Preventing recurrence in Sonic Hedgehog Subgroup Medulloblastoma using the OLIG2 inhibitor CT-179.

Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.

Publisher Correction: A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Q-Cell Glioblastoma Resource: Proteomics Analysis Reveals Unique Cell-States are Maintained in 3D Culture.

Glioblastoma (GBM) is a treatment-refractory central nervous system (CNS) tumour, and better therapies to treat this aggressive disease are urgently needed. Primary GBM models that represent the true disease state are essential to better understand disease biology and for accurate preclinical therapy assessment. We have previously presented a comprehensive transcriptome characterisation of a panel (n = 12) of primary GBM models (Q-Cell). We have now generated a systematic, quantitative, and deep proteome abundance atlas of the Q-Cell models grown in 3D culture, representing 6167 human proteins. A recent study has highlighted the degree of functional heterogeneity that coexists within individual GBM tumours, describing four cellular states (MES-like, NPC-like, OPC-like and AC-like). We performed comparative proteomic analysis, confirming a good representation of each of the four cell-states across the 13 models examined. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified upregulation of a number of GBM-associated cancer pathway proteins. Bioinformatics analysis, using the OncoKB database, identified a number of functional actionable targets that were either uniquely or ubiquitously expressed across the panel. This study provides an in-depth proteomic analysis of the GBM Q-Cell resource, which should prove a valuable functional dataset for future biological and preclinical investigations.

Simultaneous targeting of DNA replication and homologous recombination in glioblastoma with a polyether ionophore.

BACKGROUND: Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. METHODS: In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. RESULTS: Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell-like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. CONCLUSION: Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

The dystroglycan receptor maintains glioma stem cells in the vascular niche.

Glioblastomas (GBMs) are malignant central nervous system (CNS) neoplasms with a very poor prognosis. They display cellular hierarchies containing self-renewing tumourigenic glioma stem cells (GSCs) in a complex heterogeneous microenvironment. One proposed GSC niche is the extracellular matrix (ECM)-rich perivascular bed of the tumour. Here, we report that the ECM binding dystroglycan (DG) receptor is expressed and functionally glycosylated on GSCs residing in the perivascular niche. Glycosylated αDG is highly expressed and functional on the most aggressive mesenchymal-like (MES-like) GBM tumour compartment. Furthermore, we found that DG acts to maintain an MES-like state via tight control of MAPK activation. Antibody-based blockade of αDG induces robust ERK-mediated differentiation leading to reduced GSC potential. DG was shown to be required for tumour initiation in MES-like GBM, with constitutive loss significantly delaying or preventing tumourigenic potential in-vivo. These findings reveal a central role of the DG receptor, not only as a structural element, but also as a critical factor promoting MES-like GBM and the maintenance of GSCs residing in the perivascular niche.

A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma.

Low-passage, serum-free cell lines cultured from patient tumour tissue are the gold-standard for preclinical studies and cellular investigations of glioblastoma (GBM) biology, yet entrenched, poorly-representative cell line models are still widely used, compromising the significance of much GBM research. We submit that greater adoption of these critical resources will be promoted by the provision of a suitably-sized, meaningfully-described reference collection along with appropriate tools for working with them. Consequently, we present a curated panel of 12 readily-usable, genetically-diverse, tumourigenic, patient-derived, low-passage, serum-free cell lines representing the spectrum of molecular subtypes of IDH-wildtype GBM along with their detailed phenotypic characterisation plus a bespoke set of lentiviral plasmids for bioluminescent/fluorescent labelling, gene expression and CRISPR/Cas9-mediated gene inactivation. The cell lines and all accompanying data are readily-accessible via a single website, Q-Cell (qimrberghofer.edu.au/q-cell/) and all plasmids are available from Addgene. These resources should prove valuable to investigators seeking readily-usable, well-characterised, clinically-relevant, gold-standard models of GBM.

Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma.

Glioblastomas are the most common and lethal neoplasms of the central nervous system. Neighbouring glioma cells maintain extreme degrees of genetic and phenotypic variation that form intratumoural heterogeneity. This genetic diversity allows the most adaptive tumour clones to develop treatment resistance, ultimately leading to disease recurrence. We aimed to model this phenomenon and test the effectiveness of several targeted therapeutic interventions to overcome therapy resistance. Heterogeneous tumour masses were first deconstructed into single tumour cells, which were expanded independently as single-cell clones. Single nucleotide polymorphism arrays, whole-genome and RNA sequencing, and CpG methylation analysis validated the unique molecular profile of each tumour clone, which displayed distinct pathologic features, including cell morphology, growth rate, and resistance to temozolomide and ionizing radiation. We also identified variable sensitivities to AURK, CDK, and EGFR inhibitors which were consistent with the heterogeneous molecular alterations that each clone harboured. These targeted therapies effectively eliminated the temozolomide- and/or irradiation-resistant clones and also parental polyclonal cells. Our findings indicate that polyclonal tumours create a dynamic environment that consists of diverse tumour elements and treatment responses. Designing targeted therapies based on a range of molecular profiles can be a more effective strategy to eradicate treatment resistance, recurrence, and metastasis.

Regulation of liver metabolism by the endosomal GTPase Rab5.

The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.

Uncovering global SUMOylation signaling networks in a site-specific manner.

SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution MS, we have studied global SUMOylation in human cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites in >1,600 proteins. To our knowledge, this is the first time that >1,000 SUMOylation sites have been identified under standard growth conditions. We quantitatively studied SUMOylation dynamics in response to SUMO protease inhibition, proteasome inhibition and heat shock. Many SUMOylated lysines have previously been reported to be ubiquitinated, acetylated or methylated, thus indicating cross-talk between SUMO and other post-translational modifications. We identified 70 phosphorylation and four acetylation events in proximity to SUMOylation sites, and we provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, precursor-mRNA splicing and ribosome assembly.

Ultradeep human phosphoproteome reveals a distinct regulatory nature of Tyr and Ser/Thr-based signaling.

Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.

Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-ß.

Transforming growth factor-ß (TGF-ß) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-ß. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-ß. We identified several proteins known to be involved in TGF-ß-induced cellular processes, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-ß signaling. Although many TGF-ß responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-ß receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMAD-independent TGF-ß signaling. Combined analysis of changes in protein abundance and phosphorylation and knowledge of protein interactions and transcriptional regulation provided a comprehensive representation of the dynamic signaling events underlying TGF-ß-induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-ß, early signaling is a mixture of both pro- and antiproliferative signals, whereas later signaling primarily inhibits proliferation.

Super-SILAC allows classification of diffuse large B-cell lymphoma subtypes by their protein expression profiles.

Correct classification of cancer patients into subtypes is a prerequisite for acute diagnosis and effective treatment. Currently this classification relies mainly on histological assessment, but gene expression analysis by microarrays has shown great promise. Here we show that high accuracy, quantitative proteomics can robustly segregate cancer subtypes directly at the level of expressed proteins. We investigated two histologically indistinguishable subtypes of diffuse large B-cell lymphoma (DLBCL): activated B-cell-like (ABC) and germinal-center B-cell-like (GCB) subtypes, by first developing a general lymphoma stable isotope labeling with amino acids in cell culture (SILAC) mix from heavy stable isotope-labeled cell lines. This super-SILAC mix was combined with cell lysates from five ABC-DLBCL and five GCB-DLBCL cell lines. Shotgun proteomic analysis on a linear ion trap Orbitrap mass spectrometer with high mass accuracy at the MS and MS/MS levels yielded a proteome of more than 7,500 identified proteins. High accuracy of quantification allowed robust separation of subtypes by principal component analysis. The main contributors to the classification included proteins known to be differentially expressed between the subtypes such as the transcription factors IRF4 and SPI1/PU.1, cell surface markers CD44 and CD27, as well as novel candidates. We extracted a signature of 55 proteins that segregated subtypes and contained proteins connected to functional differences between the ABC and GCB-DLBCL subtypes, including many NF-κB-regulated genes. Shortening the analysis time to single-shot analysis combined with use of the new linear quadrupole Orbitrap analyzer (Q Exactive) also clearly differentiated between the subtypes. These results show that high resolution shotgun proteomics combined with super-SILAC-based quantification is a promising new technology for tumor characterization and classification.

Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation.

Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has proven to be particularly successful ("high-low" strategy). Here we investigate if the improved sensitivity of higher energy collisional dissociation (HCD) on an LTQ-Orbitrap Velos instrument allows a "high-high" strategy. A high resolution MS scan was followed by up to 10 HCD MS/MS scans, and we achieved cycle times of about 3 s making the method compatible with chromatographic time scales. Fragment mass accuracy increased about 50-fold compared to the "high-low" strategy. Unexpectedly, the HCD approach mapped up to 16,000 total phosphorylation sites in one day's measuring time--the same or better than the standard high-low strategy. Reducing the target values from a standard of 30,000 to 5000 ions did not severely affect identification rates but did decrease identification and localization scores for phosphorylation sites. We conclude that HCD in the new configuration is now a viable method for large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD).

PknH, a transmembrane Hank's type serine/threonine kinase from Mycobacterium tuberculosis is differentially expressed under stress conditions.

Serine/threonine protein kinases (STPKs) represent a burgeoning concept in prokaryotic signaling and have been implicated in a range of control mechanisms. This paper describes the enzymatic and molecular characterization of PknH, a mycobacterial STPK. After cloning and expression as a Glutathione-S-transferase fusion protein in E. coli, PknH was found to phosphorylate itself and exogenous substrates like myelin basic protein and histone. The kinase activity of PknH was inhibited by the kinase inhibitors staurosporine and H-7. The results confirmed that PknH is a transmembrane protein and is restricted to members of the Mycobacterium tuberculosis complex. In addition, transcriptional analysis of pknH in M. tuberculosis under various stress conditions revealed that exposure to low pH and heat shock decreased the level of pknH transcription significantly. This is the first report describing differential expression of a mycobacterial kinase in response to stress conditions which can indicate its ability to regulate cellular events promoting bacterial adaptation to environmental change.