39 40 41 42 43 Running title: Novel mutations in TOP2A in gliomas 44 45 46 47 48 High grade gliomas (HGGs) are aggressive, primary brain tumors with poor clinical outcomes. 49To better understand glioma pathobiology and find potential therapeutic susceptibilities, we 50 designed a custom-panel 664 cancer-and epigenetics-related genes and employed targeted 51 next generation sequencing to study the genomic landscape of somatic and germline variants in 52 182 glioma samples of different malignancy grades. Besides known alterations in TP53, IDH1, 53 ATRX, EGFR genes, we found several novel variants that can be potential drivers in gliomas. In 54 four patients from the Polish glioma cohort, we identified a novel recurrent mutation in the 55 TOP2A gene coding for Topoisomerase 2A in glioblastomas (GBM, WHO grade IV gliomas). 56The mutation results in a substitution of glutamic acid (E) 948 to glutamine (Q) of TOP2 A and 57we predicted this E948Q substitution may affect DNA binding and a TOP2A enzymatic activity. 58Topoisomerases are enzymes that control the higher order DNA structure by introducing 59 transient breaks and rejoining DNA strands. Using recombinant proteins we demonstrated 60 stronger DNA binding and DNA supercoil relaxation activities of the variant proteins. 61 Glioblastoma (GBM) patients with the mutated TOP2A had shorter overall survival than wild 62 type TOP2A GBM patients. Computational analyses of transcriptomic data showed that the 63 GBM samples with the mutated TOP2A have different transcriptomic patterns suggesting higher 64 transcriptomic activity. The results suggest that TOP2A E948Q variant strongly binds to DNA 65 and is more active in comparison to the wild-type protein. Altogether, our findings suggest that 66 the E948Q substitution leads to gain of function by TOP2A. 67 68 69 4

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Glioblastomas (GBM) are the most common, primary brain tumors in adults. Despite advances in neurosurgery and radio- and chemotherapy, the median survival of GBM patients is 15 months. Recent large-scale genomic, transcriptomic and epigenetic analyses have shown the cellular and molecular heterogeneity of GBMs, which hampers the outcomes of standard therapies. We have established 13 GBM-derived cell cultures from fresh tumor specimens and characterized them molecularly using RNA-seq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1R132H, TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), and the expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, β-Tubulin III) markers revealed the striking intertumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at the mRNA/protein levels suggested increased epithelial-to-mesenchymal transition (EMT) in most studied cell cultures. The effects of temozolomide (TMZ) or doxorubicin (DOX) were tested in three GBM-derived cell cultures with different methylation status of the MGMT promoter. Amongst TMZ- or DOX-treated cultures, the strongest accumulation of the apoptotic markers caspase 7 and PARP were found in WG4 cells with methylated MGMT, suggesting that its methylation status predicts vulnerability to both drugs. As many GBM-derived cells showed high EGFR levels, we tested the effects of AG1478, an EGFR inhibitor, on downstream signaling pathways. AG1478 caused decreased levels of phospho-STAT3, and thus inhibition of active STAT3 augmented antitumor effects of DOX and TMZ in cells with methylated and intermediate status of MGMT. Altogether, our findings show that GBM-derived cell cultures mimic the considerable tumor heterogeneity, and that identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations.