Tor-Christian Aase Johannessen

DNA repair and cancer stem-like cells - Potential partners in glioma drug resistance?

Glioblastoma is the most malignant and frequent primary brain tumour in adults. Current treatment remains insufficient as these tumours display a diffuse infiltrative growth pattern and tend to recur despite extensive debulking surgery followed by radio- and chemotherapy. The alkylating agents carmustine (1,3-bis-(2-chloroethyl)-1-nitrosourea, or BCNU) and temozolomide (TMZ) are the drugs of choice for adjuvant glioma chemotherapy. However, several independent DNA repair mechanisms can restore the integrity of alkylated DNA bases, and thus contribute to drug resistance and subsequent therapy failure. Recent work suggests that glioblastomas develop as cellular and functional hierarchies through small subpopulations of stem cell-like cancer cells that are responsible for tumour initiation and maintenance. Such cells also appear to possess enhanced DNA repair capacity compared to other cells within the tumours. Challenges in glioblastoma therapy are to determine (1) whether the cancer stem-like cell subpopulations represent a clinically novel target for therapy, and (2) which additional treatment strategies should be applied to improve quality of life and prolong survival of glioblastoma patients. This review addresses clinically relevant mechanisms which contribute to glioma resistance towards current alkylating agent-based chemotherapy, and discusses related mechanisms and treatment strategies in the light of the cancer stem cell hypothesis.

Molecular mechanisms of temozolomide resistance in glioblastoma multiforme

Glioblastoma multiforme (GBM; WHO astrocytoma grade IV) is considered incurable owing to its inherently profound resistance towards current standards of therapy. Considerable effort is being devoted to identifying the molecular basis of temozolomide resistance in GBMs and exploring novel therapeutic regimens that may improve overall survival. Several independent DNA repair mechanisms that normally safeguard genome integrity can facilitate drug resistance and cancer cell survival by removing chemotherapy-induced DNA adducts. Furthermore, subpopulations of cancer stem-like cells have been implicated in the treatment resistance of several malignancies including GBMs. Thus, a growing number of molecular mechanisms contributing to temozolomide resistance are being uncovered in preclinical studies and, consequently, we are being presented with a broad range of potentially novel targets for therapy. A substantial future challenge is to successfully exploit the increasing molecular knowledge contributing to temozolomide resistance in robust clinical trials and to ultimately improve overall survival for GBM patients.

Highly infiltrative brain tumours show reduced chemosensitivity associated with a stem cell‐like phenotype

Aims: Cancer stem‐like cells might have important functions in chemoresistance. We have developed a model where highly infiltrative brain tumours with a stem‐like phenotype were established by orthotopic transplantation of human glioblastomas to immunodeficient rats. Serial passaging gradually transformed the tumours into a less invasive and more angiogenic phenotype (high‐generation tumours). The invasive phenotype (low‐generation tumours) was characterized by an increase in stem cell markers and increased phosphorylation of kinases in the phosphatidylinositol 3‐kinase (PI3K)/AKT pathway. These markers were reduced in the serially passaged vascular tumours. The present study was aimed at investigating how the two phenotypes responded in vitro to doxorubicin, a clinically potent cytotoxic drug for solid tumours. Methods: Biopsy spheroids were implanted and passaged intracranially in nude rats. Gene expression and protein analyses were performed, and drug sensitivity was assessed. Results: Microarray analysis revealed gene ontology categories connected to developmental aspects and negative regulators of differentiation, especially in the infiltrative stem cell‐like tumours. The highly invasive stem‐like phenotype was chemoresistant compared with the angiogenic phenotype. By interfering with the PI3K it was possible to sensitize tumour spheroids to chemotherapy. Real‐time quantitative polymerase chain reaction showed downregulation of the stem cell markers Nestin and Musashi‐1 in low‐generation biopsy spheroids following PI3K inhibition. Conclusions: Highly invasive tumours with a stem‐like phenotype are more chemoresistant than angiogenic tumours derived from the same patients. We suggest that treatment resistance in glioblastomas can be related to PI3K/AKT activity in stem‐like tumour cells, and that targeted interference with the PI3K/AKT pathway might differentiate and sensitize this subpopulation to chemotherapy.

The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells

INTRODUCTION Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most frequent and most malignant primary brain tumor in adults. Despite multimodal therapy, all such tumors practically recur during the course of therapy, causing a median survival of only 14.6 months in patients with newly diagnosed GBM. The present study was aimed at examining the expression of the DNA repair protein AlkB homolog 2 (ALKBH2) in human GBM and determining whether it could promote resistance to temozolomide chemotherapy. METHODS ALKBH2 expression in GBM cell lines and in human GBM was determined by quantitative real-time PCR (qRT-PCR) and gene expression analysis, respectively. Drug sensitivity was assessed in GBM cells overexpressing ALKBH2 and in cells in which ALKBH2 expression was silenced by small-interfering (si)RNA. ALKBH2 expression following activation of the p53 pathway was examined by western blotting and qRT-PCR. RESULTS ALKBH2 was abundantly expressed in established GBM cell lines and human GBM, and temozolomide exposure increased cellular ALKBH2 expression levels. Overexpression of ALKBH2 in the U87 and U251 GBM cell lines enhanced resistance to the methylating agents temozolomide and methyl methanesulfonate but not to the nonmethylating agent doxorubicin. Conversely, siRNA-mediated knockdown of ALKBH2 increased sensitivity of GBM cells to temozolomide and methyl methanesulfonate but not to doxorubicin or cisplatin. Nongenotoxic activation of the p53 pathway by the selective murine double minute 2 antagonist nutlin-3 caused a significant decrease in cellular ALKBH2 transcription levels. CONCLUSION Our findings identify ALKBH2 as a novel mediator of temozolomide resistance in human GBM cells. Furthermore, we place ALKBH2 into a new cellular context by showing its regulation by the p53 pathway.

Tumor vasculature: the Achilles' heel of cancer?

Introduction: Tumor-associated angiogenesis is one of the essential hallmarks underlying cancer development and metastasis. Anti-angiogenic agents accordingly aim to restrain cancer progression by blocking the formation of new vessels, improving the delivery of chemotherapeutic agents to the tumor site and reducing the shedding of metastatic cells into the circulation. This review article addresses some key issues in the use of angiogenesis inhibitors in cancer. Areas covered: The authors review the complex interactions between cell signaling pathways involved in tumor angiogenesis, and focus in particular on the molecular mechanisms that may induce resistance to angiogenesis inhibitors. They will also discuss some novel therapeutic strategies evolving within anti-angiogenic therapy such as the targeting of VEGFR-3, endothelial integrins and hepatocyte growth factor-MET signaling. Expert opinion: Although anti-angiogenic therapy is targeted at the non-malignant part of the tumor, the intricate network of growth promoting signaling pathways and in particular the redundancy when single pathways are targeted in endothelial cells represents a major therapeutic obstacle. A key challenge will be to develop more efficient inhibitors, combined with an individualized approach based on each tumors own endothelial signaling profile. Furthermore, reliable biomarkers which pinpoint those patients that will benefit from anti-angiogenic therapy need to be identified.

Rapid Conversion of Mutant IDH1 from Driver to Passenger in a Model of Human Gliomagenesis

Missense mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. Although mutant IDH1 expression is thought to drive the gliomagenesis process, the extent to which it remains a viable therapeutic target remains unknown. To address this question, we exposed immortalized (p53/pRb deficient), untransformed human astrocytes to the mutant IDH1 inhibitor AGI-5198 prior to, concomitant with, or at intervals after, introduction of transforming mutant IDH1, then measured effects on 2-HG levels, histone methylation (H3K4me3, H3K9me2, H3K9me3, or H3K27me3), and growth in soft agar. Addition of AGI-5198 prior to, or concomitant with, introduction of mutant IDH1 blocked all mutant IDH1-driven changes, including cellular transformation. Addition at time intervals as short as 4 days following introduction of mutant IDH1 also suppressed 2-HG levels, but had minimal effects on histone methylation, and lost the ability to suppress clonogenicity in a time-dependent manner. Furthermore, in two different models of mutant IDH1–driven gliomagenesis, AGI-5198 exposures that abolished production of 2-HG also failed to decrease histone methylation, adherent cell growth, or anchorage-independent growth in soft agar over a prolonged period. These studies show although mutant IDH1 expression drives gliomagenesis, mutant IDH1 itself rapidly converts from driver to passenger. Implications: Agents that target mutant IDH may be effective for a narrow time and may require further optimization or additional therapeutics in glioma. Mol Cancer Res; 14(10); 976–83. ©2016 AACR.

Mutant IDH1 Expression Drives TERT Promoter Reactivation as Part of the Cellular Transformation Process

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR.

Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide: Thioridazine blocks autophagy in glioblastoma cells

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14–15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome‐wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late‐stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy‐lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

EXTH-47. RAPID CONVERSION OF MUTANT IDH1 FROM DRIVER TO PASSENGER IN A MODEL OF HUMAN GLIOMAGENESIS

Missense mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. Although mutant IDH1 expression is thought to drive the gliomagenesis process, the extent to which it remains a viable therapeutic target remains unknown. To address this question, we exposed immortalized (p53/pRb deficient), untransformed human astrocytes to the mutant IDH1 inhibitor AGI-5198 prior to, concomitant with, or at intervals after, introduction of transforming mutant IDH1, then measured effects on 2-HG levels, histone methylation (H3K4me3, H3K9me2, H3K9me3, or H3K27me3), and growth in soft agar. Addition of AGI-5198 prior to, or concomitant with, introduction of mutant IDH1 blocked all mutant IDH1-driven changes, including cellular transformation. Addition at time intervals as short as 4 days following introduction of mutant IDH1 also suppressed 2-HG levels, but had minimal effects on histone methylation, and lost the ability to suppress clonogenicity in a time-dependent manner. Furthermore, in two different models of mutant IDH1-driven gliomagenesis, AGI-5198 exposures that abolished production of 2-HG also failed to decrease histone methylation, adherent cell growth, or anchorage-independent growth in soft agar over a prolonged period. These studies show although mutant IDH1 expression drives gliomagenesis, mutant IDH1 itself rapidly converts from driver to passenger. IMPLICATIONS Agents that target mutant IDH may be effective for a narrow time and may require further optimization or additional therapeutics in glioma. Mol Cancer Res; 14(10); 976-83. ©2016 AACR.

Abstract 1725: Activation of the p53 pathway by the MDM2 antagonist nutlin-3 downregulates the DNA repair protein hABH2 and sensitizes glioma cells to chemotherapy

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastoma is the most frequent and most malignant primary brain tumor in adults. Standard first line treatment for glioblastoma patients includes surgery followed by focal fractionated radiotherapy with concomitant and adjuvant administration of the alkylating agent temozolomide. During the course of therapy, however, radio- and chemoresistance typically become evident through local tumour recurrence, and median survival time for glioblastoma patients remains around 15 months. In order to improve this poor prognosis, there is a critical need to recognize the molecular basis for the low sensitivity of glioblastomas towards chemotherapeutic treatment. We have previously reported that elevated expression levels of the DNA repair protein hABH2 may provoke temozolomide resistance in glioma cells. In this work, we explored whether activation of the p53 (encoded by TP53) pathway by the MDM2 antagonist nutlin-3 would influence cellular levels of hABH2 and subsequently chemoresistance. Immunoblotting following nutlin-3 exposure for 24 hours showed increased protein levels of p53, MDM2, p21 and PUMA in glioma cell lines expressing wild type p53, but decreased hABH2 levels in a dose-dependent manner. Real-time quantitative PCR confirmed reduced hABH2 mRNA levels after nutlin-3 therapy, suggesting that hABH2 downregulation occurred at a transcriptional level. Combination therapy with nutlin-3 and temozolomide further enhanced activation of the p53 pathway, diminished levels of hABH2 protein and increased cellular cytotoxicity compared to either agent alone. Our results show that levels of hABH2 can be downregulated by activation of the p53 pathway in glioma cells. This suggests that p53 pathway activation may increase glioma sensitivity to chemotherapy not only by increased transcription of pro-apoptotic genes such as PUMA, but also by reducing levels of DNA repair proteins such as hABH2 that would otherwise promote drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2011-1725