Cytotoxicity of DCA against IDH1 wild-type and mutant cells for the U87 (A) and NHA (B) models (* indicates significant difference (p<0.05) relative to control).

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.

Effect of 2-HG on cell proliferation of IDH1 wild-type cells for the U87 (A) and NHA (B) models.

Comparison of PDH activity in the various models used in this study.

Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.

Abstract BACKGROUND Imagining ways to prevent or treat glioblastoma (GBM) have been hindered by a lack of understanding of its pathogenesis. Although platelet derived growth factor-A (PDGFA) overexpression may be an early event, critical details of the biology of GBM, and tools to study its initiation have been lacking. Indeed, many PDGF-driven models replicate its microscopic appearance, but not genomic architecture. Recently, we reported an in vitro model of GBM initiation that overcomes this barrier to authenticity. METHODS We used a method developed to establish neural stem cell cultures to investigate the effects of PDGF-A on cells derived from the subventricular zone (SVZ), a putative region where the cells of origins for GBM are derived. We micro-dissect SVZ tissue from p53-null and wild-type adult mice, culture cells in media supplemented with PDGF-A, and assess cell viability, proliferation, mitotic capacity, and genome stability. RESULTS Paradoxical to its canonical role as a growth factor, we observe abrupt and substantial cell death in PDGF-A. Abnormal mitosis was the first observable alteration and occurred immediately in cells of both p53 wild-type and null genotypes: wild-type cells did not survive in PDGF-A, whereas a fraction of null cells evade apoptosis. Evading cells displayed attenuated proliferation accompanied by early chromosomal gains and losses. After approximately 100 days in PDGF-A, surviving cells suddenly proliferate rapidly, acquire growth factor independence, and become tumorigenic in immune-competent mice. Transformed cells continue to display highly abnormal mitotic phenotypes with complex karyotypes similar to GBM, had a neural progenitor cell (NPC) lineage profile, and were resistant to PDGFR-alpha inhibition. CONCLUSION Abnormal mitosis induced by PDGF-A initiates and perpetuates the genome instability that transforms p53-null neural progenitor cells to yield cancers with the types of recurring chromosomal gains and losses that characterize human GBM.

Glioblastomas (GBMs) are aggressive primary malignant brain tumors characterized by extensive levels of inter- and intra-tumor genetic and phenotypic heterogeneity. Patient-derived organoids (PDOs) have recently emerged as useful models to study such heterogeneity. Here, we present bulk exome as well as single-cell genome and transcriptome profiles of primary IDH wild type GBMs from ten patients, including two recurrent tumors, as well as PDOs and brain tumor-initiating cell (BTIC) lines derived from these patients. We find that PDOs are genetically similar to and variably retain gene expression characteristics of their parent tumors. At the phenotypic level, PDOs appear to exhibit similar levels of transcriptional heterogeneity as their parent tumors, whereas BTIC lines tend to be enriched for cells in a more uniform transcriptional state. The datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived organoids, especially in the context of studying cellular heterogeneity.

Glioblastomas (GBMs) that occur in adults are well described but how they begin—the earliest event—is still unknown, making it difficult to envision preventative or risk reduction strategies. Several observations hint that platelet-derived growth factor (PDGF) signaling,1,2 especially by PDGFA, might cause isocitrate dehydrogenase (IDH) wild-type (WT) GBM. Ozawa et al. used mathematical modeling to predict that PDGFA initiates IDH-WT GBM3 and murine models confirm that PDGFA can induce GBM-like cancers.3,4 Recently, we reported that p53-null sub-ventricular zone murine cells cultured chronically in PDGFA acquire gains and losses of whole chromosomes and become tumorigenic,5 reproducing the types of genomic alterations that occur in human IDH-WT GBM; a phenomenon that does not occur when cultures are supplemented with other GBM-associated growth factors. Although these findings demonstrate that exposure to PDGFA can transform neural progenitor cells, an in situ source for overexpression of PDGFA has not been identified and is unexplained by PDGFA gene mutations, which are rare in GBM.6 Chromosome 7 amplification, a hallmark of IDH-WT GBM,7 is a potential source of PDGFA, but Ozawa et al. suggested that overexpression of PDGFA precedes not follows gain of 7.3 Following up on their hypothesis, we analyzed PDGFA expression in WHO grade II-IV IDH-WT astrocytic gliomas reasoning that GBMs (ie, grade IV), like most adult cancers, evolve over time and that grade II and III tumors offer a rare glimpse into their beginning; rare, because most IDH-WT GBMs appear suddenly after an indolent phase that is usually hidden from view. We assessed the relative expression levels of PDGFA by comparing IDH-WT astrocytomas from The Cancer Genome Atlas (TCGA) glioma dataset to normal brain from the GenotypeTissue Expression (GTEx) portal using the TCGAbiolinks R package. In contrast to normal brain, median PDGFA expression is high in all grades of astrocytoma and not limited to GBM (Fig. 1A). To explore the relationship between histological grade and amplification of chromosome 7, copy number values by chromosomal arm processed by GISTIC2 on TCGA SNP6 arrays were downloaded from Broad GDAC Firehose. Values for 7p were utilized to determine amplification status based on a threshold of +0.3. We found that the percentage of patients with chromosome 7p amplification increased with grade (Fig. 1B). In grade II IDH-WT astrocytomas, harbingers of WT GBM, PDGFA overexpression was a frequent finding, whereas amplification of 7p was not. What then does amplification of chromosome 7 signify? PDGFA-induced genomic instability, as reported by our group,5 might select for gain of 7 if it increases cellular fitness. To explore this thinking, we assessed EGFR expression in relation to chromosome arm 7p. EGFR is relevant in this context for two reasons: it is overexpressed in most IDH-WT GBMs,6 and like PDGFA, is located on chromosome 7p. Unlike PDGFA, however, we found that EGFR expression increased significantly with histological grade (Fig. 1A). Indeed, increasing expression of EGFR and gain of 7p emerged as associated progressive events (P < .05, Wilcoxon rank-sum test), whereas overexpression of PDGFA was often present at the earliest visible stages of IDH-WT tumors. If PDGFA is central to the genesis of IDH-WT GBM by inducing genomic instability in progenitor cells, where does it come from, when and how is it overexpressed, and how might research proceed? Learning more about PDGFA biology could be a fruitful line of inquiry. Does PDGFA originate in GBM cells or from cells that surround the cell of origin of GBM? Moreover, since the incidence of IDH-WT GBM increases with age, does altered PDGFA signaling or overexpression occur as the brain ages? Such questions about PDGFA and other members of the PDGF family of ligands and receptors, several of which have been associated with cancers of the central nervous system in adults and children,1,8 merit further study. Any prospect for reducing the risk of GBM surely lies in understanding its beginning. Although preventing GBM may seem fanciful, curing the full-blown disease is equally difficult to imagine.

BACKGROUND:Imagining ways to prevent or treat glioblastoma (GBM) has been hindered by a lack of understanding of its pathogenesis. Although overexpression of platelet derived growth factor with two A-chains (PDGF-AA) may be an early event, critical details of the core biology of GBM are lacking. For example, existing PDGF-driven models replicate its microscopic appearance, but not its genomic architecture. Here we report a model that overcomes this barrier to authenticity.METHODS:Using a method developed to establish neural stem cell cultures, we investigated the effects of PDGF-AA on subventricular zone (SVZ) cells, one of the putative cells of origin of GBM. We microdissected SVZ tissue from p53-null and wild-type adult mice, cultured cells in media supplemented with PDGF-AA, and assessed cell viability, proliferation, genome stability, and tumorigenicity.RESULTS:Counterintuitive to its canonical role as a growth factor, we observed abrupt and massive cell death in PDGF-AA: wild-type cells did not survive, whereas a small fraction of null cells evaded apoptosis. Surviving null cells displayed attenuated proliferation accompanied by whole chromosome gains and losses. After approximately 100 days in PDGF-AA, cells suddenly proliferated rapidly, acquired growth factor independence, and became tumorigenic in immune-competent mice. Transformed cells had an oligodendrocyte precursor-like lineage marker profile, were resistant to platelet derived growth factor receptor alpha inhibition, and harbored highly abnormal karyotypes similar to human GBM.CONCLUSION:This model associates genome instability in neural progenitor cells with chronic exposure to PDGF-AA and is the first to approximate the genomic landscape of human GBM and the first in which the earliest phases of the disease can be studied directly.

ABSTRACTLow grade astrocytomas (LGAs) are classified based on the mutational status of the isocitrate dehydrogenase (IDH) gene. While IDH wild-type (WT) LGAs evolve rapidly to glioblastoma, mutant tumors generally have a more indolent course. To identify potential drivers of the differential progression of LGAs, we analyzed transcriptomes from The Cancer Genome Atlas. Compared to mutant LGAs, platelet-derived growth factor (PDGF) signaling is enriched in WT cases, andPDGFAis the top overexpressed gene in the pathway. Putative mechanisms for differentialPDGFAexpression included copy number gains of chromosome 7 in WT cases and methylation of thePDGFApromoter in mutant LGAs. Additionally, we found that highPDGFAexpression is associated with aneuploidy, immunosuppressive features, and worse prognosis, and that WT LGAs use multiple means to inactivate the p53 pathway to progress to GBM. Our work highlights the contribution of PDGF gene family towards the unique behaviour of LGAs.STATEMENT OF SIGNIFICANCEThis study of gene expression in LGAs suggests that differential regulation of the PDGF pathway may underlie the different natural histories ofIDHWT andIDHmutant LGAs including divergent evolutionary trajectories to GBM. This insight may inspire new therapeutic strategies to suppress the transformation of LGAs to higher-grade cancers.

Gliomas with wild type (WT) isocitrate dehydrogenase (IDH) are considerably more aggressive than those with mutant IDH. To identify putative drivers of the distinct progression trajectories of IDH WT and mutant disease, we analyzed transcriptomes of lower grade astrocytomas (LGAs; grade 2–3) with retention of 1p and 19q from The Cancer Genome Atlas (TCGA; n = 347). Compared to IDH mutant LGAs, we found that PDGF signaling was significantly enriched in IDH WT LGAs and that PDGFA was the top overexpressed gene in this pathway. We identified copy number gains of chromosome 7 in WT LGAs, and methylation of the PDGFA promoter in mutant LGAs, as candidate mechanisms for the differential expression of PDGFA. High PDGFA expression and low PDGFA promoter methylation were significantly associated with poor survival in all LGAs. We also found that PDGFA expression was positively associated with aneuploidy and extracellular matrix-related immunosuppressive features in WT LGAs, underscoring the role of PDGFA as a secreted mitogen. Finally, we show that the proportion of p53 pathway mutations increase significantly with grade in IDH WT gliomas. Taken together, our findings suggest that IDH WT LGAs evolve to higher grades, and ultimately to GBM, by progressive inactivation of the p53 pathway - functioning in concert with a background of increased PDGFA expression. These data emphasize the scope of genomic reprogramming that occurs in gliomas in relation to IDH mutations and further highlight the role of PDGFA in glioma formation, progression, and prognosis. Going forward, this work provides critical biological insight that may inspire new therapeutic strategies to suppress the transformation of IDH WT LGAs to higher-grade cancers.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

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.

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligoden-droglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neuro-development and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.

Abstract IDH-wildtype GBM is the most common variant of this cancer and occurs in older adults. Unfortunately patients’ tumors are either inherently resistant to standard treatment, which includes radio- and chemo-therapy, or acquire resistance during the therapeutic process. Additionally, although effective in other cancers, targeted therapies have yielded disappointing results in GBM, perhaps because the fully developed disease has significant cellular and molecular heterogeneity, allowing the tumour to adapt to treatments. Better insight into managing GBM might result from a detailed knowledge of its initiating events, which have not yet been elucidated. With this in mind, we developed a mouse model of GBM in which the earliest stages can be studied. This ex vivo model recreates GBM by culturing subventricular zone (SVZ) cells, the putative ‘cell of origin’ of GBM in platelet-derived growth factor A (PDGFA). Under this condition SVZ cells from p53 null mice transform, becoming exogenous growth factor independent and tumorigenic in immune-competent mice. In contrast, wildtype SVZ cells do not proliferate in PDGFA and null cells in EGF/FGF do not transform. To discover why p53 null SVZ cells uniquely transform in PDGFA, we performed array comparative genomic hybridization (aCGH) on cells before and after transformation in PDGFA and whole genome sequencing (WGS) on transformed cells and tumours generated from PDGFA-transformed cells. aCGH and WGS revealed that the genomic landscape of transformed cells displayed a striking similarity to that observed in primary human GBM. Specifically, these studies showed that chromosomal alterations are a hallmark of culturing SVZ cells in PDGFA, an intriguing finding considering GBM is also characterized by a specific landscape of copy number alterations. This model may resemble the pathogenesis of human GBM and be leveraged to investigate the early stages of tumorigenesis, further leading to the development of preventative strategies and novel therapeutics.

Background Imagining ways to prevent or treat glioblastoma (GBM) have been hindered by a lack of understanding of its pathogenesis. Although PDGF-AA overexpression may be an early event, critical details of the core biology are lacking. Existing PDGF-driven models replicate its microscopic appearance but not the genomic architecture characteristic of the human disease. Here we report a new model of GBM that overcomes this barrier to authenticity. Methods Using a method developed to study neural stem cells, we investigated the effects of PDGF-AA on subventricular zone (SVZ) cells, the putative cell of origin of GBM. We micro-dissected SVZ tissue from p53-null and wild-type adult mice, established primary cultures in media supplemented with PDGF-AA, and assessed cell viability, proliferation, genome stability, and tumour forming potential. Results Counterintuitive to its canonical role as a growth factor, we observed immediate and massive death of SVZ cells in PDGF-AA. Wild-type cells did not survive in PDGF-AA. However, a small fraction of null cells evaded apoptosis, displayed attenuated proliferation, gradually accumulated whole chromosome gains and losses, and, signalled by sudden rapid proliferation and growth factor independence, became tumorigenic in immune-competent syngeneic mice. Transformed cells had an OPC-like profile, were resistant to PDGFR-α inhibition, and harboured highly abnormal karyotypes similar to those seen in human GBMs. Conclusion This model associates genome instability in SVZ cells with chronic exposure to PDGF-AA; it is the first model to replicate the genomic landscape of GBM and first in which the earliest phases of GBM can be directly observed. IMPORTANCE OF STUDY We have developed a mouse model in which the initiation, evolution and genomic landscape of GBM can be thoroughly studied thus paving the way for ideas about how this deadly brain cancer might be prevented, interrupted at an occult stage, or treated with very different therapies.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

Adult Glioblastomas (GBMs) remain one of the least curable brain cancers despite the discovery and use of DNA alkylating agent Temozolomide (TMZ). TMZ provides a moderate survival benefit to sensitive patients whose O6-methylguanine-methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately, TMZ potential is stunted because of the rapid onset of tumour recurrence and acquired resistance believed to result from the upregulation of DNA damage repair by the base excision repair (BER), mismatch repair (MMR), or homologous recombination (HR) systems. Our laboratory previously demonstrated that cell lines obtained from recurrent, TMZ-resistant GBMs could be re-sensitized to TMZ when treated with an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1) – a protein instrumental in the recruitment of BER machinery. From this preliminary research, we postulate that PARP-1 inhibition may not only be used to overcome established resistance in GBM but may also be used to prevent its emergence altogether. To test this hypothesis, we utilized the MGMT-methylated GBM cell line U251N and developed an in vitro model of inducible TMZ resistance. We verified that prolonged treatment of U251N cells with TMZ resulted in the emergence of resistant colonies that resembled recurrent GBM clinically observed in TMZ-treated patients. However, when the parental U251N line was co-treated with TMZ and PARP-1 inhibitor ABT-888, resistant colonies failed to appear. Therefore, PARP-1 inhibition may possess the potential to maintain tumour sensitivity to TMZ as well as evade the otherwise inevitable development of resistance in GBM.

Despite the discovery and widespread use of the chemotherapeutic drug temozolomide (TMZ), glioblastoma (GBM) remains a fatal cancer. TMZ, a DNA alkylating agent, provides a moderate survival benefit to patients whose tumours do not express the O6-methylguanine-methyltransferase (MGMT) gene. However, even these TMZ-sensitive GBMs recur and upon doing so, many are resistant to TMZ. The development of TMZ resistance is commonly associated with mutations in mismatch repair (MMR) and the re-expression of MGMT. Upregulation of homologous recombination (HR) and base excision repair (BER) have also been implicated as mechanisms of acquired TMZ resistance. To better characterize these mechanisms and to develop strategies to prevent or overcome resistance, our laboratory has implemented an in vitro model of inducible resistance in which frequent exposure to TMZ (100µM) yields multiple resistant colonies in the MGMT-methylated GBM cell line U251N (Yip et. al). These colonies displayed varying methods of resistance to TMZ, including those that have been clinically observed in recurrent, TMZ-treated GBMs. Several colonies harboured mutations in MMR genes MSH6, MSH2, and MLH1 with low or absent expression of their respective proteins. In addition, some MMR wild-type colonies had increased expression of poly-ADP ribose (a polymer required for the recognition of DNA breaks by the BER machinery), suggesting that upregulation of BER may be driving resistance. Furthermore, Western Blot analysis revealed that occasional colonies re-expressed MGMT. Interestingly, a few colonies did not possess these alterations, suggesting that their resistance may result from further downstream modifications of MMR or BER, or by mutations in HR. With a more comprehensive characterization of these U251 colonies, we hope to learn more about TMZ resistance in GBM, and refine treatments or preventative therapies for molecularly distinct, TMZ-resistant, recurrent tumours.