemotherapy resistance, and decreased overall survival. The molecular mechanism and phenotypic features that facilitate peritoneal metastasis are poorly understood, although mucin-expressing tumors exhibit especially high predilection for peritoneal spread. Preliminary analyses determined in a pan-cancer patient cohort that mucinous GI tumors are highly enriched in a specific mutation in GNAS that leads to pathogenic gain-of- function in the encoded G protein alpha subunit (GNAS). GNAS mutations are associated with pancreatic and small cell lung tumor development, yet the pathogenic mechanism mobilized by GNAS to facilitate metastasis is unknown. Preliminary data demonstrates that patients with GNAS-mutated GI cancers exhibit increased burden of peritoneal metastases, decreased response to first-line chemotherapy, and poor overall survival. Analysis of tumor DNA and RNA from independent groups of patient tumors shows that mutated GNAS may operate within a distinct gene-regulatory network that activates PI3K and MAPK signaling. The PI and collaborators established GNAS-mutated, patient-derived-organoids (PDOs) and a peritoneal metastasis mouse model to evaluate the hypothesis that GNAS is a key molecular driver that governs the signaling pathway involved in metastatic peritoneal seeding and growth. To test this hypothesis, the study will (1) define the GNAS-induced gene regulatory networks and phenotypic features that facilitate tumor progression in patient peritoneal metastasis and CRISPR-Cas9 gene- edited PDOs and (2) determine the impact of GNAS modulation on metastasis distribution and pathogenicity in vivo using xenograft metastatic models. Investigations will integrate multi-omic analyses with PDO experimental validation to improve the fundamental understanding of metastasis and validate GNAS signaling as a therapeutically-relevant target. The applicant, Dr. Michael Foote, is a rising Assistant Attending in the GI Oncology Service at Memorial Sloan Kettering Cancer Center (MSKCC). Dr. Foote has defined a 5-year plan to integrate his background in targeted drug development and computational bioinformatics with new expertise in experimental modeling and molecular biology. Dr. Foote will be mentored by a complementary advisory committee led by Dr. Luis Diaz, an international expert in genomics with a strong background in training successful independent physician scientists. Dr. Foote’s development plan includes supportive workshops and mentoring from an advisory committee of experts in molecular biology, cell signaling, and bioinformatics at MSKCC, a world-renowned translational center of excellence. Completion of the project goals will facilitate new therapeutic approaches for treating metastatic GI cancer and Dr. Foote’s development into an independent physician scientist and expert leader in GI metastasis.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Intellectual Disability (ID) and Autism Spectrum Disorders (ASD) are the most common developmental disorders, affecting 3-4% of children in the U.S, with few therapeutic options. Although insights into the mechanisms that cause these heterogeneous disorders remains very limited, genetic studies of ID/ASD have revealed a central role for mutations in genes encoding transcriptional regulatory proteins, including multiple subunits of the SWI/SNF ATP-dependent nucleosome remodeling complex (BAF complexes). For example, heterozygous loss-of-function mutations in Arid1b, the largest subunit of the canonical BAF complex (cBAF), are among the most frequent mutations observed in de novo ID/ASD cases. However, the function of ARID1B/cBAF complexes in gene regulation during normal brain development and the specific developmental processes that are disrupted by Arid1b loss-of-function mutations remain significant gaps in knowledge. Characterizing the specific functions of transcriptional regulatory complexes in cell type-specific gene regulation in the dynamic and heterogeneous cellular environment of the embryonic brain remains difficult using current model systems and experimental tools. Our long-term goal is to develop pluripotent stem cell- based model systems and experimental tools to characterize gene regulatory networks that control cell fate specification during brain development. Towards this end, my lab recently developed a robust, reproducible protocol to make forebrain organoids from mouse pluripotent stem cells. This reduced complexity model maintains key features of the developing brain and can enable experimental approaches that are not possible in vivo. Here, we propose to 1) perform the first in depth transcriptomic and epigenomic characterization of cerebral cortex development in our novel mouse organoid model using single cell genomics approaches, 2) define the impact of Arid1b loss-of-function mutations on cortical development in vivo and in organoids, 3) implement chemical genetic approaches (dTAG) to parse stage-specific effects of ARID1B loss, 4) define direct effects of ARID1B loss on gene regulation during cortical development, 5) determine which gene expression changes are reversible upon reintroduction of ARID1B into post-mitotic cortical neurons. These data will help to establish mouse cortical organoids as a model system that can complement and extend upon in vivo approaches for studying molecular and cellular mechanisms of brain development. Our findings will provide new insight into the mechanisms by which loss-of-function mutations in Arid1b give rise to changes in gene regulation during early stages of cortical neurogenesis and reveal specific genes, cell types, and developmental stages that are susceptible to reduction of cBAF complexes. Given the relevance of this complex to common developmental disorders, our findings may also reveal novel therapeutic opportunities for ID and ASD.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT: The polycomb repressive complex 2 (PRC2) complex establishes and maintains di- and tri-methylation of the histone H3 at lysine 27 (H3K27me2/3) in the genome and regulates chromatin structure, transcription, cellular stemness and differentiation. PRC2 is a context-dependent tumor suppressor whose core components (e.g., EZH2, EED, or SUZ12) are inactivated in various cancer types. Among these, high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma with no effective therapies, has a high prevalence (≥80%) of biallelic inactivation of EED or SUZ12, leading to complete loss of the PRC2 function. PRC2 loss in cancer results in aberrant transcriptional activation of developmentally silenced master regulators, which leads to enhanced cellular plasticity and aberrant activation of multiple signaling pathways. We recently uncovered that PRC2 loss in cancer leads to an immune-desert tumor microenvironment and resistance to immune checkpoint blockade. Nevertheless, we observed that, in murine models of PRC2-loss MPNST and mammary tumors, response to immunotherapy can be enhanced by infection with immunogenic virus, which activates double-stranded RNA (dsRNA) signaling responses. Moreover, we have identified and validated a lethal interaction between PRC2 loss and DNA methyltransferase 1 (DNMT1) knockdown in MPNST. In vitro and in vivo treatment with a demethylating agent (decitabine) or selective DNMT1 inhibitors (GSK862, GSK032) led to enhanced cytotoxicity and antitumor effects in PRC2-loss compared to PRC2 wild-type (wt) MPNST models. Mechanistically, DNMT inhibitor (DNMTi) treatment in the PRC2-loss context amplified the expression of endogenous retrotransposons and subsequently led to activation of innate immune responses, which could be explained by retrotransposons forming dsRNA and triggering cytotoxicity via PKR-dependent dsRNA sensing. we hypothesize that PRC2 loss in cancer may create therapeutic opportunities for agents (e.g., DNMTi, synthetic dsRNA, immunogenic viruses) that activate the dsRNA signaling pathways in tumor cells to induce cytotoxicity and enhance immunogenicity. Here, we propose integrative multi-omics analysis (e.g., transcriptome, epigenome, single-cell [sc]RNA-seq and scATAC-seq) and innovative approaches (e.g., novel immunogenic viruses, a novel lineage-tracking system) to evaluate novel therapeutic strategies and their mechanisms of activating dsRNA responses and anti-tumor effects in relevant PRC2-loss cancers, focusing on specific aims: (1) dissect the molecular mechanisms that underlie the selective sensitivity to DNMTi in different PRC2-loss cancers; (2) evaluate exogenous dsRNAs as a strategy to induce cytotoxicity in PRC2-loss tumors; and (3) evaluate novel therapeutic strategies of activating dsRNA-responses in relevant cancer models. These studies will generate insights into exploiting dsRNA-sensing and activation of innate immune responses as therapeutic strategies in PRC2-loss cancers and provide the pivotal preclinical data for biomarker-driven clinical trials.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY/ABSTRACT Non-Hodgkin’s lymphoma (NHL) is among the most common cancers and despite current therapies many patients relapse from their disease. Recent discoveries have implicated epigenetic mechanisms and non- classical oncogenic programs as dysregulated in patients with B-cell lymphoma. Protein arginine methyltransferase-5 (PRMT5), the major enzyme responsible for the arginine symmetric dimethylation of histones and non-histone proteins, plays an important role in lymphomagenesis, controlling the growth of transformed B cells. PRMT5 is overexpressed in lymphoma and may represent a novel therapeutic target for this disease. In order to assess what regulators drive resistance, we performed a genome wide CRISPR screen. Surprisingly, we uncovered the RNA binding protein MUSASHI2 (MSI2) as the top hit. MSI2 is an RNA binding protein that has been implicated as a stem related protein that is the most highly expressed in the most aggressive cancers but its role in B-cell lymphoma is not known. Our preliminary data suggests that MSI2 is highly expressed in lymphomas and reduction reduces proliferation which is further reduced by PRMT5 inhibition. We also discovered new post-translational modifications mediated by PRMT1 and PRMT5 in B-cell lymphoma and demonstrated a functional requirement for these newly discovered modifications. We propose to study this new PRMT-MSI2 axis in driving lymphomagenesis using genetic mouse models, human lymphoma cell lines and patient samples. Furthermore, we utilize technological innovations to study direct MSI2 targets and the mechanism for how MSI2 mediates resistance to PRMT5 inhibition in lymphoma. These studies have broad implication to how RBPs can become dysregulated and their function controlled by PRMTs in B-cell lymphoma.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT Nominating candidate risk genes and gene sets underlying disease-critical processes is of utmost importance for developing drug targets and informing CRISPR screening experiments. To this end, large scale single-cell genomic and epigenomic data (from RNA-seq, ATAC-seq, Perturb-seq) can be integrated with genome wide association studies (GWAS) to enhance our understanding of the genetic architecture of human complex diseases and traits. In this proposal, I plan to develop new computational approaches to integrate single- cell functional genomic and epigenomic data with GWAS data for complex diseases and traits to identify and rank disease-critical genes and gene sets characterizing functional processes, as well as pinpoint short genomic regions linked to these disease-associated genes. My K99 training will be conducted at the Harvard T.H. Chan School of Public Health, as well as the Broad Institute, under the mentorship of Dr. Alkes Price. The key areas of my training will be to develop and evaluate approaches for gene-level and gene set-level functional architecture of diseases and traits and integrative analysis of single-cell, as well as bulk, functional genomics data with human disease genetics. My proposed approaches will attempt to bridge the gap between functional genomics and human genetics and downstream clinical drug/gene intervention experiments. The long- term goal of this research is to produce a set of computational tools that identify and rank top disease-critical genes, top disease-critical gene sets characterizing cell types or cellular processes and gene-linked genomic regions for each disease/trait. These approaches will reshape our understanding of the functional architecture of human diseases at cellular level and will inform future drug perturbation and CRISPR screening experiments. The first aim of this proposal is to develop methods to identify and rank disease-critical genes by integrating common and rare variant disease associations with gene-level functional information derived from single-cell genomics experiments. Here I will develop, compare and contrast multiple gene prioritization strategies that differ in how they annotate SNPs for a gene, how they aggregate variant level associations at gene level and how they use functional data in performing the gene prioritization. The second aim of this proposal is to develop new computational strategies to assess disease information in sets of genes that underlie a cell type or cellular processes active within or across cell types in a tissue. The third aim of this proposal is to pinpoint and prioritize short genomic regions that are either proximally or functionally linked (for example, as an enhancer) to disease- critical genes and gene sets from Aims 1 and 2. Here, I plan to integrate GWAS association signal near these gene-linked regions with deep learning models that can infer allelic effects at base pair resolution and single-cell ATAC-seq data. All disease-critical genes, gene sets and gene-linked regions along with relevant computational tools will be distributed publicly to the scientific community.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT Breast cancer (BC) is the most common cancer in women and the most common cause of female cancer deaths. While breast conserving surgery (lumpectomy) with neo/adjuvant therapy is the best desirable treatment option, about 25% of cases require secondary surgery when tumors are incompletely removed, with possible additional complications, patient anxiety, and conversion to mastectomy. Re-excision rates are especially high among patients with more aggressive tumors, which are characterized by acidic tumor microenvironment (TME). We propose to develop and implement a holistic approach for tumor acidity imaging with pre-operative multi- parametric MRI (mpMRI) and novel pHLIP® ICG near infrared fluorescent (NIRF) intra-operative imaging. pHLIP- ICG is an ICG, FDA-approved NIRF indocyanine green dye, conjugated to a pH-sensitive pH-Low Insertion Peptide (pHLIP) for targeting tumor cell surface acidity and for marking the invasive acidic tumor-stroma interface, thereby allowing pHLIP-ICG to target cancer lesions and to delineate positive tumor margins. Our clinical study consists of a prospective first-in-human phase I trial and a feasibility phase IIa trial. Aim 1 (phase I and IIa): To prospectively develop, implement, and optimize novel mpMRI sequences in consecutive BC patients undergoing lumpectomy for clinical non-contrast non-invasive assessment of the TME and tissue acidity including intravoxel incoherent motion diffusion-weighted imaging, diffusion tensor imaging, lactate MR spectroscopy, and chemical exchange saturation transfer imaging. Aim 2 (phase I): To evaluate the safety and tolerability of intravenous pHLIP-ICG administration in 4 dose levels in patients with primary BC undergoing lumpectomy, and to establish the “optimal” dose which will be used in the phase IIa trial. This optimal dose is defined as the lowest dose that is safe and that allows the best intra-operative visualization of BC with pHLIP- ICG NIRF imaging. Aim 3 (phase IIa): To establish the feasibility of pHLIP-ICG targeting and intra-operative imaging of primary BC and margin delineation at the selected optimal dose of pHLIP-ICG using histopathology as the reference standard. Aim 4 (phase IIa): To correlate TME acidity imaging (non-contrast non-invasive pre- operative mpMRI, and intra-operative and ex vivo pHLIP-ICG NIRF imaging) with standard histopathology and investigational immunohistochemistry for concordance and accuracy of BC visualization, margin status, and properties of the TME including different levels of acidity, lesion extent, and TME structure. The overarching long-term goal is to improve the standard of care: i) pre-operative non-invasive, non-contrast TME acidity imaging with mpMRI will allow the identification of more aggressive tumor phenotypes that require intensified treatment, improve planning of surgical and treatment strategies, and enable monitoring spatial-longitudinal of tumor biology with treatment; and ii) intra-operative pHLIP-ICG NIRF imaging will allow an improved up-front resection of primary breast cancer, which could allow a shift from more extensive surgeries to highly accurate tissue-sparing lumpectomies – therefore improving the quality of patients’ lives.

NIH Research Projects · FY 2026 · 2023-01

Project Summary Natural killer (NK) cells are cytotoxic innate lymphocytes that protect the host against viruses. Newborns and immunocompromised individuals lacking NK cells and are extremely susceptible to viral infection, including herpesviruses such as human cytomegalovirus (HCMV). HCMV can be accurately modeled using mouse cytomegalovirus (MCMV) infection in mice, which represents a robust system for investigating antiviral NK cell responses. From the previous R01 funding period, my lab has discovered new cellular and molecular mechanisms underlying NK cell responses against MCMV. This current R01 renewal seeks to understand the transcriptomic, epigenetic, and metabolic control of the antiviral NK cell response, with studies centering around a novel role for the transcription factor IRF4. In exciting preliminary data, we find IRF4 is rapidly induced in NK cells during MCMV infection and plays a critical role in their effector response. For the proposed experiments, we have generated new transgenic mice with conditional Irf4 ablation. In Aim 1, we will determine how IRF4 is transcriptionally and epigenetically regulated in activated NK cells, and test how IRF4-deficiency impacts the function of NK cells against MCMV infection. Aim 2 will identify novel gene targets and binding partners of IRF4 in NK cells by integrating transcriptomic, epigenetic, and proteomic approaches. In Aim 3, we will investigate whether IRF4 drives antiviral NK cell responses by controlling overall metabolism and mitochondrial health. Altogether, the studies in this R01 renewal will advance our understanding of the molecular basis by which these powerful effector cells can mediate protection against pathogen invasion, and establish innovative clinical paradigms for how NK cells may be harnessed for therapeutic strategies against infectious disease.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY CD8 T cells specific for cancer cells are found within human tumors, but despite their presence, tumors progress, suggesting that T cells become unresponsive. To design predictably effective immunotherapies, we must elucidate the mechanisms controlling tumor-specific T cell dysfunction. We previously demonstrated that T cells in tumors enter an epigenetically encoded dysfunction state that becomes resistant to therapeutic reprogramming and found that TOX, a DNA-binding protein, is a key regulator enforcing the dysfunctional state. The factors that drive TOX expression, and how TOX precisely establishes the dysfunction program remain largely unknown. TCR signal strength impacts T cell differentiation, and while we know that antigen chronicity is a key driver of TOX-driven T cell dysfunction, we do not know how signal strength of chronic tumor antigen impacts TOX-driven dysfunction and amenability to immunotherapeutic reprogramming. In this application, we will determine how signal strength regulates TOX and TOX-driven dysfunction programs in mouse and human tumors, ask how TOX induces and maintains dysfunction, and target TOX and its downstream mediators to uncover the mechanisms underlying dysfunction imprinting. To achieve these goals, we will utilize clinically relevant genetic cancer mouse models and track T cells longitudinally within progressing tumors while encountering tumor antigens with varying signal strength. We will employ transcriptomic and epigenomic methods and innovative protein degradation strategies to determine what controls TOX, how TOX induces and/or maintains dysfunction, and how TOX downstream mediators regulate the epigenetic programs associated with plasticity and dysfunction imprinting. We will leverage human neoantigen- specific tumor-infiltrating T cell resources to understand how TCR signal strength determines TOX- dependent molecular signatures and functional states of T cells in human tumors. Importantly, we will test and design strategies to target TOX and TOX downstream mediators to improve cancer immunotherapy.

NIH Research Projects · FY 2026 · 2023-01

SUMMARY Lung adenocarcinoma (LUAD), the most common subtype of non-small cell lung cancer, results in ~55,000 deaths in the US every year. Despite the recent advancements in LUAD treatment, the disease remains highly intractable. Thus, a critical unmet clinical need exists for novel and effective therapeutic strategies for LUAD patients. Cancer cell plasticity – the capacity to differentiate and adapt to cell-extrinsic pressure – drives tumor progression and is a major cause of treatment failure in LUAD. Thus, targeting plasticity in LUAD is a promising therapeutic concept. Realizing the therapeutic potential of targeting cancer cell plasticity requires fundamental understanding of the cell states that promote plasticity in LUAD as well as the molecular mechanisms that drive them. Using a genetically engineered mouse model (GEMM) of LUAD and single-cell mRNA sequencing (scRNA-Seq) to investigate LUAD evolution we identified a high-plasticity cell state (HPCS) that is acquired by a subset of LUAD cells in early stages of tumor evolution. The HPCS was ubiquitously maintained in mouse and human LUAD in vivo irrespective of stage and considerable intra- and inter-tumoral genetic and phenotypic diversity. Further, the HPCS gene expression signature correlated with particularly poor patient outcomes. Prospectively isolated HPCS cells were endowed with robust capacity for differentiation (plasticity) and proliferation, and the HPCS was strongly enriched following chemotherapy. Our preliminary work strongly supports plasticity is concentrated in the HPCS and that it is associated with high growth potential and chemoresistance. However, the contribution or essentiality of the HPCS for LUAD growth, treatment resistance, or emergence of new malignant cell states within LUAD tumors is not known. Similarly, little is known of the transcriptional drivers of LUAD plasticity. We hypothesize that the HPCS is essential for progression of premalignant neoplasias to LUAD as well as for LUAD growth, cell state transitions, and chemoresistance. To address this hypothesis, we will interrogate HPCS in LUAD progression and treatment resistance using lineage- ablation and lineage-tracing using a novel reporter system that we have generated, which we will combine with scRNA-seq. To address molecular HPCS drivers, we will inactivate or overexpress two candidate transcription factors in the LUAD GEMM and in human patient-derived xenograft (PDX) LUAD models, followed by gene expression and chromatin accessibility profiling. Our proposed study will allow us to establish the HPCS, a previously unknown cell state, as key to eradicating plasticity in LUAD. This would lead to a new treatment paradigm, motivating targeting of high-plasticity cell states across solid tumors. Furthermore, our work will contribute a novel platform for the in situ investigation of cell state heterogeneity in cancers in vivo.

NIH Research Projects · FY 2026 · 2023-01

Abstract The research project proposed here addresses the pressing need for better statistical models and methods to analyze the spatial architecture of the tumor microenvironment (TME). TME data has demonstrated there is clear clinical and biological importance in the spatial architecture, e.g. as a determinant of response to treatment and metastasis. Given the recognized importance of the TME in cancer, technology has advanced at pace to profile the spatial properties of tumors using high resolution measurements including spatial transcriptomics and proteomics. However, the requisite computational methods to fully interpret these measurements are lagging. Accordingly, in Aim 1 we will develop a statistical framework, which we call BayesTME to model the TME at multiple scales, ranging from the level of individual cells to top-level patient stratification. We will develop BayesTME as a suite of innovative statistical methods for Bayesian multiscale spatial modeling that would enable a new class of spatial statistical models to quantitatively evaluate the properties of the TME. We have gathered a diverse and scaled collection of spatial profiling datasets upon which to test, benchmark and evaluate BayesTME. In developing BayesTME, we will create modular tools in Aim 2 that can use the same statistical concepts across different technologies such as multiplexed immunofluorescence, imaging mass cytometry and spatial transcriptomics. This will permit statistical integration of datasets that may have been generated with diverse sets of technology. In addition, we will include an extension to the base BayesTME to identify recurrent spatial properties across a cohort of samples, enabling discovery and quantitative description of spatial communities that related to specific cancer phenotypes. Finally, in Aim 3 we propose a validation experiment that will generate parallel spatial profiling data–in the form of spatial transcriptomics and imaging mass cytometry from the same ovarian cancer specimens. This dataset will help to address a critical question in ovarian cancer which is how TME dynamics enable bowel metastasis - a major determinant of morbidity and mortality for women with ovarian cancer. In summary, the goals of this proposal are to develop a robust, new class of statistical methods for analyzing the spatial architecture of the TME, generate robust open-source software enabling application of our methods across multiple spatial profiling techniques, and validate our methods and software by using them to conduct large-scale data analyses investigating novel biological hypotheses regarding the spatial architecture of the TME. Accomplishing these goals will lead to new quantitative encoding of the properties of the TME that are statistically grounded that will in turn lead to a new class of spatial biomarkers to define malignant phenotypes in cancer.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT This proposal is significant because we aim to study the cellular mechanisms that regulate the increased risk of breast cancer-related lymphedema (BCRL) development in Black women. This is important because BCRL is a highly morbid disease that causes chronic and progressive arm swelling. Patients who develop BRCL have diminished quality of life, require life-long care with compression garments, and can develop recurrent infections that require hospitalization. Due to the high prevalence of breast cancer, BCRL is the most common form of lymphedema in developed countries, afflicting 20–35% of women who undergo axillary lymph node dissection (ALND). To identify risk factors for BCRL, our group has prospectively followed 276 women with arm measurements before and after ALND for 2 years. We have found that Black women have the highest risk of BCRL even after adjusting for confounding variables. In our study, Black race increased the risk of BCRL development by >3.6 fold compared with White race. These findings are supported by two other published studies reporting increased risk of BCRL development in Black women who undergo ALND for breast cancer. Thus, while there is strong evidence that Black women have a significantly increased risk of developing BCRL, the cellular mechanisms that regulate this risk remain unknown. This gap in our knowledge is important and a major barrier to developing novel therapies that prevent or treat lymphedema in this patient population. In addition, understanding how Black race increases the risk of BCRL may shed light on the mechanisms that regulate the pathophysiology of this disease in general. Based on prior research and our preliminary studies, our central hypothesis is that Black women have an increased risk of developing BCRL due to a baseline increased propensity for inflammation and fibrosis. We propose to test this hypothesis using two Specific Aims. In Aim 1, we will analyze how racial disparities modulate inflammatory responses following lymphatic injury. This hypothesis is based on the finding that the pathophysiology of lymphedema is linked to chronic inflammation and development of T-helper 2 (Th2)-biased immune responses. Black patients have a propensity for inflammation in other pathological settings, suggesting that these differences may also contribute to an increased risk for developing BCRL. In Aim 2, we will test the hypothesis that Black women have an increased fibrotic response to lymphedema. This hypothesis is based on the observation that fibrosis is a key pathological feature of lymphedema and plays a major role in regulating lymphatic function. Black individuals have an increased potential for fibrosis in a variety of pathological settings including inflammatory skin disorders.

NIH Research Projects · FY 2026 · 2023-01

SUMMARY It is critically important to establish the causes of organ-specific metastasis; without this knowledge, prevention and timely treatment of metastatic cancer will likely remain limited. This application aims to develop novel mathematical models to understand how a rewired cellular metabolism enables cancer cells that originate in one organ such as the breast to colonize distal organs such as the lung, the brain, and the bone, which have distinct microenvironments. We will study metabolic rewiring in parental cells and their metastatic derivatives and ask how metabolic gradients in the primary tumor can generate and maintain diverse lineages with specific metabolic adaptations for organ-specific metastasis. Our central hypotheses are 1) that metabolic adaptations are key to the match between the seed (the disseminated cell) and the soil (the distal site) in metastatic breast cancer, and 2) that the metabolic microenvironment in a primary tumor drives metabolically diverse subpopulations. The hypotheses have been formulated based on 1) published data detailing metabolic heterogeneity and that metabolic adaptations can promote metastasis, 2) preliminary data and analysis of RNA expression, metabolomics, and flux measurements, revealing different metabolic adaptations in breast tumor cells that home to different tissues, and 3) preliminary data showing that metastatic lineages respond differently to hypoxia and nutrient gradients, indicating a role for the metabolic microenvironment in maintaining diverse subpopulations within the same heterogeneous primary tumor. Mathematical modeling is critical to integrate experimental data and infer changes in metabolic fluxes that cannot be directly measured. The application proposes a research strategy that combines experimental, clinical, and mathematical analysis to identify new vulnerabilities in metastatic cancer cells. We will also develop novel mathematical models to study the ecological interactions between cell lines and their microenvironment and determine the conditions that lead to coexistence of metabolically distinct pre-metastatic subpopulations in the primary tumor.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY/ABSTRACT The mechanisms underlying evolution of tumor-associated stroma remain poorly understood. In solid tumors featuring a prominent stromal reaction, an improved understanding of the functions and origins of abundant stromal cell types may facilitate the development of new and effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is the quintessence of a fibro-inflammatory malignancy, with 50-90% of tumor volume occupied by a dense, desmoplastic stroma. Cancer-associated fibroblasts (CAFs) are the key cell type which drives the stromal reaction in PDAC, and recent reports suggest that stromal CAFs represent a heterogeneous population of cells from diverse origins, potentially including cell types which support and others which suppress tumor growth. Pancreatic stellate cells (PSCs) are lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype. PSCs have been suggested as the predominant source of fibroblasts in the PDAC tumor microenvironment. However, proper lineage tracing studies have never been performed, such that the relative contribution and specific functions of PSCs in the tumor microenvironment are unknown. Here we will take advantage of a novel mouse model we have developed to track PSC differentiation and function during pancreatic tumor progression in vivo. We hypothesize that PSC-derived fibroblasts in the PDAC microenvironment are a pro-inflammatory and tumor-supportive subset of PDAC CAFs, and thus represent a viable therapeutic target. Our preliminary data support the notion that PSCs contribute to only a subset of the CAF population in the tumor microenvironment, and the functions of these distinct populations are entirely unknown. Our data to date also highlight a potential role for genetic alterations in the epithelial compartment in orchestration of stromal fibroblast evolution. PDAC stromal heterogeneity and functional significance will be interrogated with the following specific aims. Aim 1: Determine the role of PSC- derived CAFs in pancreatic tumorigenesis. A novel mouse model will be used to ablate PSC-derived CAFs for the first time and analyze the impact on tumor growth, survival, and organization of the tumor microenvironment. Aim 2: Assess the consequence of tumor genotype in pancreatic cancer stromal evolution. Motivated by preliminary data, we will use our reporter mouse model and patient samples to analyze the interaction between p53 status in tumor cells and stromal CAF evolutionary routes, with important potential implications for tumor phenotype and therapy responses. Aim 3: Define the role of PSC-derived CAFs in therapy response and resistance. As PSC-derived CAFs express a transcriptional program associated with resistance to chemotherapy and immunotherapy, we will determine the effect of these CAFs on treatment response in vivo. These findings will shed light on mechanisms and consequences of stromal evolution during pancreatic tumorigenesis, and potentially identify distinct CAF populations of relevance in additional solid tumors.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Highly disruptive truncating mutations to protein-coding genes in mitochondrial DNA (mtDNA) affect nearly 10% of all cancers and predominantly arise heteroplasmically, affecting a fraction of the total mtDNA pool. Although decades of investigation into pathogenic mtDNA variants in the germline have established that they profoundly disrupt normal mitochondrial oxidative phosphorylation, the effects of such mutations in cancer cells are largely unknown. The fundamental barrier to rigorous interrogation of mtDNA mutations in cancer cells has been a lack of tools for genetically engineering mtDNA. Recently, a new mtDNA-editing technology pairing TALE binding domains to a DddAtox cytosine base editor (DdCBE) has been successfully used to introduce point mutations into mtDNA, revolutionizing the ability to genetically manipulate mtDNA with high precision. In parallel, our team recently discovered that truncating mtDNA mutations are under strong positive selection in specific genetic contexts (subunits of NADH dehydrogenase/Complex I, “CI”) and cancer lineages (colorectal, kidney, and thyroid cancers), and that the heteroplasmic dosage and transcriptional phenotype of these mutations are readily detectable in single cell sequencing data. These convergent discoveries motivated our team to engineer DdCBEs to introduce truncating mutations to several CI and non-CI mtDNA genes in cell lines, enabling for the first time a functional interrogation of truncating mtDNA mutations in cancer cells. Using these tools, we propose integrative computational/experimental studies to test the overarching hypothesis that CI-truncating mutations produce physiologically significant and therapeutically actionable metabolic changes in tumors. In Aim 1, we will computationally investigate mtDNA mutation patterns across ~100,000 tumor samples, identifying recurrent mutant alleles and co-incident driver mutations in nuclear DNA. In parallel, we will express DdCBEs to model CI- and non-CI truncating mutations in colorectal cancer cell lines, and define the molecular phenotypes conferred by CI truncating mutations using transcriptomic, metabolomic, and isotope tracing experiments. Our Preliminary Data indicates that the phenotype of CI-truncating mutations depends on their heteroplasmic dosage. Thus, in Aim 2 we will use transient expression of DdCBEs to produce isogenic panels of colorectal cancer cell lines at characteristically distinct mutation dosages. Using a combination of single cell and bulk molecular profiling, we will test the hypothesis that CI-truncating mutations rewire tumor cell metabolism towards a pro-proliferative configuration in a dosage-sensitive manner. Finally, hypothesizing that CI-truncating mutations induce genetic dependencies absent in mtDNA-wild-type cells, Aim 3 will use DdCBE-engineered cell line models to identify, validate, and mechanistically study novel synthetic lethalities associated with CI-truncating mutations. The results of these studies will deliver a new, detailed understanding of the function, dosage sensitivity, and therapeutic vulnerability of one of the most common genetic insults in the cancer genome.

NIH Research Projects · FY 2026 · 2022-12

Defining Mechanisms of Progression and Treatment Resistance in Localized Bladder Cancer PI: Eugene Pietzak, MD SUMMARY Our overall goal is to develop therapies that selectively target molecular alterations responsible for progression of bladder cancers from non-invasive to the often-lethal muscle-invasive disease state. For patients with non- muscle invasive bladder cancer (NMIBC), the current standard is bacillus Calmette-Guérin (BCG), a nonspecific immunotherapy instilled directly into the bladder lumen. While BCG can reduce the risk of disease recurrence, a proportion of patients subsequently progress to muscle-invasive bladder cancer (MIBC). Our preliminary results indicate that this disease state, termed “secondary MIBC”, is resistant to cisplatin-based chemotherapy. The goals in the current proposal are to understand the genomic basis for treatment resistance to BCG and to identify alternative molecularly directed treatments that can achieve disease cure without the need for radical surgery. The studies proposed are based on preliminary data indicating that cytotoxic chemotherapy sensitivity in bladder cancer is influenced by somatic and germline genomic profiles, in particular mutations in DNA damage response (DDR) pathway genes, most commonly within the nucleotide excision repair gene ERCC2. As our preliminary data suggest that mutations in DDR pathway genes may also confer sensitivity to BCG, we hypothesize that prior treatment with BCG results in cross-resistance to subsequent systemic chemotherapy. To test this hypothesis, we will leverage several prospectively assembled bladder cancer cohorts, including tumor pairs collected pre-BCG and following progression to MIBC. These cohorts will be used to validate DDR mutations as predictors of BCG and cisplatin-based chemotherapy sensitivity and to identify mechanisms of progression from NMIBC to secondary MIBC. As genomic heterogeneity is common in bladder cancer, we will supplement bulk sequencing studies with multi-regional sequencing and analysis of cell-free DNA from urine to define the influence of tumor heterogeneity on cancer outcomes in early-stage bladder cancer. Our preliminary analyses of high-risk NMIBC and secondary MIBC have also identified ERBB2 mutations/amplifications as potential mediators of progression to muscle-invasive disease. Several unique patient cohorts will be used to define the frequency of ERBB2 mutation/ amplification and HER2 overexpression in high-risk NMIBC and secondary MIBC. Prior functional studies of the role of HER2 in bladder cancer pathogenesis have been impeded by a lack of patient-derived models with ERBB2 mutations and gene amplification. We will thus leverage a recently developed biobank of patient-derived organoid models containing ERBB2 mutation/amplification to study the associations between ERBB2 mutational status/HER2 expression, oncogenic dependence on HER2, and sensitivity to HER2-directed antibody drug conjugate therapy, a promising breakthrough therapy for metastatic bladder cancer. In sum, our long-term translational goals are to use integrated clinical and laboratory studies to develop more effective and less toxic treatments for patients with localized bladder cancer, a frequently fatal yet understudied disease.

NIH Research Projects · FY 2026 · 2022-12

Alzheimer’s disease (AD), a progressive neurodegenerative disease and leading cause of dementia, exacts a tremendous toll on both the healthcare system and society broadly. Although the pathogenesis of AD is poorly understood, it centers on the production of so-called β-amyloid (Aβ) peptides, which are produced from amyloid precursor protein (APP) by the intramembrane protease γ-secretase. Indeed, mutations in the catalytic subunit of γ-secretase alter Aβ production profile and cause familial AD, making the protease a promising target for developing therapies to treat or prevent Alzheimer’s Disease. Specifically, developing agents that can modify γ- secretase activity to selectively reduce the formation of pathogenic β-amyloid species without affecting γ- secretase’s overall activity represent an attractive strategy to target AD. Despite significant progress in studying γ-secretase, we still do not understand how its catalytic activity and specificity are modulated at a mechanistic level. In the proposed project, we will apply a new approach and leverage novel reagents to investigate γ- secretase and its modulation. Using an interdisciplinary approach, we aim to elucidate how the cellular context and external factors modulate γ-secretase at the molecular level. We will also characterize new tools and reagents for γ-secretase and define their mechanism of actions. Collectively, the proposed studies will provide much needed insights into γ-secretase modulation and offer a molecular basis for Alzheimer’s disease pathogenesis and therapeutic development.

NIH Research Projects · FY 2025 · 2022-10

PROJECT SUMMARY GENOMIC BIOMARKERS OF SPLENIC LYMPHOMA Splenic marginal zone lymphoma (SMZL) is the most common form of primary cancer in the spleen. SMZL typically involves the bone marrow and peripheral blood (PB) at presentation. The diagnosis is invariably established in late stages of disease via a splenectomy which is a surgical procedure carrying major risk. Additionally, the diagnosis of SMZL is subjective because there are no specific histopathologic or immunohistochemical biomarkers of the disease. Consequently, early diagnosis of SMZL is challenging and not achieved in most clinical scenarios. Further, SMZLs are among the least reproducibly diagnosed category of lymphomas. The suboptimal diagnostic accuracy necessitates the development of qualitative and objective biomarkers of SMZL. Importantly, while all cases of SMZL are characterized by PB involvement at diagnosis, however this biologic feature has not been leveraged for the reliable early detection of the disease. Using whole genome and exome sequencing, we and others defined the genomic landscape of SMZL and identified recurrently mutated genes in SMZL. An unmet clinical need is the development of reliable biomarkers for the early and accurate diagnosis of the disease based on the characteristic genomic alterations of SMZL. We propose in this application to develop a genomic-based approach that utilizes and validates peripheral blood as for early and accurate diagnosis of the disease. The overall impact of the application is the establishment of a paradigm for early, sensitive and accurate disease diagnosis by analysis of peripheral blood, thereby permitting early and appropriate treatment for the disease.

NIH Research Projects · FY 2024 · 2022-10

Project Summary Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase encoded by the ALK gene located on 5q35. Structural alterations including translocations, copy number gains and activating mutations targeting ALK occur in many types of human cancer, including lung cancer, non- Hodgkin lymphomas, Spitzoid melanocytic lesions, neuroblastoma and inflammatory myofibroblastic tumor. In over 80% of pediatric anaplastic large cell lymphoma (ALCL), the most common form of mature T cell lymphoma in this population, the chromosomal aberration t(2;5)(p23;q35) results in the expression of the constitutively active tyrosine kinase NPM-ALK. NPM-ALK positive lymphoma has served as a model for understanding ALK-mediated oncogenesis and development of targeted therapies, thus NPM-ALK related studies carry profound implications for the cancer field in general. Unfortunately, even with current intensive combined chemotherapy, approximately 30% of patients experiences disease progression or recurrence within two years of treatment. However, clinical or genetic factors that cause ALCL relapse are not known. Furthermore, prognostic biomarkers that can be easily obtained using non-invasive methods have not been clearly defined in patients receiving targeted therapies in ALCL. Our central hypothesis is that sensitive and specific quantitative assessment of circulating NPM-ALK transcript using digital droplet (dd)PCR in conjunction with plasma levels of ALK auto- antibody will serve as unique disease-specific biomarkers that will provide an opportunity for assessment of response to therapy and lead to prognostic biomarkers that may identify patients with high risk for relapse. Using plasma samples from uniformly treated patients enrolled in a Children's Oncology Group (COG) Phase II study of Brentuximab Vedotin and Crizotinib with newly diagnosed ALCL, we address our hypothesis through the following specific aims: 1) Determine the utility of ddPCR for minimal disease detection and disease monitoring in ALK+ ALCL, 2) Determine the prognostic utility of anti-ALK immune response in ALK+ ALCL 3) Investigate the prognostic significance of a multivariate model combining ddPCR and immune response to ALK in patients with ALK+ ALCL. The development of sensitive and precise assessment of minimal disseminated disease and/or minimal residual disease will play an important role in management of patients with ALK+ ALCL and facilitate decisions about discontinuation of treatment and identifying patients at risk of relapse/progression.

NIH Research Projects · FY 2025 · 2022-09

Testing for prostate-specific antigen (PSA) in blood has enabled early detection of prostate cancer and reduced metastasis and death from disease—but also contributed to overdetection of low-risk cancers. Although no PSA concentration confers zero risk of finding cancer at prostate biopsy, a single PSA measurement at midlife is a remarkably strong predictor of the risk of developing lethal prostate cancer decades later. PSA is a proteolytic enzyme that is non-catalytic in blood, and it occurs in multiple forms. A statistical model based on four kallikrein (4K) markers (free, total, and intact PSA, plus human kallikrein-related peptidase-2 [hK2]) improves specificity in detecting high-grade prostate cancer among men with elevated PSA (reducing unnecessary biopsies) and is also a strong predictor of the risk of lethal prostate cancer decades later. While intra-individual fluctuations in PSA levels are common, an excessive degree of variability is highly problematic, as temporary “false positive” elevations reduce the specificity of PSA as a cancer marker, attenuate the diagnostic value of PSA kinetics, and lead to the use of unnecessary antibiotics. Less studied but similarly abundant in prostatic fluid as PSA, the concentration of microseminoprotein-ß (MSP, MSMB) in blood is inversely associated with prostate cancer risk, and a single nucleotide polymorphism (SNP, rs10993994) in the promoter region of the MSMB gene is also associated with prostate cancer risk, but the role of these markers in clinical decision-making is unclear. Similarly, a SNP in the SERPINA3 gene is significantly associated with blood levels of PSA, and the encoded protein, alpha-1-antichymotrypsin (ACT), is the predominant stable complexing ligand to PSA in the blood. However, the clinical value of these makers is undetermined, and it remains unclear whether ACT levels in blood influence the predictive value of a baseline PSA value or affect intra-individual variation in PSA. Additionally, the intra-individual variation of the 4K-panel is currently unknown but could be determined using high-quality serial samples. As the role of these different molecular markers in combined risk-prediction models of aggressive prostate cancer is not well understood, we plan to delineate the influence of intra-individual variability in serial screening samples on clinical decision- making for risk stratification and biopsy by a single PSA value and additional markers. Using blood samples from the PLCO, Göteborg-1 & -2 trials, and Multiethnic Cohort (MEC), we plan to: 1) quantify the patterns of variation in the 4K markers + MSP in serial measurements; 2) determine the relationship between a statistical model based on 4K markers + MSP and subsequent risk of lethal prostate cancer, then independently validate the clinical utility of the markers in decision-making and risk stratification before treatment decisions in a randomized trial of prostate cancer treatments (ProtecT); and 3) compare head-to-head the clinical utility of pre-biopsy biomarkers versus magnetic resonance imaging on cancer detection rates. The resulting insights will shed light on how to improve the specificity of prostate cancer screening and early detection.