The role of BLM and RecQL4 helicases in defining the response of glioma cells to chemotherapeutics : PhD thesis

Wojnicki, Kamil

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The role of BLM and RecQL4 helicases in defining the response of glioma cells to chemotherapeutics : PhD thesis

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Title: The role of BLM and RecQL4 helicases in defining the response of glioma cells to chemotherapeutics : PhD thesis

Abstract:

Malignant gliomas are primary tumours of the central nervous system. They remain one of the hardest to treat brain tumours due to their invasive phenotype, the immunosuppressive microenvironment and anatomical localisation in vital areas of the brain. The intratumoural heterogeneity and overexpression of enzymes involved in DNA replication and repair, impair the effectiveness of commonly used therapies, leading to an inevitable relapse of the tumour.The RecQ helicases are considered ‘guardians of the genome’, as they play the fundamental roles in DNA replication, repair and maintaining genome stability. The RecQ family is composed of several structurally related helicases, including BLM (Bloom Syndrome) helicase. The involvement of RecQ helicases in tumorigenesis and responses to therapies in malignant brain tumours is not fully understood, thus we aimed to elucidate the role of BLM in these processes.The Cancer Genome Atlas (TCGA) dataset analyses and immunostaining of numerous tumour sections demonstrated that BLM is overexpressed in high grade gliomas (WHO grade 3 and 4). In malignant gliomas BLM localisation was detected in the cytoplasm whereas in benign tumours BLM was present mostly in the nuclear compartment. High BLM levels were detected in several glioma cell lines and primary patient derived cultures. To decipher the role of BLM in gliomas, BLM deficient LN18 and LN229 cells were generated using CRISPR/Cas9 genome editing technology. BLM deficiency (KO) had minor effects on basal cell viability and proliferation. However, the cell responses to chemotherapeutics, in particular to the combination of temozolomide (TMZ, a DNA methylating agent common used in glioblastoma therapy) and olaparib (OLA, a PARP inhibitor) were changed. Unexpectedly, BLM KO cells were more resistant to the combined treatment than wild type (WT) cells that underwent apoptosis. Moreover, this effect was exclusive for TMZ combined with PARP inhibitors. The BLM KO cells were not sensitive to PARP inhibitors, in contrast to expected the synthetic lethality. BLM KO cells displayed the therapy induced cellular senescence (LN229) or polyploidy (LN18). The polyploidy in p53-deficient LN18 cells was reversed by forced p53 expression. Interestingly, RecQL4-depleted LN18 and LN229 cells responded to TMZ+OLA similarly to WT cells which indicates specialised and non-overlapping functions of RecQ family helicases.To obtain more insights into the functions of distinct RecQ helicases we generated blm and recql4 knock-out in Xenopus frogs to evaluate roles of the helicases in the embryonic development. Considerable mortality of tadpoles was noted and helicase-depleted tadpoles displayed morphological and functional developmental abnormalities.Altogether, the present study demonstrates important and specific functions of BLM and RecQL4 helicases in glioma cell responses to chemotherapy. As BLM expression was highly elevated and inversely correlated with survival of patients with malignant gliomas, we postulate that BLM could be a therapeutic target. The assessment of BLM levels, p53 and MGMT, a DNA repair enzyme, status might be helpful in choosing the proper therapy against malignant gliomas