Chase Experiments Research Articles

Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway.

Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. An in vitro experimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver.

Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

TP53 (p53) and MYC are amongst the most frequently altered genes in cancer. Both are thus attractive targets for new anticancer therapies. Historically, however, both genes have proved challenging to target and currently there is no approved therapy against either. The aim of this study was to investigate the effect of the mutant p53 reactivating drug, COTI-2 on MYC. Total MYC, pSer62 MYC and pThr58 MYC were detected using Western blotting. Proteasome-mediated degradation was determined using the proteasome, inhibitor MG-132, while MYC half-life was measured using pulse chase experiments in the presence of cycloheximide. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Treatment of 5 mutant p53 breast cancer cell lines with COTI-2 resulted in dose-dependent MYC degradation. Addition of the proteasome inhibitor, MG132, rescued the degradation, suggesting that this proteolytic system was at least partly responsible for the inactivation of MYC. Using cycloheximide in pulse chase experiments, COTI-2 was found to reduce the half-life of MYC in 2 different mutant p53 breast cancer cell lines, i.e., from 34.8 to 18.6 min in MDA-MB-232 cells and from 29.6 to 20.3 min in MDA-MB-468 cells. Co-treatment with COTI-2 and the MYC inhibitor, MYCi975 resulted in synergistic growth inhibition in all 4 mutant p53 cell lines investigated. The dual ability of COTI-2 to reactivate mutant p53 and degrade MYC should enable this compound to have broad application as an anticancer drug.

Stress granules affect the sensitivity of renal cancer cells to sorafenib by sequestering and stabilizing COX‑2 mRNA.

Most patients with renal cancer will develop resistance to sorafenib therapy and will therefore exhibit disease progression. Effective therapies for these patients are extremely limited. Cyclooxygenase-2 (COX-2) promotes the malignant transformation of cancer cells and drug resistance. The potential of COX-2 inhibitor (celecoxib) administration in combination with sorafenib for the treatment of renal cancer is unclear. The present study demonstrated that sorafenib rapidly increased the expression of COX-2 in renal cancer cells, as determined using reverse transcription-quantitative PCR and western blotting. The results of the MTT assay and cell apoptosis experiment demonstrated that the cytotoxicity of sorafenib was also affected by COX-2 expression and celecoxib enhanced the cytotoxicity of sorafenib against renal cell carcinoma. Immunofluorescence analysis indicated that sorafenib induced the formation of stress granules (SGs) in renal cancer cells. In addition, COX-2 expression was associated with the formation of SGs, and SGs could capture and stabilize COX-2 mRNAs in renal cancer cells; this was confirmed using RNA fluorescence in situ hybridization and an actinomycin D chase experiment. The protective effect of SGs was further demonstrated in cell experiments and xenograft tumor models. Thus, the present study indicated that the use of celecoxib may significantly enhance the sensitivity of renal cancer cells to sorafenib and improve efficacy. Sorafenib-induced SGs may contribute to critical events that promote COX-2 expression and survival in renal cancer cells. Therefore, the present study may provide novel ideas for the treatment of renal cancer.

Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

Molecular mechanisms of PBX1 regulation of CD4 +T cell inflammatory phenotypes

Abstract The pre-B cell leukemia homeobox1 (Pbx1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, Pbx1-d that lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4 +T cells from lupus-prone mice and SLE patients than healthy controls. We have shown that overexpression of Pbx1-d in CD4 +T cells impaired Treg cell homeostasis and expanded inflammatory CD4 +T cells. Here, we investigated the molecular mechanisms by which PBX1 regulates T cells. We showed that PBX1 is preferentially expressed in Treg cells over other CD4+ T cell subsets, and that it is negatively regulated by TCR activation. Furthermore, PBX1-D protein was less stable than the normal isoform, PBX1-B, in a pulse chase experiment. PBX1-B but not PBX1-D has two predictive ubiquitin binding sites, K292 and K293, located in the DNA binding domain. Mutation of these two sites reduced PBX1-B stability to a level similar to PBX1-D, suggesting that a loss of function in the lupus associated allele through the ubiquitin-proteasome dependent pathway. In addition, contrary to PBX1-B, PBX1-D is retained in the cytoplasm without the help of the MEIS or PREP1 co-factors, indicating different requirements for nuclear translocation. Overall, these findings suggest that multiple transcriptional and post-transcriptional mechanisms are responsible for PBX1-D loss of function and induction of CD4 +T cell inflammatory phenotypes in SLE. This work was supported by National Institutes of Health Grants AI045050

Inhibition of IGF2BP1 attenuates renal injury and inflammation by alleviating m6A modifications and E2F1/MIF pathway.

Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3'-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis in vivo. In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.

P.03 Myosin dysregulation in nemaline myopathy

Nemaline myopathy (NM) is one of the most common congenital myopathies. NM is often related to pathogenic variants in the <i>NEB, ACTA1, TPM2</i> and <i>TPM3</i> genes. Even though the exact disease mechanisms remain to be elucidated, histological analyses of NM patients' muscle biopsies may reveal unexplained accumulation of glycogen and abnormal mitochondria. Thus, the aim of the present work was to identify what could cause such change in muscle energetics. For that, we applied mant-ATP chase experiments on skeletal muscle fibres from patients diagnosed with NM. We observed that the myosin-stabilising conformational state, known as the super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting patient muscle fibres expressing NEB and ACTA1 mutations. Such alteration was linked to hyper-phosphorylation of myosin-binding protein C, a protein linking myosin molecules to the thin filaments. Hence, our results may indicate a contribution of myosin to some of the histopathological aberrations related to NM.

The START domain mediates Arabidopsis GLABRA2 dimerization and turnover independently of homeodomain DNA binding.

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding HD in conjunction with a lipid-binding domain termed steroidogenic acute regulatory-related lipid transfer (START). Previous work established that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis (Arabidopsis thaliana), is required for TF activity. Here, we addressed the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) resulted in phenotypic defects in leaf trichomes, root hairs, and seed mucilage, similar to those observed for START domain mutants, despite nuclear localization of the respective proteins. In vitro and in vivo experiments demonstrated that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, protein interaction assays revealed impaired GL2 dimerization for multiple alleles of START mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provided evidence for the role of START, but not HD, in maintaining protein stability. This work advances our mechanistic understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

703 Tracing the proliferative dynamics of dermal papilla (DP) progenitors during adult hair follicle (HF) regeneration

The dermal papilla (DP) instructs the cyclical regeneration of the adult hair follicle (HF). The HF grows in anagen, regresses in catagen and rests in telogen. The DP expands in cell number as the HF regenerates, which is critical for HF growth and regulating hair shaft size. Currently how the DP expands is not clear. As the DP is considered to be a quiescent population, the prevailing theory is that the DP expands from proliferative cells that reside outside the DP. We performed EdU pulse chase experiments to track when and where DP progenitors divide and contribute to the expanding DP.

Abstract 2972: Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung cancer

Abstract Standard care of lung cancer treatment has shifted away from non-specific, cytotoxic chemotherapy in favor of targeted therapies based on genetic mutations within tumors. In 2014, somatic mutations in the small GTPase RIT1 (Ras-like in all tissues) were discovered as oncogenic drivers of lung adenocarcinoma. Thousands of patients per year are diagnosed with RIT1-driven cancer, but treatment options are limited. A targeted therapy for RIT1-driven tumors would address a major unmet clinical need. Little is known about how RIT1 drives cellular transformation. To genetically dissect RIT1 function, we performed a genome-wide CRISPR/Cas9 screen in isogenic PC9 lung adenocarcinoma cells. This screen took advantage of the observation that RIT1-mutant cells are resistant to EGFR inhibition. We leveraged this drug resistance phenotype to identify genetic dependencies (gene knockouts that are detrimental to cell growth) and cooperating factors (gene knockouts that are beneficial to cell growth) in RIT1-mutant cells. From this screen, we found that one of the top essential genes was the deubiquitinase USP9X. This is intriguing given that the protein abundance of RIT1 is known to be important for its function. Therefore, we sought out to test the hypothesis that USP9X regulates RIT1 abundance and that inhibition of USP9X could be an effective therapeutic strategy for abrogating RIT1-driven tumor growth. Our model suggests that USP9X promotes proteasome-mediated degradation of RIT1. To test this, we assessed RIT1 abundance in the context of USP9X knockout (KO). We found that RIT1 protein abundance was decreased in USP9X KO PC9 cells compared to parental cells. Furthermore, cycloheximide (CHX)-chase experiments revealed that RIT1 stability was decreased in USP9X KO cells, and RIT1 degraded faster than in parental cells. The average half-life of RIT1 in USP9X KO cells was 3.4 hrs while the average half-life in parental PC9 cells was 12.3 hrs (95% CI = -12.5 to -5.4 hrs). Treatment with the proteasome inhibitor bortezomib (BTZ) rescued RIT1 degradation by 99% in parental cells and 190% in USP9X KO cells (95% CI = 57 to 126%). In addition to assessing protein abundance and stability, we performed co-immunoprecipitation experiments in RIT1-expressing HEK293T cells and found that RIT1 and USP9X physically interact. Taken together, these data support the hypothesis that RIT1 is a substrate of USP9X. In addition to providing better insight on the protein regulation of RIT1, this work has crucial therapeutic implications. The protein abundance of RIT1 is important for its function, and our model suggests that USP9X inhibition could be an effective means of reducing RIT1 protein abundance and abrogating tumor growth. Overall, this work is poised to significantly impact the field of RIT1 biology and address a major unmet clinical need for the treatment of RIT1-driven diseases. Citation Format: Amanda K. Riley, Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, Alice H. Berger. Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2972.

Abstract 5723: JDP2 interacts with p53 and enhances p53 transactivation and stability

Abstract USAAP-1 protein mainly consists of proteins belonging to c-Jun, c-Fos, activating transcription factor (ATF), and Jun dimerization protein (JDP) family. In addition to AP-1 site, JDP2 also binds to cAMP responsive element (CRE) site in numerous cis-elements of the target genes. JDP2 has been shown to be involved in cancer development and cell-cycle regulation, suppression of adipocyte differentiation, and controlling bone homeostasis and antibacterial immunity. ATF3 and JDP2 share high similarity in C-terminal domains with identities of 65%. Previous studies have demonstrated that ATF3 can bind to p53 and regulate p53 transcriptional activity and stability. Though couple of studies have shown that JDP2 inhibits expression of p53 and p53 represses the transcriptional activity of JDP2 promoter; however, whether JDP2 directly interacts and regulates p53 transactivation remains largely unknown. Herein, we demonstrate for the first time that JDP2 can directly interact with p53. First, we found that both ATF3 and JDP2 can bind to p53 and the C-terminus of JDP2 is required for p53 binding. Secondly, in p53-null H1299 cells, JDP2 dose-dependently enhances p53 transactivation using p53RE-Luc (14X). Moreover, we demonstrated that JDP2 dose-dependently decreases p53 protein level in consistence with the previous reports. Finally, JDP2 increases p53 stability in CHX chase experiments. Taken together, our results demonstrate that JDP2 directly binds to p53 and enhances p53 transactivation and stability, suggesting that JDP2 is a novel regulator of p53. Citation Format: Wei-Hsiung Yang, Kasey Price, Leticia Cardoso, William Yang, Chiung-Min Wang, Richard Yang. JDP2 interacts with p53 and enhances p53 transactivation and stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5723.

Spatial Learning Promotes Adult Neurogenesis in Specific Regions of the Zebrafish Pallium.

Adult neurogenesis could be considered as a homeostatic mechanism that accompanies the continuous growth of teleost fish. As an alternative but not excluding hypothesis, adult neurogenesis would provide a form of plasticity necessary to adapt the brain to environmental challenges. The zebrafish pallium is a brain structure involved in the processing of various cognitive functions and exhibits extended neurogenic niches throughout the periventricular zone. The involvement of neuronal addition as a learning-related plastic mechanism has not been explored in this model, yet. In this work, we trained adult zebrafish in a spatial behavioral paradigm and evaluated the neurogenic dynamics in different pallial niches. We found that adult zebrafish improved their performance in a cue-guided rhomboid maze throughout five daily sessions, being the fish able to relearn the task after a rule change. This cognitive activity increased cell proliferation exclusively in two pallial regions: the caudal lateral pallium (cLP) and the rostral medial pallium (rMP). To assessed whether learning impinges on pallial adult neurogenesis, mitotic cells were labeled by BrdU administration, and then fish were trained at different periods of adult-born neuron maturation. Our results indicate that adult-born neurons are being produced on demand in rMP and cLP during the learning process, but with distinct critical periods among these regions. Next, we evaluated the time course of adult neurogenesis by pulse and chase experiments. We found that labeled cells decreased between 4 and 32 dpl in both learning-sensitive regions, whereas a fraction of them continues proliferating over time. By modeling the population dynamics of neural stem cells (NSC), we propose that learning increases adult neurogenesis by two mechanisms: driving a chained proliferation of labeled NSC and rescuing newborn neurons from death. Our findings highlight adult neurogenesis as a conserved source of brain plasticity and shed light on a rostro-caudal specialization of pallial neurogenic niches in adult zebrafish.

The interaction of O-GlcNAc-modified NLRX1 and IKK-α modulates IL-1β expression in M1 macrophages.

NOD-like receptor (NLR)X1 (NLRX1) is a negative regulator of inflammation by inhibiting nuclear factor-κB (NF-κB) signaling and downstream pro-inflammatory factors. However, its post-translational modification and how it participates in regulating the inflammatory responses in macrophages are still unclear. Here, we found that NLRX1 was modified with O-linked N-acetylglucosamine (O-GlcNAc). The interaction and co-localization between NLRX1 and O-GlcNAc transferase (OGT) was validated by co-immunoprecipitation and confocal microscopy analysis, and the nucleotide-binding domain (NBD) region of NLRX1 was required for its interaction with OGT. NLRX1 protein increased significantly after treatment with a high dose of OGT inhibitor OSMI-1. Elevated O-GlcNAcylation level promoted NLRX1 ubiquitination and decreased NLRX1 stability proved by ubiquitination and cycloheximide (CHX) chase experiments, and enhanced the interaction between NLRX1 and inhibitor of nuclear factor kappaB kinase-α (IKK-α), thus reducing the expression of inflammatory cytokine IL-1β in M1 macrophages. Together, our results indicate that the interaction between NLRX1 and O-GlcNAcylation coordinates and modulates the inflammatory process in macrophages.

Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.

Root Absorption and Xylem Movement of Ethephon in Tomato

Although ethephon is commonly used as a plant growth regulator during commercial production of horticultural crops, information on its movement within plants is limited. In this study, we developed a method to detect ethephon in plant tissues, and determined ethephon localization and movement using tomato (Solanum lycopersicum L.) as a model system. Tissues were ground in an acidic buffer that preserved ethephon intact. Ethylene was released from the extracts by adding sodium hydroxide and was determined subsequently by gas chromatography. Ethephon was detected in leaves within 1 hour of application to peat-based root zones and within 10 minutes in hydroponics. In a pulse–chase experiment, ethephon levels increased initially, then decreased after the plants were returned to ethephon-free solutions. Ethephon was present in directly collected xylem fluid; fluid collected from petiole stumps (after leaf blade excision) had similar ethephon levels between the different petioles. Stem girdling had no effect on ethephon accumulation in leaves. Together, these data indicate ethephon is readily mobile in the xylem stream and provides insight into the commercial use of ethephon as a root zone-applied growth regulator.

Myofibre Hyper-Contractility in Horses Expressing the Myosin Heavy Chain Myopathy Mutation, MYH1E321G.

Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1-related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses (MYH1E321G−/−) and Quarter Horses homozygous (MYH1E321G+/+) or heterozygous (MYH1E321G+/−) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca2+ sensitivity for MYH1E321G+/+ in comparison with MYH1E321G+/− and MYH1E321G−/− horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.

Osmotrophy of dissolved organic carbon by coccolithophores in darkness.

Summary The evolutionary and ecological story of coccolithophores poses questions about their heterotrophy, surviving darkness after the end‐Cretaceous asteroid impact as well as survival in the deep ocean twilight zone. Uptake of dissolved organic carbon might be an alternative nutritional strategy for supply of energy and carbon molecules.Using long‐term batch culture experiments, we examined coccolithophore growth and maintenance on organic compounds in darkness. Radiolabelled experiments were performed to study the uptake kinetics. Pulse–chase experiments were used to examine the uptake into unassimilated, exchangeable pools vs assimilated, nonexchangeable pools.We found that coccolithophores were able to survive and maintain their metabolism for up to 30 d in darkness, accomplishing about one cell division. The concentration dependence for uptake was similar to the concentration dependence for growth in Cruciplacolithus neohelis, suggesting that it was taking up carbon compounds and immediately incorporating them into biomass. We recorded net incorporation of radioactivity into the particulate inorganic fraction.We conclude that osmotrophy provides nutritional flexibility and supports long‐term survival in light intensities well below threshold for photosynthesis. The incorporation of dissolved organic matter into particulate inorganic carbon, raises fundamental questions about the role of the alkalinity pump and the alkalinity balance in the sea.

Small Molecule Activators of Protein Phosphatase 2A (SMAPs) Cause c-Myc Degradation and Tumor Growth Suppression in Preclinical Models of Lymphoid Malignancies

Introduction: c-myc is an oncogene implicated in the initiation and progression of multiple hematologic malignancies. Characteristic translocations of the c-myc gene with the immunoglobulin heavy chain gene (in Burkitt lymphoma and “double-hit” non-Hodgkin lymphoma) and with the T cell receptor gene (in T cell acute lymphoblastic leukemia, T-ALL) lead to constitutive expression of the c-myc transcription factor, ultimately driving dysregulated proliferation (Boxer & Dang, Oncogene 2001; 20(40):5595-5610). Strategies for inhibition of c-myc remain an area of active research; however, to date there are no approved myc-targeted agents within the cancer armamentarium, and cytotoxic chemotherapy remains the standard of care for c-myc-driven lymphoid malignancies. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase tumor suppressor protein that contributes to c-myc degradation through both direct dephosphorylation of an identified phosphodegron as well as by indirect mechanisms (Farrell & Sears, Cold Spring Harbor Perspect Med 2014; 4(3):1-15). Our laboratory has developed a series of first in class, orally bioavailable small molecule activators of PP2A (SMAPs) by derivatizing tricyclic antipsychotics to maximize antiproliferative activity and minimize psychotropic properties (Sangodkar et al., J Clin Invest 2017;127(6):2081-2090). We hypothesized that activation of PP2A with SMAPs would lead to c-myc degradation and tumor growth inhibition in models of c-myc-driven lymphoid malignancies.Methods: We investigated the effects of SMAP treatment on c-myc content, c-myc half-life (t1/2), cell viability, and xenograft tumor growth in Burkitt lymphoma (BL) and T-ALL cell lines with wild type and mutant c-myc phosphodegrons.Results: SMAP treatment induced apoptosis in Daudi (BL) cells, as evidenced by decreased viability and increased PARP and caspase cleavage. In Daudi (BL) and CCRF-CEM (T-ALL) cell lines, SMAP treatment led to rapid loss of phosphorylated and total c-myc protein. Cycloheximide (CHX)-chase experiments performed in CCRF-CEM cells confirmed that SMAP treatment affects c-myc degradation without affecting protein synthesis. This effect was abrogated by Ser to Glu mutation of residue 62 within the c-myc phosphodegron, suggesting that SMAP-mediated c-myc degradation is PP2A-dependent. Treatment of NOD/SCID mice bearing Daudi xenografts with twice daily oral SMAP decreased tumor c-myc content and inhibited tumor growth without obvious toxicity. Xenografts overexpressing c-myc with phosphodegron mutations (S62D, T58I) were resistant to SMAP treatment.Conclusions: These in vitro and in vivo observations establish PP2A activation as a potential pharmacologic strategy to target c-myc in cancers such as BL, T-ALL, and “double hit” non-Hodgkin lymphoma. SMAPs are well-tolerated, orally bioavailable activators of PP2A poised for further exploration in the treatment of c-myc-driven malignancies, either alone or in combination with other agents. Sequencing of the c-myc phosphodegron prior to initiation of treatment may facilitate patient selection in future clinical trials of PP2A activators. [Display omitted] DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

Dual Inhibition of Aurora Kinase and BRD4 Leads to Improved Anti-Tumor Activity in Myc-Overexpressing Lymphoma Cells

Background:Myc has proven extremely difficult to target therapeutically. Therefore, we hypothesized that optimal inhibition of multiple key pathways involved in Myc expression could overcome this long-standing problem. We identified aurora A kinase (AURKA) and bromodomain protein (BRD)4 as therapeutic targets to counteract Myc deregulation based on strong evidence that these pathways are essential for tumor maintenance in Myc-driven malignancies.Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines with high and low Myc mRNA expression. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. Cycloheximide chase followed by immunoblotting was used to examine Myc protein degradation kinetics, and real-time RT-PCR was used to examine Myc mRNA levels. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined inhibition.Results: Myc-overexpressing Burkitt lymphoma (Raji) cells were treated with increasing concentrations of small molecule inhibitors against AURKA (MLN-8237), BRD4 (I-BET-151), or both drugs for 48 to 72 hours and cell viability was evaluated. MLN-8237 and I-BET-151 inhibited cell growth individually with IC-50 of 10 nM, and 650 nM, respectively. As compared to treatment with the single agent alone, dual treatment induced more significant cell growth inhibition with IC-50 achieved at drug concentrations of < 5 nM and 500 nM for MLN-8237 and I-BET-151, respectively. The combination index (CI) values were less than 1 at various drug concentrations, indicating that different combinations were synergistic in terms of inhibitory effect on tumor cell viability. Superior activity of the dual inhibition was also noted in different Myc-overexpressing lymphoma cells. Combination treatments increased apoptosis and induced more pronounced cell cycle arrest compared to the single agent treatment alone.We then analyzed protein expression by Western blot. MLN-8237 reduced the expression of p-HisH3 and p-AURKA while increasing the expression of p-S6K. Treatment with I-BET-151 resulted in significant reduction of Myc total protein. Cycloheximide chase experiments demonstrated that inhibition of AURKA increased the rate of Myc protein degradation, while BRD4 inhibition had no effect on degradation kinetics. Conversely MLN-8237 did not decrease Myc transcription, while I-BET-151 significantly reduced the levels of Myc mRNA. The combination of the two drugs demonstrated additive/synergistic decreases of both Myc protein degradation and Myc transcription at higher drug doses.Lastly, athymic nude mice bearing Ramos lymphoma xenografts were treated with MLN-8237, I-BET-151, or with the combination of both agents (n = 8 per group). The mean tumor volumes in mice treated with negative control, I-BET-151, and MLN-8237 as single agents were 3480, 2320, and 671 mm3, respectively, at Day 28. Mice treated with MLN-8237/I-BET-151 combinations had mean tumor volume of 461 mm3 at Day 28. The survival rates for mice treated with negative control, I-BET-151, and MLN-8237 as single agents were 0%, 10%, and 70%, respectively, at Day 35 (Figure 1). Mice treated with the MLN-8237 and I-BET-151 combination demonstrated a 100% survival rate at Day 35.Conclusion: Our data demonstrated that Myc-overexpressing lymphoma can be successfully targeted by inhibition of Myc transcription and/or enhancement of Myc protein degradation. Specifically, MLN-8237, a small molecule inhibitor against AURKA, induced apoptosis of Myc-overexpressing tumor cells in vitro and showed the most promising anti-tumor activity in mice bearing Myc-overexpressing lymphoma, especially when combined with I-BET-151, a BRD4 inhibitor. [Display omitted] DisclosuresPark:Cornerstone Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Speakers Bureau; Seattle Genetics: Research Funding; Teva: Consultancy, Research Funding; Takeda: Research Funding. Ghosh:TG Therapeutics: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jassen: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Sharpless:Unity Biotechnology: Other: NA; HealthSpan Diagnostics: Other: NA; Pfizer: Other: NA; G1 Therapeutics: Equity Ownership.

GTSE1 promotes SNAIL1 degradation by facilitating its nuclear export in hepatocellular carcinoma cells.

Snail family transcriptional repressor 1 (SNAIL1) is a master inducer of the epithelial-to-mesenchymal transition (EMT) process, contributing to tumor metastasis and recurrence. Our previous study reported that G2 and S phase-expressed-1 (GTSE1) served a role in regulating SNAIL1 expression in hepatocellular carcinoma (HCC). However, the underlying mechanism remains unknown. Therefore, the present study aimed to reveal the regulatory mechanism of GTSE1 on SNAIL1 expression using in vitro assays performed in HCC cell models. It was demonstrated that endogenous SNAIL1 expression was downregulated and upregulated by GTSE1 overexpression or small interfering RNA-mediated knockdown, respectively. Via cycloheximide chase experiments, it was identified that GTSE1 overexpression increased the protein turnover of SNAIL1, while knockdown of GTSE1 reduced its degradation rate. Furthermore, it was demonstrated that GTSE1 overexpression induced the cytoplasmic expression of SNAIL1 using immunofluorescence and subcellular fractionation methods. The nuclear export inhibitor leptomycin B was able to decrease the cytoplasmic retention of SNAIL1 caused by GTSE1 overexpression. In addition, TGF-βI treatment increased both the mRNA and protein expression levels of GTSE1, and decreased the protein expression level of SNAIL1 without affecting its mRNA transcription in Huh7 cells. It was also found that TGF-β signaling could upregulate the transcription of GTSE1 expression by transactivating the Smad binding elements in the GTSE1 promoter. Moreover, the TGF-βI-induced decrease in SNAIL1 protein expression was GTSE1-dependent in Huh7 cells. In conclusion, the current study provides a novel mechanism via which GTSE1 affects the stability of SNAIL1 by regulating its subcellular localization in HCC cells.