Neuroplastic changes in dorsal striatum participate in the transition from casual to habitual drug use and might play a critical role in the development of methamphetamine (METH) addiction. We examined the influence of METH self-administration on gene and protein expression that may form substrates for METH-induced neuronal plasticity in the dorsal striatum. Male Sprague-Dawley rats self-administered METH (0.1 mg/kg/injection, i.v.) or received yoked saline infusions during eight 15-h sessions and were euthanized 2 h, 24 h, or 1 month after cessation of METH exposure. Changes in gene and protein expression were assessed using microarray analysis, RT-PCR and Western blots. Chromatin immunoprecipitation (ChIP) followed by PCR was used to examine epigenetic regulation of METH-induced transcription. METH self-administration caused increases in mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and the synaptic protein, synaptophysin (Syp) in the dorsal striatum. METH also caused changes in ΔFosB, BDNF and TrkB protein levels, with increases after 2 and 24 h, but decreases after 1 month of drug abstinence. Importantly, ChIP-PCR showed that METH self-administration caused enrichment of phosphorylated CREB (pCREB), but not of histone H3 trimethylated at lysine 4 (H3K4me3), on promoters of c-fos, fosb, Bdnf and Syp at 2 h after cessation of drug intake. These findings show that METH-induced changes in gene expression are mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that mediate alterations in expression of genes and proteins serving as substrates for addiction-related synaptic plasticity.

The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.folate | one-carbon metabolism | thymidine | deoxyribouridine

Activation of the retinoic acid (RA) signaling pathway is important for controlling embryonic stem cell differentiation and development. Modulation of this pathway occurs through the recruitment of different epigenetic regulators at the retinoic acid receptors (RARs) located at RA-responsive elements and/or RA-responsive regions of RA-regulated genes. Coactivator-associated arginine methyltransferase 1 (CARM1, PRMT4) is a protein arginine methyltransferase that also functions as a transcriptional coactivator. Previous studies highlight CARM1's importance in the differentiation of different cell types. We address CARM1 function during RA-induced differentiation of murine embryonic stem cells (mESCs) using shRNA lentiviral transduction and CRISPR/Cas9 technology to deplete CARM1 in mESCs. We identify CARM1 as a novel transcriptional coactivator required for the RA-associated decrease in Rex1 (Zfp42) and for the RA induction of a subset of RA-regulated genes, including CRABP2 and NR2F1 (Coup-TF1). Furthermore, CARM1 is required for mESCs to differentiate into extraembryonic endoderm in response to RA. We next characterize the epigenetic mechanisms that contribute to RA-induced transcriptional activation of CRABP2 and NR2F1 in mESCs and show for the first time that CARM1 is required for this activation. Collectively, our data demonstrate that CARM1 is required for transcriptional activation of a subset of RA target genes, and we uncover changes in the recruitment of Suz12 and the epigenetic H3K27me3 and H3K27ac marks at gene regulatory regions for CRABP2 and NR2F1 during RA-induced differentiation.

e16234 Background: Pancreatic ductal adenocarcinoma (PDAC) will be the second leading cause of cancer death by 2030 through early onset dedifferentiation and metastasis that results in limited treatments. FDA-approved targeted therapies–including proteasome, mitogen-activated protein kinase (MEK), histone deacetylase (HDAC), and mammalian target of rapamycin (mTOR) inhibitors–have shown preclinical benefit but failed in clinic. However, combination targeted therapy has been largely unexplored in clinical trials. We hypothesized based on prior drug screening efforts in the lab on aggressive subtypes of pancreatic cancer that combinations of these targeted therapies could be synergistically active in preclinical PDAC models. Methods: We optimized combinations of proteasome, MEK, mTOR, and HDAC inhibitors by performing cell viability experiments in human PDAC lines in 2D and 3D cell culture conditions in vitro. Optimized drug combinations from these experiments were then trialed in two different in vivo animal models–human FG xenografts in immunocompromised mice (NSG) and spontaneous primary PDAC tumors from transgenic pancreatic-specific Kras-overexpressing, P53-deficient mice (KPC). RNA-sequencing and Seahorse metabolic assays were utilized to analyze molecular mechanisms of drug synergy. Dedifferentiation was assessed by histology of KPC tumors. Results: The HDAC inhibitor Panobinostat and mTOR inhibitor Everolimus synergistically killed human pancreatic cancer cell lines grown in 2D and 3D cell culture in vitro. Synergy was highest at low concentrations of Panobinostat. In vivo, low-dose Panobinostat and Everolimus synergistically blocked growth of human FG xenografts in NSG mice. In vivo synergy of low-dose Panobinostat and Everolimus was highest in the KPC mouse model, where only combination therapy could significantly reduce tumor growth, stall dedifferentiation, and improve survival. Mechanistically, RNA-sequencing of Panobinostat/Everolimus-treated FG cells revealed that Panobinostat increased tumor suppressor genes opposing dedifferentiation such as p21, EGR1, and CCN2. Everolimus complemented Panobinostat by decreasing expression of enzymes metabolizing acetyl groups such as fatty acid synthase, stearoyl-CoA desaturase, and sterol response element binding factors. Functionally, Everolimus’ activity to reduce oxidative consumption increased histone acetylation specifically in the presence of Panobinostat. Conclusions: A combination of FDA-approved, targeted oral therapies (low-dose Panobinostat/Everolimus) is synergistically active in preclinical models of pancreatic cancer in vitro and in vivo. mTOR inhibitors create drug synergy by specifically increasing histone acetylation in HDAC inhibitor-treated cells by reversing HDAC inhibitor de-repression of acetyl/oxidative metabolism, illustrating a novel connection between cancer epigenetics and metabolism.