Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines.

INTRODUCTION: Patients with pediatric low-grade gliomas (pLGGs), the most common primary brain tumors in children, can often benefit from MAPK inhibitor (MAPKi) treatment. However, rapid tumor regrowth, also referred to as rebound growth, may occur once treatment is stopped, constituting a significant clinical challenge. METHODS: Four patient-derived pediatric glioma models were investigated to model rebound growth in vitro based on viable cell counts in response to MAPKi treatment and withdrawal. A multi-omics dataset (RNA sequencing and LC-MS/MS based phospho-/proteomics) was generated to investigate possible rebound-driving mechanisms. Following in vitro validation, putative rebound-driving mechanisms were validated in vivo using the BT-40 orthotopic xenograft model. RESULTS: Of the tested models, only a BRAFV600E-driven model (BT-40, with additional CDKN2A/Bdel) showed rebound growth upon MAPKi withdrawal. Using this model, we identified a rapid reactivation of the MAPK pathway upon MAPKi withdrawal in vitro, also confirmed in vivo. Furthermore, transient overactivation of key MAPK molecules at transcriptional (e.g. FOS) and phosphorylation (e.g. pMEK) levels, was observed in vitro. Additionally, we detected increased expression and secretion of cytokines (CCL2, CX3CL1, CXCL10 and CCL7) upon MAPKi treatment, maintained during early withdrawal. While increased cytokine expression did not have tumor cell intrinsic effects, presence of these cytokines in conditioned media led to increased attraction of microglia cells in vitro. CONCLUSION: Taken together, these data indicate rapid MAPK reactivation upon MAPKi withdrawal as a tumor cell intrinsic rebound-driving mechanism. Furthermore, increased secretion of microglia-recruiting cytokines may play a role in treatment response and rebound growth upon withdrawal, warranting further evaluation.

Unraveling the Complexity of the Senescence-Associated Secretory Phenotype in Adamantinomatous Craniopharyngioma Using Multi-Modal Machine Learning Analysis.

BACKGROUND: Cellular senescence can have positive and negative effects on the body, including aiding in damage repair and facilitating tumor growth. Adamantinomatous Craniopharyngioma (ACP), the most common pediatric sellar/suprasellar brain tumor, poses significant treatment challenges. Recent studies suggest that senescent cells in ACP tumors may contribute to tumor growth and invasion by releasing a Senesecence-Associated Secretory Phenotype (SASP). However, a detailed analysis of these characteristics has yet to be completed. METHODS: We analyzed primary tissue samples from ACP patients using single-cell, single-nuclei, and spatial RNA Sequencing. We performed various analyses, including gene expression clustering, inferred senescence cells from gene expression, and conducted cytokine signaling inference. We utilized LASSO to select essential gene expression pathways associated with senescence. Finally, we validated our findings through immunostaining. RESULTS: We observed significant diversity in gene expression and tissue structure. Key factors such as NFKB, RELA, and SP1 are essential in regulating gene expression, while senescence markers are present throughout the tissue. SPP1 is the most significant cytokine signaling network among ACP cells, while the Wnt signaling pathway predominantly occurs between epithelial and glial cells. Our research has identified links between senescence-associated features and pathways, such as PI3K/Akt/mTOR, MYC, FZD, and Hedgehog, with increased P53 expression associated with senescence in these cells. CONCLUSIONS: A complex interplay between cellular senescence, cytokine signaling, and gene expression pathways underlies ACP development. Further research is crucial to understand how these elements interact to create novel therapeutic approaches for patients with ACP.

Implications of cellular senescence in paediatric pituitary tumours.

The long-standing view of senescent cells as passive and dysfunctional biological remnants has recently shifted into a new paradigm where they are main players in the development of many diseases, including cancer. The senescence programme represents a first line of defence that prevents tumour cell growth but also leads to the secretion of multiple pro-inflammatory and pro-tumourigenic factors that fuel tumour initiation, growth, and progression. Here, we review the main molecular features and biological functions of senescent cells in cancer, including the outcomes of inducing or targeting senescence. We discuss evidence on the role of cellular senescence in pituitary tumours, with an emphasis on adamantinomatous craniopharyngioma (ACP) and pituitary adenomas. Although senescence has been proposed to be a tumour-preventing mechanism in pituitary adenomas, research in ACP has shown that senescent cells are tumour-promoting in both murine models and human tumours. Future studies characterizing the impact of targeting senescent cells may result in novel therapies against pituitary tumours.

COPI vesicle formation and N-myristoylation are targetable vulnerabilities of senescent cells.

Drugs that selectively kill senescent cells (senolytics) improve the outcomes of cancer, fibrosis and age-related diseases. Despite their potential, our knowledge of the molecular pathways that affect the survival of senescent cells is limited. To discover senolytic targets, we performed RNAi screens and identified coatomer complex I (COPI) vesicle formation as a liability of senescent cells. Genetic or pharmacological inhibition of COPI results in Golgi dispersal, dysfunctional autophagy, and unfolded protein response-dependent apoptosis of senescent cells, and knockdown of COPI subunits improves the outcomes of cancer and fibrosis in mouse models. Drugs targeting COPI have poor pharmacological properties, but we find that N-myristoyltransferase inhibitors (NMTi) phenocopy COPI inhibition and are potent senolytics. NMTi selectively eliminated senescent cells and improved outcomes in models of cancer and non-alcoholic steatohepatitis. Our results suggest that senescent cells rely on a hyperactive secretory apparatus and that inhibiting trafficking kills senescent cells with the potential to treat various senescence-associated diseases.

Effects of senescence on the tumour microenvironment and response to therapy.

Cellular senescence is a state of durable cell arrest that has been identified both in vitro and in vivo. It is associated with profound changes in gene expression and a specific secretory profile that includes pro-inflammatory cytokines, growth factors and matrix-remodelling enzymes, referred to as the senescence-associated secretory phenotype (SASP). In cancer, senescence can have anti- or pro-tumour effects. On one hand, it can inhibit tumour progression in a cell autonomous manner. On the other hand, senescence can also promote tumour initiation, progression, metastatic dissemination and resistance to therapy in a paracrine manner. Therefore, despite efforts to target senescence as a potential strategy to inhibit tumour growth, senescent cancer and microenvironmental cells can eventually lead to uncontrolled proliferation and aggressive tumour phenotypes. This can happen either through overcoming senescence growth arrest or through SASP-mediated effects in adjacent tumour cells. This review will discuss how senescence affects the tumour microenvironment, including extracellular matrix remodelling, the immune system and the vascular compartment, to promote tumourigenesis, metastasis and resistance to DNA-damaging therapies. It will also discuss current approaches used in the field to target senescence: senolytics, improving the immune clearance of senescent cells and targeting the SASP.

Clearance of senescent macrophages ameliorates tumorigenesis in KRAS-driven lung cancer.

The accumulation of senescent cells in the tumor microenvironment can drive tumorigenesis in a paracrine manner through the senescence-associated secretory phenotype (SASP). Using a new p16-FDR mouse line, we show that macrophages and endothelial cells are the predominant senescent cell types in murine KRAS-driven lung tumors. Through single cell transcriptomics, we identify a population of tumor-associated macrophages that express a unique array of pro-tumorigenic SASP factors and surface proteins and are also present in normal aged lungs. Genetic or senolytic ablation of senescent cells, or macrophage depletion, result in a significant decrease in tumor burden and increased survival in KRAS-driven lung cancer models. Moreover, we reveal the presence of macrophages with senescent features in human lung pre-malignant lesions, but not in adenocarcinomas. Taken together, our results have uncovered the important role of senescent macrophages in the initiation and progression of lung cancer, highlighting potential therapeutic avenues and cancer preventative strategies.

An expression and function analysis of the CXCR4/SDF-1 signalling axis during pituitary gland development.

The chemokine SDF-1 (CXCL12) and its receptor CXCR4 control several processes during embryonic development such as the regulation of stem cell proliferation, differentiation, and migration. However, the role of this pathway in the formation of the pituitary gland is not understood. We sought to characterise the expression patterns of CXCR4, SDF-1 and CXCR7 at different stages of pituitary gland development. Our expression profiling revealed that SDF-1 is expressed in progenitor-rich regions of the pituitary anterior lobe, that CXCR4 and CXCR7 have opposite expression domains and that CXCR4 expression is conserved between mice and human embryos. We then assessed the importance of this signalling pathway in the development and function of the murine pituitary gland through conditional deletion of CXCR4 in embryonic pituitary progenitors. Successful and specific ablation of CXCR4 expression in embryonic pituitary progenitors did not lead to observable embryonic nor postnatal defects but allowed the identification of stromal CXCR4+ cells not derived from HESX1+ progenitors. Further analysis of constitutive SDF-1, CXCR7 and CXCR4 mutants of the pathway indicates that CXCR4 expression in HESX1+ cells and their descendants is not essential for normal pituitary development in mice.

BH3 mimetics targeting BCL-XL impact the senescent compartment of pilocytic astrocytoma.

BACKGROUND: Pilocytic astrocytoma (PA) is the most common pediatric brain tumor and a mitogen-activated protein kinase (MAPK)-driven disease. Oncogenic MAPK-signaling drives the majority of cells into oncogene-induced senescence (OIS). While OIS induces resistance to antiproliferative therapies, it represents a potential vulnerability exploitable by senolytic agents. METHODS: We established new patient-derived PA cell lines that preserve molecular features of the primary tumors and can be studied in OIS and proliferation depending on expression or repression of the SV40 large T antigen. We determined expression of anti-apoptotic BCL-2 members in these models and primary PA. Dependence of senescent PA cells on anti-apoptotic BCL-2 members was investigated using a comprehensive set of BH3 mimetics. RESULTS: Senescent PA cells upregulate BCL-XL upon senescence induction and show dependency on BCL-XL for survival. BH3 mimetics with high affinity for BCL-XL (BCL-XLi) reduce metabolic activity and induce mitochondrial apoptosis in senescent PA cells at nano-molar concentrations. In contrast, BH3 mimetics without BCL-XLi activity, conventional chemotherapy, and MEK inhibitors show no effect. CONCLUSIONS: Our data demonstrate that BCL-XL is critical for survival of senescent PA tumor cells and provides proof-of-principle for the use of clinically available BCL-XL-dependent senolytics.

Contemporary Biological Insights and Clinical Management of Craniopharyngioma.

Craniopharyngiomas (CPs) are clinically aggressive tumors because of their invasive behavior and recalcitrant tendency to recur after therapy. There are 2 types based on their distinct histology and molecular features: the papillary craniopharyngioma (PCP), which is associated with BRAF-V600E mutations and the adamantinomatous craniopharyngioma (ACP), characterized by mutations in CTNNB1 (encoding ß-catenin). Patients with craniopharyngioma show symptoms linked to the location of the tumor close to the optic pathways, hypothalamus, and pituitary gland, such as increased intracranial pressure, endocrine deficiencies, and visual defects. Treatment is not specific and mostly noncurative, and frequently includes surgery, which may achieve gross total or partial resection, followed by radiotherapy. In cystic tumors, frequent drainage is often required and intracystic instillation of drugs has been used to help manage cyst refilling. More recently targeted therapies have been used, particularly in PCP, but also now in ACP and clinical trials are underway or in development. Although patient survival is high, the consequences of the tumor and its treatment can lead to severe comorbidities resulting in poor quality of life, in particular for those patients who bear tumors with hypothalamic involvement. Accordingly, in these patients at risk for the development of a hypothalamic syndrome, hypothalamus-sparing treatment strategies such as limited resection followed by irradiation are recommended. In this review, we provide an update on various aspects of CP, with emphasis on recent advances in the understanding of tumor pathogenesis, clinical consequences, management, and therapies.

Hypothalamic syndrome.

Hypothalamic syndrome (HS) is a rare disorder caused by disease-related and/or treatment-related injury to the hypothalamus, most commonly associated with rare, non-cancerous parasellar masses, such as craniopharyngiomas, germ cell tumours, gliomas, cysts of Rathke's pouch and Langerhans cell histiocytosis, as well as with genetic neurodevelopmental syndromes, such as Prader-Willi syndrome and septo-optic dysplasia. HS is characterized by intractable weight gain associated with severe morbid obesity, multiple endocrine abnormalities and memory impairment, attention deficit and reduced impulse control as well as increased risk of cardiovascular and metabolic disorders. Currently, there is no cure for this condition but treatments for general obesity are often used in patients with HS, including surgery, medication and counselling. However, these are mostly ineffective and no medications that are specifically approved for the treatment of HS are available. Specific challenges in HS are because the syndrome represents an adverse effect of different diseases, and that diagnostic criteria, aetiology, pathogenesis and management of HS are not completely defined.

Adamantinomatous craniopharyngioma as a model to understand paracrine and senescence-induced tumourigenesis.

Cellular senescence is a process that can prevent tumour development in a cell autonomous manner by imposing a stable cell cycle arrest after oncogene activation. Paradoxically, senescence can also promote tumour growth cell non-autonomously by creating a permissive tumour microenvironment that fuels tumour initiation, progression to malignancy and metastasis. In a pituitary tumour known as adamantinomatous craniopharyngioma (ACP), cells that carry oncogenic ß-catenin mutations and overactivate the WNT signalling pathway form cell clusters that become senescent and activate a senescence-associated secretory phenotype (SASP). Research in mouse models of ACP has provided insights into the function of the senescent cell clusters and revealed a critical role for SASP-mediated activities in paracrine tumour initiation. In this review, we first discuss this research on ACP and subsequently explore the theme of paracrine tumourigenesis in other tumour models available in the literature. Evidence is accumulating supporting the notion that paracrine signalling brought about by senescent cells may underlie tumourigenesis across different tumours and cancer models.

Cell senescence in neuropathology: A focus on neurodegeneration and tumours.

The study of cell senescence is a burgeoning field. Senescent cells can modify the cellular microenvironment through the secretion of a plethora of biologically active products referred to as the senescence-associated secretory phenotype (SASP). The consequences of these paracrine signals can be either beneficial for tissue homeostasis, if senescent cells are properly cleared and SASP activation is transient, or result in organ dysfunction, when senescent cells accumulate within the tissues and SASP activation is persistent. Several studies have provided evidence for the role of senescence and SASP in promoting age-related diseases or driving organismal ageing. The hype about senescence has been further amplified by the fact that a group of drugs, named senolytics, have been used to successfully ameliorate the burden of age-related diseases and increase health and life span in mice. Ablation of senescent cells in the brain prevents disease progression and improves cognition in murine models of neurodegenerative conditions. The role of senescence in cancer has been more thoroughly investigated, and it is now accepted that senescence is a double-edged sword that can paradoxically prevent or promote tumourigenesis in a context-dependent manner. In addition, senescence induction followed by senolytic treatment is starting to emerge as a novel therapeutic avenue that could improve current anti-cancer therapies and reduce tumour recurrence. In this review, we discuss recent findings supporting the role of cell senescence in the pathogenesis of neurodegenerative diseases and in brain tumours. A better understanding of senescence is likely to result in the development of novel and efficacious anti-senescence therapies against these brain pathologies.

Biological Behaviour of Craniopharyngiomas.

Jakob Erdheim (1874-1937) first described craniopharyn-giomas (CPs) as "hypophyseal duct tumours" and postulated the existence of two tumour types based on their histological features: (1) an aggressive type showing similarities to adamantinomas (tumours of the jaw) and (2) a more benign form characterised by the presence of papillary structures. More than a century later, these initial observations have been confirmed; based on their distinct genetic, epigenetic, and histological features, the WHO classifies CPs into two types: adamantinomatous CPs (ACPs) and papillary CPs (PCPs). Considerable knowledge has been generated on the biology of CPs in the last 20 years. Mutations in CTNNB1 (encoding ß-catenin) are prevalent in ACP, whilst PCPs frequently harbour mutations in BRAF (p.BRAF-V600E). The consequence of these mutations is the activation of either the WNT/ß-catenin (ACP) or the MAPK/ERK (PCP) pathway. Murine models support a critical role for these mutations in tumour formation and have provided important insights into tumour pathogenesis, mostly in ACP. A critical role for cellular senescence has been uncovered in murine models of ACP with relevance to human tumours. Several gene profiling studies of human and murine ACP tumours have identified potential targetable pathways, and novel therapeutic agents are being used in clinical and pre-clinical research, in some cases with excellent results. In this review, we will present the accumulated knowledge on the biological features of these tumours and summarise how these advances are being translated into potential novel treatments.

Galactose-modified duocarmycin prodrugs as senolytics.

Senescence is a stable growth arrest that impairs the replication of damaged, old or preneoplastic cells, therefore contributing to tissue homeostasis. Senescent cells accumulate during ageing and are associated with cancer, fibrosis and many age-related pathologies. Recent evidence suggests that the selective elimination of senescent cells can be effective on the treatment of many of these senescence-associated diseases. A universal characteristic of senescent cells is that they display elevated activity of the lysosomal ß-galactosidase, and this has been exploited as a marker for senescence (senescence-associated ß-galactosidase activity). Consequently, we hypothesized that galactose-modified cytotoxic prodrugs will be preferentially processed by senescent cells, resulting in their selective killing. Here, we show that different galactose-modified duocarmycin (GMD) derivatives preferentially kill senescent cells. GMD prodrugs induce selective apoptosis of senescent cells in a lysosomal ß-galactosidase (GLB1)-dependent manner. GMD prodrugs can eliminate a broad range of senescent cells in culture, and treatment with a GMD prodrug enhances the elimination of bystander senescent cells that accumulate upon whole-body irradiation treatment of mice. Moreover, taking advantage of a mouse model of adamantinomatous craniopharyngioma (ACP), we show that treatment with a GMD prodrug selectively reduced the number of ß-catenin-positive preneoplastic senescent cells. In summary, the above results make a case for testing the potential of galactose-modified duocarmycin prodrugs to treat senescence-related pathologies.

Cardiac glycosides are broad-spectrum senolytics.

Senescence is a cellular stress response that results in the stable arrest of old, damaged or preneoplastic cells. Oncogene-induced senescence is tumor suppressive but can also exacerbate tumorigenesis through the secretion of pro-inflammatory factors from senescent cells. Drugs that selectively kill senescent cells, termed senolytics, have proved beneficial in animal models of many age-associated diseases. Here, we show that the cardiac glycoside, ouabain, is a senolytic agent with broad activity. Senescent cells are sensitized to ouabain-induced apoptosis, a process mediated in part by induction of the pro-apoptotic Bcl2-family protein NOXA. We show that cardiac glycosides synergize with anti-cancer drugs to kill tumor cells and eliminate senescent cells that accumulate after irradiation or in old mice. Ouabain also eliminates senescent preneoplastic cells. Our findings suggest that cardiac glycosides may be effective anti-cancer drugs by acting through multiple mechanism. Given the broad range of senescent cells targeted by cardiac glycosides their use against age-related diseases warrants further exploration.

Craniopharyngioma.

Craniopharyngiomas are rare malformational tumours of low histological malignancy arising along the craniopharyngeal duct. The two histological subtypes, adamantinomatous craniopharyngioma (ACP) and papillary craniopharyngioma (PCP), differ in genesis and age distribution. ACPs are diagnosed with a bimodal peak of incidence (5-15 years and 45-60 years), whereas PCPs are restricted to adults mainly in the fifth and sixth decades of life. ACPs are driven by somatic mutations in CTNNB1 (encoding ß-catenin) that affect ß-catenin stability and are predominantly cystic in appearance. PCPs frequently harbour somatic BRAFV600E mutations and are typically solid tumours. Clinical manifestations due to increased intracranial pressure, visual impairment and endocrine deficiencies should prompt imaging investigations, preferentially MRI. Treatment comprises neurosurgery and radiotherapy; intracystic chemotherapy is used in monocystic ACP. Although long-term survival is high, quality of life and neuropsychological function are frequently impaired due to the close anatomical proximity to the optic chiasm, hypothalamus and pituitary gland. Indeed, hypothalamic involvement and treatment-related hypothalamic lesions frequently result in hypothalamic obesity, physical fatigue and psychosocial deficits. Given the rarity of these tumours, efforts to optimize infrastructure and international collaboration should be research priorities.

Genetic Deletion of Hesx1 Promotes Exit from the Pluripotent State and Impairs Developmental Diapause.

The role of the homeobox transcriptional repressor HESX1 in embryonic stem cells (ESCs) remains mostly unknown. Here, we show that Hesx1 is expressed in the preimplantation mouse embryo, where it is required during developmental diapause. Absence of Hesx1 leads to reduced expression of epiblast and primitive endoderm determinants and failure of diapaused embryos to resume embryonic development after implantation. Genetic deletion of Hesx1 impairs self-renewal and promotes differentiation toward epiblast by reducing the expression of pluripotency factors and decreasing the activity of LIF/STAT3 signaling. We reveal that Hesx1-deficient ESCs show elevated ERK pathway activation, resulting in accelerated differentiation toward primitive endoderm, which can be prevented by overexpression of Hesx1. Together, our data provide evidence for a novel role of Hesx1 in the control of self-renewal and maintenance of the undifferentiated state in ESCs and mouse embryos.

Conditional Dicer1 depletion using Chrnb4-Cre leads to cone cell death and impaired photopic vision.

Irreversible photoreceptor cell death is a major cause of blindness in many retinal dystrophies. A better understanding of the molecular mechanisms underlying the progressive loss of photoreceptor cells remains therefore crucial. Abnormal expression of microRNAs (miRNAs) has been linked with the aetiology of a number of retinal dystrophies. However, their role during the degenerative process remains poorly understood. Loss of cone photoreceptors in the human macula has the greatest impact on sight as these cells provide high acuity vision. Using a Chrnb4-cre; Dicerflox/flox conditional knockout mouse (Dicer CKO) to delete Dicer1 from cone cells, we show that cone photoreceptor cells degenerate and die in the Dicer-deleted retina. Embryonic eye morphogenesis appeared normal in Dicer CKO mice. Cone photoreceptor abnormalities were apparent by 3 weeks of age, displaying either very short or absent outer segments. By 4 months 50% of cones were lost and cone function was impaired as assessed by electroretinography (ERG). RNAseq analysis of the Dicer CKO retina revealed altered expression of genes involved in the visual perception pathway. These data show that loss of Dicer1 leads to early-onset cone cell degeneration and suggest that Dicer1 is essential for cone photoreceptor survival and homeostasis.

Learning from cases: Analysis of two cases of craniopharyngioma from the 19 th to the 21 st centuries.

This manuscript describes the study of two cases of craniopharyngioma, which have been examined repeatedly over three separate centuries. This includes analysis by Josef Engel in 1839, who sought to uncover the physiological role of the pituitary gland; Jacob Erdheim in 1904, who initially described the disease we now call craniopharyngioma, and recent high resolution MRI and micro-CT imaging and attempted DNA analyses of the tumours. The cases highlight how, rightly or wrongly, our interpretation of data is shaped by the technologies, methodologies and prevailing theories of a given time.