Targeting intrinsic apoptosis and other forms of cell death by BH3-mimetics in glioblastoma.

INTRODUCTION: Novel approaches to treat malignant brain tumors are necessary since these neoplasms still display an unfavorable prognosis. Areas covered: In this review, the authors summarize and analyze recent preclinical data that suggest that targeting intrinsic apoptosis may be a suitable strategy for the treatment of malignant gliomas. They focus on the anti-apoptotic Bcl-2 family members of proteins and the recent drug developments in that field with a special focus on BH3-mimetics. With the discovery of BH3-mimetics that interfere with anti-apoptotic Bcl-2 family members in the low nanomolar range significant excitement has been generated towards these class of inhibitors, such as ABT-737, ABT-263 and the most recent successor, ABT-199 which is most advanced with respect to clinical application. The authors discuss the more recent selective inhibitors of Bcl-xL and Mcl-1. Concerning Mcl-1, these novel classes of inhibitors have the potential to impact malignant gliomas since these tumors reveal increased levels of Mcl-1. Expert opinion: The recent development of certain small molecules raises significant hope that intrinsic apoptosis might soon be efficiently targetable for malignancies of the central nervous system. That being said, additional studies are necessary to determine which of the BH3-mimetics might be most suitable.

BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma.

Drug combination therapies remain pivotal for the treatment of heterogeneous malignancies, such as glioblastomas. Here, we show a novel lethal interaction between Bcl-xL and c-myc inhibition accomplished by bromodomain protein inhibitors. Established, patient-derived xenograft and stem cell-like glioma cells were treated with the novel bromodomain protein inhibitors, JQ1 and OTX015, along with BH3-mimetics, ABT263 or Obatoclax. Synergy was assessed by calculation of CI values. Small interfering RNAs (siRNAs) were used for gene silencing and mechanistic studies. In vivo experiments were performed in a glioblastoma xenograft model. Single treatments with JQ1 and OTX015 had only moderate effects on the reduction of cellular viability. However, the combination treatment of BH3-mimetics along with JQ1 or OTX015 resulted in a highly synergistic reduction of cellular viability in a broad range of different model systems of malignant glioma. Similarly, knockdown of c-myc sensitized glioma cells for ABT263 mediated cell death. The enhanced loss of cellular viability in the combination treatment was mediated by activation of apoptosis with dissipation of mitochondrial membrane potential and caspase cleavage. The combination treatment led to a modulation of anti- and pro-apoptotic Bcl-2 family members with an increase in pro-apoptotic Noxa mediated by ATF4. Small interfering RNA mediated knockdown of Bak and Noxa protected glioma cells from ABT263/JQ1 mediated apoptosis. Finally, the combination treatment of ABT263 and OTX015 resulted in a regression of tumors and a significantly smaller tumor size as compared to single or vehicle treated tumors. Thus, these results warrant clinical testing for the drug combination of BH3-mimetics along with bromodain protein inhibitors.

Immune phenotypes predict survival in patients with glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM), a common primary malignant brain tumor, rarely disseminates beyond the central nervous system and has a very bad prognosis. The current study aimed at the analysis of immunological control in individual patients with GBM. METHODS: Immune phenotypes and plasma biomarkers of GBM patients were determined at the time of diagnosis using flow cytometry and ELISA, respectively. RESULTS: Using descriptive statistics, we found that immune anomalies were distinct in individual patients. Defined marker profiles proved highly relevant for survival. A remarkable relation between activated NK cells and improved survival in GBM patients was in contrast to increased CD39 and IL-10 in patients with a detrimental course and very short survival. Recursive partitioning analysis (RPA) and Cox proportional hazards models substantiated the relevance of absolute numbers of CD8 cells and low numbers of CD39 cells for better survival. CONCLUSIONS: Defined alterations of the immune system may guide the course of disease in patients with GBM and may be prognostically valuable for longitudinal studies or can be applied for immune intervention.

Combined inhibition of HER1/EGFR and RAC1 results in a synergistic antiproliferative effect on established and primary cultured human glioblastoma cells.

Glioblastoma is the most frequent brain tumor of glial origin in adults. With the best available standard-of-care, patients with this disease have a life expectancy of only approximately 15 months after diagnosis. Because the EGF receptor (HER1/EGFR) is one of the most commonly dysregulated oncogenes in glioblastoma, HER1/EGFR-targeted agents, such as erlotinib, were expected to provide a therapeutic benefit. However, their application in the clinical setting failed. Seeking an explanation for this finding, we previously identified several candidate genes for resistance of human glioblastoma cell lines toward erlotinib. On the basis of this panel of genes, we aimed at identifying drugs that synergistically enhance the antiproliferative effect of erlotinib on established and primary glioblastoma cell lines. We found that NSC23766, an inhibitor of RAC1, enhanced the antineoplastic effects of erlotinib in U87MG, T98MG, and A172MG glioblastoma cell lines for the most part in a synergistic or at least in an additive manner. In addition, the synergistic antiproliferative effect of erlotinib and NSC23766 was confirmed in primary cultured cells, indicating a common underlying cellular and molecular mechanism in glioblastoma. Therefore, agents that suppress RAC1 activation may be useful therapeutic partners for erlotinib in a combined targeted treatment of glioblastoma.

Erlotinib in glioblastoma: lost in translation?

Glioblastoma represents the most common primary brain tumor in adults. Despite improvements of multimodal therapy, the prognosis of this disease remains unfavorable. Thus, great efforts have been made to identify therapeutic agents directed against those specific molecular targets whose presence was shown to be associated with worse clinical outcomes. The epidermal growth factor receptor (HER1/EGFR) has been identified as one such target, and different compounds were developed to inhibit HER1/EGFR and/ or its mutant form, EGFRvIII. However, clinical trials did not confirm the initial enthusiasm conveyed by promising results from experimental studies. Therefore, a therapeutic approach directed at inhibiting solely HER1/EGFR does not seem to translate into a clinical benefit. This review discusses the current therapeutic situation in the setting of glioblastoma while putting the spotlight on erlotinib, a HER1/EGFR-targeted small molecule tyrosine kinase inhibitor.

A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src.

Cell migration on extracellular matrix requires the turnover of integrin-dependent adhesions. The nonreceptor tyrosine kinases Src and FAK regulate focal-adhesion turnover by poorly understood mechanisms. ERK/MAP kinase-mediated activation of the protease Calpain 2 also promotes focal-adhesion turnover; however, it is not known if this is linked to the activities of Src and FAK. Calpain 2 has previously been demonstrated to colocalize with focal-adhesion structures and can cleave several focal-adhesion complex components, including FAK. Studies utilizing Calpain inhibitors or Calpain-deficient cells confirm that Calpain's role in regulating focal-adhesion turnover is necessary for cell migration. We have identified a novel and kinase-independent function for FAK as an adaptor molecule that mediates the assembly of a complex consisting of at least Calpain 2 and p42ERK. Mutation of proline residues (Pro2) in the amino-terminal region of FAK blocks direct binding with Calpain 2 and also prevents formation of the Calpain 2/p42ERK complex in cells. We show that both complex formation and MEK/ERK activity are associated with Calpain-mediated proteolysis of FAK and focal adhesion turnover during transformation and migration. Furthermore, FAK is necessary for recruiting both Calpain 2 and p42ERK/MAPK to peripheral adhesion sites facilitating maximal Calpain activity.

Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling.

Although Src expression and activity are often elevated in colon cancer, the precise consequences of overexpression of the non-catalytic Src homology (SH) domains, or enhanced catalytic activity, are unknown. We show that, in KM12C colon cancer cells, elevated Src activity causes the components of adherens junctions, including vinculin, to be redistributed to Src-induced integrin adhesion complexes. Specifically, elevated Src activity blocks proper assembly of cell cell contacts after cells are switched from media containing a low level of calcium to media containing a high level of calcium, and E-cadherin remains internalized. In contrast, although elevated expression of the non-catalytic domains of Src is sufficient to induce assembly of integrin adhesion complexes, it does not induce disorganization of E-cadherin-associated intercellular contacts. Surprisingly, Src-induced disruption of E-cadherin localization requires specific integrin signalling, because E-cadherin redistribution is blocked by loss of cell-matrix interaction, or by inhibitory antibodies to alpha(v) or beta(1) integrin subunits. Furthermore, phosphorylation of the integrin-regulated focal adhesion kinase (FAK) on Src-specific sites is required for Src-induced de-regulation of E-cadherin, demonstrating interdependence between integrin-induced signals and cadherin-associated adhesion changes induced by Src.