Background The development of rational combination therapies is key to overcome inherent treatment resistance of glioblastoma. We aim at identifying new druggable targets by disturbing glioblastoma cells with inhibitors of Bromodomain and Extra-Terminal motif (BET) proteins to reveal cancer relevant vulnerabilities that may sensitize to a second drug. BET-proteins are epigenetic modulators and have been associated with proto-oncogene overexpression in cancer. Methods A glioblastoma derived sphere-line was treated with the BET inhibitor JQ1 over a time-course of 48h, followed by RNA-sequencing. Four chromatin marks were investigated by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Signatures of interest were functionally validated in vitro and in orthotopic xenografts. Combination therapies were evaluated for synergistic effects. Results Cancer relevant pathways significantly modulated by JQ1 comprised interferon-alpha (IFN-α) response genes and response signatures to histone deacetylase inhibitors (HDACi). The IFN-signature was reminiscent of a glioblastoma-derived IFN-signature comprising CD274 (PD-L1). Functional pathway analysis suggested that JQ1 was acting directly on the transcriptional level of IFN-response genes and not via the canonical JAK-STAT pathway. This was in line with JQ1 modulated expression and BRD4 and POL2 occupancy at IFN-signature genes, supporting a direct mechanistic interaction. Finally, we showed that combining HDACi with JQ1 acts synergistically in reducing cell viability of GS-lines. Conclusions Our approach identified BETi-induced vulnerabilities in cancer relevant pathways, potentially amenable to synergistic combinatorial therapy, such as combination with HDACi. The direct inhibitory effect of BETi on IFN-responsive genes in GBM cells, including CD274, indicates modulation of the tumor immune landscape and warrants further studies.

DNA methylome analysis of glioblastoma (GBM) identified the HIV-1 Tat interactive protein 2 (HTATIP2) gene as aberrantly methylated and silenced. HTATIP2 is a negative regulator of importin β-mediated (KPNB1) cytoplasmic-nuclear translocation of proteins and its deregulation may alter the functionality of cancer relevant nuclear proteins. We propose N-Methylpurine-DNA Glycosylase (MPG), responsible for removing alkylated bases and initiating base excision repair (BER), as a potential GBM relevant candidate. Here we investigated the role of epigenetic silencing of HTATIP2 on the subcellular localization of MPG, and MPG-mediated DNA repair. Induction of HTATIP2 expression in GBM cells lead to a significant shift of predominantly nuclear to cytoplasmic MPG, while depletion of endogenous levels of HTATIP2 resulted in enhanced nuclear MPG localization. We observed exclusion of MPG from the area exhibiting co-localization of HTATIP2 and KPNB1 in proximity to the nuclear membrane, suggesting competition of HTATIP2 with MPG to bind to KPNB1. In accordance, pharmacologic inhibition of KPNB1 similarly induced cytoplasmic retention of MPG as HTATIP2 expression. Reduced nuclear MPG localization, induced by HTATIP2 expression or depletion of MPG, yielded less P-H2AX-positive cells upon treatment with an alkylating agent. This suggested reduced MPG-mediated formation of apurinic/apyrimidinic (AP) sites, leaving behind unrepaired DNA lesions, hence, reflecting a reduced capacity of BER in response to the alkylating agent. Taken together, these results suggest that epigenetic silencing of HTATIP2 may increase nuclear localization of MPG, thereby increasing the capacity of the tumor cells to repair treatment related lesions and eventually contributing to treatment resistance.

In a genome-wide DNA methylation analysis of glioblastoma we identified aberrant methylation of the HIV-1 Tat interactive protein 2 (HTATIP2) gene promotor. This was strongly correlated with downregulation of HTATIP2 gene expression, suggesting a potential tumor suppressor function in glioblastoma. HTATIP2 has been shown to inhibit nuclear translocation through interaction with β-importins. We hypothesize that deregulation of HTATIP2 expression inhibits nuclear entry of cancer-relevant proteins, thereby disturbing their specific function in the nucleus. We identified N-Methylpurine-DNA Glycosylase (MPG) as a potential cancer relevant candidate with the observation of nuclear and/or cytoplasmic localization in glioblastoma. MPG is a DNA repair protein that recognizes DNA lesions (N7-meG, N3-meA) and initiates base excision repair. We have shown that nuclear MPG expression contributes to resistance of glioblastoma to treatment with the alkylating agent temozolomide. Here we investigated the effect of HTATIP2 on cellular localization of MPG using (i) an inducible system (TET-ON) to express HTATIP2 in non-expressing glioblastoma cells, and (ii) HTATIP2-targeting siRNAs to knock down HTATIP2 in cells with endogenous expression. Results from confocal microscopy or Imagestream analyses showed a significant cytoplasmic retention of MPG in presence of HTATIP2, while knock-down of HTATIP2 resulted in nuclear MPG localization. Cytoplasmic retention of MPG in the presence of HTATIP2 was associated with a significant increase in γ-H2Ax signal after treatment with the alkylating agent: methyl methanesulfonate (MMS), suggesting increase in DNA damage. Mechanistically, we found that HTATIP2 co-localizes with importin β1, and excludes MPG localization. Furthermore, HTATIP2 displayed a similar effect on cytoplasmic retention of MPG as pharmacologic inhibition of Importin β 1. Taken together, these results suggest that epigenetic silencing of HTATIP2 may increase nuclear localization of MPG, thereby increasing the capacity of the tumor cells to repair treatment related lesions and thereby contributing to treatment resistance.

Glioblastoma (GBM) is the most aggressive brain tumor in adults with a median overall survival of only 15 months. Despite multiple attempts, single agent therapies have failed in clinical trials and new strategies for combination treatments are urgently needed. Here, our aim was to predict rational combination therapies with BET inhibitors (BETi) that target bromodomain and extra-terminal tail (BET) proteins and are currently evaluated as anti-cancer drugs. In order to identify BETi-induced vulnerabilities in cancer relevant pathways that may be targeted with a second drug, we obtained differential expression profiles of glioma sphere lines (GS-lines) treated with the BETi JQ1. Gene set enrichment analysis revealed several significantly disturbed pathways that included IFN-alpha response genes and signatures of response to histone deacetylase inhibitors (HDACi). In order to validate the observed down regulation of the interferon response gene signature, we primed the GS-lines with IFN-alpha and confirmed that interferon-stimulated genes (ISGs), such as MX1, OAS1, and CD274 are down regulated after a 4-hour exposure to JQ1. Importantly, the levels of pSTAT1 in the nucleus remained unchanged upon JQ1 treatment, suggesting that JQ1 was acting directly on the transcriptional level of ISGs and not on the canonical JAK-STAT pathway. Similar results were obtained in adherent GBM cell lines that constitutively express ISGs. Moreover, in U87MG orthotropic xenografts in mice, a single i.p. injection of JQ1 downregulated OAS1 and CD274 expression. Currently we are studying the mechanism of how BETi represses transcription of ISGs by chromatin immuno-precipitation. Finally, we show that HDACi and JQ1 synergize to reduce cell viability of GS-lines in vitro. Further experiments will test HDACi and BETi drug combinations in mouse orthotopic xenografts of GS-lines. Citation Format: Olga Gusyatiner, Minh Diêu Thanh Pham, Yvonne Lei, Jungyeon Park, Pierre Bady, Mauro Delorenzi, Monika Hegi. BET inhibitors synergize with HDAC inhibitors and downregulate expression of interferon response genes in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2923.

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