Core pathway mutations induce de-differentiation of murine astrocytes into glioblastoma stem cells that are sensitive to radiation but resistant to temozolomide

Abstract

Background: Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype.

Methods: To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling.

Results: Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal.

Conclusion: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.

Keywords: Astrocytes; glioblastoma; glioma stem cells; radiation; temozolomide.

Fig. 1.

TRP astrocytes acquire glioma stem cell features in vitro and in vivo. Neurosphere self-renewal of TRP astrocytes was indistinguishable from wild-type neural stem cells (P ≥ .1). Scale bar = 50 µm (A). Serum-induced differentiation of nestin⁺ TRP neurospheres into glial fibrillary acidic protein-positive astrocytes (B), NG2⁺ oligodendroglia, and Tuj1⁺ neurons (C). Scale bar = 10 µm. Primary TRP allografts were serially transplantable into secondary and tertiary host brains. Median survival (26–27 d) was indistinguishable (P = .8), and all mice developed glioblastoma (D). A cell dose-dependent (10²–10⁶) increase in median survival (23–45 d) was evident (P < .0001, E).

Fig. 2.

G1/S ablation and activating MAPK and PI3K mutations are required to induce efficient astrocyte de-differentiation and tumorigenesis. TRP astrocytes contained significantly more neurosphere-forming stem cells than T and TR astrocytes but less than neural stem cells (1 in 21) (Supplementary material, Fig. S2A) (A). Serum-induced differentiation of T, TR, and TRP neurospheres into glial fibrillary acidic protein-positive astrocytes, NG2⁺ oligodendroglia, and Tuj1⁺ neurons (B). TRP astrocytes developed into astrocytomas in all mice, but T and TR astrocytes failed to develop tumors in >70% (P < .0001, C). Mice injected with 10⁵ TRP astrocytes required significantly less time (26.0 ± 0.6 d) to develop tumors than T (345 ± 13.6 d) and TR (216 ± 10.6 d) astrocytes (P < .0001, D). T astrocyte tumors were significantly smaller (mean 0.4 ± 0.2 mm²) than TR (mean 7.4 ± 2.8 mm²) or TRP (mean 8.9 ± 0.6 mm²) astrocyte tumors (P = .016, E). N, number of mice examined; NL, normal; A2, diffuse astrocytoma (WHO grade II); A3, anaplastic astrocytoma (WHO grade III); GBM, glioblastoma (WHO grade IV). Scale bars = 10 µm.

Fig. 3.

TRP mutations induce genome-wide alterations in the chromatin landscape of astrocytes. Highly variable intervals (300 bp) of open chromatin were analyzed by hierarchical clustering. Regions of increased chromatin accessibility were enriched in TRP, T and TRP, TR, or wild-type astrocytes (AC) (A). Principal component analysis confirmed that chromatin landscapes of these cells were distinct (B). Representative genes from TRP-enriched (CD) and AC-enriched (E) regions are shown. Target motifs specific to particular transcription factor (TF) families were enriched in each class, but only the TRP class harbored motifs from multiple families of stem-cell TF (F).

Fig. 4.

TRP mutations induce downregulation of astrocyte markers and upregulation of stemness genes. Differential expression analysis showed that TRP mutations induced significant transcriptome alterations (10.9%, q < 0.001) relative to wild-type astrocytes (A), including downregulation of published astrocyte gene signatures (B, FDR < 0.01). Decreased expression of the astrocyte-specific gene Aqp4 (C) and increased expression of the Hoxa locus of stem cell transcription factors (D) anti-correlated with chromatin accessibility alterations within 70KB of their transcriptional start sites. An aggregated gene ontology analysis showed upregulation of cell growth and embryonic development (E) and downregulation of neural and cell adhesion-related processes (F). FDR values are colored; diameter indicates term frequency.

Fig. 5.

TRP astrocytes are sensitive to XRT but resistant to TMZ due to MGMT. TRP astrocytes and U87 cells were sensitive to XRT (P = .26) (A). U87 cells (IC₅₀ 21 µM, 95% CI: 13–33 µM) but not TRP astrocytes (IC₅₀ 647 µM, 95% CI: 409–1024 µM) were sensitive to TMZ (P < .0001) (B). MGMT levels were 3.5-fold higher in TRP astrocytes (P < .0001) (C). The MGMT inhibitor O⁶-BG produced a dose-dependent increase in TMZ sensitivity in TRP astrocytes (P < .0001) (D). TMZ IC₅₀ fold change from D is shown in E.

Fig. 6.

Radiation therapy (XRT), but not temozolomide (TMZ), is effective in TRP allografts and induces a proneural-to-mesenchymal transcriptome shift. XRT (33 days, P < .0001), but not TMZ (22 days, P = .8), significantly prolonged median survival of TRP allograft mice (21 d). Adding TMZ did not extend survival (32 d) relative to XRT alone (P = .5) (A). XRT produced a significant decrease in TRP allograft growth rate by longitudinal MRI (doubling time 3.3 vs 1.8 d, P = .002) (B). XRT induced a similar decrease in TRP-Luc allograft growth by longitudinal BLI (doubling time 35 vs 7.7 d, P < .0001) (C). Transcriptomes of control, untreated TRP allografts were most similar to human proneural (PN) glioblastoma (GBM) (P = .0005). XRT induced a shift to mesenchymal (MES) GBM (P = .01) rather than classical (CL) or neural (NL) subtypes (P ≥ .2).