SUMMARY Developmental Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of medulloblastoma. We show here that Hedgehog promotes polyamine biosynthesis in GCPs by engaging a non-canonical axis leading to the translation of ornithine decarboxylase (ODC). This process is governed by AMPK, which phosphorylates threonine 173 of the zinc finger protein CNBP in response to Hedgehog activation. Phosphorylated CNBP increases its association with Sufu, followed by CNBP stabilization, ODC translation and polyamine biosynthesis. Notably, CNBP, ODC and polyamines are elevated in Hedgehog-dependent medulloblastoma and genetic or pharmacological inhibition of this axis efficiently blocks Hedgehog-dependent proliferation of medulloblastoma cells in vitro and in vivo. Together, these data illustrate an auxiliary mechanism of metabolic control by a morphogenic pathway with relevant implications in development and cancer.

The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

SHH Medulloblastoma (SHH-MB) is a pediatric brain tumor characterized by an inappropriate activation of the developmental Hedgehog (Hh) signaling. SHH-MB patients treated with the FDA-approved vismodegib, an Hh inhibitor that targets the transmembrane activator Smoothened (Smo), have shown the rapid development of drug resistance and tumor relapse due to novel Smo mutations. Moreover, a subset of patients did not respond to vismodegib because mutations were localized downstream of Smo. Thus, targeting downstream Hh components is now considered a preferable approach. We show here that selective inhibition of the downstream Hh effectors HDAC1 and HDAC2 robustly counteracts SHH-MB growth in mouse models. These two deacetylases are upregulated in tumor and their knockdown inhibits Hh signaling and decreases tumor growth. We demonstrate that mocetinostat (MGCD0103), a selective HDAC1/HDAC2 inhibitor, is a potent Hh inhibitor and that its effect is linked to Gli1 acetylation at K518. Of note, we demonstrate that administration of mocetinostat to mouse models of SHH-MB drastically reduces tumor growth, by reducing proliferation and increasing apoptosis of tumor cells and prolongs mouse survival rate. Collectively, these data demonstrate the preclinical efficacy of targeting the downstream HDAC1/2-Gli1 acetylation in the treatment of SHH-MB.

Regulation of gene expression in response to mitogenic stimuli is a critical aspect underlying many forms of human cancers. The AP-1 complex mediates the transcriptional response to mitogens, and its deregulation causes developmental defects and tumors. We report that the coactivator CRTC1 cyclic AMP response element-binding protein (CREB)-regulated transcription coactivator 1 is a potent and indispensable modulator of AP-1 function. After exposure of cells to the AP-1 agonist 12-O-tetradecanoylphorbol-13-acetate (TPA), CRTC1 is recruited to AP-1 target gene promoters and associates with c-Jun and c-Fos to activate transcription. CRTC1 consistently synergizes with the proto-oncogene c-Jun to promote cellular growth, whereas AP-1-dependent proliferation is abrogated in CRTC1-deficient cells. Remarkably, we demonstrate that CRTC1-Maml2 oncoprotein, which causes mucoepidermoid carcinomas, binds and activates both c-Jun and c-Fos. Consequently, ablation of AP-1 function disrupts the cellular transformation and proliferation mediated by this oncogene. Together, these data illustrate a novel mechanism required to couple mitogenic signals to the AP-1 gene regulatory program.T he cyclic AMP response element-binding protein (CREB)-regulated transcription coactivators (CRTCs, originally called TORCs) are a novel class of signal-dependent CREB coactivators identified using a high-throughput expression screen of a mammalian cDNA library (1, 2). CRTCs associate with the bZIP region of CREB via their N-terminal region and activate transcription through interactions with components of the basal transcriptional apparatus (1, 3). Under resting conditions, CRTCs are phosphorylated by sucrose nonfermenting1/AMP-activated protein kinase (AMP/SNF) kinases and sequestered in the cytoplasm (4, 5). When the intracellular levels of calcium or cAMP rise, CRTCs are dephosphorylated, travel to the nucleus and bind to CREB, thereby activating transcription. Consistent with their role as CREB activators, CRTCs have been shown to be key regulators of gluconeogenesis (5-8), adaptive mitochondrial biogenesis (9), ␤ cell survival (10), and long-term synaptic plasticity (11).Recent observations have suggested that CRTCs also promote activation of other transcription factors besides those of the CREB/ ATF1 family. Indeed, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) causes CRTC1 nuclear translocation in HeLa cells (12), and deletion of the TPA responsive element (TRE) from the IL-8 promoter abrogates CRTC1-mediated enhancement (2), suggesting that CRTC1 can stimulate transcriptional output of a TPA-regulated pathway. Recently, it was reported that CRTC1 can be phosphorylated and activated by MEKK1 (13), a critical kinase activated by several mitogenic stimuli, including TPA.TPA is a tumor-promoting drug that activates transcription of a number of genes that typically contain a TPA response element (TRE ϭ TGACTCA) in their promoter regions (14). In turn, the TRE is bound by the dimeric AP-1 transcription factor complex, comprising a...