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Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2(+) malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called ‘Don’t eat me’ signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic ‘Eat me’ signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

Survivors of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection frequently experience lingering neurological symptoms, including impairment in attention, concentration, speed of information processing and memory. This long-COVID cognitive syndrome shares many features with the syndrome of cancer therapy-related cognitive impairment (CRCI). Neuroinflammation, particularly microglial reactivity and consequent dysregulation of hippocampal neurogenesis and oligodendrocyte lineage cells, is central to CRCI. We hypothesized that similar cellular mechanisms may contribute to the persistent neurological symptoms associated with even mild SARS-CoV-2 respiratory infection. Here, we explored neuroinflammation caused by mild respiratory SARS-CoV-2 infection – without neuroinvasion - and effects on hippocampal neurogenesis and the oligodendroglial lineage. Using a mouse model of mild respiratory SARS-CoV-2 infection induced by intranasal SARS-CoV-2 delivery, we found white matter-selective microglial reactivity, a pattern observed in CRCI. Human brain tissue from 9 individuals with COVID-19 or SARS-CoV-2 infection exhibits the same pattern of prominent white matter-selective microglial reactivity. In mice, pro-inflammatory CSF cytokines/chemokines were elevated for at least 7-weeks post-infection; among the chemokines demonstrating persistent elevation is CCL11, which is associated with impairments in neurogenesis and cognitive function. Humans experiencing long-COVID with cognitive symptoms (48 subjects) similarly demonstrate elevated CCL11 levels compared to those with long-COVID who lack cognitive symptoms (15 subjects). Impaired hippocampal neurogenesis, decreased oligodendrocytes and myelin loss in subcortical white matter were evident at 1 week, and persisted until at least 7 weeks, following mild respiratory SARS-CoV-2 infection in mice. Taken together, the findings presented here illustrate striking similarities between neuropathophysiology after cancer therapy and after SARS-CoV-2 infection, and elucidate cellular deficits that may contribute to lasting neurological symptoms following even mild SARS-CoV-2 infection.

Epigenetic dysregulation is frequently observed in the disease pathology of pediatric cancers, including neuroblastoma, the most common extracranial solid tumor in pediatric patients. Neuroblastoma tumors co-opt developmentally linked adrenergic or mesenchymal super-enhancer landscapes that rewire their transcriptional programs. Here, we describe that the lineage commitment to a mesenchymal epigenetic state is an important mechanism of resistance to anti-GD2 therapy through loss of GD2 antigen, a ganglioside glycolipid expressed on the cell surface. Low GD2 expression was significantly correlated with the mesenchymal state in a large panel of neuroblastoma cell lines and a forced adrenergic-to-mesenchymal transition conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2 expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Genome-wide CRISPR/Cas9 screening to identify regulators of GD2 in neuroblastoma revealed that the ablation of the polycomb repressive complex 2 (PRC2) significantly upregulates GD2 expression in GD2-low cells. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells into an adrenergic-like state, re-expressed ST8SIA1, and restored surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential PRC2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma. Citation Format: Nathaniel W. Mabe, Min Huang, Daniel A. Schaefer, Guillermo N. Dalton, Giulia Digiovanni, Gabriela Alexe, Anna C. Geraghty, Delan Khalid, Marius M. Mader, Michal Sheffer, Miles H. Linde, Nghi Ly, Maria Caterina Rotiroti, Benjamin A. H. Smith, Marius Wernig, Carolyn R. Bertozzi, Michelle Monje, Constantine Mitsiades, Ravindra Majeti, Ansuman T. Satpathy, Kimberly Stegmaier, Robbie G. Majzner. Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR003.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

Human central nervous system myelin development extends well into the fourth decade of life, and this protracted period underscores the potential for experience to modulate myelination. The concept of myelin plasticity implies adaptability in myelin structure and function in response to experiences during development and beyond. Mounting evidence supports this concept of neuronal activity-regulated changes in myelin-forming cells, including oligodendrocyte precursor cell proliferation, oligodendrogenesis and modulation of myelin microstructure. In healthy individuals, myelin plasticity in associative white matter structures of the brain is implicated in learning and motor function in both rodents and humans. Activity-dependent changes in myelin-forming cells may influence the function of neural networks that depend on the convergence of numerous neural signals on both a temporal and spatial scale. However, dysregulation of myelin plasticity can disadvantageously alter myelin microstructure and result in aberrant circuit function or contribute to pathological cell proliferation. Emerging roles for myelin plasticity in normal neurological function and in disease are discussed. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 123-135, 2018.

Stress can exert long-lasting changes on the brain that contribute to vulnerability to mental illness, yet mechanisms underlying this long-term vulnerability are not well understood. We hypothesized that stress may alter the production of oligodendrocytes in the adult brain, providing a cellular and structural basis for stress-related disorders. We found that immobilization stress decreased neurogenesis and increased oligodendrogenesis in the dentate gyrus (DG) of the adult rat hippocampus, and that injections of the rat glucocorticoid stress hormone corticosterone (cort) were sufficient to replicate this effect. The DG contains a unique population of multipotent neural stem cells (NSCs) that give rise to adult newborn neurons, but oligodendrogenic potential has not been demonstrated in vivo. We used a nestin-CreER/YFP transgenic mouse line for lineage tracing and found that cort induces oligodendrogenesis from nestin-expressing NSCs in vivo. Using hippocampal NSCs cultured in vitro, we further showed that exposure to cort induced a pro-oligodendrogenic transcriptional program and resulted in an increase in oligodendrogenesis and decrease in neurogenesis, which was prevented by genetic blockade of glucocorticoid receptor (GR). Together, these results suggest a novel model in which stress may alter hippocampal function by promoting oligodendrogenesis, thereby altering the cellular composition and white matter structure.

Reproductive senescence, the point in time when females cease to show estrous cyclicity, is associated with endocrine changes in the hypothalamus, pituitary, and gonads. However, the mechanisms triggering this transition are not well understood. To gain a better understanding of the top-down control of the transition from reproductive competence to a state of reproductive senescence, we investigated middle-aged female rats exhibiting varying degrees of reproductive decline, including individuals with normal cycles, irregular cycles, and complete cessation of cycles. We identified hormonal changes in the brain that manifest before ovarian cycles exhibit any deterioration. We found that females exhibit an increase in RFamide-related peptide-3 (RFRP3) mRNA expression in the hypothalamus in middle age prior to changes in estrous cycle length. This increase is transient and followed by subsequent decreases in kisspeptin (KiSS1) and gonadotropin-releasing hormone (GnRH) mRNA expression. Expression of RFRP3 and its receptor also increased locally in the ovaries with advancing age. While it is well known that aging is associated with decreased GnRH release and downstream disruption of the hypothalamic–pituitary–gonadal (HPG) axis, herein, we provide evidence that reproductive senescence is likely triggered by alterations in a network of regulatory neuropeptides upstream of the GnRH system.

It is well accepted that stress, measured by increased glucocorticoid secretion, leads to profound reproductive dysfunction. In times of stress, glucocorticoids activate many parts of the fight or flight response, mobilizing energy and enhancing survival, while inhibiting metabolic processes that are not necessary for survival in the moment. This includes reproduction, an energetically costly procedure that is very finely regulated. In the short term, this is meant to be beneficial, so that the organism does not waste precious energy needed for survival. However, long-term inhibition can lead to persistent reproductive dysfunction, even if no longer stressed. This response is mediated by the increased levels of circulating glucocorticoids, which orchestrate complex inhibition of the entire reproductive axis. Stress and glucocorticoids exhibits both central and peripheral inhibition of the reproductive hormonal axis. While this has long been recognized as an issue, understanding the complex signaling mechanism behind this inhibition remains somewhat of a mystery. What makes this especially difficult is attempting to differentiate the many parts of both of these hormonal axes, and new neuropeptide discoveries in the last decade in the reproductive field have added even more complexity to an already complicated system. Glucocorticoids (GCs) and other hormones within the hypothalamic-pituitary-adrenal (HPA) axis (as well as contributors in the sympathetic system) can modulate the hypothalamic-pituitary-gonadal (HPG) axis at all levels-GCs can inhibit release of GnRH from the hypothalamus, inhibit gonadotropin synthesis and release in the pituitary, and inhibit testosterone synthesis and release from the gonads, while also influencing gametogenesis and sexual behavior. This chapter is not an exhaustive review of all the known literature, however is aimed at giving a brief look at both the central and peripheral effects of glucocorticoids on the reproductive function.

It is well accepted that stress, measured by increased glucocorticoid secretion, leads to profound reproductive dysfunction. In times of stress, glucocorticoids activate many parts of the fight or flight response, mobilizing energy and enhancing survival, while inhibiting metabolic processes that are not necessary for survival in the moment. This includes reproduction, an energetically costly procedure that is very finely regulated. In the short term, this is meant to be beneficial, so that the organism does not waste precious energy needed for survival. However, long-term inhibition can lead to persistent reproductive dysfunction, even if no longer stressed. This response is mediated by the increased levels of circulating glucocorticoids, which orchestrate complex inhibition of the entire reproductive axis. Stress and glucocorticoids exhibits both central and peripheral inhibition of the reproductive hormonal axis. While this has long been recognized as an issue, understanding the complex signaling mechanism behind this inhibition remains somewhat of a mystery. What makes this especially difficult is attempting to differentiate the many parts of both of these hormonal axes, and new neuropeptide discoveries in the last decade in the reproductive field have added even more complexity to an already complicated system. Glucocorticoids (GCs) and other hormones within the hypothalamic-pituitary-adrenal (HPA) axis (as well as contributors in the sympathetic system) can modulate the hypothalamic-pituitary-gonadal (HPG) axis at all levels—GCs can inhibit release of GnRH from the hypothalamus, inhibit gonadotropin synthesis and release in the pituitary, and inhibit testosterone synthesis and release from the gonads, while also influencing gametogenesis and sexual behavior. This chapter is not an exhaustive review of all the known literature, however is aimed at giving a brief look at both the central and peripheral effects of glucocorticoids on the reproductive function.

The subjective experience of stress leads to reproductive dysfunction in many species, including rodents and humans. Stress effects on reproduction result from multilevel interactions between the hormonal stress response system, i.e., the hypothalamic–pituitary–adrenal (HPA) axis, and the hormonal reproductive system, i.e., the hypothalamic–pituitary–gonadal (HPG) axis. A novel negative regulator of the HPG axis known as gonadotropin-inhibitory hormone (GnIH) was recently discovered in quail, and orthologous neuropeptides known as RFamide-related peptides (RFRPs) have also been identified in rodents and primates. It is currently unknown, however, whether GnIH/RFRPs influence HPG axis activity in response to stress. We show here that both acute and chronic immobilization stress lead to an up-regulation of RFRP expression in the dorsomedial hypothalamus (DMH) of adult male rats and that this increase in RFRP is associated with inhibition of downstream HPG activity. We also show that adrenalectomy blocks the stress-induced increase in RFRP expression. Immunohistochemistry revealed that 53% of RFRP cells express receptors for glucocorticoids (GCs), indicating that adrenal GCs can mediate the stress effect through direct action on RFRP cells. It is thought that stress effects on central control of reproduction are largely mediated by direct or indirect effects on GnRH-secreting neurons. Our data show that stress-induced increases in adrenal GCs cause an increase in RFRP that contributes to hypothalamic suppression of reproductive function. This novel insight into HPA-HPG interaction provides a paradigm shift for work on stress-related reproductive dysfunction and infertility, and indicates that future work on stress and reproductive system interactions must include investigation of the role of GnIH/RFRP.