cancer

Targeting disulfidptosis in cancer: mechanisms and preclinical translation

Project Summary Studying regulated cell death is critical for our understanding of cellular homeostasis and tumor suppression. We recently discovered disulfidptosis as a new form of regulated cell death induced by disulfide stress under NADPH-depleting conditions in SLC7A11-high cancer cells. However, in contrast to our deep understanding of other cell death modalities such as apoptosis and ferroptosis, the molecular and metabolic underpinnings of disulfidptosis, along with its therapeutic implications, remain largely unexplored. The objectives of this application are to elucidate the mechanisms underlying disulfidptosis and to therapeutically target this form of cell death in SLC7A11-high cancers. The proposed studies will make extensive use of human cancer cell lines and integrated human cellbased molecular analyses, including metabolomics, proteomics, CRISPR screening, and biochemical studies, to define the metabolic and signaling mechanisms governing disulfidptosis. In addition, select in vivo studies are incorporated in the therapeutic validation components of the project, where tumor growth response, systemic drug exposure and tolerability, tumor microenvironmental influences, and host immune/stromal interactions must be evaluated in an organismal context to ensure translational rigor. Alternative in vitro systems such as organoids may provide useful complementary information on tumor-intrinsic responses, but they cannot fully recapitulate the systemic metabolic stress, pharmacologic exposure, and organism-level therapeutic efficacy required for these studies. It is expected that our proposed studies will reveal novel mechanisms underlying disulfidptosis and identify effective therapies to induce this form of cell death in SLC7A11-high cancers. Our proposal is highly innovative because it focuses on a previously unexplored cell death pathway in cancer therapy. Our proposed studies will have significant impact on both our understanding of the fundamental mechanisms of disulfidptosis and our ability to target this cell death pathway in cancer treatment.

Optimizing CD45-Targeted Astatine-211-Radioimmunotherapy for Malignant and Non-Malignant Blood Disorders

ABSTRACT CD45 is expressed on almost all normal and neoplastic hematopoietic cells but not on non-blood cells and has, therefore, been pursued as a drug target. Initially centered on augmenting conditioning before hematopoietic cell transplantation (HCT) for blood cancers, there is increasing interest in expanding CD45-directed therapies into other settings, with radioimmunotherapy (RIT) being the major therapeutic modality so far. Investigators at our institution pioneered CD45 RIT with b-emitters such as iodine-131 (131I) using the murine monoclonal antibody (mAb), BC8. A phase 3 trial testing 131I-BC8 (131I-apamistamab [Iomab-B]) with allogeneic HCT in older adults with relapsed/refractory acute myeloid leukemia showed improved outcomes over conventional care, validating this approach. More recently, attention has shifted toward a-emitters that deliver substantially higher decay energies over much shorter distances than b-emitters, rendering them more suitable for precise and potent target cell killing. In our work, we focus on astatine-211 (211At) for its ideal half-life and decay without a-emitting daughters. For clinical application, mAbs are conjugated with the bifunctional boron cage molecule, isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10-NCS), to enable stable protein astatination. Three early-phase trials testing 211At-BC8-B10 as augmentation of HCT conditioning for patients with malignant and non-malignant blood disorders are ongoing, with emerging data indicating significant anti-tumor efficacy. Nonetheless, relapses still occur. Other important limitations include marked infusion toxicities and human antimouse antibody (HAMA) responses related to the murine nature of BC8 and dimer formation after 211At labeling of mAb-B10 conjugates with tissue residualization from 211At atom oxidation. The latter may contribute to the risk of liver cell injury, the dose limiting extramedullary toxicity of CD45 RIT. As a first step toward our goal of optimizing CD45 RIT, we have raised new, fully human CD45 mAbs as basis for novel therapeutics. In preliminary in vivo studies in immunodeficient mice, we found some of these mAbs to have greater anti-tumor efficacy than a humanized version of BC8 (HuBC8) we generated as a reference mAb. We will now conduct comparative in vivo CD45+ cell targeting (“biodistribution”) and anti-tumor efficacy studies to select a lead candidate mAb for clinical application and use protein engineering to maximize the selectivity and efficacy of targeted radiation delivery. We will use immunodeficient mice xenotransplanted with human leukemia cells for this purpose as no human approaches are available and in vitro testing is inadequate to measure both the targeting and biologic RIT effects on human leukemia cells. Mice provide the in vivo milieu needed for comprehensive evaluation. Development of improved mAb astatination methodologies to minimize off-target toxicities of 211At-RIT will further increase therapy specificity and reduce toxicity. In parallel, we will conduct genome-scale, unbiased target identification/validation studies to identify partner drugs for rational combination therapies aimed at enhancing the anti-tumor efficacy of 211At-CD45 RIT.

Targeting VIP–VPAC Signaling to Reverse Immune Exclusion and Enhance Immunotherapy Response in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that is largely unresponsive to chemotherapy and current immune checkpoint blockade drugs, highlighting a critical need for the development of innovative therapeutic strategies. This R01 proposal targets vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide overexpressed in PDAC, which signals through VIP receptors (VPAC) on cancer cells, T cells, and myeloid cells within the tumor microenvironment. Based on our recent success in developing selective and potent VPAC receptor antagonists, we hypothesize that blocking VPAC signaling will reverse immunosuppression in the PDAC TME by reducing immune checkpoint expression, enhancing chemokine-driven infiltration of cytotoxic T cells, and disrupting immunosuppressive interactions between T cells and myeloid cells, ultimately leading to durable anti-cancer immunity. We propose three specific aims to explore the immunosuppressive roles of VPAC signaling in PDAC. Aim 1 will identify the primary sources of VIP in PDAC tumors and characterize the effects of VPAC signaling on immune cell function and phenotype within the tumor microenvironment. Aim 2 will investigate how VPAC signaling influences immune cell migration into tumors by modulating chemokine receptors and directional signaling. Aim 3 will determine how VPAC signaling regulates interactions between T cells and immunosuppressive myeloid cells, particularly tumor-associated macrophages, and the resulting impact on anti-cancer immune responses and immunological memory. Our preliminary findings indicate that combined inhibition of VPAC signaling and PD-1 significantly enhances the regression of PDAC tumors in multiple mouse models, generating lasting protective immunity in cured mice without triggering autoimmune responses. We will use novel methods to pursue our aims, including inducible genetically engineered mouse models (GEMM) of PDAC, long-acting VPAC antagonists engineered with immunoglobulin Fc domains to improve their plasma half-life, and advanced microfluidics technologies to analyze immune cell movement within tumors. Animal experiments will be used to validate the translational potential of observations from in vitro organoids and microfluidic experiments. The GEMM and orthotopic mouse models of PDAC are necessary to provide critical insights into the 3-D structure of the TME and tumor regression in response to our novel immunotherapy. This research will be conducted by a multidisciplinary team with complementary expertise that will clarify the therapeutic potential of VPAC signaling inhibition in PDAC using sophisticated experimental tools and single-cell RNA sequencing. Ultimately, these findings could significantly improve the development of immunotherapeutic strategies for PDAC, potentially enhancing patient outcomes in pancreatic cancer and other malignancies expressing high VIP levels.

Communication and Hospice Online with Optimal Support and Engagement (CHOOSE)

Abstract Drawing upon the principles of social identity theory, existing literature, and our initial findings from family caregiver (FCG) online support groups (OSGs), our objective is to identify fundamental facilitator communication strategies that promote safe communication engage participants, and strengthen mechanisms of action (MOAs) within OSGs, ultimately enhancing health outcomes for hospice FCGs. Our pioneering initiative, Communication and Hospice Online with Optimal Support and Engagement (CHOOSE) is backed by compelling evidence highlighting the critical role of facilitator communication in reinforcing MOAs (a shared identity, social support, and social networks) in OSGs. Preliminary research underscores the transformative power of these MOAs in improving health outcomes for FCGs, yet current studies lack generalizability and statistical robustness. CHOOSE represents the first major, multisite, rigorously designed, and theoretically informed OSG intervention explicitly tailored for hospice FCGs of cancer patients. We aim to strengthen MOAs to enhance FCG well-being, reduce depression and anxiety, improve quality of life, and diminish loneliness. By advancing this critical research, we seek to provide a well-founded, evidence-based solution to the urgent needs of FCGs, making a significant impact on their health and well-being. We have outlined the following study aims: Aim 1. Determine the effect of the CHOOSE intervention on FCGs’ health outcomes compared to usual OSGs and usual hospice care. Aim 2. Examine direct and mediational relationships between CHOOSE participation, MOAs, and health outcomes. Aim 3. Explore the relationship between facilitator communication strategies and the FCG experience of the MOA to allow for future calibration of the intervention 1

Targeting the Molecular Crosstalk Between EZHIP and PRC2 in PFA Ependymoma

Project Summary: PFA ependymoma is a rare and aggressive pediatric brain tumor with a poorly understood molecular mechanism. Unlike many cancers, PFA ependymoma exhibits very few genetic alterations. Instead, it is thought to be driven primarily by epigenetic dysregulation. A key player in this disease is the EZH1/2 inhibitory protein EZHIP, which is normally expressed only in germ cells. EZHIP is aberrantly expressed in PFA ependymoma, where it disrupts the function of Polycomb Repressive Complex 2 (PRC2), a master epigenetic regulator of developmental gene repression through deposition of the trimethylated histone H3 lysine 27 (H3K27me3) repressive histone mark. EZHIP-mediated dysregulation of PRC2 involves both enzymatic inhibition and physical stalling of PRC2 on CpG island (CGI) chromatin, leading to a global loss of H3K27me3 levels, an epigenetic hallmark of PFA ependymoma. PRC2 itself is a highly dynamic and intricate complex that assembles into two functional variants, PRC2.1 and PRC2.2. These two variants share a core composed of the catalytic subunits EZH1/2, along with EED, SUZ12, and RBBP4/7, and differ by incorporating distinct accessory subunits. PRC2.1 includes PHF1/MTF2/PHF19, EPOP, and PALI1/2, while PRC2.2 features AEBP2 and JARID2. Our preliminary data reveal intriguing molecular crosstalk between EZHIP and multiple PRC2 components, suggesting potential competitive or cooperative interplay. The ability of EZHIP to inhibit PRC2 partly stems from its mimicry of the oncohistone H3K27M, which harbors a lysine-to-methionine mutation that causes diffuse midline glioma, another devastating brain tumor in children, where PRC2 activity is also globally suppressed. However, the precise, EZHIP-specific mechanisms behind PRC2 dysregulation in PFA ependymoma remain largely unexplored. Our work aims to uncover these elusive mechanisms using a powerful combination of structural biology, biochemistry, and genomics approaches. Ultimately, we aim to identify therapeutic strategies that disrupt the pathogenic EZHIP–PRC2 crosstalk and restore the normal H3K27me3 epigenetic landscape. Specifically, in Aim 1, we will determine the structural and biochemical mechanisms underlying the enzymatic inhibition of the PRC2 core complex by EZHIP. In Aim 2, we will elucidate the molecular basis of EZHIP-mediated stalling of PRC2 on CGI chromatin, involving PRC2 functional variants. In Aim 3, we will explore an exciting mechanism-based therapeutic strategy to overcome PRC2 enzymatic inhibition and chromatin stalling induced by EZHIP.

Short-wave infrared Cerenkov imaging to better visualize targeted radiotherapy and diagnostic radiotracers

SUMMARY. The problem: Cerenkov luminescence (CL) imaging (CLI) is a new imaging method that utilizes light emitted during decay of radiotracers. CLI merges optical and nuclear imaging by utilizing affordable yet highly sensitive optical cameras with clinical radiotracers. It provides fast and cheap clinical optical imaging to explore radiotracer distribution in patients. While not tomographic, CLI systems have a lower price, smaller footprint and higher resolution than nuclear imaging scanners. Yet, due to the very low signal intensity of CL its versatility remains limited since CLI requires strict exclusion of ambient light with an enclosure. Therefore, CLI requires novel approaches to make clinical imaging more feasible. We hypothesized that we could explore the short-wave infrared (SWIR) part of CL to enable CLI under ambient light without enclosure, providing improved and facile CLI, particularly of isotopes used for therapy that cannot be imaged otherwise. SWIR imaging (900- 1300 nm) has almost no autofluorescence, absorption or scatter but provides significantly higher depth penetration, yielding images with higher contrast and resolution compared to the visible range. Since typical LEDs do not emit light beyond 850 nm, they do not interfere with the SWIR camera. We can therefore perform CLI in the SWIR range (SWIR-CLI) without the limiting light-tight box and under ambient LED light and also achieve better signal penetration and accuracy. We will investigate if SWIR-CLI can be used to monitor distribution of therapeutic isotopes for targeted radiotherapy (TRT), a fast-expanding field as highlighted by Novartis’ acquisition of Lutathera and Pluvicto for the price of $6 bn. These agents are targeting 177Lu as therapy to neuroendocrine and prostate cancers. For TRT α-emitting isotopes are particularly attractive due to the α- particle’s short path length with high linear energy transfer. However, α-emitters are very difficult to image with conventional equipment. The α-emitter could be swapped with an imaging isotope, but this can alter the agent’s biodistribution. The α-particle itself does not have sufficient energy to produce CL but several daughters in the decay chains of most α-emitters produce electrons with sufficient energy to create CL. We have already imaged the α-emitter 223Ra in patients and have recently shown that CLI of α-emitters in the SWIR is possible. SWIR- CLI could therefore provide a facile imaging approach for α-emitters. We will answer with our three independent Aims the following questions: (1) Can we image diagnostic isotopes with SWIR-CLI? (2) Can we image therapeutic emitters with SWIR-CLI? (3) Can we use SWIR-CLI to image patients undergoing PET and/or TRT? Animal studies will employ established mouse cancer models to optimize imaging parameters and validate findings, directly informing the co-clinical Aim 3 trial. By eliminating the requirement for a light-tight enclosure and enabling CLI under ambient light, SWIR-CLI represents a significant shift in the practical deployment of CLI rather than an incremental improvement. Our study will broaden the reach of CLI by enabling imaging under ambient lighting, unlocking innovative new opportunities for CLI (monitoring TRT) in research & clinical settings.

Increasing Lung Cancer Screening Uptake Among High-Risk Emergency Department Patients

PROJECT SUMMARY/ABSTRACT Lung cancer is the leading cause of cancer death in the US. Although lung cancer screening (LCS), using low- dose CT scan, decreases lung cancer mortality through early disease identification, fewer than 1 in 6 eligible individuals get screened, with significant differences based on demographic and socio-economic factors. LCS is a process, not just a test. The critical first steps in this process are (1) identification of high-risk individuals who are eligible for LCS, and (2) recruitment of these individuals into an LCS program. The Emergency Department (ED) setting is optimal for an intervention to promote LCS by accomplishing these steps. Individuals at high risk for lung cancer are over-represented in the ED population, including: individuals that smoke, non-White individuals, patients with lower education levels, and the under-insured. In fact, over 2.3 million high-risk people pass through EDs every year who are eligible for LCS but have never been screened. The investigators’ long-term goal is to develop a low-cost, scalable intervention that increases LCS uptake among ED patients and is deployable in any ED with a regionally referrable LCS program. The objective of the proposed randomized clinical trial is to test the efficacies of text messaging and a facilitated referral strategy to promote uptake of LCS in order to achieve this goal. Step 1 of the approach is to identify participants that are eligible for LCS. Step 2 is to randomize eligible participants, using a 2x2 design, among four study arms: (1) basic referral for LCS (i.e. verbal referral with written materials; comprising an enhanced control arm), (2) basic referral plus a subsequent series of text messages, grounded in behavioral change theory, aimed at generating intention and motivation to get screened, (3) facilitated referral for LCS (i.e. submission of a requisition to LCS program by staff), and (4) facilitated referral plus text messages. The investigators’ pilot work demonstrated the feasibility and efficacy of the proposed approach. A total of 1036 individuals eligible for LCS will be recruited from a high-volume urban ED and a low-volume rural ED, randomized among study arms, and followed-up at 120 days to assess interval LCS uptake. The Specific Aims of the proposed project are, (1) Compare LCS program uptake among study arms that receive text messages to study arms that do not, (2) Compare LCS program uptake among study arms with basic referral to study arms with facilitated referral, (3) Investigate the interaction between receipt of text messages (yes/no) and referral type (basic/facilitated), and (4) Evaluate participant feedback on (a) differential barriers to LCS across sub-groups and (b) acceptability and appropriateness of ED-based promotion of LCS. The study team is at the forefront of developing ED-based interventions to promote cancer screening. This project leverages the universal access setting of the ED to identify individuals at greatest risk for lung cancer and get them screened. A scalable ED-based intervention that increases LCS uptake would save lives.

Urothelial Resurfacing with Irreversible Electroporation for Adjuvant Therapy of Bladder Cancer

PROJECT SUMMARY Over 70% of bladder cancer (BCa) patients are diagnosed with early-stage and localized non-muscle invasive disease (NMIBC), yet achieving durable cancer-free survival remains a significant challenge. Most of these patients will experience local tumor recurrence within five years following standard of care (SoC) transurethral resection of bladder tumor (TURBT) and intravesical adjuvant chemo- or immunotherapy. Recurrence is driven by microscopic tumors and premalignant lesions dispersed within the urothelial layer that survive and escape these treatments. As TURBT effectively treats tumors visible on imaging, current research has predominantly focused on drugs and biologics for improving intravesical adjuvant therapy. In this proposal we pose the provocative question whether a TURBT-like ablative technique can be extended to debulk malignancy in the entire bladder and investigate the synergy with intravesical adjuvant therapy in improving outcomes. Our objective is to address this technology and knowledge gap by developing and validating whole bladder urothelial resurfacing (WBUR) using irreversible electroporation (IRE). During IRE, microsecond-long pulsed electric fields (PEF) are used to induce rapid cell death by catastrophic permeabilization of the cell membrane, without affecting the extracellular matrix (ECM) within the treated tissue. In prior work, we designed devices that utilized this unique mechanism of IRE for performing penetrative ablation in the ureter, bile duct and bronchus of swine while preserving lumen function. Our findings provided strong rationale for IRE being an ideal candidate for WBUR as alternate techniques such as thermal ablation or ionizing radiation must be performed with extreme care in the bladder to avoid perforation or fistula formation. In subsequent preliminary work we developed technology to demonstrate the feasibility and safety of WBUR with IRE in a rat model of BCa and scalability in human-sized swine bladder. In Aim 1, we will investigate the cancer treatment efficacy of combination WBUR and intravesical adjuvant therapy. In Aim 2, validate WBUR derived liquid biopsy for monitoring cancer status. In Aim 3, engineer PEF delivery strategy to enhance the safety and specificity of WBUR. The innovation of our proposed work is defined by developing whole bladder ablation as a debulking strategy and examining its synergy with SOC adjuvant therapy (Aim 1), enabled by new electrode paradigm and PEF delivery strategy (Aim 3), monitoring by an unconventional liquid biopsy approach (Aim 2). Our work can immediately aid the management of NMIBC patients who cannot undergo radical cystectomy, with future application as a cancer prevention strategy in high-risk patients. Success of individual aims will result in major contributions to the topics of IRE, BCa treatment and diagnosis.

Role of cellular physical interactions in pancreatic cancer progression

Pancreatic cancer, with a 5-year overall survival rate of 13%, has the lowest survival rate of all cancers. The goal of this project is to better understand the biological processes of pancreatic cancer progression and discover their potential as targets for efficient therapies. Pancreatic ductal adenocarcinoma (PDAC) underdoes epithelial architecture changes during its progression. However, the underlying mechanisms for these changes are largely unknown. Interestingly, our recent data demonstrate the recapitulation of the distinct epithelial architectures in the organoid culture of cells derived from the human normal pancreas, primary tumor, and metastatic lesions, thereby developing a unique organoid model for the in vitro studies of PDAC epithelial architecture changes. The primary objective of this project is to understand the regulation of the differential PDAC epithelial architectures as well as their contribution to PDAC progression. Our central hypothesis is that disruption in lumen structure drives PDAC epithelial architecture transition and promotes PDAC progression. We will combine experimental and computational approaches to test our central hypothesis by pursuing the following two specific aims: (Aim 1) define the regulators of PDAC epithelial architecture that drives PDAC progression and (Aim 2) determine the functional consequences of PDAC epithelial architecture on PDAC progression. With the completion of this aims, we expect: (Aim 1) to identify ion and water channels that are important for lumen structure as well as PDAC progression, revealing potential novel targets for therapeutic intervention, and (Aim 2) to uncover YAP’s role in PDAC progression and guide the development of YAP- targeted therapies.

Regulation of neutrophil endoplasmic reticulum stress response by IRE1a

Project Summary/Abstract: The lungs are exposed to pathogens and environmental toxins that trigger stress and cause numerous respiratory diseases. Effective host defenses against lung infection by bacterial pathogens, including methicillin- resistant Staphylococcus aureus (MRSA), rely on innate immune cells including neutrophils, prominent early responders to sites of infection. If host defenses are ineffective, MRSA causes serious lung infection, resulting in severe morbidity and a significant economic burden on healthcare facilities, where it is endemic. MRSA infections have a mortality rate of up to 14% and an estimated $500 million in healthcare costs in the US alone. Increasing resistance to vancomycin, the last resort antibiotic for MRSA infections, underscore the urgent need for innovative treatment approaches. Although directly targeting pathogens with antibiotics has been a successful approach for treating infections, many pathogens, including MRSA, eventually will become resistant to these drugs. As an alternative, immunomodulatory strategies to enhance host defenses, such as those shown to be effective against cancer cells, have the potential for treating drug-resistant pathogen infections. Recently, we showed that the inositol-requiring enzyme 1-α (IRE1α), an endoplasmic reticulum (ER) stress sensor, is required for clearance of MRSA in a murine skin abscess model, where neutrophils are robustly recruited to the site of infection. Further, IRE1α coordinates signaling events upstream of calcium (Ca2+) mobilization, histone citrullination, and production of mitochondrial reactive oxygen species (mitoROS), all of which are important for neutrophil inflammatory responses including the formation of antimicrobial neutrophil extracellular traps (NETs). Because excessive neutrophil activation and NET release can be detrimental to vital organs, it is not clear whether neutrophil IRE1α-mediated stress responses aid or impede the resolution of infection in the lungs. While IRE1α activation has been linked to the development of lung fibrosis through the regulation of alveolar epithelial- to-mesenchymal transition in the context of chronic inflammatory diseases, its role in pulmonary neutrophil defenses is unknown. Thus, there is a gap in our knowledge of how cellular stress responses modulate pulmonary neutrophil defenses and infection outcomes in the lungs. The overarching goal of this proposal is to elucidate the mechanisms by which neutrophil IRE1α signaling influences production of mitoROS and Ca2+ mobilization to drive NET release, injure lungs, and regulate pulmonary host defense against MRSA. We will accomplish the following Aims: (1) Define the molecular mechanisms underlying IRE1α-mediated mitoROS hyperactivation of human and mouse primary neutrophils and excessive NET release, and (2) Elucidate the role of neutrophil IRE1α signaling in excessive NET release, lung injury, and immunity in vivo using a MRSA pneumonia infection mouse model. These studies will yield mechanistic insight into how IRE1α-driven ER stress responses impact pulmonary neutrophil defenses and lung injury revealing potential targets for anti-microbial immunotherapies.

The role of GPR132 in regulating T cell responses in infection and cancer

PROJECT SUMMARY. CD8 T cells play a critical role in protection from a variety of infectious microorganisms, and pathogen-specific CD8 T cells undergo robust expansion, with an individual T cell clones expanding up to 10,000-fold in a matter of days. After infection is resolved, the majority of these T cells die, leaving a small population of memory cells to provide protective immunity from secondary challenge. T cell expansion and contraction are tightly orchestrated processes that involve a delicate balance between stimulatory and inhibitory signals to ensure proper immune function. Dysregulation of the T cell response can have detrimental effects; too little proliferation and the host fails to mount a successful immune response, while excessive proliferation and persistence of effector T cell populations can lead to tissue damage. This proposal aims to determine the role of the G protein coupled receptor GPR132 in the regulation of CD8 T cell responses during infection and tumorigenesis. GPR132 detects oxidized endogenous and microbial lipids, and this can lead to cell cycle arrest; however, the role of GPR132 in CD8 T cells remains unexplored. Here we identify GPR132 as a critical regulator of CD8 T cell expansion and memory differentiation. Completion of the proposed aims will: 1) uncover the temporal role of GPR132 in regulating T cell accumulation and function during infection and tumorigenesis, 2) examine the abundance of GPR132-activating ligands within the tissue during health and disease, and 3) determine how altering GPR132 ligand availability could be used to enhance/inhibit T cell responses. Overall, these studies will provide fundamental insights into the regulatory mechanisms that dictate the magnitude of T cell responses and how they can be modulated therapeutically, which would allow us to boost responses to pathogens/tumors or inhibit pathogenic responses in the context of autoimmune disease.

Weak Cell Adhesion is a Prognostic Signature of Invasive Cancer

Project Summary Despite early detection, low-grade and localized breast cancers such as ductal carcinoma in situ (DCIS) can relapse in up to 20% of cases despite standard of care. For DCIS, relapse affects over 12,000 U.S. women annually and has increased 60% in the last 40 years. Current diagnostic assessments including histopathological markers often miss early disseminating cells, lack specificity, or cannot distinguish cancer from non-cancer cells in the stroma. Hence there is an unmet need for cancer diagnostic technologies that employ radically different characterization methods. For example, significant physical differences exist between metastasizing and benign breast cancer cells, owing to metastasizing cells detaching from the primary tumor, migrating through the surrounding stroma, intravasating and extravasating, and ultimately engrafting in distant tissues. We recently demonstrated that cancer cells with weaker adhesion migrate faster and metastasize more frequently in murine breast cancer models than strongly adherent cells. In a small pilot study of human breast tumors, we also observed that the abundance of weakly adherent (WA) cells scales with disease severity; subpopulations from invasive carcinomas were the least adherent. However, a subset of DCIS cases displayed much less adhesion, suggesting that these patients may have a tumor subpopulation that progresses to metastatic disease despite standard-of-care treatment. Weak adhesion is a defining physical characteristic of tumors, but to establish their role in initiation, metastasis, and patient outcomes, we will leverage model systems and our newly patented adhesion technology to answer these fundamental questions of cancer biology and clinical translation. To understand the impact of adhesion on cancer progression, we will evaluate the tumor-initiating potential of WA versus strongly adherent (SA) tumor cells in a murine breast cancer model before confirming how weak adhesion advantages cells to cause secondary disease using bioengineered in vitro models. In dissecting the stages of metastasis where WA cells exhibit advantages, e.g., recapitulating stromal niche, transendothelial migration, and tissue-specific colonization, we will identify mechanisms that enable WA cells to thrive and evaluate therapeutic targets that disrupt these pathways. Finally, we will analyze the adhesion profiles of resected tumors and stroma from 80 breast cancer patients with DCIS or invasive disease. Adhesion data will be correlated with conventional assessment methods and ultimately with patient outcomes, e.g., disease-free and progression-free intervals. We anticipate that the DCIS subpopulation that aligns with the adhesion signature of invasive carcinomas will have shorter intervals and survival time. This integrated study design bridges mouse models, mechanistic bioengineering assays, and human samples to clarify the metastatic potential and prognostic value of WA breast cancer cells. Our use of mouse models in this grant is required to study the interactions among tumor cells, immune cells, vasculature, and stromal tissues that drive tumor formation in vivo. Bioengineered in vitro systems lack the complexity to ask such questions and using injected tumor cells is not possible in humans.

Improved Surgical Visibility and Navigation during Endoscopic Treatment of Upper Tract Urothelial Carcinoma

Project Summary The importance of localizing and treating all upper tract urothelial cancer (UTUC) tumors during a renal sparing, endoscopic treatment is emphasized by the high risk of cancer progression from inadequate tumor treatment. Insufficient treatment necessitates kidney and ureteral removal (i.e., nephroureterectomy). Nephroureterectomy permanently compromises renal function, and increases morbidity and mortality, while negatively impacting a patient’s quality of life. In contrast, endoscopic treatment (i.e., using a laser to ablate only the tumors) improves long-term outcomes by sparing healthy kidney tissue. However, endoscopic treatment is underutilized compared to nephroureterectomy because it is difficult to accomplish. Successful endoscopic treatment is dependent on the surgeon’s ability to create a mental 3D map of the branched, intrarenal endoscopic anatomy intraoperatively from preoperative 2D imaging, which is extremely difficult. Since mental mapping relies on hand-eye coordination, memory, and spatial reasoning, it is inherently imprecise and its impact on accuracy and tumor treatment is dependent on the surgeon’s experience. To make matters worse, even when tumors are successfully visualized, the surgeon often cannot accurately assess the location of tumor margins or infer pathologic grade due to the limited field of view and depth of field (10mm and 6mm on average, respectively) of current scopes. The scopes only provide visualization of a small part of the surgical field at any instant. These inherent challenges prevent many surgeons from attempting endoscopic tumor treatment since incomplete treatment leads to a devastating, oncologic outcome. Our overall goal is to create an enhanced visualization and navigational system that makes endoscopic UTUC tumor treatment easier and more accurate for all surgeons, enabling wider utilization. Toward this goal, our specific objective in this proposal is to test the hypothesis that our system can make endoscopic UTUC surgery more accurate and efficient. To test this hypothesis, we propose three Specific Aims: Aim 1 involves the development of an automatic, real-time segmentation and grading system of UTUC tumors during endoscopic treatment. Aim 2 integrates a 3D navigational map of collecting system anatomy, which includes tumor and endoscope location, during endoscopic surgery. Aim 3 evaluates the system in patients, with zero risk to the human subjects. The endpoint of this R01 will be a fully validated enhanced visualization and navigational system for endoscopic UTUC surgery, which would provide the necessary experimental data towards a large-scale, multi-center clinical trial and future FDA approval. As our system would require only software integration to current endoscopic surgical cameras, all existing endoscopic surgical systems could in principle immediately benefit from the results of this project. In this way, we believe the success of our project will facilitate improved UTUC treatment and mitigate progression to a higher risk extirpative surgery.

Structural and functional characterization of autoimmune antibodies against NMDAR

Project Summary. The goal of this project is to understand the origins and molecular mechanisms underlying the anti-cancer autoimmune response against the N-methyl-D-aspartate receptor (NMDAR) and its correlation with anti-N-methyl-D-aspartate receptor autoimmune encephalitis (NMDARAE). While anti-cancer immune responses can promote tumor elimination, they may also lead to the production of self-reactive antibodies that trigger autoimmune diseases. NMDARAE is the most common form of immune-mediated encephalitis, which results in prominent neuropsychiatric symptoms, including seizures, psychosis, and memory deficits. NMDARs belong to a family of ligand-gated ion channels expressed exclusively in the central nervous system. They are involved in various aspects of brain development and function, including learning and memory. They respond to the neurotransmitter glutamate and a co-agonist, glycine or D-serine, to mediate excitatory neurotransmission, which plays a central role in synaptic plasticity. NMDARAE is associated with ovarian teratomas, where aberrant NMDAR expression is believed to trigger an autoimmune response. In NMDARAE, anti-NMDAR antibodies, as well as B cells and antibody-secreting cells, cross the blood-brain barrier via unknown mechanisms, resulting in the presence of anti-NMDAR antibodies at high titers within the brain and cerebrospinal fluid (CSF). These antibodies target NMDARs, modulating their function and contributing to disease pathology. Emerging evidence, supported by our preliminary data, suggests that NMDARs are also expressed in triple-negative breast cancer (TNBC), extending the relevance of anti-NMDAR autoimmunity beyond ovarian teratomas. In our TNBC mouse model, which ectopically expresses NMDARs (TNBC-NMDAR), we observed the onset of anti-NMDAR autoimmunity, where the produced antibodies cause both anti-tumor activity and symptoms such as lowered seizure threshold, mirroring key features of NMDARAE. Here, we will establish this TNBC mouse model as we develop molecular methods to characterize it. Aim 1 will focus on establishing and characterizing the TNBC- NMDAR mouse model. We will develop a detection method utilizing the intact tetrameric NMDAR channel proteins and a method to isolate B cells expressing B cell receptors against NMDAR from biological samples by using fluorescently labeled intact NMDAR proteins, followed by single-cell RNA sequencing. Aim 2 will utilize single-particle cryo-electron microscopy (cryo-EM) to investigate the interactions between NMDAR and the cloned antibodies, providing insights into epitope recognition, NMDAR subtype specificity, and conformational changes induced by antibody binding. Aim 3 will assess the impact of the cloned antibodies on NMDAR channel activity using electrophysiology. We will also assess anti-tumor activity and NMDARAE onset by each antibody clone. Together, the proposed research will gain insights into the link between anti-cancer anti-NMDAR autoimmunity and NMDARAE. It will also elucidate which functional properties of the cloned antibodies promote anti-tumor activity while contributing to NMDARAE, thereby informing potential therapeutic strategies.

Multimodal computational models for early prediction of peritoneal recurrence in gastric cancer

ABSTRACT Gastric cancer represents a significant disease burden and is a leading cause of cancer-related deaths in the United States and globally. Approximately 80% of gastric cancer patients are diagnosed at an advanced stage, with the peritoneum being the most common site of relapse (peritoneal recurrence) after radical surgery. Nearly 50% of patients with advanced-stage gastric cancer develop peritoneal recurrence post-surgery, resulting in a median survival of only 3–6 months and a markedly reduced quality of life. Early peritoneal recurrence is primarily characterized by micro-metastasis, which traditional imaging techniques struggle to detect due to the small size of metastatic nodules. Predicting the likelihood and timing of peritoneal recurrence is crucial for identifying at- risk patients, enabling timely interventions that could improve survival rates and quality of life. Unfortunately, reliable predictive biomarkers and models for peritoneal recurrence in gastric cancer are lacking in clinical practice, highlighting an urgent need for innovative predictive tools. This proposal aims to develop and validate novel predictive models for early peritoneal recurrence in gastric cancer, leveraging advanced deep learning techniques and multimodal integration of clinical, radiological (CT), and histopathological (hematoxylin and eosin, H&E) data. In Aim 1, we will develop a rational approach for predicting peritoneal recurrence by creating a novel deep learning multimodal method guided by genomics knowledge. Additionally, we will integrate both deep learning-extracted features and traditional hand-crafted radiomics features with clinical data to improve prediction accuracy. Aim 2 focuses on developing a robust prediction model of peritoneal recurrence utilizing a pre-trained foundation model from large-scale H&E image data. Aim 3 will combine CT, H&E, and clinical data to further enhance predictive capabilities, employing an innovative cross-modal collaborative optimization approach for multimodal data integration. All models will be trained and internally validated using a retrospective cohort from Atrium Health Wake Forest Baptist Comprehensive Cancer Center and externally validated in two independent cohorts from additional institutions to ensure robustness across populations and imaging protocols. Additionally, we will compare our models with existing methods, including clinical staging and alternative fusion strategies. If successful, these models will enhance risk stratification and prediction of peritoneal recurrence in gastric cancer patients, significantly improving survival rates and quality of life by identifying those likely to develop peritoneal recurrence post-surgery and facilitating timely intervention. Furthermore, they can help avoid the risk of complications and extra medical costs associated with overtreatment. Since the information is derived from routinely examined CT, H&E and clinical data, they could be seamlessly integrated into current clinical workflows. The AI technology developed through this project has the potential to benefit underserved populations in low- resource settings and reduce healthcare disparities in the U.S.

Utilizing integrin-targeted PET imaging and therapeutics to predict and treat radiation-induced pulmonary fibrosis

Project Summary/Abstract. Lung cancer is the leading cause of cancer death in the US, with over 125,000 deaths annually. Radiation therapy (RT) is a critical component of curative lung cancer treatment for many patients. However, radiationinduced pulmonary fibrosis (RIPF) is a common side effect that carries a poor prognosis with limited treatment options. Up to 40% of patients with lung cancer who receive RT may experience RIPF. RIPF is a late effect of RT, typically occurring 3 or more months after treatment. The symptoms of RIPF can include shortness of breath, pleural effusions, decreased lung function, and respiratory failure. Cell surface integrin heterodimers play a key role in the pathogenesis of RIPF. In particular, the integrin αvβ6, which is expressed at a low level in the alveolar epithelium at baseline, is significantly upregulated upon RT damage. The key role of integrin αvβ6 in RIPF is illustrated by studies in which mice lacking integrin αvβ6, or treated with an αvβ6-blocking antibody, do not develop RIPF. Here, we propose to translate this mechanistic understanding of RIPF into novel approaches for monitoring and treating RIPF. We hypothesize that non-invasive αvβ6 PET imaging will be safe and can specifically bind to αvβ6 in patients with RIPF. Additionally, we hypothesize that a novel small-molecule integrin antagonist, IDL2965, can mitigate and treat RIPF in mice. In this project, we are utilizing mice to model RIPF, as mice develop RIPF that mimics human disease. In addition, cellular and in vitro models do not approximate the complex biology leading to the development of RIPF. Our data using [64Cu]Cu-DOTA-αvβ6-BP to detect early RIPF in mice are compelling in both single-fraction high-dose RT and lower dose-larger volume RT models (Lo et. al, IJROBP 2025). However, to progress to clinical trials in patients with cancer, we will obtain data to submit an Investigational New Drug (IND) application to the FDA. Importantly, we propose translating [64Cu]Cu-DOTA-αvβ6-BP PET imaging into patients with lung cancer, allowing us to better identify RIPF and develop a tool to determine the efficacy of IDL-2965 in future clinical studies. The specific aims of the proposal are: (1) Characterize the utility of [64Cu]Cu-DOTA-αvβ6-BP in mice with conventionally fractionated RT and identify circulating biomarkers of RIPF, and determine the in vivo toxicology of [64Cu]Cu-DOTA-αvβ6-BP to prepare and submit an exploratory Investigational New Drug (eIND) application to the FDA, (2) Conduct a first-in-human clinical trial of [64Cu]Cu-DOTA-αvβ6-BP to determine its safety and human dosimetry in patients with evidence of RIPF from computed tomography or in healthy controls, and (3) Determine the effect of integrin antagonism using IDL-2965 on mitigating RIPF in preclinical mouse models. The goals of this proposal are two-fold: (1) demonstrate safety and target specificity for [64Cu]Cu-DOTA-αvβ6-BP so that it can be used in future studies to identify RIPF and evaluate the efficacy of anti-fibrotic therapies, and 2) determine the ability of IDL-2965 to prevent RIPF in preclinical mouse models.

Transcriptional control of activation induced deaminase (AID) function

SUMMARY Somatic hypermutation (SHM) and class switch recombination (CSR) are vital for the generation of high affinity antibodies with appropriate effector function, protection against infection, and vaccine efficiency. They are initiated when the activation induced deaminase (AID) deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase 2 (Pol2). Aberrant DNA deamination by AID is an important driver of genetic instability and the development of B cell malignancies. Understanding the factors and mechanisms that coordinate AID-mediated deamination with Pol2 transcription is an important objective in the study of humoral immunity and the central goal of research under this grant. Our preliminary data demonstrate that Pol2 pause factor NELF, Super Elongation Complex (SEC) components MLLT1/3, and the phosphatase module of the Integrator-protein phosphatase complex (INT-PP2A) are required for SHM, with MLLT1/3 but not NELF being required for AID binding to its chromatin targets. Our findings yield a new conceptual framework and model for AID-Pol2 collaboration in which NELF and a balance between kinase and phosphatase activities of SEC and INT-PP2A regulate Pol2 pausing/elongation to generate the critical stalled Pol2 complex on which AID acts. Further, our work has yielded major methodological advances that allow us to overcome obstacles that have stymied progress in the field. In this proposal, we take advantage of these conceptual and technical advances to pursue our central goal through the following two aims: Aim 1: Determine the molecular mechanisms by which NELF and other Pol2 regulatory factors enable AID-Pol2 collaboration and SHM/CSR. It has previously been very difficult to assess the role of cell-essential factors in SHM. By combining our new Rapid Assay for SHM (RASH) cells with degron technology, we will determine the mechanism of action of our newly discovered regulators of SHM using genomic, transcriptomic, and interaction assays that assess Pol2 distribution, phosphorylation, and activity, and the chromatin binding profiles of and interactions between AID and components of NELF, SEC, and INT-PP2A. AID and MLLT1 appear to co-associate in a complex and we will test for a direct interaction between AID and MLLT1/3. Factors will be tested for roles in CSR and validated in human cell line and germinal center B cell models and in mice. Aim 2: Hypothesis testing and deep mechanistic analysis through perturbation of the balance between Pol2 pause/arrest and elongation. We will rigorously test our new model for AID-Pol2 collaboration using degron, reconstitution, mutagenesis, and small molecular inhibitor approaches to perturb the balance between Pol2 pausing and elongation, revealing how altering NELF-Pol2 interactions and the balance between SEC kinase and INT-PP2A phosphatase activities influences SHM efficiencies and AID binding. Together, our proposed studies are significant for the development of new technologies and for understanding mechanisms of antibody gene diversification and causes of genome instability and cancer.

Cytoskeletal connectors: Deciphering the fundamental mechanisms of cytoskeletal dynamics and transport

PROJECT SUMMARY The cytoskeleton is a dynamic network of filamentous structures, including microtubules and actin, that regulate essential cellular processes such as cell shape, growth, and signaling. Cytoskeleton also serves as tracks for molecular motors, which transport a variety of cellular cargoes, including organelles, macromolecules, and vesicles. These cargoes are linked to motors by specialized connector proteins. Disruptions in connector proteins are implicated in a range of neurodevelopmental and neurodegenerative diseases, as well as cancers. Despite their importance, these proteins continue to be understudied, primarily due to their perceived role as passive linkers and the technical challenges in working with them. However, recent discoveries suggest that connector proteins may play more active roles, in some cases even have enzymatic functions. This proposal aims to uncover mechanisms of connector protein functions through a detailed investigation of actin-microtubule and motor-cargo interactions. Actin and microtubules are linked by the spectraplakin family of large and evolutionarily conserved proteins, critical for neuronal development and differentiation. Recent discoveries of ATPase domains within these proteins suggest they may haves beyond simply linking cytoskeletal components. One goal of this proposal is to investigate the role of spectraplakin’s ATPase domains via structural, biochemical, and cell biology approaches. Another goal is to explore how dynamic changes in motor-cargo connectors facilitate the transport of diverse cargoes along microtubule tracks. The focus will be on the cytoplasmic dynein-1 (dynein) and the connectors (adaptors) that activate and link dynein to cargo. Dynein is a microtubule minus-end directed motor that plays essential roles in cell division, and transports hundreds of different cellular cargoes. While several motor-cargo connectors have been identified, the regulatory mechanisms enabling cargo transport are not fully understood. We are investigating whether connector proteins work together to activate dynein movement and/or facilitate cargo handoff between different dynein complexes. Using innovative approaches, including time- resolved cryo-EM, complex in-vitro reconstitutions, and live-cell imaging in induced neurons, we are uncovering critical mechanisms that govern cytoskeletal connector proteins, furthering our understanding of how the cytoskeleton regulates essential cellular processes.

Molecular Mechanism of Immunoglobulin Class Switch Recombination

Antibodies produced by B cells are a critical component of the adaptive immune system in mammals that can respond to and clear a plethora of different pathogens. A key property of B cells is their ability to alter the coding sequence of the immunoglobulin heavy and light chain genes, via VDJ-recombination, somatic hypermutation (SHM) and class switch recombination (CSR). While VDJ-recombination and SHM alter the variable regions of antibodies that directly contact pathogen antigens, CSR changes the constant region of the antibody, which dictates its effector function to optimally respond to the antigen recognized by the antibody. CSR occurs via targeted DNA double strand break (DSB) induction in the switch regions preceding the distinct constant region coding sequences. DSB induction requires active transcription of the switch regions and is initiated by activation-induced cytidine deaminase (AID) induced cytosine deamination (converting cytosine to uracil) within the switch regions. Fusion of the DSBs in the switch regions results in deletion of intervening genomic sequence, completing CSR. Since AID is inherently a mutagenic enzyme that can trigger both point mutations and genomic translocations, its activity has to be tightly controlled, and aberrant AID activity has been directly implicated in the genetic changes that lead to B cell lymphoma formation. Thus, define the molecular mechanism of CSR is critical to understand our adaptive immune system and B cell cancer development, both highly relevant to human health. To study CSR in living B cells, cellular models have been developed to analyze AID function and switch region transcription at the single molecule level. With this new methodology, the critical unanswered question of how AID is specifically recruited to the immunoglobulin heavy chain locus and not other genomic locations will be addressed. In addition, the overall kinetics of CSR will be determined and how transcription controls specific DSB induction in switch regions will be defined. The results of these works will significantly advance our understanding of CSR and provide new insights on how AID contributes to B cell lymphoma formation.

Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections

Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes essential for various cellular processes, including survival, proliferation, differentiation, self-renewal, angiogenesis, and immune response. Abnormal and persistent STAT3 activation is detected in diverse human cancers, driving multiple pro- oncogenic functions. Multiple antitumor drug development targets the inhibition of STAT3 to treat various types of cancer. Unfortunately, downregulated STAT3 significantly increases host susceptibility to recurrent infections, especially pneumonia. Additionally, individuals with genetic polymorphisms associated with lower STAT3 expression are more susceptible to severe tuberculosis. Furthermore, patients with autosomal dominant hyper- IgE syndrome (AD-HIES), also known as Job Syndrome, which is caused by de novo STAT3 mutations and substantially decreased STAT3 expression, have a significantly increased susceptibility to bacterial and fungal infections, with high mortality rates and a shortened life span often associated with Pseudomonas aeruginosa infections. Gram-negative bacteria, particularly P. aeruginosa, are opportunistic pathogens that frequently cause hospital-acquired infections. The problems are worsened by the emerging P. aeruginosa with multidrug resistance (MDR), especially in patients with repeated antibiotic treatments, such as Job Syndrome sufferers. Notably, airway epithelial cell-derived proteins play a significant role in the antimicrobial milieu, promoting effective host defense against invading pathogens. One of the most critical STAT3-regulated antimicrobial molecules is bactericidal permeability-increasing protein fold A1 (BPIFA1, also known as SPLUNC1), a multifunctional innate immunity molecule and indispensable host defense protein that is abundantly secreted in the lungs. This application aims to elucidate how STAT3 deficiency impairs host epithelial defense against microbial infections and whether BPIFA1-mediated innate immune responses can sufficiently restore effective antimicrobial protection to prevent pneumonia. The long-term objective is to advance our understanding of the respiratory innate immune response, particularly in relation to epithelial cell-specific antimicrobial defense. We characterized BPIFA1 as an airway lining fluid protein secreted apically in the airway lumen and in primary human airway epithelial cultures. In this study, we hypothesize that mucosal BPIFA1 is an essential antimicrobial protein that plays a critical role in host defense against microbial infections in STAT3-deficiency- associated pneumonia. Our proposed studies will assess innate immunity mechanisms regulating the antimicrobial activity of the airway epithelium in STAT3 deficiency-associated lung infections. By focusing on the crucial epithelial-derived protein product, BPIFA1, our study will provide an alternative treatment for respiratory infections by augmenting native host defense mechanisms in high-risk individuals, including AD-HIES, cancer, and immunocompromised patients.

Role of stress signals in the pathogenesis of pulmonary veno-occlusive disease

PROJECT SUMMARY/ABSTRACT Pulmonary veno-occlusive disease (PVOD) is a subclass of pulmonary hypertension characterized by preferential remodeling of the pulmonary venules and capillaries, and currently, there are no efficacious drug therapies. The clinical presentations and the radiographic findings of PVOD are indistinguishable from PAH, and therefore, it is often misclassified as PAH. However, the application of PAH therapeutics to PVOD patients leads to life-threatening pulmonary edema, thus, there is a critical need for diagnostic methods that accurately differentiate PVOD from PAH. Genetically, PVOD is associated with biallelic loss of function (LOF) mutations in the EIF2AK4 gene encoding GCN2. GCN2 phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α), shuts down protein synthesis, and activates the integrated stress response (ISR). However, the molecular mechanisms connecting the loss of GCN2 with pulmonary vascular remodeling are poorly understood. Recent studies find that biallelic EIF2AK4 mutations are identified in ~9% of PAH patients. Conversely, heterozygous mutations in the BMPR2 gene, a leading cause of PAH, have been reported in PVOD patients. These results suggest that (i) PVOD and PAH share some of the pathophysiological mechanisms, and (ii) the presence of EIF2AK4 or BMPR2 mutations does not provide an accurate genetic diagnosis for PVOD. The long-term goal of this proposal is to elucidate the pathophysiological mechanisms involved in remodeling not only pulmonary arterioles but also venules and capillaries and develop those pathways as potential therapies for POVD. It has been observed that cancer patients administered with the chemotherapeutic agent mitomycin-C (MMC) rapidly develop PVOD. Rats administered with MMC develop PVOD-like phenotypes, including right ventricular (RV) hypertrophy, increased RV systolic pressure, and pulmonary vascular lesions in arteries and veins. We found that Rad51, an essential enzyme for double-strand DNA break repair, associates with VE-Cad in the vascular endothelium; however, upon MMC treatment, Rad51 and VE-Cad complex (VRC) were released into the circulation, resulting in increased vascular permeability and reduced barrier integrity. MMC treatment also mediates the depletion of GCN2, which recapitulates the genetic cause of PVOD (LOF EIF2AK4 mutations). Based on these data, this proposal will test the hypothesis that the vascular remodeling in PVOD involves (i) the release of VRC, (ii) the aberrant protein synthesis due to the activation of ISR, and (iii) the mechanism of maladaptive ISR activation. Finally, we will explore the potential application of the circulating VRC as a blood biomarker for PVOD.

Multiplex single-cell chemical genomics to identify small molecule modulators of tumor cell-intrinsic immunogenicity in glioblastoma

PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme is the most common and aggressive primary brain cancer. Despite a multimodal treatment regimen of surgical resection, chemotherapy, radiotherapy, and tumor-treating fields, most patients succumb to the disease within two years of diagnosis. Cancer immunotherapy strategies have emerged as a powerful tool for treating aggressive solid tumors such as melanoma and non-small cell lung cancer. However, current strategies have led to low response rates in glioblastoma, resulting from its low immunogenicity. The proposed research program aims to identify small molecules capable of increasing the immunogenicity of glioblastoma cells, focusing on altering gene expression programs associated with recognition by the immune system and the ability of cytotoxic immune cells to target glioblastoma for destruction. We will use highly multiplex chemical transcriptomic profiling to determine the molecular consequence of exposing glioblastoma neurosphere models to 3,792 small molecules, targeting the majority of cellular activities and clinically relevant drug targets as well as a collection of previously identified immunomodulators. We will then determine how each exposure alters the expression of gene programs associated with tumor cell immunogenicity and response to therapy, including the expression of genes associated with the recognition by the immune system and those associated with immune checkpoints, as well as programs more broadly correlated with resistance to anti-cancer therapies. Chemical hits that meet specific criteria will be subjected to a medicinal chemistry review to further classify compounds by their suitability for treating malignancies in the brain. We will then screen chemical hits to determine their ability to modulate immune-mediated tumor cell killing using tumor- immune cell co-culture. Lastly, we will leverage gene editing and flow cytometry to validate hits based on on- target molecular effects and further refine the mechanism of action by inspecting the ability of drugs to modulate immunogenic programs at the protein level. Our chemical genomics screens aim to provide crucial information regarding the link between pathway activity and immunomodulation in GBM, a critical step to guide future efforts in GBM immunotherapy. More broadly, our study will establish single-cell chemical genomics as a scalable platform for phenotype-based screening for preclinical prioritization of chemical modulators of complex transcriptional phenotypes and provide a framework for hit prioritization, establishment of pipeline robustness and hit validation in the context of single- cell chemical genomics screens.

Targeted Prodrug Cytokines for Metastatic Breast Cancer Immunotherapy

Project Summary. Our approach directly addresses key limitations in targeting and treating metastatic breast cancer, where we propose the selective activation of modular immune-modulating cytokines within the hypoxic and ROS-active TME for delivery across the BBB, providing the necessary pre-clinical data for future clinical translation. The in vitro and in vivo investigations of this novel immunotherapeutic in immunocompetent models will allow our team to study the interplay between tumor-driven immune activation, cytokine signaling, and anti-tumor immunity in both primary and metastatic sites, and establish a robust groundwork for subsequent clinical validation within the OSUCCC. This proposal addresses two key challenges in developing a novel immunotherapy strategy for breast cancer by answering two hypotheses: (1) can a modular immunotherapy platform with tumor-selective activation of prodrug recombinant cytokines overcome these limitations in drug delivery, and (2) can the development of nanobody-cytokine fusions that can selectively target primary breast cancer tumors and cross the BBB to reach metastatic tumor sites? The first hypothesis focuses on achieving tumor environment-specific activation of prodrug-based recombinant cytokines. Protein cytokines are highly potent, and while others have tried to block their activity using a fused genetic linker to ‘mask’ functionality, no one has yet attempted to use a non-canonical-based chemical strategy to achieve this inhibition. Immune-modulating cytokines will be recombinantly expressed with integrated ncAAs that block cytokine activity until the function is regenerated in the breast cancer TME. Once the cytokine activity is controlled, our second hypothesis will be to achieve selective delivery of the cytokine via fusion to nanobodies. While success has been found in targeting primary tumors in drug and protein delivery, a key challenge remains in reaching secondary metastatic tumors in hard-to-reach sites (i.e., brain). Engineered nanobodies, with affinity for breast cancer tumors and the ability to bind to BBB transcytosis receptors, will enable selective delivery to metastatic breast-to-brain tumors, resulting in tumor- specific activation, immune responses, and improved therapeutic outcomes. This system can significantly improve therapeutic outcomes for patients with mBC by integrating selective activation and delivery mechanisms to reduce off-target effects and enhance tumor-specific immune responses in both primary and secondary metastatic tumor sites. Optimizing drug delivery systems to tune immune responses could offer more effective and less invasive treatment options when compared to traditional and engineered cell-based approaches. Our momentum towards precision medicine and targeted therapies holds significant promise for improving outcomes for mBC patients, and has the potential to serve as a pan-cancer treatment for aggressive metastatic cancers from the following aims: (1) generating a modular platform for tumor-specific activation of prodrug cytokines, (2) evaluating cytokine delivery and anti-cancer immune phenotypes in mBC.

Overcoming Treatment Resistance by Targeting Polyploid Breast Cancer Cells with AI assisted Single-Cell Analysis

Therapy resistance remains a formidable challenge in breast cancer treatment, with emerging evidence identifying polyploid giant cancer cells (PGCCs) as key drivers. These cells, arising through whole-genome doubling (WGD) events, exhibit enhanced resistance to therapies, contributing to disease relapse. PGCCs are characterized by enlarged cell and nuclear sizes, increased DNA content, and greater resilience compared to non-PGCCs. Their prevalence escalates with disease progression and therapeutic stress, underscoring their critical role in treatment resistance. As such, we hypothesize that inhibiting polyploid cancer cells can effectively reduce therapeutic resistance. Despite this, effective strategies targeting PGCCs are limited, hindered by the lack of high-throughput methods to assess PGCC viability and abundance. Traditional screening assays lack the sensitivity to detect the elimination of small populations of PGCCs, while current detection methods, such as visual inspection and flow cytometry, are not suited for high-throughput compound screening. Our preliminary work has established a high-throughput single-cell morphological analysis pipeline capable of quantifying PGCCs, and we successfully screened 2,726 compounds for their efficacy on PGCCs. Based on the preliminary success, we aim to further improve its robustness and accuracy under diverse staining and imaging conditions, ensuring consistent performance across multiple labs for widespread use in PGCC/WGD studies, with deep learning to accelerate the discovery of therapeutic strategies targeting PGCCs. In addition to empirical screening, our scRNA-Seq analysis of PGCCs has revealed altered gene expression, particularly in genes associated with FOXM1, a transcription factor critical in cell cycle regulation and linked to poor outcomes in various cancers. PGCCs also show altered ferroptosis regulators and elevated reactive oxygen species (ROS), indicating susceptibility to ferroptosis. Here, we propose two independent and complementary aims. Aim 1: We will develop and validate a robust deep learning–based single-cell morphological analysis pipeline for accurate PGCC/non-PGCC discrimination across variable staining, imaging, and lab settings. The model will be benchmarked on independent datasets from external labs and released as open-source, version-controlled software with full documentation to support reproducibility and broad adoption in PGCC/WGD research. Aim 2: Leveraging our screen of 2,726 FDA-approved compounds and mechanistic studies of FOXM1 and ferroptosis, we will prioritize and validate therapies that eradicate PGCCs and reduce treatment resistance. Using patient- derived cells, 3D spheroids, and syngeneic/xenograft models, we will rigorously assess top candidates as monotherapy and in combination with standard-of-care agents. Successful completion of this project will accelerate PGCC/WGD research, advance therapeutic strategies to overcome breast cancer resistance, and especially deliver benefits to patients with high PGCC burden. Given the prevalence of WGD across solid tumors and its induction by standard therapies, our approach holds broad clinical relevance and translational impact.

A NOVEL GEMM TO ELUCIDATE THE ROLE OF CHAF1A IN NEUROBLASTOMA DEVELOPMENT

PROJECT SUMMARY: This proposal focuses on the fundamental understanding on how the CHAF1A oncogene drives molecular mechanisms, cellular signaling, and metabolic processes in the oncogenesis of neuroblastoma (NB). NB is an aggressive pediatric cancer, which accounts for 15% of pediatric cancer mortalities. High-risk NB is thought to arise from a small number of recurrent genetic alterations that block the ability of neural crest cells (NCCs) to differentiate. To assess the molecular mechanisms governing NC differentiation, our laboratory has established a definitive role of the epigenetic regulator CHAF1A in blocking NC differentiation and driving NB oncogenesis. In this proposal, we will determine the impact of CHAF1A on NB initiation and progression. To accomplish this goal, we propose to develop a novel CHAF1A-driven genetically-engineered mouse model (GEMM) of NB and test the impact of CHAF1A on NB incidence, histology and metastasis, and the tumor immune microenvironment (TIME). We hypothesize that CHAF1A will increase de novo incidence of NB, reduce mouse survival, and promote a suppressive TIME. By developing a novel GEMM of NB and employing innovative technology (including ATAC-seq, lipidomics, and scRNA-seq), we will: 1- elucidate the role of CHAF1A in NB tumor initiation and progression; and 2- determine the impact of CHAF1A on MYCN-induced oncogenesis. These findings will provide a novel view on the molecular mechanisms driving NB initiation, and will have high clinical implications, informing future differentiation-based interventions for high-risk NBs.

Optimizing gamma-delta T cell receptor-mediated signaling to improve cancer immunotherapy

PROJECT SUMMARY The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell subpopulations, such as the gd T cells which are known to possess antitumor activities. Further, gd T cells are mostly independent of MHC-restriction, unconstrained by neoantigen burden, preferential homing to peripheral tissues and possess unique properties of T cells as well as natural killer cells making them an extremely attractive cancer immunotherapy target. One way of gd T cell activation involves the gd T cell receptor (gdTCR)-CD3 signaling pathway. gd T cell recognition of antigen by the gdTCR and the resulting proximal signaling through surrounding CD3 subunits are key steps of gd T cell activation. Even though the individual components of the gdTCR-CD3 and abTCR-CD3 complexes remain the same except for the TCRs, the complete gdTCR-CD3 complex extracellular structure is unknown. Identification of the specific extracellular interactions between the gdTCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate gdT cell immunity by targeting signaling through the gdTCR-CD3 complex. Our previous data showed that mutating residues in the constant domain of the abTCR resulted in altered ab T cell cytokine responses. Based on this data, our hypothesis is that gdTCR-CD3 signaling can also be modulated by targeting specific regions of the gdTCR by mutagenesis to improve gd T cell antitumor activities. To test our hypothesis, in Aim 1, we will use a novel photo-crosslinking and computational docking methodology to solve the complete extracellular structure of a gdTCR-CD3 complex. Further, we will use an in silico structure-based TCR design approach to identify gdTCR mutants that enhance signaling. In Aim 2, we will use an in vitro retroviral TCR display method using degenerate primers to create gdTCR mutant libraries at specific gdTCR sites such as Cg helix 3 and connecting peptide (CP) regions. In both instances, identified mutants will be tested for improved functionalities in an MHC-independent gd TCR (G115 Vg9Vd2 TCR) using in vitro cytokine and tumor-killing assays. Overall, the newly identified enhanced gd T cell clones could potentially lead to a new wave of effective cancer immunotherapy strategy by leaning into the largely untapped potential of gd T cells.

Personalized Spatial Regulatory Networks to Decode Breast Cancer Microenvironments

PROJECT SUMMARY Triple-negative breast cancer (TNBC) is an aggressive subtype with early recurrence, high metastatic burden, and limited treatment options. While genomic alterations contribute to its progression, epigenetic plasticity and spatial organization within the tumor microenvironment (TME) play critical roles in intra-tumor heterogeneity, immune evasion, and therapy resistance, yet remain poorly understood. To address this, we will develop a cost- effective and scalable methodology that integrates spatial ATAC-seq, spatial in situ transcriptomics (Xenium), and single-nucleus (sn) Epi Multiome sequencing (snRNA-seq + snATAC-seq) from core-needle biopsies, enabling high-resolution mapping of gene regulatory networks within the intact TME. Our preliminary data from six TNBC biopsies demonstrate that spatial in situ transcriptomics and spatial ATAC-seq provide critical insights into tissue architecture but suffer from data sparsity, necessitating the integration of single-nucleus Epi Multiome data to enhance cell-type annotation and impute missing genomic features. In Aim 1, we will establish a multi- modal workflow that maximizes molecular insights from limited biopsy material by optimizing tissue-preserving and multiplexed sequencing approaches. This includes leveraging patient-specific genetic variation to deconvolute nuclei-derived data and linking it to spatial transcriptomic and spatial chromatin accessibility profiles. In Aim 2, we will develop a computational framework to integrate these multi-layered datasets, enabling spatially resolved epigenomic-transcriptomic analysis that identifies key regulatory chromatin elements and transcriptional programs associated with TNBC progression, immune infiltration, and therapy resistance. This project will generate the first comprehensive, patient-specific spatial regulatory atlas of TNBC, providing fundamental insights into how chromatin accessibility and gene expression interact within the TME. Ultimately, this work will pave the way for novel precision oncology strategies, biomarker discovery, and the development of targeted therapies that address TNBC’s spatial and molecular heterogeneity.

Structure-Based Development of Nucleotide-Competing Inhibitors Against HIV-1 and LINE-1 Reverse Transcriptases

PROJECT SUMMARY Reverse transcriptases (RTs) from retroviruses and endogenous retroelements are essential polymerases that catalyze RNA- and DNA-dependent DNA synthesis. Nucleoside inhibitors (NIs) remain central to HIV-1 therapy and are also used against other viral infections and in cancer, but toxicity, limited selectivity, pharmacokinetic (PK) liabilities, and the emergence of drug resistance highlight the need for alternative RT inhibitor mechanisms. In contrast to NIs, nucleotide-competing inhibitors (NCIs) block the polymerase active site without requiring incorporation into nucleic acids. Structural studies by PI Ruiz have defined the NCI mechanism of action for HIV- 1 RT and revealed conserved binding modules shared across multiple polymerase families. These advances now enable rational discovery of improved NCIs. LINE-1 (L1) ORF2 RT is an emerging therapeutic target in cancer, autoimmunity, and aging, yet NIs are the only inhibitors known to act against L1 RT. Notably, the NCI-binding region is structurally similar between HIV-1 RT and L1 RT, suggesting that NCI recognition principles may extend across these two biologically distinct polymerases. This R21 seeks to establish proof-of-concept for NCI development against both enzymes. Aim 1 will discover and structurally optimize NCIs targeting HIV-1 RT by combining binding modules from known NCI chemotypes and determining their biochemical activity and co-crystal structures. Aim 2 will determine whether HIV-1 RT NCI principles translate to L1 RT by solving L1 RT/nucleic acid/NCI structures, evaluating enzymatic inhibition, and applying AI-based structure prediction and generative design to propose L1-specific NCI candidates. Cellular retrotransposition assays will test mechanism of action. Aim 3 will develop a fragment library tailored to protein–nucleic acid interfaces and perform fragment screening of HIV-1 and L1 RT/nucleic acid complexes to identify additional chemotypes that engage the NCI binding region. Successful completion will yield NCI scaffolds and mechanistic insights applicable to HIV-1 RT and L1 RT, define structural principles governing NCI recognition across two evolutionarily related polymerases, and establish new avenues for RT inhibitor development. The PI is highly qualified to lead this work, with extensive expertise in RT structural biology, drug design, and fragment-based discovery.

Post-diagnosis changes in body composition and renal cell cancer survival

ABSTRACT Significance. Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and most lethal subtype, and there is great interest in the identification of potentially modifiable prognostic factors. Although weight status seems to be relevant, the relationship between body mass index (BMI) at diagnosis and survival among ccRCC patients indicates that mortality is lowest among those classified as overweight or obese at the time of diagnosis by BMI. This has resulted in confusion in clinical guidance for weight management among ccRCC patients. Recent work involving body composition features (adipose and muscle tissue) has provided some insight, but we do not understand how weight or body composition changes after diagnosis relate to survival, nor how these changes relate to pathological and molecular tumor features— information which is needed to resolve this controversy. Rigorous analytical approaches are further required to accurately address these questions. Innovation. Our study is highly innovative in that 1) we will be the first to leverage a large-scale cohort of ccRCC patients with multiple assessments of weight and body composition from diagnosis onward; 2) we will examine tumor characteristics, including molecular features, as potential drivers of these changes; and 3) we will use a rigorous joint modeling approach to simultaneously model the post-diagnosis trajectories of weight and body composition and their relationships with cancer outcomes in the most statistically sound manner. Our findings will inform clinical management of, and identify modifiable body composition features to improve survival for the growing number of ccRCC patients. Approach. We will use available data from the RESOLVE cohort, an NCI-funded retrospective cohort of 1,239 Stage I-III clear-cell renal cell carcinoma (ccRCC) patients diagnosed between 2000-2020 at Memorial Sloan Kettering Cancer Center. These data include clinical and patient-level factors collected from the medical record, including repeated height and weight assessments, body composition measures from existing computed tomography scans, pathological and molecular tumor characteristics, and overall survival (OS) and disease-free survival (DFS). We will use a joint modeling approach to simultaneously model changes in post-diagnosis body weight (Aim 1) and OS and DFS, as well as post-diagnosis changes in muscle and adipose tissue features (Aim 2) and OS and DFS. Models will include molecular tumor characteristics as predictors of these longitudinal trajectories. Impact. These results will provide crucial insight into the relationship between body composition changes and outcomes among ccRCC patients, and potentially identify tumor-related characteristics driving these associations. These results will resolve apparent paradoxes around the relationship between obesity and ccRCC mortality and identify potential targets for nutrition and physical activity interventions on body composition.

RECONJOINT: A Preference Elicitation Tool to Improve Shared Decision Making for Breast Reconstruction Surgery

PROJECT SUMMARY/ABSTRACT Breast reconstruction is a critical component of comprehensive breast cancer care, offering physical and emotional restoration after mastectomy. However, 40% of women undergoing breast reconstruction report dissatisfaction and decisional regret due to low involvement with treatment decisions and poor alignment between treatment preferences and the chosen reconstructive technique. Current approaches to shared decision-making (SDM) often fail to elicit and integrate individual-level preferences into treatment planning. This serves as a barrier to effective SDM and patient-centered care. To address this gap, we developed a web- based decision tool that uses adaptive choice–based conjoint (ACBC) analysis to elicit patient-level preferences for breast reconstruction. Preliminary studies indicate that the decision tool is acceptable and usable; patients wanted to view their results and use the tool in clinic, which we could not accommodate at the time because the decision tool currently lacks a structured method for clinical integration. We propose to develop an implementation toolkit for the decision tool to facilitate clinical integration and then test the feasibility, acceptability, and implementation of the intervention, RECONJOINT (decision tool and toolkit). In Aim 1, we will design an implementation toolkit informed by focus groups and developed with input from key partners, including patients, providers, and patient advocates. Candidate elements for the implementation toolkit include components developed for site-level implementation: treatment preferences report, video introducing the tool and existing evidence, and recommendations for patients and providers to incorporate preferences into SDM. In Aim 2, we will evaluate the feasibility, acceptability, and preliminary efficacy of the intervention in a pilot cluster-randomized hybrid type 1 trial conducted at two cancer centers (Memorial Sloan Kettering and Duke University). Our primary outcome of interest is the feasibility of the intervention. Secondary outcomes include acceptability and preliminary efficacy. Using a hybrid design, we will simultaneously evaluate facilitators, barriers, and strategies to implementation and how these factors influence the feasibility and acceptability of the intervention. The Consolidated Framework for Implementation Research and the Theoretical Domains Framework will serve as conceptual frameworks. This study is innovative as it leverages ACBC analysis to elicit patient preferences, designs an intervention with multilevel input from clinical and community partners, and uses a hybrid trial design to simultaneously evaluate feasibility, acceptability, preliminary efficacy, and implementation. By addressing critical barriers to SDM and enhancing patient–provider communication, this research aligns with the goals of PA-25-253 and the National Cancer Plan to deliver high quality, patient-centered cancer care. Findings from this study will inform a full-scale multi-site trial to evaluate the efficacy of the intervention and implementation outcomes (e.g., reach).

Structure-function and mechanistic studies of a specific glycosyltransferase complex in fusion-driven pediatric gliomas

Abstract Glycosylation is a co/post-translational modification involved in cell-matrix interactions, antigen-antibody interactions, tumor invasion, and cell motility. Abnormal glycosylation is a hallmark of cancer, with various glycosylation-related genes linked to glioma prognosis and tumor heterogeneity. Pediatric low-grade gliomas (pLGGs) stand as the most common childhood central nervous system tumor, accounting for 30%-40% of all CNS tumors in children. Despite its relatively low mortality rate, pLGGs are associated with devastating lifelong morbidity. The most common alteration found in 75% of tumors is the KIAA1549:BRAF fusion, causing an aberrant activation of the MAPK/ERK signaling pathway. Current treatments, such as traditional chemotherapies and targeted therapies, have limitations such as resistance, lack of specificity, toxicity and paradoxical activation of the MAPK pathway. This highlights the urgent need for novel therapeutic approaches. Investigations into KIAA1549:BRAF-driven pLGGs identified their dependency on the protein-O-mannosyl transferase (POMT) complex for survival. In contrast, BRAFV600E-mutant cells did not show dependency, suggesting the POMT complex as a vulnerability and promising target in KIAA1549:BRAF-driven pLGGs. Therefore, our goal is to characterize the POMT complex structurally and biochemically and study its roles in KIAA1549:BRAF-driven pLGGs. In this proposal, we aim to 1) determine the high-resolution structures of the complex in its unbound, substrate-bound, and inhibitor-bound forms and 2) elucidate the POMT complex mechanisms in KIAA1549:BRAF-driven pLGGs. We will define the critical functional domains, active sites, interaction interfaces and translational modifications crucial for enzymatic activity using cryo-EM techniques, mutagenesis, and functional studies. To study biological pathways and molecular events modulated by the POMT complex, we will implement global proteomics and transcriptomics analysis in well-characterized disease models. In parallel, we will assess the effect of the POMT complex on the MAPK/ERK signaling pathway. This study will guide the structure-based design of probes and drugs targeting the POMT complex and will unveil glycosylation-mediated oncogenesis in pediatric gliomas. It will aid in the development of new targeted therapies and the identification of new biomarkers for pLGGs harboring the KIAA1549:BRAF fusion. The research will be conducted in the Fischer lab at Dana-Farber Cancer Institute, which provides a collaborative and resource-rich environment. The career development plan includes training in scientific writing, mentoring, and presentation skills, as well as interdisciplinary networking with experts in structural biology and pediatric oncology. The candidate’s career goal is to establish an independent research laboratory focused on developing new therapeutic modalities for pediatric neurooncology. The training provided through this fellowship represents a critical step toward achieving this goal.

Dual mRNA Therapeutics for Liver Metastatic Uveal Melanoma

Abstract Uveal melanoma (UM) is the most common primary intraocular cancer in adults, accounting for approximately 70% of all ocular malignancies. Current treatments for primary UM include surgical tumor removal, transpupillary thermotherapy, and radiotherapy. Unfortunately, both surgical enucleation and brachytherapy have shown similar survival outcomes and carry an equivalent risk of metastasis. While the survival rate for patients with primary, non-metastatic UM is relatively high, metastatic uveal melanoma (MUM), especially when it spreads to the liver, remains universally fatal. The liver is the first site of metastasis in 80 to 90 percent of cases, and about 50 percent of UM patients develop liver metastases within 15 years of initial diagnosis. Median survival following liver metastasis is only 5 to 7 months, with an almost zero percent five-year survival rate. Currently, no available therapy significantly improves outcomes for patients with liver MUM. This R21 project addresses this urgent unmet need by developing liver-tropic mRNA therapeutics targeting two key drivers of MUM progression and metastasis: (1) constitutive activation of Gαq/11 caused by single-point mutations, and (2) loss-of-function mutations in BAP1. Both alterations occur in over 80 percent of UM patients and are associated with poor prognosis. We hypothesize that inhibition of constitutively active Gαq/11 and/or restoration of BAP1 tumor suppressor function will significantly suppress MUM progression and improve survival outcomes. Aim 1 focuses on delivering mRNA encoding a novel protein trap designed to specifically inhibit constitutively active Gαq/11 and its downstream oncogenic signaling pathways. Aim 2 seeks to restore wild-type BAP1, which is mutated or lost in approximately 84 percent of MUM cases, through liver-tropic mRNA delivery using a liver MUM model established via splenic inoculation. We will also evaluate the potential synergy between Gαq/11 inhibition and BAP1 restoration. The success of this project will not only advance our understanding of the disease mechanisms underlying MUM but also provide clinically viable strategies for treating liver metastases in uveal melanoma.

Identifying host-interacting proteins of Sneathia vaginalis

PROJECT SUMMARY AND ABSTRACT Sneathia vaginalis is a member of the human normal flora of the vagina. It is also a human pathogen that is associated with preterm birth and amniotic fluid infections as well as the most common bacterial species associated with HPV infection and cervical cancer. The identification of S. vaginalis as a human pathogen is recent and little is known about how this bacterium interacts with the host in either its commensal or pathogenic lifestyles. With the exception of a single toxin, no additional virulence factors have been identified. Our preliminary data demonstrates that S. vaginalis grows to high cell density in rich media; however, it fails to grow planktonically in other media unless host cells are present indicating that S. vaginalis relies on host cells for nutrients. This is consistent with its reduced genome. In addition, bacterial proliferation requires close proximity with the host cells and previous studies demonstrate that S. vaginalis can bind a variety of host cells. The proteins that mediate contact are unknown. We hypothesize that proteins on the surface of S. vaginalis are critical for host cell adhesion. We will use two Aims to examine this. Aim 1 will use mass spectrometry to identify S. vaginalis surface proteins. Aim 2 generates deletion strains of potential adhesins identified in Aim 1 as well as predicted host-interacting proteins that have already been identified bioinformatically based on those in other bacteria. The mutant bacteria are then tested in host-cell adhesion assays. Together, these aims will identify for the first time the proteins found on the surface of S. vaginalis while identifying proteins that interact with host cells that would be expected to contribute to either its commensal or pathogenic lifestyles or both. Moreover, these studies would be used to inform clinical lab practice as surface-expressed proteins could be used to identify identifying markers of S. vaginalis detection.

Targeting subtype specification as a driver of PDAC health disparities

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is refractory to current treatment strategies due in part to adaptive mechanisms of chemoresistance. Racial health disparities also confound the treatment and care of these patients. Blacks (people with African genetic ancestry) have significantly higher incidence rates of PDAC and decreased survival times compared to Caucasians (White genetic ancestry) even after socioeconomic status and tumor stages are controlled. Therefore, it is possible different racial groups exhibit unique molecular characteristics in PDAC tumors that contribute to these health disparities. The unique molecular characteristics that distinguish PDAC tumors between racial groups exhibiting disparities have the potential to identify new therapeutic targets. In a previous study, we identified 4 distinct subtypes of PDAC (Metabolic, Progenitor-like, Proliferative, and Inflammatory) that can be distinguished using multivariate analysis of quantitative proteomic data. While these PDAC subtypes are predictive of therapeutic response, this has not yet been analyzed in disparity factor balanced studies. We have examined the proteomes of primary PDAC tumors using quantitative mass spectrometry and identified unique protein signatures for Blacks and Whites. PDAC tumors from Black patients display features consistent with the Inflammatory subtype of PDAC, which is characterized by an inflamed microenvironment expressing complement proteins that can promote resistance to chemotherapy. Therefore, it is possible that race influences subtype and Blacks could preferentially develop the more aggressive and treatment refractory Inflammatory subtype. Strategies are needed to modulate subtype to improve response to chemotherapy. Toward this goal, our proteomic analysis identified polycomb repressor complex 1 (PRC1) protein RNF2 as being upregulated in PDACs from Blacks compared to Whites. We have also discovered that RNF2 regulates mRNA expression of the PDAC subtype specification factor GATA6 and inhibiting RNF2 promotes a molecular shift toward the more chemosensitive Classical subtype of PDAC. Therapeutic targeting can be achieved with Tazemetostat that inhibits the upstream PRC2 to prevent RNF2 binding the GATA6 promoter leading to its increased expression. Additionally, the Inflammatory subtype characterized by innate immune complement protein activation could be targeted with another FDA approved drug, Avacopan, which has not previously been studied in PDAC. Therefore, the Specific Aims of this proposal are designed to: 1) Evaluate the extent to which Tazemetostat treatment impacts chemotherapy-induced subtype plasticity in patient derived organoids; and 2) To determine the extent to which strategies targeting pathways associated with PDAC disparities affect progression and subtype characteristics in vivo. The successful completion of these aims has the potential to be moved quickly into phase I clinical trials since both Tazemetostat and Avacopan are FDA approved drugs. Furthermore, if successful, this project has the potential to mitigate health disparities in PDAC and broadly improve patient outcomes by implementing new precision interventions. The mouse models we propose faithfully recapitulate pancreatic cancer's clinical syndrome, histopathology and molecular properties, including the often-unique features of the stromal and immune responses that constitute the complex desmoplasia of this disease, which cannot be addressed using in vitro model systems

I3-BC: Image-Based Infiltrating Immune Cell Detection and Outcomes in Breast Cancer Clinical Trials

PROJECT SUMMARY Tumor infiltrating lymphocytes (TILs) represent an accessible biomarker of the tumor-immune microenvironment (TIME) in breast cancer, demonstrating consistent association with response to neoadjuvant chemotherapy and outcomes in HER2-positive and triple-negative breast cancer. Despite efforts to standardize TIL enumeration from hematoxylin and eosin stained tumor slides, TILs have not gained widespread adoption due to inter- observer variability, and time limitations in pathologic assessment, among others. Further, other key elements of the microenvironment, such as tumor-associated macrophages (TAMs), do not yet have standardized approaches for quantification or characterization. As a result, there is no assessment of the TIME for the vast majority of breast cancers diagnosed in the US and around the world. However, the rapid growth of digital pathology offers the potential to leverage computational approaches to overcome these limitations and democratize access to TIL and TAM enumeration. The overall goal of this project is to determine if computational approaches to TILs (existing) and TAMs (to be developed within this grant) are comparable to pathologist- enumerated TILs and TAMs and, further, associated with relevant patient outcomes from two phase III breast cancer clinical trials. Prior to project initiation, we have developed both a compute-intensive artificial intelligence- based TILs approach, an open source software (QuPath)-based TILs approach, and expertise in RNAseq-based immune quantification. We will first focus on TILs - benchmarking the two computational and RNAseq immune approaches against pathologist TIL counts (‘gold standard’) then evaluating association of each with event-free survival in two completed clinical trials (Aim 1). In parallel, we will develop a novel computational approache to enumerate and phenotype TAMs by using immunohistochemical staining for macrophage markers on the same slide with standard H&E, then apply in the same two clinical trials (Aim 2). Our approach is innovative because we will benchmark diverse approaches at scale in relevant clinical studies. The study is significant because we will determine if computational approaches to TILs/TAMs align with pathologist estimates and clinical outcomes, then ensure these algorithms are available to the community. Our long-term goal is to democratize computational TIL and TAM enumeration as pathology decision-support to facilitate integration of accessible tumor-immune microenvironment into clinical trials and care.

Augmented-reality guided lumpectomy

Abstract Far too many women with a newly diagnosed breast cancer must undergo repeat surgery because positive margins were found at the time of their initial lumpectomy. Supine volumetric MRI has potential to improve surgical accuracy, and reduce re-excision rates by nearly 50%. Spurred by our preliminary results improving depth perception via projected apertures and integrating intra-operative marker tracking into commercial Augmented Reality systems, we have developed a highly accurate initial prototype Augmented Reality system to project volumetric MRI data inside the breast to guide surgery. In Aim 1, we will compare methods of projecting apertures in a phantom model of lumpectomy. In Aim 2, we will test the final prototype system in a pilot study of 30 women with new breast cancer. Standardized use of cavity- and shave-margins will enable paired comparisons between standard and AR-guided techniques in the same patients, including ability to reduce positive margin rates and minimize overexcision. Ultimately the system will be ready for future randomized controlled trials to measure efficacy as the next step toward broad clinical adoption.

2026 Thiol-Based Redox Regulation and Signaling Gordon Research Conference and Gordon Research Seminar

PROJECT SUMMARY This proposal requests support for the 10th meeting of the biennial Gordon Research Conference (GRC) and associated Gordon Research Seminar (GRS) on Thiol-Based Redox Regulation and Signaling to be held at the Rey Don Jaime Grand Hotel, Castelldefels, Spain on July 11-12 (GRS) and July 12-17 (GRC), 2026. Regulation of protein function through the post-translational modification of specific cysteine residues (thiol oxidation) plays an important role in cellular adaptation to local and global changes to endogenous and environmental oxidants. A key challenge for the redox-signaling field is to understand how thiol-based signaling mechanisms are integrated into cellular redox homeostasis and how these events facilitate communication between molecules, organelles, cells, and tissues to initiate and coordinate a specialized biological outcome. Significant emphasis for the 2026 meeting will be placed on an exploration of a wider range of cysteine thiol chemistry placed within a cellular context of other, often competing, oxidative or acyl modifications, some of which derive from environmental exposures, and contribute to cancer, aging and the progression of disease. In addition, we will discuss new insights into how cellular redox status impacts metabolic disease and new mathematical and analytical approaches to understand how redox gradients or “waves” impact the spatial and temporal aspects of signaling. A long-term objective is to use this new information to develop diagnostics and therapeutics for a wide range of redox-associated diseases that impact public health. This meeting provides a unique forum for extensive and immersive interaction among chemists, biologists, structural biologists and redox tool-builders, interested in a range of animal and cellular model systems, with clinical researchers and physicians focused on disease processes. While the thematic area of the conference is intentionally broad, its relevance to specialized NIH institutes is highly significant. Not only is redox toxicity proposed as a primary driver of chemically-induced pathology in humans, notably in aging and age-associated diseases, protection from these pathologies by “supersulfides” holds considerable promise. In keeping with the GRC tradition, the 2026 meeting will highlight presentations that emphasize unpublished work, creating a distinctive intellectual experience that enhances the excitement of the meeting. Investigators new to the meeting, junior investigators and graduate and post-graduate trainees will be welcomed. The associated GRS will provide a more intimate forum where graduate and postdoctoral trainees present their research to their peers, while receiving constructive comments from a few senior investigators who serve as mentors. We intend that the GRS/GRC meetings will attract and increase retention of junior scientists in the field of redox biology. We anticipate that the GRC will enhance the education of researchers at all career levels, generate new ideas and collaborations aimed at understanding thiol-based redox regulation and dysfunction, and enable future progress in the prevention, detection, and treatment of a wide-range of human diseases associated with perturbations in redox homeostasis.

Gene regulation networks in nervous system cancers: identification of novel drug targets

Astrocytes release glutamate by regulated exocytosis in health and disease

Astrocytes release glutamate by regulated exocytosis in health and disease Vladimir Parpura, International Translational Neuroscience Research Institute, Zhejiang Chinese Medical University, Hangzhou, P.R. China Parpura will present you with the evidence that astrocytes, a subtype of glial cells in the brain, can exocytotically release the neurotransmitter glutamate and how this release is regulated. Spatiotemporal characteristic of vesicular fusion that underlie glutamate release in astrocytes will be discussed. He will also present data on a translational project in which this release pathway can be targeted for the treatment of glioblastoma, the deadliest brain cancer.

Genetic and epigenetic underpinnings of neurodegenerative disorders

Pluripotent cells, including embryonic stem (ES) and induced pluripotent stem (iPS) cells, are used to investigate the genetic and epigenetic underpinnings of human diseases such as Parkinson’s, Alzheimer’s, autism, and cancer. Mechanisms of somatic cell reprogramming to an embryonic pluripotent state are explored, utilizing patient-specific pluripotent cells to model and analyze neurodegenerative diseases.

Spatial Organization of Cellular Reactive States in Human Brain Cancer

CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers

IL1beta+ macrophages fuel pathogenic inflammation in pancreatic cancer

Neuromodulation of subjective experience

Many psychoactive substances are used with the aim of altering experience, e.g. as analgesics, antidepressants or antipsychotics. These drugs act on specific receptor systems in the brain, including the opioid, serotonergic and dopaminergic systems. In this talk, I will summarise human drug studies targeting opioid receptors and their role for human experience, with focus on the experience of pain, stress, mood, and social connection. Opioids are only indicated for analgesia, due to their potential to cause addiction. When these regulations occurred, other known effects were relegated to side effects. This may be the cause of the prevalent myth that opioids are the most potent painkillers, despite evidence from head-to-head trials, Cochrane reviews and network meta-analyses that opioids are not superior to non-opioid analgesics in the treatment of acute or chronic non-cancer pain. However, due to the variability and diversity of opioid effects across contexts and experiences, some people under some circumstances may indeed benefit from prolonged treatment. I will present data on individual differences in opioid effects due to participant sex and stress induction. Understanding the effects of these commonly used medications on other aspects of the human experience is important to ensure correct use and to prevent unnecessary pain and addiction risk.

Mechanisms Underlying the Persistence of Cancer-Related Fatigue

Cancer-related fatigue is a prominent and debilitating side effect of cancer and its treatment. It can develop prior to diagnosis, generally peaks during cancer treatment, and can persist long after treatment completion. Its mechanisms are multifactorial, and its expression is highly variable. Unfortunately, treatment options are limited. Our research uses syngeneic murine models of cancer and cisplatin-based chemotherapy to better understand these mechanisms. Our data indicate that both peripherally and centrally processes may contribute to the developmental of fatigue. These processes include metabolic alterations, mitochondrial dysfunction, pre-cachexia, and inflammation. However, our data has revealed that behavioral fatigue can persist even after the toxicity associated with cancer and its treatment recover. For example, running during cancer treatment attenuates kidney toxicity while also delaying recovery from fatigue-like behavior. Additionally, administration of anesthetics known to disrupt memory consolidation at the time treatment can promote recovery, and treatment-related cues can re-instate fatigue after recovery. Cancer-related fatigue can also promote habitual behavioral patterns, as observed using a devaluation task. We interpret this data to suggest that limit metabolic resources during cancer promote the utilization of habit-based behavioral strategies that serve to maintain fatigue behavior into survivorship. This line of work is exciting as it points us toward novel interventional targets for the treatment of persistent cancer-related fatigue.

Aging promotes reactivation from metastatic melanoma dormancy

How does the primary tumor imprint a dormancy signature in disseminated tumor cells?

T cells specific for alpha-myosin drive immunotherapy-related myocarditis

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor

Neuron-glial interactions in health and disease: from cognition to cancer

In the central nervous system, neuronal activity is a critical regulator of development and plasticity. Activity-dependent proliferation of healthy glial progenitors, oligodendrocyte precursor cells (OPCs), and the consequent generation of new oligodendrocytes contributes to adaptive myelination. This plasticity of myelin tunes neural circuit function and contributes to healthy cognition. The robust mitogenic effect of neuronal activity on normal oligodendroglial precursor cells, a putative cellular origin for many forms of glioma, suggests that dysregulated or “hijacked” mechanisms of myelin plasticity might similarly promote malignant cell proliferation in this devastating group of brain cancers. Indeed, neuronal activity promotes progression of both high-grade and low-grade glioma subtypes in preclinical models. Crucial mechanisms mediating activity-regulated glioma growth include paracrine secretion of BDNF and the synaptic protein neuroligin-3 (NLGN3). NLGN3 induces multiple oncogenic signaling pathways in the cancer cell, and also promotes glutamatergic synapse formation between neurons and glioma cells. Glioma cells integrate into neural circuits synaptically through neuron-to-glioma synapses, and electrically through potassium-evoked currents that are amplified through gap-junctional coupling between tumor cells This synaptic and electrical integration of glioma into neural circuits is central to tumor progression in preclinical models. Thus, neuron-glial interactions not only modulate neural circuit structure and function in the healthy brain, but paracrine and synaptic neuron-glioma interactions also play important roles in the pathogenesis of glial cancers. The mechanistic parallels between normal and malignant neuron-glial interactions underscores the extent to which mechanisms of neurodevelopment and plasticity are subverted by malignant gliomas, and the importance of understanding the neuroscience of cancer.

Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer

Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells

PHGDH heterogeneity potentiates cancer cell dissemination and metastasis

Identifying central mechanisms of glucocorticoid circadian rhythm dysfunction in breast cancer

The circadian release of endogenous glucocorticoids is essential in preparing and synchronizing the body’s daily physiological needs. Disruption in the rhythmic activity of glucocorticoids has been observed in individuals with a variety of cancer types, and blunting of this rhythm has been shown to predict cancer mortality and declines in quality of life. This suggests that a disrupted glucocorticoid rhythm is potentially a shared phenotype across cancers. However, where this phenomenon is driven by the cancer itself, and the causal mechanisms that link glucocorticoid rhythm dysfunction and cancer outcomes remain preliminary at best. The regulation of daily glucocorticoid activity has been well-characterized and is maintained, in part, by the coordinated response of the hypothalamic-pituitary-adrenal (HPA) axis, consisting of the suprachiasmatic nucleus (SCN) and corticotropin-releasing hormone-expressing neurons of the paraventricular nucleus of the hypothalamus (PVNCRH). Consequently, we set out to examine if cancer-induced glucocorticoid dysfunction is regulated by disruptions within these hypothalamic nuclei. In comparison to their tumor-free baseline, mammary tumor-bearing mice exhibited a blunting of glucocorticoid rhythms across multiple timepoints throughout the day, as measured by the overall levels and the slope of fecal corticosterone rhythms, during tumor progression. We further examined how peripheral tumors shape hypothalamic activity within the brain. Serial two-photon tomography for whole-brain cFos imaging suggests a disrupted activation of the PVN in mice with tumors. Additionally, we found GFP labeled CRH+ neurons within the PVN after injection of pseudorabies virus expressing GFP into the tumor, pointing to the PVN as a primary target disrupted by mammary tumors. Preliminary in vivo fiber photometry data show that PVNCRH neurons exhibit enhanced calcium activity during tumor progression, as compared to baseline (no tumor) activity. Taken together, this suggests that there may be an overactive HPA response during tumor progression, which in turn, may result in a subsequent negative feedback on glucocorticoid rhythms. Current studies are examining whether tumor progression modulates SCN calcium activity, how the transcriptional profile of PVNCRH neurons is changed, and test if manipulation of the neurocircuitry surrounding glucocorticoid rhythmicity alters tumor characteristics.

Radiopharmaceutical evaluation of novel bifunctional chelators and bioconjugates for tumour imaging and therapy

Bispidines (3,7-diazabicyclo[3.3.1]nonane) and their derivatives act as bifunctional chelators (BFC), combining the advantages of multidentate macrocyclic and acyclic ligands e.g. high kinetic inertness, rapid radiolabelling under mild conditions. This bicyclic chelator system shows a great diversity in terms of its denticity and type of functional groups, yielding a wide range of multidentate ligands that can bind a variety of different metal ions. In addition, they allow a facile functionalisation of targeting molecules such as peptides, peptidomimetics, and bispeci􀄀c antibodies. Herein, examples of various bispidine complexes labelled with [64Cu]Cu2+, [111In]In3+, [ 177Lu]Lu3+ or [ 225Ac]Ac3+ will be presented which provide a picture of how different substituents in􀄁uence the coordination mode. Target-speci􀄀c radiolabelled bispidine-based conjugates (e.g. peptides, antibody fragments, antibodies) investigated in vivo by positron emission or single-photon emission computed tomography will be presented and discussed in terms of their suitability for nuclear medicine applications.

Malignant synaptic plasticity in pediatric high-grade gliomas

Pediatric high-grade gliomas (pHGG) are a devastating group of diseases that urgently require novel therapeutic options. We have previously demonstrated that pHGGs directly synapse onto neurons and the subsequent tumor cell depolarization, mediated by calcium-permeable AMPA channels, promotes their proliferation. The regulatory mechanisms governing these postsynaptic connections are unknown. Here, we investigated the role of BDNF-TrkB signaling in modulating the plasticity of the malignant synapse. BDNF ligand activation of its canonical receptor, TrkB (which is encoded for by the gene NTRK2), has been shown to be one important modulator of synaptic regulation in the normal setting. Electrophysiological recordings of glioma cell membrane properties, in response to acute neurotransmitter stimulation, demonstrate in an inward current resembling AMPA receptor (AMPAR) mediated excitatory neurotransmission. Extracellular BDNF increases the amplitude of this glutamate-induced tumor cell depolarization and this effect is abrogated in NTRK2 knockout glioma cells. Upon examining tumor cell excitability using in situ calcium imaging, we found that BDNF increases the intensity of glutamate-evoked calcium transients in GCaMP6s expressing glioma cells. Western blot analysis indicates the tumors AMPAR properties are altered downstream of BDNF induced TrkB activation in glioma. Cell membrane protein capture (via biotinylation) and live imaging of pH sensitive GFP-tagged AMPAR subunits demonstrate an increase of calcium permeable channels at the tumors postsynaptic membrane in response to BDNF. We find that BDNF-TrkB signaling promotes neuron-to-glioma synaptogenesis as measured by high-resolution confocal and electron microscopy in culture and tumor xenografts. Our analysis of published pHGG transcriptomic datasets, together with brain slice conditioned medium experiments in culture, indicates the tumor microenvironment as the chief source of BDNF ligand. Disruption of the BDNF-TrkB pathway in patient-derived orthotopic glioma xenograft models, both genetically and pharmacologically, results in an increased overall survival and reduced tumor proliferation rate. These findings suggest that gliomas leverage normal mechanisms of plasticity to modulate the excitatory channels involved in synaptic neurotransmission and they reveal the potential to target the regulatory components of glioma circuit dynamics as a therapeutic strategy for these lethal cancers.

MBI Webinar on preclinical research into brain tumours and neurodegenerative disorders

WEBINAR 1 Breaking the barrier: Using focused ultrasound for the development of targeted therapies for brain tumours presented by Dr Ekaterina (Caty) Salimova, Monash Biomedical Imaging Glioblastoma multiforme (GBM) - brain cancer - is aggressive and difficult to treat as systemic therapies are hindered by the blood-brain barrier (BBB). Focused ultrasound (FUS) - a non-invasive technique that can induce targeted temporary disruption of the BBB – is a promising tool to improve GBM treatments. In this webinar, Dr Ekaterina Salimova will discuss the MRI-guided FUS modality at MBI and her research to develop novel targeted therapies for brain tumours. Dr Ekaterina (Caty) Salimova is a Research Fellow in the Preclinical Team at Monash Biomedical Imaging. Her research interests include imaging cardiovascular disease and MRI-guided focused ultrasound for investigating new therapeutic targets in neuro-oncology. - WEBINAR 2 Disposition of the Kv1.3 inhibitory peptide HsTX1[R14A], a novel attenuator of neuroinflammation presented by Sanjeevini Babu Reddiar, Monash Institute of Pharmaceutical Sciences The voltage-gated potassium channel (Kv1.3) in microglia regulates membrane potential and pro-inflammatory functions, and non-selective blockade of Kv1.3 has shown anti-inflammatory and disease improvement in animal models of Alzheimer’s and Parkinson’s diseases. Therefore, specific inhibitors of pro-inflammatory microglial processes with CNS bioavailability are urgently needed, as disease-modifying treatments for neurodegenerative disorders are lacking. In this webinar, PhD candidate Ms Sanju Reddiar will discuss the synthesis and biodistribution of a Kv1.3-inhibitory peptide using a [64Cu]Cu-DOTA labelled conjugate. Sanjeevini Babu Reddiar is a PhD student at the Monash Institute of Pharmaceutical Sciences. She is working on a project identifying the factors governing the brain disposition and blood-brain barrier permeability of a Kv1.3-blocking peptide.

Plasticity in gut microbe-host interactions

Plasticity in gut microbe-host interactions

Emerging Treatment Options in Psychiatry

The World Health Organization (WHO) estimates that untreated mental disorders accountfor 13% of the total global burden of disease, and by 2030, depression alone will be the leadingcause of disability around the world – outpacing heart disease, cancer, and HIV. This grim pictureis further compounded by the mental health burden delivered by the coronavirus pandemic.The lack of novel treatment options in psychiatry is restricted by a limited understanding in theneuroscience basis of mental disorders, availability of relevant biomarkers, poor predictability inanimal models, and high failure rates in psychiatric drug development. However, theannouncement in 2019 from the Federal Drug Administration (FDA) for approvals of newinterventions for treatment-resistant depression (intranasal esketamine) and postpartumdepression (i.v. brexanolone), demand critical attention. Novel public-private partnerships indrug discovery, new translational data on co-morbid biology, in particular the ascendance ofpsycho-immunology, have highlighted the arrival of a new frontier in biological psychiatryresearch for depressive disorders.

Directing the timing of maturation in human pluripotent stem cell-derived neurons

Retinoblastoma: Canadian global leadership

Swarms for people

As tiny robots become individually more sophisticated, and larger robots easier to mass produce, a breakdown of conventional disciplinary silos is enabling swarm engineering to be adopted across scales and applications, from nanomedicine to treat cancer, to cm-sized robots for large-scale environmental monitoring or intralogistics. This convergence of capabilities is facilitating the transfer of lessons learned from one scale to the other. Cm-sized robots that work in the 1000s may operate in a way similar to reaction-diffusion systems at the nanoscale, while sophisticated microrobots may have individual capabilities that allow them to achieve swarm behaviour reminiscent of larger robots with memory, computation, and communication. Although the physics of these systems are fundamentally different, much of their emergent swarm behaviours can be abstracted to their ability to move and react to their local environment. This presents an opportunity to build a unified framework for the engineering of swarms across scales that makes use of machine learning to automatically discover suitable agent designs and behaviours, digital twins to seamlessly move between the digital and physical world, and user studies to explore how to make swarms safe and trustworthy. Such a framework would push the envelope of swarm capabilities, towards making swarms for people.

Developing metal-based radiopharmaceuticals for imaging and therapy

Personalised medicine will be greatly enhanced with the introduction of new radiopharmaceuticals for the diagnosis and treatment of various cancers, as well as cardiovascular disease and brain disorders. The unprecedented interest in developing theranostic radiopharmaceuticals is mainly due to the recent clinical successes of radiometal-based products including: • 177LuDOTA-TATE (trade name Lutathera, FDA approved in 2018), a peptide-based tracer that is used for treating metastatic neuroendocrine tumours • Ga 68 PSMA-11 (FDA approved in 2020), a positron emission tomography agent for imaging prostate-specific membrane antigen positive lesions in men with prostate cancer. In this webinar, Dr Brett Paterson and PhD candidate Mr Cormac Kelderman will present their research on developing the chemistry and radiochemistry to produce new radiometal-based imaging and therapy agents. They will discuss the synthesis of new molecules, the optimisation of the radiochemistry, and results from preclinical evaluations. Dr Brett Paterson is a National Imaging Facility Fellow at Monash Biomedical Imaging and academic group leader in the School of Chemistry, Monash University. His research focuses on the development of radiochemistry and new radiopharmaceuticals. Cormac Kelderman is a PhD candidate under the supervision of Dr Brett Paterson in the School of Chemistry, Monash University. His research focuses on developing new bis(thiosemicarbazone) chelators for technetium-99m SPECT imaging.

Toxic effect of pathogenic tau on the nucleus

The nuclear envelope is a lipid bilayer that encases the genome and provides a physical boundary between the cytoplasm and the nucleoplasm. While the nucleus is typically depicted as a sphere encircled by a smooth surface of nuclear envelope, the smooth exterior can be interrupted by tubular invaginations of the nuclear envelope into the deep nuclear interior. Such structures are termed the "nucleoplasmic reticulum." Increased frequency of nuclear envelope invagination occurs in disease states including various cancers, viral infections, and laminopathies, a group of heterogeneous disorders that arise due to mutations in the gene encoding lamin A. A significant increase in the frequency of nuclear envelope invaginations in the human Alzheimer's disease brain has recently been reported. Nuclear envelope invaginations are caused by pathogenic tau, one of the two major pathological hallmarks of Alzheimer's disease. Pathogenic tau-induced dysfunction of the lamin nucleoskeleton drives nuclear envelope invagination and consequent accumulation of polyadenylated RNA within invaginations, both of which drive neuronal death. Our ongoing studies suggest that maintaining proper cytoskeletal, nucleoskeletal, and genomic architecture are critical for survival and function of adult neurons.

Brain cancer and the single-cell architecture of human brain development

The assembly of a functional neocortex

Sparks, flames, and inferno: epileptogenesis in the glioblastoma microenvironment

Glioblastoma cells trigger pharmacoresistant seizures that may promote tumor growth and diminish the quality of remaining life. To define the relationship between growth of glial tumors and their neuronal microenvironment, and to identify genomic biomarkers and mechanisms that may point to better prognosis and treatment of drug resistant epilepsy in brain cancer, we are analyzing a new generation of genetically defined CRISPR/in utero electroporation inborn glioblastoma (GBM) tumor models engineered in mice. The molecular pathophysiology of glioblastoma cells and surrounding neurons and untransformed astrocytes are compared at serial stages of tumor development. Initial studies reveal that epileptiform EEG spiking is a very early and reliable preclinical signature of GBM expansion in these mice, followed by rapidly progressive seizures and death within weeks. FACS-sorted transcriptomic analysis of cortical astrocytes reveals the expansion of a subgroup enriched in pro-synaptogenic genes that may drive hyperexcitability, a novel mechanism of epileptogenesis. Using a prototypical GBM IUE model, we systematically define and correlate the earliest appearance of cortical hyperexcitability with progressive cortical tumor cell invasion, including spontaneous episodes of spreading cortical depolarization, innate inflammation, and xCT upregulation in the peritumoral microenvironment. Blocking this glutamate exporter reduces seizure load. We show that the host genome contributes to seizure risk by generating tumors in a monogenic deletion strain (MapT/tau -/-) that raises cortical seizure threshold. We also show that the tumor variant profile determines epilepsy risk. Our genetic dissection approach sets the stage to broadly explore the developmental biology of personalized tumor/host interactions in mice engineered with novel human tumor mutations in specified glial cell lineages.

Targeting the Endocannabinoid System for Management of Chemotherapy, HIV and Antiretroviral-Induced Neuropathic Pain

Chemotherapeutic drugs (used for treating cancer), HIV infection and antiretroviral therapy (ART) can independently cause difficult-to-manage painful neuropathy. Paclitaxel, a chemotherapeutic drug, for example is associated with high incidence of peripheral neuropathy, around 71% of the patients of which 27% of these develop neuropathic pain. Use of cannabis or phytocannabinoids has been reported to improve pain measures in patients with neuropathic pain, including painful HIV-associated sensory neuropathy and cancer pain. Phytocannabinoids and endocannabinoids, such as anandamide and 2-arachidonoylglycerol (2-AG), produce their effects via cannabinoid (CB) receptors, which are present both in the periphery and central nervous system. Endocannabinoids are synthesized in an “on demand” fashion and are degraded by various enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Various studies, including those from our group, suggest that there are changes in gene and protein expression of endocannabinoid molecules during chemotherapy-induced neuropathic pain (CINP), HIV and antiretroviral-induced neuropathic pain. Analysis of endocannabinoid molecule expression in the brain, spinal cord and paw skin using LC-MS/MS show that there is a specific deficiency of the endocannabinoids 2-AG and/or anandamide in the periphery during CINP. Various drugs including endocannabinoids, cannabidiol, inhibitors of FAAH and MGL, CB receptor agonists, desipramine and coadministered indomethacin plus minocycline have been found to either prevent the development and/or attenuate established CINP, HIV and antiretroviral-induced neuropathic pain in a CB receptor-dependent manner. The results available suggest that targeting the endocannabinoid system for prevention and treatment of CINP, HIV-associated neuropathic pain and antiretroviral-induced neuropathic pain is a plausible therapeutic option.

Anti-PD1 immunotherapy exacerbates cognitive deficits induced by immunogenic cancer in mice

Cognitive effects in non-CNS cancer survivors before and 1-year after standard anthracycline-based chemotherapy treatment

The Effect of Social Isolation on Cancer Metastases and the Nucleus Accumbens in Rats

Identifying novel mediators of tumor-nerve interactions in cancer pain

Nerve guidance in cancer: a new role for Netrin-1

Sleep macro- and microstructure in breast cancer survivors

Sympathetic axonal sprouting induces changes in macrophage populations and protects against pancreatic cancer

Yoga alleviates cognitive impairment and cardiac autonomic dysfunction in breast cancer patients receiving chemotherapy: a randomized controlled study

Cognitive performance is enhanced by aerobic-strength training, and related to physical fitness and reduced platinum levels in testicular germ cell cancer survivors

FENS Forum 2024

Polymeric nanoparticles for targeted cancer therapy

FENS Forum 2024

Smart Glia: Investigating the nervous system plasticity upon cancer

FENS Forum 2024

In vitro modelling of immune effector cell-associated neurotoxicity syndrome (ICANS) resulting from CAR T-cell therapy treating haematological cancer

FENS Forum 2024