Abstract Glioblastoma (GBM) is resistant to many therapies including immunotherapies. There is an urgent need to explore the role of the microenvironment in resistance to therapy with tailored pre-clinical models. Using multi-omic approaches we describe a syngeneic cancer stem cell mouse model of GBM, with a spontaneous amplification of Igf2. We investigate whether Igf2 influences tumour and microenvironment cells to promote immunosuppression in GBM. Three cell lines were previously established from spontaneous brain tumours in C57Bl/6 Trp53+/-/Nf1+/- mice and maintained under neural stem cell culture conditions. Whole genome sequencing (WGS) was used to determine single nucleotide, copy number, and structural variation, revealing loss of both copies of Trp53 and Nf1 in all three cell lines. However only one cell line, mBT0309, developed tumors when orthotopically allografted. Analysis of genomic alterations and transcriptomics revealed that mBT309 exhibits amplification of the Igf2 loci and overexpression was confirmed at the RNA and protein level. Spatial and single cell transcriptomics showed that Igf2 is overexpressed in mBT0309 allografted tumours and correlated with specific transcriptomic programs. A high-parameter Imaging Mass Cytometry (IMCTM) panel was used for spatial proteomic analysis, to monitor the development of tumours in a time-course experiment. In vitro growth characteristics and stem marker expression in both transcriptomic and spatial IMC data suggest that high levels of Igf2 may regulate GBM stemness features. Analysis of human GBM datasets revealed that Igf2 amplified tumours have reduced CXCL11 expression. These findings suggest that high levels of Igf2 may influence immunosuppression by reducing T-cell recruitment. This syngeneic immunocompetent GBM stem cell model harbouring Igf2 amplification adds to the suite of models to study the GBM microenvironment and its role in immunosuppression and resistance to therapy.

Abstract Using a newly described in vitro murine model of GBM [Bohm et al., Neuro-Oncol 2020 and Omairi et al., Neuro-Oncol 2023] in which P53 null neural progenitor cells (NPCs) divide abnormally and evolve a GBM-like genome during exposure to Platelet-Derived Growth Factor-AA (PDGFA), we asked how a brain-abundant mitogen could transform NPCs. By analyzing gene and protein expression over time, we found that PDGFA fails to induce the transcription of kinetochore and spindle assembly checkpoint genes, while simultaneously driving NPCs to enter mitosis. These dual effects caused chromosome miss-segregation in continuously dividing NPCs; moreover, they occurred in both WT and null NPCs, although only null cells survived defective mitosis. These surviving cells gradually expanded in PDGFA accumulating both random and clonal chromosomal re-arrangements. Transcriptome analysis of NPCs in PDGFA revealed significant under-expression of Foxm1, the major regulator of kinetochore transcription, together with over-expression and phosphorylation of the immediate early response gene, FOS. Analysis of signalling downstream of PDGFRα identified the Ras-MAPK pathway, especially ERK, as responsible for FOS activation. As surviving null cells gradually expanded, they accumulated random and recurrent chromosomal rearrangements. Expansion and subsequent PDGFA-independent proliferation and tumorigenicity were associated with re-expression of Foxm1 and kinetochore proteins, and accompanied by over-expression of Egfr, an RTK-signature that defines human GBM. By stimulating proliferation without setting the stage for error-free mitosis, exposure to PDGFA transforms p53 null NPCs and generates Egfr amplified GBM-like cancer cells.

Background Chromosome instability (CIN) with recurrent copy number alterations is a feature of many solid tumors, including glioblastoma (GBM), yet the genes that regulate cell division are rarely mutated in cancers. Here, we show that the brain-abundant mitogen, platelet-derived growth factor-A (PDGFA) fails to induce the expression of kinetochore and spindle assembly checkpoint genes leading to defective mitosis in neural progenitor cells (NPCs). Methods Using a recently reported in vitro model of the initiation of high-grade gliomas from murine NPCs, we investigated the immediate effects of PDGFA exposure on the nuclear and mitotic phenotypes and patterns of gene and protein expression in NPCs, a putative GBM cell of origin. Results NPCs divided abnormally in defined media containing PDGFA with P53-dependent effects. In wild-type cells, defective mitosis was associated with P53 activation and cell death, but in some null cells, defective mitosis was tolerated. Surviving cells had unstable genomes and proliferated in the presence of PDGFA accumulating random and clonal chromosomal rearrangements. The outcome of this process was a population of tumorigenic NPCs with recurrent gains and losses of chromosomal regions that were syntenic to those recurrently gained and lost in human GBM. By stimulating proliferation without setting the stage for successful mitosis, PDGFA-transformed NPCs lacking P53 function. Conclusions Our work describes a mechanism of transformation of NPCs by a brain-associated mitogen, raising the possibility that the unique genomic architecture of GBM is an adaptation to defective mitosis that ensures the survival of affected cells.

Diffuse, histologically lower grade astrocytomas of adults (LGAs) are classified based on the mutational status of the isocitrate dehydrogenase (IDH) genes. While wild-type (WT) LGAs often evolve quickly to glioblastoma (GBM), mutant tumors typically follow an indolent course. To find possible effectors of these different behaviors, we compared their respective transcriptomes. Unlike mutant LGAs, platelet-derived growth factor (PDGF) signaling was significantly enriched in WT tumors, and PDGFA was the top overexpressed gene in the pathway. Moreover, methylation of the PDGFA and PDGFD promoters emerged as a possible mechanism for their low expression in mutant tumors. Copy number gain of chromosome 7 co-occurred with high expression of PDGFA in WT cases, and high expression of PDGFA was associated with aneuploidy, extracellular matrix (ECM)-related immunosuppressive features and poor prognosis. We also noted that high PDGFA expression in WT cases occurred irrespective of tumor grade and that multiple mechanisms of p53 pathway inactivation accompanied progression to GBM in PDGFA-overexpressing tumors. Conversely, TP53 point mutations were an early and constant feature of mutant LGAs. Our results suggest that members of the PDGF gene family, in concert with different p53 pathway alterations, underlie LGA behaviors.

INTRODUCTION:Temozolomide (TMZ) is a life prolonging DNA alkylating agent active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately acquired TMZ resistance severely undermines its clinical efficacy. Using an in vitro model, we tested whether poly (ADP-ribose) polymerase-1 and -2 (PARP) inhibition could suppress the emergence of resistance to enhance the effectiveness of TMZ.METHODS:Using the MGMT-methylated GBM line U251N, in which TMZ resistance can be induced, we developed a method to rapidly recreate mechanisms of TMZ resistance seen in GBMs, including MMR mutations and MGMT re-expression. We then assessed whether TMZ resistant U251N sub-clones could be re-sensitized to TMZ by co-treatment with the PARP inhibitor ABT-888, and also whether the emergence of resistance could be suppressed by PARP inhibition.RESULTS:U251N cultures chronically exposed to TMZ developed discrete colonies that expanded during TMZ treatment. These colonies were isolated, expanded further as sub-clones, and assessed for mechanisms of TMZ resistance. Most resistant sub-clones had detectable mutations in one or more mismatch repair (MMR) genes, frequently MSH6, and displayed infrequent re-expression of MGMT. TMZ resistance was associated with isolated poly(ADP-ribose) (pADPr) up-regulation in one sub-clone and was unexplained in several others. TMZ resistant sub-clones regressed during co-treatment with TMZ and ABT-888, and early co-treatment of U251N parental cultures suppressed the emergence of TMZ resistant colonies.CONCLUSION:In a model of acquired resistance, co-treatment with TMZ and a PARP inhibitor had two important benefits: re-sensitization of TMZ resistant cells and suppression of TMZ resistance.