Understanding, inducing and exploiting actionable vulnerabilities in experimental glioma

Abstract

Glioblastoma (GB) are aggressive, primary brain tumors, for which standard therapy comprises surgical resection followed by radio-chemotherapy [1-3]. Despite this multimodal and aggressive treatment approach, overall survival of patients remains in the range of 1.5 years and almost all patients will suffer from progressive disease [1, 2, 4]. Hence, novel treatment options are urgently needed, however, identifying novel exploitable tumor vulnerabilities in GB remains challenging. Consequently, this thesis focuses on understanding, inducing and exploiting actionable vulnerabilities in experimental glioma. This thesis includes the following three projects: First, Argyrin F a cyclic peptide that inhibits the proteasome from downregulating p27Kip1 [5], is evaluated for its anti-glioma efficacy. Second, the ATR inhibitor AZD6738 is molecularly analyzed to identify potential combination partners opening new therapeutic strategies. Lastly, this thesis discusses the accumulation of Fanconi Anemia (FA) germline mutations in the molecular tumor board (MTB) neuro-oncology cohort Tübingen. Argyrin F showed promising anti-glioma efficacy in acute cytotoxicity and clonogenic survival assays in vitro (Figure 12). This anti-glioma efficacy was also detected in the ex vivo model of patient derived microtumors (PDMs) (Figure 14). Treating VM/Dk mice harboring SMA560 tumors with Argyrin F led to a significant prolongation of time until onset of neurological symptoms in vivo (Figure 12Figure 13Figure 14). Interestingly, brains harboring SMA560 tumors that were treated with Argyrin F displayed an increased influx of T cells into the tumor tissue (Figure 13), highly suggestive for an immunogenic activation upon Argyrin F treatment. This hypothesis was further validated using HLA ligandome analyses that showed treatment induced changes upon Argyrin F treatment (Figure 15). Also, PDMs co-cultured with autologous TILs that were treated with Argyrin F displayed a significant induction of cytotoxicity read-out, combination therapy using Argyrin F together with the PD-1 checkpoint inhibitor Nivolumab could significantly increase the cytotoxicity read-out compared to either monotherapy (Figure 16). In an animal experiment that also tested the combination of Argyrin F together with PD-1 checkpoint inhibition, symptom onset was delayed by ten days in the combination therapy compared to the mono-therapeutic setting (Figure 17). ATR inhibition by AZD6738 and Berzosertib, respectively, lead to anti-glioma activity in vitro and displayed a modest phenotype in vivo (Figure 18, Figure 19, Figure 20). Further characterization of cells treated with ATR inhibition, showed differences in cell cycle regulation upon treatment (Figure 21). To elucidate these differences in more detail, transcriptomic analyses using bulk RNASeq were done and revealed shared, as well as distinctly regulated pathways (Figure 22). These findings could be validated using DigiWest protein profiling (Figure 23). Based on this, ATR inhibition was combined with standard therapy Temozolomide, the PARP inhibitor Olaparib and PI3K/mTOR inhibitors Paxalisib and Everolimus (Figure 24, Figure 25, Figure 26, Figure 27, Figure 28, Figure 29). The analyses lead to different synergism read-outs depending on the cell line used, which can be explained with the distinct transcriptomic and proteomic signatures identified. Lastly, genetic analyses of 216 glioblastoma patients revealed 23 germline mutations of which 9 were part of the FA pathway (Table 4). Somatically also an accumulation of FA mutations in GB patients could be detected (Figure 31). To elucidate the influence of FA mutations on GB development and/or propagation, five FA genes were used to model glioma development in the RCAS/tv-a mouse model in vivo. In one of the five genes a reduction of time until onset of neurological symptoms was seen (Figure 33). This was accompanied by a significantly higher level of Ki67 positive cell nuclei in the tumor tissue as well as histological features of high-grade glioma (Figure 34). An analysis of proliferation capacity of stable glioma cell lines did not lead to any significant changes upon FA knockdown (Figure 36), however, in clonogenic survival assays a significantly higher treatment sensitivity could be detected (Figure 38, Figure 39, Figure 40). Taken together, this thesis presents novel insights into vulnerabilities in experimental glioma and might inform future clinical trials.