Publications by authors named "Terrance G Johns"

74 Publications

Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells.

Cell Death Discov 2021 Apr 16;7(1):81. Epub 2021 Apr 16.

Charles Perkins Centre and School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2006, Australia.

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.
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http://dx.doi.org/10.1038/s41420-021-00456-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052442PMC
April 2021

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies.

J Vis Exp 2020 07 28(161). Epub 2020 Jul 28.

Telethon Kids Institute, University of Western Australia;

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery. Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.
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http://dx.doi.org/10.3791/60882DOI Listing
July 2020

Lower Tubulin Expression in Glioblastoma Stem Cells Attenuates Efficacy of Microtubule-Targeting Agents.

ACS Pharmacol Transl Sci 2019 Dec 30;2(6):402-413. Epub 2019 Jul 30.

Faculty of Medicine and Health, Charles Perkins Centre, The University of Sydney, John Hopkins Drive, Sydney, New South Wales 2006, Australia.

Sensitivity to microtubule-targeting agents (MTAs) varies among cancers and predicting the response of individual cancer patients to MTAs remains challenging. As microtubules possess vast molecular heterogeneity generated by tubulin isotypes and their post-translational modifications, we questioned whether this heterogeneity can impact MTA sensitivity. We investigated microtubule heterogeneity in 15 glioblastoma cell lines and measured sensitivity of orthogonal MTAs using a per-division growth rate inhibition method that corrects for the confounding effects of variable cell proliferation rates. We found that the tubulin profile is unique for each glioblastoma cell line and that the total α- and β-tubulin levels impact on MTA sensitivity. The baseline levels of α- and β-tubulin were up to 20% lower in cells that were not effectively killed by MTAs. We report that lower α/β-tubulin expression is associated with lack of cell differentiation and increased expression of stemness markers. The dedifferentiated stem-like cells with low α/β-tubulin levels survive MTAs treatment via reversible nonmutational dormancy. Our findings provide novel insights into the relationships between microtubules and MTAs and lay a foundation for better understanding of the sensitivity of cancer cells to MTAs.
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http://dx.doi.org/10.1021/acsptsci.9b00045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089104PMC
December 2019

MK2 Inhibition Induces p53-Dependent Senescence in Glioblastoma Cells.

Cancers (Basel) 2020 Mar 11;12(3). Epub 2020 Mar 11.

School of Medical Sciences, Charles Perkins Centre and Faculty of Medicine and Health, The University of Sydney, NSW 2006, Australia.

MAPK-activated protein kinase 2 (MK2) has diverse roles in cancer. In response to chemotherapy, MK2 inhibition is synthetically lethal to p53-deficiency. While deletion is rare in glioblastomas, these tumors often carry mutations. Here, we show that MK2 inhibition strongly attenuated glioblastoma cell proliferation through p53 stabilization and senescence. The senescence-inducing efficacy of MK2 inhibition was particularly strong when cells were co-treated with the standard-of-care temozolomide. However, MK2 inhibition also increased the stability of p53 mutants and enhanced the proliferation of p53-mutant stem cells. These observations reveal that in response to DNA damaging chemotherapy, targeting MK2 in p53-mutated cells produces a phenotype that is distinct from the p53-deficient phenotype. Thus, MK2 represents a novel drug target in 70% glioblastomas harboring intact gene. However, targeting MK2 in tumors with mutations may accelerate disease progression. These findings are highly relevant since mutations occur in over 50% of all cancers.
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http://dx.doi.org/10.3390/cancers12030654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139970PMC
March 2020

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

Cells 2020 01 21;9(2). Epub 2020 Jan 21.

Cell and Molecular Biology Department, QIMR Berghofer Medical Research Institute, Sid Faithfull Brain Cancer Laboratory, Brisbane, QLD 4006, Australia.

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.
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http://dx.doi.org/10.3390/cells9020267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072469PMC
January 2020

Whole genome and biomarker analysis of patients with recurrent glioblastoma on bevacizumab: A subset analysis of the CABARET trial.

J Clin Neurosci 2019 Dec 30;70:157-163. Epub 2019 Sep 30.

Prince of Wales Clinical School, Cure Brain Cancer Biomarkers and Translational Research Group, University of New South Wales, Sydney, NSW, Australia.

The CABARET trial (ACTRN12610000915055) reported no difference in overall survival (OS) between patients with recurrent glioblastoma (GBM) randomized to either bevacizumab monotherapy or bevacizumab plus carboplatin. However, a subset of patients showed durable responses and prolonged survival, with recorded survival times of over 30 months in five of 122 patients (4%). Patient selection for bevacizumab therapy would be enhanced if a predictive biomarker of response or survival could be identified; this biomarker sub-study attempted to identify novel biomarkers. Patients who opted to participate in this sub-study and who had adequate biospecimens for analysis (n = 54) were retrospectively evaluated for the expression of a series of tumor proteins. Immunohistochemistry (IHC) was used to measure the expression of 19 proteins previously implicated in cancer treatment response to bevacizumab. MGMT promoter methylation was also assessed. Tumor DNA from five patients with outlying survival duration ('poor' and 'exceptional' survivors) was subjected to whole genome sequencing (WGS). No single protein expression level, including VEGF-A, predicted OS in the cohort. WGS of poor and exceptional survivors identified a gain in Chromosome 19 that was exclusive to the exceptional survivors. Validation of this finding requires examination of a larger independent cohort.
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http://dx.doi.org/10.1016/j.jocn.2019.08.044DOI Listing
December 2019

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

Neuro Oncol 2020 02;22(2):216-228

Cell and Molecular Biology Department, QIMR Berghofer MRI, Queensland, Australia.

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.
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http://dx.doi.org/10.1093/neuonc/noz159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442340PMC
February 2020

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

Acta Neuropathol 2019 12 28;138(6):1033-1052. Epub 2019 Aug 28.

Department of Cell and Molecular Biology, Sid Faithfull Brain Cancer Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, 4006, Australia.

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.
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http://dx.doi.org/10.1007/s00401-019-02069-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851226PMC
December 2019

Most clinical anti-EGFR antibodies do not neutralize both wtEGFR and EGFRvIII activation in glioma.

Neuro Oncol 2019 08;21(8):1016-1027

Brain Cancer Discovery Collaborative, New South Wales, Australia.

Background: Although epidermal growth factor receptor (EGFR) and its truncated, autoactive mutant EGFR variant (v)III are bona fide drivers of tumorigenesis in some gliomas, therapeutic antibodies developed to neutralize this axis have not improved patient survival in a limited number of trials. Previous studies using cells transduced to exogenously express EGFRvIII may have compromised mechanistic studies of anti-EGFR therapeutics. Therefore, we re-assessed the activity of clinical EGFR antibodies in patient-derived gliomaspheres that endogenously express EGFRvIII.

Methods: The antitumor efficacy of antibodies was assessed using in vitro proliferation assays and intracranial orthografts. Receptor activation status, antibody engagement, oncogenic signaling, and mechanism of action after antibody treatment were analyzed by immunoprecipitation and western blotting. Tracking of antibody receptor complexes was conducted using immunofluorescence.

Results: The EGFR domain III-targeting antibodies cetuximab, necitumumab, nimotuzumab, and matuzumab did not neutralize EGFRvIII activation. Chimeric monoclonal antibody 806 (ch806) neutralized EGFRvIII, but not wild-type (wt)EGFR activation. Panitumumab was the only antibody that neutralized both EGFRvIII and wtEGFR, leading to reduction of p-S6 signaling and superior in vitro and in vivo antitumor activity. Mechanistically, panitumumab induced recycling of receptor but not degradation as previously described. Panitumumab, via its unique avidity, stably cross-linked EGFRvIII to prevent its activation, while ch806 induced a marked reduction in the active EGFRvIII disulphide-bonded dimer.

Conclusions: We discovered a previously unknown major resistance mechanism in glioma in that most EGFR domain III-targeting antibodies do not neutralize EGFRvIII. The superior in vitro and in vivo antitumor activity of panitumumab supports further clinical testing of this antibody against EGFRvIII-stratified glioma.
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http://dx.doi.org/10.1093/neuonc/noz073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682217PMC
August 2019

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

Sci Rep 2019 03 20;9(1):4902. Epub 2019 Mar 20.

QIMR Berghofer Medical Research Institute, Brisbane, Australia.

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.
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http://dx.doi.org/10.1038/s41598-019-41277-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427001PMC
March 2019

Activation of ERBB4 in Glioblastoma Can Contribute to Increased Tumorigenicity and Influence Therapeutic Response.

Cancers (Basel) 2018 Jul 25;10(8). Epub 2018 Jul 25.

Oncogenic Signalling Group, Hudson Institute of Medical Research, 21⁻37 Wright Street, Clayton, VIC 3168, Australia.

Glioblastoma (GBM) is often resistant to conventional and targeted therapeutics. ErbB2 Receptor Tyrosine Kinase 4 (ERBB4) is expressed throughout normal brain and is an oncogene in several pediatric brain cancers; therefore, we investigated ERBB4 as a prognostic marker and therapeutic target in GBM. Using RT-qPCR, we quantified mRNA encoding total ERBB4 and known ERBB4 variants in GBM and non-neoplastic normal brain (NNB) samples. Using immunohistochemistry, we characterized the localization of total and phosphorylated ERBB4 (p-ERBB4) and EGFR protein in archived GBM samples and assessed their association with patient survival. Furthermore, we evaluated the effect of ERBB4 phosphorylation on angiogenesis and tumorigenicity in GBM xenograft models. Total mRNA was significantly lower in GBM than NNB samples, with the juxtamembrane JM-a and cytoplasmic CYT-2 variants predominating. ERBB4 protein was ubiquitously expressed in GBM but was not associated with patient survival. However, high p-ERBB4 in 11% of archived GBM samples, independent of p-EGFR, was associated with shorter patient survival (12.0 ± 3.2 months) than was no p-ERBB4 (22.5 ± 9.5 months). Increased ERBB4 activation was also associated with increased proliferation, angiogenesis, tumorigenicity and reduced sensitivity to anti-EGFR treatment in xenograft models. Despite low mRNA in GBM, the functional effects of increased ERBB4 activation identify ERBB4 as a potential prognostic and therapeutic target.
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http://dx.doi.org/10.3390/cancers10080243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116191PMC
July 2018

Changes in cell morphology guide identification of tubulin as the off-target for protein kinase inhibitors.

Pharmacol Res 2018 08 23;134:166-178. Epub 2018 Jun 23.

The University of Sydney, Faculty of Medicine and Health, Charles Perkins Centre, NSW 2006, Australia. Electronic address:

In the field of kinase inhibitors for applications in cancer research, tubulin is emerging as a targeted cellular protein that can significantly contribute to their activities. However, investigation of kinase inhibitors beyond the kinome is an area often neglected. Herein, we describe the results of pharmacological studies using drugs targeting kinases, tubulin or both. A key finding is that if cells are treated with a kinase inhibitor unintentionally targeting tubulin, their characteristic shape will diminish within a short timeframe. These changes in cell morphology are not seen when cells are treated with bona fide kinase inhibitors that do not directly target tubulin. Thus, early changes in cell morphology upon treatments are a strong indication that the inhibitor is directly targeting tubulin. Recognizing tubulin as a target of kinase inhibitors will build confidence in the future mechanistic studies using kinase inhibitors.
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http://dx.doi.org/10.1016/j.phrs.2018.06.023DOI Listing
August 2018

Gefitinib and Methotrexate to Treat Ectopic Pregnancies with a Pre-Treatment Serum hCG 1000-10,000 IU/L: Phase II Open Label, Single Arm Multi-Centre Trial.

EBioMedicine 2018 Jul 22;33:276-281. Epub 2018 Jun 22.

MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom.

Background: Ectopic pregnancies are a leading cause of maternal mortality. Most are treated surgically. We evaluated the efficacy and safety of combining oral gefitinib (epidermal growth factor receptor inhibitor) with methotrexate to treat larger ectopic pregnancies.

Methods: We performed a phase II, single arm, open label study across four hospitals in Edinburgh and Melbourne. We recruited women with a stable tubal ectopic pregnancy and a pre-treatment serum hCG between 1000 and 10,000 IU/L. We administered intramuscular methotrexate (50 mg/m) once, and oral gefitinib (250 mg) for seven days. The primary outcome was the percentage successfully treated without needing surgery. To show the treatment is at least 70% effective, 28 participants were required, and 24 or more successfully treated without surgery. Secondary outcomes were safety, tolerability, and time to resolution. This study is registered (ACTRN12611001056987).

Findings: 30 participants with stable tubal ectopic pregnancies were recruited but two withdrew, leaving 28 participants. The median (± range) pre-treatment serum hCG was 2039 (1031-8575) IU/L and nine had pre-treatment hCGs levels >3000 IU/L. The treatment successfully resolved 86% (24/28) cases with a median (±range) time to resolution of 32 (18-67) days. The treatment caused transient rash and diarrhoea, but no serious adverse events.

Interpretation: Combination gefitinib and methotrexate is at least 70% effective in resolving ectopic pregnancies with a pre-treatment serum hCG 1000-10,000 IU/L. This may be a new way to treat most stable ectopic pregnancies, but needs to be validated via a randomised clinical trial.
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http://dx.doi.org/10.1016/j.ebiom.2018.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085507PMC
July 2018

Differential Sensitivity of Human Hepatocellular Carcinoma Xenografts to an IGF-II Neutralizing Antibody May Involve Activated STAT3.

Transl Oncol 2018 Aug 22;11(4):971-978. Epub 2018 Jun 22.

CSIRO Manufacturing, Parkville, VIC 3052, Australia. Electronic address:

Hepatocellular carcinoma (HCC) is highly refractory to current therapeutics used in the clinic. DX-2647, a recombinant human antibody, potently neutralizes the action of insulin-like growth factor-II (IGF-II), a ligand for three cell-surface receptors (IGF-IR, insulin receptor A and B isoforms, and the cation-independent mannose-6-phosphate receptor) which is overexpressed in primary human HCC. DX-2647 impaired the growth of tumor xenografts of the HCC cell line, Hep3B; however, xenografts of the HCC cell line, HepG2, were largely unresponsive to DX-2647 treatment. Analysis of a number of aspects of the IGF signaling axis in both cell lines did not reveal any significant differences between the two. However, while DX-2647 abolished phospho (p)-IGF-IR, p-IR and p-AKT signaling in both cell lines, HepG2 showed high levels of p-STAT3, which was unaffected by DX-2647 treatment and was absent from the Hep3B cell line. The driver of p-STAT3 was found to be a secreted cytokine, and treatment of HepG2 cells with a pan- JAK kinase inhibitor resulted in a loss of p-STAT3. These findings implicate the activation of STAT3 as one pathway that may mediate resistance to IGF-II-targeted therapy in HCC.
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http://dx.doi.org/10.1016/j.tranon.2018.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020079PMC
August 2018

PI3K activation in neural stem cells drives tumorigenesis which can be ameliorated by targeting the cAMP response element binding protein.

Neuro Oncol 2018 09;20(10):1344-1355

Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.

Background: Hyperactivation of phosphoinositide 3-kinase (PI3K) signaling is common in cancers, but the precise role of the pathway in glioma biology remains to be determined. Some understanding of PI3K signaling mechanisms in brain cancer comes from studies on neural stem/progenitor cells (NSPCs), where signals transmitted via the PI3K pathway cooperate with other intracellular pathways and downstream transcription factors to regulate critical cell functions.

Methods: To investigate the role of the PI3K pathway in glioma initiation and development, we generated a mouse model targeting the inducible expression of a PIK3CAH1047A oncogenic mutant and deletion of the PI3K negative regulator, phosphatase and tensin homolog (PTEN), to NSPCs.

Results: Expression of a Pik3caH1047A was sufficient to generate tumors with oligodendroglial features, but simultaneous loss of PTEN was required for the development of invasive, high-grade glioma. Pik3caH1047A-PTEN mutant NSPCs exhibited enhanced neurosphere formation which correlated with increased Wnt signaling, while loss of cAMP response element binding protein (CREB) in Pik3caH1047A-Pten mutant tumors led to longer symptom-free survival in mice.

Conclusion: Taken together, our findings present a novel mouse model for glioma demonstrating that the PI3K pathway is important for initiation of tumorigenesis and that disruption of downstream CREB signaling attenuates tumor expansion.
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http://dx.doi.org/10.1093/neuonc/noy068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140786PMC
September 2018

A Pre-Clinical Assessment of the Pan-ERBB Inhibitor Dacomitinib in Pediatric and Adult Brain Tumors.

Neoplasia 2018 05 22;20(5):432-442. Epub 2018 Mar 22.

Brain Tumor Research Program, Telethon Kids Cancer Centre, Telethon Kids Institute, University of Western Australia, Perth, Western Australia, Australia; Department of Pediatric Oncology and Hematology, Princess Margaret Hospital for Children, Subiaco, Western Australia, Australia; Division of Paediatrics, School of Medicine, University of Western Australia, Crawley, Western Australia, Australia. Electronic address:

Glioblastoma in adults, and medulloblastoma and pineoblastoma that mainly affect children, are aggressive brain tumors. The survival for patients with glioblastoma remains dismal. While the cure rate for medulloblastoma exceeds 70%, this figure has stagnated over the past few decades and survivors still contend with significant long-term debilitating side effects. The prognosis for pineoblastoma is age-dependent, with little chance of a cure for children younger than three years. More effective molecularly targeted strategies are urgently required to treat these cancers. Hyper-activation of epidermal growth factor receptor (EGFR) signaling is characteristic of several different classes of human cancers, including a subset of glioblastoma and medulloblastoma. This has provided the impetus for the development of a suite of EGFR pathway blockers, including second generation irreversible inhibitors, such as dacomitinib. We have developed a comprehensive drug evaluation pipeline, including in vitro interaction analyses and orthotopic xenograft mouse models, to address the efficacy of drugs for brain tumor treatment, enabling the exclusion of potentially ineffective treatments and prioritization of truly beneficial novel treatments for clinical trial. We used this system to examine the effects of dacomitinib as a single agent, or in combination with conventional chemotherapeutics, on the growth of human adult and pediatric brain tumor cell lines. Dacomitinib inhibited EGFR or EGFRvIII activity in vitro in all three tumor types tested, and as a single agent induced a modest increase in survival time for mice bearing glioblastoma, which accurately predicted human clinical trial data. For pediatric medulloblastoma, dacomitinib blocked EGFR/HER signalling in orthotopic xenografts and extended median survival as a single agent, however was antagonistic when used in combination with standard frontline medulloblastoma chemotherapies. The findings caution against the use of dacomitinib for pediatric brain tumor clinical trials.
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http://dx.doi.org/10.1016/j.neo.2018.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916087PMC
May 2018

Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma.

Cancer Immunol Immunother 2017 Sep 13;66(9):1217-1228. Epub 2017 May 13.

Department of Medicine/Cardiology (Austin Health, Heidelberg), University of Melbourne, Lance Townsend Building, Level 10, Austin Campus, Studley Road, Heidelberg, VIC, 3084, Australia.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.
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http://dx.doi.org/10.1007/s00262-017-2013-zDOI Listing
September 2017

Development and Biological Evaluation of a Photoactivatable Small Molecule Microtubule-Targeting Agent.

ACS Med Chem Lett 2017 Apr 15;8(4):395-400. Epub 2017 Mar 15.

School of Medical Sciences and Charles Perkins Centre, The University of Sydney, Sydney, NSW 2006, Australia.

Photoremovable protecting groups added to bioactive molecules provide spatial and temporal control of the biological effects. We present synthesis and characterization of the first photoactivatable small-molecule tubulin inhibitor. By blocking the pharmacophoric OH group on compound with photoremovable 4,5-dimethoxy-2-nitrobenzyl moiety we developed the photocaged prodrug that had no effect in biological assays. Short UV light exposure of the derivative or UV-irradiation of cells treated with resulted in fast and potent inhibition of tubulin polymerization, attenuation of cell viability, and apoptotic cell death, implicating release of the parent active compound. This study validates for the first time the photoactivatable prodrug concept in the field of small molecule tubulin inhibitors. The caged derivative represents a novel tool in antitubulin approaches.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392771PMC
April 2017

Structural Optimization and Pharmacological Evaluation of Inhibitors Targeting Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases (DYRK) and CDC-like kinases (CLK) in Glioblastoma.

J Med Chem 2017 03 28;60(5):2052-2070. Epub 2017 Feb 28.

Oncogenic Signaling Laboratory, Centre for Cancer Research, Hudson Institute of Medical Research , 27 Wright Street, Clayton, Victoria 3168, Australia.

The DYRK family contains kinases that are up-regulated in malignancy and control several cancer hallmarks. To assess the anticancer potential of inhibitors targeting DYRK kinases, we developed a series of novel DYRK inhibitors based on the 7-azaindole scaffold. All compounds were tested for their ability to inhibit DYRK1A, DYRK1B, DYRK2, and the structurally related CLK1. The library was screened for anticancer efficacy in established and stem cell-like glioblastoma cell lines. The most potent inhibitors (IC ≤ 50 nM) significantly decreased viability, clonogenic survival, migration, and invasion of glioblastoma cells. Target engagement was confirmed with genetic knockdown and the cellular thermal shift assay. We demonstrate that DYRK1A's thermal stability in cells is increased upon compound treatment, confirming binding in cells. In summary, we present synthesis, structure-activity relationship, and efficacy in glioblastoma-relevant models for a library of novel 7-azaindoles.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01840DOI Listing
March 2017

Axitinib Has Antiangiogenic and Antitumorigenic Activity in Myxoid Liposarcoma.

Sarcoma 2016 16;2016:3484673. Epub 2016 Oct 16.

Centre for Cancer Research, Hudson Institute for Medical Research, 27-31 Wright Street, Clayton, VIC 3168, Australia; Monash University, Wellington Road, Clayton, VIC 3168, Australia.

Myxoid liposarcoma is a rare form of soft-tissue sarcoma. Although most patients initially respond well to treatment, approximately 21% relapse, highlighting the need for alternative treatments. To identify novel treatment regimens and gain a better understanding of myxoid liposarcoma tumor biology, we screened various candidate and approved targeted therapeutics and chemotherapeutics against myxoid liposarcoma cell lines. Therapeutics that target angiogenesis showed antitumor activity. The small molecule inhibitor axitinib, which targets angiogenesis by inhibiting the VEGFR and PDGFR families and c-Kit, inhibited cell cycle progression and induced apoptosis , as well as having significant antitumor activity against MLS 1765 myxoid liposarcoma xenografts in mice. Axitinib also displayed synergistic antitumor activity when combined with the potassium channel ionophore salinomycin or the BH3 mimetic ABT-737. Another angiogenesis-targeting therapeutic, 4EGI-1, which targets the oncoprotein eIF4E, significantly decreased angiogenic ligand expression by myxoid liposarcoma cells and reduced tumor cell growth. To verify this oncogenic addiction to angiogenic pathways, we utilized VEGFR-derived ligand traps and found that autocrine VEGFR signaling was crucial to myxoid liposarcoma cell survival. Overall, these findings suggest that autocrine angiogenic signaling through the VEGFR family is critical to myxoid liposarcoma cell survival and that further study of axitinib as a potential anticancer therapy is warranted.
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http://dx.doi.org/10.1155/2016/3484673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5086398PMC
October 2016

EGFRvIII: the promiscuous mutation.

Cell Death Discov 2016 4;2:16049. Epub 2016 Jul 4.

Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia; Monash University, Clayton, Victoria 3800, Australia.

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http://dx.doi.org/10.1038/cddiscovery.2016.49DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4979472PMC
August 2016

Incomplete target neutralization by the anti-cancer antibody rilotumumab.

MAbs 2016 ;8(2):246-52

a Oncogenic Signalling Laboratory and Brain Cancer Discovery Collaborative, Centre for Cancer Research, Hudson Institute of Medical Research , 27-31 Wright Street, Clayton , VIC 3168 , Australia.

The antibody rilotumumab, which has been tested in multiple Phase 2 and Phase 3 trials, has been reported to neutralize hepatocyte growth factor (HGF), the ligand for the oncogene MET. However, we report that rilotumumab does not prevent HGF from directly binding to MET on conventional and primary patient-derived human gliomasphere lines, a trait driven by the HGF α-chain, which remains free to engage cell-surface glycosaminoglycans and the receptor MET. This binding induces MET phosphorylation, initiates robust AKT and ERK signaling and potentiates biological effects such as cell scattering. This partial antagonism was highly exacerbated in the presence of activated epidermal growth factor receptor, which is common in several cancers. Hence, we confirm that rilotumumab is only a partial antagonist of HGF activity, a finding that has considerable implications for the therapeutic use of rilotumumab.
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http://dx.doi.org/10.1080/19420862.2015.1122149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966628PMC
December 2016

Targeted drug delivery using genetically engineered diatom biosilica.

Nat Commun 2015 Nov 10;6:8791. Epub 2015 Nov 10.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Future Industries Institute, University of South Australia, Mawson Lakes Boulevard, Mawson Lakes, South Australia 5095, Australia.

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.
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http://dx.doi.org/10.1038/ncomms9791DOI Listing
November 2015

Pharmacology of novel small-molecule tubulin inhibitors in glioblastoma cells with enhanced EGFR signalling.

Biochem Pharmacol 2015 Dec 28;98(4):587-601. Epub 2015 Oct 28.

School of Medical Sciences, The University of Sydney, NSW 2006, Australia. Electronic address:

We recently reported that CMPD1, originally developed as an inhibitor of MK2 activation, primarily inhibits tubulin polymerisation and induces apoptosis in glioblastoma cells. In the present study we provide detailed pharmacological investigation of CMPD1 analogues with improved molecular properties. We determined their anti-cancer efficacy in glioblastoma cells with enhanced EGFR signalling, as deregulated EGFR often leads to chemoresistance. Eight analogues of CMPD1 with varying lipophilicity and basicity were synthesised and tested for efficacy in the cell viability assay using established glioblastoma cell lines and patient-derived primary glioblastoma cells. The mechanism of action for the most potent analogue 15 was determined using MK2 activation and tubulin polymerisation assays, together with the immunofluorescence analysis of the mitotic spindle formation. Apoptosis was analysed by Annexin V staining, immunoblotting analysis of bcl-2 proteins and PARP cleavage. The apoptotic activity of CMPD1 and analogue 15 was comparable across glioblastoma cell lines regardless of the EGFR status. Primary glioblastoma cells of the classical subtype that are characterized by enhanced EGFR activity were most sensitive to the treatment with CMPD1 and 15. In summary, we present mechanism of action for a novel small molecule tubulin inhibitor, compound 15 that inhibits tubulin polymerisation and mitotic spindle formation, induces degradation of anti-apoptotic bcl-2 proteins and leads to apoptosis of glioblastoma cells. We also demonstrate that the enhanced EGFR activity does not decrease the efficacy of tubulin inhibitors developed in this study.
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http://dx.doi.org/10.1016/j.bcp.2015.10.014DOI Listing
December 2015

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity.

Hypertension 2015 Dec 28;66(6):1251-9. Epub 2015 Sep 28.

From the Translational Obstetrics Group (K.R.P., T.J.K.-L., R.H., N.J.H., L.Y., N.B., P.C., L.T., S.T.) and Department of Obstetrics and Gynaecology(K.R.P., T.J.K.-L., R.H., N.J.H., L.Y., N.B., P.C., L.T., A.S., S.T.), University of Melbourne, Mercy Hospital for Women, Heidelberg, Victoria, Australia; and Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria, Australia (T.G.J.).

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a-specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.115.05883DOI Listing
December 2015

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

Anat Cell Biol 2015 Mar 20;48(1):25-35. Epub 2015 Mar 20.

Department of Neurosurgery, University of Florida, Gainesville, FL, USA.

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.
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http://dx.doi.org/10.5115/acb.2015.48.1.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371178PMC
March 2015

DYRK1A in neurodegeneration and cancer: Molecular basis and clinical implications.

Pharmacol Ther 2015 Jul 17;151:87-98. Epub 2015 Mar 17.

Department of Pharmacology, School of Medical Sciences, University of Sydney, NSW 2006, Australia. Electronic address:

Protein kinases are one of the most studied drug targets in current pharmacological research, as evidenced by the vast number of kinase-targeting agents enrolled in active clinical trials. Dual-specificity Tyrosine phosphorylation-Regulated Kinase 1A (DYRK1A) has been much less studied compared to many other kinases. DYRK1A primary function occurs during early development, where this protein regulates cellular processes related to proliferation and differentiation of neuronal progenitor cells. Although most extensively characterised for its role in brain development, DYRK1A is over-expressed in a variety of diseases including a number of human malignancies, such as haematological and brain cancers. Here we review the accumulating molecular studies that support our understanding of how DYRK1A signalling could underlie these pathological functions. The relevance of DYRK1A in a number of diseases is also substantiated with intensive drug discovery efforts to develop potent and selective inhibitors of DYRK1A. Several classes of DYRK1A inhibitors have recently been disclosed and some molecules are promising leads to develop DYRK1A inhibitors as drugs for DYRK1A-dependent diseases.
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http://dx.doi.org/10.1016/j.pharmthera.2015.03.004DOI Listing
July 2015

Quantitative intratumoural microdistribution and kinetics of (131)I-huA33 antibody in patients with colorectal carcinoma.

EJNMMI Res 2014 30;4:22. Epub 2014 May 30.

Ludwig Institute for Cancer Research, Austin Health, Melbourne 3084, Australia ; Department of Nuclear Medicine and Centre for PET, Austin Health, Melbourne 3084, Australia.

Background: The ability of recombinant antibodies to adequately penetrate into tumours is a key factor in achieving therapeutic effect; however, the behaviour of antibodies at a cellular level in tumours is poorly understood. The purpose of this study was to investigate those factors that influence the macroscopic and microscopic intratumoural distribution of an IgG1-humanized antibody, huA33, in colorectal tumours.

Methods: Twelve patients were infused with radiolabelled huA33 at 7 days prior to elective surgery for colorectal carcinoma. Macroscopic huA33 uptake was determined by both gamma well counter and autoradiography measurements of the resected tumour specimens. Microscopic uptake was then quantitated at a cellular level and compared to vascular penetrance. The impact of variation in tumour antigen (GPA33) expression, tumour size, specimen type (primary vs metastatic), presence of macroscopic necrosis, and tumour vasculature on huA33 uptake were examined.

Results: The I-huA33 uptake in whole tumour sections was (mean ± SD) 5.13 ± 2.71 × 10(-3)% injected dose per gram (ID/g). GPA33 was expressed in all viable tumour cells, and huA33 uptake was excellent regardless of tumour size and specimen type. In tumours with macroscopically evident central necrosis (n = 5), huA33 uptake in tumour necrotic centres was lower than in viable peripheries (0.606 ± 0.493 vs 2.98 ± 2.17 × 10(-3)%ID, p = 0.06). However, when corrected for low cell viability in necrotic centres, uptake of huA33 at the cellular level was highly comparable to that in the more viable tumour periphery (7.10 ± 5.10 × 10(-9) vs 3.82 ± 3.67 × 10(-9)%ID/cell, p = 0.4). In the five patients who exhibited macroscopic necrosis in their tumours, huA33 showed excellent tissue penetration, with a maximum penetration distance of 26 μm in peripheral tumour regions and 118 μm in central regions. No correlation was observed between (131)I-huA33 uptake in tumour on a cellular basis and tumour vascularity.

Conclusions: In patients with colorectal carcinoma, monoclonal antibody huA33 effectively targets viable tumour cells in all cellular milieus examined, including effective penetration into necrotic tumour centres, a novel and therapeutically important finding.
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http://dx.doi.org/10.1186/s13550-014-0022-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070025PMC
July 2014

The identification of mitochondrial DNA variants in glioblastoma multiforme.

Acta Neuropathol Commun 2014 Jan 2;2. Epub 2014 Jan 2.

Mitochondrial Genetics Group, Centre for Genetic Diseases, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.

Background: Mitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis.

Results: Using next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas.

Conclusions: These novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation.
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http://dx.doi.org/10.1186/2051-5960-2-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912901PMC
January 2014