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NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Despite the dismal five-year overall survival rate, a moderate response rate to treatments, and one of the highest suicide rates among cancer patients, human papillomavirus-negative head and neck squamous cell carcinomas (HNCs) are curable if diagnosed early. Oral epithelial dysplasias (OEDs) and oral lichen planus (OLP) are potentially premalignant lesions that offer a window for disease eradication. The current standard of care for these precursor lesions involves H.&E. histologic grading and long-term clinical follow-ups. Most of OEDs and OLP do not progress to cancer. However, a significant challenge is that it is impossible to maintain high-frequency follow-ups for every patient with OED or OLP. Emerging adjunct clinical technologies often evaluate diagnostic success based on their power to detect “high-grade” OEDs. However, the WHO histologic grading of OED has little, if any, prognostic value in determining the transformation risks. In addition, the histologic grading of OEDs has low inter-observer and intra-observer consistency with the kappa-values and strength of agreement rated slight-to-poor. As a result, the American Dental Association has not recommended any adjunct diagnostic modalities for OED/OLP. Before we can deploy impactful early detection technologies, we must improve our understanding of the biology of high-risk OEDs. We first learn from decades of clinical observations. During the clinical examinations, erythematous color change and induration warrant a biopsy. These features indicate early inflammatory and mechanical changes in the microenvironment of initiating HNCs. Thus, we generated high-fidelity, genetically engineered mouse models to recapitulate these immune and mechanical alterations over the course of HNC initiation. These models are uniquely poised to establish the high-risk markers due to their 100% malignant transformation rate in the oral mucosa. Through robust longitudinal monitoring, we have uncovered an initial set of immunometabolic markers whose signals emerge before the HNC histology appears. This program will discover a comprehensive set of high-risk features and employ advanced machine learning to generate a weighted risk score, which will be validated through our extensive collections of low-risk leukoplakia and transformed OED/OLP human specimens. To support the robust on-slide technology, we also developed an optical biopsy tool, approved by the Food and Drug Administration, to perform non-invasive monitoring of molecular markers at a microscopic resolution below oral mucosal surfaces. This milestone-driven program will leverage the strengths of precision in high-fidelity modeling for transforming OEDs, the extensive translational resources, a cutting-edge optical biopsy platform, and single-cell technologies to extend the human senses in conventional histology and clinical examination of OED to unprecedented molecular levels. This integrated effort will inform transformative on-slide and optical biopsy ancillary tools to capture high-risk OEDs at the earliest phase for HNC prevention.

NIH Research Projects · FY 2026 · 2024-07

Project Summary Mammalian embryos have the potential to develop both male and female reproductive tract organs. However, they typically only differentiate one sex type depending on the presence or absence of fetal gonadal hormones. Two paired epithelial ducts form within the mesonephroi (fetal kidneys) in both genetically male and female embryos. The Wolffian ducts form first and with the adjacent mesenchyme differentiate into male organs, including the vasa deferens, epididymis, and seminal vesicle. The Müllerian ducts then form adjacent to the Wolffian ducts. The Müllerian ducts and their adjacent mesenchyme give rise to female organs, including oviduct, uterus, vagina. In male embryos, the testes secrete testosterone to induce the differentiation of the Wolffian ducts. The testes also secrete anti-Müllerian hormone (AMH) that binds receptors expressed in the mesenchyme adjacent to the Müllerian duct, resulting in elimination of the Müllerian duct epithelium. Alterations in Wolffian and Müllerian duct formation or differentiation to a male or female phenotype lead to Differences of Sex Development (DSD). DSDs are conditions that vary from the typical male and female phenotypes. In female embryos, once the two Müllerian ducts have elongated and connected to the urogenital sinus the posterior regions migrate to the midline and fuse to create the bipartite uterus in the mouse and simplex uterus in human. Alterations in these processes are thought to lead to defects in uterine morphogenesis, associated with infertility, high risk pregnancy, and miscarriage. Much progress has been made in understanding the development of the female reproductive tract organs and the elimination of the Müllerian system in male embryos but there are still many basic questions to be answered. Wnt7a encodes a secreted protein that is expressed in the Müllerian duct epithelium. We have discovered that the Müllerian ducts of Wnt7a-null females do not fuse at the midline, resulting in the formation of two separate uterine horns and two vaginal openings, i. e. a duplex uterus. This suggests that WNT7A-induced genes are required for Müllerian fusion. In males, the Müllerian ducts must be eliminated by AMH. No discrete cis-regulatory elements have been identified for Müllerian duct epithelium- or mesenchyme-specific transcription. Previously, we identified Osterix (Osx) as an AMH-induced gene expressed in the male Müllerian duct mesenchyme. Our preliminary studies demonstrate that a 39 kb region surrounding the Osx locus contains Müllerian duct mesenchyme transcriptional regulatory sequences. Little is known about the factors that establish the competence of the Müllerian duct mesenchyme to AMH for the elimination of the epithelial ducts during male differentiation. We have preliminary results that indicate that the tumor suppressor gene Wt1 may be one of these factors. This proposal focuses on Müllerian biology to determine the molecular, cellular, and developmental mechanisms that regulate female reproductive tract morphogenesis, Müllerian duct transcription, and the elimination of the Müllerian system during male differentiation.

NIH Research Projects · FY 2025 · 2024-07

Cancer in adolescents and young adults (AYA) is defined as a diagnosis in individuals aged 15-39 years and is diagnosed in approximately 90,000 AYAs each year. Current treatment regimens for AYAs diagnosed with cancer often include cardiotoxic treatment regimens, which have been associated with increased risk of cardiovascular dysfunction in survivors. Our preliminary data indicates that hearts in AYA cancer patients exposed to cardiotoxic treatment modalities have a functional phenotype that appears to mirror heart function in non-treatment exposed patients who are decades older. The incidence of this possible accelerated cardiac aging phenotype in the survivor AYA cancer population is unknown, potential differences by patient subgroups are unclear, and how known biological markers of aging correlate with heart function and potentially further influence the development of cardiotoxicity has not been investigated. To address this gap in our understanding of cardiac aging in survivors of AYA cancer, we will test the overall hypothesis that an accelerated cardiac aging phenotype contributes to the development of poor cardiovascular health in survivors of AYA cancer, and that other markers of aging can further identify those at greatest risk for cardiovascular disfunction. Towards this, the proposal has two objectives: 1) to elucidate and define accelerated cardiac aging in survivors of AYA cancer (N=1,200) and 2) to identify predictors of an accelerated cardiac aging phenotype and the impact of patient characteristics and established aging biomarkers on this phenotype. To achieve these objectives, this proposal will leverage a survivors of AYA cancer cohort anchored to echocardiographic assessments of cardiac function: Project DANCES. Data and biospecimens will enable investigation into accelerated cardiac aging and multi-level assessment (individual, neighborhood, and population) of drivers of this phenotype. Established aging biomarkers will also be analyzed to identify relationships with accelerated cardiac aging. With the overall 5-year survival rate for AYA cancer patients remaining over 84%, there is a growing segment of survivors of AYA cancer who have the potential to enjoy several more decades of life, cancer-free, highlighting the importance of prioritizing their long-term health post-cancer treatment. Cardiac dysfunction and morbidity is a major late effect in this population and the proposed research efforts are designed to identify risk factors and drivers of these events among survivors of AYA cancer.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Glioblastoma (GBM) is the most common and lethal primary central nervous system cancer in adults, where treatments are ineffective and often debilitating. Unlike many other peripheral solid tumors, GBM is highly resistant to cancer immunotherapy. The absence of immune infiltrates in GBM results in an immunologically “cold” tumor and intrinsic immune resistance mediated by GBM cells is an additional factor contributing to the failure of immunotherapy in GBM thus far. Chimeric antigen receptor (CAR) T-cell therapy has recently shown remarkable success in the treatment of hematologic cancers, and there have been attempts made to explore CAR T cell strategies against GBM. However, CAR T cells for GBM face challenges including intratumor heterogeneity, dynamic expression of target receptors, and often the inability of T cells to traffic to tumors to mediate the desired antitumor effect. In contrast to the lack of T cell infiltrates, many solid tumors, especially GBM, are abundant in immune suppressive myeloid cells including macrophages. Therefore, converting these immune suppressive cells into tumoricidal phenotype represents a promising strategy for cell-based therapy. There is now strong interest in generating CAR macrophages in which autologous macrophages are transduced with CARs delivered by viral vectors ex vivo to enhance their effector functions. However, ex vivo preparation of CAR macrophages is complex, time consuming, and due to the non-dividing nature of macrophages, is often inefficient. We propose an innovative strategy that represents a revolutionary way to produce CAR macrophages in vivo and offers a promising new approach for cell therapy against GBM. This will be the first study to evaluate the feasibility of producing CAR macrophages in vivo using an mRNA delivery platform and to assess the antitumor efficacy of CAR macrophages for GBM immunotherapy. We hypothesize that generating CAR macrophages targeting the Epidermal Growth Factor Receptor Variant III (EGFRvIII) in vivo using mRNA-loaded exosomes will have significant activity against GBM. EGFRvIII is a mutant protein that is expressed on the surface of 50% of GBM cells. Our previous study showed that we can efficiently produce mRNA-loaded exosomes to restore protein expression in solid tumors in vivo. Furthermore, our preliminary experiments showed that the exosomes loaded with EGFRvIII CAR mRNA can produce CAR macrophages in vivo with enhanced effector functions. Our current study will test the overall hypothesis through the following specific aims. In Aim 1, we will evaluate the dynamics and toxicity of CAR macrophage production in vivo using CAR mRNA exosomes. In Aim 2, we will evaluate transcriptomic and functional profiles of in vivo generated CAR macrophages. Finally, in Aim 3, we will assess the antitumor effect of in vivo generated CAR macrophages as a monotherapy or in combination with other myeloid-directed therapy against both murine and human EGFRvIII expressing GBMs. If successful, our proposed research can overcome a major technical hurdle that has limited cell therapy in GBM and greatly expand its utility for cancer treatment.

NIH Research Projects · FY 2024 · 2024-07

ABSTRACT A drawback of genome-wide association studies (GWAS) for breast cancer risk and related phenotypes is their limited insights into genotype-to-phenotype mechanisms for identified genomic regions. Although integrating GWAS with functional genomics datasets, like the Genotype-Tissue Expression Project (GTEx) and The Cancer Genomic Atlas (TCGA), has yielded promising results in identifying candidate target genes for many traits1–3, these approaches overlook the complexity of alternative splicing and isoform diversity within the transcriptome. Indeed, recent studies of long read RNA-sequencing (RNA-seq) data across tissues reveal that as much as 40- 60% of the human transcriptome is unannotated6–8 due to overlooked isoforms. We propose to re-align existing breast-specific short-read RNA-seq datasets using novel isoform annotations developed from long-read RNA- seq data. We will then integrate these with existing breast cancer and mammographic density GWAS data to identify isoform- and splice-site-specific mechanisms underlying genetic associations for breast cancer and mammographic density phenotypes. We will build on our recent work where we developed and showcased the promise of isoform-level transcriptome-wide association studies (isoTWAS), an innovative machine learning framework that integrates genetics, all expressed isoforms of a gene, and phenotypic associations. Specifically, we will first quantify isoform expression and alternative splicing events in GTEx and TCGA using novel transcript assemblies from long-read RNA-seq datasets (Aim 1). We will benchmark multiple statistical approaches for alignment of isoforms by conducting extensive evaluation studies. We will then leverage these newly aligned isoforms and alternative splicing events in breast tissue to pinpoint isoforms and alternative splicing events likely to mediate germline genetic associations with breast cancer risk and mammographic density phenotypes (Aim 2). This innovative proposal aligns with the NCI strategic objective of Understanding the Mechanisms of Cancer and Detecting and Diagnosing Cancer and addresses a critical challenge in studying the genetic etiology of breast cancer: prioritizing potential causal biological mechanisms for further follow-up. Our proposal is unique in that it will re-quantify and integrate multi-tissue, multi-level transcriptomic reference panels (both short- and long-read RNA-seq) with robust GWAS summary statistics using cutting-edge computational tools for transcriptomics and a novel integrative framework. By combining publicly available multi- level `omic datasets in a systemic genomic epidemiology framework, our work will provide both molecular data resources and reproducible computational frameworks that can be easily expanded to other tissues and traits. Specifically, we will develop open-source computational pipelines for developing tissue-specific, novel isoform annotations for short-read RNA-seq alignment and expression quantification and create and maintain a publicly available portal to host iso- and splice-QTL summary statistics and predictive models allowing for the broader research community to explore similar investigations across traits and tissues.

NIH Research Projects · FY 2025 · 2024-07

Summary Cancer cells harbor signs of genomic instability, including genome rearrangements, which can contribute to tumor development. Faithful DNA repair combats this instability and is a powerful tumor suppressor mechanism whereas erroneous repair leads to pathological outcomes. The inherent genomic instability of cancer cells makes them more vulnerable to a high damage burden and DNA damaging therapies are amongst the most widely used and most successful cancer therapies. DNA repair is enabled within nuclear compartments or repair domains. Spatial organization of repair facilitates biochemical reactions, restricts reactions to specialized compartments while bringing DNA lesions in close proximity, thus favoring rare aberrant genome rearrangements. We have made initial progress in revealing the mechanisms underlying the 3D re-organization of the genome following DNA damage. Based on our seminal discovery of mechanical forces driving the 3D arrangement of the genome following damage, we propose to better understand the mechanisms responsible for the assembly of DNA repair domains and to evaluate the physio-pathological consequences of assembling these domains following treatment with chemotherapeutic drugs. We will also explore the connection between DNA replication and DNA repair in 3D. Our studies should reveal common rules governing the organization of DNA transactions in 3D, which in turn have the potential to identify novel vulnerabilities for the most common modalities of cancer treatment.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT Chronic intestinal inflammation is a major risk factor for the development of colorectal cancer (CRC). It is well- acknowledged that pro-inflammatory cytokines derived from immune cells play critical roles in almost every developmental stage of inflammation-induced cancers including CRC; however, molecular and cellular mechanisms underlying chronic intestinal inflammation leading to tumorigenesis are still being uncovered. Thus, understanding the mechanisms that control inflammatory responses of mucosal immune cells can be beneficial for the treatment of inflammation-associated CRC. Our preliminary results have revealed the potential role of E3 ligase, gene related to anergy in lymphocyte (Grail), in maintaining intestinal homeostasis and controlling carcinogenesis. Studies in both CRC murine models and patients show a correlation between Grail expression in colon and reduction of intestinal inflammation and tumor growth, suggesting the role of Grail in controlling intestinal inflammation and importantly projecting Grail as a potential marker for diagnosis of CRC. We detected more severe CRC growth in Grail knockout (KO) mice compared to wild-type (WT) mice, which was associated with increased expression of immune cell derived pro-inflammatory cytokines in the colon. Moreover, Grail expression in hematopoietic cells, particular in T cells, is important to control CRC. Remarkably, we detected a higher number of interleukin (IL)-17 producing T follicular helper (Tfh)17 cells in colons of tumor-bearing Grail KO mice and their percentage positively correlated with cancer severity. Similarly, we detected a higher number of Grail-insufficient Tfh17 cells in patients with advanced CRC compared to patients with early stage of disease, suggesting a new role for Tfh17 cells in promoting intestinal chronic inflammation and neoplastic diseases. Interestingly, elevated numbers of Tfh17 cells in Grail KO mice were associated with the accumulation of Th17 and B cells, indicating that Tfh cells could engage in crosstalk with other pro-carcinogenic immune cells. We propose a central hypothesis that Grail maintains intestinal homeostasis by controlling the activity of pro-inflammatory mucosal Tfh17 cells. In Aim 1, we will determine the link between Grail expression in Tfh cells and intestinal tumorigenesis. In Aim 2, we will examine the mechanism(s) whereby Grail deficient Tfh17 cells contribute to colon cancer development. In both aims, we will utilize gene KO approaches and Bcl6/IL17/IFN-reporter mice. The implication from this work is significant since it will provide novel insights into the genetic link between inflammation and cancer, define a new marker for early detection of inflammation-associated CRC, and guide the development of effective colon cancer immunotherapies.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Immune checkpoint receptor blockade (ICB) has been approved recently for the treatment of metastatic, unresectable, or recurrent head and neck squamous cell carcinoma (HNC) in the first-line setting. Multiple priming strategies have entered into trials, aiming to turn cold HNCs, which account for about 85% of the cases, into T-cell inflamed hot tumors and expand the pool of patients who can benefit from immunotherapy. Mechanistically, many priming strategies activate innate immune sensors to launch the production of type-I interferons (IFN-I) by cancer cells and myeloid cells. The activation of IFN-I and its target genes promotes antigen-presenting cell (APC) and effector cell trafficking to the tumor bed and enhances APC cross-priming efficiency. A central converging point of the current priming approaches is an adaptor molecule located at the endoplasmic reticulum and its associated membranes, stimulator of interferon genes (STING). Lead cold tumor sensitization treatments, such as irradiation, inhibition of DNA damage repair, induction of DNA replication stress, and STING agonists all engage the STING pathway, further validating the promise of STING-priming in restoring the immunogenicity of cancers. However, recent trials of STING agonists showed a high resistance rate in patients with solid tumors, even in combination with ICB. The mechanism of HNC resistance to STING priming is poorly understood, and few strategies are available to overcome cancer resistance to innate immune sensing. The long-term goal of this program is to establish the biochemical and metabolic regulatory network of HNC immunogenicity and improve HNC prevention and immunotherapy by releasing the checkpoints on innate immune sensors. During the initial funding period, we have uncovered driver oncogenes that disable the STING pathway and promote immune tolerance, we have engineered the first-generation nanoparticles to improve the intracellular delivery of STING agonists, we have streamlined our single-cell immune analysis pipelines to render intra-lesional immune landscape as a function of time. Recently, we discovered a new pathway that suppresses HNC initiation through IFN-I activation. This renewal project will parlay our previous accomplishments and our recent discovery into a cohesive program that addresses the mechanisms of HNC resistance to STING stimulation and optimizes the engineering of a new generation of nanoparticles for innate immune priming in hosts insensitive to free STING agonists. To support this goal, we have established comprehensive modeling for HNC initiation and response plasticity to STING stimulation, including carcinogen-induced models, implantable models, and genetically engineered models. Resistance to STING stimulation disqualifies a spectrum of cold cancer priming strategies. This renewal project will uncover a pivotal molecular mechanism underpinning the fitness of innate immune sensors and optimize robust nanotechnology overcoming cancer resistance in individuals insensitive to STING stimulation.

NIH Research Projects · FY 2025 · 2024-07

Cervical cancer remains the second most common cancer killer of women worldwide, with an annual incidence of more than 600,000 and an annual death rate of 300,000. Further, cervical cancer disproportionately affects communities of medically underserved and minority women within the US and abroad, and improved therapies are urgently needed. Primary and secondary prevention approaches are also variably effective – even prior to the COVID-19 pandemic, just 1 in 8 girls was vaccinated against human papillomavirus (HPV), the cause of 90% of cervical cancers. Vaccination rates also dropped sharply during the pandemic, even in the United States. Current World Health Organization (WHO) estimates of HPV vaccine uptake rates is 21%. The vast majority of cervical cancers that are diagnosed after primary and secondary prevention fail are locally advanced cervical cancer (LACC). Approximately 40% of women diagnosed with LACC will relapse and die of disease even with standard-of-care curative treatment, cisplatin-based chemoradiotherapy (CRT). CRT has remained the standard-of-care for more than two decades, and novel approaches have failed to improve outcomes. We have identified a critical prognostic factor, a bacteria called Lactobacillus iners (L. iners), in the cervical tumor microbiome, which rewires tumor metabolism to utilize lactate and is associated with treatment resistance and poor survival. Further, commensal Lactobacilli (lactic acid bacteria) in other tumor sites often driven by lactate, such as head and neck and lung cancers, also appear to lead to treatment resistance and poor survival. Our objective is to understand specifically how L. iners and other lactic acid bacteria influence cancer cell and immune cell metabolism using state-of-the-art proximity proteomics and mass cytometry (Aim 1). We will also test novel therapeutic approaches to target either tumor resident bacteria by eliminating or replacing specific bacterial species (Aim 2), or metabolic effects of tumor resident bacteria via local bacterial engineering or systemic metabolism targeting anti-cancer therapies (Aim 3). Targeting cervical tumor bacteria as a therapeutic (“Bugs as Drugs”) is a paradigm-shifting idea, capitalizing on the relative simplicity of the cervicovaginal microbiome and its tendency to be dominated by Lactobacillus species, and not only will this study lead to improved microbiome- based therapeutics to improve outcomes in cervical cancer, but this proof-of-concept model could be used to inform tumor microbiome-based therapeutics across cancer types.

NIH Research Projects · FY 2023 · 2024-06

PROJECT SUMMARY/ABSTRACT Fusobacterium nucleatum (Fn) is a key member of the human oral microbiota, where it serves a fundamental role as a bridge between early dental surface colonizers and microbes associated with mature plaque. However, Fn has also been implicated as a causative agent in several oral diseases (periodontitis and oral squamous cell carcinoma; OSCC) and extra-oral pathologies including colorectal cancer (CRC), appendicitis, osteomyelitis, and adverse pregnancy outcomes such as chorioamnionitis, placental infections, and pre-term birth. Yet, despite decades of clinical relevance across disease states and human niches, Fn remains almost entirely beyond the power of modern genetics for fundamental interrogation of its physiology, metabolism, and pathogenesis. Recently, our team demonstrated that the intratumoral microbiota, dominated by Fn, colonizes specific microniches of human oral and colorectal tumors and contributes to tumor spatial and cellular heterogeneity. However, a mechanistic understanding of how Fn has gained such a fitness advantage that it can predominate in CRC to reach >80% relative abundance in tumors, but is absent within the healthy colon, is severely lacking and represents a critical barrier to progress for therapies. This proposal objective, leveraging pan-epigenome data from 158 distinct Fn strains including clinical CRC isolates, is to make Fn strains systematically tractable and to use advanced genetic systems to interrogate its pathogenesis in the context of CRC. In Aim 1 we will apply two state-of-the-art approaches (SyngenicDNA and Plasmid Artificial Modification) to tackle the inherent issue of RM heterogeneity across Fn strains of diverse origin. We will design, construct, and validate Fn- optimized genetic tools for transposon-based random mutagenesis (pFnTn), CRISPR-Cas9 targeted mutagenesis (pFnCas9), and complementation studies (pHS30MCSyn). In Aim 2 we will construct a genome- scale library of 100,000s of barcoded transposon mutants of Fna SB010, a clinical CRC isolate. Using this library, we will define the essential genes of Fna, both those that are absolutely required for survival, and those that are required in specific environments, including during colonization of GI tumors within the ApcMin/+ murine model of CRC. We will also create an ordered library of individual mutants representing the non-essential genome for validation of pooled results and further screening. Our multidisciplinary team brings together expertise in genetic engineering, pangenomics, (PI Johnston), and the intratumoral microbiota, preclinical cancer models, host- microbial in situ imaging (Co-I Bullman), in addition to extensive knowledge and experience in barcoded transposon library construction, screening, and analysis as well as generation of arrayed libraries (Sub-PI Huang). Completion of these aims will provide critical tools for interrogating Fn physiology, metabolism, and pathogenesis. Our long-term goal is to discover new strategies to prevent or treat bacterial-associated cancers. Moreover, these tools are broadly applicable and will be made openly available to advance research across human niches, including oral, gastrointestinal, and urogenital disease states.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY/ABSTRACT Myeloproliferative Neoplasms (MPNs) are chronic, progressive hematopoietic disorders characterized by aberrant proliferation of myeloid lineage constituents, pro-inflammatory sequelae, progressive bone marrow fibrosis, and increased risk of leukemic transformation. Gain-of-function mutations of the JAK/STAT pathway, including JAK2VF, are present in the majority of MPN patients and are amenable to targeted inhibition; however, clinically-improved JAK2 inhibitors fail to reduce mutant allele burden, and response wanes over time. Somatic alterations in high-risk chromatin modifiers ASXL1 and EZH2 co-occur frequently with JAK2VF in MPN and confer adverse prognosis, myelofibrotic progression, and reduced response to JAK2 therapy. The mechanisms by which chromatin dysregulation secondary to ASXL1/EZH2 alterations enhance clonal evolution and pro-fibrotic inflammatory pathways to promote fibrosis progression and JAK2 inhibitor resistance have not been elucidated. Recently, we demonstrate an absolute requirement for mutant Jak2VF in MPN maintenance using a dual- recombinase knock-in/knock-out mouse model of Jak2VF. I hypothesize that Jak2VF cooperates with Asxl1 or Ezh2 loss to alter dependency on JAK/STAT signaling for disease maintenance and promote pro-inflammatory signaling pathways favoring fibrotic progression. In this proposal, I will investigate the requirement for oncogenic Jak2VF signaling and reversibility of epigenetic dysregulation and pro-inflammatory mediated fibrosis/niche remodeling in high-risk dual-mutant MPN using my dual-recombinase Jak2VF allele. I will complement this with single-cell DNA sequencing + immunophenotyping/cytokine analysis of clinical MPN specimens evaluating cytokine production, clonal evolution, and order of mutation acquisition in MF progression. Further understanding of the cooperative effects of chromatin dysregulation in fibrotic progression and therapeutic resistance in MPNs might lead to the identification of therapeutically tractable dependencies for this high-risk MPN patient subset. Andrew Dunbar, MD, an Assistant Attending at MSKCC, will conduct this project as part of a 5-year career development plan, dedicating >75% of his time to research with remainder on clinical work. Dr. Dunbar is mentored by Dr. Ross Levine, a world expert in the study of hematologic malignancies. Dr. Dunbar is advised by Drs. Raajit Rampal, Omar Abdel-Wahab, Richard Koche and Andriy Derkach at MSKCC, Dr. P. Brent Ferrell at Vanderbilt University Medical Center, and Dr. Rebekka Schneider at University Hospital RWTH Aachen, Germany. Andrew’s training will include gaining technical skills in performing and analyzing single-cell genotypic assays with formal courses in bioinformatics, modeling the bone marrow microenvironment, and methods to investigate the epigenetic regulation of hematopoietic stem cells. In the short term, the project goal is to publish two papers on the findings from this research. In the long term, the goal is for developing a research program and obtaining R01 funding to become an independent laboratory investigator in hematologic malignancies.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY: B cells require somatic hypermutation (SHM) and class switch recombination (CSR) to develop high affinity, isotype switched immunoglobulins (Igs). Human patients deficient in these processes suffer from hyper IgM syndrome, a primary immunodeficiency characterized by recurrent and severe infections. They have low IgG levels and are often unable to develop high-affinity antibodies. Both CSR and SHM are initiated by activation- induced cytidine deaminase (AID) which deaminates cytidines, creating uracils in Ig genes. Uracil is removed by Uracil DNA glycosylase (UNG) and repaired in an error-prone fashion to complete SHM and CSR. UNG is normally associated with high-fidelity base excision repair. The mechanism that directs uracil removal to error- prone repair during SHM is not understood. However, mis regulation could threaten genome integrity through inappropriate mutation or suppress SHM and the generation of high-affinity antibodies. To determine if UNG directly influences repair outcome, we analyzed various mutants of UNG in cell-line, AID induced mutation reporter assay. We found that UNG with point mutations in the Replication Protein (RPA) binding domain efficiently supported high-fidelity repair, while suppressing the frequency of mutation. Furthermore ,RPA mutants unable to interact with UNG also suppressed mutation frequency. Our hypothesis is that UNG-RPA interaction governs error-prone repair during SHM in B cells. The cell line models available do not completely recapitulate B cell SHM in terms of frequency and mutational spectrum. Therefore, our objective is to create a mouse model with a UNG mutant deficient in RPA binding to investigate error-prone repair. We will accomplish this in Aim 1 by using CRISPR gene editing technology to create a mouse with mutant UNG. In Aim2 this mouse will be used to define how UNG-RPA interaction in B cells results in SHM. We expect with the completion of these goals to have a mouse model deficient in normal SHM and a system to mechanistically investigate how UNG and RPA regulate error-free or mutation repair outcome during base excision repair of uracils.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT For cancer immunotherapies such as immune checkpoint therapy (ICT), success depends on sustained activation of intratumoral effector T cells recognizing tumor antigens. While many of the molecular changes required for effector T cells to control cancer are known, many key epigenetic and transcriptional features required for ICT-induced anti-tumor immunity are unknown. We recently found in preclinical sarcoma and melanoma models that anti-PD-1 or anti-CTLA-4 ICT induces strong upregulation of the transcription factor, Bhlhe40, in tumor antigen-specific CD8 and CD4 T cells, which are crucial for ICT-induced tumor rejection and elimination. We further discovered conditional deletion of Bhlhe40 in CD4+ T regulatory cells (Tregs), CD4 T cells, and CD8 T cells rendered mice treated with ICT incapable of rejecting ICT-sensitive tumors. Bhlhe40- deficient CD4 and CD8 T cells exhibited notable reductions in ICT-driven interferon gamma (IFN-γ) production associated with defects in ICT-induced remodeling of intratumoral macrophages. Gene set enrichment analysis indicated dysregulated metabolism within certain subpopulations of CD4+ and CD8+ T cells in the absence of Bhlhe40. However, these analyses were done in mice lacking Bhlhe40 in Tregs and conventional CD4 and CD8 T cells. Therefore, it is important to delineate how Bhlhe40 contributes to not only CD8 but also CD4 T cell (which are now acknowledged to be critical for effective anti-tumor immunity) effector function. We will test the central hypothesis that Bhlhe40 is a pivotal transcriptional regulator of both CD4 and CD8 T cell anti-tumor effector function during anti-PD-1/anti-CTLA-4 ICT and tumor-specific cancer vaccine therapies. In Aim 1, we will use conventional CD4 or CD8 T cell-specific Bhlhe40 knockout (KO) mice, newly generated mouse melanoma models that express defined neoantigens, and mouse sarcoma lines to determine how cell- specific deletion of Bhlhe40 impacts response to anti-PD-1 and/or anti-CTLA-4 ICT or tumor-specific neoantigen cancer vaccine treatment. We will then analyze tumors from CD4 or CD8 T cell specific Bhlhe40 KO mice and Bhlhe40 reporter mice by single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF), and CODEX multiplex imaging, to assess how loss of Bhlhe40 in CD4 or CD8 T cells alters the immune tumor microenvironment. In Aim 2, we will dissect the mechanism by which Bhlhe40 regulates CD4 and CD8 T cell function in the context of different immunotherapies by several approaches such as functional assays, single- cell sequencing assay for transposase-accessible chromatin (scATACseq), chromatin immunoprecipitation sequencing (ChIPseq), and luciferase reporter assays. In Aim 3, we will relate our findings in mice to humans by first manipulating the expression of Bhlhe40 in human CD4 and CD8 T cells to determine how Bhlhe40 regulates their effector function and then analyzing pre- and post-ICT treatment patient samples. These studies will contribute to our understanding of how T cells recognizing tumor antigens maintain effector functions during ICT and therapeutic neoantigen cancer vaccines and could identify Bhlhe40 as a novel therapeutic target.

NIH Research Projects · FY 2026 · 2024-05

Project Summary/Abstract Neuropathy associated with type 2 diabetes (T2D) is frequently associated with pain and sensory loss. Our incomplete mechanistic understanding of the pathophysiology of nerve injury in T2D hinders our ability to develop more effective drugs and therapeutic strategies. Given the emerging role of myeloid cells, particularly macrophages, across various types of neuropathy, their apparent involvement in diverse forms of nerve injury pain and the known increase in production of the peptide hormone angiotensin II (Ang II) in diabetes, we posit that elevated Ang II in T2D precipitates neuropathy, pain and sensory loss by disrupting myeloid cell development and phenotype. However, many knowledge gaps persist. Our preliminary studies indicate that circulating immature myeloid cell numbers are increased in diet-induced and genetic models of T2D, a change also associated with elevated levels of Ang II in lymphoid tissues. Furthermore, the sensory gain observed in diet-induced T2D is reversible by inhibitors of Ang II production. The overall goal of this project is to identify how Ang II disrupts myeloid cells in T2D, culminating in neuropathy, sensory loss and pain. The first specific aim will assess the extent of alterations to myelopoiesis across the progression from metabolic syndrome to early stage T2D to late-stage T2D, and whether increases in Ang II are causally related. The second specific aim will determine the degree to which T2D modifies tissue-resident myeloid cell function in ways that promote sensory dysfunction. Finally, the third specific aim will determine if opposing T2D-related changes in angiotensin signaling can normalize pathological changes to myeloid cells, thereby reducing sensory dysfunction. Our multidisciplinary approach involves preclinical T2D models, reflexive and voluntary pain-behavioral studies, immunohistochemistry, in vitro live cell functional imaging, single cell RNA and ATAC-sequencing and transcriptomic analysis of human skin biopsies. Our project will unambiguously dissect the nature of Ang II-based myeloid cell disruption to determine the role of the renin-angiotensin system and macrophage-driven inflammation in the development of peripheral neuropathy and chronic pain in T2D. Elucidating the mechanistic underpinnings of sensory loss and pain in T2D neuropathy is critical to advancing our understanding of the underlying mechanism(s), as well as rapid translation into development of new- generation, efficacious, non-opioid analgesics that target such neuro-immune crosstalk.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Dyspnea is one of the most common and distressing symptoms associated with cancer, occurring in nearly 70% of patients with advanced cancer. Dyspnea is associated with impaired function, decreased quality of life, and shortened survival. Previous trials suggest that existing palliative interventions, such as low flow supplemental oxygen and opioids, have limited efficacy. Due to the paucity of high-quality evidence, there are currently no U.S. Food and Drug Administration (FDA)-approved treatment options. Multiple organizations, including the Institute of Medicine and National Hospice and Palliative Nurses Association, identified dyspnea as a priority for research. The long-term goal of our research is to develop evidence-based therapies for the palliation of dyspnea in patients with cancer. The overall objective of our proposed high risk, high impact, two-arm, parallel-group, wait- list control, randomized clinical trial is to compare the efficacy of a respiratory therapist (RT)-led Structured Personalized Oxygen and support Therapies for dyspnea in ONcology patients (SPOT-ON) intervention with enhanced usual care on dyspnea. Based on our preliminary data, we hypothesize that a personalized RT-led intervention incorporating time-limited trials of oxygen and support therapies would be effective in reducing dyspnea in acutely ill hospitalized patients with cancer. The two primary specific aims of this study are to compare the effect of SPOT-ON and enhanced usual care on the change in intensity of dyspnea at 24 h in hospitalized cancer patients with hypoxemia (Primary Aim 1) and without hypoxemia (Primary Aim 2). The third aim is to compare the effect of SPOT-ON and enhanced usual care on patient outcomes over 72 h, including dyspnea intensity, dyspnea unpleasantness, dyspnea response, vital signs, symptom burden, health-related quality of life, adverse events, pattern of device use, and hospital outcomes. The fourth aim is to assess factors associated with efficacy of the SPOT-ON intervention, including patient demographics, preferences, and level of usage of oxygen delivery modalities. After obtaining informed consent, a RT will administer the structured study interventions and monitor the participants closely for 72 hours. This study is highly innovative because (1) it will be the first clinical trial to specifically examine a RT-led intervention for palliation of dyspnea; (2) it will examine how time-limited trials can be used to personalize dyspnea management; (3) it will provide treatment based on patient preference instead of physician prescription; (4) it will test the effect of various oxygen modalities in patients without hypoxemia; (5) it adopts personalized dyspnea goal as a novel response criteria; and (6) it employs an innovative personalized trial design. Successful completion of this definitive study is expected to identify the optimal strategy to reduce dyspnea with oxygen- based and support therapies. Results of this study are expected to enhance quality of life for patients with advanced cancer, transform the management of dyspnea, and shift the paradigm of symptom research. .

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Melanoma is a devastating skin cancer that kills nearly 8,000 patients in the US annually. Patients with thicker melanomas, invading deeper and closer to lymphatics and blood vessels, are more likely to have metastasis to regional lymph nodes (LNs). For patients without clinically evident LN metastases, the presence of occult metastases in the sentinel LN (SLN) or first tumor-draining LN (TDLN) is the most important prognostic factor. However, why melanoma metastasizes to LNs in some patients and not in others remain poorly understood; therefore, it is critical to determine the factors regulating metastasis to inform the development of novel therapeutic approaches to prevent and target metastasis. We demonstrated that not only is the gut microbiome a critical modulator of response to immune checkpoint blockade (ICB) but also that a favorable gut microbiome can augment anti-tumor immunity at distant sites, including the skin. We have also identified differences in the gut microbiome of melanoma patients with early-stage (0-II) compared to late-stage (III-IV) disease. The gap in knowledge is the effect of the gut microbiome on the natural evolution of melanoma at the primary tumor and on metastasis to regional LNs. Our hypothesis is that microbes in the gut of melanoma patients influence primary tumor development and risk for LN metastasis, such that targeted modulation of the gut microbiome can prevent melanoma LN metastases. To test this hypothesis, we will: 1) Identify gut microbial and immunologic features associated with tumor progression and LN metastasis in melanoma patients. We will compare gut microbiome profiles and paired tissue immune profiles using GeoMx digital spatial profiling to characterize the immune microenvironment (IME) of the primary melanoma and SLN, and 16S gene amplicon sequencing and/or whole metagenomic sequencing to analyze fecal samples of melanoma patients with: 1.1) Low-risk vs High-risk primary tumors; 1.2) Negative vs Positive SLNs; and 1.3) Clinical Stage III melanoma receiving neoadjuvant ICB. Statistical correlation analyses will be applied to identify gut microbial features associated with immune modulation, melanoma progression and metastasis. 2) Delineate strategies to modulate gut microbes to prevent melanoma progression and LN metastasis. We will investigate the causal relationship between the gut microbiome, tumor progression, and LN metastasis using an inducible model of murine melanoma (cKit:ERT2;BrafV600E;Ptenfl/fl) by assessing the: 2.1) Impact of FMT from responder and non- responder patients on primary tumor progression and LN metastasis; 2.2) IME in both the primary tumor and SLN to identify the microbiome-dependent immunological mechanisms of tumor progression; 2.3) Effect of perturbation of the gut microbiota on the development of regional LN metastasis. The impact of this work will be recognition of the gut microbiome as a critical determinant of melanoma progression at the primary and metastasis to regional LNs, aiding the development of next generation approaches to prevent LN metastasis in melanoma and other cancers.

NIH Research Projects · FY 2026 · 2024-04

SUMMARY/ ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of only ~12%. Therefore, there is a critical unmet need for new treatment options. Chimeric antigen receptor (CAR)-T cells have led to a paradigm shift in the treatment of some hematologic cancers, but efficacy in solid tumors remains limited, partly due to the lack of highly specific targets and the immunosuppressive tumor microenvironment (TME). Moreover, the time required and the high cost of manufacturing an autologous cell product, and the toxicity of CAR-T cells call for novel products that are universal, safe, and potent. CAR-NK cells have emerged as a promising cell therapy for cancer due to the NK cells’ innate ability to kill tumor cells and their safety in the allogeneic setting. In a first-in-human study, our group showed the safety and efficacy of cord blood (CB)-derived CAR-NK cells targeting CD19 in B-lymphoid malignancies. This proposal aims to build on this platform to develop the next-generation NK cell therapies for PDAC by enhancing NK cell potency and persistence through optimal co-stimulatory signaling, cytokine armoring and checkpoint inhibition. We have identified TROP2 as a promising therapeutic target in PDAC and developed a novel strategy to target TROP2 by genetically modifying CB-NK cells with a retroviral vector encoding: (i) the humanized RS7 scFv targeting TROP2; (ii) DAP10 as an NK- specific co-stimulatory domain; (iii) IL-15 to support their survival and proliferation; and (iv) inducible caspase-9 (iC9) as a safety switch (iC9/TROP2CAR/IL-15). Our data show the efficacy and safety of this approach in vitro and in vivo and support its translation to the clinic. In addition, we have developed a robust strategy for the cryopreservation of CAR-NK cells, enabling the generation of a biobank of off-the-shelf engineered NK cells that could be thawed and infused at bedside, thus reducing cost and increasing accessibility. We have manufactured and cryopreserved 125 patient doses of GMP-grade iC9/TROP2CAR/IL-15 NK cells and a clinical study to test the safety and efficacy of this off-the-shelf product in PDAC was recently approved by the FDA (Protocol 2022- 0687; IND 29348). Finally, we have devised a novel strategy to target the immune metabolic checkpoint CREM to modulate the metabolic fitness and potency of CAR-NK cells in the PDAC TME. We hypothesize that targeting TROP2 with iC9/TROP2CAR/IL-15 NK cells will greatly improve outcomes in PDAC and that by deleting the metabolic immune checkpoint CREM we can further enhance the fitness and potency of NK cells. We will test our hypothesis in three specific aims: In Aim 1 we will conduct a Phase I/II clinical trial to test the safety and efficacy of intraperitoneally delivered iC9/TROP2CAR/IL-15 NK cells in patients with TROP2+ PDAC. In Aim 2 we will apply innovative single-cell transcriptomics and spatial proteomics to comprehensively characterize the fate of the adoptively transferred CAR-NK cells and their interaction with other cells within the TME, to uncover key mechanisms of efficacy and resistance. In Aim 3 we will investigate the mechanism by which targeting CREM enhances the antitumor activity of iC9/TROP2CAR/IL-15 NK cells in an orthotopic PDX mouse model of PDAC.

NIH Research Projects · FY 2024 · 2024-04

SUMMARY The ability to distinguish harmful and beneficial microbes is critical for the survival of an organism. Increasing evidence indicates that gut distension caused by bacterial colonization activates a broad innate immune response. We propose that microbial colonization and bloating of the intestine may be perceived as a danger signal that activates an immune fight-and-flight response. This innate immune activation depends on inputs from the intestine that can aid in the recognition of a broad range of microbes and can modulate host responses using a neural-gut axis that controls immune homeostasis. This proposal describes experiments designed to elucidate the mechanisms by which the nervous system may sense overall changes in host physiology during pathogen infections and coordinate innate immune responses. Using the nematode Caenorhabditis elegans, we have demonstrated that specific genes and neurons in the nervous system of the animal control immune responses, indicating that cell non-autonomous signals from different neurons may act on non-neural tissues to regulate innate immune responses at the organismal level. We propose the use of a variety of molecular and genetic techniques to explore the general hypothesis that alterations in host physiology caused by bacterial infections trigger innate immune responses against bacterial infections that are controlled at that whole animal level by the nervous system.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY/ABSTRACT Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for patients with hematologic malignancies. It is believed that alloreactivity upon HSCT is initiated by lymphoid cells that express rearranging receptors upon recognizing the alloantigens on the graft. Unlike lymphoid cells, cells from the innate immune system do not express rearranging receptors and are nonspecifically induced by “danger” molecules. However, recent studies demonstrated that the innate immune system could specifically distinguish the non-self graft and subsequently enhance the alloimmune response. Signal regulatory protein α (SIRPα) is a polymorphic immunoglobulin receptor that is exclusively expressed on the surface of innate cells. The interaction between SIRPα and its ubiquitously expressed ligand, CD47, suppresses the macrophage's phagocytic function. As shown in the murine transplant model, mismatches of SIRPα between donor and recipients can upregulate the allorecognition response. Our group recently investigated the role of SIRPα variant mismatch in recipients of allo-HSCT from a human leukocyte antigen (HLA)-matched related donors (MRD) for the treatment of different hematological malignancies. We found that for the first time, donor/recipient SIRPα mismatch is commonly detected and associated with a significantly increased risk of chronic graft-versus-host disease (cGVHD) and a lower rate of early relapse (Blood Advances 2021; Frontier in Immunology 2022). We proposed a large registry-based study to validate our results in allo-HSCT recipients with MRD, which is one of the most common donor sources in allo-HSCT. The study is approved by the Center for International Blood and Marrow Transplant Research (CIBMTR) immunobiology working committee. CIBMTR and the National Marrow Donor Program (NMDP) will facilitate the study by providing scientific and statistical expertise, as well as associated clinical outcome information and biospecimens. We hypothesize that the mismatched SIRPα elicits a non-self recognition which will further promote adaptive immunity leading to a higher risk of cGVHD and a lower risk of relapse. In Aim 1, we will perform a retrospective analysis of the SIRPα mismatch in allo-HSCT with MRD using the CIBMTR data and specimens. Aim 2 is to determine the relative clinical significance of SIRPα mismatch in the entire cohort and the subgroups of allo-HSCT. The study will not only define the clinical role of SIRPα variant mismatch in the allo-HSCT setting but also significantly advance our knowledge of GVHD/ Graft versus Leukemia (GVL) and the allorecognition of the innate immune system in the context of stem cell transplant.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY The loss of the tumor suppressor gene SMARCB1, a core member of the SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex, defines a group of malignancies characterized by a particularly aggressive clinical course and prominent metastatic behavior. Renal medullary carcinoma (RMC) is the most commonly diagnosed SMARCB1-deficient renal malignancy and predominantly afflicts young individuals of African descent carrying the sickle cell trait. SMARCB1-deficient renal malignancies are extremely challenging diseases that fail to respond to standard-of-care therapeutic regimens used for other renal tumors. In our recent work, we found that SMARCB1-deficient renal malignancies such as RMC are characterized by a highly inflamed phenotype, yet demonstrate accelerated tumor progression (hyperprogression) in response to standard immune checkpoint inhibition (ICI). Our preliminary data in prospectively collected tissue specimens and immunocompetent genetically engineered mouse models (GEMMs) of RMC suggest that this hyperprogression following ICI is due to the engagement of myeloid-affiliated transcriptional programs in tumor cells. Our overarching hypothesis is that the hijacking of myeloid-affiliated transcriptional circuits by SMARCB1-deficient renal malignancies is the major mechanism driving ICI-induced hyperprogression, which can be prevented by targeting master myeloid regulators such as S100A9 and the CEBPB/p300 complex. In the first aim, we will perform immunogenomic profiling studies at the single-cell level in our clinical trial tissue specimens and immunocompetent GEMMs to determine how ICIs, using anti-PD-1 alone or in combination with either anti- CTLA-4 or anti-LAG-3, modulate the tumor immune microenvironment to drive hyperprogression via the engagement of myeloid-affiliated transcriptional pathways. In the second aim, we will perform translational genomic and pharmacologic experiments in our GEMMs of RMC to determine whether targeting S100A9 or the CEBPB/p300 complex can induce sensitivity to ICIs in SMARCB1-deficient renal malignancies. In the third aim, we will utilize a first-in-class technology that enables the retrieval of ICI therapy-resistant clones to investigate clonal dynamics in our GEMMs and determine the role of SMARCB1 loss in the engagement of myeloid-related transcriptional pathways and subsequent hyperprogression of tumor cells following ICI treatment. This project is realistic and feasible within the proposed timeline and budget because it utilizes technology, tissue samples, and models of SMARCB1-deficient renal malignancies already available to the multidisciplinary research team of experts and a dedicated translational pipeline led by the PI, Dr. Pavlos Msaouel. Our approach can provide fundamental information about the mechanisms of ICI-induced hyperprogression in SMARCB1-deficient renal malignancies, which will substantially improve our understanding of how to optimally elicit antitumor immune responses and thus will open new therapeutic avenues for these and other diseases driven by loss of SMARCB1 or of other subunits of the SWI/SNF chromatin remodeling complex.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Tamoxifen (TMX) is a selective estrogen receptor modulator (SERM) approved by FDA for treatment of estrogen receptor positive (ER+) early stage and advanced breast cancer. Although TMX is the mainstay of breast cancer treatment, 20-30% of of breast cancer patients exhibit an existing or developing resistance to TMX therapy with cancer metastsis, by poorly understood mechanisms. Therefore an understanding of molecular mechanisms and predictive correlates of these pathological effects associated with TMX treatment will help design improved adjuvant therapies to overcome TMX resistance and related pathologies for effective management of breast cancer. In this regard, we recently reported that TMX induces robust formation of neutrophil extracellular traps (NETs), which are DNA fibrils exuded from activated neutrophils that can trap and kill extracellular pathogens to boost antimicrobial host defense in chronic granulomatous disease. However, unconstrained NET release has been linked to several immunopathologies including cancer. Because breast cancer patients are typically prescribed TMX as the first or second line therapy, we hypothesize that long-term TMX treatment induces NET formation in these patients, which correlates with and can account for TMX resistance and cancer metastasis. This hypothesis is supported by our preliminary data showing that the extent of NET formation in breast cancer patients is directly proportional to the duration of TMX treatment and that NETs purified from TMX treated breast cancer patients increase cancer cell survival. Leveraging a characterized large cohort of breast cancer patients treated with TMX at MD Anderson Cancer Center, in this clinical exploratory study we will quantify and characterize NETs in blood samples from pre- and post- menopausal women diagnosed with ERα+ breast cancer receiving TMX therapy for varying durations of time, and correlate it with clinical data on co-morbidities, cancer metastasis and recurrence (Aim 1a). We will examine the phenotype and transcriptional landscape of neutrophils from these patients with a focus on TMX-activated NET pathway dentified by us (Aim 1b). Lastly, we will determine the direct impact of TMX-induced NETs on survival and transcriptional reprogramming of breast cancer cells in response to tamoxifen exposure (Aim 2). Because the NET-inducing action of TMX in breast cancer patients and its impact on breast cancer cells as it relates to tamoxifen resistance is completely unknown, our proposed studies will provide novel insights into the mechanism of tamoxifen resistance and future therapeutic opportunities to prevent cancer metastasis and other adverse effects of TMX.

NIH Research Projects · FY 2026 · 2024-04

Tobacco use is the leading cause of preventable and premature disease, disability, and death in the US. Training a workforce to understand, conduct, and evaluate the rigor of addictions research is critical to expanding innovations in tobacco use prevention and treatment and enabling the rapid translation of evidence-based interventions in routine healthcare delivery, ultimately reducing tobacco-related morbidity and mortality in the US. By leveraging extant NIDA investments and creating new courses and activities, the Supporting Tobacco-Related Ongoing Education and Research (STRONGER) Scholar Program will provide research and education experiences to prepare the next generation of clinician-scientists to address tobacco/nicotine addiction in research and practice. The STRONGER Scholar Program will provide research education in tobacco addiction science to medical students and clinically-focused doctoral trainees, matriculating a total of 51 STRONGER Scholars. The STRONGER Scholar Program will build scholars’ competencies in: 1) tobacco dependence treatment knowledge, 2) tobacco research knowledge and skills; 3) resiliency for a career that incorporates research; and 4) responsible conduct of research through participation in a mentored research project and courses for skills development. The Specific Aims of the STRONGER Scholar Program are to: 1) identify, select, train, and mentor qualified trainees, resulting in an over-time increase in knowledge, skills, and interest in conducting tobacco addiction research; 2) increase scholars’ knowledge of tobacco/nicotine dependence and treatment through a competency-based curriculum, fostering skill development and interprofessional learning experiences; 3) systematically evaluate all aspects of the research education program using a mixed methods approach to enhance and strengthen the program iteratively over time; and 4) achieve >51 professional conference presentations and >51 peer-reviewed publications; disseminating information about the program, its research, and its scholars through newsletters, a website, and health campaign messaging. The program aligns with the University of Texas MD Anderson Cancer Center’s strategic planning goals and its scholars will benefit from the rich resources for scholarly and professional development at the nation’s #1 cancer center. The goal of the STRONGER Scholar program is to enhance clinically-focused trainees’ interest in, ability to be successful within, and – ultimately – pursuit of a career in tobacco addiction science that incorporates research activities.

NIH Research Projects · FY 2026 · 2024-04

Genetic variations in HLA genes in humans are associated with over 200 diseases, and large-scale genomic sequencing projects are now generating data on HLA genes from millions of individuals. Despite their immense clinical relevance, next-generation sequencing based computational inference of short (SNP or insertion/deletion) in HLA genes is difficult because of their highly polymorphic nature, inter-HLA gene similarity, and strong linkage disequilibrium. Existing tools for HLA variant detection are error-prone, not designed for scalability, not interoperable across sequencing formats, and the developers have no formal mechanisms to provide support after publication. The objective of this application is to develop highly accurate, robust, scalable, and deployment-ready pipelines for identifying germline and somatic variants in HLA genes through integration and enhancement of our previously developed tools. To achieve this goal, we aim to (1) Develop tools for detecting short germline and somatic HLA variants by enhancing our Polysolver tool for allele inference across all HLA genes, and further developing the Mutect3 pipeline for mutation detection; (2) Establish a reference dataset for benchmarking performance of HLA variant detection tools; and (3) Use the widely used GATK4 framework and Workflow Definition Language (WDL) to create and disseminate robust, scalable and well-supported HLA variant detection pipelines. This will be the first such comprehensive HLA analysis toolkit, which we expect will be widely used by both individual researchers and sequencing consortia in multiple disease communities. Mutect3, which internally employs a “deep sets” architecture, will be the first mutation detection tool capable of jointly calling germline and somatic short variants and handling multiple references at a genomic locus. If successful, this project will unlock the hitherto untapped potential of rapidly growing sequencing datasets by enabling discovery of new HLA alleles, variations in known HLA alleles, and novel HLA-disease associations which can directly be harnessed for personalized preventive and therapeutic applications.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Approximately 120,000 Americans with neurofibromatosis type I (NF1) suffer a constellation of clinical manifestations, including aberrant proliferation of neural tissues leading to malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are highly aggressive sarcomas with a high risk of recurring and spreading, resulting in a 5-year survival rate of less than 25%. Early diagnosis and intervention offer the best outcomes. However, the lack of convenient and specific diagnostic tools is a major barrier to optimal therapeutic intervention. Conventional imaging modalities such as computed tomography (CT scan) and 18F-fluoro-d-glucose positron emission tomography scans (PET scan) are insufficiently specific. Further, lesion biopsies are extremely painful and have limited value due to the heterogeneity of these tumors. Therefore, accurate biomarkers for diagnosis and post-treatment monitoring of MPNSTs are critically needed. The long-term goal of this proposal is to deploy cfDNA-based biomarkers in patients with NF1 into routine clinical use to improve early detection of malignant transformation and monitor responses to therapy. The objective of this proposal is to establish a diagnostic panel of DNA methylation markers, cfDNA-derived tumor fraction, and aneuploidy profiles that correlate with and complement imaging-based modalities for a clinically effective tool to distinguish the presence of MPNSTs from plexiform neurofibromas. Our central hypothesis is that MPNSTs arising in patients with NF1 have unique DNA methylation and somatic chromosomal copy number profiles (aneuploidy) that can be detected in the plasma and serve as clinical biomarkers for early diagnosis or guide therapeutic strategies. We propose 4 Specific Aims: 1) Demonstrate methylation profiles as biomarkers of MPNST in plasma samples; 2) Establish aneuploidy and tumor fraction as biomarkers of MPNST; 3) Evaluate the role of advanced MRI in the early detection of MPNST; 4) Determine the feasibility of cfDNA biomarkers to predict malignant transformation and recurrent disease. Upon conclusion, we anticipate the development and validation of cfDNA methylation markers, cfDNA-derived tumor fraction, and aneuploidy profiles that can complement imaging-based modalities and can help physicians in the early detection of MPNST. Our studies are significant because they will facilitate early and accurate diagnosis of these aggressive tumors and, consequently, expedite appropriate treatment, thereby improving patient quality of life, including survival.

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Pancreatic cancer is the most common form of pancreatic cancer and is currently incurable. Unraveling essential cellular reprograming required for PDAC development and progression, and further identifying new drug targets to treat the disease is crucial to impact the outcomes for patients with this devastating disease. In this application, we propose to study the function of transmembrane protein in regulating major nutrient supply routes by which cells scavenge “food” to fuel their growth. It stands to reason that targeting such transmembrane protein will block the nutrient supply of pancreatic cancer cells and result in cell death. Building on our strong expertise in study transmembrane proteins in pancreatic cancer cell survival and growth, the overarching objective of this proposed research is to gain a comprehensive understanding of the role of transmembrane protein in mediating the nutrient supply routes and evaluate the potential of such transmembrane protein as a targeting node for pancreatic cancer treatment. The major advantage of studying transmembrane proteins is their accessibility by therapeutic agents, such as antibodies. To achieve the translational potential of our study, we will further develop and characterize novel therapeutic agents direct target such membrane proteins in various new preclinical models of pancreatic cancer. Upon completion of the proposed research, we will have more complete knowledge of the extent to which pancreatic cancer is reliant upon transmembrane protein for nutrient salvage, how the nutrient supply machinery is regulated by transmembrane protein on the surface in pancreatic cancer, and whether targeting such membrane protein-mediated metabolic reprogramming may be potent to eradicate pancreatic cancer cells. Answering these questions will significantly impact our ability to determine whether a rational approach to therapeutically intervene in nutrient supply routes in pancreatic cancer exists. Furthermore, our research is significant because it may have broad implications for other types of human cancers depending similar nutrient supply mechanisms. In the short term, findings from this project will advance our understanding of the nutrient supply routes required to sustain pancreatic cancer cells and provide strong preclinical evidence for targeting the nutrient supply routes as therapeutic strategy in treating pancreatic cancer. In the long term, findings of our research are expected to guide the development of clinical trials for achieving clinical benefits for patients with pancreatic cancer. We believe that the results of our studies will have a positive impact on the general public in the United States, benefiting individuals affected by pancreatic cancer and potentially leading to improved outcomes and enhanced quality of life.