592 we have also shown previously in that context that E2F1 is required for expression of the key lipogenic enzymes including fatty acid synthase (FASN) and acetyl CoA carboxylase (ACC), and that abrogation of E2F1 activity in vivo results in impaired lipogenesis in tumors. This ﬁrmly linked E2F1 activity downstream of Shh to de novo lipid synthesis in medulloblastoma and their proposed cells-of- origins. PPARc is a pivotal transcription factor that is triggered by fatty acid ligands and a key regulator of lipogenesis. Here, using immunostaining and blotting techniques in mouse NeuroD2-SmoA1 medulloblastomas, we detected very robust levels of PPARc in the tumors as opposed to the adjacent non-tumor cerebellar tissues (Fig. 1a, b). We next examined whether in medulloblastomas expression of PPARc, like that of FASN, requires E2F1 activity. PPARc has been reported to be transcriptionally controlled by E2F1 in adipocytes [7]. We treated medulloblastoma- bearing mice with the cdk inhibitor olomoucine (6 mg/kg) for 10 days. As we have previously reported, this treatment regimen markedly prolongs survival of tumor-bearing mice and is associated with reduced E2F1 and FASN expression (Fig. 1c) and with diminished proliferation in the tumors, as indicated by reduced levels of cyclin D2 (Fig. 1c). Indeed, olomoucine treatment resulted in a striking reduction of PPARc levels in the NeuroD2-SmoA1 medulloblastomas (Fig. 1c). These results suggest that PPARc lies downstream of the Shh ? E2F1 axis in Shh- driven medulloblastomas and further, that investigating pathways downstream of PPARc could lead to a better insight into the biology of medulloblastoma and their cancer associated metabolic patterns. Glycolytic enzymes are elevated in NeuroD2-SmoA1 medulloblastomas Substrate utilization pathways are functionally intertwined. Cells in anabolic tissues have a large capacity to generate anew and store fats from excessive glucose inﬂux. We have shown that Shh-driven medulloblastomas and their pro- posed cells-of-origin, cerebellar granule neuron precursors (CGNPs), exhibit high levels of lipogenesis, and here we report that these tumors feature increased PPARc. In order to determine whether glycolysis is increased in NeuroD2- SmoA1 medulloblastomas, we carried out immunostaining and western blot analysis of medulloblastomas and adja- cent, non-tumorous cerebellum. As shown in Fig. 2a, b, the medulloblastomas are marked by a robust accumulation of many key glycolytic enzymes, including hexokinase II (HKII), pyruvate kinase M2 (PKM2), and glucose trans- porter type 4 (Glut4), which control glucose transport, especially into muscle and adipocytes in response to insulin stimulation. Notably such patterns appear to be speciﬁc to 123 Acta Neuropathol (2012) 123:587–600 HKII as hexokinase I (HKI) levels were higher in normal cerebellar tissue than in the medulloblastomas. We next asked whether the expression of these glyco- lytic enzymes, like that of lipogenic markers, is dependent upon Rb inactivation and E2F1 activity. To this end we used western blot analysis of cerebellar tissue and medul- loblastomas prepared from DMSO- or olomoucine-treated mice. As shown in Fig. 2c, levels of HKII, PKM2, and Glut4 were all reduced in olomoucine-treated tumors, while levels of HKI were up-regulated in the olomoucine- treated medulloblastomas. These results indicate that the glycolytic markers are associated with E2F1 activity and proliferation, and further suggest that HKI plays a role distinct from glycolysis in differentiated (e.g. normal cer- ebellum) or non-proliferating tissue (e.g. olomoucine- treated medulloblastoma). E2F1 is required for PPARc and the glycolytic markers expression in neural precursors of the developing cerebellum Medulloblastomas associated with increased activity of the Shh pathway have been proposed to arise from proliferat- ing neural precursors in the developing cerebellum [6]. During perinatal development, these cells proliferate rap- idly in the external germinal layer (EGL) response to Shh secreted from the underlying Purkinje cells (PN) [4, 44, 45]. Upon exiting the cell cycle they migrate inward to the internal granule layer (IGL), where they undergo terminal differentiation. Shh-associated medulloblastomas share many characteristics of these cerebellar granule neuron precursors (CGNPs), including high expression of N-myc, microRNA miR 17/92, increased lipogenesis, and as we previously reported and show again here (Fig. 3a, b) ele- vated E2F1 [2, 29, 33]. To determine whether increased glycolysis is also a shared feature of Shh-associated medulloblastomas and CGNPs, and whether this is, like lipogenesis, an E2F1-dependent event, we carried out immunoﬂuorescence and western blot analysis of wild-type and E2F1-null mouse cerebella at postnatal day 7 (PN7), when CGNP proliferation is at its peak. As shown in Fig. 3a (bottom panels) and in Fig. 3b, PPARc is found in the EGL, as are HKII, PKM2, and Glut4. We also observed some expression of Glut4 in the IGL. In contrast, HKI was absent from the EGL and was highly expressed in the IGL, where differentiating CGNPs are found. Ablation of E2F1 resulted in loss of expression of PPARc, HKII, PKM2, and Glut4, but did not affect levels of HKI. These results suggest that in the developing cerebellum, as in medulloblastoma, PPARc and glycolytic markers lie downstream of Sonic hedgehog and E2F1. We next asked whether CGNPs carry out glycolysis in vitro, and whether acute manipulation of E2F1 abrogates