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Glioblastoma multiforme continues to have a dismal prognosis. Even though detailed information on the genetic aberrations in cell signaling and cell-cycle checkpoint control is available, no effective targeted treatment has been developed. Despite the advanced molecular defects, glioblastoma cells may have remnants of normal growth-inhibitory pathways, such as the bone morphogenetic protein (BMP) signaling pathway. We have evaluated the growth-inhibitory effect of BMP4 across a broad spectrum of patient samples, using a panel of 40 human glioblastoma initiating cell (GIC) cultures. A wide range of responsiveness was observed. BMP4 sensitivity was positively correlated with a proneural mRNA expression profile, high SOX2 activity, and BMP4-dependent upregulation of genes associated with inhibition of the MAPK pathway, as demonstrated by gene set enrichment analysis. BMP4 response in sensitive cells was mediated by the canonical BMP receptor pathway involving SMAD1/5/9 phosphorylation and SMAD4 expression. SOX2 was consistently downregulated in BMP4-treated cells. Forced expression of SOX2 attenuated the BMP4 sensitivity including a reduced upregulation of MAPK-inhibitory genes, implying a functional relationship between SOX2 downregulation and sensitivity. The results show an extensive heterogeneity in BMP4 responsiveness among GICs and identify a BMP4-sensitive subgroup, in which SOX2 is a mediator of the response.

Tumor cell heterogeneity is a crucial characteristic of malignant brain tumors and underpins phenomena such as therapy resistance and tumor recurrence. Advances in single-cell analysis have enabled the delineation of distinct cellular states of brain tumor cells, but the time-dependent changes in such states remain poorly understood. Here, we construct quantitative models of the time-dependent transcriptional variation of patient-derived glioblastoma (GBM) cells. We build the models by sampling and profiling barcoded GBM cells and their progeny over the course of 3 weeks and by fitting a mathematical model to estimate changes in GBM cell states and their growth rates. Our model suggests a hierarchical yet plastic organization of GBM, where the rates and patterns of cell state switching are partly patient-specific. Therapeutic interventions produce complex dynamic effects, including inhibition of specific states and altered differentiation. Our method provides a general strategy to uncover time-dependent changes in cancer cells and offers a way to evaluate and predict how therapy affects cell state composition.

Glioblastoma (GBM) is a lethal disease and curative treatment is still lacking. BMP4 was initially suggested as a differentiation treatment of GBM, since it has been shown to induce astrocytic differentiation, but other reports have demonstrated that the response is reversible, variable among patient samples, and heterogeneous within the same cell line. To further interrogate the nature of the BMP4 proliferation-inhibitory response, we focused on cellular growth and senescence. BMP4 was found to induce a senescence-like phenotype in a SMAD-dependent manner in a subpopulation of GBM cells, demonstrated by induction of senescence-associated (SA)-β-gal, downregulation of lamin B1, as well as increased lysosomal mass, cell size and granularity, paralleled by an increase in p21 levels. To zoom in on the heterogeneity in BMP4 response within a cell culture, we used a therapy-sensitive/proneural-like (SENS/PN) clone and a therapy-resistant/mesenchymal-like (RES/MES) clone from the same tumor and saw a more prominent induction of a senescence-like phenotype in the RES/MES clone. Since the RES/MES clone showed higher basal levels of p21 and lower lamin B1 than the SENS/PN clone, we suggest that the expression of senescence markers is a component of the mesenchymal profile. Senolytic treatment ablated the SA-β-gal positive cells and normalized the p21 levels, and this enabled us to couple p21 upregulation to the senescence-like phenotype. In p21 knockout cells, BMP4-induced cell growth and SA-β-gal were abolished and lamin B1 was not down-regulated. SOX2, which in part mediates the inhibition of proliferation by BMP4, was down-regulated but still BMP4-treated p21 knockout cells proliferated faster than BMP4-treated wild-type cells. Altogether, this demonstrates that p21 signaling is crucial for BMP4-mediated induction of a senescence-like phenotype in GBM.

Glioblastoma (GBM) is a lethal disease with no curative treatment. SOX2 is a stem cell transcription factor which is widely expressed across human GBM tumors. Downregulation of SOX2 inhibits tumor formation and its depletion leads to a complete stop of cell proliferation. Despite its known important role in GBM, there is a lack of SOX2 overexpression studies in human GBM cells cultured under stem cell conditions. Previous work in our lab suggests that SOX2 levels need to be precisely maintained for GBM cells to thrive. In this project, we have investigated how altered SOX2 expression affects primary human GBM lines. We found that elevated SOX2 expression inhibited proliferation in a dose-dependent manner in three out of four GBM cell lines. Global gene expression in the resistant line was shifted towards that of the proliferation-inhibited lines upon SOX2 induction. However, SOX2 induction also led to an increase in a GBM stem cell injury response phenotype, which was not present in proliferation-inhibited lines. Furthermore, CRISPR/Cas9-mediated SOX2 knockout revealed a SOX2 independence in the resistant cell line, where SOX2-negative cells could be propagated both in vitro and in vivo.