Ligand-dependent hedgehog signaling maintains an undifferentiated, malignant osteosarcoma phenotype.

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.

Pharmacological inhibition of TBK1/IKKε blunts immunopathology in a murine model of SARS-CoV-2 infection.

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.

A STAT3 protein complex required for mitochondrial mRNA stability and cancer.

Signal transducer and activator of transcription 3 (STAT3) is a potent transcription factor necessary for life whose activity is corrupted in diverse diseases, including cancer. STAT3 biology was presumed to be entirely dependent on its activity as a transcription factor until the discovery of a mitochondrial pool of STAT3, which is necessary for normal tissue function and tumorigenesis. However, the mechanism of this mitochondrial activity remained elusive. This study uses immunoprecipitation and mass spectrometry to identify a complex containing STAT3, leucine-rich pentatricopeptide repeat containing (LRPPRC), and SRA stem-loop-interacting RNA-binding protein (SLIRP) that is required for the stability of mature mitochondrially encoded mRNAs and transport to the mitochondrial ribosome. Moreover, we show that this complex is enriched in patients with lung adenocarcinoma and that its deletion inhibits the growth of lung cancer in vivo, providing therapeutic opportunities through the specific targeting of the mitochondrial activity of STAT3.

ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma.

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. SIGNIFICANCE: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

Atypical Teratoid Rhabdoid Tumours Are Susceptible to Panobinostat-Mediated Differentiation Therapy.

Atypical teratoid rhabdoid tumour (ATRT) is a rare but highly aggressive undifferentiated solid tumour arising in the central nervous system and predominantly affecting infants and young children. ATRT is exclusively characterized by the inactivation of SMARCB1, a member of the SWI/SNF chromatin remodelling complex that is essential for the regulation of large sets of genes required for normal development and differentiation. Histone deacetylase inhibitors (HDACi) are a promising anticancer therapy and are able to mimic the normal acetylation functions of SMARCB1 in SMARCB1-deficient cells and drive multilineage differentiation in extracranial rhabdoid tumours. However, the potential efficacy of HDACi in ATRT is unknown. Here, we show that human ATRT cells are highly responsive to the HDACi panobinostat and that sustained treatment leads to growth arrest, increased cell senescence, decreased clonogenicity and induction of a neurogenesis gene-expression profile. Furthermore, in an orthotopic ATRT xenograft model, continuous panobinostat treatment inhibits tumour growth, increases survival and drives neuronal differentiation as shown by the expression of the neuronal marker, TUJ1. Collectively, this preclinical study supports the therapeutic potential of panobinostat-mediated differentiation therapy for ATRT.

STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1ß expression.

Detection of microbial components such as lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) on macrophages induces a robust pro-inflammatory response that is dependent on metabolic reprogramming. These innate metabolic changes have been compared to aerobic glycolysis in tumour cells. However, the mechanisms by which TLR4 activation leads to mitochondrial and glycolytic reprogramming are unknown. Here we show that TLR4 activation induces a signalling cascade recruiting TRAF6 and TBK-1, while TBK-1 phosphorylates STAT3 on S727. Using a genetically engineered mouse model incapable of undergoing STAT3 Ser727 phosphorylation, we show ex vivo and in vivo that STAT3 Ser727 phosphorylation is critical for LPS-induced glycolytic reprogramming, production of the central immune response metabolite succinate and inflammatory cytokine production in a model of LPS-induced inflammation. Our study identifies non-canonical STAT3 activation as the crucial signalling intermediary for TLR4-induced glycolysis, macrophage metabolic reprogramming and inflammation.

Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling.

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

A Sexually Dimorphic Role for STAT3 in Sonic Hedgehog Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children and represents 20% of all pediatric central nervous system neoplasms. While advances in surgery, radiation and chemotherapy have improved overall survival, the lifelong sequelae of these treatments represent a major health care burden and have led to ongoing efforts to find effective targeted treatments. There is a well-recognized male bias in medulloblastoma diagnosis, although the mechanism remains unknown. Herein, we identify a sex-specific role for the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3) in the Sonic Hedgehog (SHH) medulloblastoma subgroup. Specific deletion of Stat3 from granule cell precursors in a spontaneous mouse model of SHH medulloblastoma completely protects male, but not female mice from tumor initiation. Segregation of SHH medulloblastoma patients into high and low STAT3 expressing cohorts shows that low STAT3 expression correlates with improved overall survival in male patients. We observe sex specific changes in IL-10 and IL-6 expression and show that IL-6 stimulation enhances SHH-mediated gene transcription in a STAT3-dependent manner. Together these data identify STAT3 as a key molecule underpinning the sexual dimorphism in medulloblastoma.

Deep multi-region whole-genome sequencing reveals heterogeneity and gene-by-environment interactions in treatment-naive, metastatic lung cancer.

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma.

BACKGROUND: Different cell types possess different copies of mtDNA to support their specific requirements for cellular metabolism. Cell-specific mtDNA copy numbers are established through cell-specific mtDNA replication during cell differentiation. However, cancer cells are trapped in a "pseudo-differentiated" state as they fail to expand mtDNA copy number. Global DNA methylation can regulate this process, as induced DNA demethylation promotes differentiation of cancer cells and expansion of mtDNA copy number. RESULTS: To determine the role that mtDNA methylation plays in regulating mtDNA replication during tumorigenesis, we have characterized the patterns of mtDNA methylation using glioblastoma and osteosarcoma tumor models that have different combinations of mtDNA genotypes and copy number against common nuclear genome backgrounds at different stages of tumor progression. To ensure the reliability of the findings, we have applied a robust experimental pipeline including three approaches, namely whole-mtDNA bisulfite-sequencing with mtDNA-genotype-specific analysis, pyrosequencing, and methylated immunoprecipitation against 5mC and 5hmC. We have determined genotype-specific methylation profiles, which were modulated through tumor progression. Moreover, a strong influence from the nuclear genome was also observed on mtDNA methylation patterns using the same mtDNA genotype under different nuclear genomes. Furthermore, the numbers of mtDNA copy in tumor-initiating cells affected mtDNA methylation levels in late-stage tumors. CONCLUSIONS: Our findings highlight the influences that the nuclear and mitochondrial genomes have in setting mtDNA methylation patterns to regulate mtDNA copy number in tumorigenesis. They have important implications for assessing global DNA methylation patterns in tumorigenesis and the availability of mtDNA template for mtDNA replication.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy.

Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53.

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

Cre-dependent DNA recombination activates a STING-dependent innate immune response.

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies.

Low-Dose Histone Deacetylase Inhibitor Treatment Leads to Tumor Growth Arrest and Multi-Lineage Differentiation of Malignant Rhabdoid Tumors.

PURPOSE: Malignant rhabdoid tumor (MRT) and atypical teratoid rhabdoid tumors (ATRT) are rare aggressive undifferentiated tumors primarily affecting the kidney and CNS of infants and young children. MRT are almost exclusively characterized by homozygous deletion or inactivation of the chromatin remodeling gene SMARCB1 SMARCB1 protein loss leads to direct impairment of chromatin remodeling and we have previously reported a role for this protein in histone acetylation. This provided the rationale for investigating the therapeutic potential of histone deactylase inhibitors (HDACi) in MRT. EXPERIMENTAL DESIGN: Whereas previously HDACis have been used at doses and schedules that induce cytotoxicity, in the current studies we have tested the hypothesis, both in vitro and in vivo, that sustained treatment of human MRT with low-dose HDACi can lead to sustained cell growth arrest and differentiation. RESULTS: Sustained low-dose panobinostat (LBH589) treatment led to changes in cellular morphology associated with a marked increase in the induction of neural, renal, and osteoblast differentiation pathways. Genome-wide transcriptional profiling highlighted differential gene expression supporting multilineage differentiation. Using mouse xenograft models, sustained low-dose LBH589 treatment caused tumor growth arrest associated with tumor calcification detectable by X-ray imaging. Histological analysis of LBH589-treated tumors revealed significant regions of ossification, confirmed by Alizarin Red staining. Immunohistochemical analysis showed increased TUJ1 and PAX2 staining suggestive of neuronal and renal differentiation, respectively. CONCLUSIONS: Low-dose HDACi treatment can terminally differentiate MRT tumor cells and reduce their ability to self-renew. The use of low-dose HDACi as a novel therapeutic approach warrants further investigation. Clin Cancer Res; 22(14); 3560-70. ©2016 AACR.

The mouse endometrium contains epithelial, endothelial and leucocyte populations expressing the stem cell marker telomerase reverse transcriptase.

STUDY HYPOTHESIS: The mouse endometrium harbours stem/progenitor cells that express the stem cell marker mouse telomerase reverse transcriptase (mTert). STUDY FINDING: We used a mouse carrying a transgenic reporter for mTert promoter activity to identify rare endometrial populations of epithelial and endothelial cells that express mTert. WHAT IS KNOWN ALREADY: Stem/progenitor cells are hypothesized to be responsible for the remarkable regenerative capacity of the endometrium, but the lack of convenient endometrial stem/progenitor markers in the mouse has hampered investigations into the identity of these cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: A mouse containing a green fluorescent protein (GFP) reporter under the control of the telomerase reverse transcriptase promoter (mTert-GFP) was used to identify potential stem/progenitor cells in the endometrium. mTert promoter activity was determined using fluorescence microscopy and flow cytometry to identify GFP(+) cells. GFP(+) cells were examined for epithelial, stromal, endothelial, leucocyte and proliferation markers and bromodeoxyuridine retention to determine their identity. The endometrium of ovariectomized mice was compared to that of intact cycling mice to establish the role of ovarian hormones in maintaining mTert-expressing cells. MAIN RESULTS AND THE ROLE OF CHANCE: We found that mTert-GFP is expressed by rare luminal and glandular epithelial cells (0.3% of epithelial cells by flow cytometry), rare CD45(-) cells in the stromal compartment (0.028 ± 0.010% of stromal cells by microscopy) and many CD45(+) leucocytes. Ovariectomy resulted in significant decrease of mTert-GFP(+) epithelial cells (P = 0.029 for luminal epithelium; P = 0.034 for glandular epithelium) and a decrease in the percentage of mTert-GFP(+) CD45(+) leucocytes in the stromal compartment (P = 0.015). However, CD45(-) mTert-GFP(+) cells in the stromal compartment were maintained in ovariectomized mice. This population is enriched for cells bearing the endothelial marker CD31 (10.3% of CD90(-) CD45(-) and 97.8% CD90(+) CD45(-) by flow cytometry). CD45(-) mTert-GFP(+) cells also immunostained for the endothelial marker von Willebrand factor. These results suggest that the endometrial epithelium and vasculature are foci of stem/progenitor activity and provide a system to investigate molecular mechanisms involved in endometrial regeneration and repair. LIMITATIONS, REASONS FOR CAUTION: The stem/progenitor activity of endometrial mTert-GFP(+) cells needs to be experimentally verified. WIDER IMPLICATIONS OF THE FINDINGS: The identification and characterization of mTert-expressing progenitor cells in the mouse will facilitate the identification of equivalent populations in the human endometrium that are likely to be involved in endometrial function, fertility and disease. LARGE-SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was funded by National Health and Medical Research Council (NHMRC) of Australia grants (1085435, C.E.G., J.A.D.), 1021127 (C.E.G.), NHMRC Senior Research Fellowship (1042298, C.E.G.), the Victorian Infrastructure Support Program, U.S. National Institutes of Health grant R01 DK084056 (D.T.B.) and the Harvard Stem Cell Institute (D.T.B.). The authors have no conflicts of interest to declare.

Sustained Low-Dose Treatment with the Histone Deacetylase Inhibitor LBH589 Induces Terminal Differentiation of Osteosarcoma Cells.

Histone deacetylase inhibitors (HDACi) were identified nearly four decades ago based on their ability to induce cellular differentiation. However, the clinical development of these compounds as cancer therapies has focused on their capacity to induce apoptosis in hematologic and lymphoid malignancies, often in combination with conventional cytotoxic agents. In many cases, HDACi doses necessary to induce these effects result in significant toxicity. Since osteosarcoma cells express markers of terminal osteoblast differentiation in response to DNA methyltransferase inhibitors, we reasoned that the epigenetic reprogramming capacity of HDACi might be exploited for therapeutic benefit. Here, we show that continuous exposure of osteosarcoma cells to low concentrations of HDACi LBH589 (Panobinostat) over a three-week period induces terminal osteoblast differentiation and irreversible senescence without inducing cell death. Remarkably, transcriptional profiling revealed that HDACi therapy initiated gene signatures characteristic of chondrocyte and adipocyte lineages in addition to marked upregulation of mature osteoblast markers. In a mouse xenograft model, continuous low dose treatment with LBH589 induced a sustained cytostatic response accompanied by induction of mature osteoblast gene expression. These data suggest that the remarkable capacity of osteosarcoma cells to differentiate in response to HDACi therapy could be exploited for therapeutic benefit without inducing systemic toxicity.