[Show abstract][Hide abstract] ABSTRACT: Previous work has demonstrated that the hormone prolactin promotes oligodendrocyte precursor proliferation and remyelination following lysolecithin-induced demyelination of the mouse spinal cord. Prolactin, however, can elicit pro-inflammatory responses, and its use in the prototypical demyelinating and inflammatory condition, multiple sclerosis (MS), should thus be approached cautiously. Here, we sought to determine whether recombinant prolactin could alter the course of experimental autoimmune encephalomyelitis (EAE), an inflammatory animal model of MS. Consistent with previous literature, we found that prolactin activated leukocytes in vitro. Daily treatment with prolactin from around the time of onset of clinical signs, for 9 (days 9 to 17) or 25 (days 9 to 33) days did not increase clinical or histological signs of EAE over that of vehicle-treated mice. Instead, the combination of prolactin and a suboptimal dose of recombinant murine interferon-beta resulted in (days 9 to 17 group) or trended towards (days 9 to 33 group), a greater amelioration of clinical signs of EAE, compared to either treatment alone or to vehicle controls. Histological analyses corroborated the clinical EAE data. These results suggest that prolactin may be beneficial when administered in combination with interferon-beta in MS.
Preview · Article · Dec 2015 · Journal of Neuroinflammation
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GBM), characterized by an aggressive clinical course, therapeutic resistance and striking molecular heterogeneity, remains incurable. A large number GBMs have EGFR alterations and, despite poor clinical translation to date, EGFR inhibition remains of therapeutic relevance. Recent evidence, from our group and others, indicates that JAK2/ STAT3 pathway is an important mediator of tumor cell survival, growth, and invasion in GBM. Interestingly, EGFR inhibition leads to activation of survival-signalling pathways such as STAT3, diminishing effectiveness of EGFR inhibition. We investigated the efficacy of a novel JAK2 inhibitor, pacritinib, in brain tumour initiating cell (BTIC) lines to evaluate potential use in the treatment of GBM patients. In a Phase III study of patients with myelofibrosis, pacritinib demonstrated manageable toxicity and clinically and statistically improved patient spleen volume and patient reported outcomes. Pacritinib results in on-target JAK2/STAT3 inhibition at 1-2 mM and dramatically reduces BTIC proliferation, regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. Pacritinib in combination with temozolomide prolongs survival, over either drug alone, in orthopically xenografted NOD-SCID. We are testing the hypothesis that concurrent inhibition of JAK/STAT and EGFR signalling may be an effective, clinically relevant therapeutic, strategy for GBM. We examined the in vitro actions of pacritinib on BTICs, in combination with three clinically approved EGFR inhibitors, erlotinib, afatinib and lapatinib and are investigating other clinically relevant EGFR inhibitors for GBM. Combinatorial treatment with pacritinib and EGFR inhibitors shows striking responses, with lowered IC50s and BTIC viability. The combinato-rial actions of EGFR and STAT3 inhibition were particularly effective in BTIC lines with EGFR activating vIII and missense point mutations. On target activity was demonstrated with reduced phospho-EGFR, phospho-STAT3 and effectors of both pathways. Current studies are aimed at investigating the combined actions of Pacritinib and EGFR inhib-itors, over single agents, in orthotopic xenograft animal survival.
[Show abstract][Hide abstract] ABSTRACT: In glioblastoma multiforme (GBM), brain tumor initiating cells (BTICs) with cancer stem cell characteristics have been identified and proposed as primordial cells responsible for disease initiation, recurrence and therapeutic resistance. However, the extent to which individual, patient-derived BTIC lines reflect the heterogeneity of GBM remains poorly understood. Here, we applied a stem cell biology approach and compared self-renewal, marker expression, label retention and asymmetric cell division in 20 BTIC lines. Through cluster analysis, we identified two subgroups of BTIC lines with distinct precursor states, stem- or progenitor-like, predictive of survival after xenograft. Moreover, stem and progenitor transcriptome signatures were identified, which showed a strong association with the proneural and mesenchymal subtypes, respectively, in the TCGA cohort. This study proposes a new framework for the study and use of BTIC lines and provides novel precursor biology insights into GBM.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma Multiforme (GBM) is the most aggressive subtype of brain tumour with a median survival of 15 months. Currently, GBM is managed by a combination of maximal safe resection followed by radiation and chemotherapy. However, GBM invariably recurs, highlighting the need to better delineate the basis of recurrent disease and develop novel more effective and targeted therapies. The Signal Transducer and Activator of Transcription 3 (STAT3) is abnormally active in GBM. A growing body of evidence supports the hypothesis that STAT3 is a major oncogenic molecular hub in patient-derived Brain Tumor Initiating Cells (BTICs) leading to stemness, proliferation, invasion and therapeutic resistance. Recently, there has been an increasing awareness that Epithelial to Mesenchymal Transition (EMT) is an essential process promoting the generation of highly tumorigenic cells with stem-like characteristics. The mesenchymal GBM profile, suggests that an EMT-like process has clinical relevance in GBM. Moreover, recent studies support a possible role for STAT3 in an EMT-like process in GBM. Here we show in that SLUG (encoded by SNAI2) appears to be expressed at higher levels than other EMT regulators in patient samples and matching BTICs. High SLUG protein level is observed in a large number of BTIC lines and positively correlates with activated STAT3. Moreover, SLUG is significantly decreased after pharmacological inhibition of STAT3 signalling using a well-characterized STAT3 inhibitor (Stattic) and a novel clinically relevant JAK2 inhibitor. Furthermore, IL-6 treatment or overexpression of a constitutively active form of STAT3 promote SLUG expression. SLUG and the subsequent EMT-like process may play an important role in mesenchymal GBM and the archetypal characteristics of BTICs: self-renewal, invasion, tumorigenicity and therapeutic resistance. We propose that STAT3-dependent EMT-like process and the key regulator SLUG are interesting novel therapeutic targets in GBM.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The EGFR and PI3K/mTORC1/2 pathways are frequently altered in glioblastoma (GBM), but pharmacologic targeting of EGFR and PI3K signaling has failed to demonstrate efficacy in clinical trials. Lack of relevant models has rendered it difficult to assess whether targeting these pathways might be effective in molecularly defined subgroups of GBMs. Here, human brain tumor-initiating cell (BTIC) lines with different combinations of endogenous EGFR wild-type, EGFRvIII, and PTEN mutations were used to investigate response to the EGFR inhibitor gefitinib, mTORC1 inhibitor rapamycin, and dual mTORC1/2 inhibitor AZD8055 alone and in combination with temozolomide (TMZ) EXPERIMENTAL DESIGN: In vitro growth inhibition and cell death induced by gefitinib, rapamycin, AZD8055, and TMZ or combinations in human BTICs were assessed by alamarBlue, neurosphere, and Western blotting assays. The in vivo efficacy of AZD8055 was assessed in subcutaneous and intracranial BTIC xenografts. Kaplan-Meier survival studies were performed with AZD8055 and in combination with TMZ.
We confirm that gefitinib and rapamycin have modest effects in most BTIC lines, but AZD8055 was highly effective at inhibiting Akt/mTORC2 activity and dramatically reduced the viability of BTICs regardless of their EGFR and PTEN mutational status. Systemic administration of AZD8055 effectively inhibited tumor growth in subcutaneous BTIC xenografts and mTORC1/2 signaling in orthotopic BTIC xenografts. AZD8055 was synergistic with the alkylating agent TMZ and significantly prolonged animal survival.
These data suggest that dual inhibition of mTORC1/2 may be of benefit in GBM, including the subset of TMZ-resistant GBMs.
No preview · Article · Oct 2014 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The current standard of care for glioblastoma (GBM) involves a combination of surgery, radiotherapy, and temozolomide chemotherapy, but this regimen fails to achieve long-term tumor control. Resistance to temozolomide is largely mediated by expression of the DNA repair enzyme MGMT; however, emerging evidence suggests that inactivation of MSH6 and other mismatch repair proteins plays an important role in temozolomide resistance. Here, we investigate endogenous MSH6 mutations in GBM, anaplastic oligodendroglial tumor tissue, and corresponding brain tumor-initiating cell lines (BTIC).
MSH6 sequence and MGMT promoter methylation were determined in human tumor samples and BTICs. Sensitivity to temozolomide was evaluated in vitro using BTICs in the absence and presence of O(6)-benzylguanine to deplete MGMT. The influence of MGMT and MSH6 status on in vivo sensitivity to temozolomide was evaluated using intracranial BTIC xenografts.
We identified 11 previously unreported mutations in MSH6 in nine different glioma samples and six paired BTIC lines from adult patients. In addition, MSH6 mutations were documented in three oligodendrogliomas and two treatment-naïve gliomas, both previously unreported findings. These mutations were found to influence the sensitivity of BTICs to temozolomide both in vitro and in vivo, independent of MGMT promoter methylation status.
These data demonstrate that endogenous MSH6 mutations may be present before alkylator therapy and occur in at least two histologic subtypes of adult glial neoplasms, with this report serving as the first to note these mutations in oligodendroglioma. These findings broaden our understanding of the clinical response to temozolomide in gliomas.
No preview · Article · Jul 2014 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Despite intense investigation, the ability to treat high-grade glioma (HGG) has remained dismal, in part due to molecular and phenotypic heterogeneity. The cancer stem cell, a cell believed to be the clonogenic core of tumors including human glioma, has been a major focus for the development of new therapies but emerging evidence has exposed the dynamic and heterogenous characteristics of this stem-like population that facilitates evasion from current therapies.
[Show abstract][Hide abstract] ABSTRACT: "Therapeutic Targeting of Glioblastoma" is a new pan-Canadian research team of the Terry Fox Research Institute and the Canadian Stem Cell Network funded to discover efficacious therapeutics for GBM. The team's goals are also to discover novel signaling pathways regulating GBM cell survival and genetic alterations that mediate drug resistance. As a platform, we use our collection of over 100 primary GBM tumor-initiating lines (BTIC) that are subjected to drug screening by over 1400 compounds, and to genetic and phosphoproteomic analysis.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM) is a devastating disease and the most lethal of adult brain tumours. Despite standard surgery, radiation and chemotherapy, the median survival is 15 months, thought to be due, in part, to recurrence from a small reservoir of brain tumour initiating cells. Our laboratory discovered adult neural stem cells, now found to be present in the brains of all adult mammals, through the development of the clonal neurosphere assay. This assay has contributed to the identification of adult human brain tumour initiating cells (BTICs), which may represent a reservoir that leads to GBM recurrence and death. Building upon the identification of growth factors and cytokines that converge on the cytoplasmic signal transducer and activator of transcription 3 (STAT3) to maintain the adult neural stem cell undifferentiated state, and the fact that STAT3 is abnormally active in GBM and may be one of the causes of tumour growth and therapeutic resistance, targeting the janus kinase (JAK)/STAT3 signalling pathway has become a major research focus for our laboratory. Here, we report the GBM translational potential of R333, a JAK/SYK inhibitor in development for other indications by Rigel Pharmaceuticals.
[Show abstract][Hide abstract] ABSTRACT: Background
Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects.Methods
We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant ((mt)) and IDH wild-type ((wt)) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas.ResultsWe found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH(mt) gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH(mt) derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH(wt)), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH(mt) glioblastomas.Conclusion
To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH(mt) gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM) is the most common and deadly malignant brain cancer, with a median survival of <2 years. GBM displays a cellular complexity that includes brain tumour-initiating cells (BTICs), which are considered as potential key targets for GBM therapies. Here we show that the transcription factors FOXG1 and Groucho/TLE are expressed in poorly differentiated astroglial cells in human GBM specimens and in primary cultures of GBM-derived BTICs, where they form a complex. FOXG1 knockdown in BTICs causes downregulation of neural stem/progenitor and proliferation markers, increased replicative senescence, upregulation of astroglial differentiation genes and decreased BTIC-initiated tumour growth after intracranial transplantation into host mice. These effects are phenocopied by Groucho/TLE knockdown or dominant inhibition of the FOXG1:Groucho/TLE complex. These results provide evidence that transcriptional programmes regulated by FOXG1 and Groucho/TLE are important for BTIC-initiated brain tumour growth, implicating FOXG1 and Groucho/TLE in GBM tumourigenesis.
Full-text · Article · Dec 2013 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Brain tumor initiating cells (BTICs) contribute to the genesis and recurrence of gliomas. We examined whether the microglia and macrophages that are abundant in gliomas alter BTIC growth. We found that microglia derived from non-glioma human subjects markedly mitigated the sphere-forming capacity of glioma patient-derived BTICs in culture by inducing the expression of genes that control cell cycle arrest and differentiation. This sphere-reducing effect was mimicked by macrophages, but not by neurons or astrocytes. Using a drug screen, we validated amphotericin B (AmpB) as an activator of monocytoid cells and found that AmpB enhanced the microglial reduction of BTIC spheres. In mice harboring intracranial mouse or patient-derived BTICs, daily systemic treatment with non-toxic doses of AmpB substantially prolonged life. Notably, microglia and monocytes cultured from glioma patients were inefficient at reducing the sphere-forming capacity of autologous BTICs, but this was rectified by AmpB. These results provide new insights into the treatment of gliomas.
Full-text · Article · Dec 2013 · Nature Neuroscience
[Show abstract][Hide abstract] ABSTRACT: The STAT3 gene is abnormally active in glioblastoma and is a critically important mediator of tumour growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant im-portance. Herein, we report a most potent, small molecule, non-phosphorylated STAT3 inhibitor, 31 that displayed potent STAT3 binding affinity (KD = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells, effec-tively suppresses STAT3 phosphorylation and downstream transcriptional targets at low nM concentrations. In vivo studies with 31 in mice orthotopically xenografted with glioma and analyzed by immunohistochemical staining demonstrated that 31 exhibited blood brain barrier permeability, in vivo potency, on target anti-STAT3 activity and potent inhibition of tumour cell proliferation and increased apoptosis. This work demonstrates the clinical efficacy of a STAT3 inhibitor for clinical application in glioblastoma.
[Show abstract][Hide abstract] ABSTRACT: The propagation of neural precursors in culture is an essential tool for the study of the signaling matrix that regulates their proliferation, self-renewal, and generation of terminally differentiated progeny. Neural precursors can be expanded in vitro using both adherent and non-adherent culture protocols. The culture of fetal human neural precursors in the absence of serum as free-floating clusters of cells termed neurospheres is described here.
No preview · Article · Aug 2013 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Early life events can modulate brain development to produce persistent physiological and behavioural phenotypes that are transmissible across generations. However, whether neural precursor cells are altered by early life events, to produce persistent and transmissible behavioural changes, is unknown. Here, we show that bi-parental care, in early life, increases neural cell genesis in the adult rodent brain in a sexually dimorphic manner. Bi-parentally raised male mice display enhanced adult dentate gyrus neurogenesis, which improves hippocampal neurogenesis-dependent learning and memory. Female mice display enhanced adult white matter oligodendrocyte production, which increases proficiency in bilateral motor coordination and preference for social investigation. Surprisingly, single parent-raised male and female offspring, whose fathers and mothers received bi-parental care, respectively, display a similar enhancement in adult neural cell genesis and phenotypic behaviour. Therefore, neural plasticity and behavioural effects due to bi-parental care persist throughout life and are transmitted to the next generation.
[Show abstract][Hide abstract] ABSTRACT: Background
Intratumoral heterogeneity in glioblastoma multiforme (GBM) poses a significant barrier to therapy in certain subpopulation such as the tumor-initiating cell population, being shown to be refractory to conventional therapies. Oncolytic virotherapy has the potential to target multiple compartments within the tumor and thus circumvent some of the barriers facing conventional therapies. In this study, we investigate the oncolytic potential of myxoma virus (MYXV) alone and in combination with rapamycin in vitro and in vivo using human brain tumor-initiating cells (BTICs).Methods
We cultured fresh GBM specimens as neurospheres and assayed their growth characteristics in vivo. We then tested the susceptibility of BTICs to MYXV infection with or without rapamycin in vitro and assessed viral biodistribution/survival in vivo in orthotopic xenografts.ResultsThe cultured neurospheres were found to retain stem cell markers in vivo, and they closely resembled human infiltrative GBM. In this study we determined that (i) all patient-derived BTICs tested, including those resistant to temozolomide, were susceptible to MYXV replication and killing in vitro; (ii) MYXV replicated within BTICs in vivo, and intratumoral administration of MYXV significantly prolonged survival of BTIC-bearing mice; (iii) combination therapy with MYXV and rapamycin improved antitumor activity, even in mice bearing "advanced" BTIC tumors; (iv) MYXV treatment decreased expression of stem cell markers in vitro and in vivo.Conclusions
Our study suggests that MYXV in combination with rapamycin infects and kills both the BTICs and the differentiated compartments of GBM and may be an effective treatment even in TMZ-resistant patients.
[Show abstract][Hide abstract] ABSTRACT: The emerging field of targeted molecular therapeutics holds significant promise for treatment of glioblastoma (GBM), but has failed to achieve more than modest efficacy in select patient subsets in recent clinical trials. Data from the Cancer Genome Atlas and other large genomic studies demonstrate that the epidermal growth factor receptor (EGFR) and PI3Kinase/mTORC1/2 pathways are frequently altered in GBM. However, pharmacological targeting of EGFR and PI3Kinase signaling in GBM has been not been promising. A lack of relevant models has rendered it difficult to assess whether targeting these pathways might be effective in molecularly defined subgroups of GBMs. In this study, human brain tumor stem cell (BTSC) lines with different combinations of endogenous EGFR wild type, EGFRvIII and PTEN mutations were used to investigate response to the EGFR inhibitor Iressa, the mTORC1 inhibitor rapamycin and the dual mTORC1/2 inhibitor AZD8055. We confirm that Iressa and rapamycin have modest effects in most BTSC lines, but AZD8055 was highly effective at inhibiting Akt/mTORC2 activity and dramatically reduced the viability of BTSCs regardless of their EGFR and PTEN mutational status. Moreover, AZD8055 was synergistic with the alkylating agent temozolomide (TMZ). These data suggest that dual inhibition of the mTORC1/2 may be of benefit in GBM including the subset of TMZ-resistant GBMs. Funding acknowledgements: Alberta Cancer Foundation and Stem Cell Network