Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM

Abstract

Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

Figure 1

(A) Schematic diagram of a model to explain TMZ-response in GBM neoplastic cells on the perspective of clonal heterogeneity. (B) The experimental design for TMZ-treatment and post-treatment recovery of GBM neurospheres in vitro to address this hypothetical model of TMZ-response.

Figure 2. Radiological images of responder GBM patient A49910 and MGMT mRNA expressions of 6 GBM patient-derived tumor biopsy tissues and isolated neurospheres.

(a), Diagnostic MRI scan of the brain showing a space-occupying lesion (SOL) in insular cortex in A49910. (b), Postoperative CT scan showing gross total resection in A49910. (c), MRI scan image after the 2^nd cycle of chemotherapy showing recurrence of the tumor in A49910. (d), Follow-up scan after 6 cycles of chemotherapy showing decrease in the size of the lesion in A49910. qRT-PCR estimation of MGMT mRNA expression relative to GAPDH mRNA expression in (e), GBM tumor biopsies and (f), in corresponding neurospheres isolated from the tumors. (^✶ p-value < 0.05, ^✶✶ p-value < 0.01 and ^✶✶✶ p-value < 0.001).

Figure 3

(A) Light microscopic images (200 μm bar) and viable cell count of the neurospheres following TMZ treatment and post-treatment recovery. (a), DMSO treated control and (b), 5 days TMZ-treated neurospheres in A49910. (c), % viable cells in A49910 with DMSO- and 5 days of TMZ- treatment. (d), DMSO-treated control and (e), 5 days TMZ-treated neurospheres in M45481. (f), % viable cells in M45481 with DMSO- and 5 days of TMZ- treatment. (g), DMSO-treated control and (h), 28 days of post-TMZ-treated neurospheres in A49910. (i), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in A49910. (j), DMSO treated control and (k), 28 days post-TMZ-treated neurospheres in M45481. (l), % viable cells of DMSO-treated controls and 28 days of post-TMZ-treated cells in M45481. (^✶ p-value < 0.05, ^✶✶ p-value < 0.01 and ^✶✶✶ p-value < 0.001). (B) Growth curves (MTS assay) following 5 days of TMZ treatment and 28 days of post-treatment recovery. (a), A49910 DMSO-treated control and 5 days of TMZ-treated cells; (b), M45481 DMSO-treated control and 5 days of TMZ-treated cells; (c), A49910 DMSO-treated control and 28 days of post-TMZ-treated cells; (d), M45481 DMSO-treated control and 28 days of post-TMZ-treated cells. (C), flow cytometry analysis of apoptosis using annexin V and propidium iodide staining. (a), DMSO-treated A49910 cells, (b), 5 days of TMZ-treated A49910 cells, (c), DMSO treated M45481 cells and (d), 5 days of TMZ-treated M45481 cells. (D) Tracking cell division by CFSE staining. (a), growth arrest of TMZ-treated A49910 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated control (blue line). The TMZ-treated A49910 cells remained arrested till day 28 post-treatment recovery (pink line) while the DMSO-treated control cells proliferated (green line). (b), Growth arrest in TMZ-treated M45481 cells at day 5 of treatment (yellow line) compared to corresponding DMSO-treated controls (blue line). At day 28 post-treatment recovery one subpopulation of TMZ-treated M45481 cells showed growth arrest while other subpopulation proliferated (pink line) showing a staining intensity almost similar to their corresponding DMSO-treated control cells (green line).

Figure 4

(A) Light microscopic images (50 μm bar) at single cellular level. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (B) Light microscopic images (50 μm bar) of the cells showing SA-β-Gal staining following TMZ treatment and post-treatment recovery. (a), day 5 DMSO-treated control cells of A49910, (b), day 28 DMSO-treated control cells of A49910. (c), day 5 DMSO-treated control cells of M45481, (d), day 28 DMSO-treated control cells of M4548, (e), day 5 TMZ-treated cells of A49910, (f), day 28 post-TMZ-treated cells of A49910, (g), day 5 TMZ-treated cells of M45481, and (h), day 28 post-TMZ-treated cells of M45481. (C) VAFs showing biphasic trends where the first phase is the comparison of VAFs between C5 to T5 and second phase is the comparison of VAFs between T5 to T28. Red arrows are showing enriching VAFs in upward direction, blue showing downward direction and black showing no significant change. The green and purple horizontal bars on the right side represent the number of genes (N) in each category in A49910 and in M45481 respectively. (D) Sanger sequencing chromatogram showing a G to A transition on STK36 gene (arrow mark) in M45481 (lower panel) but not in A49910 (upper panel). (E) Showing mRNA expression patterns of Hh-pathway component genes, STK36 (a), GLI1 (b), GLI2 (c), GLI3 (d), Hh-pathway target gene SNAI1 (e) and MGMT (f) following TMZ-treatment and post-treatment recovery (C5, DMSO treated control; T5, day-5 TMZ-treated; T28, day-28 post-treatment recovery, ^✶p-value < 0.05, ^✶✶p-value < 0.01 and ^✶✶✶p-value < 0.001).

Figure 5

(a), mRNA expressions of GLI1, SNAI1and MGMT in 6 neurospheres. (b), Correlation matrix of 11 Hh-pathway component genes and MGMT (|r| = 0.96 FDR 0.1) on 5 cells except M45481. (c), Regression model fitting with the expressions of GLI1 with MGMT and (d), SNAI1 with MGMT. Red dots representing non-responder cells and black dots responder cells.

Figure 6. Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere.

a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). (^✶ p-value < 0.05, ^✶✶ p-value < 0.01 and ^✶✶✶ p-value < 0.001).