Roswell Park Cancer Institute Corp

NIH Research Projects · FY 2025 · 2024-03

A developing body of preclinical evidence suggests that the integrity and composition of the tumor immune microenvironment (TIME) is highly plastic and is modulated by host lifestyle exposures. Unhealthful exposures including obesity, inactivity and a high fat diet can lead to a loss of CD8+ T cell effector function and tumor progression in preclinical breast tumor models, while healthful exposures, such as voluntary wheel running, can offset tumor growth and dysregulated conditions in the TIME leading to increased trafficking and enhanced func- tion of CD8+ T cells. Although emerging pre-clinical evidence is compelling, several gaps in epidemiological knowledge regarding the link among lifestyle factors, tumor immunity and BC outcomes remain. First, despite a suggestion to adhere to The American Institute for Cancer Research (AICR) and The Ameri- can Cancer Society (ACS) cancer prevention lifestyle guidelines after a cancer diagnosis, which lifestyle factors and whether they work together to impact BC outcomes has not been well established. Second, research on the role of lifestyles and immunity in BC patients has primarily focused on immune cell subsets in the circulating peripheral blood. To our knowledge, whether lifestyles are associated with distinct immune cell phenotypes in the breast TIME, the site most relevant for treatment response and BC prognosis, has remained uninvestigated. Building off the extant preclinical evidence and multiple lines of published and preliminary evidence from our group, we designed the current study to address these critical gaps in knowledge. Leveraging data from 1160 primary invasive BC patients enrolled in the Data Bank and BioRepository (DBBR) at Roswell Park, we devel- oped a composite lifestyle index score (LIS) reflecting adherence to seven cancer prevention recommendations from The AICR and The ACS including: 1) maintaining a healthy weight; 2) being physically active; 3) eating a variety of fruits and vegetables; 4) limiting red and processed meats; 5) limiting or avoiding sugar-sweetened beverages; 6) limiting or avoiding alcohol consumption; and 7) avoiding smoking. Our preliminary analyses re- veals that patients with the highest versus lowest LIS (e.g., tertiles) experience striking survival advantages. Based on these and other preliminary data, we formulated our central hypothesis that CD8+ T cell composition in the breast TIME mediates the observed association between the LIS and BC outcomes. To test this hypothesis, we will link detailed lifestyle data with multispectral immunohistochemical immune profiling from 1160 BC patients enrolled in DBBR with available FFPE tumor tissue to execute formal mediation pathway analyses via three Specific Aims. In aim 1, we will define associations of the LIS with CD8+ T cell composition in the breast TIME (cell densities, ratios and clusters of total CD8+ T cells, exhausted CD8+ T cells and activated CD8+ T cells). Next, in aim 2, we will identify associations of CD8+ T cell composition in the breast TIME with BC outcomes. In aim 3, we will delineate the contributions of CD8+ T cells in the TIME as biological intermediaries underlying the observed association between the LIS and BC outcomes.

NIH Research Projects · FY 2025 · 2024-03

Project Summary/Abstract Given the high mortality of castration-resistant prostate cancer (CRPC) as a result of relapse after androgen deprivation therapy (ADT), novel treatment strategies are urgently needed. Tumor suppressor gene- phosphatase and tensin homolog ( PTEN) is mutated in approximately 20% of primary prostate cancers, and in as many as 40~60% of CRPC. In addition to activation of PI3K oncogenic signaling pathway, loss of PTEN is also associated with cytokine and chemokine signaling that creates an immunosuppressive microenvironment. Both defects in antigen presentation in tumor cells and the immunosuppressive tumor microenvironment (TME) have been implicated in CRPC progression after ADT and therapeutic resistance in prostate cancer in the clinic. Genetic or pharmacologic inactivation of β isoform of PI3K (p110β) in PTEN-deficient prostate cancer has been reported to significantly inhibit tumor growth and progression. Ataxia telangiectasia and Rad3-related (ATR) exhibits essential functions in controlling DNA replication stress and DNA damage response in cancer cells. PTEN-deficient cells have been reported to be more sensitive to ATR inhibition. PI3Kβ is also functionally associated with DNA damage response and genomic integrity in tumor growth. By using a syngeneic genetically engineered mouse model of CRPC driven by co-deletion of PTEN and TP53 (PP-CRPC), we found that addition of p110β inhibitor significantly reduces the viability of ATR inhibitor-treated PP-CRPC cells. Moreover, our preliminary data show that inhibition of p110β or ATR alone significantly increases MHC class I antigen presentation in mouse or human CRPC cells. Based on these novel preliminary data, we hypothesize that p110β inhibitor in combination with ATR inhibitor will show promising anti-tumor activity in PTEN-deficient CRPC via inducing cell death in cancer cells and anti-tumor immunity in the tumor microenvironment. We also hypothesize that inhibition of p110β will increase DNA replication stress and sensitize PTEN-deficient CRPC cells to ATR inhibition. In this study, we will 1) evaluate the therapeutic efficacy and antitumor immune responses of combined ATR inhibition and p110β inhibition i n syngeneic PTEN-deficient CRPC mouse models; 2) investigate the molecular mechanisms of the combined effect of ATR inhibitor and p110β inhibitor in replication stress and DNA damage-induced cell death in PTEN-deficient CRPC cells. Our study will not only provide fundamental information for translational research of p110β inhibitors and ATR inhibitors in cancer treatment, but also provide a novel treatment strategy for patients with PTEN-deficient advanced CRPC.

NIH Research Projects · FY 2026 · 2023-12

Abstract. DNA-targeting drugs are a mainstream therapy for cancer, but it is becoming clearer that some of their anti-cancer activity is due not only to DNA damage, but also chromatin decondensation, which we call chromatin damage (CD). Moreover, it has been shown that some drugs cause only CD without DNA damage, and these "CD-only" drugs are as potent anti-cancer drugs as compounds causing both CD and DNA damage, but lack the toxicity associated with DNA damage. The focus of our study is to understand how CD activates IFN, and how this property can be used in the clinic to gauge the potency of CD drugs. Our ultimate goal of our research is to show that CD is a superior mechanism to DNA damage for the next generation of anti-cancer chemotherapy, which is more effective, less toxic, and more powerful in activating an anti-tumor immune response. To achieve this goal, we have developed a new group of "CD-only" chemicals, curaxins. The anti-cancer activity of curaxin clinical lead, CBL0137, has been demonstrated in multiple mouse models. Phase I trial has shown manageable toxicities and evidence of antitumor activity. These studies confirmed induction of IFN signaling by CBL0137 in mice and humans. Notably, curaxins cause stronger and faster IFN induction than DNA damaging agents, DNA methyltransferase inhibitors, and HDAC inhibitors. It is well established now that IFN induction significantly improves their anti-cancer efficacy of DNA damaging therapy. CD causes IFN induction via different mechanisms, but our data showed that IFN activation enhances anti-cancer efficacy of CD. Therefore, we propose that anti-cancer activity of CD agents is mediated in vivo via two complementary mechanisms: direct killing of tumor cells and engagement of anti-tumor immune attack. This proposal focuses on the hypothesis that IFN signaling in tumor and/or non-tumor cells is induced by CD via a mechanism different from DNA damage and potentiates the anti-cancer activity of CD drugs. We will focus on triple negative breast cancer (TNBC), a challenging disease to treat due to its aggressive behavior and lack of actionable targets. Our specific aims are: 1. Determine the impact of the IFN activation in tumor and non-tumor cells on the anti-tumor activity of CD therapy. 2. Define the mechanisms of IFN activation by CD agents. 3. Develop strategies for reversing the immune suppressive environment in triple-negative breast cancer using CD agents.

NIH Research Projects · FY 2025 · 2023-09

Abstract The current application seeks salary support for Aimee Stablewski, PhD who serves as the Director of the Gene Targeting and Transgenic Shared Resource (GeTT) of Roswell Park Comprehensive Cancer Center (Roswell Park). This position was created in 1999 to oversee the research applications of the various genome editing platforms available in the resource and to apply her extensive experience in these areas in collaborations and consultations with the GeTT investigator user base. With over 25 years of experience in research applications of the creation of genetically modified animals and cells (23 years at Roswell Park), Dr. Stablewski throughout her career has collaborated with many investigators at the Cancer Center on NIH funded projects. The GeTT is an integral part of Roswell Park's Cancer Center Support grant (P30CA016056) for which the cancer center Director, Dr. Johnson, serves as the Principal Investigator. All three CCSG programs (Developmental Therapeutics (DT), Tumor Immunology and Immunotherapy (TII) and Cancer Stress Biology (CSB) equally make up the user base of the GeTT; and numerous NIH-funded researchers in these programs actively collaborate and consult with Dr. Stablewski. Dr. Stablewski' s contributions to Roswell Parks research enterprise have been significant and impactful specifically with regards to the NIH-funded research of the members of the TII program. As of September 2022, there are thirty-nine grant applications that cited use of the GeTT shared resources. There were twenty-six NIH/ NCI, two DOD, and the remaining applications include private and public funding sponsors, eleven of whom were TII Program members, fourteen are CSB members and nine are DT members. The cancer center has also made significant investments in the GeTT with regards to advancing technology in genetic modifications. With the changes in the R50 program now making the distinction between Laboratory (PAR-20-288) and Core (PAR-20-287) based scientists and with the GeTT being an integral part of the Roswell Park CCSG infrastructure, Dr. Johnson as the PI of the CCSG grant, will now serve as the Unit Director for the application.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Immune checkpoint inhibitors (ICIs) have markedly transformed the therapeutic landscape for many types of advanced malignancies over the past decade. A sizable proportion of patients with advanced cancer derive durable benefits from ICIs and achieve longer periods of progression-free survival or remission than previously possible. Yet we still know little about the determinants of durable response to ICI treatment and the symptom trajectory and survivorship needs of this growing patient population. We thus propose a multi-institute study with two sister cohorts of patients living with advanced cancers. First, a retrospective EHR data-only cohort will include 8,860 patients with advanced disease, inclusive of all cancer types, who have been treated with ICI- based immunotherapy in 2014-2022. This large cohort will allow us to identify and study durable responders to ICIs, defined as patients who achieve partial or complete response to ICI treatment and live at least one year after ICI treatment initiation. Second, a prospective cohort will enroll and actively follow an estimated 1,200 patients with durable response to ICI treatment for advanced lung cancer, kidney cancer, and melanoma, the three most common cancers treated with ICIs. Clinical and patient-reported outcome data will be collected at baseline and every 6 months during follow up. This prospective cohort will allow us to study long-term survival and physical and psychosocial symptom trajectories in patients with durable response to ICIs, and to identify clinical and modifiable behavioral factors predictive of long-term survival and common side effects of ICI treatment. The predictors identified in these analyses will be independently validated in the DiRECT Cohort, a large ongoing study of racial disparities in ICI treatment led by the study team. Our Specific Aims are: 1. In the retrospective EHR data-only cohort, 1a) Determine the proportion of patients who had durable response to ICI treatment (partial/complete response and alive ≥1 year since initial ICI treatment) and chart their survival trajectory; 1b) Identify clinical predictors for durable response to ICI treatment; 1c). In the independent DiRECT Cohort, validate the clinical predictors for durable response to ICI treatment. 2. In the prospective cohort, 2a) Identify long-term survival and longitudinal trajectories of patients' physical and psychosocial symptoms after ICI treatment; 2b) Investigate the relationships of long-term survival and common side effects from ICI treatment with multidimensional predictors; 2c). In the independent DiRECT Cohort, validate the predictors for survival and common side effects in patients with durable response. Findings from our study will provide much-needed data that can inform new evidence-based intervention strategies as the next step to optimize survivorship care and extend and improve quality of life for the growing population of survivors living after a diagnosis of advanced cancer due to ICI treatment.

NIH Research Projects · FY 2025 · 2023-09

Project Abstract/Summary For decades, animals in biomedical research have yielded significant scientific and medical breakthroughs by generating the essential preclinical data that ultimately support the discovery and development of treatments for human diseases, including cancer. However, while we to rely on animal models to investigate the complexity of cancer and cancer therapies, these preclinical studies have alarmingly low success in reproducibility, and even lower preclinical-to-clinical success rates. As per the Guide for the Care and Use of Laboratory Animals 8th Edition, research institutions have standardized, minimum guidelines for the housing, husbandry, and overall care for laboratory animals that they must adhere to. A mildly cool ambient temperature is a critical aspect of animal housing that has been shown to elicit significant physiological changes to research rodents, driven by the activation of the sympathetic nervous system and increased β- adrenergic receptor (β-AR) signaling as a result of the systemic release of norepinephrine. This is due to the compensatory response, known as non-shivering thermogenesis, employed by rodents housed at temperatures that fall below their thermoneutral zone (which is the range of ambient temperatures at which heat generated by basal metabolism is sufficient for maintaining homeostatic core temperature. ) Our lab has previously established that standard (ST), subthermoneutral laboratory housing temperatures result in significant impairment to the murine CD8+ T cell-dependent anti-tumor immune response compared to mice house at thermoneutral temperatures (TT). Additionally, we have shown that the immune checkpoint inhibitor αPD-1, an immunotherapy that has recently seen success as a front-line approach to treating cancers like melanoma, has improved efficacy in treating tumor-bearing mice housed at TT in a β-AR-dependent manor. Although published and preliminary data indicate a role for the co-receptor, CD28, in the diminished anti-tumor function of CD8+ T cells as a result of increased β-AR signaling, a gap exists in our understanding of the mechanisms underlying the reduced CD8+ T cell activation and effector function in mice housed at ST. Therefore, we propose using genetically engineered mouse models to precisely interrogate CD28 signaling and test hypothesis that standard housing temperatures impairs CD8+ T cell anti-tumor immunity and the in vivo efficacy of αPD-1 via impaired CD28 co-stimulation. We will use in vitro and in vivo approaches to examine the effects of housing temperature on CD8+ T cell CD28 expression and signaling, as well as tumor- infiltrating lymphocytes in mice treated with αPD-1 therapy. The studies outline in this proposal have the potential to identify a previously undefined mechanism by which subthermoneutral laboratory animal housing temperatures influence experimental outcomes of cancer and immunotherapy models, while also characterizing a widely underappreciated variable that exists in our animal models.

NIH Research Projects · FY 2025 · 2023-09

Project Summary / Abstract: In preclinical models, norepinephrine released by sympathetic nerves during chronic stress have been demonstrated to promote an immunosuppressive tumor microenvironment (TME) through activation of the β2- adrenergic receptor (β2AR) on various cells including immune cells. This sequence of events could be detrimental to the treatment outcome in cancer patients who are experiencing increased levels of stress. Innate lymphoid cells (ILCs), specifically type II ILCs (ILC2s), have been demonstrated as a small but critical cell population within the TME. However, there is little known about the mechanisms that regulate ILC plasticity and function in the TME. Even though ILC2s express high levels of β2AR, how stress impacts ILC2 activity within the TME is not yet known. In new preliminary data, we have observed a correlation between increased ILC2s and decreased tumor volume in knock out mice lacking the β2AR. Furthermore, our data suggests a shift in ILC2 plasticity toward an anti-tumor phenotype upon loss of β2AR signaling by single-cell RNA sequencing. Therefore, we hypothesize that β2AR signaling activated by chronic stress drives the immunosuppressive function of ILC2s, suppressing the anti-tumor immune response within the tumor microenvironment. We will interrogate the role β2AR signaling plays as a rheostat in ILC development and plasticity into the helper ILC subsets by the two aims proposed here. In Aim 1, we will determine the effect of β2AR signaling on common lymphoid progenitors (CLPs) differentiation into ILC2s and ILC2 plasticity. Utilizing in vitro cultures of wildtype and β2AR-/- CLPs and ILC2s with a β-AR agonist treatment, we will analyze the changes in ILC subset ratios due to β2AR signaling. We will also elucidate the molecular mechanism by which these changes occur using both targeted and high-throughput methodologies. In Aim 2, we will determine the impact of β2AR signaling on ILC2-mediated tumor progression using IL-5creβ2-ARfl/fl conditional knockout mice. The changes in the TME and tumor growth in mice with a conditional β2AR knock out in ILC2 will be analyzed using spectral flow cytometry. Overall, this project will utilize in vitro and in vivo models and other state-of-the-art techniques to understand how chronic stress through β2- AR signaling hampers effective ant-tumor immune response in tumor microenvironment.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Genetic alterations in the tumor suppressor p53 gene (TP53) are found in most colorectal adenocarcinomas (CRC) and contributes to poor prognosis. The p53 protein encoded by TP53 is a key element of DNA damage checkpoints that are activated by DNA damage response and control genome integrity. Although decades of research generated immense information on the functional consequences of p53 mutations, therapeutic efforts targeted to mutant p53 have proven largely unfruitful. Existing therapeutic options for p53-deficient CRC are ineffective and cause toxic side effects stressing the need for better therapeutics. We developed a conceptually novel treatment strategy for selectively targeting p53-deficient cancer cells that takes advantage of their unique DNA repair deficiencies. Our preclinical research revealed that p53-deficient tumors accumulate DNA damage upon incorporation into DNA of a thymidine analogue (i.e., trifluorothymidine, a component of FDA-approved drug called TAS102). The thymidine analogue does not interrupt DNA replication but rather prompts DNA repair that requires p53-dependent checkpoint. We found that p53-deficient cells lacking the p53-dependent checkpoint selectively accumulate DNA breaks. Importantly, this DNA damage is strongly enhanced by inhibitors of poly (ADP) ribose polymerase (PARP) leading to cell death. This novel inducer- amplifier strategy was extensively validated in multiple preclinical models. Our preclinical data demonstrated a superior anti-tumor activity of TAS102 in combination with PARP inhibitor (PARPi) compared to either drug alone in p53-deficient cancer models. Based on our preclinical data, we developed a first-in-human Phase I clinical trial for advanced CRC with two FDA-approved drugs TAS102 and PARPi talazoparib. Thus, we hypothesize that the combination of TAS102 with PARPi talazoparib is an effective biomarker-driven treatment for patients with p53-deficient CRC. The current proposal is aimed to define the efficacy of our combination therapy strategy for the first time in humans with advanced CRC in a collaborative effort of basic, translational, and clinical scientists. The study will generate the biomarkers to guide clinical implementation and further development of our inducer-amplifier strategy by using patient-derived material from our ongoing clinical trial. The study will employ state-of-the art next-generation sequencing approaches to define genome-wide changes in response to the TAS102-PARPi combination in CRC models. Importantly, we will examine the antitumor efficacy of our novel two-drug therapy in p53-deficient CRC patients. Together, this work will provide mechanistic insights in the action of our two-drug therapy and will serve as a platform for development of better treatments. Our study matters for thousands of patients with aggressive p53-deficient CRC.

NIH Research Projects · FY 2025 · 2023-07

Pancreatic ductal adenocarcinoma (PDAC) represents a therapy recalcitrant disease with 5-year survival rate of approximately 10%. Multiple failed clinical trials reinforce the need for new approaches to treatment that employ rationally developed therapies targeting key genetic features of PDAC tumors as well as the tumor microenvironment. Fundamentally PDAC is a KRAS-driven tumor; however, approaches to target KRAS either genetically or pharmaceutically have led to the realization that tumors can evolve mechanisms to continue cell division in the presence of such interventions. Using a combination of unbiased analyses and patient derived models we find that most tumors have evolved mechanisms to deregulate the retinoblastoma tumors suppressor (RB) pathway that limits the effectiveness of targeting RAS or effector pathways (e.g. MEK inhibition). Consequently, the activation of RB is sufficient to limit the proliferation and tumorigenic growth of PDAC models. In addition to effects on the proliferation, we have found that RB activation is sufficient to coordinate changes in gene expression that impact on genes related to classical vs. basal subtypes of PDAC and immunological gene expression programs (e.g. antigen presentation and interferon response). Unlike cell cycle responses, these changes in gene expression are more heterogeneous and the underlying mechanisms and key regulatory elements remain poorly understood. In Aim 1 we will delineate the RB regulation and mechanisms of gene expression regulation in PDAC models. While RB status clearly has effects on tumor cell division, an important element of RB-pathway activation are effects related to the tumor microenvironment. Preliminary and published data indicate that RB activation has broad effects on the tumor stroma and immunological milieu. Much of these studies have employed systemic strategies that impact on both the tumor and the host. In preliminary data, we have found that activation of RB selectively within the tumor is sufficient to change the fibroblastic and immunological cell subtypes with the tumor, albeit not at the same level of systemic treatments. Here we will interrogate the intersection between the tumor and the microenvironment and how these impact tumor-static response and can be exploited by immunotherapy approaches. Together, Aim 2 will define the impact of RB- activation on the tumor microenvironment using a combination of immune competent models and clinical specimens.

NIH Research Projects · FY 2025 · 2023-06

The liver is a frequent site of metastasis for several cancers and when this occurs, it is associated with poor response to immunotherapy. Liver-directed radiotherapy (RT) is a non-traditional immune modulating therapy that improves immune checkpoint blockade (ICB, αPD-1 or αPD-1/αCTLA-4) mediated control of liver metastasis and non-irradiated abscopal tumors. Unfortunately, enhanced tumor control following liver-directed radiotherapy and ICB is often short-lived for irradiated tumors and unpredictable for non-irradiated tumors. Thus, novel immunotherapeutic approaches that can stimulate antitumor immunity in the liver have the potential to boost liver-directed RT plus ICB mediated control of irradiated and non-irradiated abscopal tumors. Toward this goal, we are harnessing the ability of the toll-like receptor (TLR) 5 pathway to signal in the liver more so than other sites. Hepatocytes sense Salmonella flagellin and our derivative called entolimod through cell surface TLR5 and cytoplasmic NAIP5 inflammasome. Importantly, systemically administered entolimod was shown to be safe in rodents, non-human primates, and Phase I safety trials in healthy volunteers and cancer patients cumulatively involving nearly 200 subjects. Our prior work showed that entolimod protects normal tissues but not tumors from radiation toxicities and stimulates CD8+ T cell dependent antitumor immunity against preclinical models of liver metastasis. Here, we showed that entolimod enhances liver-directed RT in pre-clinical models mirroring advanced liver metastasis and that this occurs via a poorly resolved Nφ dependent mechanism. The outstanding questions that this proposal seeks to address are: 1) what is the impact of entolimod on liver-directed RT plus ICB mediated control of liver metastasis and abscopal tumors?; 2) how does the TLR5-NAIP5 pathway in Nφ support antitumor immunity post liver-directed RT and ICB?; and 3) what is the translational relevance of the TLR5-NAIP5 pathway plus RT in human tumors and Nφ? Our central hypothesis is that entolimod enhances liver-directed RT plus ICB via Nφ-dependent release of IFN-γ, a cytokine that can be released by activated Nφ to support antitumor immunity. To test our central hypothesis, we propose three interactive aims: 1) To unveil the impact of entolimod on liver-directed RT plus ICB mediated control of liver metastasis and abscopal tumors; 2) To elucidate the mechanism by which the TLR5-NAIP5 pathway in Nφ triggers antitumor immunity post liver- directed RT and ICB; and 3) To determine the translational relevance of the TLR5-NAIP5 pathway plus RT in fresh human colorectal cancer and melanoma samples and patient-matched Nφ. The impact of this work lies in the fact that all components of this therapy are in the clinic for cancer patients thereby ensuring the feasibility to combine these components into a novel and innovative therapy to improve the dire survival outcomes associated with liver metastasis.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY / ABSTRACT DNA replication follows a defined spatiotemporal program in which different parts of the genome replicate at different times during S phase. The DNA replication timing program interfaces with genome regulation while also influencing the genome’s stability and mutational landscape. Abnormal replication timing is associated with genetic diseases and oncogenesis. Despite the importance of DNA replication regulation to cell and organismal biology, we have very limited understanding of the DNA sequences and molecular mechanisms that specify the eukaryotic DNA replication timing program. Similarly, it remains unclear how replication timing control intersects with the regulation of gene expression and with the epigenome in general. My lab has, and continues to, develop novel experimental and computational approaches for measuring genome-wide replication timing. We also pioneered a unique approach of linking replication timing to human genetic variation in order to reveal the sequence elements that control replication timing and study their mechanisms of action (Koren et al., Cell 2014; Ding et al., Nature Communications 2021; NIH Director’s New Innovator Award DP2- GM123495). In this MIRA application, we will: 1) generate a new dataset of human replication timing, the largest so far, and use a computational approach to link replication timing to gene expression and epigenomic regulation in a novel and powerful way. 2) Utilize our previous and concurrent replication timing genetic mapping in order to experimentally edit DNA sequences controlling replication timing. This will be followed by several epigenomic assays and genetic experiments that will reveal the mechanisms and consequences of replication timing regulation. As part of this, we will also develop a new approach, “perturb-RT”, to systematically screen for novel sequence elements controlling replication timing. 3) Continue our recent success of profiling replication timing in single cells by jointly measuring replication timing and gene expression in the same single cells. We will use this approach to study how replication timing and gene expression co-vary during cellular differentiation. Our work will substantially contribute to the field of DNA replication timing and beyond, by providing unprecedented genomic resources, introducing novel genetic and genomic approaches, deciphering the genetic basis of human replication timing, and revealing new insights into replication timing regulation and its consequences.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Overexpression of Cyclin D1 (CCND1) is recognized as a major resistance mechanism to HER2 targeting agents. Patients with CCND1 amplification have the worst response to trastuzumab-containing therapy, and CCND1 overexpression is enough to render cancer cells insensitive to HER2 inactivation. Furthermore, tumors that evade HER2 targeted therapy by reducing HER2 expression rely on CCND1 overexpression and are extremely sensitive to its ablation. Thus, targeting CCND1 is a promising strategy for overcoming resistance to HER2-targeting agents in breast cancer patients. Although CCND1 itself is not a druggable target, this protein has a rapid turnover, and its steady-state level in cells is tightly regulated by ubiquitin-mediated degradation. Perturbation of CCND1 deubiquitination accelerates its degradation. We have recently discovered a novel, previously uncharacterized deubiquitinating enzyme – USP27X, whose higher expression significantly correlates with poor survival of HER2 positive breast cancer patients. Importantly, our studies identify USP27X as a critical regulator of CCND1 protein stability in HER2 therapy-resistant breast cancer cells. Ablation of USP27X severely reduces xenograft tumor growth and significantly increases cell sensitivity to HER2 and CDK4/6 inhibitors. We hypothesize that USP27X can be a key therapeutic target in HER2 therapy-resistant or recurrent breast cancer through CCND1 degradation. This project focuses on understanding the functions of USP27X in CCND1 regulation and elucidating the roles of this deubiquitinating enzyme in cancer development and progression. We will utilize several engineered cell lines as well as a novel and clinically relevant mouse model carrying a conditional Usp27X allele to 1) Elucidate the molecular mechanism underlying the reduced CCND1 steady-state levels in USP27X ablated cells 2) Determine how the loss of USP27X will affect tumor development and progression in vivo, and 3) Define how abrogation of USP27X activity alters the therapeutic response of HER2 therapy-resistant cells to HER2 inhibition. USP27X is a druggable target, and our studies will illuminate new avenues for therapeutic intervention in HER2 therapy-resistant and CCND1 dependent cancers. Although there are no USP27X specific inhibitors currently available, the studies proposed in this project are essential for establishing this deubiquitinating enzyme as a drug target as they will provide a rationale for developing USP27X specific inhibitors.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Post-transplant cyclophosphamide (PTCy) has substantially reduced the risk of lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (alloHCT). However, like other forms of GVHD prophylaxis, PTCy still relies upon passive graft-versus-leukemia (GVL) to prevent disease relapse. Progression-free survival after alloHCT is largely limited to 41-50%. Thus, concurrent GVHD and leukemia relapse prevention remains a critical unmet need in transplantation. Innovation in alloHCT must now maintain GVHD prevention at the level of PTCy and add tools to directly reduce relapse and not simply maintain or preserve GVL. Distinct from pharmacologic immune suppression, we have developed novel, human, CD83-targeted chimeric antigen receptor (CAR) T cells for concurrent protection against relapse of CD83+ leukemia as well as GVHD. CD83 is a member of the immunoglobulin superfamily. We show CD83 is expressed on human myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and alloreactive T cells implicated in GVHD. Importantly, we have demonstrated that CD83 CAR T cells kill leukemia in vivo and eradicate GVHD without impairing antiviral immunity. Further, CD83 is largely absent from hematopoietic stem cells, myeloid progenitors, and neutrophils, limiting the risk for on-target/off-tumor toxicity or myeloid aplasia. We also developed an ‘OR’ logic gated CD19/CD83 CAR T that can kill B cell ALL that expresses either CD19 OR CD83 via a shared activating endodomain. We show that our ‘OR’ gated CD19/CD83 CAR T can overcome CD19 antigen loss, which is clinically seen in 25% of ALL patients after treatment with CD19 mono- CAR T. In this application, we will (Aim 1, mouse experiments) test whether human CD83-targeted CAR T cells can concurrently prevent leukemia relapse and GVHD, as compared to standard PTCy. To parallel expected initial clinical trials, we will also investigate the sequential use of CD83 CAR T consolidation after PTCy to eliminate leukemia relapse and GVHD. Our preliminary data demonstrates that CD83 is expressed on CD4+ conventional T cells during acute GVHD, as well as B cells and T helper follicular cells during chronic GVHD. Thus, (Aim 2, biomarker validation study) we will investigate whether CD83 is a biomarker and therapeutic target among effectors of acute and chronic GVHD onset and therapeutic response. Successful completion of this work will guide the seamless transition from discovery to clinical translation of human CD83 CAR T cells to concurrently eliminate life-threatening relapse and GVHD after alloHCT.

NIH Research Projects · FY 2026 · 2023-02

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers with a survival rate of 11% due to its aggressive nature and resistance to therapies. While nearly all PDA cases are driven by mutations in the KRAS gene, efforts to target KRAS or its effectors (e.g., MEK and ERK) are met with adaptive resistance. Pancreatic cancer stem cells (PCSCs), a subpopulation of transcriptionally-plastic cancer cells that are especially drug resistant and particularly tumorigenic, are a critical component of the aggressive and therapy- resistant nature of PDA. There are currently no strategies to target PCSCs, as well as a lack of information regarding their drivers. We previously identified HNF1A, a gastrointestinal-lineage transcription factor, as a novel master regulator of the PCSC state. Our preliminary data show that HNF1A expression can be potently blocked by BET-inhibitors (BETi), a class of drugs which inhibit the epigenetic reader protein BRD4. Interestingly, re-expression of HNF1A rescues cell cycle progression and PCSC-properties in BETi-treated PDA cells, suggesting that HNF1A is a novel and critical target for these drugs. We have also found that HNF1A is a novel driver of resistance to targeting KRAS and downstream MEK/ERK-signaling. Importantly, the use of BETi in combination with MEK- and ERK-inhibitors (MEKi/ERKi) increases growth arrest and cell death in an HNF1A-dependent manner. We hypothesize that HNF1A is directly regulated by BRD4, and is therefore targetable with BETi, and that the inhibition of HNF1A expression with BETi will nullify HNF1A-dependent PCSCs and adaptive resistance to KRAS-ablation. In this proposal, we aim to characterize the regulation of HNF1A and its role in therapeutic response and resistance. In Specific Aim 1, we will characterize the regulation of HNF1A by BETi-target BRD4. Specific Aim 1 will combine genetic manipulation of BRD4, ChIP- PCR, and reporter assays to demonstrate regulation of HNF1A by BRD4. Re-expression of HNF1A in BRD4- depleted cells will 1) define the role of HNF1A in BRD4-mediated cell growth and survival in vitro and in vivo, as well as 2) determine the contribution of HNF1A to the BRD4 transcriptome using RNA-seq/ChIP-seq to identify how HNF1A determines response to BETi. In Specific Aim 2, we will establish BETi as a means to overcome HNF1A-mediated resistance to KRAS-ablation. Specific Aim 2 will use PDA cells with and without ectopic HNF1A expression to examine the contribution of HNF1A to BETi and KRAS-pathway inhibitor activity; we will utilize in vitro and in vivo assays to examine how these drugs affect PCSCs and use RNA-seq/ChIP- seq to determine how HNF1A promotes resistance to targeting KRAS-signaling. Both aims will utilize next- generation bromodomain-selective inhibitors that have not been explored in PDA or combinatorial therapies. The completion of the above studies will dramatically improve our understanding of PDA biology and uncover novel therapeutic targets. By improving our understanding of resistance to KRAS suppression and expanding the therapeutic spectrum of PDA to include PCSCs, more effective treatment of PDA can be achieved.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY There is a growing trend in the use of vaping devices used to administer liquid concentrates containing Δ9- tetrahydrocannabinol (THC). However, there is a gap in our knowledge and understanding of the pharmacokinetics (PK) and pharmacodynamics (PD) of THC delivered from this new generation of cannabis products. The literature on nicotine vaping has clearly shown that vaping product characteristics and user behaviors influence the PK/PD of vaped substances. Additionally, most cannabis consumers who vape liquid cannabis concentrates also report concurrent use of nicotine-containing vaping products (e-cigarettes). Building on strategies used to establish the evidence base on the PK/PD of nicotine-containing vaping products, this project will examine the PK/PD profile of THC-containing liquid concentrates delivered through vaping devices WITH and WITHOUT co-use of nicotine through completion of three specific aims: Aim 1 is to examine how vaping product characteristics and user behaviors impact the PK/PD of THC. Aim 1 will characterize the PK/PD variability in real-world settings. This will be achieved by using a mobile laboratory bus to recruit current vapers of THC-containing liquids and examine their PK/PD. Study participants will use their own commercially obtained THC cartridges ad lib through their usual vaping device. In Aim 2, a controlled laboratory paradigm will be used to compare the PK/PD profiles of equivalent standard THC units delivered from vaping liquids vs smoked cannabis. Aim 3 will assess the PK/PD profiles of THC vaping liquids sequentially administered with nicotine using a double-blind, placebo-controlled, randomized within-subjects trial design. This highly innovative approach will offer a significant advantage over the existing literature by combining naturalistic observational methods and controlled laboratory assessments to provide a depiction of the PK/PD of THC delivered through vaping liquids. The findings will be directly relevant for the existing cannabis market and consumers who use these products. This project will improve our understanding of the PK/PD effects of THC by characterizing the PK/PD profiles of THC vaping liquid formulations and devices used by cannabis consumers, establishing differences in the effects of these products relative to smoked cannabis, and providing necessary data on how co-use of vaped nicotine and THC may impact THC PK/PD. Data from this project will be highly relevant for informing future regulatory and consumer safety efforts for THC vaping products.

NIH Research Projects · FY 2025 · 2022-09

In the United States, Down syndrome occurs in approximately one in every 691 newborns and presents with a constellation of clinical manifestations. It results from human trisomy 21, the most common chromosomal abnormality associated with intellectual disabilities. Currently, there are no effective treatments for the intellectual disabilities linked to Down syndrome, underscoring the urgent need for innovative strategies to develop therapeutic interventions. The mouse remains the premier model organism for studying Down syndrome due to the presence of highly conserved orthologous regions between human chromosome 21 and three mouse genomic segments located on chromosomes 10, 16, and 17. A prevailing hypothesis in Down syndrome research posits that its phenotypes may be causally linked to dosage increases of genes located on human chromosome 21. In line with this hypothesis, extensive efforts have focused on generating and analyzing mouse models that carry an extra copy of these orthologous regions—typically achieved by duplicating the corresponding segments in the mouse genome. These duplication models are often combined with mice carrying targeted deletions of smaller genomic intervals within the duplicated regions to help identify critical dosage-sensitive loci. Progress in this field has been greatly accelerated by advances in Cre/loxP-mediated chromosome engineering technology. To generate long-range chromosomal duplications and deletions in mice via Cre/loxP-mediated recombination, two independent gene targeting events must be performed in mouse embryonic stem cells, followed by Cre-mediated recombination. To support this process, several genetic cassettes are employed, including those encoding antibiotic resistance. In this supplement project, we aim to re-examine an underexplored area of knowledge related to chromosome engineering technology. The success of our efforts will have significant implications for Down syndrome research and broader applications in genetic modeling and analysis.

NIH Research Projects · FY 2025 · 2022-09

Project Summary The goal of our proposed Coordinating and Data Management Center (CDMC) application is to coordinate the activities across the Acquired Resistance to Therapy Network (ARTNet), and to manage, integrate and disseminate the data and resources generated through the network. Leveraging cutting-edge multi-disciplinary team approaches, we will support the ARTNet to inform new strategies that can be better translated to overcome significant challenges in acquired resistance to cancer therapies. Our strategy is to enhance the productivity of ARTNet investigators by fostering a collaborative and supportive research community, accelerate the progress of ARTNet research by reducing barriers to accessing analytical expertise, ensure the reproducibility of ARTNet data by deploying best practices for data acquisition and harmonization, and unleash the full potential of ARTNet activities by developing enhanced tools to enable resource sharing to the broader scientific community. First, we will provide a centralized administrative infrastructure to coordinate ARTNet activities, building upon our well-functioning infrastructure that currently coordinates network studies under the umbrella of the NCI Cancer Moonshot initiatives. Second, we will actively promote the ARTNet and engage in trans-consortium interactions, where we will leverage our demonstrable experience in Cross-Moonshot outreach and Bioconductor’s decades-long record in community engagement. Third, we will provide multidisciplinary analytical expertise to support ARTNet collaborative research, leveraging five of Roswell Park CCSG’s shared resources: Biostatistics, Bioinformatics, Biomedical Informatics, Pharmacokinetics/Pharmacodynamics, and Data Bank and BioRepository. Our analytical support will be provided at no cost to ARTNet investigators in need of analytical expertise, based on collaboration, transparency, and sharing. Fourth, we will develop improved data integration software and workflows to enhance ARTNet’s research capacity, capitalizing on our extensive track-record in developing NIH-supported Moonshot, Bioconductor and AnVIL ecosystems. The main deliverables from the proposed aims will be administrative and outreach support to coordinate network activities, facilitate network collaboration, and engage in interaction with the broader community (Aim 1); polices and infrastructures to ensure that all resources generated by the ARTNet will be findable in a centralized virtual resource sharing repository, and that all resources will be shared with the broader scientific community (Aim 2); workflows to ensure that all data generated by the ARTNet will be harmonized using standards interoperable with the broader cancer data ecosystem, analysis tools to integrate ARTNet data and facilitate cross-study analysis within and beyond the ARTNet, and multidisciplinary analytical supports to accelerate ARTNet research progress.(Aim 3).

NIH Research Projects · FY 2025 · 2022-08

Abstract Epithelial ovarian cancer (OC) is the leading cause of death from gynecological malignancies in the United States. While OC is immunogenic and increased infiltration of cytotoxic T lymphocytes (CTL) correlates with longer survival, single agent checkpoint inhibitors are largely ineffective in the relapsed/refractory setting. Our long-term goal is to develop novel approaches to overcome obstacles to durable antitumor immunity to make immunotherapy more effective. Our results point to previously unrecognized mechanisms for mature neutrophils acquiring a suppressor phenotype within the tumor microenvironment (TME). Neutrophil suppressors inhibited stimulated proliferation of circulating naïve, central memory, and effector memory T cells, and of tumor-associated lymphocytes (TAL) from patients with newly diagnosed OC. Induction of the neutrophil suppressor phenotype required complement signaling and NADPH oxidase activation. A similar complement- dependent neutrophil suppressor phenotype was induced by ascites from patients with recurrent OC and from malignant effusions from patients with a number of metastatic cancers, underscoring the generalizability of our findings. These results in human samples and work by others in tumor-bearing mice support targeting complement to enhance anti-tumor immunity. We will evaluate APL-2, a peptide C3 inhibitor, in a randomized phase 2 clinical trial in patients with recurrent OC and persistent malignant effusions. APL-2 was safe and superior to eculizumab for paroxysmal nocturnal hemoglobinuria (PNH) and is FDA-approved for this indication; it’s use in cancer is novel. Following a safety lead-in, patients will be randomized to the following cohorts: (i) bevacizumab (anti-VEGF); (ii) APL-2 + pembrolizumab (anti-PD1); and (iii) APL-2 + pembrolizumab + bevacizumab. Specific Aim 1: To evaluate safety and control of malignant effusions as primary endpoints and progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), overall survival (OS), and quality of life (QoL) as secondary endpoints. Specific Aim 2: To determine the effects of APL-2-based therapy in modulation of immune responses in the TME. Patient samples (blood, effusion, tumor) will be collected at baseline and at pre-specified time points during the trial. We will test the extent that APL-2 will abrogate neutrophil suppressor activity and expand activated T cells in the TME. A combination of functional studies and transcriptional and mass cytometry profiling of circulating and ascites neutrophils and T cells will be performed. The impact of our proposal is to gain safety and preliminary efficacy data on APL-2- based regimens in recurrent OC with malignant effusions and a detailed understanding of how these regimens modulate the immune landscape in the TME. This research is expected to establish the foundation for larger randomized trials to definitively test the efficacy of C3 inhibition in enhancing cancer immunotherapy.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Some of the largest disparities in human papillomavirus vaccination (HPVV) occur in rural communities, which may represent missed opportunities for cancer prevention since incidence of HPV associated cancers have been increasing in rural areas in the U.S. While there are evidence-based strategies to improve adolescent HPVV for cancer prevention in clinical practices, current gaps include a lack of tools available for community-based practices and rural communities. Targeted approaches are needed by rural community-based primary care practices to increase HPVV for cancer prevention and meet the needs of low-resourced rural communities. To address this gap, we developed an implementation science focused strategies for rural primary care practices (PC TEACH) that considers community, culture, and health systems as upstream factors in adolescent HPVV for cancer prevention by engaging the entire medical team using a practice facilitation approach. In the current proposal, we will test the effectiveness of our innovative practice-level approach (PC TEACH) to address: (i) HPVV for cancer prevention in adolescent boys and girls, and (ii) the average vaccination age in primary care practices. We hypothesize that HPVV for cancer prevention will depend on practice facilitation of evidence-based PC TEACH strategies. We will use a stepped-wedge cluster randomized trial design, which conserves sample size while maintaining power, with an integrated implementation framework, and process and outcome evaluation, to address the following aims: Aim 1 - Implement and test the effectiveness of practice facilitation of PC TEACH, using an implementation science approach, on HPVV for cancer prevention in rural community-based primary care practices. Aim 2 - Measure and monitor practice-level characteristics (e.g., size, scope, socioeconomic, and vaccination experiences during the COVID-19 pandemic), and patient-level characteristics (e.g., sex, age, insurance type, HPV knowledge, and child vaccination history), to identify factors that influence HPVV for cancer prevention. Aim 3 - Evaluate the extent to which practice facilitation activities most readily lead to adoption and implementation of the evidence-based PC TEACH strategies as delivered by the primary care practice site staff. The proposed study is innovative in that our use of community-engaged methods enhance scalability of the implementation science intervention in low-resource rural primary care settings to enable system changes related to HPVV for cancer prevention. Findings from this study will be essential in understanding how to implement systematic practice-level changes in primary care practices in rural communities where there is increasing incidence of HPV-associated cancers.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Dissecting the Mechanism of SETDB1 and its K867 Monoubiquitination in Lung Cancer Progression Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality worldwide and associated with high mutation burdens. During the last decade, biomarker-driven targeted therapies have achieved clear clinical benefits in patients with several oncogenic driver mutations such as EGFR, BRAF and ALK. However, the treatments for tumors with the most prevalent KRAS mutations remain challenging despite of recent breakthrough in targeting KRAS-G12C mutation. In KRAS mutant NSCLC, the constitutively activated downstream signaling ultimately leads to transcription reprogramming to sustain tumor growth and progression. This process is orchestrated by a cohort of crucial transcription factors, whose temporal and spatial expression is associated with distinct epigenetic changes. Thus, dissecting epigenetic mechanisms in this process could open new avenues for targeting strategies. SETDB1 is a principal methyltransferase catalyzing histone H3K9 methylation, a major repressive epigenetic modification. The focal amplification and upregulation of SETDB1 have been observed in a wide range of cancers including NSCLC, suggesting it has pro-oncogenic function. However, the mechanisms of action of SETDB1 in NSCLC remain elusive. In preliminary study, we uncovered that SETDB1 upregulation in lung adenocarcinoma (ADC) correlates with poorer prognosis and significantly co-occurs with KRAS gene alternations. In NSCLC cells harboring oncogenic RAS, SETDB1 loss attenuates migration and invasion, invadopodia formation and ECM degradation. These phenotypic changes correlate with reactivation of transcription factors FOXA2 and CDX2 which function as key regulatory nodes to impede metastatic progression of KRAS mutant lung ADC. More importantly, Setdb1 loss in tumor cells derived from metastatic KrasG12D;p53fl/fl lung ADC abolished their spontaneous ability to metastasize from the subcutaneous tumor to the lungs. These findings argue for a novel concept that SETDB1-mediated epigenetic mechanisms are critical for oncogenic KRAS-induced transcription reprogramming during lung ADC progression. Moreover, we have demonstrated that SETDB1 is monoubiquitinated at lysine-867 by the UBE2E family of E2 enzymes independent of E3s and this monoubiquitination is essential for SETDB1-mediated H3K9 methylation and ERV silencing. Accordingly, one objective of this proposal is to delineate SETDB1's pro-metastatic activity in KRAS mutant lung ADC using various genetically engineered mouse models and xenograft models. Another objective is to dissect the mechanisms of lysine-867 monoubiquitination in SETDB1's pro-metastatic activity in vivo and in vitro. Our central hypothesis is that SETDB1 plays important roles in KRAS mutant lung ADC progression through epigenetic mechanisms that require its monoubiquitination dependent enzymatic activity. This project is significant because it will characterize SETDB1 as an actionable drug target in KRAS mutant lung cancer and identify novel molecular interfaces for targeting. Our long-term goal is to develop better and more effective therapeutic strategies by delineating epigenetic mechanisms in lung cancer.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT: In the classical mammalian cell cycle model, CDK and cyclin complexes are responsible for driving specific events in a sequential fashion. Mitogenic or oncogenic signals drive the activation of CDK4/6 complexes that initiate cell cycle progression. These complexes promote RB phosphorylation that leads to the expression of a highly conserved cadre of genes that are required for progression through the remainder of the cell cycle. The concept put forward by this model is that cell cycle control is linear and highly predictable. However, recent findings related to the inter-dependencies of CDK/cyclins illustrate the need for better understanding the cell cycle repertoires that are operable in tumors. In preliminary data using unbiased and targeted approaches we have interrogated the extent to which the “utopian” simple version of the cell cycle breaks-down. This work indicates that in cancer models there are multiple different cell cycle modes, which have significance for tumorigenic proliferation and therapeutic interventions. Here we will take an integrated approach to fundamentally understand “dystopian” cell cycle states (Aim 1) and define mechanisms of collateral therapeutic resistance and new vulnerabilities (Aim 2) which associate with non-canonical cell cycle states.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY Major depressive disorder and other mood disorders are heterogeneous, debilitating illnesses that affect millions of individuals worldwide. Antidepressants that target the serotonergic system (e.g., SSRIs) remain the most effective treatment for these disorders and are widely used. However, emerging data show that maternal antidepressant treatment affects the long-term health and neurodevelopment of offspring through, as yet poorly understood epigenetic mechanisms. Recent pioneering discoveries from my lab have allowed us to develop the model organism C. elegans to dissect the molecular mechanisms by which maternal stress, through the serotonergic system, impacts chromatin in the pre-fertilized oocyte. Notwithstanding the differences between the C. elegans and mammalian serotonergic systems (5-HT source being neurons, enterochromaffin cells and maternal placenta in mammals and only neurons in C. elegans), an acute elevation of serotonin levels is a stress signal in both mammals and C. elegans and acts through conserved receptor-mediated signal transduction pathways to induce the ancient, conserved transcription factor, HSF1. Our working hypothesis, premised on preliminary data, is that the stress-induced release of serotonin from maternal neurons in C. elegans enables HSF1 to recruit chromatin remodeling proteins in a piRNA (21U-RNA)-dependent manner in the germline and modify chromatin in pre-fertilized oocytes. The result is that threat perception by the mother alters the development, behavior and physiology of future offspring. In Aim 1, we will identify the neural bases and signaling mechanisms of the serotonergic defense survival circuit in the parent. In Aim 2, we will identify the epigenetic changes in pre-fertilized germ cells caused by maternal serotonin, and understand how they impact offspring neurodevelopment, behavior, and stress resilience.

NIH Research Projects · FY 2026 · 2022-03

ABSTRACT Epidemiological evidence suggests that environmental exposures during development may play a role in disease susceptibility later in life, and researchers have hypothesized that epigenetic changes induced by common toxicants such as pesticides, herbicides, endocrine disruptors, and heavy metals may be facilitating this link 1,2. The mechanisms by which these changes are induced and propagated remain challenging to dissect, largely because environmental toxicant induced changes are often 1) subtle when assayed across the bulk cell population, 2) transient in nature and therefore difficult to reproducibly detect, and/or 3) randomly distributed throughout the genome, making reproducibility and measurement of statistical significance challenging. In fact, most studies trying to link toxicant exposures directly to frank cellular transformation, including our own, have been relative failures. In virtually all cases, in order to see overt transformation, exposure studies need to be conducted in cell or animal models that already have baseline genetic or epigenetic changes that facilitate the progression to malignancy. We have exciting preliminary data that demonstrates that Ewing sarcoma cells demonstrate a significantly elevated level of transcriptional and replication stress (RS), and we propose that environmental exposures may not only induce RS and activate the RSR, but also cause epigenetic changes that precondition cells to allow for survival following expression of driver fusion proteins despite elevated levels of RS. This proposal will focus our efforts on understanding the downstream effects of TCDD exposures in mesenchymal stem cells (MSC) when paired with 2,4-D and 2,4,5-T and investigate the role of STAG2 in modulating downstream molecular events associated with environmental toxicant exposures. Our overall hypothesis is that environmental toxicant exposures cooperate with STAG2 loss to increase replication stress, ultimately leading to genomic and epigenomic instability, and creating a permissive epigenome for fusion gene expression. Three Specific Aims are proposed: SPECIFIC AIM 1: To determine whether environmental toxicant exposures increase baseline levels of replication stress in iMSC and cooperate with STAG2 loss to lead to epigenomic remodeling. SPECIFIC AIM 2: To determine whether environmental toxicant exposures cooperate with STAG2 loss to lead increased clonal genetic and epigenetic heterogeneity. SPECIFIC AIM 3: To determine whether environmental toxicant exposure leads to a permissive epigenome for survival of pre-malignant cells following fusion protein expression.

NIH Research Projects · FY 2026 · 2021-12

In pediatric health care non-adherence to medications is a significant driver of avoidable suffering and death. Over half of children do not adhere to prescribed medications, and non-adherence is the leading cause of treatment failure in pediatrics. Non-adherence can lead to worsening illness, death, preventable hospitalization, increased health care cost, and morbidity. Even in pediatric cancer, when the consequences of non-adherence to chemotherapy are potentially life threatening, over 40% of patients have clinically significant non-adherence. For the most common pediatric cancer, Acute Lymphoblastic Leukemia (ALL), children who miss just 10% of chemotherapy doses have a nearly 4-fold risk of cancer relapse. Despite decades of research we do not have effective strategies to meaningfully increase pediatric medication adherence. The goal of the proposed research is to reduce preventable pediatric morbidity and mortality through testing a novel target – behavioral parenting skills – as a modifiable mechanism to improve medication adherence in young children (ages 3-9). Based upon our preliminary data we have begun to develop CareMeds, a parenting skills-focused adherence intervention. The goal of this project is to use stages 0 and 1 of the NIH Stage Model to further develop and evaluate the feasibility of the CareMeds intervention. Evidence is converging on family functioning and parenting style as critical factors that shape child medication adherence. Yet, previous studies typically rely on one-time global measures, making it difficult to discern the precise parenting skills that improve medication adherence. For example, we know very little about what exactly “supportive” or “cohesive” families are doing to promote medication adherence. In Aim 1 we will use direct observation of medication administration at home to understand common episode-level barriers and identify the behavioral parenting skills that are most successful in achieving medication administration in young children. In Aim 2 we will use daily diary data collection to examine how daily parenting experiences influence the risk of medication non-adherence. We will use data from Aims 1 and 2 and input from diverse parents to refine the final CareMeds intervention package. In Aim 3 we will conduct a pilot RCT of the intervention versus usual care with 100 families of young children ages 3-9 with ALL within 1 month of initiation of oral chemotherapy prescription. Findings from this program of research will make significant conceptual contributions through providing nuanced understanding of the aspects of parenting at the episode and daily levels that shape medication adherence in young children. It will make innovative methodological advances through use of direct observation of medication administration, daily diary data on transient parenting experiences, and rigorous measurement of adherence through objective behavioral measures (electronic pill bottle monitoring) and pharmacological measures (validated biomarkers of drug metabolites). Finally, it will have significant translational impact through setting the stage for a full-scale, multi- center, RCT to examine the efficacy of the CareMeds intervention.

NIH Research Projects · FY 2024 · 2021-09

Radiation Therapy (RT) is a common form of cancer treatment that can be effective in treating numerous malignancies. Two key components of an effective RT regimen are a dose of irradiation that is sufficient to cause tumor cell death, and an innate immune response, driven by dendritic cells and fueled by the debris from dying tumor cells, that goes on to activate anti-tumor adaptive immunity. Collectively, this process has come to be known as the in situ vaccine effect of radiation. Unfortunately for many patients, a deficiency in one of these two key components can occur from the onset of treatment, or develop over time, and result in resistance to RT. For example, if an insufficient amount of tumor cell death occurs from a given dose of radiation, not only will more live cancer cells remain within the tumor, but this lack of cell death will also ultimately limit the activation and recruitment of adaptive immune cells. Without adaptive immune activation, the remaining live cells within the tumor, and potential metastases that could be present throughout the body, can survive and proliferate. We have determined that chronic stress mediated by β-adrenergic signaling is capable of inducing tumor cell resistance to irradiation induced cell death in vitro, and we have also determined that this same stress results in a subdued anti-tumor immune response generated from RT in vivo. The goal of this proposal is to resolve the mechanism through which adrenergic stress induces tumor cell radioresistance, and to determine whether this change in cell death is driving the immunologic changes observed in vivo, in addition to the direct effects of stress on immune cells. To address these goals, we will use pharmacologic and genetic approaches to induce or inhibit signaling cascades downstream of the β1, β2, and β3-ARs, and determine which receptor, and which signaling pathways, are responsible for the observed increase in tumor cell survival after irradiation. We will define how this signaling drives survival by evaluating cell death pathways including apoptosis, necrosis, and necroptosis, and determine whether inhibiting this signaling also leads to a potentially more immune stimulating tumor microenvironment. To do so, we will assess cGAS/STING signaling and damage associated molecular pattern (DAMP) production (including ATP, HMGB1, and Calreticulin) in vitro. Using a series of co-culture experiments where dendritic cells (DCs) are cultured with irradiated tumor cells experiencing varying levels of β-AR signaling, we will evaluate whether changes in the radiation induced cell death processes described above affect DC maturation and function. In vivo, we will utilize various β-AR deficient mouse strains to evaluate whether increased β-AR signaling in tumor cells alone is sufficient to drive resistance to therapy and impaired anti-tumor immunity. Changes in DAMP production in vivo will also be evaluated. Taken together, this project has the potential to produce paradigm shifting discoveries which outline a new and important mechanism of radiation resistance that is driven by the human physiologic response to chronic stress and anxiety, β-adrenergic signaling. Ultimately, these discoveries could enhance the efficacy RT, improve patient outcomes, and increase patient quality of life.