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NIH Research Projects · FY 2026 · 2022-03

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the USA. An estimated 53,200 people will die from CRC in 2020. Another disturbing fact is that the rates of incidence and mortality from young adults have been increasing since the 1990s for unknown reasons. The mortality of CRC patients is mainly attributed to tumor metastasis, a complex, multistep process. This proposal aims to uncover the underlying mechanisms that control CRC metastasis and the key pathways and environmental factors that contribute to it. One possible factor is sugar-sweetened beverages (SSBs), the consumption of which has dramatically increased in the past four decades. SSBs are any liquids that are sweetened with refined sugars, such as sucrose and high-fructose corn syrup (HFCS), both of which consist of glucose and fructose in a ratio of approximately 1:1. More than half of the adults in the USA consume SSBs on a given day. Epidemiological studies have shown a positive correlation between SSBs consumption and the recurrence of and mortality from advanced-stage CRC. However, it is still unclear whether SSB consumption can directly affect metastasis on advanced-stage CRC and, therefore mortality, independently of obesity. This question is critical because certain CRC patients regularly consume SSBs. Moreover, oncologists and nutritionists in the US recommend that their CRC patients drink energy drinks and concentrated juices—which are, in fact, SSBs with high amounts of added sugar—because these SSBs also contain calories, protein, and/or micronutrients. Our preliminary data show that a medium containing both glucose and fructose (hereafter, the HFCS condition) increased the migration and invasion of CRC cell lines (hereafter, CRC motility), which was correlated with an increased level of sorbitol. The deletion of a NAD(H)-dependent sorbitol dehydrogenase (SORD) decreased CRC motility and sorbitol level in the HFCS condition. Our xenograft mice models also showed that HFCS treatment increased CRC metastasis (local invasion and liver metastasis), and SORD was necessary for the metastasis. Notably, the loss of SORD also inhibited CRC motility in the high-glucose condition, in which glucose converts to fructose via the polyol pathway. It is known that chronic consumption of SSBs causes a high concentration of glucose in the blood. Thus, we hypothesize that SORD plays a key role in accelerating CRC metastasis by interacting with sugars. We propose three aims: Aim 1: Determine how SORD accelerates CRC motility in the HFCS condition. Aim 2: Determine how SORD increases CRC motility in the high-glucose condition. Aim 3: Understand the role of SORD in CRC metastasis. Successful completion of these proposed studies will characterize SORD as a novel therapeutic target for CRC metastasis. Further, our results will identify metabolic pathways and metabolites that may serve as new biomarkers or targets for preventing CRC metastasis. Finally, our study will highlight the danger of HFCS consumption, especially in the form of SSBs, which may accelerate CRC metastasis. This will change current clinical practices and dietary guidelines for CRC patients, significantly reducing CRC mortality.

NIH Research Projects · FY 2026 · 2022-03

Abstract This multi-site study aims to establish best practice recommendations and clinical value of a new consensus protocol for dynamic susceptibility contrast (DSC) MRI. DSC-MRI is one of the most widely used physiologic imaging techniques in neuro-oncology, with a reported use in 85% of all routine brain tumor scans at sites across the US and Europe. DSC-MRI measures of relative cerebral blood volume (rCBV) can differentiate glioma grades, identify tumor components in non-enhancing glioma, distinguish tumor recurrence from post-treatment effects (including pseudoprogression and radiation necrosis) and predict treatment response and patient survival after targeted therapy. Despite this demonstrated value, translation of consistent clinical DSC-MRI guidelines across multiple institutions remains challenging owing to variability in acquisition and post-processing protocols. To improve the accuracy, repeatability, and multi-site consistency of DSC-MRI, the team of investigators on this proposal identified and validated a DSC-MRI protocol that has since become a consensus recommendation for patients with glioma. Accordingly, we are well positioned to address critical gaps in this field that, once solved, will accelerate the widespread adoption of the consensus protocol and direct its use in clinical practice. We first aim to establish best practices for the pre- and post-processing methods for DSC-MRI. We will then leverage our expertise with image-localized biopsies and matched image-tissue correlations to establish guidelines for interpreting rCBV maps generated with the consensus protocol, specifically applied to the differentiation of low- from high-grade glioma, and progression of high-grade glioma from post-treatment effects. Finally, we aim to establish the clinical value added of the consensus DSC-MRI protocol for impacting brain tumor patient management. These studies will address key practical and clinical unmet needs that will directly facilitate the future use of DSC-MRI in glioma patients, thereby promoting widespread adoption of the consensus DSC-MRI protocol.

NIH Research Projects · FY 2026 · 2022-03

ABSTRACT In 2021, approximately 76,080 individuals in the United States will be diagnosed with kidney cancer, with renal cell carcinoma (RCC) accounting for >90% of all cases. Approximately 25% of patients with RCC present with metastasis at the time of initial diagnosis and up to 20-30% of patients develop recurrent disease after nephrectomy. Immunotherapy is a new standard-of-care option for the first-line treatment of intermediate-risk or poor-risk patients with advanced RCC. However, the immunotherapy outcomes are heterogeneous, with some patients achieving a complete remission, and others having no benefit at all. Therefore, it is important to identify modifiable factors and biomarkers that could help with patient selection and risk stratification, and to detect patients who will experience treatment-related adverse events and disease progression. Growing evidence supports that the gut microbiome contributes to cancer therapy toxicities and may modulate response to cancer therapy. Therefore, we propose to 1) identify and validate pre-treatment gut microbiome profiles, specific bacterial species, and bacterial functional pathways associated with circulating T-cell activation, cancer immunotherapy response, adverse events, and progression-free survival among patients with advanced RCC; 2) identify and validate cancer immunotherapy-associated changes in the gut microbiome profiles and bacterial functional pathways, and their association with circulating T-cell activation, cancer immunotherapy response, adverse events, and progression-free survival among patients with advanced RCC, 3) explore the role of bacterial metabolites and related biomarkers in cancer immunotherapy response, and 4) obtain preliminary data on the gut microbiome profiles and bacterial functional pathways and outcomes by sex and race. The study results will contribute considerably to our understanding of the role of the gut microbiome in cancer immunotherapy response, efficacy, and adverse events, and will be relevant not only for advanced RCC patients, but also for a larger group of cancer patients treated with cancer immunotherapy, which is considered to be most promising recent development in cancer therapy.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a 5-year overall survival (OS) rate of 7% for metastatic disease and less than 20% for locally advanced disease. Benefit from current therapies including chemoradiation and surgery is often modest and transient. The significant challenge in the field is how to turn immunologically cold PDAC into hot tumors that respond to immune checkpoint blockade (ICB) therapy. We recently showed that irreversible electroporation (IRE), a tumor ablative technique currently used in the clinics, significantly sensitized PDAC to anti-PD-1 ICB, leading to long-term survival in ~40% mice in an aggressive orthotopic PDAC model. The remarkable anti-PDAC activity was attributed to efficient induction of immunogenic cell death and stromal perturbation in favor of tumor infiltration of CTLs. As part of an effort to define approaches to further enhance the efficacy of IRE + anti-PD-1 combination against PDAC, we uncovered novel immune suppressive mechanism through time-of-flight mass cytometry (CyTOF) immune profiling and single cell RNAseq of PDACs, which showed significant infiltration of CXCR2-expressing myeloid suppressive cells (MDSCs). Furthermore, we found that IRE collapsed glycolysis and oxidative phosphorylation (OxPhos) while upregulated glutaminase and glutamate, suggesting glutaminolysis as a compensatory mechanism to satisfy energy and biosynthesis needs of IRE-treated cells. These data, taken together with the known critical role of MDSCs and heightened glutamine metabolism in immune suppression, the findings by others that the anti-diabetic drugs metformin and phenformin fundamentally change the tumor metabolic program to sensitize tumors to ICB therapy, and our preliminary findings that both IRE and Re-Phen, a newly developed analogue of phenformin, downregulated the OxPhos pathway while displaying an opposite effect on glutamate production, lead us to hypothesize that attenuation of the immunosuppressive TME by depletion of MDSCs or suppression of glutaminolysis by Re-Phen potentiates IRE + ICB to further prolong overall survival and increase the rate of durable response. To test our hypothesis, we will pursue the following specific aims: 1) To identify immunosuppressive factors associated with long-term versus short-term response to IRE + ICB. We will use CyTOF immune profiling, scRNAseq, and cytokine array analyses to fully characterize the impact of IRE in the presence and absence of anti-PD-1 on the immunosuppressive TME. 2) To determine the extent to which therapies directed at MDSCs potentiate IRE + ICB. 3) To determine the extent to which disruption of the metabolic program by theranostic agent Re-Phen potentiates IRE + ICB. The findings from this project are expected to reveal previously undefined roles of MDSCs and deregulated metabolic programming in immune suppression in the context of combined IRE + ICB therapy. Success of this project will have exceptional impact because it will offer a potentially effective therapy for PDAC.

NIH Research Projects · FY 2026 · 2022-01

Glioblastoma (GBM) is a malignant brain cancer that is resistant to all treatment modalities. This resistance is due, in large part, to a population of high invasive and low proliferative cancer cells that elude surgical resection and are refractory to chemotherapy and radiation. While a great deal is known about oncogenes, tumor suppressors, and other pathways that promote GBM cell proliferation, we understand relatively little about mechanisms that drive GBM cell invasion in the brain microenvironment. Therefore, the PI's group performed genetic and biochemical screens to identify adhesion and signaling factors that regulate invasive cell growth in GBM. These efforts identified the non-receptor protein tyrosine phosphatase PTP-PEST/PTPN12 as a critical signaling effector in invasive GBM cells. Here, we present a significant amount of supporting data showing that PTP-PEST promotes GBM cell invasion by regulating the stability of key focal adhesion signaling proteins, particularly Crk-associated substrate (p130Cas). In particular, we have discovered that PTP-PEST in focal adhesions mediates interactions between p130Cas and valosin containing protein (Vcp), a ubiquitin-dependent segregase and key component of the ubiquitin proteasome system. These findings have led to our working hypothesis that PTP-PEST is essential for GBM cell invasion by regulating the phosphorylation- dependent ubiquitination of focal adhesion protein substrates. To test this hypothesis, we will (1) characterize protein domains and motifs that mediate interactions between PTP-PEST, p130Cas, and Vcp, as well as determine how these interactions modulate focal adhesion protein stability in GBM cells; (2) identify PTP-PEST-generated phosphodegron sequences in p130Cas and determine how they regulate focal adhesion dynamics by recruiting Vcp and facilitating p130Cas degradation by the proteasome; (3) genetically mutate Vcp at specific sites required for the phosphorylation-dependent ubiquitination of p130Cas and analyze cell invasion using three-dimensional culture systems and pre-clinical mouse models of GBM; and (4) quantify levels and spatial patterns of PTP-PEST signaling via p130Cas and Vcp in human GBM samples and primary cancer cell culture systems. We will correlate these data with patient survival as well as response to therapies such as temozolomide and bevacizumab. Collectively, these experiments will not only elucidate signaling pathways that control the GBM cell invasive state, but may lead to new strategies to target invasive cells and block tumor progression.

NIH Research Projects · FY 2025 · 2022-01

ABSTRACT We respond to Notice of Special Interest: Research in the Emergency Setting, and will build upon the Compre- hensive Oncologic Emergency Research Network (CONCERN). For many cancer patients, Immune Check- point Inhibitors (ICPIs) can be life-saving. However, the immune-related adverse events (irAEs) from ICPIs can be debilitating, and can quickly become severe, or even be fatal. Often irAEs will precipitate visits to the emer- gency department (ED). Therefore, early recognition and the decision to admit, observe or discharge these pa- tients from the ED can be key to a cancer patient’s morbidity and mortality. ED clinicians typically make their decision for disposition (admit, observe or discharge) within 2-6 hours from their patient’s ED presentation. However, irAEs are particularly challenging in the ED because of atypical presentations, the absence of classic symptoms, delayed availability of diagnostic tests during the ED encounter, and the fast pace in the ED setting. At present, there is no single sufficiently large ED data source with clinical, biological, laboratory, and imaging data that will allow for the development of a tool that will guide early recognition and appropriate ED dispo- sition of patients with potential irAEs. Therefore, we propose to capitalize on a multi-site collaboration among 4 CONCERN EDs (MD Anderson Cancer Center, Ohio State University, Northwestern University and University of California San Diego) in different States, to achieve the following aims: 1) To develop a probability model [the Immune-related Emergency Disposition Index (IrEDi)] to risk stratify ED patients on ICPIs for ED disposition. We will leverage our existing data (n=~2000) of unique ED patients who received ICPIs within 3 months of ED presentation at the 4 research sites. We hypothesize that host immune response underlie the development of irAEs and that inflammation/immune biomarkers available during the ED encounter will im- prove the prediction of Hospital admission; Observation; or Discharge, along with traditional factors, i.e. epide- miological factors (age, race/ethnicity), biological factors (sex, BMI), cancer (type, stage/metastases) and treat- ment-related variables (class of ICPI, monotherapy versus combination, dose/duration), and clinical status (comorbidities, preexisting autoimmune diseases, vital signs, laboratory results, imaging study results); and 2) To validate IrEDi using prospective data and determine the predictive validity of irEDi. We will conduct a pro- spective cohort study of ED cancer patients who had received ICPIs, recruiting 1500 total from 4 sites over a 3- year period. A common limitation of ED studies is that patients may receive care from multiple EDs. Thus, we will conduct follow-up calls in 30 days to assess ED revisits or hospitalization. We hypothesize that IrEDi devel- oped in aim 1 will have high sensitivity (≥90%) and high specificity (≥90%) for predicting appropriate ED dispo- sition (hospital admission, observation, or discharge). If our aims are achieved, the IrEDi will be the first risk stratification tool derived from a large racial/ethnically and geographically diverse population of cancer patients. Our future goal is to validate irEDi in general EDs to improve emergency care of cancer patients on ICPIs.

NIH Research Projects · FY 2026 · 2021-12

Abstract The central nervous system (CNS), comprised of the brain, spinal cord and retina, is the most vascularized organ system in the human body. Neurons and glial cells closely contact blood vessels and communicate with vascular endothelial cells and pericytes to control normal CNS development and physiology. Blood vessel dysfunction occurs in multiple CNS diseases, including developmental brain disorders, retinal deficits, and age-related neurodegeneration. We understand surprisingly little about mechanisms that regulate normal CNS vascular development and physiology or how these events go awry during disease pathogenesis. To characterize new and potentially targetable factors that control blood vessel morphogenesis in the developing CNS, we queried open-source databases to identify genes with putative roles in vascular endothelial cell growth, differentiation and sprouting. These efforts have led to the current project focused on Prnd, a member of the prion gene family that is expressed in the CNS vascular endothelium. We present a substantial amount of data that bolster our working hypothesis that glycophosphatidylinositol (GPI)-linked Prnd activates endothelial cell signaling pathways to regulate angiogenesis and blood vessel permeability in the brain and retina. Furthermore, we propose that abnormal Prnd expression and function contributes to brain and retinal blood vessel pathologies. To test our hypotheses, we will (1) utilize biochemical strategies and high-resolution cell imaging methods to analyze Prnd-dependent signaling pathways that promote endothelial cell differentiation, growth and migration; (2) analyze roles for Prnd in developmental and pathological CNS angiogenesis; and (3) identify and characterize cues in the CNS microenvironment that promote Prnd expression and functions in angiogenic endothelial cells. In summary, these experiments will reveal novel functions for Prnd in regulating CNS blood vessel morphogenesis and may identify new targets for therapeutically inhibiting pathological angiogenesis in the CNS.

NIH Research Projects · FY 2025 · 2021-12

Summary [Print using "Actual size" (Acrobat) or "Scale: 100%" (Preview)] for proper font size (11)] Glioblastoma (GBM), a lethal human brain tumor, is made up of multiple molecular subtypes, suggesting that therapy could be targeted to particular subtypes. Yet all newly diagnosed GBM patients are treated with a similar therapeutic regimen, which results in overall poor patient outcomes. GBM tumors contain stem-like cells (GSCs) that contribute to tumor initiation, growth, and resistance to standard-of-care temozolomide (TMZ) and ionizing radiation (IR). Thus, GSCs present an excellent system in which to study the biology of GBM and develop and evaluate targeted therapeutic approaches to GBM. Our long-term goal is to develop mechanism- based therapeutic approaches to significantly advance the care of GBM patients. Our laboratory discovered the transcriptional repressor REST as a stem cell promoter, and thus a critical oncogenic regulator, in medulloblastoma. We and others discovered that REST also regulates oncogenesis in GBM and that tumors with GSCs expressing high levels of REST (HR-GSCs) are molecularly and biologically distinct from tumors with GSCs expressing low levels of REST (LR-GSCs). Further, GBM patients with an HR tumor transcriptome signature have shorter survival than patients with an LR tumor signature, similar to our results with HR-GSC versus LR-GSC tumors in mouse models. These studies have suggested that REST is a potential therapeutic target in HR-GBM tumors. Yet there is no REST-specific therapeutic approach for stratified HR-GSC tumors. The goal of this project is to determine therapeutic approaches for HR-GSC tumors using mouse intracranial tumor models. First, information obtained here will determine whether targeting HR-GBM tumors with REST- specific inhibitor, REST-VP16, is a valuable therapeutic approach for HR-GBM. Second, Information obtained here will determine whether targeting HR-GBM tumors with REST downstream miR targets via exosome- mediated delivery would promote therapeutic approaches for HR-GBM. Third, we will determine the underlying regulatory network changes and transcriptome signatures in selected tumors with and without treatment conditions. Such regulatory networks will provide information about changes in treatment-dependent downstream pathways and targets. The transcriptome signatures could be useful to measure treatment progression in a clinical setting. Fourth, we will determine the homing mechanism of Exosome-mediated delivery of miRs to HR-tumors. Information obtained here will aid in designing exosomes with enhanced homing capabilities to HR-GBM. Thus, the project has the potential to produce a novel, mechanism-based therapeutic approach for the HR-GBM subtype, for which such approaches are limited.

NIH Research Projects · FY 2025 · 2021-09

* * * * PROJECT SUMMARY * * * * Abstract: Technical batch effects pose a fundamental challenge to quality control and reproducibility of even single-laboratory research projects, but the possibilities for serious error are greatly magnified in complex, multi- institutional enterprises such as the cancer molecular profiling projects being undertaken by the NCI Center for Cancer Genomics (CCG). To aid in detection, quantitation, interpretation, and (when appropriate) correction for technical batch effects in such data, we have developed the MBatch software system. MBatch proved indispensable for quality-control “surveillance” of data in The Cancer Genome Atlas (TCGA) and ongoing CCG projects. But detecting and quantitating batch effects (or trend effects or statistical outliers) are just the first steps in a process. The next steps involve detective work in collaboration with those who generated the data, drawing upon expertise in integrative analysis across data types, pathways, and systems-level biology. That detective work usually succeeds in diagnosing the cause of a batch effect as technical or biological. If technical, then computational methods to ameliorate the batch effect can be applied (judiciously). The primary aim of the proposed Genome Data Analysis Center (GDAC) is to continue to translate that successful quality-control model to the CCG’s other current and future large-scale molecular profiling projects We will be ready to do that on Day 1. We will continue to enhance and extend the power of MBatch and incorporate a number of innovative new algorithms, tools, and interactive visualizations into it (OmicPioneer-sc, MutBatch, CarDEC, and CorNet). Evaluating and correcting batch effects is a complex process, so we will collaborate with other GDACs and data generating centers to determine the influence of artifacts on any analysis results they produce. The second aim is to contribute and enhance additional competencies. We are prepared to (i) provide integrated cluster solutions to segregate cases into biologically relevant groups; (ii) provide tools and expertise for high-level visualization of omic data (including single-cell data); and (iii) analyze RPPA proteomic data from the subset of projects that generate such data. Our final aim is to communicate results and distribute corrected data back to other network members, project stakeholders, and the scientific community. We bring a number of assets to the table, including multidisciplinary expertise in bioinformatics, biostatistics, software engineering, cancer biology and cancer medicine; PIs with a combined 40+ years of experience in molecular profiling of cancers; expertise gained in 10 years of doing the batch effects surveillance for TCGA and other CCG projects; a highly professional software engineering team with a track record of producing high-end bioinformatics tools; extensive computing resources, including one of the most powerful academic clusters in the world; and close working relationships with first-class basic, translational, and clinical researchers across MD Anderson, one of the foremost cancer centers in the U.S. The bottom-line mission of the GDAC will be to aid the research community’s effort to understand cancer and to prevent, detect, diagnose, and treat it more effectively.

NIH Research Projects · FY 2025 · 2021-09

Project Abstract/Summary The identification of targetable driver oncogenes, such as EGFR, has been a revolutionary advance in the treatment of lung cancer and other malignancies. In tumors with such genetic drivers, the vast majority of cells are dependent on specific oncogenes for survival (commonly referred to as oncogene “addicted” or oncogene-dependent). Effective inhibitors can dramatically improve clinical outcomes for patients but most of the times these drugs do not completely eradicate tumors leaving residual cells that are not killed by the initial treatment. These cells, termed drug-tolerant persister cells (DTPCs), may remain quiescent or clinically invisible for prolonged periods of time and eventually progress to become resistant cells with enhanced metastatic potential. My laboratory research efforts over the past decade have been focused, in large part, on elucidating mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) and developing strategies to overcome resistance, efforts that have been translated into new drugs and combinations into the clinic (e.g. poziotinib, Axl+EGFR inhibitors, VEGF+EGFR inhibitors, etc). Dr. Monique Nilsson has been leading some of these studies including the identification of stress hormones and IL-6 as drivers of EGFR TKI resistance and the characterization of the YAP/FOXM1 axis as a critical pathway in the development of EGFR TKI resistance. She also identified novel targets for EGFR mutant TKI resistant tumors such as aurora kinases. More recently she was able to demonstrate pre-clinically the mechanisms underlying the VEGF dependency of EGFR mutant lung tumors. Importantly, Dr. Nilsson’s studies led to several new clinical trials that are currently ongoing in our institution. In order to prevent the emergence of drug resistance of EGFR mutant tumors, Dr. Nilsson’s goal in this proposal is to identify novel treatment strategies to eliminate DTPCs in the early stages. There is little known about the signaling pathways and potential therapeutic vulnerabilities of DTPCs and we aimed to perform a deep analysis at the single cell level of a large collection of preclinical models that my laboratory and Dr. Nilsson have developed during the past years. We believe that these studies will also help to advance in the understanding of other molecularly defined oncogene-driven lung subtypes and other solid tumors.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Lynch Syndrome (LS) is the most common cause of hereditary colorectal cancer (CRC), affecting >1 million Americans. LS is caused by germline mutations in the DNA mismatch repair (MMR) genes. Normal colorectal epithelial cells in LS patients become MMR deficient upon acquisition of a ‘second’ somatic hit in the alternative allele of the same MMR gene that harbors the germline mutation, thus triggering the accumulation of hundreds to thousands of base-to-base mismatches and insertion-deletion mutations (indels) in microsatellite sequences. These mutations generate frameshift peptides (FSP) that become neoantigens (neoAg) and stimulate the adaptive immune system. We have reported that LS pre-cancers are immune activated and present strikingly high levels of expression of adaptive immune genes. Therefore, LS patients constitute a well-defined and prevalent population that has the potential to benefit from immune-interception strategies to prevent CRC. We have acquired a substantial amount of genomic data from LS colorectal pre-cancers and tumors to catalog and to identify the most frequent recurrent neoAg present in these lesions. In addition, we have been studying chemopreventive strategies that could augment the immune response and observed increased activation of the resident immune cells in the colorectal mucosa upon exposure to naproxen, a non-steroidal anti-inflammatory drug (NSAID), from our biomarker analysis of our NCI-sponsored Phase Ib clinical in LS patients. Furthermore, we have performed a co-clinical trial in a humanized LS mouse model that has observed that peptide vaccination with neoAg is highly effective in preventing LS CRC with the activity that is further enhanced by its combination with naproxen, thus laying the foundations for this grant proposal. The central hypothesis of this proposal is that naproxen is an immune-modulator that activates resident immune cells in the colorectal mucosa, and these will increase the recognition of NeoAg and activation of resident T-cells eliciting tumor cell killing. To explore this hypothesis, we propose three specific aims: 1. To characterize the immune cell types that are regulated after the administration of chemopreventive naproxen and aspirin in LS patients using single-cell genomics and imaging mass cytometry within a randomized phase II clinical trial; 2. To assess the immunogenicity of candidate shared neoAg identified LS patients pre-cancers and tumors for personalized immunoprevention using tetramer bound to magnetic beads in ELISpots, Tetramer stain, and cytotoxicity assays of co-cultured patient-derived organoids and autologous CD8+ T cells; 3. To profile the T cell Receptor (TCR) of neoantigen-specific CD8+ T cell clones for tracking tumor immunogenicity in LS patients. The proposed research will significantly impact the field by developing a combination of a peptide vaccination and an NSAID for immune-interception in hereditary cancers for the first time. The proposal is highly innovative by combining a chemoprevention trial using imaging mass cytometry, single-cell genomics, and systems biology to assess trial endpoints, and using tetramers bound to magnetic beads for positive selections of clones in immunology experiments.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Lung cancer is the leading cause of cancer-related mortality in the United States and worldwide. The efficacy of immune checkpoint inhibitors (ICIs) in patients with metastatic non-small lung cancer (NSCLC) prompted the clinical investigation of these agents in the early-stage operable setting. Several theoretical advantages exist when we administer ICIs before surgery (neoadjuvant) rather than postoperatively (adjuvant), including an opportunity to address micrometastases early in the course of treatment, and may impart immunologic memory to prevent tumor recurrence. Indeed, the results from our preclinical models of resectable NSCLC demonstrated that combined neoadjuvant ICIs resulted in fewer lung metastases, greater immune infiltration of tumors, and longer overall survival compared with mice treated with monotherapy or adjuvant combined ICIs. Those results informed the first reported randomized phase 2 study testing neoadjuvant ICI combinations in patients with resectable NSCLC using major pathologic response (MPR, ≤10% viable tumor) as a surrogate endpoint for clinical efficacy (NEOSTAR, PI: Cascone). Neoadjuvant chemoimmunotherapy has been shown to be highly promising for resectable NSCLC, and is now being tested in one of the phase 3 randomized studies in patients with operable NSCLC (CheckMate-77T, Lead PI: Cascone). However, a major shortcoming of all of the neoadjuvant trials, is that no validated biomarker exists that can be used to stratify patients. Consequently, many of these patients on these trials do not achieve an MPR at surgery, indicating that limited benefit may be gained from induction ICIs. By delaying surgery in patients who may not benefit, the risks of disease progression and of eliminating a chance to offer potentially curative surgery upfront occur. The ongoing evaluation of molecular biomarkers of clinical benefit to ICIs has proved disappointing as evidenced by the significant intertrial variability, possibly related to intratumor heterogeneity. By contrast, radiologic imaging provides a holistic view of tumor characteristics and interactions with the adjacent tissue. Built on our promising preliminary data, we propose to spearhead radiographic and radiogenomics strategies to address this unmet clinical need. We hypothesize that imaging phenotypes reflect tumor microenvironment, and quantitative imaging phenotyping will shed light on our understanding of the mechanisms of response to ICIs and yield surrogates of clinical efficacy. We will leverage the parallel assessment of well-curated data from unique clinical trials and immunocompetent mouse models to develop new imaging biomarkers and validate their clinical and biological relevance. The strength of this proposal is our interdisciplinary team with the requisite expertise and ability to treat patients, obtain and analyze high- quality, longitudinal imaging and biospecimens and rapidly evaluate putative imaging biomarkers for therapeutic response and clinical outcomes. The advent of imaging biomarkers will: 1) identify those patients most likely to benefit from neoadjuvant ICIs, 2) maximize the clinical effectiveness, and 3) lead to the development of new therapies that will improve outcomes for a greater number of patients with resectable NSCLC.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract Most clinical trial designs use \one-size- ts-all" rules for treatment assignment and evaluation based on models that ignore patient heterogeneity. This is disconnected from medical practice, where physicians use each patient's diagnosis and prognostic variables to make personalized, precision medicine treatment decisions. Modern precision medicine exploits biotechnologies such as proteomics, genomics, gene sequencing, mass spectrometry, or cytometry methods that evaluate multiple cell surface markers. These generate vectors of biomarkers that may be used to re ne existing disease subgroup de nitions, construct new disease classi cations, and formulate clinical trial designs and statistical rules for personalized/precision treatment assignment. In oncology and other disease areas, there is rapidly increasing development of new biotherapies, including cell therapies, immunotherapies, and targeted molecular agents. A biotherapy may be administered once or in multiple cycles; used in combination with conventional treatments such as cytotoxic chemotherapy, radiation, or surgery; and often generates complex outcomes, such as repeatedly evaluated tumor status, multiple biological variables, and occurrence times of both early and late onset toxicities. This complicates the de nitions of \response" and \toxicity," and produces multidimensional treatment e ects that may di er between subgroups. An example is a phase I-II trial to optimize subgroup-speci c doses of donor derived natural killer (NK) cells for treating B-cell hematologic malignancies, where donated NK cells are engineered using chimeric antigen receptors to enhance their cancer killing e ects, then expanded using growth factors to obtain cell doses large enough for therapeutic use. Subgroups may be de ned using disease subtypes and prognostic variables. Co-primary outcomes may include ordinal disease status, including complete or partial remission, stable disease, or disease progression, evaluated either once or at monthly intervals; time to severe NK cell-related toxicity, such as cytokine release syndrome; and a binary indicator of 100-day survival. Considering (biotherapy, dose, administration schedule) a treatment regime, a clinical trial of one or more new biotherapies may include a subgroup-speci c risk-bene t tradeo based dose or schedule optimization for each biotherapy, randomization among regimes restricted to achieve balance within subgroups, and subgroup-speci c group sequential rules to select superior regimes or drop unsafe or ine ective regimes. The proposed research will construct robust Bayesian regression models for regime-outcome e ects that account for patient heterogeneity, including possible regime-subgroup interactions. These will be the basis for sequential decision making and regime assignment, and they may include latent variables to adaptively combine subgroups with similar regime-outcome e ects. Each clinical trial design will be tailored to address a combination of these goals in speci c biotherapy settings. For each design, user-friendly computer software will be provided, including programs for trial simulation to establish design operating characteristics, trial conduct, and use by practicing physicians to choose optimal regimes for their patients. The overarching goal of the proposed research is to develop and identify optimal personalized biotherapy regimes, spanning a variety of di erent diseases and clinical settings, for greater anti-disease e ects, increased safety, and improved survival.

NIH Research Projects · FY 2026 · 2021-09

Project Summary Alzheimer’s disease (AD) is a major health concern defined by pathologic changes in the brain that produce altered behavior and cognitive function. There is a need for primate models of AD because they naturally recapitulate some neuropathological features of AD with advanced age whereas other model organisms (i.e., rodents) do not. For instance, while amyloid-beta (Aβ) deposition occurs in a few mammals, tau-positive neurofibrillary tangles have only been identified in a very limited nonhuman species studied to date. Additionally, elderly nonhuman primates, develop cerebral amyloid angiopathy (CAA), a neurovascular condition found in almost 100% of AD patients and associated with cognitive decline. Here, we are proposing to further develop squirrel monkeys as a model species for current and future studies on the biology of aging and AD research. In the R21 component, we propose to train a cohort of group living squirrel monkeys on the use of an automated cognitive testing system (ACTS) that is designed to assess a variety of cognitive functions including learning, memory and executive control. Creating large cohorts of squirrel monkeys trained on the ACTS system will provide animals with established cognitive phenotypes for use in preclinical studies and allow for examining of their association with potential age-related differences in neuroanatomical, neuropathological and biomarker data. In the R33 component of the proposed studies, we will test for associations between age-related changes in cognition and measures of blood/CSF biomarkers, neural organization and integrity and neuropathology. Additionally, we will test for the effect of ACTS training on aged related changes in neuroanatomy, neuropathology and AD-related biomarkers. Specifically, during Years 3 to 5, we will obtain magnetic resonance images (MRI) and biological samples from 40 elderly and geriatric monkeys trained on the ACTS system. With this cohort, 20 monkeys will receive continued cognitive training (ACTS+) during year 3 to 5 while the remaining 20 individuals will not receive training on any new cognition asks (ACTS-). In a subset of ACTS+ and ACTS- monkeys, we will obtain postmortem measures of neuropathology. In one set of analyses within the ACTS cohort, we will test for longitudinal changes in cognition and their association with variation in (1) neural organization and integrity quantified form MRI scans and (2) several key biomarkers of AD-related neuropathology. Additionally, to examine whether cognitive stimulation slows down the normal brain aging process, we will compare age-related changes in cognition and the brain between the ACTS+ and ACTS- monkeys. The proposed studies, in their entirety, will fill an important gap in our knowledge about the comparative biology of aging and disease in squirrel monkeys and provide critical translational insight into how those processes contribute to the progression of CAA and AD in humans. This information will provide crucial direction for future AD therapeutic trials using nonhuman primate models and enhance the potential of successful translation to patients.

NIH Research Projects · FY 2026 · 2021-09

ABSTRACT Over 40,000 U.S. women will die of breast cancer each year. Screening mammography saves lives but also results in potential harms. Personalized screening regimens tailored to a woman's individual risk can both improve early detection of lethal cancers through more intensive regimens for high-risk women, and reduce over-screening and over-treatment of low-risk women. However, the current clinical breast cancer risk prediction models are insufficiently accurate for discriminating high-risk and low-risk women. New radiomic deep learning algorithms, which automatically mine troves of breast tissue features from a woman's screening mammogram to predict her future cancer risk, have enormous potential to transform breast cancer screening, but have not been independently validated. New polygenic risk scores (PRS) for breast cancer also show promise for improving risk prediction, although still costly to implement on a population scale. We propose to examine whether adding radiomic and genomic risk scores can significantly improve current clinical risk prediction models in a large, diverse population-based cohort of 178K women enrolled in Kaiser Permanente's Research Program on Genes, Environment and Health (RPGEH) who were screened with 2D full-field digital mammography (FFDM). We also propose to extend the best performing radiomic deep learning algorithms to diverse screening mammography systems utilized in two large health care settings in California and New York, including a cohort of 50K women screened with 3D digital breast tomosynthesis (DBT) in the Mount Sinai Health System (MSHS). The specific aims are to: (1) Evaluate the performance of radiomic deep learning breast cancer risk prediction models, estimate their associations with 5-year and 10-year breast cancer risk, and determine the extent to which the associations are independent of known clinical risk factors; (2) Determine whether radiomic and genomic risk scores independently predict breast cancer risk, and explore potential differences by race/ethnicity and other clinical risk factors; and (3) Transfer the best radiomic deep learning algorithm(s) from 2D FFDM to 3D tomosynthesis. The proposed research will fill essential knowledge gaps needed to realize the potential of radiomics and genomics by validating new radiomic algorithms, quantifying the improvements in model performance above traditional risk factor models and new polygenic risk scores, exploring differences by race/ethnicity, and extending the best radiomic tools to diverse mammography systems utilized in two large multi-ethnic health care settings. 1

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Studies in healthy individuals have shown that acquired DNA mutations are surprisingly common in normal- appearing tissues. Clonal hematopoiesis (CH) mutations are acquired mutations in the blood present in the majority of older individuals. CH mutations are also frequent among individuals with solid malignancy; are associated with past receipt of oncologic therapy, in particular radiation therapy; and are associated with worse overall survival. This project will address key knowledge gaps by investigating whether CH mutations are associated with adverse oncologic treatment toxicity and outcomes in patients with solid malignancy (Aim 1), how oncologic therapy impacts the frequency and characteristics of CH mutations in patients with solid malignancy (Aim 2), and whether there are actionable intermediary biomarkers of adverse outcomes among individuals with CH mutations (Aim 3). We will address these aims by undertaking targeted DNA sequencing using existing biospecimens from prospective cohorts of patients undergoing oncologic therapy for solid malignancy (Aims 1 and 2) and using existing clinical and genetic data from a large prospective biobank (Aim 3). Our approach leverages unique patient resources and novel methodology. Completion of these aims will provide key insight regarding the importance of CH mutations in patients with solid malignancy and guide future investigations to determine whether knowledge of CH mutations can improve cancer care. The applicant, Dr. Kevin T. Nead, MD, MPhil, is an assistant professor in epidemiology and radiation oncology at MD Anderson Cancer Center. This study is in line with Dr. Nead’s long-term career goal to become an independent, R01 funded investigator studying the use of inherited and acquired patient-level genetic data to improve cancer prevention and treatment paradigms. The overall career development objectives of Dr. Nead’s proposal are to 1) expand his knowledge and expertise in genetics/genomics, bioinformatics, somatic mutation analysis, and study design; 2) complete an interdisciplinary research plan and establish a published body of work on acquired mutations in normal tissues and oncology care; and 3) build a platform for his successful transition to an independent investigator. During the award period Dr. Nead will devote at least 75% of his effort to the proposed project and career development activities. Dr. Paul Scheet, the candidate’s primary mentor, is chair of the Department of Epidemiology, an international expert in the study of acquired mutations in normal tissue, and has received multiple awards for the quality of his mentorship and teaching. Dr. Nead will also benefit from the rich training environment and outstanding resources and mentorship available at MD Anderson Cancer Center for the proposed project. Completion of the proposed research and career development plan will give Dr. Nead the necessary knowledge and skills to transition to independence and to pursue an impactful career improving cancer prevention and care.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Successful reproduction through the fusion of the sperm and oocyte is essential for the perpetuation of species. In human females, oocytes complete meiosis I at birth, and enter a long period of meiotic II arrest until onset of meiotic maturation at puberty. Because the oocytes are quiescent and arrested during this period, RNAs are loaded into the developing oocytes prior to the arrest and these RNAs are critical for early embryonic development. Mechanisms that regulate generation and protection (from degradation) of maternal RNAs during the long meiotic arrest as well as mechanisms that regulate the degradation of these RNAs in the embryo remain an active area of investigation. Our work in C. elegans and work from mammalian models in the past few years turned the light on regulation of the maternal transcriptome which dictates oocyte quality and impacts progeny development. Specifically, we uncovered a direct link between RAS/ERK growth factor signaling and the small RNA biogenesis factors Dicer1, Drosha and DIS3 (an RNA exosomal component) which regulates distinct populations of small non-coding RNAs and thus the maternal transcriptome and proteome. We propose a model wherein ERK-mediated phosphorylation of Dicer1 (and a subsequent arginine methylation of Dicer1), phosphorylation of Drosha and DIS3 results in a regulatory circuit that fine tunes the generation of small non-coding RNAs in specific subsets and regulates the maternal and zygotic transcriptome and proteome. We investigate this model in vivo during oocyte development and oocyte-to-embryo transition using a combination of live imaging, next generation sequencing, single oocyte sequencing, mass spectrometric and proteomic methods, CRISPR Cas9 genome editing and cell biological assays. We find that Dicer1, Drosha and Dis3 are phosphorylated in mammals as well. Additionally, we identified arginine methylation of Dicer1 adjacent to the phosphorylation event in mammalian cell culture system. Given their conserved role in RNA biology, reproduction and their aberrations associated with cancer onset and progression, we expect this work to have direct relevance to human biology.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Gastric cancer (GC) is one of the most common and lethal cancers worldwide. GC surgery is highly morbid, and responses to the limited array of treatment options are poor. There is hope that recent genomic sequencing data can be leveraged to develop newer, improved molecular therapies for GC, but rigorous mechanistic testing is still needed to validate the therapeutic potential of targeting any newly proposed oncogenes. Immunotherapy is an exciting new therapy that has revolutionized oncology and shows tremendous potential. In contrast to cytotoxic chemotherapies, which exhibit fractional killing invariably leading to resistance, immune cells can infiltrate almost all anatomic sites to recognize and completely eliminate malignant cells in primary and wide-spread metastatic disease. However, the immune system’s full anti-tumor killing potential can be restricted by evasive measures by the tumor and/or intrinsic immunosuppressive mechanisms that limit collateral damage to normal tissues during anti-tumor inflammatory reactions. In GC, little is known about how cancer cells evade the system, and studies investigating the molecular mechanisms underlying tumor- immune interactions have been limited by a lack of physiologically relevant in vitro human systems where state-of-the-art genetic approaches can be applied. These mechanisms are important because they would be essential to our understanding of GC tumorigenesis and the regulation of immunotherapeutic responses. Such mechanistic insight on the immune system to GC is fundamental and significant to advance and improve GC therapies. In this proposal, we utilize a series of CRISPR/Cas9 genome editing tools to create novel forward genetically engineered models of the four major GC subtypes as defined by The Cancer Genome Atlas project, including chromosomal instability (CIN), genomic stability (GS), microsatellite instability (MSI) and Epstein-Barr virus-associated (EBV) in primary 3D human gastric organoids (Aim1 and Aim2). In a parallel translational aim, we propose to use a second-generation patient-derived organoid model that allows tumor and stroma to be preserved alongside each other to study interactions between tumor cells and their veritable ecosystem of cohabiting immune cells in primary human gastric cancer (Aim 3). The overall goal of this project is to investigate how genetic alterations contribute to gastric tumorigenesis and immunotherapeutic responses using synergistic next-generation in vivo and in vitro models. Collectively, the results of this project will provide new insights into fundamental aspects of the molecular mechanisms underlying the tumor-immune interaction and enhance current GC immunotherapies. A team of expert mentors, advisors and collaborators will train Dr. Lo in new methods that are critical to the success of this research. The combination of mentoring support, skills, and data obtained in the K99 phase will provide Dr. Lo a springboard to achieving independence as an investigator in the R00 phase and beyond.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Oncogenic KRASG12C (KG12C) mutations underpin the development of ~13% of non-squamous non-small cell lung cancer (NSCLC) and account for ~10,000 deaths annually in the U.S. The development of potent, selective and clinically active covalent inhibitors of the KG12C oncoprotein represents one of the most exciting recent advances in the field of targeted cancer therapy, yet strategies to circumvent the development of adaptive resistance and improve the durability of individual responses to KG12C inhibitors are urgently needed in order to transform clinical outcomes for patients. In addition to their tumor cell-intrinsic effects, KG12C inhibitors recondition the tumor immune microenvironment in preclinical models and synergize with anti-PD-1 therapy to promote long-term tumor regressions and immunological memory. The mechanisms that underpin KG12C inhibitor-triggered immune pathway activation in KG12C NSCLC are poorly understood. Furthermore, the optimal combinations of KG12C inhibitors with standard of care (SOC) first-line NSCLC systemic therapies including platinum-doublet chemotherapy, PD-1 inhibitor monotherapy and chemo-immunotherapy in order to maximize antitumor immunity have not been established. Furthermore, the impact of co-occurring genomic alterations in STK11/LKB1, KEAP1, TP53 and RBM10 – that shape the immune contexture of KRAS-mutant NSCLC and modify its response to PD- 1 axis blockade – on the clinical efficacy and immunological sequelae of KG12C inhibitor-based therapeutic combinations has not been systematically examined. Based on our preliminary findings and previous work we hypothesize that: 1. Induction of immunogenic cell death contributes to KG12C inhibitor-triggered immune pathway activation in KG12C NSCLC; 2. KG12C inhibitors exhibit immune-sensitizing effects that can be further enhanced with the addition of chemotherapy and/or immune checkpoint inhibitors (ICI); 3. Co-occurring genomic alterations impact both clinical and immunological responses to KG12C inhibitor mono- and combination therapy in KG12C NSCLC. In Aim 1, we will dissect mechanisms and molecular determinants of KG12C inhibitor-mediated immune sensitization in KG12C NSCLC. In Aim 2, we will evaluate synergistic KG12C inhibitor interactions with standard of care first-line systemic therapies (including platinum-doublet chemotherapy, PD-(L)1 inhibitor monotherapy and chemo-immunotherapy) in immune-competent mouse models of KG12C-mutant NSCLC. In Aim 3, we will validate treatment-induced changes in the KG12C NSCLC immune contexture at whole-tumor and single-cell resolution using clinical samples from patients with surgically resected KG12C NSCLC who were treated with neo-adjuvant AMG 510 in combination with platinum-doublet chemotherapy in an investigator-initiated phase 2 clinical trial. Clinical significance: This work will yield fresh insights into mechanisms and determinants of immune pathway activation in response to KG12C inhibitor mono- and combination therapy and will facilitate the development of personalized co-mutation-tailored combination therapeutic strategies that aim to maximize the immune- sensitizing potential and long-term clinical benefit from KG12C inhibitors.

NIH Research Projects · FY 2024 · 2021-09

With current treatment regimens, long-term remission rates for adult patients with high risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) are 15-25%. RUNX1 is the DNA- binding subunit of the core-binding factor (CBF) complex and a master-regulator transcription factor involved in hematopoiesis. Majority of mutant (mt) RUNX1 are missense, large deletions or truncation- mutations, behaving mostly as loss of function (LOF) mutations. Presence of mtRUNX1 confers relative therapy-resistance and poorer survival in patients with MDS/AML. The germline mutations and deletions in RUNX1 cause the highly penetrant (~40%) Familial Platelet Disorder with a propensity to evolve into MDS or AML. Lack of specific targeted therapy, coupled with resistance to standard therapy may account for poorer prognosis and outcome in MDS/AML expressing somatic or germline mtRUNX1. Therefore, there is an unmet need to develop novel targeted therapies for MDS/AML expressing mtRUNX1. Our preliminary studies demonstrate that knockdown of RUNX1 induces significantly more in vitro lethality in AML blasts expressing mtRUNX1 versus wild type (wt) RUNX1. Utilizing RNA-Seq signature of RUNX1 knockdown and querying the LINCS1000-CMap (Connectivity-Mapping) datasets, we identified homoharringtonine (HHT) among the top expression mimickers (EMs). Consistent with observations that presence of mtRUNX1 impairs ribosomal biogenesis (RiBi), treatment with HHT or its semisynthetic analog omacetaxine mepesuccinate (OM), which inhibit protein translation, preferentially exerted more lethality in vitro and efficacy in vivo in models of AML expressing mtRUNX1. This was associated with repression of RUNX1 and its targets, as well as attenuation of short-lived proteins including c-Myc and MCL-1. Notably, co-treatment with OM and venetoclax (Ven) induced synergistic lethality and superior in vivo efficacy in xenograft models of AML expressing mtRUNX1. Therefore, our Overarching hypothesis motivating studies proposed is that targeted combination of OM and Ven will yield high remission rates and improved survival, correlating with specific genetic and gene-expression signatures in patients with high-risk MDS/AML expressing mtRUNX1. Specific aims of studies proposed are: AIM 1: To conduct a Phase Ib/II clinical trial of co-treatment with OM and Ven in patients with high risk MDS or AML expressing mtRUNX1. AIM 2: To determine correlates of efficacy/resistance to co-treatment with OM and Ven, including genetic-lesions architecture (via NextGen and scDNA sequencing), epigenetic and gene- expression signature (via RNA-Seq, RPPA and CyTOF analyses) and impaired RiBi features in MDS/AML cells of patients enrolled on the Phase Ib/II trial. AIM 3: To determine pre-clinical efficacy of additional OM-based combinations with BET or CDK9 inhibitor, as well as with novel targeted agents directed against druggable hits nominated through an in vitro protein domain-specific CRISPR-gRNA screen.

NIH Research Projects · FY 2025 · 2021-08

Transcriptomic mechanisms underlying the immune modulating function and therapeutic efficacy of PARP inhibitors Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are approved for the treatment of ovarian cancer, as well as BRCA1 or BRCA2 (BRCA1/2) mutant breast and pancreatic cancers. Our current understanding is that one of the main mechanisms responsible for the efficacy of PARPis is through synthetic lethality, specifically in cancers with homologous recombination repair defects (‘BRCAness’). While PARP1-mediated PARylation is an essential regulator of gene transcription, it remains unknown how PARPi-induced transcriptomic changes contribute to its therapeutic efficacy. While our preclinical and clinical studies assessing PARPis in combination with programmed cell death-ligand 1 (PD-L1)/PD-1 inhibitors (PD-1/L1is) showed durable responses in different cancers, the responses were heterogeneous between patients, and surprisingly, the therapeutic benefit of this combination did not correlate with known predictive biomarkers for PARPis, such as BRCA1/2 mutations. These data suggest that the immunomodulating function of PARPis may be different from or independent of the existing ‘BRCAness’ paradigm underlying the therapeutic efficacy of PARPis. To determine the molecular mechanisms underlying the immunomodulating function of PARPis, we utilized single-cell RNA sequencing to assess the transcriptomic impact of PARPis on tumor cells and the tumor immune microenvironment. Surprisingly, we identified that PARPi-induced PARP1-trapping to DNA may upregulate B7-H3 (CD276), a key immune checkpoint protein. Based on our preliminary studies, we hypothesize that PARPis transcriptionally regulate cancer-cell intrinsic B7-H3 expression by trapping the PARP1 protein to the B7-H3 promoter region, which may serve as a key regulatory node for the immunomodulating function and therapeutic efficacy of PARPis. We will use cell and animal models, as well as patient specimens from clinical trials of PARPi-based therapies to test this hypothesis. We will test three aims: Aim 1: Determine mechanisms by which PARPis transcriptionally induce cancer cell intrinsic B7-H3 expression through PARP1-chromatin trapping. Aim 2: Determine if B7-H3 functions as a key regulatory node for the immunomodulating function and efficacy of PARPis in preclinical animal models. Aim 3: Validate PARPi-induced B7-H3 expression as a biomarker in determining the efficacy of PARPis as immunomodulating agents by analysing patient tumor and blood samples from multiple clinical trials. We believe that our proposal is highly innovative because it fills key gaps in our knowledge of the therapeutic efficacy of PARPis as immunomodulating agents through transcriptional regulation, which goes beyond the current mechanistic paradigm of PARPis. If successful, our study will have a significant impact on expanding the clinical applications of PARPis as immunomodulating agents by promoting antitumor immunity and enhancing the efficacy of immunotherapy. This may also lead to the clinical development of rational PARPi combinations with an B7-H3 inhibitor and/or a PD-1/L1i, depending on the immune characteristics of the individual patient tumor.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Cure rates for childhood cancers have improved. Unfortunately, many survivors now live with life-long side effects from treatment itself. Radiation therapy, used for brain tumors, is particularly damaging. The most serious side effect is necrosis which can result in weakness, paralysis or even death. Proton therapy is an increasingly popular radiation modality. Proton therapy reduces exposure to normal tissues and the reby may decrease the incidence of cognitive deficits following radiation. However, recent studies, including our own suggest that certain areas of proton beams may be more damaging to brain tissue than others potentially leading to higher rates of necrosis. Here we will develop high accuracy models to correlate necrosis with the physical parameters of proton beams. These models will include multi-cell type human brain “organoids” as well as rodent animal models. Using these models as well as clinical data, we will identify the physical factors of proton therapy which may lead to necrosis. This is significant in that this data may be used to design safer proton therapy treatments in which the most biologically effective portions of beams are solely placed within the tumor. This should reduce necrosis and improve disease control. In a second component of our study, we will examine the molecular mechanisms of necrosis. Rather than being simple dis-organized death, we will determine if radiation induces an orderly programmed cell death pathway. We will conduct the following aims; (1) relate the physical factors of proton beams with biological response, (2) explore the cellular and molecular mechanisms of radiation induced brain damage and (3) validate the clinical consequences of variability in the effectiveness of proton beams. The knowledge gained will quickly influence the treatment of brain tumor patients and expedite the clinical introduction of agents and approaches to combat the negative effects of radiation on the brain.

NIH Research Projects · FY 2025 · 2021-08

Our goal in this application is to test the hypothesis that neutralizing the newly identified immune-suppressive regulator fibrinogen-like protein 2 (Fgl2) in glioblastoma (GBM) following standard care chemotherapy will trigger tumor-specific resident memory T cells in the brain (bTrm cells), which allows immunological clearance of gliomas within the central nervous system and prevention of GBM recurrence. This hypothesis was raised based on our recently published papers and newly established preliminary data. In brief, we have discovered that Fgl2 is highly expressed in GBM tissues (Yan et al, JNCI, 2015) and can transform low-grade brain tumors to GBM (Latha et al, JNCI, 2018). Knockout of Fgl2 in tumor cells completely eliminates tumor progression in the brains of immune-competent mice but not in immune-deficient mice (Yan et al, Nat Commun, 2019). Our unpublished preliminary data have shown that neutralizing Fgl2 via administering T cells armed with a membrane-anchored anti-Fgl2 scFv induces bTrm cells that reject intracranial tumor cell challenge directly or after intracranial transplantation into naïve mice (see preliminary data section); the same mice are unable to reject tumors from peripheral tissue challenge. To test our central hypothesis, the following aims are proposed: Aim 1: Determine how T-aFgl2– neutralizing T-cell therapy induces bTrm cells in brains; Aim 2: Optimize the T-aFgl2–neutralizing cell therapy and develop a next-generation T-aFgl2 cell therapy for boosting safety and therapeutic efficacy. Impact: This study will yield a therapeutic candidate—an Fgl2-neutralizing cell therapy that may permanently prevent tumor recurrence—the key deadly cause of GBM patient death. Considering that Fgl2 can be detected in almost all GBMs, with most having very high levels, this candidate therapeutic will be important. This study will also further mechanistically elucidate how Fgl2-neutralizing cell therapy induces bTrm cells and how we can make additional improvements to move this therapy into the next phase. Ultimately, this novel field will transform the treatment of GBM.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY/ABSTRACT The long-term goals of this project are to define the mechanisms of resistance to immunotherapy and to develop effective therapies for patients with metastatic bladder cancer (BC). The overall objective of this proposal is to establish successful combination therapies for patients with a specific genomic subset of metastatic BC harboring homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene from the chromosome 9p21 region. Although novel immune checkpoint therapy (ICT), including anti-PD1/PD-L1, provides substantial benefits to patients with metastatic BC, response rates are usually modest at 15% to 25%. This is partly because this biologically heterogeneous cancer is still treated clinically as a uniform disease. Therefore, identification of specific genomic subtypes of BC that confer insensitivity to ICT may provide novel opportunities to improve clinical responses. We have confirmed that ~1/4 of BC contain homozygous deletion of MTAP (MTAPdef) from the 9p21region. The MTAP gene encodes for an essential enzyme to catalyze methylthioadenosine (MTA) in the salvage pathway for adenine synthesis. Tumor MTAPdef leads to both immunologic and metabolic consequences. Immunologically, tumor MTAPdef results in accumulation of its substrate MTA, which acts through the adenosine 2B receptor (A2BR) to inhibit IFN signaling and T cell function. Therefore, MTAPdef BC may foster a “cold” tumor immune microenvironment (TIME) unfavorable to ICT. Metabolically, tumor MTAPdef results in a lack of salvage pathway adenine synthesis; thus, MTAPdef BC should be very sensitive to the cytotoxic effects of anti-folate agents (e.g., pemetrexed), which effectively inhibit de novo adenine synthesis. This concept is confirmed by pre-clinical and clinical data to be presented. Importantly, our data also indicate that pemetrexed increases tumor immune cell infiltration and PD-L1 expression and thus may sensitize BC to ICT. Based on these data, we hypothesize that, by targeting the metabolic vulnerability of MTAPdef BC and directly modulating its tumor immune microenvironment, effective combination therapies can be established for MTAPdef BC. To test this hypothesis, we proposed two Specific Aims: (1) Define the immunological consequences of MTAPdef in BC; (2) Identify successful combination therapies specifically targeting MTAPdef BC. Patient-derived BC tissues, gene knockout and “rescue” mouse BC models, and samples from an IRB-approved clinical trial will be used to address these goals. At completion, we expect to establish the contribution of MTAPdef and/or loss of adjacent genes such as CDKN2A in the 9p21 region to the BC TIME. In addition, we will determine the extent of TIME modulation by pemetrexed +/- avelumab (anti-PD-L1) in relation to their therapeutic efficacy in patients with metastatic BC. Furthermore, we will define the preclinical therapeutic benefits of triple combination treatment with pemetrexed, anti-PD-L1, and A2RB inhibitor on mouse MTAPdef BC. These findings are important for the establishment of novel, biomarker-guided, highly effective combination therapies that can be tested in clinical trials. These data could be extrapolated to 14% of all cancers containing MTAPdef in the 9p21 region.

NIH Research Projects · FY 2025 · 2021-08

Human papillomavirus (HPV) prevention medicine is important because it protects against cancers caused by HPV infection. Nearly 79 million people are currently infected in the United States and about 14 million people become infected with HPV each year. HPV infection can cause cancers and the financial burdens of these HPV-related diseases cost more than $8 billion per year in the US. Recently approved HPV prevention medicine can prevent up to 74% of HPV-associated invasive cancers. The Centers for Disease Control and Prevention (CDC) recommends HPV prevention medicine through age 26. Among adults18–26 years, HPV series completion is unacceptably low whereas HPV infection rates are unacceptably high. There is also a lack of interventions to improve HPV prevention medicine uptake among young adults who were not protected during childhood. This study aims to fill in the important gaps by using a 2 by 3 factorial design to test the independent and combined effects of a multilevel intervention: school-based HPV prevention services (no enhanced access vs. enhanced access) at the system level and web-based narratives (no enhanced access vs. video vs. written) at the individual level. This intervention is developed based on pilot studies and socio-ecological models that recognize the impact multi-level factors have on behaviors. The 2 by 3 factorial design results in six groups: standard CDC information about HPV prevention (control); 2) video narratives about HPV prevention; 3) written narratives about HPV prevention; 4) access to HPV prevention medicine at school combined with standard CDC information, 5) access to HPV prevention medicine at school combined with video narratives, or 6) access to HPV prevention medicine at school combined with written narratives . This design allows us to investigate the independent and combined effects of tailored narratives and access to school-based HPV prevention medicine on its uptake. College students aged 18-26 years who are not previously administered HPV prevention medicine will be randomly assigned to one of the six groups. Primary outcomes are series initiation and completion at 3- and 9-month follow-ups, respectively. This study has implications for creating a new paradigm in HPV prevention services by inspiring future new research directions that include targeting multi-level factors to improve the uptake of HPV prevention medicine. This study will make a significant positive impact on public health, as it is using evidence-based strategies tailored for young adults aged 18-26 years. If successful, the web-based intervention can be easily disseminated because it is brief and scalable. School-based HPV prevention services can be implemented on college campuses in the future to improve healthcare delivery. Success and lessons learned in this study will inform future strategies to develop tailored narrative messages for different groups.