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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.

Factory-treated, long-lasting permethrin baby wraps for the prevention of malaria: A phase III randomized controlled trial

PROJECT SUMMARY/ABSTRACT Progress against malaria has stalled. Novel interventions – particularly those targeting outdoor and daytime biting – are needed. In a randomized, placebo-controlled trial of permethrin- vs. sham-treated baby wraps in Uganda, we found a significant reduction in clinical malaria incidence among children carried in permethrin- as compared to sham-treated wraps (Boyce et al, NEJM, 2025). Despite these promising results, our trial incorporated a monthly re-treatment strategy that would be difficult to operationalize at scale. Furthermore, we only followed participants for 6 months, which is shorter than the expected period of use. Therefore, implementation studies - and specifically trials of long-lasting, factory-treated textiles - are now needed. Factory-treated materials would not only eliminate the need for retreatment for up to 12 months, but because the chemicals are more tightly bound, result in less absorption across the skin. Therefore, we now propose to conduct a randomized, double-blind trial of factory-treated, long-lasting (FTLL) wraps. AIM 1: Determine the effectiveness of FTLL permethrin wraps in combination with existing interventions for the prevention of malaria in children. We will enroll 750 mother-infant pairs from routine immunization visits (~3 months of age) at 3 sites of varying transmission intensity across Uganda. All participants will receive new dual active ingredient (AI) bed nets and be randomized (1:1) to either FTLL or untreated wraps. The primary outcome will be clinical malaria incidence during the period of wrap use, defined as fever a positive malaria rapid diagnostic test (RDT) between the FTLL and untreated arms. AIM 2: Confirm the safety of extended exposure to FTLL permethrin wraps for use in young children. Although a review of factory-treated clothing by the US Environmental Protection Agency, including clothing for children and toddlers, did not identify scenarios of concern, the frequency of use envisioned here may be beyond that modeled. To accomplish this, we will perform semi-annual assessments of growth (e.g., height-for-weight) and neurodevelopment (ND) during the period of use and 12-months after discontinuation. AIM 3: Assess the effect of FTLL permethrin wraps on Anopheles mosquito indices and blood-meal seeking behaviors. We will conduct longitudinal entomological surveillance, including CDC-light trap and aspirator collections, supplemented by human landing catches at sentinel households (~10-15%) from both the FTLL and untreated arms. This work tests a novel intervention, which leverages technology developed by the US military, to reduce the burden of malaria in endemic countries. Addressing malaria in these countries minimizes the risk of importation into the US. If successful, the project will provide additional evidence for treated textiles, which may be used to protect American travelers and deployed military servicemembers. The project will be conducted in Uganda, where malaria is highly endemic and it will be possible to enroll at-risk women-infant pairs.

Perturbation of mammary immunoglobulins during maternal antibiotic administration

Project Summary Prescribed in up to 40% of pregnancies, antibiotics represent the most commonly used class of medication during pregnancy. Although this practice is often necessary for maternal health, accumulating evidence suggests that antibiotic exposure may have unintended consequences for the mother-infant dyad. Epidemiologic studies associate maternal antibiotic exposure, especially in the absence of infection, with increased risk of neonatal complications including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), yet the mechanisms driving these associations remain poorly understood. Secretory IgA (sIgA) in milk is an essential component of neonatal mucosal immunity, shaping early gut microbial colonization and providing protection against enteric pathogens. The mechanisms by which maternal physiology regulates the abundance and microbial specificity of these antibodies in milk remain poorly understood. In animal models, the maternal gut–mammary axis governs the generation of milk IgA: IgA-committed lymphocytes from the maternal intestine migrate to the mammary gland during advancing pregnancy via CCL- 28/CCR10 signaling. Our preliminary data suggest that maternal antibiotic exposure disrupts this process leading to a decrease in milk IgA. However, the timing and extent of antibody dysbiosis are undefined; the downstream effects on neonatal intestinal health are unknown; and the underlying mechanisms—whether due to altered microbial stimulation, impaired recruitment of IgA⁺ cells to the mammary gland, or both—remain to be elucidated. Our central hypothesis is that maternal antibiotic exposure reduces pathogen-reactive IgA in milk by impairing gut-to-mammary immune cell trafficking thereby compromising neonatal mucosal immunity and increasing infection susceptibility. We will address this hypothesis through three integrated aims: (1) Determine the magnitude and duration of antibiotic-mediated mammary antibody dysbiosis in women who deliver preterm and at term; (2) Identify microbial targets of mammary antibodies diminished by maternal antibiotic exposure and (3 Determine the role of maternal antibiotics in the disruption of mammary resident IgA+ plasma cells in animal models. This integrative human and animal study will uncover critical mechanisms by which maternal antibiotic use alters the maternal-infant immune axis. The results will provide mechanistic insight into the risks associated with perinatal antibiotic exposure and inform clinical strategies to mitigate risk to neonatal health.

Systems Biology of Early Atopy: Role of Human Milk (SunBEAm-Milk)

Surprisingly little is known about the effect of breastfeeding (BF) on infant immune system development besides an effect on the gut microbiome, but its impact on metabolites and Tregs could support protection against food allergy (FA). BF is currently recommended to prevent the development of allergic diseases, especially asthma/recurrent wheezing and AD in early childhood, but firm conclusions could not be drawn regarding FA due to high heterogeneity and low quality of studies. Reverse causation, recall bias and the poor accuracy of outcome assessment are significant limitations. Most are inadequately powered to specific FA; however, a recent study showed that exclusively BF infants had lower odds of egg, sesame, and peanut allergies. Importantly, immunomodulatory composition of HM varies between mothers, which has not been taken into consideration. For over two decades we have been developing methods to assess immunomodulatory factors in the complex matrix of HM and their association with infant FA. We have shown that high levels of HM total and specific IgA are associated with protection against cow’s milk allergy, but it is unclear whether HM IgA is responsible for or is a biomarker of the vertical transfer of protection. Infant fecal and systemic IgA levels during breastfeeding and after weaning are also elevated in infants at low risk for atopic disease raising the question of whether HM factors such as cytokines can promote IgA production in infants. Consistent with this, we showed that HM cytokines, such as APRIL, induce IgA production in naïve infant B cells, and infants receiving HM with higher levels of APRIL had lower incidence of allergic disease. Finally, lower levels of several HM fatty acids including short-chain fatty acids and DHA were associated with FA. While some these factors were are associated with maternal atopic disease, several of them are not and suggest a role for diet instead. The System Biology of Early Atopy (SunBEAm) population-based cohort of 2500 mother-infant pairs is >50% recruited and provides an unprecedented opportunity to assess association of HM feeding and immune factors in HM with development of infant immune system and FA/AD. The Common Sample comprises a subset of 100 dyads with FA, 100 with FA+AD, 100 with AD, 100 with no FA or AD and more extensively profiled biological data. Utilizing all 2-month HM samples available in the Common Sample, we will assess levels of immune factors in HM and their association with maternal/infant characteristics (Aim 1). Utilizing data from the whole cohort, we will assess the association between HM vs formula feeding on well-defined FA/AD further adjusted based on high vs low levels of HM immune components in the Common Sample (Aim 2b). Finally, we will examine the immune cell and epithelial effects of HM on infant immune markers and intestinal organoids (Aim 3). Key findings will be validated in an independent birth cohort. The ultimate goal is to uncover protective properties of BF and HM in FA and subsequent design of policies and prevention strategies to address the increasing rates of FA.

Metabolic Assessment of Metformin in Pregnancy (MoM-P)

PROJECT SUMMARY The objective of the “Metabolic Assessment of Metformin in Pregnancy “(MoM-P) proposal is to assess the physiological effect of metformin on maternal and neonatal metabolism during pregnancy in individuals developing gestational diabetes (GDM). Metformin is increasingly being used for medical treatment of GDM not adequately treated with nutrition and physical activity. There is inconsistency among various organizations (Society for Maternal Fetal Medicine, American College of Obstetrics and Gynecology and the American Diabetes Association) as to metformin’s role in the medical management of GDM. We will examine the metabolic action of metformin in GDM pregnancies and effect on mothers and their offspring. We plan to recruit 50 participants from Massachusetts General Hospital (MGH) for Specific Aims 1, 2 and 3 and 100 participants from Ohio State University college of Medicine (OSUCOM) for Specific Aims 2 and 3. Participants for the study will have been diagnosed with GDM requiring medical management of GDM as part of the DECIDE multicenter randomized controlled trial. The primary site for DECIDE is OSUCOM, with Dr. Mark Landon as the PI. The MoM-P study will recruit participants from the DECIDE trial at MGH and OSUCOM. The MoM-P study aims are: Aim 1: To establish metformin’s effects on endogenous (primarily hepatic) glucose production (EGP) and insulin sensitivity in late pregnancy. We hypothesize that metformin does not lower EGP in pregnancy and hence the need of additional insulin in the medical management of GDM. We will perform infusion of a stable isotope of glucose (6,6 2H2 glucose) to estimate EGP and a HOMA-IR prior to initiation of medical management and again at 37 weeks gestation. Aim 2: Metformin increases GDF15 levels in human GDM pregnancy and is associated with lower nutrient intake, gestational weight gain (GWG) and increased resting energy expenditure (REE). We hypothesize that metformin increases GDF15 concentrations which lead to GI upset, lower caloric intake/GWG and increases REE. In DECIDE participants randomized to metformin vs. insulin, we will measure GDF15 and examine the relationship to ASA-nutrition records, REE with indirect calorimetry and maternal body composition using air displacement plethysmography (ADP) prior to initiation of medication and again at 37 weeks. Aim 3: To compare fetal growth and body composition in neonates exposed and unexposed to metformin in utero. We hypothesize that metformin treatment of GDM decreases fetal weight: 1) directly based on metformin’s effect on neonatal metabolism (fetal AMPK and mTOR pathways) and 2) indirectly by lowering maternal nutritional intake, fat free mass (FFM) and increasing maternal REE, resulting in decreased neonatal FFM and increased fat mass in childhood. In DECIDE participants, we will measure neonatal body composition with 72 hours of delivery using pediatric ADP and a planned follow-up of children at 2 years in the DECIDE protocol with estimates of male and female children’s body composition.

Maternal Depression and Antidepressant Effects on Fetal Brain Structure and Function (FABMOMS)

PROJECT ABSTRACT Major depressive disorder (MDD) is one of the most common diseases in childbearing women, with a prevalence of 12.7% in pregnancy and 21.9% the year after birth. Exposure to maternal stress and depressive symptoms alters fetal/infant neurodevelopment, functional brain connectivity, and networks implicated in stress processing. About 5% of pregnant women are prescribed a serotonin selective or serotonin norepinephrine reuptake inhibitor (collectively, SRI). Remission of maternal MDD is crucial to the health and functioning of the mother and family. In observational studies typical of this field, differentiating the effects of drug exposure on offspring from the sequelae of the underlying psychiatric disease, both physiological and psychosocial, is challenging. Substantial progress has been made using sophisticated study designs and analytic approaches with large pregnancy cohorts that reduce the risk of spurious associations. Increased rates of overall and cardiac defects, stillbirth, preterm birth, and fetal growth have been largely explained by confounding by factors associated with both MDD and these outcomes rather than SRI exposure. Assessing the neurobehavioral development of children exposed in utero to SRI is the current research priority in this field. Our team pioneered the development of novel and safe fetal and neonatal quantitative magnetic resonance imaging (qMRI) tools, which will be combined with an evaluation of maternal heart rate variability to explore associations between exposures to stress, psychiatric symptoms and SRI on fetal and neonatal brain structure and function. The overarching goal of this project is to evaluate the separate and interactive effects of exposure to antidepressants in utero and maternal MDD on fetal and infant brain structure and function, with a specific focus on the hippocampus. We will accomplish this by evaluating four groups of pregnant women who have: 1) MDD treated with SRI to remission), 2) MDD treated with SRI (non-remitted, with both depressive symptom and SRI exposure), 3) MDD untreated with antidepressants, and 4) no current MDD or SRI treatment. Maternal assessments will occur at intake and in the early third trimesters and in then newborn period (at the time of fetal/newborn MRI) after birth. Maternal and infant evaluations will continue at 6 and 12 months postpartum. Maternal psychosocial and psychiatric status will provide extensive data on the context in which mothers experience pregnancy and infant care and allow adjustment for factors that will inevitably differ across groups. Lastly, we will explore the effects of maternal choline on MDD and offspring brain development. As these exposures and neurodevelopmental studies are conducted, exploring primary preventive strategies is a public health imperative. We will explore a potential mediator, poor maternal choline intake, a modifiable risk factor for both maternal MDD and altered fetal hippocampal growth and infant neurobehavior.

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.

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.

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

Stability in disrupted maternal representations over the perinatal period: Contributors and consequences

Abstract High-quality mother-infant relationships promote social, emotional, and cognitive development while protecting against poor child behavioral, health, and psychological adaptation that create risk for long- term negative outcomes. As mothers transition to parenthood, their own experiences of being cared for influence their emerging views of parenting and representations of their developing child. Evidence suggests that ‘disrupted’ maternal representations of the child, i.e., representations characterized by mixed communication, role merging, extreme withdrawal, and other unusual psychological processes, are tied to both poor child socioemotional adjustment and both insecure and disorganized attachment. However, it is unclear whether disrupted representations that emerge during pregnancy remain stable across the first several years of the child’s life. In addition, to date, research has not examined how change/stability in these representations may affect maternal caregiving and subsequent child adaptation. Using data from a longitudinal, multi-method study, this proposed project will examine the stability of maternal representations of the child for 99 women living in high risk contexts using the Working Model of the Child Interview during the third trimester and again when the child is two years of age. Mothers’ demographic characteristics (i.e. SES and relationship status), interpersonal violence experiences (i.e. child maltreatment or intimate violence exposure), psychological health (i.e. depressive, anxious, and PTSD symptoms), and parenting stress (i.e. perceptions of the child as difficult and parent-child interactions as dysfunctional) are measured as well to examine influences on representation stability. Finally, the observed quality of maternal caregiving and child adaptation are measured and examined in relation to stability in maternal representations of the child. Findings from this study have the potential to identify which mother-child dyads are at greatest risk for poor adaptation across the perinatal period and to delineate the contributors and consequences of maternal representational stability. These findings will serve as an important step towards informing the development or modification of existing prevention/intervention approaches that are targeted specifically towards mother-child dyads who are most at need.

Programming Offspring Metabolism: The Role of Milk Extracellular Vesicles in Fat Development

SUMMARY Obesity is a global health crisis, contributing significantly to the prevalence of metabolic disorders, cardiovascular diseases, and various chronic conditions. A growing body of evidence suggests that maternal obesity during pregnancy and lactation can predispose offspring to obesity and metabolic dysfunction later in life. However, the mechanisms by which maternal obesity programs these adverse outcomes in offspring remain poorly understood. Breast milk is not only a source of essential nutrients but also contains bioactive components, including extracellular vesicles (EVs), which play crucial roles in cellular communication and development. Recent studies have shown that EVs can survive digestion and enter the infant’s circulation, influencing immune and metabolic development. Despite the established link between maternal obesity and altered breast milk composition, no study has investigated the role of milk-derived EVs (mEVs) in programming offspring fat development and metabolism. Understanding this novel pathway could revolutionize our approach to preventing intergenerational transmission of obesity. Our preliminary studies using a mouse model of maternal high-fat diet-induced obesity revealed significant alterations in mEV biogenesis and cargo composition, including changes in specific miRNAs. Oral administration of mEVs from obese dams to neonatal mice increased adiposity and impaired lipid metabolism, indicating that mEVs are crucial in modulating fat development and metabolic pathways in offspring. Several key miRNAs found in mouse mEVs are conserved in human milk EVs, highlighting the potential translational relevance of our findings to human health. We hypothesize that mEVs are critical mediators of maternal obesity’s programming effects on offspring metabolism and adiposity. In specific aim 1, we will use mouse models and advanced molecular techniques (miRNA sequencing, proteomics, and lipidomics) to characterize how maternal obesity affects mEV biogenesis and the composition of their bioactive cargo. We will also evaluate how maternal dietary intake, independent of obesity, influences mEV composition. Specific aim 2 will define the programming effects of mEVs on offspring energy metabolism and obesity. In addition, we will explore whether human milk EVs from lean and obese mothers exert similar programming effects on fat development and metabolism in a mouse model. This R21 application embodies a high-risk, high-reward approach to obesity research. It ventures into uncharted territory by proposing that mEVs are novel regulators of metabolic programming, a concept that has not been explored in prior studies. The potential reward is substantial: discovering a new mechanism by which maternal obesity influences offspring health could fundamentally shift our understanding of early-life metabolic programming and lead to innovative strategies for obesity prevention. If successful, this research could open a new field of study with broad implications for maternal and child health.

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.

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.

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.

Nature over Nurture: Functional neuronal circuits emerge in the absence of developmental activity

During development, the complex neuronal circuitry of the brain arises from limited information contained in the genome. After the genetic code instructs the birth of neurons, the emergence of brain regions, and the formation of axon tracts, it is believed that neuronal activity plays a critical role in shaping circuits for behavior. Current AI technologies are modeled after the same principle: connections in an initial weight matrix are pruned and strengthened by activity-dependent signals until the network can sufficiently generalize a set of inputs into outputs. Here, we challenge these learning-dominated assumptions by quantifying the contribution of neuronal activity to the development of visually guided swimming behavior in larval zebrafish. Intriguingly, dark-rearing zebrafish revealed that visual experience has no effect on the emergence of the optomotor response (OMR). We then raised animals under conditions where neuronal activity was pharmacologically silenced from organogenesis onward using the sodium-channel blocker tricaine. Strikingly, after washout of the anesthetic, animals performed swim bouts and responded to visual stimuli with 75% accuracy in the OMR paradigm. After shorter periods of silenced activity OMR performance stayed above 90% accuracy, calling into question the importance and impact of classical critical periods for visual development. Detailed quantification of the emergence of functional circuit properties by brain-wide imaging experiments confirmed that neuronal circuits came ‘online’ fully tuned and without the requirement for activity-dependent plasticity. Thus, contrary to what you learned on your mother's knee, complex sensory guided behaviors can be wired up innately by activity-independent developmental mechanisms.

Programmed axon death: from animal models into human disease

Programmed axon death is a widespread and completely preventable mechanism in injury and disease. Mouse and Drosophila studies define a molecular pathway involving activation of SARM1 NA Dase and its prevention by NAD synthesising enzyme NMNAT2 . Loss of axonal NMNAT2 causes its substrate, NMN , to accumulate and activate SARM1 , driving loss of NAD and changes in ATP , ROS and calcium. Animal models caused by genetic mutation, toxins, viruses or metabolic defects can be alleviated by blocking programmed axon death, for example models of CMT1B , chemotherapy-induced peripheral neuropathy (CIPN), rabies and diabetic peripheral neuropathy (DPN). The perinatal lethality of NMNAT2 null mice is completely rescued, restoring a normal, healthy lifespan. Animal models lack the genetic and environmental diversity present in human populations and this is problematic for modelling gene-environment combinations, for example in CIPN and DPN , and identifying rare, pathogenic mutations. Instead, by testing human gene variants in WGS datasets for loss- and gain-of-function, we identified enrichment of rare SARM1 gain-of-function variants in sporadic ALS , despite previous negative findings in SOD1 transgenic mice. We have shown in mice that heterozygous SARM1 loss-of-function is protective from a range of axonal stresses and that naturally-occurring SARM1 loss-of-function alleles are present in human populations. This enables new approaches to identify disorders where blocking SARM1 may be therapeutically useful, and the existence of two dominant negative human variants in healthy adults is some of the best evidence available that drugs blocking SARM1 are likely to be safe. Further loss- and gain-of-function variants in SARM1 and NMNAT2 are being identified and used to extend and strengthen the evidence of association with neurological disorders. We aim to identify diseases, and specific patients, in whom SARM1 -blocking drugs are most likely to be effective.

Family Planning in Academia

PROGRAM: 16:00 - 16:30 Rebuild the Academy: Supporting academic mothers during COVID-19 and beyond with Robinson W. Fulweiler & Sarah W. Davies --- 16:30 - 17:00 Experiences with academia and family life by Siri Leknes (Norway-based) and Rachel Buckley (USA-based)--- 17:00 - 17:30 Questions and discussion

Sensing in Insect Wings

Ali Weber (University of Washington, USA) uses the the hawkmoth as a model system, to investigate how information from a small number of mechanoreceptors on the wings are used in flight control. She employs a combination of experimental and computational techniques to study how these sensors respond during flight and how one might optimally array a set of these sensors to best provide feedback during flight.

Dynamic spatial processing in insect vision

How does the visual system of insects function in vastly different light intensities, process separate parts of the visual field in parallel, and cope with eye sizes that differ between individuals? This talk will give you the answers we receive from our unique(ly adorable) model system: hawkmoths.

Australian Bogong moths use a true stellar compass for long-distance navigation at night

Each spring, billions of Bogong moths escape hot conditions in different regions of southeast Australia by migrating over 1000 km to a limited number of cool caves in the Australian Alps, historically used for aestivating over the summer. At the beginning of autumn the same individuals make a return migration to their breeding grounds to reproduce and die. To steer migration Bogong moths sense the Earth’s magnetic field and correlate its directional information with visual cues. In this presentation, we will show that a critically important visual cue is the distribution of starlight within the austral night sky. By tethering spring and autumn migratory moths in a flight simulator, we found that under natural dorsally-projected night skies, and in a nulled magnetic field (disabling the magnetic sense), moths flew in their seasonally appropriate migratory directions, turning in the opposite direction when the night sky was rotated 180°. Visual interneurons in the moth’s optic lobe and central brain responded vigorously to identical sky rotations. Migrating Bogong moths thus use the starry night sky as a true compass to distinguish geographic cardinal directions, the first invertebrate known to do so. These stellar cues are likely reinforced by the Earth’s magnetic field to create a robust compass mechanism for long-distance nocturnal navigation.

Social transmission of maternal behavior

Maternal care is profoundly important for mammalian survival, and in many species requires the contribution of non-biological parents, or alloparents. In the absence of partum and post-partum related hormonal changes, alloparents acquire maternal skills from experience, by yet unknown mechanisms. One critical molecular signal for maternal behavior is oxytocin, a hormone centrally released by hypothalamic paraventricular nucleus (PVN). Do experiences that induce maternal behavior act by engaging PVN oxytocin neurons? To answer this, we used virgin female mice, animals that in the wild live in colonies with experienced mothers and their pups, helping with pup care. We replicated this setup in the lab, and we continuously monitored homecage behavior of virgin mice co-housed for days with a mother and litter, synchronized with recordings from virgin PVN cells, including from oxytocin neurons. Mothers engaged virgins in maternal care in part by shepherding virgins towards the nest, ensuring their proximity to pups, and in part by self-generating pup retrieval episodes, demonstrating maternal behavior to virgins. The frequency of shepherding and of dam retrievals correlates with virgin's subsequent ability to retrieve pups, a quintessential mouse maternal skill. These social interactions activated virgin PVN and gated behaviorally-relevant cortical plasticity for pup vocalizations. Thus, rodents can acquire maternal behavior by social transmission, and our results describe a mechanism for adapting brains of adult caregivers to infant needs via endogenous oxytocin.

Cognition plus longevity equals culture: A new framework for understanding human brain evolution

Narratives of human evolution have focused on cortical expansion and increases in brain size relative to body size, but considered that changes in life history, such as in age at sexual maturity and thus the extent of childhood and maternal dependence, or maximal longevity, are evolved features that appeared as consequences of selection for increased brain size, or increased cognitive abilities that decrease mortality rates, or due to selection for grandmotherly contribution to feeding the young. Here I build on my recent finding that slower life histories universally accompany increased numbers of cortical neurons across warm-blooded species to propose a simpler framework for human evolution: that slower development to sexual maturity and increased post-maturity longevity are features that do not require selection, but rather inevitably and immediately accompany evolutionary increases in numbers of cortical neurons, thus fostering human social interactions and cultural and technological evolution as generational overlap increases.

Blood is thicker than water

According to Hamilton’s inclusive fitness hypothesis, kinship is an organizing principle of social behavior. Behavioral evidence supporting this hypothesis includes the ability to recognize kin and the adjustment of behavior based on kin preference with respect to altruism, attachment and care for offspring in insect societies. Despite the fundamental importance of kinship behavior, the underlying neural mechanisms are poorly understood. We repeated behavioral experiments by Hepper on behavioral preference of rats for their kin. Consistent with Hepper’s work, we find a developmental time course for kinship behavior, where rats prefer sibling interactions at young ages and express non-sibling preferences at older ages. In probing the brain areas responsible for this behavior, we find that aspiration lesions of the lateral septum but not control lesions of cingulate cortices eliminate the behavioral preference in young animals for their siblings and in older rats for non-siblings. We then presented awake and anaesthetized rats with odors and calls of age- and status-matched kin (siblings and mothers) and non-kin (non-siblings and non-mothers) conspecifics, while performing in vivo juxta-cellular and whole-cell patch-clamp recordings in the lateral septum. We find multisensory (olfactory and auditory) neuronal responses, whereby neurons typically responded preferentially but not exclusively to individual social stimuli. Non-kin-odor responsive neurons were found dorsally, while kin-odor responsive neurons were located in ventrally in the lateral septum. To our knowledge such an ordered representation of response preferences according to kinship has not been previously observed and we refer this organization as nepotopy. Nepotopy could be instrumental in reading out kinship from preferential but not exclusive responses and in the generation of differential behavior according to kinship. Thus, our results are consistent with a role of the lateral septum in organizing mammalian kinship behavior.

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.

Untitled Seminar

Mammalian neonates are born immature. Thus mothers are equipped with innate motivation to nurture them. Moreover, in species that live in a family group, fathers and older siblings may also provide extensive care to the young. By studying those highly social species, including laboratory mice, common marmosets, and humans, we are trying to elucidate the neural mechanisms of parental care. Neuronal activity mapping and site-specific functional suppression in mice identified the central part of the medial preoptic area (cMPOA) as the hub of caregiving network for both mothers and fathers.Recent findings about the neural circuit and molecular signaling involved in caregiving motivation will be discussed.

Vagal sensory neurons that guard the airways

The vagus nerve contains a diversity of sensory neurons that detect peripheral stimuli such as blood pressure changes at the aortic arch, lung expansion during breathing, meal-induced stomach distension, and chemotherapeutics that induce nausea. Underlying vagal sensory mechanisms are largely unresolved at a molecular level, presenting tremendously important problems in sensory biology. We charted vagal sensory neurons by single cell RNA sequencing, identifying novel cell surface receptors and classifying a staggering diversity of sensory neuron types. We then generated a collection of ires-Cre knock-in mice to target each neuron type, and adapted genetic tools for Cre-based anatomical mapping, in vivo imaging, targeted ablation, and optogenetic control of vagal neuron activity. We found different sensory neuron types that innervate the lung and exert powerful effects on breathing, others that monitor and control the digestive system, and yet others that innervate that innervate the larynx and protect the airways. Together with Ardem Patapoutian, we also identified a critical role for Piezo mechanoreceptors in the sensation of airway stretch, which underlies a classical respiratory reflex termed the Hering-Breuer inspiratory reflex, as well as in the neuronal sensation of blood pressure and the baroreceptor reflex.

Fate and freedom in developing neocortical circuits

During brain development, neurons are born in specialized niches and migrate to target regions where they assemble to form the circuits that underlie mammalian behaviour. During their journey, neurons follow cell-intrinsic, genetic programs transmitted by their mother cells but also environmental cues, which together drive their maturation. Here, focusing on the neocortex, I will discuss recent findings from our laboratory in which we untangle and manipulate the programs at play in progenitors and their daughter neurons to better understand the emergence of cellular diversity in the developing brain.

Brain Tumor Treatment Using Tunable Local Chemotherapy

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

Dual PI3Kδ/γ inhibitor Duvelisib prevents development of chemotherapy induced neuropathic pain

Functional imaging memories of a mother during filial imprinting in awake domestic chick newborns

Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain

Motherhood changes the processing and response to social cues of female mice

Obese mothers have a higher risk of developing depressive-like behaviour due to hormonal alterations

Odor Background Increases the Pheromone Coding Efficiency in Moth Olfactory Neurons

Preclinical mouse study on maternal separation impacting childhood chemotherapy-induced cognitive impairment

Predictive and construct validity in a mouse model of chemotherapy-induced cognitive impairment

Stimulus duration encoding by moth olfactory receptor neurons

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

AI-driven image analysis for label-free quantification of chemotherapeutic cytotoxicity in glial cells

FENS Forum 2024

Chemotherapy alters the Kolmer cell of the choroid plexus

FENS Forum 2024

Chemotherapy-induced peripheral neuropathy caused by vincristine involves high mobility group box 1 (HMGB1) released from macrophages and Schwann cells

FENS Forum 2024

Modelling chemotherapy-induced peripheral neuropathy on-a-chip

FENS Forum 2024

Modulating voltage-gated sodium channels to enhance differentiation and sensitize glioblastoma cells to chemotherapy

FENS Forum 2024

Pregnancy-responsive pools of adult neural stem cells for transient neurogenesis in mothers

FENS Forum 2024

Single-cell uniparental disomies and mother-offspring interaction: A comparative study

FENS Forum 2024

Understanding the role of microglia in ‘chemofog’ to resolve chemotherapy-induced cognitive impairment

FENS Forum 2024