Tyrosine kinase signaling-independent MET-targeting with CAR-T cells.

BACKGROUND: Recent progress in cancer immunotherapy encourages the expansion of chimeric antigen receptor (CAR) T cell therapy in solid tumors including hepatocellular carcinoma (HCC). Overexpression of MET receptor tyrosine kinase is common in HCC; however, MET inhibitors are effective only when MET is in an active form, making patient stratification difficult. Specific MET-targeting CAR-T cells hold the promise of targeting HCC with MET overexpression regardless of signaling pathway activity. METHODS: MET-specific CARs with CD28ζ or 4-1BBζ as co-stimulation domains were constructed. MET-CAR-T cells derived from healthy subjects (HS) and HCC patients were evaluated for their killing activity and cytokine release against HCC cells with various MET activations in vitro, and for their tumor growth inhibition in orthotopic xenograft models in vivo. RESULTS: MET-CAR.CD28ζ and MET-CAR.4-1BBζ T cells derived from both HS and HCC patients specifically killed MET-positive HCC cells. When stimulated with MET-positive HCC cells in vitro, MET-CAR.CD28ζ T cells demonstrated a higher level of cytokine release and expression of programmed cell death protein 1 (PD-1) than MET-CAR.4-1BBζ T cells. When analyzed in vivo, MET-CAR.CD28ζ T cells more effectively inhibited HCC orthotopic tumor growth in mice when compared to MET-CAR.4-1BBζ T cells. CONCLUSION: We generated and characterized MET-specific CAR-T cells for targeting HCC with MET overexpression regardless of MET activation. Compared with MET-CAR.4-1BBζ, MET-CAR.CD28ζ T cells showed a higher anti-HCC potency but also a higher level of T cell exhaustion. While MET-CAR.CD28ζ is preferred for further development, overcoming the exhaustion of MET-CAR-T cells is necessary to improve their therapeutic efficacy in vivo.

Bidirectional Transcription at the PPP2R2B Gene Locus in Spinocerebellar Ataxia Type 12.

BACKGROUND: Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in the PPP2R2B gene. OBJECTIVE: In this study, we tested the hypothesis that the PPP2R2B antisense (PPP2R2B-AS1) transcript containing a CUG repeat is expressed and contributes to SCA12 pathogenesis. METHODS: Expression of PPP2R2B-AS1 transcript was detected in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific reverse transcription polymerase chain reaction. The tendency of expanded PPP2R2B-AS1 (expPPP2R2B-AS1) RNA to form foci, a marker of toxic processes involving mutant RNAs, was examined in SCA12 cell models by fluorescence in situ hybridization. The apoptotic effect of expPPP2R2B-AS1 transcripts on SK-N-MC neuroblastoma cells was evaluated by caspase 3/7 activity. Western blot was used to examine the expression of repeat associated non-ATG-initiated translation of expPPP2R2B-AS1 transcript in SK-N-MC cells. RESULTS: The repeat region in the PPP2R2B gene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 mouse brains. Transfected expPPP2R2B-AS1 transcripts induce apoptosis in SK-N-MC cells, and the apoptotic effect may be mediated, at least in part, by the RNA secondary structure. The expPPP2R2B-AS1 transcripts form CUG RNA foci in SK-N-MC cells. expPPP2R2B-AS1 transcript is translated in the alanine open reading frame (ORF) via repeat-associated non-ATG translation, which is diminished by single-nucleotide interruptions within the CUG repeat and MBNL1 overexpression. CONCLUSIONS: These findings suggest that PPP2R2B-AS1 contributes to SCA12 pathogenesis and may therefore provide a novel therapeutic target for the disease. © 2023 International Parkinson and Movement Disorder Society.

The Interplay between α-Synuclein and Microglia in α-Synucleinopathies.

Synucleinopathies are a set of devastating neurodegenerative diseases that share a pathologic accumulation of the protein α-synuclein (α-syn). This accumulation causes neuronal death resulting in irreversible dementia, deteriorating motor symptoms, and devastating cognitive decline. While the etiology of these conditions remains largely unknown, microglia, the resident immune cells of the central nervous system (CNS), have been consistently implicated in the pathogenesis of synucleinopathies. Microglia are generally believed to be neuroprotective in the early stages of α-syn accumulation and contribute to further neurodegeneration in chronic disease states. While the molecular mechanisms by which microglia achieve this role are still being investigated, here we highlight the major findings to date. In this review, we describe how structural varieties of inherently disordered α-syn result in varied microglial receptor-mediated interactions. We also summarize which microglial receptors enable cellular recognition and uptake of α-syn. Lastly, we review the downstream effects of α-syn processing within microglia, including spread to other brain regions resulting in neuroinflammation and neurodegeneration in chronic disease states. Understanding the mechanism of microglial interactions with α-syn is vital to conceptualizing molecular targets for novel therapeutic interventions. In addition, given the significant diversity in the pathophysiology of synucleinopathies, such molecular interactions are vital in gauging all potential pathways of neurodegeneration in the disease state.

Genetic driver mutations introduced in identical cell-of-origin in murine glioblastoma reveal distinct immune landscapes but similar response to checkpoint blockade.

Glioblastoma (GBM) is the most aggressive primary brain tumor. In addition to being genetically heterogeneous, GBMs are also immunologically heterogeneous. However, whether the differences in immune microenvironment are driven by genetic driver mutation is unexplored. By leveraging the versatile RCAS/tv-a somatic gene transfer system, we establish a mouse model for Classical GBM by introducing EGFRvIII expression in Nestin-positive neural stem/progenitor cells in adult mice. Along with our previously published Nf1-silenced and PDGFB-overexpressing models, we investigate the immune microenvironments of the three models of human GBM subtypes by unbiased multiplex profiling. We demonstrate that both the quantity and composition of the microenvironmental myeloid cells are dictated by the genetic driver mutations, closely mimicking what was observed in human GBM subtypes. These myeloid cells express high levels of the immune checkpoint protein PD-L1; however, PD-L1 targeted therapies alone or in combination with irradiation are unable to increase the survival time of tumor-bearing mice regardless of the driver mutations, reflecting the outcomes of recent human trials. Together, these results highlight the critical utility of immunocompetent mouse models for preclinical studies of GBM, making these models indispensable tools for understanding the resistance mechanisms of immune checkpoint blockade in GBM and immune cell-targeting drug discovery.

Krüppel-like factor 4 (KLF4) induces mitochondrial fusion and increases spare respiratory capacity of human glioblastoma cells.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critical for the regulation of many cellular functions in both normal and neoplastic cells. Here, using human glioblastoma cells, we investigated KLF4's effects on cancer cell metabolism. We found that forced KLF4 expression promotes mitochondrial fusion and induces dramatic changes in mitochondrial morphology. To determine the impact of these changes on the cellular functions following, we analyzed how KLF4 alters glioblastoma cell metabolism, including glucose uptake, glycolysis, pentose phosphate pathway, and oxidative phosphorylation. We did not identify significant differences in baseline cellular metabolism between control and KLF4-expressing cells. However, when mitochondrial function was impaired, KLF4 significantly increased spare respiratory capacity and levels of reactive oxygen species in the cells. To identify the biological effects of these changes, we analyzed proliferation and survival of control and KLF4-expressing cells under stress conditions, including serum and nutrition deprivation. We found that following serum starvation, KLF4 altered cell cycle progression by arresting the cells at the G2/M phase and that KLF4 protected cells from nutrition deprivation-induced death. Finally, we demonstrated that methylation-dependent KLF4-binding activity mediates mitochondrial fusion. Specifically, the downstream targets of KLF4-mCpG binding, guanine nucleotide exchange factors, serve as the effector of KLF4-induced mitochondrial fusion, cell cycle arrest, and cell protection. Our experimental system provides a robust model for studying the interactions between mitochondrial morphology and function, mitochondrial dynamics and metabolism, and mitochondrial fusion and cell death during tumor initiation and progression.

Carbon Dots as a New Class of Diamagnetic Chemical Exchange Saturation Transfer (diaCEST) MRI Contrast Agents.

While carbon dots (C-dots) have been extensively investigated pertaining to their fluorescent, phosphorescent, electrochemiluminescent, optoelectronic, and catalytic features, their inherent chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) properties are unknown. By virtue of their hydrophilicity and abundant exchangeable protons of hydroxyl, amine, and amide anchored on the surface, we report here that C-dots can be adapted as effective diamagnetic CEST (diaCEST) MRI contrast agents. As a proof-of-concept demonstration, human glioma cells were labeled with liposomes with or without encapsulated C-dots and implanted in mouse brain. In vivo CEST MRI was able to clearly differentiate labeled cells from non-labeled cells. The present findings may encourage new applications of C-dots for in vivo imaging in deep tissues, which is currently not possible using conventional fluorescent (near-infrared) C-dots.

Krüppel-like factor 9 and histone deacetylase inhibitors synergistically induce cell death in glioblastoma stem-like cells.

BACKGROUND: The dismal prognosis of patients with glioblastoma (GBM) is attributed to a rare subset of cancer stem cells that display characteristics of tumor initiation, growth, and resistance to aggressive treatment involving chemotherapy and concomitant radiation. Recent research on the substantial role of epigenetic mechanisms in the pathogenesis of cancers has prompted the investigation of the enzymatic modifications of histone proteins for therapeutic drug targeting. In this work, we have examined the function of Krüppel-like factor 9 (KLF9), a transcription factor, in chemotherapy sensitization to histone deacetylase inhibitors (HDAC inhibitors). METHODS: Since GBM neurosphere cultures from patient-derived gliomas are enriched for GBM stem-like cells (GSCs) and form highly invasive and proliferative xenografts that recapitulate the features demonstrated in human patients diagnosed with GBM, we established inducible KLF9 expression systems in these GBM neurosphere cells and investigated cell death in the presence of epigenetic modulators such as histone deacetylase (HDAC) inhibitors. RESULTS: We demonstrated that KLF9 expression combined with HDAC inhibitor panobinostat (LBH589) dramatically induced glioma stem cell death via both apoptosis and necroptosis in a synergistic manner. The combination of KLF9 expression and LBH589 treatment affected cell cycle by substantially decreasing the percentage of cells at S-phase. This phenomenon is further corroborated by the upregulation of cell cycle inhibitors p21 and p27. Further, we determined that KLF9 and LBH589 regulated the expression of pro- and anti- apoptotic proteins, suggesting a mechanism that involves the caspase-dependent apoptotic pathway. In addition, we demonstrated that apoptosis and necrosis inhibitors conferred minimal protective effects against cell death, while inhibitors of the necroptosis pathway significantly blocked cell death. CONCLUSIONS: Our findings suggest a detailed understanding of how KLF9 expression in cancer cells with epigenetic modulators like HDAC inhibitors may promote synergistic cell death through a mechanism involving both apoptosis and necroptosis that will benefit novel combinatory antitumor strategies to treat malignant brain tumors.

Kruppel-like factor-9 (KLF9) inhibits glioblastoma stemness through global transcription repression and integrin α6 inhibition.

It is increasingly important to understand the molecular basis for the plasticity of neoplastic cells and their capacity to transition between differentiated and stemlike phenotypes. Kruppel-like factor-9 (KLF9), a member of the large KLF transcription factor family, has emerged as a regulator of oncogenesis, cell differentiation, and neural development; however, the molecular basis for the diverse contextual functions of KLF9 remains unclear. This study focused on the functions of KLF9 in human glioblastoma stemlike cells. We established for the first time a genome-wide map of KLF9-regulated targets in human glioblastoma stemlike cells and show that KLF9 functions as a transcriptional repressor and thereby regulates multiple signaling pathways involved in oncogenesis and stem cell regulation. A detailed analysis of one such pathway, integrin signaling, showed that the capacity of KLF9 to inhibit glioblastoma cell stemness and tumorigenicity requires ITGA6 repression. These findings enhance our understanding of the transcriptional networks underlying cancer cell stemness and differentiation and identify KLF9-regulated molecular targets applicable to cancer therapeutics.

c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype.

The tyrosine kinase c-Met promotes the formation and malignant progression of multiple cancers. It is well known that c-Met hyperactivation increases tumorigenicity and tumor cell resistance to DNA damaging agents, properties associated with tumor-initiating stem cells. However, a link between c-Met signaling and the formation and/or maintenance of neoplastic stem cells has not been previously identified. Here, we show that c-Met is activated and functional in glioblastoma (GBM) neurospheres enriched for glioblastoma tumor-initiating stem cells and that c-Met expression/function correlates with stem cell marker expression and the neoplastic stem cell phenotype in glioblastoma neurospheres and clinical glioblastoma specimens. c-Met activation was found to induce the expression of reprogramming transcription factors (RFs) known to support embryonic stem cells and induce differentiated cells to form pluripotent stem (iPS) cells, and c-Met activation counteracted the effects of forced differentiation in glioblastoma neurospheres. Expression of the reprogramming transcription factor Nanog by glioblastoma cells is shown to mediate the ability of c-Met to induce the stem cell characteristics of neurosphere formation and neurosphere cell self-renewal. These findings show that c-Met enhances the population of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met-responsive reprogramming transcription factors.

Lymphocyte-Activation Gene 3 Facilitates Pathological Tau Neuron-to-Neuron Transmission.

The spread of prion-like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target.

Aplp1 interacts with Lag3 to facilitate transmission of pathologic α-synuclein.

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid ß precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson's disease and related α-synucleinopathies.

Cell Therapy for Parkinson's Disease.

Parkinson's Disease (PD) is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons of the substantia nigra pars compacta with a reduction in dopamine concentration in the striatum. It is a substantial loss of dopaminergic neurons that is responsible for the classic triad of PD symptoms, i.e., resting tremor, muscular rigidity, and bradykinesia. Several current therapies for PD may only offer symptomatic relief and do not address the underlying neurodegeneration of PD. The recent developments in cellular reprogramming have enabled the development of previously unachievable cell therapies and patient-specific modeling of PD through Induced Pluripotent Stem Cells (iPSCs). iPSCs possess the inherent capacity for pluripotency, allowing for their directed differentiation into diverse cell lineages, such as dopaminergic neurons, thus offering a promising avenue for addressing the issue of neurodegeneration within the context of PD. This narrative review provides a comprehensive overview of the effects of dopamine on PD patients, illustrates the versatility of iPSCs and their regenerative abilities, and examines the benefits of using iPSC treatment for PD as opposed to current therapeutic measures. In means of providing a treatment approach that reinforces the long-term survival of the transplanted neurons, the review covers three supplementary avenues to reinforce the potential of iPSCs.

Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs underwent SMART-seq2 sequencing and multiple bioinformatic analysis tools were applied to high-resolution gene and transcript expression analyses. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Bidirectional transcription at the PPP2R2B gene locus in spinocerebellar ataxia type 12.

OBJECTIVE: Spinocerebellar ataxia type 12 (SCA12) is a neurodegenerative disease caused by expansion of a CAG repeat in the PPP2R2B gene . Here we tested the hypothesis that the PPP2R2B antisense ( PPP2R2B-AS1 ) transcript containing a CUG repeat is expressed and contributes to SCA12 pathogenesis. METHODS: Expression of PPP2R2B-AS1 transcript was detected in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific RT-PCR (SS-RT-PCR). The tendency of expanded PPP2R2B-AS1 ( expPPP2R2B-AS1 ) RNA to form foci, a marker of toxic processes involving mutant RNAs, was examined in SCA12 cell models by fluorescence in situ hybridization. The toxic effect of expPPP2R2B-AS1 transcripts on SK-N-MC neuroblastoma cells was evaluated by caspase 3/7 activity. Western blot was used to examine the expression of repeat associated non-ATG-initiated (RAN) translation of expPPP2R2B-AS1 transcript in SK-N-MC cells. RESULTS: The repeat region in PPP2R2B gene locus is bidirectionally transcribed in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 mouse brains. Transfected expPPP2R2B-AS1 transcripts are toxic to SK-N-MC cells, and the toxicity may be mediated, at least in part, by the RNA secondary structure. The expPPP2R2B-AS1 transcripts form CUG RNA foci in SK-N-MC cells. expPPP2R2B-AS1 transcript is translated in the Alanine ORF via repeat-associated non-ATG (RAN) translation, which is diminished by single nucleotide interruptions within the CUG repeat, and MBNL1 overexpression. INTERPRETATION: These findings suggest that PPP2R2B-AS1 contributes to SCA12 pathogenesis, and may therefore provide a novel therapeutic target for the disease.

Pathological Tau transmission initiated by binding lymphocyte-activation gene 3.

The spread of prion-like protein aggregates is believed to be a common driver of pathogenesis in many neurodegenerative diseases. Accumulated tangles of filamentous Tau protein are considered pathogenic lesions of Alzheimer's disease (AD) and related Tauopathies, including progressive supranuclear palsy, and corticobasal degeneration. Tau pathologies in these illnesses exhibits a clear progressive and hierarchical spreading pattern that correlates with disease severity1,2. Clinical observation combined with complementary experimental studies3,4 have shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remains poorly understood. Here, we show that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF, but not monomer, of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. Our results identify neuronal Lag3 as a receptor of pathologic Tau in the brain, and for AD and related Tauopathies a therapeutic target.

Krüppel-like family of transcription factor 9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells.

Tumor-initiating stem cells (alternatively called cancer stem cells, CSCs) are a subpopulation of tumor cells that plays unique roles in tumor propagation, therapeutic resistance, and tumor recurrence. It is becoming increasingly important to understand the molecular signaling that regulates the self-renewal and differentiation of CSCs. Transcription factors are critical for the regulation of normal and neopolastic stem cells. Here, we examined the expression and function of the Krüppel-like family of transcription factors (KLFs) in human glioblastoma (GBM)-derived neurosphere lines and low-passage primary GBM-derived neurospheres that are enriched for tumor-initiating stem cells. We identify KLF9 as a relatively unique differentiation-induced transcription factor in GBM-derived neurospheres. KLF9 is shown to induce neurosphere cell differentiation, inhibit neurosphere formation, and inhibit neurosphere-derived xenograft growth in vivo. We also show that KLF9 regulates GBM neurosphere cells by binding to the Notch1 promoter and suppressing Notch1 expression and downstream signaling. Our results show for the first time that KLF9 has differentiating and tumor-suppressing functions in tumor-initiating stem cells.

Targeting UDP-α-d-glucose 6-dehydrogenase alters the CNS tumor immune microenvironment and inhibits glioblastoma growth.

Glioblastoma (GBM, WHO grade IV glioma) is the most common and lethal malignant brain tumor in adults with a dismal prognosis. The extracellular matrix (ECM) supports GBM progression by promoting tumor cell proliferation, migration, and immune escape. Uridine diphosphate (UDP)-glucose 6-dehydrogenase (UGDH) is the rate-limiting enzyme that catalyzes the biosynthesis of glycosaminoglycans that are the principal component of the CNS ECM. We investigated how targeting UGDH in GBM influences the GBM immune microenvironment, including tumor-associated microglia/macrophages (TAMs) and T cells. TAMs are the main immune effector cells in GBM and can directly target tumor cells if properly activated. In co-cultures of GBM cells and human primary macrophages, UGDH knockdown in GBM cells promoted macrophage phagocytosis and M1-like polarization. In orthotropic human GBM xenografts and syngeneic mouse glioma models, targeting UGDH decreased ECM deposition, increased TAM phagocytosis marker expression, reduced M2-like TAMs and inhibited tumor growth. UGDH knockdown in GBM cells also promoted cytotoxic T cell infiltration and activation in orthotopic syngeneic mouse glioma models. The potent and in-human-use small molecule GAG synthesis inhibitor 4-methylumbelliferone (4-MU) was found to inhibit GBM cell proliferation and migration in vitro, mimic the macrophage and T-cell responses to UGDH knockdown in vitro and in vivo and inhibit growth of orthotopic murine GBM. Our study shows that UGDH supports GBM growth through multiple mechanisms and supports the development of ECM-based therapeutic strategies to simultaneously target tumor cells and their microenvironment.

ATRX loss promotes immunosuppressive mechanisms in IDH1 mutant glioma.

BACKGROUND: ATRX inactivation occurs with IDH1R132H and p53 mutations in over 80% of Grades II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1R132H/p53mut glioma cells and xenografts. METHODS: Isogenic astrocytoma cells (+/-IDH1R132H/+/-ATRXloss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline-inducible IDH1R132H, ATRX shRNA, or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1R132H+/-ATRXloss on cell migration, growth, DNA repair, and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays, and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization, and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1R132H/p53mut/ATRXloss cells. RESULTS: Adding ATRX loss to the IDH1R132H/p53mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune-suppressive transcriptome consisting of up-regulated immune checkpoints (e.g., PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g., IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1R132H/p53mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune-suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition. CONCLUSIONS: ATRX loss activates a BRD-dependent immune-suppressive transcriptome and immune escape mechanism in IDH1R132H/p53mut astrocytoma cells.

Edaravone activates the GDNF/RET neurotrophic signaling pathway and protects mRNA-induced motor neurons from iPS cells.

BACKGROUND: Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. METHODS: We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. RESULTS: We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. CONCLUSIONS: This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.

Frondoside A Inhibits an MYC-Driven Medulloblastoma Model Derived from Human-Induced Pluripotent Stem Cells.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the MYC oncogene combined with p53 loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non-small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.