Amelie Griveau

Oligodendrocyte-Encoded HIF Function Couples Postnatal Myelination and White Matter Angiogenesis

Myelin sheaths provide critical functional and trophic support for axons in white matter tracts of the brain. Oligodendrocyte precursor cells (OPCs) have extraordinary metabolic requirements during development as they differentiate to produce multiple myelin segments, implying that they must first secure adequate access to blood supply. However, mechanisms that coordinate myelination and angiogenesis are unclear. Here, we show that oxygen tension, mediated by OPC-encoded hypoxia-inducible factor (HIF) function, is an essential regulator of postnatal myelination. Constitutive HIF1/2α stabilization resulted in OPC maturation arrest through autocrine activation of canonical Wnt7a/7b. Surprisingly, such OPCs also show paracrine activity that induces excessive postnatal white matter angiogenesis in vivo and directly stimulates endothelial cell proliferation in vitro. Conversely, OPC-specific HIF1/2α loss of function leads to insufficient angiogenesis in corpus callosum and catastrophic axon loss. These findings indicate that OPC-intrinsic HIF signaling couples postnatal white matter angiogenesis, axon integrity, and the onset of myelination in mammalian forebrain.

Cooperative interactions of BRAFV600E kinase and CDKN2A locus deficiency in pediatric malignant astrocytoma as a basis for rational therapy.

Although malignant astrocytomas are a leading cause of cancer-related death in children, rational therapeutic strategies are lacking. We previously identified activating mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) (BRAFT1799A encoding BRAFV600E) in association with homozygous cyclin-dependent kinase inhibitor 2A (CDKN2A, encoding p14ARF and p16Ink4a) deletions in pediatric infiltrative astrocytomas. Here we report that BRAFV600E expression in neural progenitors (NPs) is insufficient for tumorigenesis and increases NP cellular differentiation as well as apoptosis. In contrast, astrocytomas are readily generated from NPs with additional Ink4a-Arf deletion. The BRAFV600E inhibitor PLX4720 significantly increased survival of mice after intracranial transplant of genetically relevant murine or human astrocytoma cells. Moreover, combination therapy using PLX4720 plus the Cyclin-dependent kinase (CDK) 4/6-specific inhibitor PD0332991 further extended survival relative to either monotherapy. Our findings indicate a rational therapeutic strategy for treating a subset of pediatric astrocytomas with BRAFV600E mutation and CDKN2A deficiency.

A Small-Molecule Smoothened Agonist Prevents Glucocorticoid-Induced Neonatal Cerebellar Injury

A Sonic hedgehog–Smoothened signaling agonist can protect against brain injury in mice caused by glucocorticoids, which are given to treat chronic lung disease in preterm infants. Tilting the Risk-Benefit Ratio for Preterm Infants Preterm babies often develop chronic lung disease, which can be treated by postnatal administration of glucocorticoids (GCs). However, GC treatment can induce permanent neurological deficits and inhibit the growth of the cerebellum, a brain center critical for coordination of movement and higher-order neurological functions. To address this problem, Heine et al. have built on previous work showing that the Smoothened–Sonic hedgehog signaling pathway drives cerebellar cell proliferation during development and that it is this pathway that is disrupted by GCs. It was known that putting this pathway into genetic overdrive can prevent GC-induced injury, and the authors therefore devised a small-molecule mimic of this effect: SAG, an agonist of Smoothened. They showed that SAG crossed the blood-brain barrier to activate Sonic hedgehog targets in the mouse cerebellum and that it prevented the growth-inhibitory effects of GCs. They also showed that this treatment did not induce the cerebellar tumor medulloblastoma or other cancers, a reasonable fear because the Smo pathway is critical for proliferation of some tumors. Most importantly, SAG did not antagonize beneficial effects of GCs for the lung. These findings suggest that adjuvant therapy with SAG is safe and might be an effective way to prevent neurotoxic side effects of GCs when they are given to preterm infants for life-threatening conditions. Further toxicity studies will need to determine the optimal dose schedule and safety parameters before clinical trials in humans, but SAG may tilt the risk-benefit ratio of neonatal GC treatment toward more benefit for preterm infants. Glucocorticoids are used for treating preterm neonatal infants suffering from life-threatening lung, airway, and cardiovascular conditions. However, several studies have raised concerns about detrimental effects of postnatal glucocorticoid administration on the developing brain leading to cognitive impairment, cerebral palsy, and hypoplasia of the cerebellum, a brain region critical for coordination of movement and higher-order neurological functions. Previously, we showed that glucocorticoids inhibit Sonic hedgehog–Smoothened (Shh-Smo) signaling, the major mitogenic pathway for cerebellar granule neuron precursors. Conversely, activation of Shh-Smo in transgenic mice protects against glucocorticoid-induced neurotoxic effects through induction of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) pathway. Here, we show that systemic administration of a small-molecule agonist of the Shh-Smo pathway (SAG) prevented the neurotoxic effects of glucocorticoids. SAG did not interfere with the beneficial effects of glucocorticoids on lung maturation, and despite the known associations of the Shh pathway with neoplasia, we found that transient (1-week-long) SAG treatment of neonatal animals was well tolerated and did not promote tumor formation. These findings suggest that a small-molecule agonist of Smo has potential as a neuroprotective agent in neonates at risk for glucocorticoid-induced neonatal cerebellar injury.

Cerebellar cortical lamination and foliation require cyclin A2

The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.

A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

Cyclin A2 promotes DNA repair in the brain during both development and aging

Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation in CCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2s DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice.

Abstract LB-206: Role of Olig2 phosphorylation in pediatric glioma formation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Malignant gliomas are a leading cause of tumor-related mortality in children. Current therapies often have significant adverse effects on brain development and are largely ineffective against aggressive gliomas. The development of rational therapies has been limited by poor information regarding the cell of origin for pediatric glioma and the relevant underlying genetic alterations. Recent findings now reveal that mutations existing in pediatric glioma are different than those seen in adult gliomas. For example, BRAFV600E activating mutations are associated with deletion of the tumor suppressor gene, p16 (Ink4a-Arf), in about 15% of pediatric gliomas. We have generated a faithful genetic mouse model in which there is concomitant activation of BRAFV600E and deletion of Ink4a-Arf in oligodendrocyte progenitor cells (OPCs) leading to the generation of pediatric high-grade glioma. OPCs have been shown to be a potential cell of origin for glioma and expression of Olig2 is found in 100 percent of human pediatric and adult high-grade gliomas. Given that Olig2 is also required for normal myelin development, targeting all Olig2-expressing cells with small molecule inhibitors would likely have detrimental effects. We have recently found that Olig2 protein is present in a phosphorylated form that promotes proliferation of adult glioma cells. In this study, we demonstrate that phospho-Olig2 is expressed in tumorigenic oligodendrocyte progenitor cells and is required for pediatric glioma generation. In addition, we found that phospho-Olig2 and Olig2 interact with different proteins, some of which are feasible drug targets. This study gives insights into new therapies by understanding key protein interactions in normal oligodendrocyte development as well as in tumorigenic oligodendrocyte progenitor cells. Citation Format: Amelie GRIVEAU, An-Chi TIEN, David H. Rowitch. Role of Olig2 phosphorylation in pediatric glioma formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-206. doi:10.1158/1538-7445.AM2014-LB-206

Abstract 1944: Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells

Glioblastoma multiforme (GBM) are heterogeneous tumors that are refractory to radiation and traditional chemotherapeutics. Separate studies have identified populations of neural stem and progenitor -like GBM cells, however the contribution of these cells types to tumor progression and the response to targeted therapy are poorly understood. Small molecules (such as vemurafenib) targeting the most common MAPK-activating mutation, BRAFV600E, are currently entering clinical trials for treatment of high-grade glioma. Understanding the contribution of different cell types to treatment responses will be necessary for the design of effective adjuvant therapeutic strategies. We show that activating MAPK pathway gene mutations in GBM correlate with elevated expression levels of oligodendrocyte precursor (OPC) and neural stem cell (NSC)-associated genes. Cells expressing CD133 and NG2 coexist in human GBM and BRafV600E expressing mouse neurosphere cultures, and maintain some of their respective neural stem and progenitor-like properties. In isogenic mouse cells expressing BRafV600E, NG2+ cells expand rapidly and symmetrically, while rates of asymmetric division are unaffected in slow-proliferating CD133+ cells. Tracing of proliferation rate with the membrane-associated dye PKH-26 reveals heterogeneity in the proliferation rate of CD133+ cells, but not proliferative NG2+ cells. BRAF-targeted inhibition of human GBM cells with PLX4720, the tool compound of vemurafenib, reduces S-phase entry of NG2+ cells, yet CD133+ cells are less affected. Interestingly targeted inhibition of components of the asymmetric division machinery selectively disrupts G2-M progression in CD133+ cells. The functionally divergent populations of CD133-expressing NSC-like cells will likely be therapeutically problematic with mono-therapeutic approaches. These data point to a differential regulation of key cell cycle processes between CD133+ stem-like cells and NG2+ OPC-like cells, that will inform the intelligent design of compound treatment strategies. Note: This abstract was not presented at the meeting. Citation Format: Robin G. Lerner, Yuichiro Ihara, Kate Lewis, Amelie Griveau, Brian Reichhold, Dian Qu, Martin McMahon, David Rowitch, Charles D. James, Claudia Petritsch. Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2014-1944

Abstract 2298: Cell-specific effects of BRafV600E expression disrupt gliogenesis and lead to astrocytoma formation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Within the adult mammalian brain the asymmetric distribution of cell-fate determinants during mitosis maintains a balance between differentiated and self-renewing cells. We have previously shown that disruption of asymmetric divisions in oligodendrocyte progenitor cells (OPC) causes cells to aberrantly self-renew and fail to differentiate, giving rise to oligodendroglioma (Sugiarto et al. Cancer Cell; 2011; 20; 320-40). Here we proposed to address whether asymmetric divisions are also disrupted in precursors of malignant astrocytoma, a highly aggressive and therapy resistant type of glioma. The constitutively active BRaf mutant BRafV600E is mutated in a subset of malignant astrocytoma alongside deletion of Cdkn2a, and can transform NSCs into astrocytoma precursors (Huillard et al. PNAS; 2012; 109; 8710-5). We used BRafCA mice (Dankort et al. Genes Dev. 2007; 21; 379-84) with Cre recombinase-activated expression of BRafV600E and deletion of Ink4a/Arf to generate an in situ model of NSC-derived malignant astrocytoma. In vivo studies to assess asymmetric division and related defects were complemented by studies of BRafV600E expressing, Ink4a/Arf-deleted neurosphere cells in cell-based assays of asymmetric division, self-renewal and differentiation. Our results provide unprecedented evidence that BRafV600E, although uniformly expressed in NSC and their progeny, disrupts asymmetric divisions in a cell-type specific manner. NSCs continue to divide asymmetrically but their progeny, including OPC, divide more symmetrically and exhibit defects in differentiation and proliferation. These defects are reversible with BRafV600E-specific inhibitor PLX4720, and are associated with increased expression of the mitotic kinase polo-like kinase 1, increased pERK and decreased pAKT. These data describe a mechanism by which NSC-targeted expression of BRafV600E may generate numerous subpopulations of tumor cells with distinct characteristics. This heterogeneity may mirror that seen in malignant astrocytoma, which has often been associated with high incidence of therapy resistance (Burger et al. Cancer. 1985; 56; 1106-11). Citation Format: Robin G. Lerner, Yuichiro Ihara, Brian Reichholf, Dian Qu, Sista Sugiarto, Amelie Griveau, David James, Martin McMahon, Claudia Petritsch. Cell-specific effects of BRafV600E expression disrupt gliogenesis and lead to astrocytoma formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2298. doi:10.1158/1538-7445.AM2013-2298