University Of Tx Md Anderson Can Ctr

NIH Research Projects · FY 2025 · 2024-02

Project Summary/Abstract New strategies to prevent breast cancer are urgently needed because: i) breast cancer incidence rates keep rising by 0.5% per year and about 13%, or 1 in 8, of U.S. women are going to develop invasive breast cancer in their life time; ii) the endocrine-targeted agents for ER+ breast cancer prevention do not reduce breast cancer mortality; iii) prevention strategy for ER- breast cancer, which has higher mortality rate, is still not available yet. Our recent study (published on Cancer Cell 2022 Jan 10;40(1):36-52) uncovered that high levels of plasma histamine and histamine receptor H1 (HRH1) expression in tumors (mainly on macrophages) are significantly correlated with reduced cytotoxic immune cell infiltration and devastating T cell function in patients with breast cancer. On the contrary, breast cancer patients who took H1-antihistamines during immunotherapy treatment had evidently improved survival. Importantly, the association between antihistamine uptake and better clinical outcome was only observed in patients treated with immunotherapies but not that with chemotherapies, suggesting that H1-antihistamines may contribute to antitumor immunity. Indeed, we found that H1-antihistamine treatment or HRH1 knockout boosted CD8+ cytotoxic T cell activities and enhanced immunotherapy responses in several mammary tumor models. More interestingly, our latest retrospective analysis of the electronic health record of a MDACC patient cohort with stage 0 or 1 breast cancers revealed that patients who took H1- antihistamines had significantly reduced tumor recurrence compared to patients who did not take them. Patients who received therapies other than surgery were excluded, suggesting that the reduced recurrence is not due to enhanced therapeutic response. These new findings led us to hypothesize that blocking histamine-HRH1 axis by H1-antihstamines enhances immune surveillance and intercepts breast cancer development. We propose two Specific Aims: 1) Test the effect of H1-antihistamines on intercepting mouse mammary tumor development. We will test i) whether H1-antihistmaines can intercept early-stage mammary tumors in mouse models that mimic patients with early stage breast cancers (i.e., the cohort in the analysis); ii) whether H1-antihistamines will prevent/delay basal-like breast cancer initiation/progression in a genetically engineered mouse model which recapitulates the natural development of breast cancer; 2) Determine the impact of blocking histamine-HRH1 axis on systemic immune landscape and tumor immune microenvironment in early stage mammary tumors. We will examine i) broad effects of H1-antihistamine on blood immune cell landscape by CyTOF; ii) impacts of H1- antihistamines on local immunity, e.g., the spatial interactions of mammary epithelial cells and diverse immune cell populations. Integrating above data, we will identify key changes of immune cells that are linked with antihistamine-mediated immunoprevention. The proposed study will explore a new strategy based on our latest findings to address the unmet challenge for breast cancer prevention. If the results are promising, our findings could be translated into clinic trials for immunoprevention of breast cancer, especially, in women at high-risk.

NIH Research Projects · FY 2026 · 2024-02

Project Summary In mammals, the uterus serves many functions including a passage for spermatozoa, embryo implantation, and fetal gestation. Congenital uterine anomalies are present in 25% of women with a history of miscarriage and infertility. It has been increasingly noted that uterine anomalies are simultaneously present with reproductive diseases. For this reason, it is necessary to further understand female reproductive development to better manage and prevent reproductive diseases in women. The female reproductive tract develops from a pair of epithelial tubes called the Müllerian ducts (MD). During embryonic development, the Müllerian ducts and adjacent mesenchyme differentiate into the oviducts, uterus, cervix, and upper part of the vagina. In many mammals, the MD must fuse at the midline for proper uterine morphogenesis. Formation of the uterus occurs with the differentiation, invagination, elongation, and fusion first, of the MD to the urogenital sinus (UGS) and secondly, the two MD fuse to each other at the body midline during uterine morphogenesis. Any divergence in MD fusion during human development can lead to uterine variation that may prevent a healthy pregnancy or delivery of a newborn. While there have been many studies on MD formation, it is currently unknown which genes and molecular mechanisms regulate the fusion of the MD ducts. Preliminary data from our lab shows that Wnt7a knockout mice have uterine abnormalities as a result of unfused MD, suggesting an essential role for Wnt7a in fusion of the two MD. Currently, the cellular behaviors and mechanisms that regulate MD fusion are poorly understood. The crosstalk between MD epithelium with the adjacent mesenchyme has not been studied in the context of MD fusion. In Aim 1, I will use ex vivo organ culture time-lapse imaging and immunofluorescence staining to determine which cellular changes coordinate MD fusion. In Aim 2, I will use spatial transcriptomics to identify differentially expressed genes downstream of Wnt7a, using wild-type and Wnt7a knockout embryos. The objective of this proposal is to determine the precise timing, position, and length of MD fusion, what cellular changes occur during MD fusion, and the downstream genes of Wnt7a that govern MD fusion for uterine morphogenesis. Our primary hypothesis is that Wnt7a directs the crosstalk between MD epithelium with the adjacent mesenchyme to instruct MD fusion to form a portion the uterus. The Behringer laboratory located at MD Anderson in the Texas Medical Center has expertise in reproductive biology, mammalian developmental genetics, and mouse genetic models. During my graduate training, I will meet with my Sponsor biweekly, advisory committee as a group biannually, and with experts on the subject individually as needed. I have written a chapter review and will write two first-author publications summarizing my findings. I will present my work at both my graduate program seminars and at two national conferences annually. Ultimately this work will facilitate my long-term goals of conducting my postdoctoral studies in reproductive sciences and becoming an independent reproductive biology researcher.

NIH Research Projects · FY 2026 · 2024-02

Project Summary / Abstract Research Plan: Targeted protein degradation using molecular glues - small molecules that bring a protein of interest in proximity to an E3 ligase - emerged as a promising therapeutic strategy for cancer, especially for proteins that have been historically challenging to target with conventional drug discovery approaches. However, the development of molecular glue degraders presents a formidable challenge due to the absence of a systematic methodology for identifying these compounds. Currently, most molecular glue degraders employed in the clinic setting have been identified through serendipitous discovery, thereby limiting their potential applications for targeting intractable oncogenic proteins. To address this challenge, I have developed a novel chemoproteomic approach, referred to as "Lysate IP", which enables the identification of novel molecular glue activities in small molecules. In my preliminary investigation using this method, I discovered that lenalidomide, an FDA-approved molecular glue degrader, induces ASS1 recruitment to CRBN, but it does not result in ASS1 degradation. Given the extensive use of lenalidomide in the clinic and in CRBN-based PROTACs, it is crucial to examine all potential activities that lenalidomide elicits. Therefore, during the mentored K99 phase, I propose to investigate the implication of ASS1 recruitment to CRBN by lenalidomide, and its role in lenalidomide resistance in multiple myeloma (Aim 1). In the subsequent independent R00 phase, I intend to utilize the developed Lysate IP assay for the large-scale, multiplexed identification of novel molecular glues against unexplored E3 ubiquitin ligases (Aim 2), as part of the anticancer drug discovery process. The primary objective of this research is to systematically identify and characterize new molecular glues with the ultimate goal of expanding the small molecule toolbox for targeted cancer therapy. Career Development Plan: I have created a 5-year career development plan that enables me to attain my goal of becoming an independent investigator in cancer biology. My extensive background in chemical biology and medicinal chemistry has positioned me in a unique position to accomplish the goals outlined in this proposal. Supported by an interdisciplinary team of advisors and collaborators, including Drs. Benjamin Ebert, Eric Fischer, Scott Armstrong, and Amit Choudhary, I aim to acquire novel proficiencies in a range of areas including large- scale chemical screenings, mass spectrometry, omics-scale data analysis and visualization, and comprehensive cancer biology. Additionally, with institutional support from the Broad Institute, as well as through formal coursework and training, I will establish an independent, cancer-oriented drug discovery program with the goal of expanding the range of druggable proteins using small molecules.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY: Two component gene regulatory systems (TCS), which generally consist of a membrane-embedded sensor kinase which controls the phosphorylation of its cognate response regulator, are a central mechanism by which bacteria modulate gene expression in response to external stimuli. The control of virulence regulator/sensor kinase (CovRS) of the major human pathogen group A Streptococcus (GAS) has long been a model for understanding how TCS influence bacterial infectivity. Phosphorylated CovR (CovR~P) primarily serves to repress virulence factor production, and CovS can either phosphorylate or dephosphorylate CovR at D53 in response to extracellular factors. Recent completion of global CovR binding/transcriptome analyses (ChIP- seq/RNAseq) showed that CovR~P repressed virulence factor encoding genes have the expected increased CovR promoter binding and decreased transcript levels at high CovR~P levels. However, these studies also identified a large number of directly CovR regulated genes with increased transcript quantity at higher CovR~P levels (i.e. CovR~P activated). By ChIP-seq analyses, these CovR~P activated genes had higher CovR promoter occupancy at lower CovR~P levels suggesting that non-phosphorylated CovR (CovR-NP) may be the predominant CovR isoform binding/acting at these promoters. Additionally, we have identified that ska, which encodes the critical GAS virulence factor streptokinase, is CovR~P repressed in emm1 strains but CovR~P activated in acapsular emm28, emm87, and emm89 strains. It is the goal of this proposal to elucidate mechanisms underlying “CovR~P activation”. In Specific Aim 1, we propose to perform comparative ChIP-exo analyses of wild-type (~80% CovR~P) and CovR-D53A (100% CovR-NP) emm1 and emm89 strains to better define the contribution of CovR-NP to global CovR binding. ChIP-exo provides an advance over ChIP-seq because of an exonuclease digestion step which greatly improves identification of the precise DNA binding location of transcriptional regulators. Additionally, we will perform focused in vitro transcription assays and luminescence-based plasmid reporter studies to definitively test the comparative function of CovR-NP and CovR~P at specific CovR~P activated promoters. In specific aim 2, we will seek to determine the mechanism by which ska is CovR~P repressed in emm1 but CovR~P activated in emm89 GAS. Specifically, we will perform in vitro transcription assays using CovR~P and CovR-NP as well as in vivo promoter activity assays using emm1 and emm89 ska promoter DNA in low and high CovR~P emm1 and emm89 strains. Moreover, we will swap the emm1 and emm89 promoters (i.e. place the emm1 promoter into an emm89 strain) to discern the role of strain background on observed transcript level variation as well as assess the role of the non-coding ska regulatory RNA fasX. Completion of the proposed research will markedly improve mechanistic understanding of how a model prokaryotic response regulator activates gene expression.

NIH Research Projects · FY 2026 · 2024-01

Abstract. This proposal is disease agnostic and patient centered, focusing on symptoms associated with side effects of immune checkpoint inhibitors (ICPis). Although ICPis are effective in the treatment of several can- cers, the risk of immune-related adverse events (irAEs) due to unrestrained activation of the immune system presents a significant challenge. Severe irAEs (grade 3 or higher) can be devastating in many ways. The symptoms associated with severe irAEs can be debilitating, resulting in frequent hospitalizations. Their impact on quality of life can be significant enough to mandate discontinuation of an ICPi that is having beneficial anti- tumor activity, resulting in progression of disease and decreased overall survival. In the absence of prompt in- tervention, severe irAEs may cause life-threatening decline in organ function and are potentially fatal. Our insti- tution (MD Anderson Cancer Center) has shown that early detection and treatment of irAEs improves treat- ment outcomes. However, biomarkers to predict a patient’s risk for irAEs that could help in developing surveil- lance strategies and early diagnosis and management of irAEs are lacking, representing a gap in knowledge. The overall objective of this application is to build and validate a risk-prediction model and develop a clinical tool to predict the risk for severe irAEs in patients treated with ICPis. In preliminary studies, we found that poly- morphism in the germline-encoded T-cell receptor beta (TCRB) variable (TRBV) gene serves as a predictive biomarker of severe irAEs. We also observed that changes in symptoms reported using the MD Anderson Symptom Inventory (MDASI)-Immunotherapy measure within the first 3 weeks of immunotherapy initiation are predictive of subsequent development of severe irAEs. Guided by our preliminary findings, we hypothesize that integration of TRBV polymorphism and early changes in MDASI-Immunotherapy symptom items are pre- dictive of severe irAEs. To test our hypothesis, we will evaluate 500 patients on treatment with ICPis at MD Anderson or the National Cancer Institute. In Aim 1, we will perform long-amplicon TCRB repertoire sequenc- ing of RNA extracted from baseline peripheral blood samples to predict risk of severe irAEs based on TRBV polymorphism. In Aim 2, we will longitudinally administer patient-reported outcome (PRO) measures to deter- mine symptom, function, and health status changes that predict the risk of severe irAEs. Finally, in Aim 3, com- bining TRBV polymorphism and PRO changes with relevant demographic, socioeconomic, treatment, and clini- cal factors, we will build a predictive model and validate the model in an independent cohort of patients. To translate our results into clinical practice, we will also develop a web-based application to predict the risk of severe irAEs during treatment with ICPis. Our contribution is significant since risk assessment using the novel, simple, web-based tool will enable patients and their treating physicians to formulate personalized irAE- monitoring care plans to mitigate irAEs. This multidisciplinary approach will facilitate early detection and prompt management of irAEs, which will reduce the chance of progression to life-threatening or fatal events.

NIH Research Projects · FY 2025 · 2024-01

Title: A novel uterine leiomyosarcoma mouse model for therapeutic development PROJECT SUMMARY: Uterine leiomyosarcoma is an extremely rare but clinically aggressive uterine cancer arising from the smooth muscle of the uterus. Each year, it is estimated to occur in 6 out of every 1,000,000 women in the United States. The average age at diagnosis is 51 and women with uterine leiomyosarcomas have a poor prognosis. Current therapies for uterine leiomyosarcoma are not very effective and the primary treatment of uterine leiomyosarcoma are surgery. Due to the intrinsic rarity of this disease, prospective randomized clinical trials examining the outcome in individuals with uterine leiomyosarcoma is limited. Moreover, a poor understanding of the pathogenesis of this disease is also a major knowledge gap to develop effective treatment strategies. Thus, preclinical uterine leiomyosarcoma models that recapitulate the human disease is desperately needed to support clinical trial readiness. Homozygous p53 mutations and homozygous Pten deletion have been observed in approximately 61% and 19% human uterine leiomyosarcomas respectively, genetic mouse models with p53 homozygous mutations and homozygous deletion of Pten will be genetically relevant to the human disease. However, to generate homozygous p53 mutations with previously developed conditional Trp53 mutant allele (such as LSL p53-R172H) in mice is not feasible. It is because, mice with homozygous LSL p53-R172H allele are p53 null and will die of mainly lymphomas at 4-6 month of age. However, the Trp53wm-R172H and Trp53wm- R245W alleles, recently developed at our Institute, express functional wild-type p53 even in mice with homozygous genotype. Thus, these novel alleles allow homologous mutant p53 expression and Pten deletion to be generated upon Amhr2-Cre-mediated recombination. Our preliminary data showed that mouse with homozygous p53 mutation and homozygous deletion of Pten developed metastatic uterine leiomyosarcoma around 24 weeks of age. RNAseq and Gene Set Enrichment Analysis (GSEA) indicated that the gene expression of uterine leiomyosarcoma from these mice is like that of human uterine leiomyosarcoma. Thus, we propose to further understand the mechanism underlying the initiation and progression of uterine leiomyosarcoma using these mouse models, which will lead to more rational drug development and testing. Since p53 mutation and Pten deletion are also very common in other cancers, what we will learn from these mouse models will potentially benefit the study of other cancer with heterozygous or homozygous p53 mutations for therapeutic development.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT Small cell lung cancer (SCLC) is an extremely deadly cancer, and current chemo/radiation therapies lack durable effects. Immunotherapy with immune checkpoint blockades is effective for only ~13% of SCLC patients and rarely results in sustained responses in extensive-stage SCLC. Targeted therapy development for SCLC is challenging as SCLC tumors typically have few actionable drivers but instead are mainly driven by loss-of- function mutations in RB1 and TP53 (>90%) and frequent loss and inactivation of other potential tumor suppressors, including a set of epigenetic regulators. Nevertheless, recent discoveries from SCLC genomics provide a new framework for understanding the biology of SCLC and identifying the molecular vulnerabilities of the disease. Functional characterization of the recurrent mutations in putative tumor suppressor genes is crucial for defining the mechanisms of tumorigenesis, which will facilitate the discovery of biomarkers for tumor prevention and intervention. We recently identified a new tumor suppressor gene, CRACD (Capping protein inhibiting Regulator of ACtin Dynamics; KIAA1211/CRAD). CRACD encodes a protein that binds to and inhibits the capping proteins, thereby facilitating actin polymerization. CRACD is frequently mutated or transcriptionally downregulated in SCLC patient tumors. Cracd knockout (KO) promotes the transformation of preneoplastic precursor lung epithelial cells. Cracd KO also significantly accelerates SCLC development in an autochthonous mouse model in which tumor initiation is induced by the deletion of Rb1, Trp53, and Rbl2 triple KO mouse models. Cracd KO SCLC tumors harbor distinct root cell clusters with aberrant cell lineage trajectories and impaired antigen presentation. Single-cell transcriptome analysis has further stratified SCLC patients by CRACD inactivation and antigen presentation pathway impairment. Intriguingly, ablation of Cracd alone induces hyperplasia of neuroendocrine cells and aberrant cell lineage plasticity in the mouse lung. These results led to the hypothesis that CRACD inactivation induces aberrant neuroendocrine cell plasticity to initiate and promote SCLC tumorigenesis via dysregulated nuclear actin dynamics and subsequent epigenetic reprogramming. The proposed study will unveil the biology of SCLC initiation and progression and determine the cells-of-origin and their relevant cell lineages inducing neuroendocrine cell plasticity, providing novel insight into SCLC tumorigenesis. Additionally, this study will establish a new model system for SCLC initiation and progression and introduce a novel somatic engineering system, which is technologically innovative. Completing this study will also lay a solid foundation to determine whether CRACD loss is a molecular signature for specific SCLC patient stratification and immunotherapy response prediction, which will be conceptually and clinically innovative.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY/ABSTRACT Lung cancer is the second deadliest cancer. Despite its prevalence and unfavorable consequences, current therapeutic approaches are limited. The proposed study aims to understand the biology of lung tumorigenesis and use that knowledge to lay a foundation to develop potential treatment regimens for lung cancer. Normal cells undergo finite division and remain quiescent. The dimerization partner, RB-like, E2F, and multi-vulval class B (DREAM) complex is a cell cycle-regulatory multiprotein complex that orchestrates cell quiescence and the cell cycle. In association with RBL2/p130 (retinoblastoma-like protein 2), E2F4, and DP1 (E2F dimerization partner 1), the DREAM complex is localized to the promoters of cell cycle-regulating genes, repressing their transcription and inducing cell quiescence via G0 and G1 arrest. However, dysregulation of such a process causes the cell quiescence exit and re-entry into abnormal cell proliferation, likely leading to tumor initiation and progression. Nonetheless, the mechanism of how the DREAM complex is aberrantly regulated in cancer cells remains elusive. Comprehensive approaches employing genetically engineered animal models and unbiased screening suggest that PAF (PCNA-associated factor/KIAA0101/PCLAF) plays the oncogenic roles in non-small cell lung carcinoma (NSCLC) by remodeling the DREAM complex. Intriguingly, we found that aberrantly regulated calcium signaling enhances the DREAM complex-mediated gene transactivation. Despite the implication of Ca2+ signaling in cancer, the oncogenic roles of Ca2+ signaling in lung cancer were poorly understood. Based on the preliminary results, we hypothesize that dysregulated calcium signaling contributes to NSCLC tumorigenesis via the DREAM complex, which appears to be a targetable vulnerability of lung cancer. The hypothesis will be tested by pursuing two specific aims: Aim 1. To determine the therapeutic impact of NFAT inhibitors on lung tumorigenesis; Aim 2. To dissect the mechanism of NFAT-mediated lung tumorigenesis. Completing the proposed research is expected to advance the development of a new approach for treating lung cancer based on a novel targetable molecular mechanism involved in lung tumorigenesis.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY There is a critical need for treatment approaches that conveniently deliver evidence-based interventions of counseling and pharmacotherapy to a wide range of smokers, and address the complex timeline of cessation attempt, failure, and re-attempt. The objective of this proposal is to identify the best individualized pharmacological treatment strategies used for both initial smoking cessation and for rescue therapy among those who fail to quit or relapse. The trial will use a SMART design (Sequential Multiple Assignment Randomized Trial) to estimate the comparative effectiveness of (1) an initial 6-week course of either standard dose varenicline or dual nicotine replacement (our two best performing pharmacotherapies), and (2) among those smokers who initially fail to quit after the first 6 weeks, to estimate the effects of either continuing on same medication for another 6 weeks, switching to the medication they did not receive initially, or augmenting current pharmacotherapy by increasing the dose or adding additional FDA-approved cessation medications. All treatments will occur via a virtual delivery method ensuring the widest possible application with the fewest barriers and will include both dynamic pharmacotherapy and counseling delivered in a unified environment to enhance uptake, effectiveness, and patient experience. The proposed trial will include 2000 adult participants from throughout Texas who are seeking to quit smoking. Participants will be initially randomly assigned to one of our two best-performing smoking cessation treatments, either dual nicotine replacement therapy (NRT; nicotine patch plus lozenge) or varenicline (2 mg/twice daily). After 6 weeks, smoking abstainers will remain on their current treatment and non-abstainers will be re-randomized to either (a) switch therapies (i.e., receive the treatment not given in the first 6 weeks), (b) augment their current therapy (change dosage and/or add other medications, e.g., bupropion), or (c) continue the same medication for 6 more weeks. The treatments will function as comparators with each other at the selected timepoints specific to each treatment phase. Our primary outcomes will be biochemically verified (carbon monoxide <6 ppm) continuous smoking abstinence at Week 6 and at end-of-treatment (EOT) + 30 days. Our secondary outcomes include 6-month abstinence, and withdrawal, craving, positive and negative affect, and depressive symptoms (anhedonia) Week 6 and at end- of-treatment (EOT) + 30 days. Our results will inform the patient-provider discussion on the optimal treatment approach of the future dissemination of our findings. This addresses a critical gap in treating nicotine dependence because while the majority of smokers relapse within two weeks of an initial cessation attempt there is little empirical evidence to guide clinicians or patients on the best subsequent treatment to enhance the likelihood of cessation. Our study sample will be representative of the general population of smokers, drawing volunteers from diverse community sources as well from disadvantaged populations (Medicaid eligible, rural, and uninsured).

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Esophageal squamous cell carcinoma (ESCC) accounts for over 80% of esophageal cancer cases and has a poor prognosis, largely due to the absence of symptoms in the early stages. Although an early diagnosis of ESCC may lead to better outcomes in treatment, early detection is challenging since ESCC originates from the basal cell layer and invades the lamina propria. Therefore, understanding the biology of ESCC initiation and developing model systems that recapitulate ESCC neoplasia is crucial. Currently, the mechanism of ESCC initiation and immune landscape remodeling during ESCC initiation and tumorigenesis remain unclear. We have established a genetically engineered murine esophageal organoid model system that has shown significant potential to study ESCC initiation. Through CRISPR-based genetic manipulation of murine esophageal organoids, we identified the key tumor suppressor genes most frequently deleted in ESCC patients and established 32 esophageal organoid lines. Transcriptomics of the neoplastic organoids indicated a transcriptional signature consistent with ESCC patients, and single-cell transcriptomics identified distinct cell lineage, multiple root cells, and critical regulons of such neoplastic organoids. Interestingly, only specific neoplastic organoid- derived cells developed tumors in immunocompetent mice. Our preliminary results suggest that the loss of tumor suppressor genes is a crucial event for ESCC initiation by inducing esophageal neoplasia (cell-autonomous) and remodeling the immune landscape (non-cell-autonomous). Therefore, we hypothesize that the combinatorial loss of tumor suppressor genes initiates ESCC by inducing esophageal neoplasia, cell plasticity, and immune evasion. This hypothesis will be tested through two specific aims: Aim 1. To determine the genetic and transcriptional network initiating ESCC using new model systems; Aim 2. To determine the impact of targeting the ESCC-driven immune landscape remodeling on ESCC initiation and tumorigenesis. The study will fill the current knowledge gap by unveiling the biology of ESCC initiation and providing new models for developing viable therapeutic applications for early ESCC.

NIH Research Projects · FY 2025 · 2023-12

Activation of wild-type (wt) p53 tumor suppressor is sufficient to kill tumor cells, even if other gene defects are present. Cisplatin is a p53-dependent drug that is heavily used in treating cancer. However, in several cancers harboring predominantly wt-p53, its benefit, if any, is short-lived. For instance, response rate to cisplatin in the Clear Cell ovarian cancer (OvCa) sub-type is a low 25%, resulting in an overall survival of only 10%. Thus, cisplatin resistance is a significant impediment, and this has created an unmet need to identify novel strategies that would lead to targeted therapeutic options for rational development against the refractory OvCa disease. A major factor in resistance is the loss of p53 function, and not necessarily through mutation. This proposal is focused on wt-p53 since there is now acute awareness from our work and those of others that this genotype is ubiquitous in many advanced clinically resistant cancers, such as mesothelioma and osteo- sarcoma. In this proposal, our focus is on refractory OvCa harboring wt-p53, which is the genotype present predominantly (~90%) in the rare OvCa sub-types, constituting 30% of all ovarian cancers. Importantly, these sub-types occur at an earlier age and, therefore, death is observed in younger women. But, current therapy of these sub-types remains empirical, due largely to profound knowledge gaps that exist in this disease. We have established that tumor cells become resistant to cisplatin when this drug fails to activate wt- p53 due to silencing of the specific kinase that is vital for p53 phosphorylation and, thereby, p53-dependent transactivation function. Normally, wt-p53 is maintained in an inactive state by its endogenous natural inhibitors. The activity of partially released p53 through pharmacological targeting of such inhibitors, however, is insufficient to exceed the critical threshold for apoptotic function. Since p53 phosphorylation is vital for fully functionalizing p53 for its apoptotic function, we propose a novel hypothesis that targeted reduction in MDM4 will fully release p53 and allow access of the critical p53-site to endogenous active kinase(s) and induce phosphorylation. We will test this hypothesis through two specific aims: 1) Establish basal phosphorylation of p53 by targeting MDM4 in refractory OvCa sub-type tumor models in vitro and in vivo; 2) Identify the active kinase that phosphorylates p53 and assess its potential role for therapy. We will utilize pharmacologic tools to define tumor sensitivity following MDM4 modulation, biochemical and molecular tools to identify and modulate the kinase and delineate the basis for reversal of cisplatin resistance, and graphically model antitumor effects of rational combinations to establish synergy. This high-risk project has the potential to establish a conceptual foundation for therapeutic activation of wt-p53 in rare OvCa that will enable follow-up studies and likely change the trajectory of treatment outcomes for large numbers of patients diagnosed with cisplatin-resistant OvCa sub- types. Importantly, there is a high potential that the outcome from the proposed studies will likely be tumor agnostic and, thereby, substantially impact a wider spectrum of wt-p53 cancers resistant to other drugs.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Lung cancer is the leading cause of cancer-related death in men and women in Western countries, with non- small cell lung cancer (NSCLC) accounting for ~85% of all lung cancer cases. Response rates to chemotherapy, newer targeted therapies, and immune checkpoint inhibitor (ICI) monotherapy remain unsatisfactory and new treatments are needed for NSCLC. Elevated levels and activation of signal transducer and activator of transcription (STAT) 3, a transcription factor that serves as a master regulator of cancer, occur in most NSCLC tumors and are associated with worse survival and ICI resistance mediated, in part, through the actions of myeloid derived suppressor cells (MDSC) in the tumor microenvironment (TME). STAT3 activation also may contribute to ICI-mediated immune-related severe adverse events (irSAE). The Tweardy group, working with a clinical-stage biotechnology company (Tvardi Therapeutics, Inc.), used computer-based docking and lead- compound optimization strategies to identify TTI-101, a potent, non-toxic and orally bioavailable inhibitor of STAT3. TTI-101 hit target and blocked tumor growth in preliminary studies using the CC-LR K-ras mutant mouse model of NSCLC and in published studies in nude mice bearing xenografts of the LKB1-null human NSCLC cell line, A549. A Phase 1, dose-escalation study of TTI-101 monotherapy in patients with advanced/refractory solid tumors (NCT03195699) completed enrollment in Q3 2022 and revealed no dose limiting toxicities or fatal treatment-related adverse events and identified a RP2D. TTI-101 administration reduced levels of activated STAT3 within tumors by 57% and was beneficial in 21 of 39 (54%) evaluable patients, including 5 confirmed partial responses. Based on these results, TTI-101 was granted FDA Fast Track designation and has emerged as the most promising small-molecule STAT3 inhibitor in clinical development to treat cancer. Our long-term translational goal is to improve outcomes in patients with recurrent or metastatic (RM) NSCLC. The main objective here is to determine if STAT3 targeting with TTI-101 is beneficial in treatment of RM NSCLC. The specific aims designed to achieve this objective are: 1) to determine if TTI-101 alone or in combination with anti- PD-1 monoclonal antibody is effective in treatment of NSCLC in the K-ras mutant (CC-LR) mouse model, 2) determine the anti-tumor efficacy and immune-modulatory effects of TTI-101 alone or in combination with anti- PD-1 in immune-competent mouse models of K-ras and Stk11/Lkb1 (KL) co-mutated NSCLC, and 3) determine the safety and anti-tumor efficacy of STAT3 inhibition with TTI-101 in combination with pembrolizumab (anti-PD- 1) in previously treated patients with KRAS and STK11/LKB1 (KL) co-mutated RM NSCLC in a Phase IB/II clinical trial. We will obtain tumor biopsies pre- and post-treatment in the Phase II trial and perform correlative studies that include determining the relationships between pharmacokinetics, pharmacodynamics, the TME, and clinical responses in patients and mice with NSCLC treated with TTI-101 alone or in combination with anti-PD-1 therapy. If successful, our research will improve survival and decrease toxicity in patients with RM NSCLC.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY/ABSTRACT DNA cytosine methylation yielding 5-methylcytosine (5mC) is a major epigenetic modification involved in the regulation of chromatin structure and gene expression. Mammalian DNA methylation is catalyzed by three DNA methyltransferases (DNMTs), belonging to two structurally and functionally distinct families. DNMT3A and DNMT3B establish the initial cytosine methylation pattern, whereas DNMT1 maintains that pattern on newly replicated DNA. Cancer cells exhibit aberrant DNA methylation patterns, including global hypomethylation, which is associated with low maintenance efficiency, and regional hypermethylation, which is mainly due to abnormal de novo methylation and/or deficient demethylation. Although the demethylating agents 5-azacytidine and 5- aza-2'-deoxycytidine (decitabine) have been approved by FDA for treating some hematological malignancies, these nucleoside analogs incorporate into DNA, leading to substantial DNA damage and cellular toxicity, and are ineffective in treating solid tumors. Recently, GlaxoSmithKline (GSK) reported the discovery of a new class of dicyanopyridine-containing DNMT1-selective inhibitors with therapeutic potential. The long-term goal of this research is to develop a highly potent and selective non-nucleoside DNMT3A/3B inhibitor for cancer treatment. The objective of this 2-year exploratory project is to identify one or more promising leads for further development. The rationale is that remedying hypermethylation by inhibiting DNMT3A/3B would reactivate abnormally silenced genes, including tumor suppressor genes, and thus provide therapeutic benefits for cancer patients. The proposal is based on preliminary data, generated in the applicants’ laboratories, showing that some dicyanopyridine-containing derivatives and quinoline-based derivatives can selectively inhibit DNMT3A/3B. The applicants propose to perform biochemical and structural studies to improve the potency and selectivity of DNMT3A/3B inhibitors (Aim 1); and validate the compounds for their potency and selectivity using wild-type and DNMT-deficient mouse embryonic stem cells (mESCs), determine their anti-cancer effects using cancer cell lines with or without mutations in components of the DNA methylation and demethylation enzymes (DNMT3A, TET2 and IDH1/2), and assess their cytotoxicity using untransformed cell lines (Aim 2). The potential impact of identifying non-nucleoside DNMT3A/3B inhibitors is likely to be very substantial..

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Shenqqing (Stan) Gu, Ph.D., is an experimental-computational biologist whose career goal is to develop personalized strategies for optimal combination immunotherapy to cure cancer. Titled “Enhancing the efficacy of immunotherapy by optimal use of SMAC mimetics”, the proposed research seeks to elucidate the context dependencies of SMAC mimetics’ effects and examine its optimal combination with existing immune checkpoint blockade therapy. Career development plan: Dr. Gu is a recipient of Sara Elizabeth O’Brien Trust Fellowship. His previous work has focused on the regulation of cancer-immune interaction, using various approaches including clonal tracing, data mining, and genome-wide CRISPR screening, which have prepared him to conduct the proposed research. Dr. Gu has outlined specific training activities to expand his skill set in four areas: 1) T cell biology, 2) machine learning, 3) single-cell technologies, 4) leadership and professional skills. This skill set is necessary to succeed in my independent research career. Collaborators/Environment: Dr. Gu’s collaboration team assembles world-leading experts in cancer immunology, computational biology, translational and clinical research, melanoma, and single-cell technology. Leveraging the extensive collaboration resources at DFCI, Harvard, and NCI, and access to a large amount of clinical samples, Dr. Gu is uniquely placed to identify the optimal usage of SMAC mimetics to enhance the efficacy of immunotherapy of cancer. Research: Our previous research identified that SMAC mimetics can upregulate MHC-I in some cancer cells and potentiate immunotherapy, but it is unclear which subset of cancer patients can benefit from SMAC mimetic treatment. To facilitate effective use of SMAC mimetics in the clinic, this proposal will interrogate SMAC mimetics regarding the optimal contexts of gene regulation and rational combination with immune checkpoint blockade. Aim 1 will integrate genetic/epigenetic feature selection and functional validation to identify the context dependencies of SMAC mimetics’ effects on cancer cells. Aim 2 will integrate multi-omic profiling at single-cell level to examine the effects of SMAC mimetic treatment on immune cells. Aim 3 will evaluate the efficacy of different scheduling of combination of SMAC mimetics and ICB using in vitro co-culture and in vivo transplantation models. Outcomes/Impact: This project will reveal the genetic/epigenetic context dependencies of SMAC mimetics’ effects, elucidate their effects on immune cells, and examine the optimal combination with immunotherapy. Data from this study can help improve the design of clinical trials testing SMAC mimetics in cancer patients. The development career transition award will enable Dr. Gu to become a leader in the new field of developing personalized combination immunotherapy strategies.

NIH Research Projects · FY 2025 · 2023-09

Comparative effectiveness research (CER) aims to inform healthcare decisions by providing evidence on the benefits and harms of different alternatives in cancer care. While randomized controlled trials are often considered to be the gold standard of research and have high internal validity due to their controlled environment, they are often not generalizable to ‘real-world’ populations. Clinical trials often exclude large segments of the population, particularly those who are older or have comorbid conditions, threatening the external validity of the findings. Furthermore, explanatory trials typically compare only two or three interventions. As the number of cancer therapies and technologies increases exponentially, additional methods are necessary to adequately compare benefits and harms across several different interventions. While the emphasis in the past was on determining whether an intervention could work under the best conditions, increasing importance is given to whether interventions also work in ‘real-world’ settings, and how they compare to other available alternatives. Real-world evidence and CER are vital to adequately evaluate and position new health interventions and technologies as they emerge. Few training programs specifically address CER methodologies. The overarching goal of this application is to implement a comprehensive national CER training program for junior cancer investigators, encompassing the broad spectrum of healthcare delivery in cancer care. Training will encompass three major CER methodologies: 1) Knowledge synthesis including systematic reviews, meta-analysis, and decision analysis; 2) Observational studies and secondary data analysis of registries and other sources of data, and 3) Pragmatic clinical trials, and. After first completing a series of interactive online courses in these topics to acquire a strong knowledge base in CER, participants will complete an intensive, hands-on training small group workshop in one or more of the three CER methodologies. Each participant will develop an individual protocol for a CER study, for a research question of their interest. Topics of interest can address any of the stages of care throughout the cancer continuum, including prevention, screening, diagnosis, treatment, supportive care, end-of-life care, and survivorship. Program faculty will provide individual methods guidance and support to participants during the 12 months after enrolling in the course. Participants will be junior investigators, recruited nationally, and affiliated with academic institutions, cancer centers or non-profit organizations (junior faculty, research scientists or post-doctoral or clinical fellows with an expectation of a research career path). Recruitment will target academic and other research institutions across the nation. This education program is designed to support the National Cancer Institute’s educational mission by arming cancer researchers with innovative tools needed to conduct CER. Expanding CER in cancer research will lead to robust real-world evidence, which can inform cancer patients, caregivers, providers and policy-makers of the benefits and harms of cancer treatment options.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Androgen indifferent prostate cancers account for a large proportion of the disease lethality and have limited therapy options, partly due to the lack of identifying biomarkers. To address the unmet need of developing effective therapies for this subset, we defined the aggressive variant prostate cancers (AVPC) criteria. Through a series of prospective trials and studies in mice, we showed that the AVPC criteria can enrich for prostate cancers that respond poorly to androgen inhibition and benefit from adding carboplatin to cabazitaxel. In recent trials we examined the contribution of PARP inhibitor maintenance, and of anti-PD1 inhibition to the chemotherapy backbone in men with AVPC. Early data show meaningful improvements in outcomes with these additions, but many men with AVPC are still progressing rapidly. Our overall goal is to arrive at rational, biologically-based combination therapies that effectively treat the AVPC. The analysis of samples from our trial participants, and preclinical studies, converge on altered arginine metabolism as a key metabolic pathway in androgen-indifferent prostate cancer biology. We also found evidence of argininosuccinate synthase 1 (ASS1) silencing with platinum chemotherapy in AVPC cell lines and patient samples. ASS1 deficiency renders cells dependent on extracellular arginine, and thus sensitive to arginine depletion with agents such as PEGylated arginine deiminase (ADI-PEG20). ADI-PEG20 depletes serum arginine levels, has activity in several malignancies (alone and in combination with chemotherapy), and has immunomodulatory effects. However, the effects of serum arginine depletion on intratumoral metabolite levels in patients are unknown, and its effects on the human immune tumor microenvironment (TME) remain poorly understood. We hypothesize that the addition of ADI-PEG20 will improve the efficacy of carboplatin+cabazitaxel by modifying intratumoral arginine metabolism and immune profiles in the AVPC TME. In AIM 1, we will conduct a phase I/II dose escalation trial to identify the optimal dose (in terms of safety and efficacy) of ADI-PEG20 to combine with carboplatin+cabazitaxel in men with AVPC. In AIM 2, peripheral blood and metastatic tumor biopsies obtained from trial participants at baseline, after 1 and after 6 cycles of treatment, and at parallel time-points in PDX and syngeneic mouse models, will be used to measure associations between: (a) serum levels of arginine and citrulline, (b) intratumoral levels of arginine, (c) ASS1 expression, and (d) the expression of other arginine metabolism enzymes, and (e) outcomes. In AIM 3 we will examine the effects of treatment on immune profiles and immune cell distribution within the TME. Our studies will provide a comprehensive evaluation of the effects of serum arginine depletion on the immune and non-immune AVPC TME, shed light on the mechanisms of synergy between ADI-PEG20 and cytotoxic chemotherapy, and ultimately, serve to prioritize rational combinations for the treatment of the AVPC.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT The SETER/PR index of sensitivity to endocrine therapy measures endocrine receptor-related transcription from formalin-fixed paraffin-embedded tumor sections and is highly reproducible within and between laboratories. SETER/PR values correlate with the ligand binding activity of estrogen receptors and progesterone receptors, predict early pharmocodynamic response to endocrine therapy and prognosis following endocrine therapy in palliative and adjuvant treatment settings. Secondly, SET2,3 index adjusts the measurements of SETER/PR index for baseline prognostic factors, enabling the assessment of endocrine-related transcriptional activity in the context of baseline prognostic risk. Both indices add independently prognostic information to contemporary genomic tests, likely due to stronger prediction of the cancer’s sensitivity to endocrine therapy. The NSABP B-42 trial compared extended duration of adjuvant endocrine therapy with letrozole for 5 additional years, versus placebo for 5 years, in patients who had completed 5 years of adjuvant endocrine therapy. Extended endocrine therapy demonstrated improved disease-free survival at 10 years from randomization, although there was no difference between treatments during the initial 4 years. To-date, correlative science studies from NSABP B-42 have evaluated the Breast Cancer Index 2-gene ratio of HOXB13/IL17BR expression, the 70-gene MammaPrint assay, and normalized ESR1 gene expression scores from the 21-gene Recurrence Score. All failed to demonstrate predictive interaction in their respective primary analyses, although exploratory analyses suggest that prediction will be possible with a more specific biomarker. We hypothesize that SETER/PR index will predict benefit from extended letrozole therapy (ELT) in hormone receptor-positive HER2-negative breast cancer. Specifically, that ELT offers most benefit to patients whose breast cancer has a moderate degree of endocrine sensitivity. Our primary analysis plan is to test whether SETER/PR index between 1.10 and 2.10 (inclusive) has significant interaction with lower rate of breast cancer- free interval (BCFI) from extended letrozole treatment. This represents an inter-quartile range from a similar population to NSABP B-42. Secondary analyses will evaluate other endpoints, subtypes defined by nodal status or prior tamoxifen treatment, and other cutpoints of SETER/PR index. Secondly, we will evaluate SET2,3 for long-term risk of late relapse in patients from each treatment arm. Clinically, a robust predictor of benefit from extended duration of endocrine therapy will be useful, as 10 years of treatment is too long for many patients to tolerate and the absolute rate of benefit is small in the overall population with hormone receptor-positive breast cancer. Current evidence shows SETER/PR index to be an independently strong predictor of tumoral sensitivity to endocrine therapy, and associated prognosis, with strong potential to predict who is likely to benefit from longer duration of endocrine treatment. 1

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Venous thromboembolism (VTE) develops in about one-fourth of patients with ovarian cancer and is associated with significant morbidity and mortality. Chemotherapy increases VTE risk, but administration of prophylactic anticoagulation to all patients on chemotherapy is associated with a substantial risk of bleeding. Therefore, it is crucial to identify patients with a higher risk of VTE. In the University of Texas MD Anderson Cancer Center (MDACC) Ovarian Cancer Moon Shot program, we have assembled a cohort of 354 patients who have received neoadjuvant chemotherapy. The availability of tumor specimens, blood samples, and an extensive clinical database from these patients provides us a unique opportunity to investigate the novel predictive biomarkers for VTE in ovarian cancer. Most previous studies on cancer thrombosis analyzed clinical, demographic, or hemostatic factors already known to be risk factors for VTE in cancer patients instead of identifying tumor-specific prothrombotic factors. We will explore cancer cell products that increase VTE risk and particularly investigate the impact of cancer cell-derived podoplanin and mitochondria on VTE. We found mitochondria in plasma samples of cancer patients and showed that ovarian cancer cells release mitochondria (both free and microvesicle-embedded). Injection of mitochondria caused venous thrombi in mice, rich in neutrophils and neutrophil extracellular trap (NETs). We speculate that mitochondria-targeted antioxidants and antibiotics blocking the synthesis of chemotactic formylmethionine(fMet)-tagged peptides reduce cancer VTE. We found that podoplanin is expressed on ovarian cancer cells and tumor-derived extracellular vesicles (EVs), and its expression is increased by chemotherapy. Podoplanin-expressing EVs activate platelets, and their injection into mice causes platelet-rich venous thrombi. We propose that a small molecule blocking podoplanin interaction with platelets reduces cancer thrombosis. We will examine whether the number of mitochondria and concentration of podoplanin in plasma predict VTE risk in ovarian cancer patients receiving chemotherapy. We will investigate the effect of a mitochondria-targeted antioxidant, an antibiotic blocking synthesis of fMet peptides, and a podoplanin inhibitor on venous thrombosis in a murine model of IVC ligation. Finally, we will compare the mutation profile and mutation burden of mitochondria and nuclear genes in tumors of ovarian cancer patients with and without VTE to identify the genetic changes in cancer cells associated with an increased VTE risk.

NIH Research Projects · FY 2025 · 2023-09

The overall goal of the MD Anderson Cancer Center SPORE in Ovarian Cancer is to test and translate novel therapeutic strategies, including those to overcome adaptive resistance to conventional cytotoxic chemotherapy, poly (ADP-ribose) polymerase inhibitors (PARPi), anti-angiogenic agents (bevacizumab) and immune checkpoint blockade. Over the last five years, 1,417 new patients with ovarian cancer received care at MD Anderson. We have prioritized ovarian cancer research through recruitment, salary support, clinical facilities, laboratory space, and philanthropic funds. Philanthropic support from the MD Anderson Ovarian Cancer Moon Shot has provided organization and infrastructure, but the work in the SPORE is completely distinct. We successfully implemented measures to increase the recruitment to our Developmental Research Program (DRP) and Career Enhancement Program (CEP). Over the last 22 years, our SPORE investigators have contributed over 1280 manuscripts regarding ovarian cancer with 155 in the last four years. Our SPORE has made significant contributions including: 1) conducted the SPORE and EDRN-supported Normal Risk Ovarian Screening Study (NROSS) where 71% of cases have been detected in stage I or II; 2) identified biomarkers that detect 18% of CA125 negative cases; 3) developed a 4-biomarker algorithm that in retrospect detects advanced stage disease 1.4 to 4.8 years earlier than the CA125-based NROSS algorithm; 4) found anti-TP53 autoantibodies elevated 8 months before CA125 and 22 months before diagnosis; 5) observed a 54% objective response rate to anti-angiogenic therapy with aflibercept and docetaxel; 6) completed a trial targeting Dll4; 7) demonstrated that CSF1R inhibitors can deplete macrophages and reduce resistance to anti-VEGF therapy; 8) demonstrated significant activity of the MEK inhibitor selumetinib in low-grade ovarian cancers and completed an international phase III trial of another potent MEK inhibitor trametinib; and 9) developed a robust biomarker panel that predicts response to PARPi and initiation of multiple trials combining PI3K and PARPi in high-grade ovarian cancer. In the proposed SPORE, Project 1 and Project 4 investigators tackle therapeutic resistance to PARP inhibitors and immune checkpoint blockers from multiple directions to speed progress and improve outcomes for women with ovarian cancer. Both projects have the potential to enhance T-cell infiltration in tumors and impart immunologic memory, which is particularly important given the likelihood of this cancer to recur. In Project 2, we will develop a novel TROP2-targeted CAR-NK therapy. In Project 3, we will develop therapy aimed at the tumor microenvironment using a novel EGFL6 targeted monoclonal antibody. Overall, our translational studies conducted in an optimal environment with a multi-institutional team are directed toward improving clinical outcomes of women with ovarian cancer.

NIH Research Projects · FY 2025 · 2023-09

Project Summary This proposal addresses three major challenges in adoptive cellular therapy: 1) T cell persistence; 2) Tumor immune resistance and 3) Target epitope identification for solid tumors, bringing together the expertise of Dr. Yee in T cell therapy, Dr. Rai in epigenetics and Dr. Morelli in clinical trials. We have established a strategy for generating memory T cells from peripheral blood of patients, via an ACT modality known as Endogenous T Cell (ETC) therapy pioneered in the Yee lab, allowing us to target HORMAD1, a cancer testis antigen broadly expressed in gastric and esophageal cancer, which in its advanced stages represents a significant unmet need. By applying rationally designed combinations of epigenetic modulators we plan to optimize the replicative capacity and memory properties of HORMAD1-specific CTL (HMD-CTL) generate ex vivo to address the challenge of limited in vivo T cell persistence (UG3 Aim 1). One of the dominant extrinsic factors mediating tumor immune resistance in gastric and esophageal cancer is the influence of TGF-β in the tumor microenvironment. In addressing this challenge, we propose the use of a dominant negative TGF-β receptor to sequester TGF-β by engineering expression of the TGFBDNR2 gene into HORMAD1-specific CTL using a retroviral transduction strategy that is well-established in the Cell Therapy Manufacturing Center (UG3 Aim 2). Finally, addressing the challenge of targeting gastric and esophageal cancers was borne out of an ongoing antigen discovery pipeline developed over 5 years examining the portfolio of tandem mass spectrometric defined epitopes eluted from MHC of over 30 tumor samples. From this pipeline we have empirically validated both an HLA-A2 and HLA-A11 epitopes of HORMAD1, demonstrated high affinity with CTL generated using the ETC workflow and propose the use of HORMAD1-specific CTL for this trial, allowing us to cover more than 25% of all gastric and esophageal patients given the prevalence of HORMAD1 expression in these tumors (> 40%) and HLA allele expression (> 65%). At completion of the UG3 portion of this proposal, we will identify an optimal epigenetic program (Aim 1) and TGFBDNR2 transduction workflow (Aim 2) that fulfills the metrics for a Go/ No-Go decision. In the clinical trial component (UH3) these strategies will be assembled to allow us to evaluate safety and duration of in vivo persistence, comparing HORMAD1-specific CTL and TGFBDNR2-engineered HORMAD-1-specitic CTL in two cohorts, while assessing for preliminary efficacy in an Expansion arm. The ETC platform provides greater flexibility than other ACT modalities in its agility to direct specificity to almost any desired target epitope, demonstrated evidence of antigen-spreading, minimal toxicities, and has an established memory potential proven to be sustained in several clinical trials. We anticipate, that with an enhanced epigenetic memory program and, endowed with a mechanism to undermine at least one feature of tumor immune resistance, ETC therapy can provide a vehicle for advancing adoptive cellular therapy for the treatment of solid tumors in a systematic and stepwise fashion.

NIH Research Projects · FY 2025 · 2023-09

Cervical cancer affects women in low socioeconomic regions, such as sub-Saharan Africa, which also have a high incidence of Human Immunodeficiency Virus (HIV) infection, and access to cervical cancer screening is less available for women living in these communities. Furthermore, People Living with Human Immunodeficiency Virus (PLWH) have evidence of premature aging, which could contribute to cervical cancer progression and responses to chemoradiation therapy (CRT). CRT is the standard of care for locally advanced cervical cancer, and older women (≥52 years of age) treated with CRT have worse side effects than younger (<52 years) women, suggesting that advanced age may influence clinical outcomes from CRT for cervical cancer. Many of the hallmarks of cancer, including tumorigenesis, tumor maintenance, therapy resistance, and immune evasion, are regulated by epigenetic changes in DNA (e.g., DNA methylation). DNA methylation levels are correlated with (1) chronological clocks, which estimate the age of a sample/patient, or (2) biological clocks, a widely accepted measure of where an individual is in their lifespan, regardless of chronological age, which can be reflective of disease morbidity and mortality risk. Indeed, the biological age of PLWH (i.e., HIV-mediated epigenetic age) is advanced up to 20 years beyond chronological age; however, studies examining epigenetic aging in PLWH have not evaluated premature aging in PLWH with cervical cancer, nor the contribution, if any, of oncologic therapy on premature aging. Preliminary data comparing women living with HIV(WHIV) vs. HIV-negative cervical cancer patients indicate that biological aging, defined using patterns of methylation that accumulate on host DNA over time, was significantly accelerated in WHIV vs. HIV-negative cervical cancer patients, and this accelerated aging was significantly associated with mortality after cancer diagnosis. The proposed study will test the hypothesis that a biomarker of aging can be identified and will correlate with systemic and tumor immunologic phenotype and function that can be used, in the future, to select WHIV and cervical cancer for novel therapeutic regimens. In Aim 1, differences in DNA methylation will be compared between WHIV vs. HIV-negative patients with cervical cancer. Aim 2 will focus on measuring systemic and tumor immune cell phenotype, function, and repertoire that will be correlated with biologic age at pre-CRT and 1 year post-CRT. Furthermore, Aim 3 will focus on determining an association of longitudinal (pre-CRT and 1-year post-CRT), biologic age changes with clinical outcomes. Results from the proposed work are expected to elucidate how oncologic treatment in the setting of immunosuppression due to HIV infection impacts the aging process and, through detailed interrogation of immune cells, to link aging to underlying biological features that may exacerbate differences in clinical outcomes observed in women living with HIV and cervical cancer.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT In the United States, cancer remains the leading cause of death by disease in those <20 years of age, and approximately 80% of survivors have at least one chronic health condition by 45 years of age. One of the strongest risk factors for cancer in children and adolescents is being born with a congenital anomaly—this is true both for chromosomal abnormalities (e.g., Down syndrome) and non-chromosomal birth defects (e.g., non- syndromic congenital heart defects), as recently validated in our registry linkage study of over 10 million live births. By linking data from population-based birth defects and cancer registries in four states included in the Genetic Overlap Between Anomalies and Cancer in Kids (GOBACK) Study, we identified multiple novel congenital anomaly-cancer associations that are not part of known cancer predisposition syndromes, including choanal atresia-acute leukemia (HR=9.2, 95% CI: 3.8-22.1) and CHD-neuroblastoma (HR=3.4, 95%: 2.6-4.5). Furthermore, consistent with our epidemiologic findings, our genomic assessments have led to the identification of a novel leukemia predisposition disorder characterized by multiple congenital anomalies (including choanal atresia) and pathogenic germline variants in USP9X. In this application, we propose to expand our integrated epidemiologic and genomic approach to address our central hypothesis that pleiotropic genetic variation generates specific congenital anomaly-pediatric cancer (CA-PC) patterns with increased risk conferred to each co-occurring phenotype. Our overall objective is to elucidate the overlap between congenital anomalies and pediatric cancer by leveraging and integrating existing datasets, including 1) the Gabriella Miller Kids First Pediatric Research Program with 44 congenital anomaly and pediatric cancer cohorts, representing 20,000 patients and 48,000 genomes; 2) the All of Us Research Program with WGS data on >90,000 individuals; and 3) the GOBACK Registry Linkage Birth Cohort, which includes population-based data on >25 million live births. In Aim 1, we will analyze an expanded population- based cohort of >25 million children to identify new CA-PC patterns and confirm previously reported patterns. In Aim 2, we will analyze germline whole-genome sequencing (WGS) data from 2,000 children with both congenital anomalies and pediatric cancer and over 20,000 children with either congenital anomalies or pediatric cancer to identify novel pleiotropic genes harboring rare, pathogenic variants responsible for specific CA-PC patterns. In Aim 3, we will describe the landscape of somatic alterations in pediatric cancers that result from pathogenic CA-PC variants through the analysis of tumor-normal WGS and RNA-Seq data in 2,000 children. This work will generate novel insights into cancer predisposition and subsequently lead to improved care for children with congenital anomalies, who comprise 120,000 births every year in the United States. In addition, insights into cancer development among at-risk populations could provide clinical utility (e.g., genetic counseling or therapeutic targets) for children and adults with cancer in the general population.

NIH Research Projects · FY 2024 · 2023-09

Project Summary This proposal addresses three major challenges in adoptive cellular therapy: 1) T cell persistence; 2) Tumor immune resistance and 3) Target epitope identification for solid tumors, bringing together the expertise of Dr. Yee in T cell therapy, Dr. Rai in epigenetics and Dr. Morelli in clinical trials. We have established a strategy for generating memory T cells from peripheral blood of patients, via an ACT modality known as Endogenous T Cell (ETC) therapy pioneered in the Yee lab, allowing us to target HORMAD1, a cancer testis antigen broadly expressed in gastric and esophageal cancer, which in its advanced stages represents a significant unmet need. By applying rationally designed combinations of epigenetic modulators we plan to optimize the replicative capacity and memory properties of HORMAD1-specific CTL (HMD-CTL) generate ex vivo to address the challenge of limited in vivo T cell persistence (UG3 Aim 1). One of the dominant extrinsic factors mediating tumor immune resistance in gastric and esophageal cancer is the influence of TGF-β in the tumor microenvironment. In addressing this challenge, we propose the use of a dominant negative TGF-β receptor to sequester TGF-β by engineering expression of the TGFBDNR2 gene into HORMAD1-specific CTL using a retroviral transduction strategy that is well-established in the Cell Therapy Manufacturing Center (UG3 Aim 2). Finally, addressing the challenge of targeting gastric and esophageal cancers was borne out of an ongoing antigen discovery pipeline developed over 5 years examining the portfolio of tandem mass spectrometric defined epitopes eluted from MHC of over 30 tumor samples. From this pipeline we have empirically validated both an HLA-A2 and HLA-A11 epitopes of HORMAD1, demonstrated high affinity with CTL generated using the ETC workflow and propose the use of HORMAD1-specific CTL for this trial, allowing us to cover more than 25% of all gastric and esophageal patients given the prevalence of HORMAD1 expression in these tumors (> 40%) and HLA allele expression (> 65%). At completion of the UG3 portion of this proposal, we will identify an optimal epigenetic program (Aim 1) and TGFBDNR2 transduction workflow (Aim 2) that fulfills the metrics for a Go/ No-Go decision. In the clinical trial component (UH3) these strategies will be assembled to allow us to evaluate safety and duration of in vivo persistence, comparing HORMAD1-specific CTL and TGFBDNR2-engineered HORMAD-1-specitic CTL in two cohorts, while assessing for preliminary efficacy in an Expansion arm. The ETC platform provides greater flexibility than other ACT modalities in its agility to direct specificity to almost any desired target epitope, demonstrated evidence of antigen-spreading, minimal toxicities, and has an established memory potential proven to be sustained in several clinical trials. We anticipate, that with an enhanced epigenetic memory program and, endowed with a mechanism to undermine at least one feature of tumor immune resistance, ETC therapy can provide a vehicle for advancing adoptive cellular therapy for the treatment of solid tumors in a systematic and stepwise fashion.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Tumor suppressor gene speckle-type POZ protein (SPOP), a substrate adaptor of cullin3-RING ubiquitin ligase, demonstrates heterozygous missense mutations in up to 15% of prostate cancers, yet the functional significance of these SPOP mutations is largely unknown. We identified SPOP-binding consensus motifs in multiple proteins of the canonical cGAS-STING and non-canonical STING-NF-κB pathways and demonstrated that human and mouse STING protein is a bona fide SPOP target. Analysis of SPOP mutant (SPOPmut) and SPOP wild-type (SPOPwt) castration-resistant prostate cancer (CRPC) clinical data sets revealed a 29-gene “SPOPmut gene signature”, which reflected STING-NF-κB signaling activity and suggested a role for the recruitment of tumor microenvironment (TME)–facilitated tumor cell growth and survival in SPOPmut CRPC. Further evaluation of the “SPOPmut gene signature” in primary, untreated prostate cancer from TCGA revealed a subset of SPOP mutant tumors, as well as a subset of CHD1 mutant (CHD1mut) tumors, that are enriched for the signature. Importantly, co-mutations in SPOP and CHD1 (chromatin remodeling factor) are well documented. In stably transduced human and mouse SPOPmut (SPOPF102C and SPOPF133V)–expressing CRPC models, we demonstrated upregulation of non-canonical STING-NF-κB-IL-6 pathway proteins, STAT3, and HMG proteins involved in promoting secretory pathway activities. We also showed that PARP inhibitor (PARPi) treatment of SPOPmut CRPC cells induces DNA damage, activates canonical cGAS-STING-TBK-IFN-ß signaling and suppresses non- canonical STING-NF-κB-IL-6/STAT3, in part through inhibitory phosphorylation (S754-STAT3), leading to growth suppression and apoptotic signaling. Olaparib (OLA) increased IFN-β secretion and reduced viability to a greater extent in SPOPmut prostate cancer cells than in control cells in coculture with macrophages versus monoculture. In addition, activation of cGAS-STING and induction of IFN-ß in macrophages were demonstrated only in OLA- treated coculture models, and neutralizing antibody experiments showed that paracrine regulation of OLA- mediated growth suppression involved IFN-β induction and IL-6 suppression of SPOPmut prostate cancer cells. We hypothesize that SPOPmut, CHD1 deletion (CHD1del) and SPOP + CHD1 co-mutations in prostate cancers promote PARPi or PARPi + anti-IL-6 therapeutic efficacy through enhanced synthetic lethality driven by increased, unrepaired DNA damage, which leads to a shift in the balance toward canonical cGAS-STING-IFN-ß signaling and suppression of IL-6/STAT3. We propose to analyze the underlying mechanisms of the SPOPmut and CHD1del prostate cancer phenotype and define these genetically driven TME alterations and their protein effectors through macrophage reprogramming using state-of-the-art proteomics (Aim 1); analyze the efficacy of PARPi and anti-IL-6 treatment in SPOPmut (F133V), CHD1del, and SPOPmut;CHD1del RM-1-BM syngeneic CRPC models (Aim 2); and analyze clinical trial tissue and blood samples from PARPi-treated SPOPmut and CHD1mut prostate cancer patients for biomarkers of this genetically driven phenotype (Aim 3).

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Given the generally incurable nature of metastatic cancer, patients and their family caregivers are at high risk of experiencing depressive symptoms and spiritual distress (e.g., lack of meaning and peace, despair, alienation). As caregivers struggle with their own fears and stressors, providing quality support and care to the patient may be difficult. Moreover, psychospiritual distress is interdependent in families coping with cancer suggesting the need for a dyadic approach to psychospiritual supportive care. Despite accumulating evidence that highlights the value of positive psychology interventions in improving depressive symptoms and spiritual wellbeing, evidence-based dyadic interventions are limited in the palliative care setting. To address critical knowledge gaps and build upon our pilot work, we propose an adequately powered efficacy trial of a 4-session family-focused meditation (FFM) intervention targeting psychospiritual outcomes. Patient-family caregiver dyads will be randomized to either the FFM intervention, a usual care (UC) control group or a dose-matched attention control (AC) group receiving a social support intervention. To enhance accessibility and scalability for future dissemination, the FFM and AC interventions will be delivered via videoconferencing. To increase generalizability of our findings, we will use a multi-site enrollment strategy and recruit families from a community hospital caring for a diverse including underserved patient population. We will administer all study procedures including the Patients and caregivers will complete assessments at baseline (prior to randomization) and then again 6, 12, and 24 weeks later. Guided by our exciting pilot findings, we propose a multi-method assessment strategy to uncover a priori intervention mechanisms. In addition to self-reports, at baseline and the 6-week follow-up, patients and caregivers will wear a device called the Electronically Activated Recorder (EAR), an innovative, in vivo naturalistic observation tool that will allow us to objectively assess changes in observed behaviors in daily life as a function of group assignment to evaluate mechanisms of intervention efficacy. We propose depressive symptoms as the primary outcome given the prognostic value of depression in the survival of metastatic cancer patients and family caregivers; and spiritual wellbeing as the secondary outcome as it protects against the desire for a hastened death, hopelessness, and suicidal ideation independent of depression in patients with metastatic cancer. For families who transition to end of life care over the course of the study, we seek to explore their experiences with this transition and if the FFM program improves patient and caregiver psychospiritual adjustment at this crucial timepoint. The knowledge gained from this randomized controlled trial will advance the science of behavioral medicine, and, ultimately, inform the clinical care of a vulnerable and understudied patient-caregiver population. intervention in English or Spanish based on participant preference.