A PDGFRα-driven mouse model of glioblastoma reveals a stathmin1-mediated mechanism of sensitivity to vinblastine

Glioblastoma multiforme (GBM) is an aggressive primary brain cancer that includes focal amplification of PDGFRα and for which there are no effective therapies. Herein, we report the development of a genetically engineered mouse model of GBM based on autocrine, chronic stimulation of overexpressed PDGFRα, and the analysis of GBM signaling pathways using proteomics. We discover the tubulin-binding protein Stathmin1 (STMN1) as a PDGFRα phospho-regulated target, and that this mis-regulation confers sensitivity to vinblastine (VB) cytotoxicity. Treatment of PDGFRα-positive mouse and a patient-derived xenograft (PDX) GBMs with VB in mice prolongs survival and is dependent on STMN1. Our work reveals a previously unconsidered link between PDGFRα activity and STMN1, and highlight an STMN1-dependent cytotoxic effect of VB in GBM.

For survival in H1703 cells, cells were placed into low serum media (0.1% Heat Inactivated FBS in DMEM), and then 24 hours later stimulated with 25 ng/mL PDGF-AA (Sigma Alrich) every 12 hours for 48 hours. Cells were then treated with either vehicle or 100nM vinblastine and counted 24 hours later for live and dead cells using trypan blue exclusion. Survival is reflected as the percentage of live cells relative to the number of total cells counted. Viability assays were conducted in biological triplicates.

CRISPR/Cas9-Mediated Knock Out of STMN1
The sequence of the sgRNA are as follows:

Gene Expression Analysis via qRTPCR
Cells pellets were isolated in biological triplicates, and total RNA was then isolated using the RNeasy Kit from Qiagen. Following RNA isolation, cDNA was then generated using Superscript III First Strand Synthesis Supermix (Thermo Fisher). Gene Expression was then analyzed using SYBR Green Real Time PCR with the following primers: 5'-TGCTGCTCCTCGGCTGCGGATA-3' and 5'-AAATGACCGTCCTGGTCTTGC-3' for the detection of Human PDGF-A and 5'-TTGGGGAGAGTGAAGTGAGCT-3' and 5'-GATGTAAATGTGCCTGCCTTC-3' for the detection of Human PDGFRα.

Immunofluorescence
Cells were cultured on chamber slides, placed into low serum media (0.1% Heat Inactivated FBS in DMEM) for 24 hours, and then 0, 0.1 or 10 µg/mL Doxycycline was added. After 48 hours, cells were treated with either vehicle or 100nM vinblastine. 24 hours later, cells were washed twice in PBS, fixed in cold methanol for 10 minutes at -20C, washed twice quickly in cold acetone, washed twice in PBS, and incubated with FITC conjugated Anti-α-Tubulin antibody (Thermo Fisher F2168) diluted 1:100 in PBS plus 3% BSA overnight at 4C. Cells were then washed in PBS and mounted with DAPI Slowfade Gold Antifade mounting media (Thermo Fisher). Images were acquired using a Zeiss LSM 880 Confocal Laser Scanning Microscope, and were analyzed and quantified using Image J Image Processing Software. At least 5 biological replicates were analyzed for each condition.

Analysis of co-occurrence
Co-occurance between PDGFRA amplification/overexpression and each mutation/deletion event that presenting in at least 5% of patients was examined by using Fisher's exact test. Overexpression cutoff of PDGFRA was determined by the 10% quantile of its expression in PDGFRA amplified patients. The gene expression, copy number variation, mutation call were obtained from the Broad GDAC Firehose (stddata__2015_06_01, https://gdac.broadinstitute.org/).

SUPPLEMENTARY MOVIE 1
Cells were treated with VB with and without hPDGFRα activation and imaged for 48 hours.

Supplementary Figure 3. PDGF-A expression in mice drives oligodendrocyte precursor cells (OPCs) proliferation. Ectopic CNS expression of PDGF-A in mice elicits a dose-dependent
proliferation of PDGFRα-positive OPCs 5, 6, 7 . Consistent with this view, the hPDGF-A;p53 2lox mice fed a high DOX diet displayed an oligodendrocytic histology and uniformly expressed (a) OPC markers PDGFRα, Olig2 8 and NG2 9 and were negative for GFAP. Representative photomicrographs of IHC from FFPE sections from hPDGF-A-Cre;p53 -/brains. Scale bar, 500 µm (H&E, OLIG2, Ki67), 250 µm (NG2, mPDGFRα), 200 µm (GFAP). (b) To substantiate the OPC nature of these cells, we profiled their transciptome of brains from hPDGF-A-Cre lentivirus injected p53 2lox mice fed a high DOX diet and performed a gene set enrichment analysis (GSEA) against profiles obtained from primary isolates of mouse astrocytes, neurons, OPCs, resting oligodendrocytes, stimulated oligodendrocytes, microglia and endothelial cells 10, 11 . The highest enrichment scores were to those of pure OPCs and oligodendrocytes, reinforcing the notion that these are composed of proliferating OPCs responding to