Oren J. Becher

Inhibition of PRC2 Activity by a Gain-of-Function H3 Mutation Found in Pediatric Glioblastoma

EZ Inhibition Missense mutations in the core constituents of the genome packaging material, chromatin, have been implicated in several of human cancers. Nucleosomes are made up of histones, and a mutation of lysine 27 (K27) to methionine in the N-terminal tail of histone variants H3.3 and H3.1 has been identified in various pediatric gliomas. Lewis et al. (p. 857, published online 28 March; see the Perspective by Morgan and Shilatifard) show that the polycomb enzyme complex, which can epigenetically modify K27 by addition of a methyl group—and which is often a silencing signal—is itself potently inhibited by replacement of the H3.3/3.1 K27 by methionine. The inhibition of the EZH2 subunit causes an overall reduction of K27 methylation. Methionine mutants of other methylated lysine residues in histone H3 cause similar reductions in methylation levels of the cognate lysine, altering the epigenetic profiles of such cancer cells. Mutations of histones in some cancers result in inhibition of enzymes that lay down epigenetic marks on chromatin. [Also see Perspective by Morgan and Shilatifard] Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall amounts of H3 with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. We propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.

PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo

Medulloblastomas are brain tumors that arise in the cerebellum of children and contain stem cells in a perivascular niche thought to give rise to recurrence following radiation. We used several mouse models of medulloblastomas in parallel to better understand how the critical cell types in these tumors respond to therapy. In our models, the proliferating cells in the tumor bulk undergo radiation-induced, p53-dependent apoptotic cell death. Activation of Akt signaling via PTEN loss transforms these cells to a nonproliferating extensive nodularity morphology. By contrast, the nestin-expressing perivascular stem cells survive radiation, activate PI3K/Akt pathway, undergo p53-dependent cell cycle arrest, and re-enter the cell cycle at 72 h. Furthermore, the ability of these cells to induce p53 is dependent on the presence of PTEN. These cellular characteristics are similar to human medulloblastomas. Finally, inhibition of Akt signaling sensitizes cells in the perivascular region to radiation-induced apoptosis.

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge

We have extended our understanding of the molecular biology that underlies adult glioblastoma over many years. By contrast, high-grade gliomas in children and adolescents have remained a relatively under-investigated disease. The latest large-scale genomic and epigenomic profiling studies have yielded an unprecedented abundance of novel data and provided deeper insights into gliomagenesis across all age groups, which has highlighted key distinctions but also some commonalities. As we are on the verge of dissecting glioblastomas into meaningful biological subgroups, this Review summarizes the hallmark genetic alterations that are associated with distinct epigenetic features and patient characteristics in both paediatric and adult disease, and examines the complex interplay between the glioblastoma genome and epigenome.

Genetically Engineered Models Have Advantages over Xenografts for Preclinical Studies

Mouse models of human cancer are valuable tools for cancer research. Although xenografts and genetically engineered models (GEMs) possess limitations as well as advantages, each system plays a significant role in preclinical testing. Tumor xenografts are easy to use, relatively inexpensive, and reproducible. The main drawback of xenografts is that the genetics and histology of the tumors are frequently not representative of the respective human tumor and, thus far, these models have not been as predictive of therapeutic success as one would like. By contrast, GEMs are histologically and genetically accurate models of human cancer but have disadvantages of heterogeneity with regard to frequency, latency, and growth. These disadvantages are reminiscent of the variable behavior of actual human tumors. Recently, these shortcomings have been partly overcome with the development of anatomic and molecular in vivo imaging techniques such as magnetic resonance imaging and bioluminescence imaging. These new technologies will hopefully support the use of GEMs in preclinical trials and help determine if trials in GEMs are more predicative than xenografts of human responses.

Sonic Hedgehog Pathway Activation Is Induced by Acute Brain Injury and Regulated by Injury-Related Inflammation

The adult mammalian brain responds to injury by activating a program of cell proliferation during which many oligodendrocyte precursors, microglia, and some astrocytes proliferate. Another common response to brain injury is the induction of reactive gliosis, a process whereby dormant astrocytes undergo morphological changes and alter their transcriptional profiles. Although brain injury-induced reactive gliosis is concurrent with the proliferation of surrounding cells, a functional relationship between reactive gliosis and this cell proliferation has not been clearly demonstrated. Here, we show that the mitogen sonic hedgehog (SHH) is produced in reactive astrocytes after injury to the cerebral cortex and participates in regulating the proliferation of Olig2-expressing (Olig2+) cells after brain injury. Using a cortical freeze injury to induce reactive gliosis in a Gli–luciferase reporter mouse, we show that the SHH pathway is maximally active 3 d after brain injury and returns to baseline levels by 14 d. SHH expression parallels Gli activation and localizes to glial fibrillary acidic protein-expressing reactive astrocytes. Inhibition of the SHH pathway with cyclopamine blocks the Gli response and significantly reduces both the proliferating and overall number of Olig2+ cells in the injured cortex. To provide mechanistic insight into SHH pathway activation in astrocytes, we show that proinflammatory stimuli activate SHH-expressing reactive astrocytes, whereas inhibition of inflammation-induced reactive gliosis by macrophage depletion abolishes SHH activation after brain injury and dampens cell proliferation after injury. Our data describes a unique reactive astrocyte-based, SHH-expressing niche formed in response to injury and inflammation that regulates the proliferation of Olig2+ cells.

Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor-induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show beta-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity.

Preclinical evaluation of radiation and perifosine in a genetically and histologically accurate model of brainstem glioma.

Brainstem gliomas (BSG) are a rare group of central nervous system tumors that arise mostly in children and usually portend a particularly poor prognosis. We report the development of a genetically engineered mouse model of BSG using the RCAS/tv-a system and its implementation in preclinical trials. Using immunohistochemistry, we found that platelet-derived growth factor (PDGF) receptor alpha is overexpressed in 67% of pediatric BSGs. Based on this observation, we induced low-grade BSGs by overexpressing PDGF-B in the posterior fossa of neonatal nestin tv-a mice. To generate high-grade BSGs, we overexpressed PDGF-B in combination with Ink4a-ARF loss, given that this locus is commonly lost in high-grade pediatric BSGs. We show that the likely cells of origin for these mouse BSGs exist on the floor of the fourth ventricle and cerebral aqueduct. Irradiation of these high-grade BSGs shows that although single doses of 2, 6, and 10 Gy significantly increased the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei, only 6 and 10 Gy significantly induce cell cycle arrest. Perifosine, an inhibitor of AKT signaling, significantly induced TUNEL-positive nuclei in this high-grade BSG model, but in combination with 10 Gy, it did not significantly increase the percent of TUNEL-positive nuclei relative to 10 Gy alone at 6, 24, and 72 hours. Survival analysis showed that a single dose of 10 Gy significantly prolonged survival by 27% (P = 0.0002) but perifosine did not (P = 0.92). Perifosine + 10 Gy did not result in a significantly increased survival relative to 10 Gy alone (P = 0.23). This PDGF-induced BSG model can serve as a preclinical tool for the testing of novel agents.

Histopathological spectrum of paediatric diffuse intrinsic pontine glioma: diagnostic and therapeutic implications

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumour-related death in children. In the majority of cases diagnosis is based on clinical and MRI findings, resulting in the scarcity of pre-treatment specimens available to study. Our group has developed an autopsy-based protocol to investigate the histologic and biologic spectrum of DIPG. This has also allowed us to investigate the terminal pattern of disease and gain a better understanding of what challenges we are facing in treating DIPG. Here, we review 72 DIPG cases with well documented clinical history and molecular data and describe the pathological features of this disease in relation to clinical and genetic features. Fifty-three of the samples were autopsy material (7 pre-treatment) and 19 were pre-treatment biopsy/surgical specimens. Upon histological review, 62 patients had high-grade astrocytomas (18 WHO grade III and 44 WHO grade IV patients), 8 had WHO grade II astrocytomas, and 2 had features of primitive neuroectodermal tumour (PNET). K27M-H3 mutations were exclusively found in tumours with WHO grade II–IV astrocytoma histology. K27M-H3.1 and ACVR1 mutations as well as ALT phenotype were only found in WHO grade III–IV astrocytomas, while PIK3CA mutations and PDGFRA gains/amplifications were found in WHO grade II–IV astrocytomas. Approximately 1/3 of DIPG patients had leptomeningeal spread of their tumour. Further, diffuse invasion of the brainstem, spinal cord and thalamus was common with some cases showing spread as distant as the frontal lobes. These findings suggest that focal radiation may be inadequate for some of these patients. Importantly, we show that clinically classic DIPGs represent a diverse histologic spectrum, including multiple cases which would fit WHO criteria of grade II astrocytoma which nevertheless behave clinically as high-grade astrocytomas and harbour the histone K27M-H3.3 mutation. This suggests that the current WHO astrocytoma grading scheme may not appropriately predict outcome for paediatric brainstem gliomas.

Cancer stem cells and survival pathways

Gliomas and medulloblastomas are the most frequent malignant brain tumors in adult and children respectively. Although both tumors arise in the CNS, there is a significant difference in their therapeutic response, resulting in medulloblastomas as being relatively curable, while glioblastomas are basically incurable. During the last decade several reports have demonstrated the existence of cancer stem cells in brain tumors, their location and their response to treatment. We have recently described the therapeutic response of medulloblastomas to radiation in their native microenvironment, describing how p53 and PI3K signaling pathway leads to nestin-expressing cells in the perivascular stem cell niche evading cell death while the tumor-bulk succumbs to apoptosis 1. It remains to be determined whether this mechanism of tumor resistance applies to the more complex stem-cell niche and tumor bulk of gliomas.