SUMMARY The histone lysine demethylase KDM4C is often overexpressed in cancers primarily through gene amplification. The molecular mechanisms of KDM4C action in tumorigenesis are not well defined. Here we report that KDM4C transcriptionally activates amino acid biosynthesis and transport, leading to a significant increase in intracellular amino acid levels. Examination of the serine-glycine synthesis pathway reveals that KDM4C epigenetically activates the pathway genes under steady-state and serine deprivation conditions by removing the repressive histone modification H3 lysine 9 (H3K9) trimethylation. This action of KDM4C requires ATF4, a transcriptional master regulator of amino acid metabolism and stress responses. KDM4C activates ATF4 transcription and interacts with ATF4 to target serine pathway genes for transcriptional activation. We further present evidence for KDM4C in transcriptional coordination of amino acid metabolism and cell proliferation. These findings suggest a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transactivation in reprogramming amino acid metabolism for cancer cell proliferation.

MYCN amplification drives the development of neuronal cancers in children and adults. Given the challenge in therapeutically targeting MYCN directly, we searched for MYCNactivated metabolic pathways as potential drug targets. Here we report that neuroblastoma cells with MYCN amplification show increased transcriptional activation of the serine-glycineone-carbon (SGOC) biosynthetic pathway and an increased dependence on this pathway for supplying glucose-derived carbon for serine and glycine synthesis. Small molecule inhibitors that block this metabolic pathway exhibit selective cytotoxicity to MYCN-amplified cell lines and xenografts by inducing metabolic stress and autophagy. Transcriptional activation of the SGOC pathway in MYCN-amplified cells requires both MYCN and ATF4, which form a positive feedback loop, with MYCN activation of ATF4 mRNA expression and ATF4 stabilization of MYCN protein by antagonizing FBXW7mediated MYCN ubiquitination. Collectively, these findings suggest a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited as a strategy for selective cancer therapy. Significance: This study identifies a MYCN-dependent metabolic vulnerability and suggests a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited for cancer therapy. See related commentary by Rodriguez Garcia and Arsenian-Henriksson, p. 3818 Materials and Methods Cell lines and cell culture Neuroblastoma cell lines BE(2)

Summary High-risk neuroblastoma remains one of the deadliest childhood cancers. Identification of metabolic pathways that drive or maintain high-risk neuroblastoma may open new avenues of therapeutic interventions. Here we report the isolation and propagation of neuroblastoma sphere-forming cells with self-renewal and differentiation potential from tumors of TH-MYCN mice, an animal model of high-risk neuroblastoma with MYCN amplification. Transcriptional profiling reveals that mouse neuroblastoma sphere-forming cells acquire a metabolic program characterized by transcriptional activation of the cholesterol and serine-glycine synthesis pathways, primarily as a result of increased expression of sterol regulatory element-binding factors and Atf4, respectively. This metabolic reprogramming is recapitulated in high-risk human neuroblastomas and is prognostic for poor clinical outcome. Genetic and pharmacological inhibition of the metabolic program markedly decreases the growth and tumorigenicity of both mouse neuroblastoma sphere-forming cells and human neuroblastoma cell lines. These findings suggest a therapeutic strategy for targeting the metabolic program of high-risk neuroblastoma.

Background: To examine the effect of CXCR4 antagonist AMD3100 on the recruitment of endogenous endothelial precursor cells (EPC) in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) and outcome of stroke. Methods: Adult male SD rats underwent pMCAO. AMD3100 was injected once at 1 hour (an early phase) or on day 14 (a later phase) or for 7 consecutive days from day 1 to day 7(3 mg/kg/day) after pMCAO. Flow cytometry analyses were performed to detect endogenous EPCs in peripheral blood (PB). Endogenous EPCs in IBZ were identified by immunofluorescence staining. SDF-1 expression levels in IBZ were measured by real time PCR dynamically. Infarct volume and neurological outcome including neurological score and body weight loss were used to estimate the outcome of stroke. Results: AMD3100-treatment could mobilize endogenous EPCs to PB of rats after pMCAO, and continuous AMD3100-treatment mobilized more EPCs to PB than single AMD3100-treatment. Single AMD3100 treatment at 1 hour after pMCAO rather than continuous AMD3100 treatment in an early phase could recruit endogenous EPCs in IBZ and improve neurological outcome after pMCAO. Single AMD3100 administration in later phase (on day 14) could not recruit endogenous EPCs to IBZ or improve neurological outcome after pMCAO. SDF-1 relative expression in IBZ increased in an early phase from day 1 to day 3, then decreased in later phase from day 7 to day 14. Conclusions: Our findings suggested that single AMD3100 treatment in an early phase could recruit endogenous EPCs to IBZ and improve the outcome of stroke, and AMD3100 might be used for the treatment of stroke if given at proper time window.