BMP4 and Temozolomide Synergize in the Majority of Patient-Derived Glioblastoma Cultures

Abstract

One of the main causes of poor prognoses in patient with glioblastoma (GBM) is drug resistance to current standard treatment, which includes chemoradiation and adjuvant temozolomide (TMZ). In addition, the concept of cancer stem cells provides new insights into therapy resistance and management also in GBM and glioblastoma stem cell-like cells (GSCs), which might contribute to therapy resistance. Bone morphogenetic protein-4 (BMP4) stimulates astroglial differentiation of GSCs and thereby reduces their self-renewal capacity. Exposure of GSCs to BMP4 may also sensitize these cells to TMZ. A recent phase I trial has shown that local delivery of BMP4 is safe, but a large variation in survival is seen in these treated patients and in features of their cultured tumors. We wanted to combine TMZ and BMP4 (TMZ + BMP4) therapy and assess the inter-tumoral variability in response to TMZ + BMP4 in patient-derived GBM cultures. A phase II trial could then benefit a larger group of patients than those treated with BMP4 only. We first show that simultaneous treatment with TMZ + BMP4 is more effective than sequential treatment. Second, when applying our optimized treatment protocol, 70% of a total of 20 GBM cultures displayed TMZ + BMP4 synergy. This combination induces cellular apoptosis and does not inhibit cell proliferation. Comparative bulk RNA-sequencing indicates that treatment with TMZ + BMP4 eventually results in decreased MAPK signaling, in line with previous evidence that increased MAPK signaling is associated with resistance to TMZ. Based on these results, we advocate further clinical trial research to test patient benefit and validate pathophysiological hypothesis.

Keywords: bone morphogenetic proteins; drug synergy; glioblastoma; temozolomide; therapy.

Figure 1

Combination therapy TMZ + BMP4 is generally more effective than sequential therapy: (a) schematic representation of the treatment protocol per day, starting on day-1 (D-1); (b) mean cell viability with standard deviation (n = 3) on day 7 of treatment normalized to untreated cells. ** p < 0.01, *** p < 0.001.

Figure 2

GBM cell culture viability after treatment with temozolomide (TMZ) and BMP4. Average cell viability (n = 3) in cultures GS755 (a), GS838 (b), and GS786 (c) after 7 days of treatment with a 3-fold dilution ranging from 180 ng down to 0.74 ng BMP4/mL (blue) and 360 µM down to 1.5 µM TMZ (red) and a combination of both (yellow). The lowest dose is depicted in white (1.5 µM TMZ; 0.75 ng BMP4/mL) increasing (4.5 µM/2.25 ng/mL; 13.5 µM/6.8 ng/mL; 40 µM/20 ng/mL; 120 µM/60 ng/mL) to the highest dose depicted in dark grey (360 µM TMZ and 180 ng BMP4/mL). (d) Heat map of HSA score per dose and culture. A low HSA score (antagonism) is depicted in blue and a high score (synergy) is shown in red. The bars above the figure represent the sensitivity (black) or resistance (white) to TMZ (EC50 > 100 µM) and BMP4 (EC50 > 60 ng/mL).

Figure 3

Effects of temozolomide (TMZ) and BMP4 on GBM culture cell proliferation and apoptosis. Average percentage of proliferating cells (EdU+) (a) and apoptotic cells (annexin-V+) (b) with standard deviation (n = 3) in untreated condition (gray) and after treatment with TMZ (red), BMP4 (blue), and TMZ + BMP4 (yellow). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. No further specified comparisons were considered statistically significant.

Figure 4

Venn diagram of DEGs (p < 0.01, log FC > 1.5) treated compared with untreated cells per culture, where upregulated genes are red and downregulated cells are blue.

Figure 5

Exclusive differentially expressed genes (DEGs) after monotherapy with BMP4. (a) Heatmap of the top 10 most up- (red) and downregulated (blue) genes after treatment with TMZ, BMP4, and TMZ + BMP4, which are unique for monotherapy with BMP4, per sequenced culture. (b) Overlap of genes that were uniquely differentially expressed after monotherapy with BMP4 per culture. Purple lines linking identical genes. (c) Heat map of gene set enrichment analysis of DEGs unique after BMP4 monotherapy.

Figure 6

Exclusive differentially expressed genes (DEGs) after combination therapy with TMZ and BMP4. (a) Heat map of the top 10 most upregulated (red) and downregulated (blue) genes after treatment with TMZ, BMP4, and TMZ + BMP4, which are unique for combination therapy of TMZ + BMP4, per sequenced culture. (b) Overlap of genes that were uniquely differentially expressed after monotherapy with BMP4 per culture. Purple lines link identical genes and blue lines link genes that belong to similar enriched ontology terms. Genes marked in red are common between GS627 and GS838, green between GS627 and GS755, and black between GS755 and GS838. (c) Heatmap of gene set enrichment analysis of unique DEGs after treatment with BMP4 and TMZ.