Specific Chemical Inhibitors Research Articles

Derivation of neural crest stem cells from human epidermal keratinocytes requires FGF-2, IGF-1, and inhibition of TGF-β1.

Neural crest (NC) cells play a central role in forming the peripheral nervous system, the craniofacial skeleton, and the pigmentation of the skin during development due to their broad multilineage differentiation potential into neurons, Schwann cells, melanocytes, and mesenchymal stem cells. Recently, we identified an easily accessible source of pluripotent NC stem cells from human inter‐follicular keratinocyte (KC) cultures (KC‐NC). In this work, we examined specific conditions for the derivation of NC from KC cultures. More specifically, we examined the role of two growth factors, FGF2 and IGF1, in NC proliferation and in expression of two potent NC transcription factors, Sox10 and FoxD3. Using specific chemical inhibitors, we uncovered that the downstream regulatory pathways AKT/PI3K, MEK/ERK, and JNK/cJun may be critical in Sox10 and FoxD3 regulation in KC‐NC. The TGF‐β1 pathway was also implicated in suppressing Sox10 expression and NC proliferation. In summary, our study shed light into the role of FGF2, IGF1, and TGF‐β1 on the induction of NC from KC cultures and the pathways that regulate Sox10 and FoxD3. We also established culture conditions for sustaining KC‐NC multipotency and, therefore, the potential of these cells for regenerative medicine and cellular therapies.

Disentangling the role of PI3K/Akt, Rho GTPase and the actin cytoskeleton on dengue virus infection

BackgroundInfection generated by Dengue Virus (DENV) does not have a specific pharmacologic treatment. Therefore, it is necessary to investigate research strategies departing from traditional approaches. Studying cellular mechanisms during early DENV infection may allow the design of a host-based approach to antivirals. Herein, we describe early/late events of DENV infection in mammalian cells related to PI3K/Akt, Rho GTPases, and the actin cytoskeleton. MethodsTo evaluate whether PI3K/Akt/Rho GTPases and the actin cytoskeleton participate in DENV replication in Huh7 cells, chemical and genetic inhibition were performed over 24 h.p.i., including early (1–12 h.p.i.) and late (12–24 h.p.i.) infection. Effects were evidenced by quantification of viral titers, activation of kinases assayed by western blot and In-Cell Western and subcellular patterns registered by quantitative fluorescence microscopy. ResultsDENV infections induced activation of PI3K/Akt with concomitant reorganization of the actin cytoskeleton, which was confirmed using specific chemical inhibitors. Additionally, inhibition of PI3K/Akt/Rho GTPases and actin microfilaments significantly reduced new viral progeny. Blocking the downstream effectors (ROCK and Rac1) of this pathway mimicked the cellular phenotype of PI3K/Akt/Rho GTPases inhibition. Furthermore, blockage of the final executor (i.e., actin) of this cellular process in infected cells also elicited molecular and viral effects. Finally, combined PI3K/Akt inhibition and Rho GTPases knockdown (Rac1, Rac2 and Cdc42), showed a similar effect on DENV-2 titer to that observed by individual treatment. ConclusionsTaken together, these findings suggest that the PI3K/Akt pathway is involved in DENV-2 infection in a Rho GTPase- and actin-dependent manner and that DENV-2 uses this signaling cascade to efficiently replicate in cells.

Inhibition of Stat3 signaling pathway decreases TNF-α-induced autophagy in cementoblasts

Autophagy is a self-digestive process that eliminates impaired or aged proteins and potentially toxic intracellular components to maintain homeostasis. We previously demonstrated that TNF-α played a critical role in cementoblast differentiation, mineralization and apoptosis; however, the effect of TNF-α on cementoblast autophagy has remained unclear. In this study, an elevated immunofluorescence signal of LC3B and autophagic vacuoles, autophagosomes and autolysosomes were detected under TNF-α stimulation in OCCM-30 cells. Autophagy-related genes and proteins, Beclin-1, LC3A and Atg-5, were significantly upregulated by TNF-α in a time- and concentration-dependent manner. During this process, the activity of Stat3 was dramatically enhanced and when the activity of Stat3 was blocked by either a specific chemical inhibitor or siRNA transfection before TNF-α stimulation, the TNF-α-induced upregulation of autophagy-related genes and proteins was strongly inhibited. Our results suggest that TNF-α induced autophagy in cementoblasts was dependent, or partially dependent on the activity of Stat3 signaling pathway.

Leucine-rich repeat kinase 2 aggravates secondary brain injury induced by intracerebral hemorrhage in rats by regulating the P38 MAPK/Drosha pathway

Leucine-rich repeat kinase 2 (LRRK2) is the genetic cause of both familial and idiopathic Parkinson's disease (PD), and it is associated with neuronal death, vesicle trafficking, mitochondrial dysfunction, and inflammation. However, its role in secondary brain injury (SBI) induced by intracerebral hemorrhage (ICH) has not been evaluated. In this study, an ICH model was induced by injecting autologous whole blood into the right basal ganglia of adult rats. Meanwhile, primary rat cortical neurons treated with Oxyhemoglobin (OxyHb) were used as an in vitro ICH model. Protein levels of LRRK2 increased significantly in brain tissues after ICH. Upregulation of LRRK2 by genetic overexpression augmented inflammatory responses, behavioral and cognitive dysfunction, brain edema, blood-brain barrier (BBB) injury, and cell death involved in SBI following ICH. Downregulation of LRRK2 by GNE7915 (a specific chemical inhibitor of LRRK2) and genetic knockdown yielded opposite effects. Additionally, inhibiting LRRK2 by GNE7915 obviously reduced OxyHb-induced neuronal apoptosis in vitro and attenuated phosphorylation of p38 MAPK and Drosha both in vivo and in vitro. Therefore, we concluded that LRRK2 participated in ICH-induced SBI by mediating inflammatory responses, behavioral and cognitive dysfunction, brain edema, and BBB injury and by modulating neuronal death and dysfunction and regulating the p38 MAPK/Drosha pathway.

RalA controls glucose homeostasis by regulating glucose uptake in brown fat

Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo.

Small Molecule Inhibitors of NFkB Reverse Iron Overload and Hepcidin Deregulation in a Zebrafish Model for Hereditary Hemochromatosis Type 3.

Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasian populations, with no viable therapeutic options except phlebotomy. We describe a zebrafish model of human HH (HH) created by targeted mutagenesis of the gene encoding transferrin receptor 2 ( tfr2). TFR2 mutations in humans lead to HH Type 3, a rare but severe form of the disease. The tfr2 mutant model in zebrafish recapitulates the defining features of HH3: iron overload and suppression of hepcidin, the iron regulatory hormone. Using in vivo chemical screens in zebrafish embryos, we identify a new small molecule inducer of hepcidin: SC-514, a specific chemical inhibitor of NFkB signaling. Using independent small molecule inhibitors of the NFkB pathway, we demonstrate that inhibition of NFkB signaling causes induction of hepcidin transcription and reduction of iron overload in the HH3 model. This first successful chemical intervention for hereditary hemochromatosis may also have relevance in treatment of other very prevalent iron regulatory iron overload disorders such as thalassemia.

Attenuation of TGFBR2 expression and tumour progression in prostate cancer involve diverse hypoxia-regulated pathways

BackgroundDysregulation of transforming growth factor β (TGF-β) signaling and hypoxic microenvironment have respectively been reported to be involved in disease progression in malignancies of prostate. Emerging evidence indicates that downregulation of TGFBR2, a pivotal regulator of TGF-β signaling, may contribute to carcinogenesis and progression of prostate cancer (PCa). However, the biological function and regulatory mechanism of TGFBR2 in PCa remain poorly understood. In this study, we propose to investigate the crosstalk of hypoxia and TGF-β signaling and provide insight into the molecular mechanism underlying the regulatory pathways in PCa.MethodsProstate cancer cell lines were cultured in hypoxia or normoxia to evaluate the effect of hypoxia on TGFBR2 expression. Methylation specific polymerase chain reaction (MSP) and demethylation agents was used to evaluate the methylation regulation of TGFBR2 promoter. Besides, silencing of EZH2 via specific siRNAs or chemical inhibitor was used to validate the regulatory effect of EZH2 on TGFBR2. Moreover, we conducted PCR, western blot, and luciferase assays which studied the relationship of miR-93 and TGFBR2 in PCa cell lines and specimens. We also detected the impacts of hypoxia on EZH2 and miR-93, and further examined the tumorigenic functions of miR-93 on proliferation and epithelial-mesenchymal transition via a series of experiments.ResultsTGFBR2 expression was attenuated under hypoxia. Hypoxia-induced EZH2 promoted H3K27me3 which caused TGFBR2 promoter hypermethylation and contributed to its epigenetic silencing in PCa. Besides, miR-93 was significantly upregulated in PCa tissues and cell lines, and negatively correlated with the expression of TGFBR2. Ectopic expression of miR-93 promoted cell proliferation, migration and invasion in PCa, and its expression could also be induced by hypoxia. In addition, TGFBR2 was identified as a bona fide target of miR-93.ConclusionsOur findings elucidate diverse hypoxia-regulated pathways including EZH2-mediated hypermethylation and miR-93-induced silencing contribute to attenuation of TGFBR2 expression and promote cancer progression in prostate cancer.

Disposition of Mianserin and Cyclizine in UGT2B10-Overexpressing Human Embryonic Kidney 293 Cells: Identification of UGT2B10 as a Novel N-Glucosidation Enzyme and Breast Cancer Resistance Protein as an N-Glucoside Transporter.

UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and detoxification of amine-containing chemicals; however, the disposition mechanisms for amines via UGT metabolism are not fully clear. We aimed to investigate a potential role of UGT2B10 in N-glucosidation and to determine the transporters for the excretion of N-glucosides. We first established a human embryonic kidney cell line 293 (HEK293) that stably overexpressed UGT2B10. Incubation of mianserin or cyclizine with the cells generated one N-glucuronide and one N-glucoside. Chemical inhibition (using specific chemical inhibitor Ko143) and biologic inhibition [using specific short hairpin RNA of breast cancer resistance protein (BCRP)] resulted in a significant reduction in efflux of N-glucuronide. Similar results were observed when multidrug resistance-associated protein (MRP4) was inhibited. Furthermore, inhibition of BCRP led to increased intracellular N-glucoside, whereas inhibition of MRP4 caused no changes in disposition of N-glucoside. Overall, the data indicated that BCRP, not MRP4, was responsible for the excretion of N-glucosides, whereas both BCRP and MRP4 contributed to excretion of N-glucuronides. Interestingly, downregulation of N-glucuronidation led to increased N-glucosidation in the cells, supporting the glucosidation as a potential complementary pathway for conventional glucuronidation. In conclusion, UGT2B10 was for the first time identified as an N-glucosidation enzyme. Generated N-glucosides were excreted primarily by the BCRP transporter.

CXCL8 is associated with the recurrence of patients with acute myeloid leukemia and cell proliferation in leukemia cell lines

Acute myeloid leukemia (AML) blasts release a wide range of chemokines in which CXCL8 has recently been recognized to be important for tumor progression. To find out the function of CXCL8 in AML, we compared blood serum of AML patients and healthy donors and found that the average level of CXCL8 was higher in AML patients. Among patients, higher expression of CXCL8 was also a positive recurrence indicator which illustrated the critical role of CXCL8 in AML. Knocking down of CXCL8 in leukemic cell lines led to significant reduction of proliferation via inducing G0/G1 cell cycle arrest and apoptosis, which was accompanied by the inactivation of ERK1/2. Furthermore, inhibition of ERK1/2 by specific chemical inhibitors reconstructed the CXCL8 knocking down phenomenon. Overall, we demonstrated that expression level of CXCL8 had a positive relationship with recurrence probability in AML. And CXCL8 was strongly implicated in AML cells growth by activating the ERK1/2 signal pathway.

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation.

SLC2A5 promotes lung adenocarcinoma cell growth and metastasis by enhancing fructose utilization

The metabolism of cancer cells is highly plastic. Cancer cells can change their preference for nutrient uptake under nutrient stress. Fructose is one of the most common carbohydrates in diet and its metabolism is also involved in the development and progression of tumors. GLUT5, encoded by SLC2A5, is the specific fructose transporter in mammalian cells. In this study, we found that SLC2A5 is significantly upregulated in lung adenocarcinoma (LUAD) patients and overexpression of SLC2A5 is highly correlated with poor prognosis of LUAD patients. The expression of SLC2A5 determined fructose uptake and utilization efficacy in LUAD cells. GLUT5 is critical for the survival of LUAD cells in fructose-containing culture medium. Depletion of SLC2A5 undermined cell proliferation and invasion meanwhile increased cell apoptosis. Overexpression of SLC2A5 enhances cell proliferation, migration, invasion, and tumorigenic. Compared to glucose, fructose is prone to strengthen intracellular-free fatty acid accumulation and ATP production. Moreover, inhibition of GLUT5 by specific small chemical inhibitor sensitizes LUAD cells to paclitaxel treatment. Taken together, our results suggest that GLUT5 could be a potential target alone or combination with other treatment for lung cancer therapy.

Nitroimidazole-based inhibitors DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit the activity of human CA IX in Xenopus oocytes

Carbonic anhydrase (CA) IX is a hypoxia inducible enzyme that is highly expressed in solid tumours. Therefore, it has been considered as an anticancer target using specific chemical inhibitors. The nitroimidazoles DTP338 and DTP348 have been shown to inhibit CA IX in nanomolar range in vitro and reduce extracellular acidification in hypoxia, and impair tumour growth. We screened these compounds for toxicity using zebrafish embryos and measured their in vivo effects on human CA IX in Xenopus oocytes. In the toxicity screening, the LD50 for both compounds was 3.5 mM. Neither compound showed apparent toxicity below 300 µM concentration. Above this concentration, both compounds altered the movement of zebrafish larvae. The IC50 was 0.14 ± 0.02 µM for DTP338 and 19.26 ± 1.97 µM for DTP348, suggesting that these compounds efficiently inhibit CA IX in vivo. Our results suggest that these compounds can be developed as drugs for cancer therapy.

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein.

BackgroundThe adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized.Methodology/Principal findingsBy infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells.Conclusions/SignificanceOur data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.

IL-4 up-regulates cyclooxygenase-1 expression in macrophages

Macrophages use various cell-surface receptors to sense their environment and undergo polarized responses. The cytokines, interleukin (IL)-4 and IL-13, released from T-helper type 2 (Th2) cells, drive macrophage polarization toward an alternatively activated phenotype (M2). This phenotype is associated with the expression of potent pro-resolving mediators, such as the prostaglandin (PG) D2-derived cyclopentenone metabolite, 15d-PGJ2, produced by the cyclooxygenase (Ptgs; Cox) pathway. Interestingly, IL-4 treatment of bone marrow-derived macrophages (BMDMs) significantly down-regulates Cox-2 protein expression, whereas Cox-1 levels are significantly increased. This phenomenon not only challenges the dogma that Cox-1 is only developmentally regulated, but also demonstrates a novel mechanism in which IL-4-dependent regulation of Cox-1 involves the activation of the mechanistic target of rapamycin complex (mTORC). Using specific chemical inhibitors, we demonstrate here that IL-4-dependent Cox-1 up-regulation occurs at the post-transcriptional level via the Fes-Akt-mTORC axis. Activation of AMP-activated protein kinase (AMPK) by metformin, inhibition of mTORC by torin 1, or CRISPR/Cas9-mediated genetic knock-out of tuberous sclerosis complex-2 (Tsc2) blocked the IL-4-dependent expression of Cox-1 and the ability of macrophages to polarize to M2. However, use of 15d-PGJ2 partially rescued the effects of AMPK activation, suggesting the importance of Cox-1 in macrophage polarization as also observed in a model of gastrointestinal helminth clearance. In summary, these findings suggest a new paradigm where IL-4-dependent up-regulation of Cox-1 expression may play a key role in tissue homeostasis and wound healing during Th2-mediated immune responses, such as parasitic infections.

Blocking autophagy enhances the apoptotic effect of 18\u03b2-glycyrrhetinic acid on human sarcoma cells via endoplasmic reticulum stress and JNK activation

Sarcoma, a rare form of cancer, is unlike the much more common carcinomas as it occurs in a distinct type of tissue. The potent antitumor effects of 18β-glycyrrhetinic acid (GA), a novel naturally derived agent, have been demonstrated in various cancers. However, the effect of GA on human sarcoma, and the underlying mechanisms, remain to be elucidated. In the current study, we show that GA inhibits sarcoma cell proliferation by inducing G0/G1-phase arrest. Exposure to GA resulted in the activation of caspase-3, -8, and -9, indicating that GA induced apoptosis through both extrinsic and intrinsic pathways. In addition, the autophagy pathway, characterized by the conversion of LC3-I to LC3- II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression, and stimulation of autophagic flux. The present findings showed that GA stimulated autophagy by inducing endoplasmic reticulum (ER) stress via the IRE1–JNK pathway. Our data supported the prosurvival role of GA-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors. Finally, GA markedly reduced sarcoma growth, with little organ-related toxicity, in vivo. The present results suggest that the combination of GA with a specific autophagy inhibitor represents a promising therapeutic approach for the treatment of sarcoma.

GPE Promotes the Proliferation and Migration of Mouse Embryonic Neural Stem Cells and Their Progeny In Vitro.

This study was designed to investigate a possible role of the N-terminal tripeptide of insulin-like growth factor-1 (IGF-I), Gly-Pro-Glu (GPE), physiologically generated in neurons following IGF-I-specific cleavage, in promoting neural regeneration after an injury. Primary cultures of mouse neural stem cells (NSCs), obtained from 13.5 Days post-conception (dpc) mouse embryos, were challenged with either GPE, growth hormone (GH), or GPE + GH and the effects on cell proliferation, migration, and survival were evaluated both under basal conditions and in response to a wound healing assay. The cellular pathways activated by GPE were also investigated by using specific chemical inhibitors. The results of the study indicate that GPE treatment promotes the proliferation and the migration of neural stem cells in vitro through a mechanism that involves the activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase PI3K-Akt pathways. Intriguingly, both GPE effects and the signaling pathways activated were similar to those observed after GH treatment. Based upon the results obtained from this study, GPE, as well as GH, may be useful in promoting neural protection and/or regeneration after an injury.

W09, a novel autophagy enhancer, induces autophagy-dependent cell apoptosis via activation of the EGFR-mediated RAS-RAF1-MAP2K-MAPK1/3 pathway

ABSTRACTThe EGFR (epidermal growth factor receptor) signaling pathway is frequently deregulated in many malignancies. Therefore, targeting the EGFR pathway is regarded as a promising strategy for anticancer drug discovery. Herein, we identified a 2-amino-nicotinonitrile compound (w09) as a novel autophagy enhancer, which potently induced macroautophagy/autophagy and consequent apoptosis in gastric cancer cells. Mechanistic studies revealed that EGFR-mediated activation of the RAS-RAF1-MAP2K-MAPK1/3 signaling pathway played a critical role in w09-induced autophagy and apoptosis of gastric cancer cells. Inhibition of the MAPK1/3 pathway with U0126 or blockade of autophagy by specific chemical inhibitors markedly attenuated the effect of w09-mediated growth inhibition and caspase-dependent apoptosis. Furthermore, these conclusions were supported by knockdown of ATG5 or knockout of ATG5 and/or ATG7. Notably, w09 increased the expression of SQSTM1 by transcription, and knockout of SQSTM1 or deleting the LC3-interaction region domain of SQSTM1, significantly inhibited w09-induced PARP1 cleavage, suggesting the central role played by SQSTM1 in w09-induced apoptosis. In addition, in vivo administration of w09 effectively inhibited tumor growth of SGC-7901 xenografts. Hence, our findings not only suggested that activation of the EGFR-RAS-RAF1-MAP2K-MAPK1/3 signaling pathway may play a critical role in w09-induced autophagy and apoptosis, but also imply that induction of autophagic cancer cell death through activation of the EGFR pathway may be a potential therapeutic strategy for EGFR-disregulated gastric tumors.

FoxO1-AMPK-ULK1 Regulates Ethanol-Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1-PIK3C3 Complex.

Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOH impairs protein synthesis in C2C12 myoblasts via a FoxO1-AMPK-TSC2-mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol-induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1-AMPK. C2C12 myoblasts were incubated with EtOH for various periods of time, and autophagy pathway-related proteins were assessed by Western blotting and immunoprecipitation. Expression of targeted genes was suppressed using electroporation of specific siRNAs and chemical inhibitors. Incubation of C2C12 myoblasts with 100mM EtOH increased the autophagy markers LC3B-II and ATG7, whereas levels of SQSTM1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with EtOH. EtOH altered ULK1 S555 and S757 phosphorylation in a time- and AMPK-dependent manner. The activation of AMPK and ULK1 was associated with increased BECN1 (S93, S14) and PIK3C3/VPS34 (S164) phosphorylation as well as increased total ATG14 and PIK3C3. These changes promoted formation of the ATG14-AMBRA1-BECN1-PIK3C3 proautophagy complex that is important in autophagosome formation. EtOH-induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK3C3 complex binding with 14-3-3θ. Reduction of AMPK using siRNA suppressed the stimulatory effect of EtOH on BECN1 S93, BECN1 S14, and PIK3C3 S164 phosphorylation in a time-dependent manner. Likewise, knockdown of AMPK or chemical inhibition of FoxO1 attenuated phosphorylation of ULK1 at both residues. Knockdown of ULK1 or BECN1 antagonized the effect of EtOH on LC3B-II, SQSTM1, and ATG7 protein expression. EtOH-induced autophagy is mediated through changes in phosphorylation and interaction of various PIK3C3 complex components. This, in turn, is regulated either directly via FoxO1-AMPK or indirectly via the FoxO1-AMPK-ULK1 signaling cascade in a mTORC1-independent or mTORC1-dependent manner.

Abstract A37: Development of screening methods to identify Translesion DNA Synthesis inhibitors

Abstract Translesion DNA synthesis (TLS) is a DNA damage tolerance process that employs specialized polymerases to bypass DNA damage during replication. Recent evidence indicates that TLS is a key process that promotes the development of resistance to cancer treatments that induce DNA damage (i.e. Cisplatin). Thus, the inhibition of TLS emerges as a promising strategy for cancer therapy. However, to date, specific chemical inhibitors of TLS are not available. The main goal of our project is to identify specific inhibitors of TLS that can be used as a proof of concept in cancer therapy by developing cell-based assays that explore TLS markers. Our rational is that since TLS polymerases recruitment to sites of DNA damage is a key step for TLS success, we can indirectly monitor TLS efficiency in a given context by studying two key markers of TLS polymerases recruitment: 1) The mono-ubiquitylation of the replication auxiliary factor PCNA and 2) the accumulation of a TLS polymerase into replication foci. We thus developed two screening methods that allow us to promptly identify inhibitors of PCNA ubiquitylation and TLS polymerases recruitment into DNA damage sites. The first marker, PCNA mono-ubiquitylation, is assessed through a western-Blot-based platform where the identification of unmodified PCNA and mono-ubiquitylated PCNA are achieved by two different antibodies coupled to fluorescent infrared detection using a fluorescence scanner. For the second marker, TLS polymerase recruitment to damage sites, we developed stable cell lines expressing TLS polymerases fused to fluorescent proteins and we analyze polymerases recruitment through an imaging-based assay. In this poster we describe the results of a pilot screening using an open source library of kinase inhibitors from GlaxoSmithKline (known as PKIS2), and the early validation of the identified hits. Citation Format: Florencia Villafañez, Alejandra Iris García, María Florencia Pansa, Sofía Carbajosa, José Luis Bocco, Gastón Soria. Development of screening methods to identify Translesion DNA Synthesis inhibitors [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A37.

Marked for death: targeting epigenetic changes in cancer.

In the past few years, it has become clear that mutations in epigenetic regulatory genes are common in human cancers. Therapeutic strategies are now being developed to target cancers with mutations in these genes using specific chemical inhibitors. In addition, a complementary approach based on the concept of synthetic lethality, which allows exploitation of loss-of-function mutations in cancers that are not targetable by conventional methods, has gained traction. Both of these approaches are now being tested in several clinical trials. In this Review, we present recent advances in epigenetic drug discovery and development, and suggest possible future avenues of investigation to drive progress in this area.