An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

Anarchy or Respect the Hierarchy? The Complexity of Glioblastoma.

Glioma stem-like cells (GSC) in glioblastoma (GBM) structure tumor cells into a hierarchical organization and are postulated to be recalcitrant to conventional treatments, resulting in fatal relapse of the disease. A better understanding of these cells would be essential for meaningful and lasting treatments. In this issue of Cancer Research, Virolle and colleagues report a fascinating phenotype whereby the extracellular signal-regulated kinase (ERK) pathway regulates a mechanism of dedifferentiation of GBM cells into a stem-like state expressing markers of pluripotency through an EGFR-ERK-EGR1-dependent axis. This aptly termed "toggle switch" may contribute to maintenance of GSCs, promote intratumor heterogeneity, and potentially provide innovative treatment options.See related article by Almairac et al., p. 3236.

Identification of Novel RAS Signaling Therapeutic Vulnerabilities in Diffuse Intrinsic Pontine Gliomas.

Diffuse intrinsic pontine gliomas (DIPG) are incurable brain tumors with an aggressive onset. Apart from irradiation, there are currently no effective therapies available for patients with DIPG, who have a median survival time of less than one year. Most DIPG cells harbor mutations in genes encoding histone H3 (H3K27M) proteins, resulting in a global reduction of H3K27 trimethylation and activation of oncogenic signaling pathways. Here we show that the H3K27M mutations contribute to RAS pathway signaling, which is augmented by additional RAS activators including PDGFRA. H3K27M mutation led to increased expression of receptor tyrosine kinases (RTK). A RAS pathway functional screen identified ERK5, but not ERK1/2, as a RAS pathway effector important for DIPG growth. Suppression of ERK5 decreased DIPG cell proliferation and induced apoptosis in vitro and in vivo. In addition, depletion or inhibition of ERK5 significantly increased survival of mice intracranially engrafted with DIPG cells. Mechanistically, ERK5 directly stabilized the proto-oncogene MYC at the protein level. Collectively, our data demonstrate an underappreciated role of H3K27M in RAS activation and reveal novel therapeutic targets for treating DIPG tumors. SIGNIFICANCE: These findings identify the H3K27M mutation as an enhancer of RAS activation in DIPG and ERK5 as a novel, immediately actionable molecular target. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4026/F1.large.jpg.

Pervasive H3K27 Acetylation Leads to ERV Expression and a Therapeutic Vulnerability in H3K27M Gliomas.

High-grade gliomas defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Comparative molecular profiling of HPV-induced squamous cell carcinomas.

Approximately 5% of all cancer incidences result from human papillomavirus (HPV) infection. HPV infection most commonly leads to cancers of the anogenital region or oropharynx. It is unknown whether different HPV-mediated cancers collectively share a molecular signature and it is important to determine if there are targetable alterations common to different types of HPV-positive tumors. We analyzed 743 p53 wild-type samples of anal, cervical, oropharyngeal, and vulvar squamous cell carcinomas which underwent multiplatform testing at a commercial molecular profiling service. Expression of 24 proteins was measured by immunohistochemistry (IHC), mutation of 48 genes was determined by next-generation and Sanger sequencing, and copy number alteration for six genes was determined by in situ hybridization. The four cohorts had remarkably similar molecular profiles. No gene had a statistically significant difference in mutation frequency or copy number change between the four different types of squamous cell carcinomas. The only significant differences between cohorts were frequency of ERCC1 and SPARC loss as determined by IHC. In all four cancer types, oncogene mutation and PD-L1 expression was relatively infrequent. The most commonly mutated gene was PIK3CA, with mutations most often affecting the helical domain of the protein and accompanied by concurrent lack of PTEN expression. Loss of MGMT and RRM1 was common among the four cohorts and may be predictive of response to cytotoxic therapies not currently being used to treat these cancer types. The similar molecular profiles of the four cohorts indicate that treatment strategies may be similarly efficacious across HPV-positive cancers.

PLK1 inhibition enhances temozolomide efficacy in IDH1 mutant gliomas.

Despite multimodal therapy with radiation and the DNA alkylating agent temozolomide (TMZ), malignant gliomas remain incurable. Up to 90% of grades II-III gliomas contain a single mutant isocitrate dehydrogenase 1 (IDH1) allele. IDH1 mutant-mediated transformation is associated with TMZ resistance; however, there is no clinically available means of sensitizing IDH1 mutant tumors to TMZ. In this study we sought to identify a targetable mechanism of TMZ resistance in IDH1 mutant tumors to enhance TMZ efficacy. IDH1 mutant astrocytes rapidly bypassed the G2 checkpoint with unrepaired DNA damage following TMZ treatment. Checkpoint adaptation was accompanied by PLK1 activation and IDH1 mutant astrocytes were more sensitive to treatment with BI2536 and TMZ in combination (<20% clonogenic survival) than either TMZ (~60%) or BI2536 (~75%) as single agents. In vivo, TMZ or BI2536 alone had little effect on tumor size. Combination treatment caused marked tumor shrinkage in all mice and complete tumor regression in 5 of 8 mice. Mutant IDH1 promotes checkpoint adaptation which can be exploited therapeutically with the combination of TMZ and a PLK1 inhibitor, indicating PLK1 inhibitors may be clinically valuable in the treatment of IDH1 mutant gliomas.

Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma.

BACKGROUND: Findings based on recent advances in next-generation sequence analysis suggest that, in some tumors, a single catastrophic event, termed chromothripsis, results in several simultaneous tumorigenic alterations. Previous studies have suggested that glioblastoma (GBM) may exhibit chromothripsis at a higher rate (39%) than other tumors (9%). Primary glioblastoma is an aggressive form of brain cancer that typically appears suddenly in older adults. With aggressive treatment, the median survival time is only 15 months. Their acute onset and widespread genomic instability indicates that chromothripsis may play a key role in their initiation and progression. GBMs are often characterized by EGFR amplification, CDKN2A and PTEN deletion, although approximately 20% of GBMs harbor additional amplifications in MDM2 or MDM4 with CDK4. METHODS: We used the chromothripsis prediction tool, Shatterproof, in conjunction with a custom whole genome sequence analysis pipeline in order to generate putative regions of chromothripsis. The data derived from this study was further expanded on using fluorescence in situ hybridization (FISH) analysis and susceptibility studies with colony formation assays. RESULTS: We show that primary GBMs are associated with higher chromothripsis scores and establish a link between chromothripsis and gene amplification of receptor tyrosine kinases (RTKs), as well as modulators of the TP53 and RB1 pathways. CONCLUSIONS: Utilizing a newly introduced bioinformatic tool, we provide evidence that chromothripsis is associated with the formation of amplicons containing several oncogenes involved in key pathways that are likely essential for post-chromothriptic cell survival.

ROS1 amplification mediates resistance to gefitinib in glioblastoma cells.

Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed EGFR, however, tumors treated with tyrosine kinase inhibitors (TKIs) will inevitably recur highlighting the need to identify signalling pathways involved in GBM resistance to these drugs. To this end, we treated GBM cells that overexpress EGFR with increasing concentrations of gefitinib and isolated resistant clones. These resistant clones were subject to RNAseq and the expression of several genes was found to be upregulated. These genes are mainly tyrosine kinase receptors and include ROS1, DDR1 and PDGFRA and are known to control several downstream targets of EGFR. The upregulation of ROS1 and DDR1 was confirmed at the protein level by western blot. Treatment with a potent and highly specific pyrazole ROS1 inhibitor in ROS1 overexpressing clones led to a sensitization of these cells to low concentrations of gefitinib. Combined treatment with gefitinib and ROS1 inhibitor induces massive cell death by apoptosis following a prolonged S phase cell cycle arrest. Our current study led to the discovery of alternative pathways used by GBM cells to evade cell death following treatment with gefitinib and identifies new therapeutic targets to prevent GBM cell resistance to the drug.