Novel therapeutic venues for glioblastoma: novel rising preclinical treatment opportunities

High grade gliomas, including anaplastic glioma WHO grade III and glioblastoma WHO IV (GBM), carry a dismal prognosis. Taking all nowadays-available therapeutics options, including radiation, chemotherapy and surgery, for GBM into consideration the prognosis after initial diagnosis is about 12 month. Despite this bad prognosis, researchers gained a tremendous insight into the molecular and genetic signatures of low and high grade gliomas. Several different subtypes of GBM were demonstrated with respect to their genetic background. These genetic alterations include p53 mutation in secondary GBMs and EGFR amplification in primary GBMs, respectively. Very recently, great excitement was raised after the discovery of IDH1 mutation in low-grade gliomas and secondary GBMs. This discovery is of great significance since it allows further categorizing of GBMs and is helpful in distinguishing low-grade gliomas from non-neoplastic adjacent brain tissue. Despite all this progress there is an urgent need for fresh additional therapeutic strategies. In addition to the identification of novel therapeutic regimens it is of utmost importance to gain an understanding about the molecular mechanisms on how GBMs manage to evade from almost any anti-cancer treatment regimen. In experimental models of glioblastoma there are a number of novel therapeutic regimens that exhibited promising results. These novel therapeutics include, but are not limited to: Apoptosis-based therapeutics (Tumor necrosis factor alpha related apoptosis inducing ligand, TRAIL), tyrosinkinase-inhibitors, Heat-shock-protein 90 (HSP90) inhibitors, polyphenols, novel drug combinations and intracranial application based strategies. This chapter will primarily review and focus on molecular mechanisms of resistance in GBM and rising new therapeutic venues for high-grade gliomas. High-grade gliomas are a group of primary heterogenous tumors of which glioblastoma World Health Organisation, WHO IV (GBM), is the most common one. Once the diagnosis of GBM is made, the average survival time is approximately 12-15 month (Hegi, Diserens et al., 2005). Treatment usually consists of temozolomide (commonly used chemotherapeutic drug for the treatment of GBM, TMZ), radiation (either alone or in combination with chemotherapeutics) and surgery (Hegi, Diserens et al., 2005)..

Dominant-negative ATF5 rapidly depletes survivin in tumor cells.

Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin

Regression/eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide.

Malignant gliomas have poor prognosis and urgently require new therapies. Activating Transcription Factor 5 (ATF5) is highly expressed in gliomas, and interference with its expression/function precipitates targeted glioma cell apoptosis in vitro and in vivo. We designed a novel deliverable truncated-dominant-negative (d/n) form of ATF5 fused to a cell-penetrating domain (Pen-d/n-ATF5-RP) that can be intraperitoneally/subcutaneously administered to mice harboring malignant gliomas generated; (1) by PDGF-B/sh-p53 retroviral transformation of endogenous neural progenitor cells; and (2) by human U87-MG xenografts. In vitro Pen-d/n-ATF5-RP entered into glioma cells and triggered massive apoptosis. In vivo, subcutaneously-administered Pen-d/n-ATF5-RP passed the blood brain barrier, entered normal brain and tumor cells, and then caused rapid selective tumor cell death. MRI verified elimination of retrovirus-induced gliomas within 8-21 days. Histopathology revealed growth-suppression of intracerebral human U87-MG cells xenografts. For endogenous PDGF-B gliomas, there was no recurrence or mortality at 6-12 months versus 66% mortality in controls at 6 months. Necropsy and liver-kidney blood enzyme analysis revealed no adverse effects on brain or other tissues. Our findings thus identify Pen-d/n-ATF5-RP as a potential therapy for malignant gliomas

Activation of LXR Receptors and Inhibition of TRAP1 Causes Synthetic Lethality in Solid Tumors

Cholesterol is a pivotal factor for cancer cells to entertain their relentless growth. In this case, we provide a novel strategy to inhibit tumor growth by simultaneous activation of liver-X-receptors and interference with Tumor Necrosis Factor Receptor-associated Protein 1 (TRAP1). Informed by a transcriptomic and subsequent gene set enrichment analysis, we demonstrate that inhibition of TRAP1 results in suppression of the cholesterol synthesis pathway in stem-like and established glioblastoma (GBM) cells by destabilizing the transcription factor SREBP2. Notably, TRAP1 inhibition induced cell death, which was rescued by cholesterol and mevalonate. Activation of liver X receptor (LXR) by a clinically validated LXR agonist, LXR623, along with the TRAP1 inhibitor, gamitrinib (GTPP), results in synergistic reduction of tumor growth and cell death induction in a broad range of solid tumors, which is rescued by exogenous cholesterol. The LXR agonist and TRAP1 inhibitor mediated cell death is regulated at the level of Bcl-2 family proteins with an elevation of pro-apoptotic Noxa. Silencing of Noxa and its effector BAK attenuates cell death mediated by the combination treatment of LXR agonists and TRAP1 inhibition. Combined inhibition of TRAP1 and LXR agonists elicits a synergistic activation of the integrated stress response with an increase in activating transcription factor 4 (ATF4) driven by protein kinase RNA-like endoplasmic reticulum kinase (PERK). Silencing of ATF4 attenuates the increase of Noxa by using the combination treatment. Lastly, we demonstrate in patient-derived xenografts that the combination treatment of LXR623 and gamitrinib reduces tumor growth more potent than each compound. Taken together, these results suggest that TRAP1 inhibition and simultaneous activation of LXR might be a potent novel treatment strategy for solid malignancies

Metabolic Reprogramming by c-MET Inhibition as a Targetable Vulnerability in Glioblastoma

The elucidation of better treatments for solid tumors and especially malignant glial tumors is a priority. Better understanding of the molecular underpinnings of treatment response and resistance are critical determinants in the success for this endeavor. Recently, a battery of novel tools have surfaced that allow to interrogate tumor cell metabolism to more precise extent than this was possible in the earlier days. At the forefront of these developments are the extracellular flux and carbon tracing analyses. Through utilization of these techniques our group made the recent observation that acute and chronic c-MET inhibition drives fatty acid oxidation that in turn can be therapeutically targeted for drug combination therapies. Herein, we summarize and comment on some of our key findings related to this study

Epigenetic Targeting of Mcl-1 Is Synthetically Lethal with Bcl-xL/Bcl-2 Inhibition in Model Systems of Glioblastoma

Apoptotic resistance remains a hallmark of glioblastoma (GBM), the most common primary brain tumor in adults, and a better understanding of this process may result in more efficient treatments. By utilizing chromatin immunoprecipitation with next-generation sequencing (CHIP-seq), we discovered that GBMs harbor a super enhancer around the Mcl-1 locus, a gene that has been known to confer cell death resistance in GBM.We utilized THZ1, a known super-enhancer blocker, and BH3-mimetics, including ABT263, WEHI-539, and ABT199. Combined treatment with BH3-mimetics and THZ1 led to synergistic growth reduction in GBM models. Reduction in cellular viability was accompanied by significant cell death induction with features of apoptosis, including disruption of mitochondrial membrane potential followed by activation of caspases. Mechanistically, THZ1 elicited a profound disruption of the Mcl-1 enhancer region, leading to a sustained suppression of Mcl-1 transcript and protein levels, respectively. Mechanism experiments suggest involvement of Mcl-1 in the cell death elicited by the combination treatment. Finally, the combination treatment of ABT263 and THZ1 resulted in enhanced growth reduction of tumors without induction of detectable toxicity in two patient-derived xenograft models of GBM in vivo. Taken together, these findings suggest that combined epigenetic targeting of Mcl-1 along with Bcl-2/Bcl-xL is potentially therapeutically feasible

Combined inhibition of Bcl-2/Bcl-xL and Usp9X/Bag3 overcomes apoptotic resistance in glioblastoma in vitro and in vivo

Despite great efforts taken to advance therapeutic measures for patients with glioblastoma, the clinical prognosis remains grim. The antiapoptotic Bcl-2 family protein Mcl-1 is overexpressed in glioblastoma and represents an important resistance factor to the BH-3 mimetic ABT263. In this study, we show that combined treatment with ABT263 and GX15-070 overcomes apoptotic resistance in established glioblastoma cell lines, glioma stem-like cells and primary cultures. Moreover, this treatment regimen also proves to be advantageous in vivo. On the molecular level, GX15-070 enhanced apoptosis by posttranslational down-regulation of the deubiquitinase, Usp9X, and the chaperone Bag3, leading to a sustained depletion of Mcl-1 protein levels. Moreover, knock-down of Usp9X or Bag3 depleted endogenous Mcl-1 protein levels and in turn enhanced apoptosis induced through Bcl-2/Bcl-xL inhibition. In conclusion, combined treatment with ABT263 and GX15-070 results in a significantly enhanced anti-cancer activity in vitro as well as in vivo in the setting of glioblastoma. Both drugs, ABT263 and GX15-070 have been evaluated in clinical studies which facilitates the translational aspect of taking this combinatorial approach to the clinical setting. Furthermore we present a novel mechanism by which GX15-070 counteracts Mcl-1 expression which may lay a foundation for a novel target in cancer therapy

Dual Inhibition of Bcl-2/Bcl-xL and XPO1 is synthetically lethal in glioblastoma model systems

XPO1 has recently emerged as a viable treatment target for solid malignancies, including glioblastoma (GBM), the most common primary malignant brain tumor in adults. However, given that tumors become commonly resistant to single treatments, the identification of combination therapies is critical. Therefore, we tested the hypothesis that inhibition of anti-apoptotic Bcl-2 family members and XPO1 are synthetically lethal. To this purpose, two clinically validated drug compounds, the BH3-mimetic, ABT263, and the XPO1 inhibitor, Selinexor, were used in preclinical GBM model systems. Our results show that inhibition of XPO1 reduces cellular viability in glioblastoma cell cultures. Moreover, addition of ABT263 significantly enhances the efficacy of XPO1 inhibition on the reduction of cellular viability, which occurs in a synergistic manner. While selinexor inhibits the proliferation of glioblastoma cells, the combination treatment of ABT263 and selinexor results in substantial induction of cell death, which is accompanied by activation of effector- initiator caspases and cleavage of PARP. Mechanistically we find that XPO1 inhibition results in down-regulation of anti-apoptotic Mcl-1 and attenuates ABT263 driven Mcl-1 up-regulation. Consistently, siRNA mediated silencing of Mcl-1 sensitizes for ABT263 mediated cell death and partially for the combination treatment. By using a human patient-derived xenograft model of glioblastoma in mice, we demonstrate that the combination treatment of ABT263 and Selinexor reduces tumor growth significantly more than each compound alone. Collectively, these results suggest that inhibition of XPO1 and Bcl-2/Bcl-xL might be a potential strategy for the treatment of malignant glial tumors

A critical evaluation of PI3K inhibition in Glioblastoma and Neuroblastoma therapy

Members of the PI3K/Akt/mTor signaling cascade are among the most frequently altered proteins in cancer, yet the therapeutic application of pharmacological inhibitors of this signaling network, either as monotherapy or in combination therapy (CT) has so far not been particularly successful. In this review we will focus on the role of PI3K/Akt/mTOR in two distinct tumors, Glioblastoma multiforme (GBM), an adult brain tumor which frequently exhibits PTEN inactivation, and Neuroblastoma (NB), a childhood malignancy that affects the central nervous system and does not harbor any classic alterations in PI3K/Akt signaling. We will argue that inhibitors of PI3K/Akt signaling can be components for potentially promising new CTs in both tumor entities, but further understanding of the signal cascade’s complexity is essential for successful implementation of these CTs. Importantly, failure to do this might lead to severe adverse effects, such as treatment failure and enhanced therapy resistance

Inhibition of HDAC1/2 Along with TRAP1 Causes Synthetic Lethality in Glioblastoma Model Systems

The heterogeneity of glioblastomas, the most common primary malignant brain tumor, remains a significant challenge for the treatment of these devastating tumors. Therefore, novel combination treatments are warranted. Here, we showed that the combined inhibition of TRAP1 by gamitrinib and histone deacetylases (HDAC1/HDAC2) through romidepsin or panobinostat caused synergistic growth reduction of established and patient-derived xenograft (PDX) glioblastoma cells. This was accompanied by enhanced cell death with features of apoptosis and activation of caspases. The combination treatment modulated the levels of pro- and anti-apoptotic Bcl-2 family members, including BIM and Noxa, Mcl-1, Bcl-2 and Bcl-xL. Silencing of Noxa, BAK and BAX attenuated the effects of the combination treatment. At the metabolic level, the combination treatment led to an enhanced reduction of oxygen consumption rate and elicited an unfolded stress response. Finally, we tested whether the combination treatment of gamitrinib and panobinostat exerted therapeutic efficacy in PDX models of glioblastoma (GBM) in mice. While single treatments led to mild to moderate reduction in tumor growth, the combination treatment suppressed tumor growth significantly stronger than single treatments without induction of toxicity. Taken together, we have provided evidence that simultaneous targeting of TRAP1 and HDAC1/2 is efficacious to reduce tumor growth in model systems of glioblastoma