Cell autonomous microglia defects in a stem cell model of frontotemporal dementia.

Neuronal dysfunction has been extensively studied as a central feature of neurodegenerative tauopathies. However, across neurodegenerative diseases, there is strong evidence for active involvement of immune cells like microglia in driving disease pathophysiology. Here, we demonstrate that tau mRNA and protein are expressed in microglia in human brains and in human induced pluripotent stem cell (iPSC)-derived microglia like cells (iMGLs). Using iMGLs harboring the MAPT IVS10+16 mutation and isogenic controls, we demonstrate that a tau mutation is sufficient to alter microglial transcriptional states. We discovered that MAPT IVS10+16 microglia exhibit cytoskeletal abnormalities, stalled phagocytosis, disrupted TREM2/TYROBP networks, and altered metabolism. Additionally, we found that secretory factors from MAPT IVS10+16 iMGLs impact neuronal health, reducing synaptic density in neurons. Key features observed in vitro were recapitulated in human brain tissue and cerebrospinal fluid from MAPT mutations carriers. Together, our findings that MAPT IVS10+16 drives cell-intrinsic dysfunction in microglia that impacts neuronal health has major implications for development of therapeutic strategies.

Long non-coding RNA SNHG8 drives stress granule formation in tauopathies.

Tauopathies are a heterogenous group of neurodegenerative disorders characterized by tau aggregation in the brain. In a subset of tauopathies, rare mutations in the MAPT gene, which encodes the tau protein, are sufficient to cause disease; however, the events downstream of MAPT mutations are poorly understood. Here, we investigate the role of long non-coding RNAs (lncRNAs), transcripts >200 nucleotides with low/no coding potential that regulate transcription and translation, and their role in tauopathy. Using stem cell derived neurons from patients carrying a MAPT p.P301L, IVS10 + 16, or p.R406W mutation and CRISPR-corrected isogenic controls, we identified transcriptomic changes that occur as a function of the MAPT mutant allele. We identified 15 lncRNAs that were commonly differentially expressed across the three MAPT mutations. The commonly differentially expressed lncRNAs interact with RNA-binding proteins that regulate stress granule formation. Among these lncRNAs, SNHG8 was significantly reduced in a mouse model of tauopathy and in FTLD-tau, progressive supranuclear palsy, and Alzheimer's disease brains. We show that SNHG8 interacts with tau and stress granule-associated RNA-binding protein TIA1. Overexpression of mutant tau in vitro is sufficient to reduce SNHG8 expression and induce stress granule formation. Rescuing SNHG8 expression leads to reduced stress granule formation and reduced TIA1 levels in immortalized cells and in MAPT mutant neurons, suggesting that dysregulation of this non-coding RNA is a causal factor driving stress granule formation via TIA1 in tauopathies.

Defects in lysosomal function and lipid metabolism in human microglia harboring a TREM2 loss of function mutation.

TREM2 is an innate immune receptor expressed by microglia in the adult brain. Genetic variation in the TREM2 gene has been implicated in risk for Alzheimer's disease and frontotemporal dementia, while homozygous TREM2 mutations cause a rare leukodystrophy, Nasu-Hakola disease (NHD). Despite extensive investigation, the role of TREM2 in NHD pathogenesis remains poorly understood. Here, we investigate the mechanisms by which a homozygous stop-gain TREM2 mutation (p.Q33X) contributes to NHD. Induced pluripotent stem cell (iPSC)-derived microglia (iMGLs) were generated from two NHD families: three homozygous TREM2 p.Q33X mutation carriers (termed NHD), two heterozygous mutation carriers, one related non-carrier, and two unrelated non-carriers. Transcriptomic and biochemical analyses revealed that iMGLs from NHD patients exhibited lysosomal dysfunction, downregulation of cholesterol genes, and reduced lipid droplets compared to controls. Also, NHD iMGLs displayed defective activation and HLA antigen presentation. This defective activation and lipid droplet content were restored by enhancing lysosomal biogenesis through mTOR-dependent and independent pathways. Alteration in lysosomal gene expression, such as decreased expression of genes implicated in lysosomal acidification (ATP6AP2) and chaperone mediated autophagy (LAMP2), together with reduction in lipid droplets were also observed in post-mortem brain tissues from NHD patients, thus closely recapitulating in vivo the phenotype observed in iMGLs in vitro. Our study provides the first cellular and molecular evidence that the TREM2 p.Q33X mutation in microglia leads to defects in lysosomal function and that compounds targeting lysosomal biogenesis restore a number of NHD microglial defects. A better understanding of how microglial lipid metabolism and lysosomal machinery are altered in NHD and how these defects impact microglia activation may provide new insights into mechanisms underlying NHD and other neurodegenerative diseases.

Long non-coding RNA SNHG8 drives stress granule formation in tauopathies.

Tauopathies are a heterogenous group of neurodegenerative disorders characterized by tau aggregation in the brain. In a subset of tauopathies, rare mutations in the MAPT gene, which encodes the tau protein, are sufficient to cause disease; however, the events downstream of MAPT mutations are poorly understood. Here, we investigate the role of long non-coding RNAs (lncRNAs), transcripts >200 nucleotides with low/no coding potential that regulate transcription and translation, and their role in tauopathy. Using stem cell derived neurons from patients carrying a MAPT p.P301L, IVS10+16, or p.R406W mutation, and CRISPR-corrected isogenic controls, we identified transcriptomic changes that occur as a function of the MAPT mutant allele. We identified 15 lncRNAs that were commonly differentially expressed across the three MAPT mutations. The commonly differentially expressed lncRNAs interact with RNA-binding proteins that regulate stress granule formation. Among these lncRNAs, SNHG8 was significantly reduced in a mouse model of tauopathy and in FTLD-tau, progressive supranuclear palsy, and Alzheimer’s disease brains. We show that SNHG8 interacts with tau and stress granule-associated RNA-binding protein TIA1. Overexpression of mutant tau in vitro is sufficient to reduce SNHG8 expression and induce stress granule formation. Rescuing SNHG8 expression leads to reduced stress granule formation and reduced TIA1 levels, suggesting that dysregulation of this non-coding RNA is a causal factor driving stress granule formation via TIA1 in tauopathies.

Inhibiting ACK1-mediated phosphorylation of C-terminal Src kinase counteracts prostate cancer immune checkpoint blockade resistance.

Solid tumours are highly refractory to immune checkpoint blockade (ICB) therapies due to the functional impairment of effector T cells and their inefficient trafficking to tumours. T-cell activation is negatively regulated by C-terminal Src kinase (CSK); however, the exact mechanism remains unknown. Here we show that the conserved oncogenic tyrosine kinase Activated CDC42 kinase 1 (ACK1) is able to phosphorylate CSK at Tyrosine 18 (pY18), which enhances CSK function, constraining T-cell activation. Mice deficient in the Tnk2 gene encoding Ack1, are characterized by diminished CSK Y18-phosphorylation and spontaneous activation of CD8+ and CD4+ T cells, resulting in inhibited growth of transplanted ICB-resistant tumours. Furthermore, ICB treatment of castration-resistant prostate cancer (CRPC) patients results in re-activation of ACK1/pY18-CSK signalling, confirming the involvement of this pathway in ICB insensitivity. An ACK1 small-molecule inhibitor, (R)-9b, recapitulates inhibition of ICB-resistant tumours, which provides evidence for ACK1 enzymatic activity playing a pivotal role in generating ICB resistance. Overall, our study identifies an important mechanism of ICB resistance and holds potential for expanding the scope of ICB therapy to tumours that are currently unresponsive.

Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor (AR) antagonism is exacerbated by HOXB13 in castration-resistant prostate cancers (CRPC). However, it is unclear when and how HOXB13 primes CRPCs for AR antagonism. By mass-spectrometry analysis of CRPC extract, we uncovered a novel lysine 13 (K13) acetylation in HOXB13 mediated by CBP/p300. To determine whether acetylated K13-HOXB13 is a clinical biomarker of CRPC development, we characterized its role in prostate cancer biology. EXPERIMENTAL DESIGN: We identified tumor-specific acK13-HOXB13 signal enriched super enhancer (SE)-regulated targets. We analyzed the effect of loss of HOXB13K13-acetylation on chromatin binding, SE proximal target gene expression, self-renewal, enzalutamide sensitivity, and CRPC tumor growth by employing isogenic parental and HOXB13K13A mutants. Finally, using primary human prostate organoids, we evaluated whether inhibiting an acK13-HOXB13 target, ACK1, with a selective inhibitor (R)-9b is superior to AR antagonists in inhibiting CRPC growth. RESULTS: acK13-HOXB13 promotes increased expression of lineage (AR, HOXB13), prostate cancer diagnostic (FOLH1), CRPC-promoting (ACK1), and angiogenesis (VEGFA, Angiopoietins) genes early in prostate cancer development by establishing tumor-specific SEs. acK13-HOXB13 recruitment to key SE-regulated targets is insensitive to enzalutamide. ACK1 expression is significantly reduced in the loss of function HOXB13K13A mutant CRPCs. Consequently, HOXB13K13A mutants display reduced self-renewal, increased sensitivity to enzalutamide, and impaired xenograft tumor growth. Primary human prostate tumor organoids expressing HOXB13 are significantly resistant to AR antagonists but sensitive to (R)-9b. CONCLUSIONS: In summary, acetylated HOXB13 is a biomarker of clinically significant prostate cancer. Importantly, PSMA-targeting agents and (R)-9b could be new therapeutic modalities to target HOXB13-ACK1 axis regulated prostate cancers.

Chronologically modified androgen receptor in recurrent castration-resistant prostate cancer and its therapeutic targeting.

Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys609 in the zinc finger DNA binding domain of AR (acK609-AR) that not only allowed AR translocation but also galvanized a distinct global transcription program, conferring enzalutamide insensitivity. Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase-mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, (R)-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.

Loss of Long Noncoding RNA NXTAR in Prostate Cancer Augments Androgen Receptor Expression and Enzalutamide Resistance.

Androgen receptor (AR) signaling continues to play a dominant role in all stages of prostate cancer, including castration-resistant prostate cancers (CRPC) that have developed resistance to second generation AR antagonists such as enzalutamide. In this study, we identified a long noncoding RNA (lncRNA), NXTAR (LOC105373241) that is located convergent with the AR gene and is repressed in human prostate tumors and cell lines. NXTAR bound upstream of the AR promoter and promoted EZH2 recruitment, causing significant loss of AR (and AR-V7) expression. Paradoxically, AR bound the NXTAR promoter, and inhibition of AR by the ACK1/TNK2 small molecule inhibitor (R)-9b excluded AR from the NXTAR promoter. The histone acetyltransferase GCN5 bound and deposited H3K14 acetylation marks, enhancing NXTAR expression. Application of an oligonucleotide derived from NXTAR exon 5 (NXTAR-N5) suppressed AR/AR-V7 expression and prostate cancer cell proliferation, indicating the translational relevance of the negative regulation of AR. In addition, pharmacologic restoration of NXTAR using (R)-9b abrogated enzalutamide-resistant prostate xenograft tumor growth. Overall, this study uncovers a positive feedback loop, wherein NXTAR acts as a novel prostate tumor-suppressing lncRNA by inhibiting AR/AR-V7 expression, which in turn upregulates NXTAR levels, compromising enzalutamide-resistant prostate cancer. The restoration of NXTAR could serve as a new therapeutic modality for patients who have acquired resistance to second generation AR antagonists. SIGNIFICANCE: This study identifies NXTAR as a tumor suppressive lncRNA that can epigenetically downregulate AR/AR-V7 expression and provides a therapeutic strategy to reinstate NXTAR expression for treating recurrent CRPC.

Culturable diversity of bacterial endophytes associated with medicinal plants of the Western Ghats, India.

Bacterial endophytes are found in the internal tissues of plants and have intimate associations with their host. However, little is known about the diversity of medicinal plant endophytes (ME) or their capability to produce specialised metabolites that may contribute to therapeutic properties. We isolated 75 bacterial ME from 24 plant species of the Western Ghats, India. Molecular identification by 16S rRNA gene sequencing grouped MEs into 13 bacterial genera, with members of Gammaproteobacteria and Firmicutes being the most abundant. To improve taxonomic identification, 26 selected MEs were genome sequenced and average nucleotide identity (ANI) used to identify them to the species-level. This identified multiple species in the most common genus as Bacillus. Similarly, identity of the Enterobacterales was also distinguished within Enterobacter and Serratia by ANI and core-gene analysis. AntiSMASH identified non-ribosomal peptide synthase, lantipeptide and bacteriocin biosynthetic gene clusters (BGC) as the most common BGCs found in the ME genomes. A total of five of the ME isolates belonging to Bacillus, Serratia and Enterobacter showed antimicrobial activity against the plant pathogen Pectobacterium carotovorum. Using molecular and genomic approaches we have characterised a unique collection of endophytic bacteria from medicinal plants. Their genomes encode multiple specialised metabolite gene clusters and the collection can now be screened for novel bioactive and medicinal metabolites.

AEBP1 down regulation induced cell death pathway depends on PTEN status of glioma cells.

Glioblastoma (GBM) is the most common aggressive form of brain cancer with overall dismal prognosis (10-12 months) despite all current multimodal treatments. Previously we identified adipocyte enhancer binding protein 1 (AEBP1) as a differentially regulated gene in GBM. On probing the role of AEBP1 over expression in glioblastoma, we found that both cellular proliferation and survival were affected upon AEBP1 silencing in glioma cells, resulting in cell death. In the present study we report that the classical caspase pathway components are not activated in cell death induced by AEBP1 down regulation in PTEN-deficient (U87MG and U138MG) cells. PARP-1 was not cleaved but over-activated under AEBP1 down regulation which leads to the synthesis of PAR in the nucleus triggering the release of AIF from the mitochondria. Subsequently, AIF translocates to the nucleus along with MIF causing chromatinolysis. AEBP1 positively regulates PI3KinaseCß by the binding to AE-1 binding element in the PI3KinaseCß promoter. Loss of PI3KinaseCß expression under AEBP1 depleted condition leads to excessive DNA damage and activation of PARP-1. Furthermore, over expression of PIK3CB (in trans) in U138MG cells prevents DNA damage in these AEBP1 depleted cells. On the contrary, AEBP1 down regulation induces caspase-dependent cell death in PTEN-proficient (LN18 and LN229) cells. Ectopic expression of wild-type PTEN in PTEN-deficient U138MG cells results in the activation of canonical caspase and Akt dependent cell death. Collectively, our findings define AEBP1 as a potential oncogenic driver in glioma, with potential implications for therapeutic intervention.

DDX5/p68 associated lncRNA LOC284454 is differentially expressed in human cancers and modulates gene expression.

Long non-coding RNAs (lncRNAs) are emerging as important players in regulation of gene expression in higher eukaryotes. DDX5/p68 RNA helicase protein which is involved in splicing of precursor mRNAs also interacts with lncRNAs like, SRA and mrhl, to modulate gene expression. We performed RIP-seq analysis in HEK293T cells to identify the complete repertoire of DDX5/p68 interacting transcripts including 73 single exonic (SE) lncRNAs. The LOC284454 lncRNA is the second top hit of the list of SE lncRNAs which we have characterized in detail for its molecular features and cellular functions. The RNA is located in the same primary transcript harboring miR-23a∼27a∼24-2 cluster. LOC284454 is a stable, nuclear restricted and chromatin associated lncRNA. The sequence is conserved only in primates among 26 different species and is expressed in multiple human tissues. Expression of LOC284454 is significantly reduced in breast, prostate, uterus and kidney cancer and also in breast cancer cell lines (MCF7 and T47D). Global gene expression studies upon loss and gain of function of LOC284454 revealed perturbation of genes related to cancer-related pathways. Focal adhesion and cell migration pathway genes are downregulated under overexpression condition, and these genes are significantly upregulated in breast cancer cell lines as well as breast cancer tissue samples suggesting a functional role of LOC284454 lncRNA in breast cancer pathobiology.

Long Noncoding RNAs in Cancer and Therapeutic Potential.

Long noncoding RNAs (lncRNAs) are the major elements of the mammalian transcriptome that is emerging as a central player controlling diverse cellular mechanisms. Most of the well-studied lncRNAs so far are found to be crucial in regulating cellular processes such as cell cycle, growth, and apoptosis that ensure homeostasis. Owing to their location and distribution in the genome, lncRNAs influence the transcription of a wide range of proteins directly or indirectly by transcriptional and posttranscriptional alterations, which opens up the "LncRNA-cancer paradigm" in a context-dependent manner, i.e., either oncogenic or tumor suppressive. Thus, this chapter is a consolidation of lncRNA association in exhibiting or suppressing the typical cancer hallmarks such as continuous proliferation, surpassing apoptosis, genomic instability, drug resistance, invasion, and metastasis studied till date. In addition, special focus has been given on the efficient application of lncRNAs as potential targets for therapeutics that holds a great promise for future cancer therapy.

GAS5 long non-coding RNA in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA "sponge". Our aim was to investigate the possible role of the GAS5 in the growth of MPM. METHODS: Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry. RESULTS: GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression. CONCLUSIONS: The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.