ALS iPSC-derived microglia and motor neurons respond to astrocyte-targeted IL-10 and CCL2 modulation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs). The loss of MNs in ALS leads to muscle weakness and wasting, respiratory failure, and death often within two years of diagnosis. Glial cells in ALS show aberrant expression of pro-inflammatory and neurotoxic proteins associated with activation and have been proposed as ideal therapeutic targets. In this study, we examined astrocyte-targeted treatments to reduce glial activation and neuron pathology using cells differentiated from ALS patient-derived iPSC carrying SOD1 and C9ORF72 mutations. Specifically, we tested the ability of increasing interleukin 10 (IL-10) and reducing C-C motif chemokine ligand 2 (CCL2/MCP-1) signaling targeted to astrocytes to reduce activation phenotypes in both astrocytes and microglia. Overall, we found IL10/CCL2NAb treated astrocytes to support anti-inflammatory phenotypes and reduce neurotoxicity, through different mechanisms in SOD1 and C9ORF72 cultures. We also found altered responses of microglia and motor neurons to astrocytic influences when cells were cultured together rather than in isolation. Together these data support IL-10 and CCL2 as non-mutation-specific therapeutic targets for ALS and highlight the role of glial-mediated pathology in this disease.

Sickle cell disease iPSC-derived sensory neurons exhibit increased excitability and sensitization to patient plasma.

ABSTRACT: Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. Challenges to gaining mechanistic insight into pathogenic SCD pain processes include differential gene expression and function of sensory neurons between humans and mice with SCD, and extremely limited availability of neuronal tissues from patients with SCD. Here, we used induced pluripotent stem cells (iPSCs), derived from patients with SCD, differentiated into sensory neurons (SCD iSNs) to begin to overcome these challenges. We characterize key gene expression and function of SCD iSNs to establish a model to investigate intrinsic and extrinsic factors that may contribute to SCD pain. Despite similarities in receptor gene expression, SCD iSNs show pronounced excitability using patch clamp electrophysiology. Furthermore, we find that plasma taken from patients with SCD during acute pain associated with a vaso-occlusive event increases the calcium responses to the nociceptive stimulus capsaicin in SCD iSNs compared with those treated with paired plasma from patients with SCD at steady state baseline or healthy control plasma samples. We identified high levels of the polyamine spermine in baseline and acute pain states of plasma from patients with SCD, which sensitizes SCD iSNs to subthreshold concentrations of capsaicin. Together, these data identify potential intrinsic mechanisms within SCD iSNs that may extend beyond a blood-based pathology.

Viral mediated knockdown of GATA6 in SMA iPSC-derived astrocytes prevents motor neuron loss and microglial activation.

Spinal muscular atrophy (SMA), a pediatric genetic disorder, is characterized by the profound loss of spinal cord motor neurons and subsequent muscle atrophy and death. Although the mechanisms underlying motor neuron loss are not entirely clear, data from our work and others support the idea that glial cells contribute to disease pathology. GATA6, a transcription factor that we have previously shown to be upregulated in SMA astrocytes, is negatively regulated by SMN (survival motor neuron) and can increase the expression of inflammatory regulator NFκB. In this study, we identified upregulated GATA6 as a contributor to increased activation, pro-inflammatory ligand production, and neurotoxicity in spinal-cord patterned astrocytes differentiated from SMA patient induced pluripotent stem cells. Reducing GATA6 expression in SMA astrocytes via lentiviral infection ameliorated these effects to healthy control levels. Additionally, we found that SMA astrocytes contribute to SMA microglial phagocytosis, which was again decreased by lentiviral-mediated knockdown of GATA6. Together these data identify a role of GATA6 in SMA astrocyte pathology and further highlight glia as important targets of therapeutic intervention in SMA.

A health survey of the reef forming scleractinian cold-water corals Lophelia pertusa and Madrepora oculata in a remote submarine canyon on the European continental margin, NE Atlantic.

Monitoring of cold-water corals (CWCs) for pathogens and diseases is limited due to the environment, protected nature of the corals and their habitat and as well as the challenging and sampling effort required. It is recognised that environmental factors such as temperature and pH can expedite the ability of pathogens to cause diseases in cold-water corals therefore the characterisation of pathogen diversity, prevalence and associated pathologies is essential. The present study combined histology and polymerase chain reaction (PCR) diagnostic techniques to screen for two significant pathogen groups (bacteria of the genus Vibrio and the protozoan Haplosporidia) in the dominant NE Atlantic deep-water framework corals Lophelia pertusa (13 colonies) and Madrepora oculata (2 colonies) at three sampling locations (canyon head, south branch and the flank) in the Porcupine Bank Canyon (PBC), NE Atlantic. One M. oculata colony and four L. pertusa colonies were collected from both the canyon flank and the south branch whilst five L. pertusa colonies were collected from the canyon head. No pathogens were detected in the M. oculata samples. Neither histology nor PCR detected Vibrio spp. in L. pertusa, although Illumina technology used in this study to profile the CWCs microbiome, detected V. shilonii (0.03%) in a single L. pertusa individual, from the canyon head, that had also been screened in this study. A macroborer was observed at a prevalence of 0.07% at the canyon head only. Rickettsiales-like organisms (RLOs) were visualised with an overall prevalence of 40% and with a low intensity of 1 to 4 (RLO) colonies per individual polyp by histology. L. pertusa from the PBC canyon head had an RLO prevalence of 13.3% with the highest detection of 26.7% recorded in the south branch corals. Similarly, unidentified cells observed in L. pertusa from the south branch (20%) were more common than those observed in L. pertusa from the canyon head (6.7%). No RLOs or unidentified cells were observed in corals from the flank. Mean particulate organic matter concentration is highest in the south branch (2,612 µg l-1) followed by the canyon head (1,065 µg l-1) and lowest at the canyon flank (494 µg l-1). Although the route of pathogen entry and the impact of RLO infection on L. pertusa is unclear, particulate availability and the feeding strategies employed by the scleractinian corals may be influencing their exposure to pathogens. The absence of a pathogen in M. oculata may be attributed to the smaller number of colonies screened or the narrower diet in M. oculata compared to the unrestricted diet exhibited in L. pertusa, if ingestion is a route of entry for pathogen groups. The findings of this study also shed some light on how environmental conditions experienced by deep sea organisms and their life strategies may be limiting pathogen diversity and prevalence.

Racial and ethnic disparities in brain tumour survival by age group and tumour type.

PURPOSE: The extent to which racial/ethnic brain tumour survival disparities vary by age is not very clear. In this study, we assess racial/ethnic brain tumour survival disparities overall by age group and type. METHODS: Data were obtained from the Surveillance, Epidemiology, and End Results (SEER) 18 registries for US-based individuals diagnosed with a first primary malignant tumour from 2007 through 2016. Cox proportional hazards regression was used to compute adjusted hazard ratios (HRs) and 95% confidence intervals (95% CIs) for the association between race/ethnicity and brain tumour survival, stratified by age group and tumour type. RESULTS: After adjusting for sex, socioeconomic status, insurance status, and tumour type, non-Hispanic (NH) Blacks (HR: 1.26; 95% CI: 1.02-1.55), NH Asian or Pacific Islanders (HR: 1.29; 95% CI: 1.01-1.66), and Hispanics (any race) (HR: 1.28; 95% CI: 1.09-1.51) all showed a survival disadvantage compared with NH Whites for the youngest age group studied (0-9 years). Furthermore, NH Blacks (HR: 0.88; 95% CI: 0.91-0.97), NH Asian or Pacific Islanders (HR: 0.84; 95% CI: 0.77-0.92), and Hispanics (any race) (HR: 0.91; 95% CI: 0.85-0.97) all showed a survival advantage compared with NH Whites for the 60-79 age group. Tests for interactions showed significant trends, indicating that racial/ethnic survival disparities disappear and even reverse for older age groups (P < 0.001). This reversal appears to be driven by poor glioblastoma survival among NH Whites (P < 0.001). CONCLUSION: Disparities in brain tumour survival among minorities exist primarily among children and adolescents. NH White adults show worse survival than their minority counterparts, which is possibly driven by poor glioblastoma biology.

Impact of loss of NF-κB1, NF-κB2 or c-REL on SLE-like autoimmune disease and lymphadenopathy in Fas(lpr/lpr) mutant mice.

Defects in apoptosis can cause autoimmune disease. Loss-of-function mutations in the 'death receptor' FAS impair the deletion of autoreactive lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus-like autoimmune disease in mice (Fas(lpr/lpr) (mice homozygous for the lymphoproliferation inducing spontaneous mutation)) and humans. The REL/nuclear factor-κB (NF-κB) transcription factors regulate a broad range of immune effector functions and are also implicated in various autoimmune diseases. We generated compound mutant mice to investigate the individual functions of the NF-κB family members NF-κB1, NF-κB2 and c-REL in the various autoimmune pathologies of Fas(lpr/lpr) mutant mice. We show that loss of each of these transcription factors resulted in amelioration of many classical features of autoimmune disease, including hypergammaglobulinaemia, anti-nuclear autoantibodies and autoantibodies against tissue-specific antigens. Remarkably, only c-REL deficiency substantially reduced immune complex-mediated glomerulonephritis and extended the lifespan of Fas(lpr/lpr) mice. Interestingly, compared with the Fas(lpr/lpr) animals, Fas(lpr/lpr)nfkb2(-/-) mice presented with a dramatic acceleration and augmentation of lymphadenopathy that was accompanied by severe lung pathology due to extensive lymphocytic infiltration. The Fas(lpr/lpr)nfkb1(-/-) mice exhibited the combined pathologies caused by defects in FAS-mediated apoptosis and premature ageing due to loss of NF-κB1. These findings demonstrate that different NF-κB family members exert distinct roles in the development of the diverse autoimmune and lymphoproliferative pathologies that arise in Fas(lpr/lpr) mice, and suggest that pharmacological targeting of c-REL should be considered as a strategy for therapeutic intervention in autoimmune diseases.

bak deletion stimulates gastric epithelial proliferation and enhances Helicobacter felis-induced gastric atrophy and dysplasia in mice.

Helicobacter infection causes a chronic superficial gastritis that in some cases progresses via atrophic gastritis to adenocarcinoma. Proapoptotic bak has been shown to regulate radiation-induced apoptosis in the stomach and colon and also susceptibility to colorectal carcinogenesis in vivo. Therefore we investigated the gastric mucosal pathology following H. felis infection in bak-null mice at 6 or 48 wk postinfection. Primary gastric gland culture from bak-null mice was also used to assess the effects of bak deletion on IFN-γ-, TNF-α-, or IL-1ß-induced apoptosis. bak-null gastric corpus glands were longer, had increased epithelial Ki-67 expression, and contained fewer parietal and enteroendocrine cells compared with the wild type (wt). In wt mice, bak was expressed at the luminal surface of gastric corpus glands, and this increased 2 wk post-H. felis infection. Apoptotic cell numbers were decreased in bak-null corpus 6 and 48 wk following infection and in primary gland cultures following cytokine administration. Increased gastric epithelial Ki-67 labeling index was observed in C57BL/6 mice after H. felis infection, whereas no such increase was detected in bak-null mice. More severe gastric atrophy was observed in bak-null compared with C57BL/6 mice 6 and 48 wk postinfection, and 76% of bak-null compared with 25% of C57BL/6 mice showed evidence of gastric dysplasia following long-term infection. Collectively, bak therefore regulates gastric epithelial cell apoptosis, proliferation, differentiation, mucosal thickness, and susceptibility to gastric atrophy and dysplasia following H. felis infection.

Sickle cell disease patient plasma sensitizes iPSC-derived sensory neurons from sickle cell disease patients.

Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. In order to develop novel therapies, it is necessary to better understand the neurobiological mechanisms underlying SCD pain. There are many barriers to gaining mechanistic insight into pathogenic SCD pain processes, such as differential gene expression and function of sensory neurons between humans and mice with SCD, as well as the limited availability of patient samples. These can be overcome by utilizing SCD patient-derived induced pluripotent stem cells (iPSCs) differentiated into sensory neurons (SCD iSNs). Here, we characterize the key gene expression and function of SCD iSNs to establish a model for higher-throughput investigation of intrinsic and extrinsic factors that may contribute to increased SCD patient pain. Importantly, identified roles for C-C Motif Chemokine Ligand 2 (CCL2) and endothelin 1 (ET1) in SCD pain can be recapitulated in SCD iSNs. Further, we find that plasma taken from SCD patients during acute pain increases SCD iSN calcium response to the nociceptive stimulus capsaicin compared to those treated with paired SCD patient plasma at baseline or healthy control plasma samples. Together, these data provide the framework necessary to utilize iSNs as a powerful tool to investigate the neurobiology of SCD and identify potential intrinsic mechanisms of SCD pain which may extend beyond a blood-based pathology.

Sorting nexin 10 sustains PDGF receptor signaling in glioblastoma stem cells via endosomal protein sorting.

Glioblastoma is the most malignant primary brain tumor, the prognosis of which remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared with NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor ß (PDGFRß) proliferative and stem cell signaling pathways through posttranscriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

Wnt5a inhibits the CpG oligodeoxynucleotide-triggered activation of human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) fulfil multiple roles in immunity, and can secrete large amounts of interferon (IFN)-α. However, the available evidence suggests that they may actually counteract efficient antitumour immunity. Thus in melanoma, pDCs are abundant, but they are anergic and deficient in IFN-α secretion. pDC anergy is thought to be caused by immunosuppressive factors secreted by melanoma cells. One factor strongly expressed by melanoma is Wnt5a, which is implicated in cancer tissue invasion. In this paper, we show that Wnt5a is able to block the upregulation of the activation markers CD80 and CD86 on naive human pDCs stimulated by CpG oligodeoxynucleotide, and CpG-triggered secretion of IFN-α by pDCs. Our results suggest that Wnt5a may not only initiate cancer invasion, but could also regulate activation of pDC. These data provide a clear rationale to investigate a role for Wnt5a in immune regulation.

Accumulation of marine litter in cold-water coral habitats: A comparative study of two Irish Special Areas of Conservation, NE Atlantic.

Cold-water corals (CWCs) have come under threat from anthropogenic activities such as fishing despite their ecological significance as biodiversity hotspots and as such are being protected in Europe under the EU Habitats Directive with some designated as Special Areas of Conservation (SACs). This study maps the distribution and sources of marine litter in CWC habitats in two SACs on the Irish margin. Data were collected with remotely operated vehicle in the SACs. The density, abundance and composition of litter were assessed, with differences observed between the two sites. The regional morphology influences the distribution of litter in the SACs, with CWC reefs and rock exposures trapping more marine litter. Fishing gear (80.7%) and plastics (55.1%) were commonly found. The observed fisheries-derived litter in the SACs exceed global averages of 10-20% fishing gear, suggesting the SACs appear to offer limited protection to the coral habitats with respect to marine litter.

CDK 4/6 inhibitors for the treatment of meningioma.

Meningiomas are the most common non-metastatic brain tumors, and although the majority are relatively slow-growing and histologically benign, a subset of meningiomas are aggressive and remain challenging to treat. Despite a standard of care that includes surgical resection and radiotherapy, and recent advances in meningioma molecular grouping, there are no systemic medical options for patients with meningiomas that are resistant to standard interventions. Misactivation of the cell cycle at the level of CDK4/6 is common in high-grade or molecularly aggressive meningiomas, and CDK4/6 has emerged as a potential target for systemic meningioma treatments. In this review, we describe the preclinical evidence for CDK4/6 inhibitors as a treatment for high-grade meningiomas and summarize evolving clinical experience with these agents. Further, we highlight upcoming clinical trials for patients meningiomas, and discuss future directions aimed at optimizing the efficacy of these therapies and selecting patients most likely to benefit from their use.

Microglia Influence Neurofilament Deposition in ALS iPSC-Derived Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here, we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin which exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte-conditioned medium and microglial-conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.

Can a bioactive interbody device reduce the cost burden of achieving lateral lumbar fusion?

OBJECTIVE: Intervertebral devices are increasingly utilized for fusion in the lumbar spine, along with a variety of bone graft materials. These various grafting materials often have substantial cost burdens for the surgical procedure, although they are necessary to overcome the limitations in healing capacity for many traditional interbody devices. The use of bioactive interbody fusion devices, which have demonstrable stimulatory capacity for the surrounding osteoblasts and osteoprogenitor cells and allow for osseointegration, may reduce this heavy reliance on osteobiologics for achieving interbody fusion. The objective of this study was to evaluate the rate of successful interbody fusion with a bioactive lateral lumbar interbody titanium implant with limited volume and low-cost graft material. METHODS: The authors conducted a retrospective study (May 2017 to October 2018) of consecutively performed lateral lumbar interbody fusions with a bioactive 3D-printed porous titanium interbody device. Each interbody device was filled with 2-3 cm3/cage of a commercially available ceramic bone extender (ß-tricalcium phosphate-hydroxyapatite) and combined with posterior pedicle screw fixation. No other biological agents or grafts were utilized. Demographic, clinical, and radiographic variables were captured. Fusion success was the primary endpoint of the study, with graft subsidence, fixation failure, and patient-reported outcomes (Oswestry Disability Index [ODI] and visual analog scale [VAS]-back and -leg pain scores) collected as secondary endpoints. The authors utilized a CT-based fusion classification system that accounted for both intervertebral through-growth (bone bridging) and ingrowth (integration of bone at the endplate-implant interface). RESULTS: In total, 136 lumbar levels were treated in 90 patients. The mean age was 69 years, and 63% of the included patients were female. Half (50.0%) had undergone previous spinal surgery, and a third (33.7%) had undergone prior lumbar fusion. A third (33.7%) were treated at multiple levels (mean levels per patient 1.51). One year after surgery, the mean improvements in patient-reported outcomes (vs preoperative scores) were -17.8 for ODI (p < 0.0001), -3.1 for VAS-back pain (p < 0.0001), and -2.9 for VAS-leg pain (p < 0.0001). Bone bridging and/or appositional integrity was achieved in 99.3% of patients, including 97.8% who had complete bone bridging. No fixation loosening or implant failure was observed at any segment. Low-grade graft subsidence (Marchi grade ≤ I) occurred in 3 levels (2.2%), and intraoperative endplate violation occurred twice (1.5%). High-grade subsidence was not found. No implant failure or revision surgery for pseudarthrosis/subsidence was necessary. CONCLUSIONS: The use of bioactive titanium interbody devices with a large surface footprint appears to result in a very high rate of effective fusion, despite the use of a small volume of low-cost biological material. This potential change in the osteobiologics required to achieve high fusion rates may have a substantially beneficial impact on the economic burden inherent to spinal fusion.

Glioblastoma stem cells reprogram chromatin in vivo to generate selective therapeutic dependencies on DPY30 and phosphodiesterases.

Glioblastomas are universally fatal cancers and contain self-renewing glioblastoma stem cells (GSCs) that initiate tumors. Traditional anticancer drug discovery based on in vitro cultures tends to identify targets with poor therapeutic indices and fails to accurately model the effects of the tumor microenvironment. Here, leveraging in vivo genetic screening, we identified the histone H3 lysine 4 trimethylation (H3K4me3) regulator DPY30 (Dpy-30 histone methyltransferase complex regulatory subunit) as an in vivo­specific glioblastoma dependency. On the basis of the hypothesis that in vivo epigenetic regulation may define critical GSC dependencies, we interrogated active chromatin landscapes of GSCs derived from intracranial patient-derived xenografts (PDXs) and cell culture through H3K4me3 chromatin immunoprecipitation and transcriptome analyses. Intracranial-specific genes marked by H3K4me3 included FOS, NFκB, and phosphodiesterase (PDE) family members. In intracranial PDX tumors, DPY30 regulated angiogenesis and hypoxia pathways in an H3K4me3-dependent manner but was dispensable in vitro in cultured GSCs. PDE4B was a key downstream effector of DPY30, and the PDE4 inhibitor rolipram preferentially targeted DPY30-expressing cells and impaired PDX tumor growth in mice without affecting tumor cells cultured in vitro. Collectively, the MLL/SET1 (mixed lineage leukemia/SET domain-containing 1, histone lysine methyltransferase) complex member DPY30 selectively regulates H3K4me3 modification on genes critical to support angiogenesis and tumor growth in vivo, suggesting the DPY30-PDE4B axis as a specific therapeutic target in glioblastoma.

Traumatic rupture of thoracic epidural capillary hemangioma resulting in acute neurologic deficit: illustrative case.

BACKGROUND: Thoracic epidural capillary hemangioma is exceedingly rare, with only a few reported cases. The typical presentation usually includes chronic, progressive symptoms of spinal cord compression in middle-aged adults. To the authors' knowledge, this case is the first report in the literature of acute traumatic capillary hemangioma rupture. OBSERVATIONS: A 22-year-old male presented with worsening lower extremity weakness and paresthesias after a fall onto his spine. Imaging showed no evidence of spinal fracture but revealed an expanding hematoma over 24 hours. Removal of the lesion demonstrated a ruptured capillary hemangioma. LESSONS: This unique case highlights a rare occurrence of traumatic rupture of a previously unknown asymptomatic thoracic capillary hemangioma in a young adult.

The RNA m6A Reader YTHDF2 Maintains Oncogene Expression and Is a Targetable Dependency in Glioblastoma Stem Cells.

Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N 6-methyladenosine (m6A) mRNA modifications in GSCs by methyl RNA immunoprecipitation followed by sequencing and transcriptome analysis, finding transcripts marked by m6A often upregulated compared with normal neural stem cells (NSC). Interrogating m6A regulators, GSCs displayed preferential expression, as well as in vitro and in vivo dependency, of the m6A reader YTHDF2, in contrast to NSCs. Although YTHDF2 has been reported to destabilize mRNAs, YTHDF2 stabilized MYC and VEGFA transcripts in GSCs in an m6A-dependent manner. We identified IGFBP3 as a downstream effector of the YTHDF2-MYC axis in GSCs. The IGF1/IGF1R inhibitor linsitinib preferentially targeted YTHDF2-expressing cells, inhibiting GSC viability without affecting NSCs and impairing in vivo glioblastoma growth. Thus, YTHDF2 links RNA epitranscriptomic modifications and GSC growth, laying the foundation for the YTHDF2-MYC-IGFBP3 axis as a specific and novel therapeutic target in glioblastoma. SIGNIFICANCE: Epitranscriptomics promotes cellular heterogeneity in cancer. RNA m6A landscapes of cancer and NSCs identified cell type-specific dependencies and therapeutic vulnerabilities. The m6A reader YTHDF2 stabilized MYC mRNA specifically in cancer stem cells. Given the challenge of targeting MYC, YTHDF2 presents a therapeutic target to perturb MYC signaling in glioblastoma.This article is highlighted in the In This Issue feature, p. 211.

Fas ligand, Bcl-2, granulocyte colony-stimulating factor, and p38 mitogen-activated protein kinase: Regulators of distinct cell death and survival pathways in granulocytes.

The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2-controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.

bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells.

Immunization with T cell-dependent antigens generates long-lived memory B cells and antibody-forming cells (AFCs). Both populations originate in germinal centers and, predominantly, produce antibodies with high affinity for antigen. The means by which germinal center B cells are recruited into these populations remains unclear. We have examined affinity maturation of antigen-specific B cells in mice expressing the cell death inhibitor bcl-2 as a transgene. Such mice had reduced apoptosis in germinal centers and an excessive number of memory B cells with a low frequency of V gene somatic mutation, including those mutations encoding amino acid exchanges known to enhance affinity. Despite the frequency of AFCs being increased in bcl-2-transgenic mice, the fraction secreting high-affinity antibody in the bone marrow at day 42 remained unchanged compared with controls. The inability of BCL-2 to alter selection of bone marrow AFCs is consistent with these cells being selected within the germinal center on the basis of their affinity being above some threshold rather than their survival being due to a selective competition for an antigen-based signal. Continuous competition for antigen does, however, explain formation of the memory compartment.

B lymphocytes differentially use the Rel and nuclear factor kappaB1 (NF-kappaB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells.

Rel and nuclear factor (NF)-kappaB1, two members of the Rel/NF-kappaB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-kappaB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1-/- mice, the level of apoptosis in cultures of quiescent nfkb1-/-, but not c-rel-/-, B cells is higher. The failure of c-rel-/- or nfkb1-/- B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel-/- and nfkb1-/- B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-kappaB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-kappaB-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel-/- B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-kappaB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-kappaB factors to control cell cycle progression and prevent apoptosis.