TRIGBM · Identification of TRAIL sensitivity/resistance mechanisms and searching for novel TRAIL-sensitizing agents in Glioblastoma Multiforme

Identification of TRAIL sensitivity/resistance mechanisms and searching for novel TRAIL-sensitizing agents in Glioblastoma Multiforme

The goal of the TRIGBM project was to explore the molecular basis of the response of a very aggressive brain tumor type, glioblastoma multiforme (GBM), to a promising therapeutic molecule, Tumor necrosis factor related apoptosis-inducing ligand (TRAIL), and identify novel TRAIL-based therapeutic approaches. TRAIL has emerged as a prime candidate for cancer treatment due to its tumor-specificity, but its success has been limited due to the acquired or inherent resistance of some cancer cells to this factor. In GBMs, TRAIL can be an excellent therapeutic candidate since it has the potential to induce tumor regression without harming the normal tissue. In this project, we aimed to test the role of a pro-apoptotic factor, harakiri (HRK), in mediating TRAIL response in GBMs. We then generated new reporter tools to monitor HRK expression as well as other TRAIL pathway components. In parallel, we generated new isogenic cell lines with differential sensitivity/resistance to TRAIL and explored the molecular determinants of TRAIL response by genome wide transcriptome analysis. In the end, we performed chemical screens to discover new molecules that had the potential to overcome TRAIL resistance. Together, these studies improved our understanding of GBM biology and laid a groundwork for new TRAIL-based combinatorial approaches for GBMs. GBM is the most common and the most aggressive type of brain tumors with extremely poor prognosis and a mean survival of 12 months. These deadly cancers display apoptotic resistance, a common attribute of all cancers. Therefore, re-activating dormant apoptotic programs in cancer is a favorable approach. To this end, soluble recombinant human TRAIL ligand (Apo2L/TRAIL/dulanermin), and TRAIL receptor agonist monoclonal antibodies (mapatumumab, lexatumumab) have been developed and tested in pre-clinical studies of solid tumors.While these systemically delivered TRAIL-based agents have clinical potential, their utility and broad applicability are limited by several factors, including the innate or acquired resistance of many tumors to TRAIL. Therefore, the main focus of this project was to address the molecular mechanisms of TRAIL resistance in GBMs and identify new agents that can overcome resistance by modulating these mechanisms. TRIGBM project had the following main objectives: 1) Elucidating the role of HRK, a pro-apoptotic BH3 only Bcl-2 protein, in modulating the response of human GBMs to TRAIL. 2) Generating tools to monitor the expression of HRK and known TRAIL pathway components, and characterizing their utility and function in a panel of GBM lines. 3) Identifying novel TRAIL-sensitizing agents and assessing their function in vitro and in vivo. During the TRIGBM project, significant progress was made towards achieving these objectives. Specifically, the expression levels and the role of HRK were tested in GBM cells using loss-of-function and gain-of-function tools in several cell lines with differential TRAIL sensitivity/resistance. In parallel, more isogenic cell line pairs with differential apoptotic thresholds were generated and HRK expression was tested as a potential marker of apoptotic threshold in these cell lines as well. Expression profiling of several cell lines with next generation sequencing revealed HRK as differentially expressed in GBM cells that have different apoptotic thresholds. These genome-wide expression-profiling experiments also discovered several other Bcl-2 family members as regulators of apoptosis, as expected. In addition to characterizing the role of HRK in GBM cells’ response to TRAIL, novel molecular players of TRAIL response were identified during this project. For example, Insulin Growth Factor Binding Protein 2 (IGFBP2) was found to be markedly overexpressed in TRAIL-resistant subpopulations of GBM cells, while HRK was among the markedly downregulated genes. Conducting loss-of-function and gain-of-function experiments, we showed that IGFBP2 was partly associated with TRAIL response. These results on the novel molecular players of TRAIL response in isogenic cell pairs are now being prepared as a mansucript that will be submitted to Cell Death and Disease as an original article. The experiments designed to test the role of HRK in GBM cell apoptosis led to the conclusion that HRK can induce apoptosis in a subset of GBM cell lines in vitro and in in vivo orthotopic models of GBM. These experiments suggested that HRK indeed plays a role in TRAIL responsiveness. However, our studies indicated that the mode-of-action of HRK is not dependent solely on its expression, but more so on its functional interaction with anti-apoptotic Bcl-2 family members, Bcl-2 and Bcl-XL. Therefore, our results suggest that HRK plays a critical regulatory role of apoptosis in GBM cells. These results were accepted to as a publication at Oncotarget journal. In parallel, we conducted chemical screens to identify secondary agents that can overcome TRAIL resistance. First, we conducted an extensive drug-re-profiling screen to identify FDA-approved compounds that can be used clinically as TRAIL-sensitizing agents. Using selected isogenic GBM cell pairs with differential levels of TRAIL sensitivity, we revealed 26 TRAIL-sensitizing compounds, 13 of which were effective as single agents. We focused on the characterization of drugs that were enhancers of TRAIL response without any effect on their own. One such drug, Mitoxantrone, a DNA-damaging agent, did not cause toxicity to non-malignant cells at the doses that synergized with TRAIL on tumor cells. We investigated the downstream changes in apoptosis pathway components upon Mitoxantrone treatment, and observed that HRK expression was significantly upregulated in favor of apoptosis. Together, our results suggested that combination of Mitoxantrone and TRAIL can be a promising therapeutic approach for GBM patients and that HRK is involved in Mitoxantrone and TRAIL-mediated apoptosis. These findings have been published as an original article at Cancer Biology and Therapy. In the second screen, we utilized a more focused compund library to identify TRAIL sensitizing agents. This library, received from Structural Genomics Consortium and Oxford University, included 70 compounds targeting chromatin modifying proteins, such as Histone Deacetylases (HDACs), Histone Demethylases (HDMs), Histone Methyltransferases (HMTs), DNA Methyltransferases (DNMTs), and Bromodomain Proteins. From this epigenome-directed screen, we identified several interesting compunds with an ability ro cooperate with TRAIL in resistant cells. These included novel HDAC inhibitors, and Chaetocin, inhibitor of an HMT, Suv39H1. We assessed transcriptome changes with RNA-seq and showed that apoptosis programme is altered in response to treatment with these compunds. We are currently concluding our in vivo experiments, that address the potential of these sensitizing agents, particularly Chaetocin, in reducing tumor growth. The results of these findings will be submitted as an original article to Cancer Research, and expected to have a profound impact in the field. Through this project, five graduate students have been trained. The results of these findings have been presented at very prestigious conferences as posters or invited talks.

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