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NIH Research Projects · FY 2025 · 2021-07

Understanding of the etiology of Alzheimer's Disease (AD) is complicated due to the existence of dysregulations at different biological scales, ranging from genetic mutations to structural and functional brain alterations. Most models for studying AD are primarily focused on unimodal analysis, but there is a lack of systematic approaches that can integrate data across multiple scales to study the longitudinal disease progression. For example, the molecular mechanisms of brain atrophy related to progression to AD is not well understood. Although the promise of integrative analysis across multiple scales is increasingly recognized, there has been limited progress in developing interpretable and systematic approaches due the fact that the neuroimaging and -omics features have unique patterns of dependence and it is not immediately clear how to combine these two modalities for modeling progression to AD. Another limitation is that most of the existing methods have focused on delineating biological causes for differences between disease specific phenotypes that does not account for heterogeneity and does not treat the disorder as a continuum, which is recommended as per current NIA guidelines. To address these critical challenges, we develop a suite of statistical methods for modeling disease progression in AD involving longitudinal neuroimaging (MRI) scans and cognitive scores, combined with baseline -omics features and demographic and clinical data. Our integrative longitudinal analysis addresses critical gaps in literature and generates more robust results that are generalizable to more inclusive populations and yields more power in detecting true signals. We use spatially distributed voxel-wise brain surface features derived from MRI scans that provides high resolution interpretations about the changes in brain shape associated with disease progression. We develop predictive models which treats AD as a continuum while integrating data across disease stages and multiple visits in a systematic manner that is able to account for heterogeneity between and within disease stages and provides interpretable insights into longitudinal neuroimaging and baseline -omics features that drive cognition. Our methods can be used for developing individualized prediction trajectories for disease progression, identify latent states that are prognostic for specific disease stages, and predict cognition at future visits that can be directly used for early detection of high-risk individuals. We will develop and train our models using longitudinal ADNI data involving several thousand individuals and validate our findings on an independent longitudinal B-SHARP dataset. The statistical tools and algorithms developed will be made widely available to the broader research community. To our knowledge, our project is one of the first to develop an integrative and interpretable statistical framework for studying the trajectory of disease progression in AD using longitudinal and heterogeneous biomarker data from multiple scales, which provides valuable computational tools for early detection in AD that is of tremendous clinical importance in delivering patent centric outcomes in precision medicine.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract CD19-targeted chimeric antigen receptor (CAR) T cell therapy has shown remarkable treatment effects in B-cell malignancies, but many patients suffer from limited response and CD19-negative tumor relapse. Recently, we demonstrated that next-generation “armored” CAR T cells constitutively expressing the immune-stimulatory molecule CD40 ligand (CD40L) exhibited superior antitumor efficacy in preclinical studies but have not yet been fully explored in the context of CAR antigen-negative tumor relapse. There remains an urgent need for the non-invasive in vivo tracking of transfused T cells to determine their biodistribution, expansion, and functionality and to increase the CAR T cells’ killing capacity in case of imminent treatment failure. To overcome these limitations, we began developing methods for monitoring the in vivo kinetics of CAR T cells based on the concept of immune cell radiohapten capture. We demonstrated for the first time that T cells can be successfully transduced with a DOTA-antibody reporter, the DAbR1, enabling their in vivo tracking via PET and SPECT. In the current proposal, we build on this work and optimize our approach for translation of immune cell radiohapten capture from animals to patients, based on greatly improved components of 1) radiohapten capture reporter scFv C825 with picomolar binding affinity, and 2) optimized radiohaptens, the next- generation Proteus-DOTA (Pr) series suitable for imaging and targeted alpha therapy (TAT). The primary objectives of this study are to develop a clinically applicable PET imaging strategy of CAR T cell trafficking in B- cell malignancies and further study the effect of CD40L on counteracting the immune inhibition in syngeneic models of B-cell malignancies. The secondary objectives are to deliver TAT to enhance T cells’ killing capacity in cases of imminent treatment failure and improve the potency of CAR T cell therapies. We project to achieve our aims by generating second-generation and armored CD 19 CAR T cells expressing cell-surface anchored scFv C825. Syngeneic and immunodeficient xenograft murine models of CD19+ B cell malignancies including antigen-loss variants will be employed. After successful transduction, we will assess in vitro functionality of the CAR and the reporter, as well as radiation toxicity, followed by in vivo functionality, imaging sensitivity, and biodistribution, and develop an armored CAR T cell-based theranostic approach with the novel pair [86Y]YPr/[225Ac]AcPr. Finally, as a prerequisite to clinical translation of this novel platform, we will conduct studies using human second-generation and armored CAR T cells in clinically relevant xenograft models. The availability of such a single platform would provide crucial information for safer, more effective clinical trials. The aims thus have immediate translational relevance for our current clinical CD19 CAR T studies and other planned CAR T cell therapy trials.

NIH Research Projects · FY 2025 · 2021-07

Abstract Head and Neck squamous cell carcinoma (HNSCC) is the seventh most common cancer world-wide and afflicts more than 50,000 individuals in the U.S. each year. Because of its anatomic location and poor survival rate HNSCC is a devastating disease. Treatment can lead to profound functional defects and disfigurement. In the U.S., HNSCC incidence is increasing in large part due to the human papillomavirus (HPV). Although HPV- driven HNSCC generally has a better outcome than its HPV-negative counterpart, smokers with HPV-driven HNSCC demonstrate inferior oncologic outcomes and are clinically defined as intermediate risk disease. The significant complexity of HNSCC biology results in a complex clinical scenario, in which some patients are overtreated while others are undertreated, leading to unnecessary long-term side effects and suboptimal clinical response. In the laboratory, we discovered that HPV+ HNSCCs have unusually high expression of genes involved in DNA damage and repair (DDR) and are highly sensitive to drugs targeting RAD51, a DDR protein functioning in homologous recombination repair (HRR) that also protects cells from replication stress (RS). We hypothesize that the HPV-driven biology of this genomic subset of HNSCC renders them more sensitive to drugs targeting RS and HRR. We further hypothesize that HPV-neg HNSCC that share the same pattern of overexpressed DDR genes may also be susceptible to drugs targeting RS/HRR. In this translational proposal, we plan to: 1) Determine the efficacy and feasibility of replacing cisplatin with a RAD51 inhibitor to sensitize tumors to radiation using preclinical models of HPV+ HNSCC; 2) Elucidate the molecular mechanisms governing sensitivity of HPV+HNSCC to RAD51 inhibition; 3) Determine if drug combinations targeting RS or HRR are efficacious in preclinical models of HNSCC. Progress in this area could lead directly to human clinical trials that may improve tumor control with less toxicity.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY The overall goal of this project is to improve activities of therapeutic antibodies against cancer metastasis through developing novel bispecific antibodies (BsAbs). The central hypothesis of this project is that BsAbs designed with one specificity for a receptor or marker overexpressed on the cancer cell surface and the other specificity for a soluble growth factor or cytokine abundant in the tumor microenvironment induce co- phagocytosis of the growth factor or cytokine in the tumor microenvironment along with the co-targeted cancer cells via antibody-mediated cellular phagocytosis (ADCP) and thereby produce stronger antitumor activities than simple combination of 2 parental antibodies. The applicant has developed a pair of BsAbs, one mouse and one human, using a new BsAb format targeting human epidermal growth factor receptor-2 (HER2), an oncogenic driver that is emerging as a promising target for genomically informed therapy across a variety of cancer types beyond breast and gastric cancer, and targeting vascular endothelial growth factor A (VEGFA), another key driver that promotes tumor angiogenesis and suppresses tumor immune responses in the tumor microenvironment. Preliminary studies with the BsAbs showed remarkable anti-metastasis activity and prolonged survival in mouse tumor models. In the work proposed, three specific aims will be rigorously pursued: Aim 1 is to test the working hypothesis that the BsAbs exert stronger antitumor activities than simple combination of the 2 parental antibodies through inducing VEGFA co-phagocytosis via ADCP. Aim 2 is to determine the extent to which adaptive immune response is involved in the mechanisms of action of the BsAbs against metastasis of syngeneic mouse tumor models. Aim 3 is to assess the translational potential of the BsAbs against colorectal cancer patient-derived xenografts (PDXs) in humanized mice. The proposed work will be carried out through 1) investigating the role of engagement of FcγR in BsAb-mediated VEGFA co- phagocytosis and in BsAb-mediated antitumor activity, 2) analyzing the immune landscape in the tumor microenvironment and detecting presence of antigen-specific T cells upon treatment with BsAb vs with simple combination of 2 parental antibodies with and without FcγR blockade, and 3) determining the therapeutic activity of the BsAbs against HER2-overexpressing colorectal cancer PDXs in humanized mice. The work is expected to demonstrate that VEGFA co-phagocytosis by the BsAbs is a key mechanism by which the BsAbs exert stronger antitumor activity than simple combination of the 2 parental antibodies in the mouse models, and that T cell-mediated activities play an additional important role in synergizing the BsAb's antitumor activity. The impact of this work is expected to be high because if the study is successful, the findings will support future clinical testing of BsAbs to treat metastasis and recurrence of HER2-overexpressing colorectal cancer and development of additional BsAbs to target other growth factor receptors or markers overexpressed on the cancer cell surface and other tumor-promoting growth factors and cytokines in the tumor microenvironment.

NIH Research Projects · FY 2025 · 2021-07

Cancer cells are embedded in a protective and nourishing “niche”, an environment that cancer cells create by secreting proteins into their surroundings. Because cancer cells depend on their niche to survive and spread to other parts of the body, we believe that therapies designed to inhibit secretion could suppress cancer spread and thereby improve the length and quality of cancer patients' lives. Developing such therapies will require a better understanding of how secretion is activated in cancer. Our proposal will address this knowledge gap. Here we show that p53 protein loss, an established driver of cancer spread, enhances secretion by reprogramming the Golgi apparatus, a master regulator of protein transport in cells. We show that p53 loss activates the formation of a Golgi protein complex that controls secretion, and we have identified secreted proteins that are essential for lung cancer growth and spread. Furthermore, we have identified a drug that can block the formation of the Golgi protein complex, reduce secretion, and inhibit lung cancer growth and spread. In this application, we seek to elucidate the molecular underpinnings and therapeutic implications of the heightened secretion driven by p53 loss. In aim 1, we propose studies to elucidate how the Golgi protein complex enhances secretion and drives lung cancer progression. In aim 2, we propose studies to determine how the Golgi protein complex increases sensitivity to the drug we have identified. These studies will provide insight into how secretion is activated in cancer and may lead to new ways to target secretion in cancer patients.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract The unavailability of culturally competent mental health care for Chinese immigrant breast cancer survivors and their unmet psychological needs characterize the health disparity experienced by Chinese immigrant breast cancer survivors. Breast cancer is the cancer with the highest incidence rate among the Chinese population and Chinese immigrant breast cancer survivors have lower quality of life compared to their non- Hispanic white counterpart. However, interventions to improve quality of life are lacking among this immigrant population. Expressive writing intervention uses writing prompts to promote health by facilitating emotional and cognitive processes. Guided by Western and Eastern theories and preliminary studies showing the benefits of expressive writing among Chinese immigrants, this study proposes to test the health effects of an innovative and brief writing intervention among Chinese immigrant breast cancer survivors using a randomized controlled trial and mixed methods design. Chinese immigrant breast cancer survivors (N=240) will be randomly assigned either to a control condition to write about neutral topics or to one of two intervention conditions, self-regulation and self-cultivation, which both aim to promote adaptive cognitive processes but differ in how they achieve this goal. The self-regulation intervention incorporates a traditional Western expressive writing paradigm, whereas the self-cultivation intervention incorporates Asian cultural values. Participants in all three conditions will be asked to write in Chinese during three weekly 30-minute sessions. The primary outcome will be QOL, and the secondary outcomes will be perceived stress, stress biomarkers, and medical appointments for cancer-related morbidities. Self-reported health outcomes (QOL and perceived stress) will be assessed at baseline and 6- and 12-month follow-ups. Stress biomarkers (salivary cortisol and alpha-amylase) will be assessed at baseline and 6-week follow-up, and perceived stress will also be self-reported at the 6-week follow-up. Medical appointments up to the 12-month follow-up will be self-recorded and verified by medical record. We hypothesize that the two intervention conditions will improve quality of life, reduce perceived stress and medical appointments for cancer-related morbidities, and normalize stress biomarkers. Few studies have tested evidence-based programs in communities of color, and even fewer have tested culturally based interventions that adopted the cultural values of the underserved communities. We expect that the proposed study, guided by theory, practices, and methods and tailored for an underserved population, will inspire new directions in research to address these scientific and practical needs in health disparities research.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Due to their hyperproliferative nature and intrinsic genomic instability, triple-negative breast cancer (TNBC) cells exhibit high levels of replication stress, which occurs when the DNA replication machinery encounters obstacles that impede the replication process. How TNBC cells adapt to these high levels of replication stress remains poorly understood. These adaptive mechanisms, if identified, would reveal specific targets in TNBC and provide an effective strategy for TNBC treatment. To this end, we generated innovative cell models and discovered that one major mechanism required for TNBC cells to survive high replication stress is an increase in the enzyme RNase H2. RNase H2 acts to remove ribonucleotides that have been improperly incorporated into the genome, a key driver of replication stress. Subsequent bioinformatic analysis revealed that RNASEH2A, the catalytic subunit of RNase H2, is overexpressed in 89% of TNBC tumors and all the TNBC cell lines that we tested. More importantly, we found that RNase H2 inhibition, by genetic depletion or by the chemical inhibitor R14, specifically kills TNBC cells both in vitro and in vivo with minimal effects on nontumorigenic mammary epithelial cells. These important findings indicate that RNase H2 inhibition may be a promising therapeutic strategy for TNBC treatment. Intriguingly, we also found that RNase H2 inhibition activated the stimulator of interferon genes (STING) pathway and increased expression of key T-cell-attracting cytokines in TNBC cells and sensitized mouse TNBC tumors to anti-PD-1 immunotherapy, suggesting that the therapeutic effects of RNase H2 inhibition may be potentiated by anti-PD-1 therapy. All of these exciting findings support the hypotheses that RNase H2 inhibition offers a promising therapeutic strategy to treat TNBC and that it may be enhanced by anti-PD-1 immunotherapy. These hypotheses will be tested via 3 specific aims: (1) To identify the underlying mechanisms of the therapeutic efficacy of RNase H2 inhibition in TNBC. We will determine if limiting levels of dNTPs leads to increased misincorporation of ribonucleotides into the genomes of TNBC cells, and if inhibition of RNase H2 in TNBC prevents removal of these misincorporated ribonucleotides, consequently leading to unsustainably high replication stress and cell death. We will also evaluate the potential mechanisms mediating the escape of TNBC from RNase H2 inhibition and strategies to overcome resistance. (2) To determine the therapeutic potential of R14 for TNBC treatment. We will determine the in vivo tolerability of R14 in mice to determine the maximum tolerated dose as well as any potential toxicities. We will then assess the efficacy of R14 treatment in 10 TNBC patient-derived xenograft models representative of 5 TNBC subtypes. (3) To determine the therapeutic efficacy of the combination of RNase H2 inhibition with PD-1 immunotherapy in TNBC. We will evaluate the therapeutic efficacy of the R14/PD-1 immunotherapy combination in TNBC using 5 syngeneic TNBC mouse models. We will also assess if and how R14-mediated therapies affect the tumor immune microenvironment.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. The disease is universally fatal with current standard treatment being ineffective and debilitating. Cancer immunotherapy has demonstrated remarkable clinical success against multiple aggressive cancers and growing evidence suggests that boosting the body’s immune system can help eliminate highly aggressive and advanced tumors, including those resistant to conventional therapies. Its effectiveness against GBM, however, remains unclear, with multiple clinical trials exploring cancer immunotherapy regimens for GBM failed to demonstrate significant improvement in patient outcomes. Our group and others have recently discovered that GBM cells overexpress innate checkpoint CD47 to evade detection and clearance by professional antigen presenting cells (APCs). The expression level of CD47 was also found to correlate with survival in GBM patients. However, multiple studies showed that blockade of CD47 provided modest survival benefit in preclinical models of human cancers and additional phagocytosis checkpoints such as the β2 microglobulin (B2m) subunit of MHC-I molecule have been identified to promote tumor immune evasion. Disruption of B2m interaction with its phagocyte receptor leukocyte Ig-like receptor B1 (LILRB1) promotes phagocytosis of a diverse collection of tumor cells that were resistant to CD47 blockade. Yet, when anti-CD47 and anti-B2m antibodies were administered independently, we did not observe improved GBM phagocytosis. Therefore, based on these findings, we hypothesize that simultaneous blockade of phagocytosis checkpoints CD47 and B2m will activate innate immune responses against GBM, leading to a potent and durable adaptive antitumor immunity. To this end, we developed a novel bispecific antibody (CD47-B2m) that readily crosses the blood brain barrier (BBB). Aim 1 of the proposal will mechanistically examine whether CD47-B2m can promote antigen-specific antitumor T cell responses by APCs through induced GBM cell phagocytosis. In Aim 2, we will investigate if innate immune sensing pathways are critical in bridging innate and adaptive antitumor immunity in the setting of phagocytosis checkpoint blockade by CD47-B2m. Finally, in Aim 3, we will evaluate the use of CD47-B2m as a novel immunotherapeutic for GBM in clinically relevant murine models of GBM as a monotherapy or in combination with radiation. We will also investigate potential molecular mechanisms that predict treatment responses. If successful, our study will provide important preclinical data supporting further investigation of a completely novel immunotherapeutic agent against GBM. Additionally, the results generated here will highlight the importance of bridging innate and adaptive immunity to produce the most optimal antitumor immune responses. The concept of targeting multiple phagocytosis checkpoints can be applied to potentially all human cancers, and if successful may provide a new strategy to enhance the effectiveness of cancer immunotherapies.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract Castration-resistant prostate cancer is associated with substantial clinical, pathologic, and molecular heterogeneity; most tumors remain driven by androgen receptor (AR) signaling, which has clinical implications for patient selection for AR-directed therapies. However, histologic and clinical resistance phenotypes can also emerge after prolonged AR pathway inhibition, in which the tumors become less dependent on AR signaling (referred to as ‘androgen indifferent’). These highly aggressive and lethal tumors, termed treatment-emergent neuroendocrine prostate cancer (t-NEPC), are clonally derived from adenocarcinoma through lineage plasticity or transdifferentiation. There is an urgent need for novel targets and therapies for t-NEPC. t-NEPC cells carry recurrent genetic and epigenetic alterations as an adaptive response, thus suggesting that key molecular pathways and drivers controlling cell fate may be used as targets for therapeutic intervention. N6- methyladenosine (m6A) is an abundant internal RNA modification in eukaryote messenger RNAs. Despite its functional importance in different types of cancer, their specific role in prostate cancer progression and therapy resistance still remains elusive. Our integrative analysis of phosphoproteome, epitranscriptome, transcriptome, and ribosome profiling using in vitro and in vivo models identified m6A as exciting new epigenetic mark underlying prostate cancer lineage transition and therapeutic resistance. We therefore hypothesize that m6A drives lineage plasticity and is dynamically regulated by antiandrogen in prostate cancer, and that targeting m6A can reverse the lineage transformation, thereby restoring sensitivity to antiandrogen therapy in t-NEPC. We will test our central hypothesis by pursuing the following specific aims: (1) Determine the functional significance of m6A RNA epigenetics for therapeutic resistance in prostate cancer; (2) Elucidate the molecular mechanisms of m6A function in prostate cancer lineage plasticity and antiandrogen resistance; and (3) Establish the therapeutic potential of inhibitors tageting m6A for treatment of t-NEPC. The outcomes of this project are expected to open new avenues for t-NEPC therapeutics in linking m6A RNA epigenetics to lineage plasticity-mediated therapy resistance, and should have a profound impact on our approach to tackle the greatest challenges facing patients with treatment-emergent maligancies.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Small cell lung cancer (SCLC) is an aggressive malignancy for which there is a critical need for improved therapeutic strategies. While targeted and immune-based therapies have demonstrated encouraging results recently, they have shown benefit in only a subset of patients and, thus, have yielded little to no impact on the survival of unselected populations and even these benefits are limited by the rapid onset of resistance. There are currently no standard markers for selecting treatment or evaluating therapeutic resistance, issues made more challenging by the dearth of available tissue for molecular assessment in SCLC. Recent evidence from our group and others suggests that SCLC is a molecularly diverse disease and can be divided into four subtypes largely defined by the differential expression of three transcription factors [ASCL1 (SCLC-A), NEUROD1 (SCLC-N), and POU2F3 (SCLC-P)], and a fourth subtype with high expression of inflammatory and mesenchymal markers [Inflamed, (SCLC-I)]. Each subtype is characterized, in vitro, by distinct therapeutic vulnerabilities. Moreover, we showed that genomic and immune intra-tumoral heterogeneity (ITH) portends poorer survival, while increasing transcriptional ITH may be associated with therapeutic resistance in SCLC. The overarching goal of this proposal is to systematically investigate heterogeneity in SCLC and its association with therapeutic response, and develop tools to evaluate these features in the clinic. More specifically, we hypothesize (1) That SCLC is heterogeneous and can be divided into major subgroups with distinct therapeutic vulnerabilities; and (2) That greater ITH- assessed either at the genomic, immune, or transcriptional level- is associated with therapeutic resistance in SCLC and can be assessed dynamically during treatment in a non-invasive manner using blood-based biomarkers. To address these hypotheses, in Aim 1, we will assess whether these four molecular subtypes can serve as predictive biomarkers in co-clinical trials in vivo and in retrospective patient tissue analyses, while also developing blood-based strategies to identify the subtypes. In Aim 2, we will assess ITH at multiple molecular levels, including genomic, transcriptomic, methylomic, and immunologic, to characterize how baseline ITH influences patient survival. Lastly, in Aim 3, we will assess dynamic changes in transcriptional ITH following treatment, using paired samples from in vivo models and patient samples, to determine if increasing ITH of molecular subtype drives resistance and whether epigenetic modification may prevent or reverse it. The overall hypothesis tested here is that careful initial molecular subtyping of SCLC tumors, paired with strategies aimed at assessing, then limiting/reversing ITH, may better optimize the rate and duration of response to therapy. The studies will be facilitated by a comprehensive library of patient-derived murine models and extensive clinical data sets and executed by a multidisciplinary team of clinical/laboratory investigators, pathologists, computational biologists, and others with a strong track record of innovation in SCLC and translating laboratory findings into the clinic.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer, accounting for approximately 85% of all lung cancers, which is one of the deadliest types of cancers. Standard NSCLC treatments include surgery, immunotherapy, chemotherapy, and radiation therapy. Radiation therapy can be delivered by either photons or protons; however, both types of radiation therapy to the chest can result in cardiac injury. To date, no available clinical tool exists to guide physicians in choosing the best type of radiation therapy according to an individual’s risk for radiation-mediated cardiac injury. To plan radiation therapy, normal tissue complication probability (NTCP) models are commonly used and take into consideration differences in geometric shape or volumes between tumor and non-tumor tissue, as well as tissue dose constraints. However, these patient population-reliant models are based only on photon radiation therapy data (not proton), do not consider the differences in radiation vulnerability of organ substructures, and do not consider the individual NSCLC patient’s risk for a specific toxicity (e.g., cardiac toxicity). Hence, this proposal tests the hypothesis that chemoradiation- related cardiac toxicity can be minimized by dose optimization and individual pre-existing cardiovascular risk- stratification for choosing appropriate radiation modality. Pre-existing cardiovascular risk factors, such as individual genetic predisposition, cardiac injury blood biomarkers, and extent of vascular calcification will be correlated with chemoradiation-associated cardiac toxicity and overall survival (OS) in Aim 1. Data on pre- existing cardiovascular risk factors will be retrospectively collected from two prospective, randomized comparisons of photons vs. protons and from a registry trial, which included proton-treated patients not enrolled into the randomized trial. Associations between pre-existing cardiovascular events and radiation therapy- mediated cardiac events as well as OS will be used as parameters to generate a one-of-a-kind NTCP model (Aim 2). In Aim 3, a prospective cohort registration trial will be developed to longitudinally assess cardiac function, cardiac fitness, and model implementation. During model implementation, two maximally optimized radiation therapy plans for each enrolled patient will be developed: 1) using standard population-based dose constraints; and 2) using personalized dose constraints based on individual risk. A predefined NTCP goal will be set to evaluate both plans. If the personalized plan improves the NTCP goal by 15%, the patient will be treated using the personalized plan. The model performance will be continuously assessed and improved using the data accumulated from the trial. The long-term objectives of this proposed project are to minimize cardiovascular injury while optimizing NSCLC patient outcomes, based on individual patient risk to cardiac injury after concurrent chemoradiation therapy by multivariable model selection of radiation therapy modality and technique. Preventing cardiovascular injury in cancer patients so that individuals can live longer, and more fulfilling lives is in direct alignment with the mission of both the National Cancer Institute and the National Heart, Lung, and Blood Institute.

NIH Research Projects · FY 2025 · 2021-06

Summary Statement/Abstract Pediatric brain tumors are the most common solid tumors in children, with approximately 5000 new cases diagnosed per year in the United States. Around 17% of brain tumors in children age 0–14 years are high-grade gliomas (HGGs), which are currently incurable. The lack of effective treatments highlights the urgent need to identify mechanism-based therapeutic approaches. Substantial experimental evidence has recently revealed that H3.3-G34R–harboring pediatric HGGs (pHGGs) exhibit high genomic instability and high-level expression of neuronal markers, indicating that these tumors represent a distinct subtype of pHGG compared with other types, including the one with an H3.3-K27M mutation. More than 90% of H3.3-G34R gliomas also harbor ATRX loss-of-function mutations. Using a newly established genetically engineered murine model (GEMM), we demonstrated that H3.3-G34R mutation and ATRX deletion in premalignant neural stem cells (PM-NSCs) with the Trp53-/- background could strongly promote gliomagenesis. These tumors exhibit typical features of human H3.3-G34R–harboring pHGGs, so this GEMM provides us with a faithful tool for studying the molecular mechanisms underlying the synergistic effects of H3.3-G34R mutation and ATRX deletion and for identifying novel therapeutic targets. We have found that H3.3-G34R mutation changes histone modifications both locally and globally and leads to high expression of FoxD1 and HoxA1, transcription factors essential for early neuronal development. Given that enrichments of FoxD1 and HoxA1 are associated with worse prognosis in glioma patients, they provide 2 novel therapeutic targets for pHGGs. In addition, we found that ATRX loss leads to ALT activation, which makes tumor cells sensitive to perturbation of their mitochondrial function. On the basis of these observations, we hypothesize that distinctive epigenetic profiles induced by H3.3-G34R mutation and ATRX loss drive gliomagenesis and lead to targetable vulnerabilities involving dysfunctional telomeres and impaired mitochondrial activity. To test this hypothesis, we plan to 1) determine the roles of FoxD1 and HoxA1 in H3.3- G34R–driven gliomagenesis, 2) define the therapeutic vulnerability induced by ATRX deficiency in pHGGs, and 3) elucidate the synergistic effect of H3.3-G34R mutation and ATRX loss on epigenetic reprogramming in gliomas. The completion of the proposed studies will not only fill the gaps in our knowledge of how H3.3-G34R and ATRX loss change the epigenome to lead to normal neuronal development and gliomagenesis, but also— and more importantly—contribute to the development of therapeutic strategies that target pHGGs and provide insights into the role of epigenetic regulation in brain development and gliomagenesis.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT The HEALTH Research Institute was established at UH to pursue a bold research agenda aimed at addressing the public health crises health disparities inflict on our most vulnerable communities. Investing in a new NIDA R25 at UH represents an excellent strategic investment in establishing a diverse pipeline of future substance abuse scientists given our: (1) central location in arguably the most ethnically diverse metropolitan area (~44% Latinx, ~26% White, ~23% Black, and ~7% Asian) in the U.S.; (2) one of the only Tier-1 research institutions with a Hispanic-Serving Institution (HSI) and Asian American and Native American Pacific Islander-Serving Institution (AANAPISI) designation; (3) ~400 health research faculty and 97 degree programs who will benefit from a research education program that focuses on substance abuse given Houston’s designation as a high-intensity drug trafficking area (HTDTA); and (4) the institutional investment in the HEALTH Research Institute which will house this proposed NIDA R25 to ensure it addresses substance abuse education and research training across the University and beyond. This HEALTH – Future Addiction Scientist Training (HEALTH-FAST) Program will focus on annually enrolling and advancing the careers of Doctoral (n=4), Postdoctoral (n=2), and Early Stage Investigators (ESI) Trainees (n=2) – with a total of 40 Trainees from underrepresented backgrounds in the health sciences across the 5-year funding period. This will be achieved by the successful completion of 4 specific aims: (1) Identify, select, educate, and mentor highly qualified Doctoral, Postdoctoral, and ESI Trainees from underrepresented groups in the health sciences for substance abuse research careers with an emphasis on linkages to ATOD-related chronic diseases and health disparities; (2) Develop a cutting-edge substance abuse research education curriculum that will include an array of diverse activities that are reflective of emerging national trends to facilitate an enriching educational experience; (3) Advance skills needed to effectively disseminate innovative scientific research at professional meetings and in peer-reviewed journals, while simultaneously accelerating Trainees’ competitiveness to secure NIDA grant funding; and (4) Systematically evaluate HEALTH-FAST processes, programming, and Trainee outcome data in real-time to demonstrate a NIDA return on investment and inform data-driven program modifications as needed. Given the proposed transdisciplinary approach to health-equity science, the HEALTH-FAST Program will leverage Program Faculty Mentors across 7 colleges and 12 academic units (i.e., Biomedical Engineering; Biomedical Sciences; Clinical Sciences; Electrical & Computer Engineering; Health & Human Performance; Health Systems & Population Health Sciences; Medicinal Chemistry; Pharmaceutical Health Outcomes and Policy; Psychological, Health, & Learning Sciences; Psychology; Biology & Biochemistry; and Social Work) to execute a cutting-edge research education program.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Background: In-depth study of neoantigens will promote our knowledge of the fundamental mechanisms of basic immunology and immune-related disease processes, such as response to cancer immunotherapy. Neoantigens play a key role in the recognition of tumor cells by T cells and are increasingly shown to be targets of checkpoint inhibitor-induced immune response. However, several missing links exist in neoantigen research. (1) Only a small proportion of neoantigens can elicit T cell responses. It is even less clear which neoantigens will be recognized by which specific T cell receptor (TCR). (2) Although neoantigens are important during the course of action of immunotherapies, how neoantigen repertoire data can be used to predict patient response is only poorly understood. (3) The lack of standardized analysis pipelines and limited sharing of neoantigen data have hindered efficient and consistent research in the tumor immunogenomics field. Aim 1: Build a transfer learning-based model to predict immunogenicity of neoantigens. So far, only a very limited number of reports have created predictive models determining whether a neoantigen/MHC complex can elicit any T cell response. Even fewer of them are capable of predicting the TCR-binding specificity of neoantigens. However, the capability to predict the overall immunogenicity and the TCR-binding specificity of neoantigens is critical for improving the benefit of immunotherapy. Aim 1 addresses this challenge with advanced transfer learning algorithms, followed by benchmarking and laboratory validations. Aim 2: Predict response to checkpoint inhibitors by integration of the immunogenicity and other properties of all neoantigens in a patient, through a Bayesian multi-instance learning model. To date, most studies have focused on the neoantigen/mutation load approach in correlation with response of patients to immunotherapy administration. This simplistic approach misses the rich information contained in the whole repertoire of neoantigens per patient and has been successful in only a few studies, but not others. Aim 2 addresses this important inadequacy by creating a Bayesian multi-instance learning model that fully considers various quality features, including immunogenicity, of all neoantigens in a patient for prediction of treatment response. Aim 3: Create a web portal to provide neoantigen-related computational services and to share neoantigen data. The PI will establish a public webserver providing cloud-based standardized services, including prediction of neoantigens and the advanced analysis methods developed in Aim 1 and 2. The webserver will openly share neoantigen/TCR and patient phenotype data, in accordance with IRB and HIPAA regulations. Expected impact: (1) This project will predict the immunogenicity of neoantigens, which could inform neoantigen vaccine development. (2) This project will predict response to checkpoint inhibitors and other forms of immunotherapy based on patient neoantigen profiles. (3) The neoantigen database will propel research and also lead to clinical applications for cancers and other immune-related diseases, such as COVID-19.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Most adults in the U.S. fail to meet national physical activity (PA) recommendations, and minorities are less likely to meet these recommendations than non-Hispanic Whites. Physical inactivity and obesity are major risk factors for cardiovascular disease, certain cancers, diabetes, and stroke and are important contributors to preventable morbidity and mortality in the U.S. Social environments are widely recognized to have an important impact on PA, yet social contexts remain understudied in intervention research. The goal of the proposed research is to evaluate the effectiveness of a 6-month behavioral dyadic intervention to promote positive and sustained change in PA among inactive predominantly Latina and African American women in Houston, TX. Dyads will first be randomly assigned to the dyadic intervention or to an individual condition. Within the individual condition, one woman from each dyad will subsequently be randomized to the individual intervention and the other woman to a health education control. The dyadic and individual interventions will consist of telephone-based health coaching, a Fitbit, and health education newsletters to enhance motivation and behavioral skills for increasing PA. The health coaching for the dyadic intervention additionally focuses on building participants' capacity to be a supportive partner by training dyads in positive communication strategies and offering support in a non-judgmental and empathetic way. The health education control will consist of a Fitbit and health education newsletters. Study participants will include 500 predominantly Latina and African American inactive women recruited and enrolled as dyads (e.g., family or friends; n=250 dyads). The intervention expands upon a pilot randomized trial conducted by the investigative team that showed preliminary evidence of support and also identified areas for improvement. Participants will be assessed at baseline, 6 months, and 12 months after baseline to evaluate both intermediate and long-term effects. The primary outcome is change in minutes per week of moderate-intensity PA. Lower body strength, anthropometric measures (i.e., BMI, waist circumference), sedentary behavior, mean daily steps, and blood pressure are secondary outcomes. Autonomous motivation, social support, autonomy support, self-efficacy, and outcome expectancies will be examined as potential mediators of changes in PA. The proposed research is innovative in its comparison of a dyadic intervention against an individual intervention and in its emphasis on dyadic social processes in addition to standard behavior change strategies. The intervention explicitly targets existing social networks to foster social environments supportive of healthy behavior change that will persist beyond the intervention period. This research is expected to yield critical insight regarding whether and how features of social contexts can be modified to support healthy lifestyle change as a means of addressing disparities in cancer and chronic disease risk among predominantly Latina and African American women.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM), a high-grade glioma (grade IV), is the most prevalent and malignant primary brain tumor in adults. There is no effective treatment of GBM. After standard treatment, the median survival of GBM patients is around 15 months. GBM is featured by enhanced cell proliferation, high propensity of invasion/diffuse infiltration throughout the brain, and resistance to chemo-/radiation therapy. Emerging evidence supports that long non-coding RNAs (lncRNAs, ~18,000 human lncRNA genes) can mediate tumor- promoting/suppressing effects and serve as independent diagnostic/prognostic biomarkers. Our previous integrative genomic study revealed that many lncRNAs show dysregulated expression, are associated with clinical prognosis, or harbor frequent somatic copy number alterations in GBM, suggesting an important role of lncRNAs in GBM pathogenesis. However, the function and mechanism of most lncRNAs in GBM are unknown. To fill this gap, our long-term goal is to leverage systematic multi-omic approaches to characterize the function and mechanism of lncRNAs in GBM, and to help develop new therapeutic strategies based on novel insight into lncRNA regulation in GBM. Our large-scale loss-of-function screen using CRISPR interference (CRISPRi) identified an antisense lncRNA, lnc-YINC (YBX1-interacting lncRNA) that was critical for GBM cell growth and was significantly up-regulated in GBM compared with normal brain tissue and low-grade glioma (LGG). The higher expression of lnc-YINC was associated with shorter overall survival of GBM patients. Functionally, lnc- YINC acted in-trans and was a key regulator of cell cycle progression of GBM cells/glioma stem cells (GSCs) and self-renewal of GSCs. It also protected GBM cells/GSCs from DNA double-strand breaks (DSBs) caused by endogenous stress or exogenous ionizing radiation. Mechanistically, lnc-YINC interacted with YBX1, and post-transcriptionally regulated the expression of key regulators of cell cycle or DSB repair, at least partially by stabilizing their mRNAs. Based on these new findings, we hypothesize that lnc-YINC is a GBM-promoting lncRNA that promotes proliferation of and modulates radio-sensitivity of GBM cells/GSCs, by enhancing YBX1 binding to key regulators of cell cycle/DSB repair and co-regulating their expression at post-transcriptional level. The objectives of this proposal are (a) to determine the role of lnc-YINC in gliomagenesis, (b) to integrate enhanced CLIP-seq (eCLIP-seq), ribosome profiling, RNA-seq and quantitative mass spectrometry data, with functional assays to identify the downstream targets of lnc-YINC/YBX1 that are key to regulating cell cycle or DSB repair, (c) to dissect the molecular mechanisms whereby lnc-YINC/YBX1 axis post-transcriptionally regulates the expression of key regulators of cell cycle or DSB repair, (d) to determine the role of lnc-YINC in modulating radio-sensitivity of GBM cells/GSCs and to investigate the therapeutic impact of combining inhibition of lnc-YINC with radiation therapy on GBM maintenance in vivo. This application is strengthened by a team of experts of basic/clinical science of GBM, DNA damage repair, RNA biology and proteomics.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Gastric cancer (GC) is the fifth most common malignancy and the third most lethal cancer worldwide, with a 5-year survival rate of 5-10% in advanced stages. The long-term goal of our research is to develop novel interventions to prevent and treat gastric cancer (GC) based on molecular targeting of crucial events in GC carcinogenesis. This application is based on an exciting finding that mice with villin-promoter– driven PPAR-d overexpression (villin-PPAR-d mice) spontaneously developed large and invasive GCs. Our published data showed that PPAR-d was upregulated in human GC tissues and this upregulation was associated with poor prognosis of patients. Villin promoter is active in a small subpopulation of gastric epithelial cells that are considered villin-positive gastric progenitor cells (V-GPCs). Cancer stem cells (CSCs) are thought to arise from transformation of normal stem/progenitor cells to drive tumor formation. We found that PPAR-d enhanced the stemness of V-GPCs and enabled V-GPCs to form tumors via activating the PPAR-d–interferon- gamma (IFNG) signaling loop. Furthermore, we also found 1) CCL20 is the most markedly upregulated chemokine by PPAR-d overexpression in V-GPCs, and 2) CCR6+CD45+ cells (CCR6 is the sole receptor of CCL20) have significantly higher IFNG mRNA expression than CCR6-CD45+ cells do. Helicobacter pylori (H. pylori) is a class I carcinogen for human GC. Chronic H. pylori infection, currently affecting nearly half of the world population, is a known strong risk factor for human GC. H. pylori infection increases PPAR-d, CCL20, and IFNG, which in turn promotes H. pylori–induced gastric inflammation, enhances GPCs’ stemness and promotes gastric epithelial proliferation in mice and humans. Whether this PPAR-d overactivation is required for H. pylori– induced GC is largely unknown. Addressing this knowledge gap is important to the public, especially for individuals who suffer from chronic H. pylori infection, because PPAR-d is a druggable protein for which both agonists and antagonists have been developed, and PPAR-d agonists are being used for noncancerous indications (e.g. enhancing muscle endurance). Thus, we hypothesize that PPAR-d overactivation in GPCs drives GC via upregulating the PPAR-d–CCL20/CCR6–IFNG signaling pathway and molecular targeting of this pathway could be a novel intervention modality for GC. Aim 1 will determine the role of PPAR-d upregulation in V-GPCs and Lgr5-positive GPCs (L-GPCs) at adult onset on GC carcinogenesis. Aim 2 will determine the effect of PPAR-d genetic deletion/loss of function in V-GPCs or L-GPCs on H. felis-induced GC tumorigenesis. H. felis is a close relative of H. pylori that has analogous effects in mice to those of H. pylori in humans. Aim 3 will determine the molecular mechanisms underlying dysregulated PPAR-d–IFNG signaling and evaluate the effects of molecular targeting of this pathway on GC carcinogenesis. We expect that completion of this proposal will not only provide new insights into the molecular pathogenesis of GC, but also advance the development of novel mechanism-based approaches for GC prevention and therapy.

NIH Research Projects · FY 2025 · 2021-04

As they progress, malignant tumors accumulate cross-linked collagen fibrils that enhance matrix stiffness, thereby activating collagen receptors that trigger cancer cell invasion and dissemination. We previously reported that a collagen modifying enzyme called lysyl hydroxylase 2 (LH2) is highly expressed in metastatic lung cancer cells and promotes metastasis by increasing the amount of a particularly stable type of collagen cross-link called hydroxylysine aldehyde-derived collagen cross-link (HLCC). Although LH family members (LH1-3) have highly conserved LH and glucosylgalactosyltransferase (GGT) domains, LH2 reportedly lacks GGT activity and is unique among LHs in its ability to hydroxylate lysine (lys) residues on collagen N- and C- termini (“telopeptides”) that are required to generate HLCCs. The structural basis for LH2's unique functional properties remains unclear. The studies proposed herein will address this crucial knowledge gap. On the basis of collagen LH and GGT domain crystal structures that we recently solved, we show that LH2 has telopeptidyl LH (t-LH) activity owing to a unique basic residue cluster that generates electrostatic interactions with acidic residues on collagen. Furthermore, by using a new collagen GGT activity assay we developed that is more sensitive than ones reported previously, we show that LH2 has GGT activity owing to an alternatively spliced exon 13a-encoded loop that resides at the entrance of the GGT active site. We show that LH2 isoforms that lack (LH2a) or contain (LH2b) exon 13a are differentially expressed in the TCGA lung cancer cohort, and that LH2b is the predominant isoform expressed in an orthotopic KMLC model in which LH2 promotes metastasis and causes widespread alterations in intra-tumoral immunity. We developed defined collagen matrices that are deficient or replete in LH2-mediated HLCCs (total or glucosylated) and used these matrices as tools to show that HLCCs influence lung cancer cell behaviors. On the basis of these preliminary results, we postulate that LH2 drives lung cancer metastasis through dual (LH- and GGT-mediated) collagen modifications and will test this hypothesis by completing 3 specific aims. Aim 1: To demonstrate a causal relationship between LH2's electrostatic interactions with collagen, HLCC formation, and lung cancer metastasis. Aim 2: To demonstrate a causal relationship between inclusion of LH2's exon 13a, collagen glucosylation, and metastasis. Aim 3: To demonstrate a causal relationship between LH2's dual (t-LH- and GGT-mediated) collagen modifications and LAIR-1-mediated immunosuppression. In summary, the novelty of our proposal rests in an hypothesis that is based on unique insight into LH2's dual enzymatic activities and the tools we developed to generate that hypothesis (e.g., crystal structures, enzymatic assays, and defined collagen matrices). Findings from these studies will provide a basis for future testing of selective LH2 antagonists that we have already identified from high-throughput screens and have begun to optimize outside the scope of this application.

NIH Research Projects · FY 2025 · 2021-04

Summary Therapeutic resistance and tumor relapse present as major barriers to achieving a definitive cure for cancer. This challenge is especially relevant for patients with pancreatic ductal adenocarcinoma (PDAC), who are largely diagnosed at advanced stages and face low survival odds. Recent studies have revealed that tumors are complex ecosystems consisting of coexisting subclonal populations that each harbor a unique genomic landscape. Indeed, tumors are constantly adapting in response to external perturbations such as therapeutics, and clones capable of surviving treatment are evidence of evolved resistance to therapeutics and may provide the foundation for relapse. While the role of mitochondria in tumors has been largely neglected, recent studies have demonstrated that OXPHOS can contribute to treatment resistance as well as several other processes such as invasion and metastatization. Here, we will leverage out our novel Clonal Replica Tumors (CRTs) platform to test the hypothesis that heterogenous mitochondrial activity across different clonal lineages plays a central role in determining tumor response to therapy, and thus contributes to the development of therapeutic resistance and tumor relapse in PDAC. Our CRT platform enables the testing of multiple pharmacological disruptors or other external factors in parallel in animals bearing patient-derived xenotransplanted (PDX) tumors with identical clonal composition. This approach allows us to explore how intra-tumor mitochondria functional diversity is shaped by genomic heterogeneity as well as whether and how this diversity contributes to therapeutic resistance. By focusing on treatment-naïve subclonal lineages with distinct responses to therapy isolated from early passage pancreatic cancer PDXs, we will investigate the following aims: 1) explore the role of genomic heterogeneity in shaping mitochondria functional diversity and define mitochondrial molecular signatures that predict treatment response; 2) elucidate the role of mitochondria in mediating pharmacological resistance; 3) determine the effects of targeting mitochondria on tumor clonal architecture and construct a 3D map of tumor resistance. Ultimately, we will explore the therapeutic benefits of targeting mitochondria to prevent therapeutic resistance and relapse in pancreatic cancer. We are confident that our study is responsive to the NIH/NCI mission to improve patient outcomes, as it addresses fundamental questions about how intratumoral mitochondria heterogeneity and distinct mitochondrial phenotypes influence treatment response to drugs and sustain tumor relapse. We further anticipate that our research will have an immediate translational impact through the identification of new biomarkers that can be used to identify patients who may benefit from the OXPHOS inhibitors currently under clinical investigation.

NIH Research Projects · FY 2025 · 2021-04

SUMMARY Surgical resection is curative in hepatocellular carcinoma (HCC). Unfortunately, surgical cases often suffer from early recurrence (up to 50% in two years), which leads to dismal outcomes. Currently, there are no approved neoadjuvant therapy options to induce pathologic response and reduce the rate of recurrence. Notably, suppression of host immunity in HCC is a hallmark of cancer establishment and progression. Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor present on T-cells that binds to specific ligands (PD-L1, PD-L2) on both tumor stroma and cancer cells. In preliminary studies, we have analyzed resected HCC tissues after preoperative immunotherapy in early HCC. We found specific clusters correlating with promising responses, including frequent pathologic complete responses at the time of surgery (Cancer Immunol Res 2019). In addition, we demonstrated that PD-1 blockade can stabilize tumor vasculature, thus enhancing anti-tumor immunity when combined with VEGF pathway blockade in preclinical models of HCC (Hepatology 2020). Our goal is to show that reprogramming of immune microenvironment in HCC will result in greater benefit for immunotherapy. We hypothesize that: 1) Neoadjuvant anti-PD-L1/VEGF therapy is feasible, active and will induce pathologic complete response in resectable HCCs, and 2) HCCs responding to neoadjuvant anti-PD-L1/VEGF therapy will have increased intratumoral CD8+ T-cell : Treg ratio, circulating cytokine levels, and favorable metabolic changes on imaging. We will test these hypotheses in 2 specific aims: Aim 1: To evaluate the safety and efficacy of anti-PD-L1/VEGF combination therapy in neoadjuvant setting in resectable HCC patients. This randomized, multi-site phase II clinical study will accrue 90 patients based on 2:1 randomization (neoadjuvant atezo/bev = 60, versus upfront surgery = 30) and has 2 sub-aims: Aim 1a will evaluate safety and pathologic response rate as the primary endpoints in the atezo/bev arm; Aim 1b will evaluate 3-year recurrence-free survival rate and OS as secondary endpoints in the atezo/bev arm versus control. Aim 2: To determine if activation of anti-tumor immunity correlates with durable responses after dual anti-PD-L1/VEGF therapy in neoadjuvant setting in resectable HCC patients, which has 3 sub-aims through studying tissue, peripheral blood, and imaging parameters. Aim 2a is to analyze immune T- cell clusters in pretreatment biopsies and posttreatment surgical resection specimens to evaluate CD8+ T- cell : Treg ratio using multiplexed immunofluorescence and single cell RNA sequencing. Aim 2b is to study changes in tissue immune cells, regulatory proteins, and plasma cytokines (multiplexed protein array) to explore a biomarker signature that may predict response. Aim 2c is to evaluate correlation between response and changes in tumor metabolic activity using PET MRI scan pre- and posttreatment. The impact of this project is that it may transform the role of immunotherapy in HCC from palliative to curative.

NIH Research Projects · FY 2025 · 2021-04

Sarcomatoid renal cell carcinoma (sRCC) represents an aggressive group of renal epithelial tumors characterized by histopathological features of epithelial–mesenchymal plasticity (EMT) and prominent metastatic behavior. These malignancies are extremely challenging in the clinic, as they fail to respond to the standard-of- care therapeutic regimens for RCC. Furthermore, in spite of the remarkable advances in cancer genomics, there are still no reliable tools or biomarkers to predict the clinical course of the disease, particularly in the context of modern therapeutic interventions. Objectives. The long-term goal of this project is to identify the genomic and molecular drivers of malignant progression in sRCC, focusing on the role of EMT in clonal evolution, in the acquisition of metastatic potential and as a mechanism of adaptation to therapy. This will lead to the identification of context-specific vulnerabilities dictated by the specific genomic and molecular landscapes that characterize this aggressive subset of kidney cancer. Rationale and Hypothesis. Preliminary studies showing the emergence of specific patterns of chromosomal alterations led us to the hypothesis that the acquisition of chromosomal instability (CIN) during tumor evolution favors the selection of clones endowed with high cellular plasticity and prominent metastatic potential. Specific Aims. In the first aim we will provide a detailed spatial and temporal annotation of epithelial and mesenchymal population dynamics during malignant progression and in response to pharmacological interventions. The second aim will define the genomic and transcriptomic landscape of the malignant subpopulation and the interplay between these cellular compartments and the components of the TME. In the third aim we will identify context-specific vulnerabilities, defining the genetic dependencies of epithelial and mesenchymal cells. Significance. The approach will provide fundamental information about clonal dynamics, tumor–host interactions at a single-cell resolution, and tumor evolution in RCC, substantially improving our understanding of the genetic and molecular bases of the disease. Translational relevance. A detailed understanding of the genetic and molecular events driving malignant cell plasticity and the evolution to sRCC will provide the framework to predict the behavior of this heterogeneous group of tumors. Furthermore, the functional genomic approach will uncover context-specific vulnerabilities and provide novel drug targets in a disease class in urgent need of effective treatments. Innovation. The project is innovative from a conceptual and technological standpoint. Targeting cancer-specific weaknesses emerging in the context of cell plasticity, increased tumor heterogeneity, and clonal diversification is a promising approach tailored to the genetic and functional hallmarks of the disease. The technological tools and approaches are unique and highly innovative. The introduction of a lineage-tracing technology and an embedded dynamic reporter will allow us to address several open questions in the field of EMT going beyond the specific tumor type. The work will therefore provide the scientific community with a valuable tool for basic and translational research.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) are the most common germinal center (GC)- derived Non-Hodgkin B cell lymphomas (BCLs). Although initially exhibiting an indolent behavior, FLs end up being mostly incurable with 40-50% eventually transforming into an aggressive and lethal form of DLBCL. Although half of DLBCLs can be cured with standard chemotherapy and immunotherapy, many patients are still refractory and succumb to progressive or relapsed disease. In addition, the strong chemotherapy regimens used, even when tolerated, have deleterious side and late effects. Thus, less toxic, targeted therapies are in urgent need for this disease. Recent advances in the molecular biology of DLBCL uncovered critical pathways in the initiation and development of these neoplasms. Of particular importance are next-generation sequencing studies that identified mutations in epigenetic modifiers that led to the development of current active clinical trials using epigenetic therapies in DLBC. Among their highest recurrent disease alleles are somatic mutations affecting two closely related histone acetyltransferase (HAT) genes, Crebbp and EP300. These are frequently monoallelic, within the HAT domain and usually mutually exclusive, suggesting that they 1) might affect a common pathway and 2) residual WT expression of CREBBP and/or EP300 is required for cell survival. We have identified the protein arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase1) as an important factor to maintain the survival of CREBBP/EP300 mutated BCLs. A potent effective small molecule inhibitor of CARM1 methylation activity has been recently developed and we hypothesize that targeting CARM1 methylation activity in CREBBP/EP300 mutated BCLs causes synthetic lethality. The major goals of this proposal are to determine how inhibition of CARM1 methylation activity affects BCLs harboring CREBBP/EP300 genetic lesions and define the molecular mechanism responsible for the sensitivity of CREBBP/EP300 BCLs to CARM1 inhibition. We anticipate that the results obtained from these studies will impact our current understanding of the pathogenesis of GC-derived BCLs, by providing new insights on the mechanisms of neoplastic transformation. Altogether we ultimately expect that these results will make a strong rationale for future clinical studies using CARM1 inhibitors in Crebbp/Ep300 mutated BCLs.

NIH Research Projects · FY 2025 · 2021-03

ABSTRACT Soft-tissue sarcomas are heterogeneous tumors that originate from cells belonging to the mesenchymal lineages, and that affect almost 200,000 individuals worldwide each year. The most aggressive and metastatic sub-types in adults are those with complex karyotypes and multiple genetic aberrations. The overall survival of these sarcoma patients has not greatly improved in recent years, and alternative approaches to chemotherapy and radiotherapy such as immunotherapy have so far provided only marginal benefits. Novel therapeutic advances for UPS are hindered by the lack of knowledge about the functional consequences of the complex genomic alterations found in patients and limited characterizations of the tumor microenvironment (TME), which would reveal non-cell-autonomous mechanisms critical to sarcoma progression. Experimental tools and available animal models currently do not address these limitations. However, appropriate models could facilitate the efficient discovery of new targets and immune- based therapies for these tumors, which have relatively low incidence and for which the development of clinical trials is often challenging. Accordingly, we propose to generate sarcoma mouse models that encompass the actual somatic aberrations observed in patients. Moreover, we will use these models to facilitate studies of treatment response and TME composition. The employment of these new models, together with newer technologies such as single-cell RNA- sequencing (scRNA-seq), CyTOF and Imaging Mass Cytometry (IMC) will ultimately illuminate the key expression profile of the single tumor cells, mechanisms of metastasis, resistance to conventional treatments and TME components that may influence such mechanisms. Ultimately, these models will translate to the clinic more effective therapeutic combinations and regimens. Successful completion of this project will i) generate new mouse models of complex sarcoma that recapitulate the genetic defects found in human sarcoma and provide a comprehensive functional characterization of these models (Aim 1), ii) illuminate the expression profile of the tumor cells and discrete sub-groups of them, to understand how these profiles influence the TME composition (Aim 2), iii) test how these models respond to different radiotherapy administration schedules in the heterogeneous settings of distinct tumor genetics, expression profiles and environmental elements (Aim 3).

NIH Research Projects · FY 2025 · 2021-03

Project Summary In addition to the classical mode of phagocytosis and intracellular oxidative killing of pathogens, a recently discovered antimicrobial function of neutrophils is the formation of extracellular traps (Neutrophil Extracellular Traps, NETs), which can trap and kill pathogens extracellularly. As NET formation (NETosis) generally requires reactive oxygen species (ROS) generation, we and others have found that neutrophils from Chronic Granulomatous Diseases (CGD) patients and Gp91phox-/- CGD mice with mutations in NADPH oxidase complex exhibit impaired NET generation in-vitro and in-vivo in response to various stimuli and pulmonary bacterial infection. We recently reported that Tamoxifen (TMX), an FDA approved selective estrogen receptor (ER) modifier for treatment of breast cancer, induces antimicrobial NETs in CGD neutrophils in a ROS independent manner. We further showed that activation of autophagy is necessary and sufficient to induce TMX-mediated NETs. In addition to this seminal report, the premise of the proposed research is derived from our preliminary data indicating a novel pathway of ROS-and ER-independent NETosis by TMX via a non-canonical autophagy activation. The two proposed specific aims will establish TMX as NET-inducing agent with antimicrobial and anti- inflammatory effect in preclinical murine CGD and human CGD neutrophils (Aim 1); and elucidate TMX-mediated non-canonical autophagy signaling pathway in neutrophils that culminates in disintegration of nuclear lamina to facilitate the release of NETs (Aim 2). Our studies provide important mechanistic insights into a novel autophagy pathway activated by TMX which will have implications not only for NET research but also for exploiting autophagy and NETs to treat infectious and autoimmune diseases. By leveraging neutrophils from a well- characterized cohort of CGD patients at NIH Clinical Center, these studies also present an exciting opportunity for preclinical testing of TMX in CGD to restore antimicrobial function of neutrophils to combat pneumonic bacterial infections, frequently observed in these patients.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT Radiation plays a central role in the management of the most lethal central nervous system malignancy, glioblastoma (GBM), yet local control rates, and hence survival, remain dismal for this disease. Even novel therapies, such as immunotherapy, have not shown efficacy in the treatment of GBM. Meanwhile, radiation dose escalation studies have demonstrated improved local control. However, dose escalated treatments are hindered by the increased incidence of radiation induced brain necrosis in surrounding tissues. High LET particle therapy holds the potential to both increase tumor cell kill and decrease normal tissue toxicity, yet the data required to develop models for clinical treatments regarding the biological effectiveness of high LET beams on normal brain tissue and GBM cells is sparse. This fact is especially true when considering results reported utilizing the appropriate environment for the origination and growth of GBM cells – the human brain. We have implemented recently developed high accuracy models which are truly beginning to recapitulate the native GBM niche in order to correlate both necrosis induction and progression and tumor cell response with the physical parameters of particle beams. These models include multi-cell type human brain organoids (cerebral organoids) as well as immune-competent orthotopic rodent models. Using these models, we will identify the physical factors of particle beams which may lead to necrosis. This is significant in that this data will aid the design of safer treatments by reducing necrosis and improving disease control. In the second component of our study, we will examine the molecular mechanisms of necrosis and neuroinflammation. Rather than being a simple accidental, disorganized death, we will determine if radiation induces an orderly programmed cell death pathway. Overall, we will conduct the following aims; (1) identify the optimal particle and fractionation for treatment of GBM, (2) explore the cellular and molecular mechanisms of radiation induced brain damage, and (3) develop biological effect models for clinical use. The knowledge gained will quickly influence the treatment of brain tumor patients and expedite the clinical introduction heavy ion therapy for glioblastoma.