Integrative analysis of therapy resistance and transcriptomic profiling data in glioblastoma cells identifies sensitization vulnerabilities for combined modality radiochemotherapy

Abstract

Background: Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance.

Methods: We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining.

Results: Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4-two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization.

Conclusions: Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Keywords: ATM; ATR; Chemosensitization; Clonogenic survival; Correlation analysis; DNA damage response; Glioblastoma; LIG4; Radiosensitization; Radiotherapy; Temozolomide; Therapy resistance.

Fig. 1

mRNA expression analysis of DNA damage response (DDR) regulator genes, and MGMT promoter methylation analysis in human GBM cell lines. A Basal mRNA expression levels of DDR regulator genes in GBM cell lines A172, LN18, LN229, T98G, U87-MG, U138-MG, and U251-MG as measured by qRT-PCR (ddCT method). Expression levels were normalized to a matrix of three reference genes (18S rRNA, β2-Microglobulin, and 5’-Aminolevulinate Synthase-1), and calibrated on the expression levels of astrocytes. Three replicates were analyzed per cell line. Expression values (log2-transformed) and samples were subjected to unsupervised hierarchical clustering. B Correlation of MGMT promoter methylation status and MGMT mRNA expression levels in the employed GBM cell lines as detected by methylome array and qRT-PCR, respectively. C Protein expression levels of MGMT in GBM cells as measured by western blot. Vinculin served as loading control

Fig. 2

Clonogenic survival of GBM cells upon treatment with X-ray IR, TMZ, and the combination thereof. A Clonogenic survival of A172, T98G, LN229, LN18, U251-MG, M138-MG, and U87-MG cells after single-shot IR (0–10 Gy), fractionated IR (0–5 fractions of 2 Gy each), TMZ treatment (0–500 μM for 24 h), and combination of 5 µM TMZ (24 h) and single-shot IR (0–10 Gy) as determined by colony formation assays. Results of at least three independent experiments are depicted. Super-imposed fitting functions are linear-quadratic in case of radiation experiments and logistic in case of TMZ treatment. B Z-transformed survival data are depicted as unsupervised hierarchical clustering. Principal component analysis (PCA)-derived resistance scores (scores of PC1 as described in [21]) are shown

Fig. 3

Correlation analysis of DDR mRNA expression levels with inherent treatment resistance scores. A Spearman's correlation analysis of DDR regulator mRNA expression levels (Fig. 1) with scores of inherent resistance (scores of PC1) to single-shot IR, fractionated IR, and TMZ treatment (Fig. 2). B PCA-Biplot of genes with |rho|> 0.4 for single-shot IR (left panel) and TMZ treatment (right panel)

Fig. 4

Validation of candidate genes by targeted inhibition and clonogenic survival assays. A Clonogenic survival of A172 and U251-MG cells after single-shot IR (0–10 Gy) and ATR inhibition by AZD-6738 (0–1.0 μM, upper panel). Immunofluorescence staining of γH2AX and 53BP1 20 h after irradiation at 4 Gy ± ATR inhibition by 1 µM AZD-6738 (lower panel). γH2AX is depicted in green, 53BP1 in red, and DNA is depicted in blue. Scale bar represents 10 µm. B Clonogenic survival of A172 and U251-MG cells after single-shot IR and LIG4 inhibition by L189 (0–50 μM, upper panel). Immunofluorescence staining of γH2AX and 53BP1 20 h after irradiation at 4 Gy ± LIG4 inhibition by 50 µM L189 as performed in A (lower panel). C Clonogenic survival of A172, U138-MG, and U251-MG cells after 24 h treatment with TMZ (0–200 μM) and ATM inhibition by KU-60019 (0–1.0 μM). Super-imposed fitting functions are linear-quadratic in case of radiation experiments and logistic in case of TMZ treatment. Results of three independent experiments are depicted for each condition, and p-values were calculated by two-way ANOVAs