Myc Induction Research Articles

Abstract 1879: Myc induces lipogenesis and suppresses fatty acid oxidation in human P493-6 B-lymphoma cells.

Abstract Myc has been known to control cell growth and proliferation through regulating thousands of genes, of which a substantial amount are involved in metabolism. We have previously shown that Myc increases ribosomal biogenesis, nucleotide metabolism, mitochondrial biogenesis, glycolysis and glutaminolysis in cancer cells. However, Myc's role in fatty acid metabolism is not well-documented, notwithstanding the expectation that Myc induction of cell growth must be accompanied by membrane biogenesis. Other studies have shown that fatty acids can be utilized as energy fuel by cancer cells or as building blocks for proliferation. The P493 human B cell line contains a tetracycline (Tet)-repressible human MYC transgene, permitting robust control of cell growth and proliferation through MYC. Having previously documented that Myc stimulates glucose and glutamine metabolism, we sought to determine whether Myc affects P493 use of fatty acids for oxidation and fatty acid synthesis. We found that resting P493 cells, in which ectopic MYC was off, can use labeled palmitate for respiration, while Myc activated P493 cells demonstrated an inability to oxidize palmitate. We also observe through microarray analysis and RT-PCR that Myc increases the levels of mRNAs for lipogenesis genes. Chromatin immunoprecipitation (ChIP) with an anti-Myc antibody documents that Myc binds to the promoters of lipogenesis genes. Moreover, immunoblot analyses with antibodies available for selected enzymes also indicate that Myc increases the protein levels of ACACA and FASN. Uniformly labeled 13C-glucose and 13C,15N-glutamine were found to be incorporated into lipids when Myc was induced in P493 cells. Cerulenin, an inhibitor of FASN, dramatically inhibits Myc-induced P493 cell proliferation. Similarly, TOFA-inhibition of ACACA suppresses P493 cell proliferation as well. Our data suggest that in P493 Burkitt lymphoma model cells, Myc inhibits fatty acid oxidation and upregulates fatty acid synthesis through transcription of target genes. Citation Format: Arvin Matthew Gouw, Anthony Mancuso, Annie Lee Hsieh, Brian Altman, Michael Wolfgang, Chi Van Dang. Myc induces lipogenesis and suppresses fatty acid oxidation in human P493-6 B-lymphoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1879. doi:10.1158/1538-7445.AM2013-1879

BACH2 Is Required for Pre-B Cell Receptor Checkpoint Control and p53-Dependent Tumor Surveillance

AbstractAbstract 1300 Background:BACH2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) is required for class-switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of mature germinal center B cells. We and others found that BACH2 is strongly upregulated in BCR-ABL1-transformed acute lymphoblastic leukemia (Ph+ ALL) cells upon treatment with tyrosine kinase inhibitors (TKI). Results:Bach2 mRNA levels are significantly lower in Ph+ ALL (n=72) compared to normal human bone marrow pre-B cells (n=10). Studying gene expression in a clinical trial for children with high risk ALL (Children’s Oncology Group, P9906; n=207), we found in a multivariate analysis that high Bach2 levels at the time of diagnosis represents an independent predictor of favorable clinical outcome (negative MRD at day and higher overall and relapse-free survival; p<0.0001). We next studied 49 sample pairs from patients with childhood ALL at diagnosis and relapse. In 44 of these sample pairs, the relapse sample showed drastically reduced mRNA levels of Bach2 (p=0.019), suggesting that loss of BACH2 expression is associated with relapse of childhood ALL. A comparison of the methylation status of BACH2 promoter of normal pre-B cells (n=5), with Ph+ ALL cells (n=70) revealed that CpG islands in the BACH2 promoter were heavily hypermethylated in the leukemia samples. These findings are also consistent with genomic analyses on patient derived samples and the identification of small deletions at 6q15 in 4 of 11 cases of childhood ALL cases that all span the BACH2 locus.To study the role of Bach2 in pre-B ALL in a genetic experiment, we transformed pre-B cells from Bach2−/− mice with BCR-ABL1. We observed that Bach2−/− normal pre-B cells lack the ability to counterselect pre-B cell clones that failed to undergo successful V(D)J rearrangement. In the absence of Bach2, a significant number of B cells survive even though they failed to rearrange immunoglobulin heavy chain genes. Besides this unexpected role in early B cell differentiation, quantitative RT-PCR and Western blot confirmed that BACH2 is also required for expression of the tumor suppressors Cdkn2a (Arf), p53 and Btg2. Consistent with extremely low protein levels of Arf and p53 in Bach2−/− leukemia cells, Bach2−/− ALL cells are more resistant to TKI-treatment, more actively proliferating (increased S-phase; p=0.02) and exhibit a ∼90-fold increased ability to form colonies in methyl cellulose (p=0.001). Studying Cre-mediated inducible deletion of p53 in p53-fl/fl leukemia cells, we found that Bach2-induced tumor suppression is largely dependent on p53 function.Forced overexpression of Myc results in oncogene-induced senescence (OIS) followed by apoptosis. Whereas Bach2+/+ leukemia cells are non-permissive to forced Myc expression and die within four days after Myc induction, Bach2−/− ALL cells tolerate forced expression of Myc and evade OIS and subsequent cell death. Similarly, overexpression of Myc alone fails to transform Bach2+/+ pre-B cells. By contrast, retroviral overexpression of Myc results in rapid transformation and growth factor-independence of Bach2−/− pre-B cells. Bach2−/− Myc-high pre-B cells cause fatal leukemia in 100% of recipient mice within 22 days, whereas all mice that received Bach2+/+ Myc-high pre-B cells survived without signs of disease until day 67, when all mice were sacrificed and analyzed for MRD by flow cytometry and PCR. No evidence of MRD was detected in most mice injected with Bach2+/+ Myc-high pre-B cells. Three mice had positive MRD PCR findings, however, at 4 log orders below findings in mice injected with Bach2−/−Myc-high pre-B cells. Conclusions:Our findings identify Bach2 as a novel tumor suppressor upstream of p53 in pre-B ALL. Bach2 is a regulator of negative selection during normal pre-B cell differentiation but also limits excessive proliferation of pre-B cell clones by induction of oncogene-induced senescence and activation of p53. In addition, our multivariate analyses identify high expression levels of Bach2 as powerful predictor of favorable clinical outcome in children, which may be useful in future approaches for risk stratification. Disclosures:No relevant conflicts of interest to declare.

B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia

Abstract SCI-25The B-cell receptor (BCR) is a flexible and variable environmental sensor with no fixed ligand. The central component is surface Ig (sIg), which appears to function at several levels, mediating a “tonic” signal essential for survival and also binding to antigens via its variable (V) regions. Expression of sIg and reliance on BCR signals generally persist in neoplastic B cells. Indolent tumors, including chronic lymphocytic leukemia (CLL), allow insight into the pathogenetic role of the BCR prior to therapy as well as revealing key proteins within these pathways for drug targeting. CLL is heterogeneous, arising at two points of differentiation, generating two major subsets, one with unmutated Ig V genes (U-CLL), another with mutated Ig V genes (M-CLL). U-CLL appears to develop from naïve B cells of the natural antibody repertoire aimed against common pathogens. The clinical behavior of the two subsets differs, with U-CLL being of poorer prognosis. Evidence for antigen drive on both subsets comes from detecting “endocytosis in vivo,” whereby sIgM expression and signal capacity in blood cells are variably downmodulated, but can recover in vitro. Mysteriously, sIgD of the same presumed antigen specificity shows no evidence for endocytic downmodulation in vivo. CLL cells apparently engage antigens via sIgM in tissue sites, leading to proliferation and downmodulation, with reexpression gradually occurring during transit through the blood. Expression of CXCR4 closely follows that of sIgM, and clonal analysis reveals subpopulations of potentially dangerous cells with high sIgM/CXCR4 primed for tissue-based proliferative stimulation. In contrast to normal B cells, this is an iterative process exposing the proliferating CLL cells to further genetic changes. Overall higher sIgM levels and increased signal capacity in U-CLL likely account for more aggressive clinical behavior. BCR-induced membrane-proximal events include LYN-mediated phosphorylation of Iga/b followed by recruitment of the tyrosine kinase Syk. Signal propagation then involves Btk and PLCg2. LYN-dependent phosphorylation of CD19 also recruits the p85 subunit of PI3K, a known survival mechanism in CLL. Downstream events include upregulation of MYC proto-oncoprotein expression and induction of MYC-regulated target genes such as cyclin D2, with both proteins detected in proliferation centers. Pathways to increased cell survival include induction of the antiapoptotic MCL1 protein and inactivation of the proapoptotic activity of BIM(EL/L) via enhanced phosphorylation. The ability to phosphorylate BIM(EL) was highly correlated with mutational status and with requirement for treatment. While these events delineate BCR-activated pathways, they provide only the skeleton. sIgM signaling is highly dependent on the polymeric nature of the antigen, with responses to solid-phase stimulus producing a higher and more prolonged signal than the soluble form. Clearly, CLL cells have to integrate BCR signals with those from other receptors for the multitude of microenvironmental factors. This is a two-way process, since BCR signals operate “inside-out” by modulating the expression of molecules involved in migration and adhesion. The fact that the glycan composition of sIgM is also modulated to a mannosylated form, potentially able to bind to mannose-binding lectins, could contribute to the latter. Clinical effects of Syk, Btk and PI3Kd inhibitors, known to affect BCR signaling and potentially other pathways, are both explicable and exciting. Disclosures:No relevant conflicts of interest to declare.

Oncostatin M Is a Growth Factor for Ewing Sarcoma

Primary bone tumors, osteosarcomas and chondrosarcomas, derive from mesenchymal stem cells committed into osteoblasts and chondrocytes; in Ewing sarcomas (ESs), the oncogenic fusion protein EWS-FLI1 prevents mesenchymal differentiation and induces neuroectodermic features. Oncostatin M (OSM) is a cytokine from the IL-6 family that modulates proliferation and differentiation in numerous cells. The basis for inhibition versus induction of proliferation by this cytokine is obscure, although MYC was described as a potent molecular switch in OSM signaling. We show herein that, in contrast to osteosarcomas and chondrosarcomas, for which OSM was cytostatic, OSM induced proliferation of ES cell lines. Knockdown experiments demonstrated that growth induction by OSM depends on both types I [leukemia inhibitory factor receptor (LIFR)] and II [OSM receptor (OSMR)] receptors, high STAT3 activation, and induction of MYC to a high expression level. Indeed, ES cell lines, mice xenografts, and patient biopsy specimens poorly expressed LIF, precluding LIFR lysosomal degradation and OSMR transcriptional induction, thus leading to a high LIFR/OSMR ratio. Because other neuroectodermic tumors (ie, glioma, medulloblastoma, and neuroblastoma) had a similar expression profile, the main role of EWS-FLI1 could be through maintenance of stemness and neuroectodermic features, characterized by a low LIF, a high LIFR/OSMR ratio, and high MYC expression. Thus, this study on rare bone malignancies gives valuable insights on more common cancer regulatory mechanisms and could provide new therapeutic opportunities.

Abstract 3080: MYC antagonizes imatinib-induced differentiation in chronic myeloid leukemia cells through downregulation of p27KIP1

Abstract Imatinib is a BCR-ABL kinase inhibitor and the most used drug in chronic myeloid leukemia (CML). MYC (c-Myc) is a transcription factor frequently deregulated in human cancer that exerts multiple biological activities, including differentiation inhibition. We found that MYC expression is higher in bone marrow cells form CML patients at diagnosis, as compared with healthy controls. Moreover, high MYC levels at diagnosis correlated with a poorer response to imatinib treatment. One of the major characteristics of CML progression is the loss of cell differentiation. We showed that low concentrations of imatinib induce erythroid differentiation in the CML-derived K562 cell line. We have studied the effect of MYC on the differentiation induced by imatinib using K562 sublines carrying inducible (by zinc cation) or activable (by 4-hydoxy-tamoxifen) MYC alleles. In both cell lines, MYC largely prevented the erythroid differentiation induced by imatinib. The differentiation inhibition mediated by MYC is not due to increased proliferation of MYC-expressing cells or enhanced apoptosis of differentiated cells in the presence of MYC. We previously reported that p27/Kip1 (p27) overexpression induces erythroid differentiation in K562. Thus, we explored the effect of imatinib on p27 levels and we found that imatinib up-regulated p27 in a time- and concentration-dependent manner. siRNA-mediated silencing of p27 antagonized the erythroid differentiation induced by imatinib, indicating that p27 up-regulation is at least in part responsible for differentiation. MYC abrogated the imatinib-induced up-regulation of p27KIP1 concomitantly with the differentiation inhibition, suggesting that MYC inhibits imatinib-mediated differentiation of K562 cells by antagonizing p27 up-regulation. We previously reported that MYC induced SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. MYC was able to induce SKP2 in the presence of imatinib. Thus, MYC-mediated inhibition of the differentiating effect of imatinib in CML cells is in part a consequence of the SKP2 induction of MYC, which in turn would down-regulate p27. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3080. doi:1538-7445.AM2012-3080

Abstract 1114: Loss of OPA1 expression results in apoptosis for cells overexpressing c-Myc

Abstract c-Myc (Myc) is a transcription factor that controls the expression of a vast array of genes that function in many essential cellular pathways, and high levels of Myc have been implicated in the development of many human cancers. One pathway that Myc controls is mitochondrial biogenesis. We have previously shown that Myc does this is in part via the regulation of the expression of proteins involved in mitochondrial fusion, including Opa1, whose levels are increased upon the over-expression of Myc. This results in a highly interconnected mitochondrial network in cells with high levels of Myc. This fused mitochondrial network is present despite the fact that Myc also increases the expression of proteins involved in mitochondrial fission as well. We sought to understand how Myc controls the expression of Opa1 and to determine if the high levels of mitochondrial fusion in Myc over-expressing cells are important in cellular transformation. We hypothesized that the increased mitochondrial fusion driven by the Myc-mediated increase in Opa1 may reduce apoptosis. Rat fibroblasts with inducible Myc expression were transfected with an OPA1-specific shRNA. These cells were characterized by a series of assays that measured different aspects of mitochondrial structure and function. After only 48h of Myc induction, mitochondrial mass increased by approximately two-fold compared to control cells; however, when Opa1 levels were reduced in this background (Myc+/Opa-), mitochondrial mass was decreased four-fold. The cells with decreased Opa1 expression but minimal Myc activity (Myc-/Opa-) had a punctate appearance to the mitochondria, which became even more dramatic in Myc+/Opa- cells. This was likely related to the ability of Myc to increase the proteins involved in fission. There was an approximate 50% decrease in the ability to perform oxidative phosphorylation (OXPHOS) when Myc-/Opa- cells were compared to Myc+/Opa- cells. This was consistent with the four-fold decrease in net ATP levels when Myc-/Opa- cells were compared to Myc+/Opa- cells. Finally, prolonged activation of Myc in cells with decreased expression of OPA1 leads to apoptosis. After 48 hours of Myc induction the Myc+/Opa- cells the cells gradually began to die and cultures were nearly devoid of viable cells within 10 days, whereas Myc-/Opa- cells remained almost completely viable. We believe that the unopposed mitochondrial fission in Myc+/Opa- cells results in an increase susceptibility to an early apoptotic death. The mechanisms involved in this phenomenon are being explored, as is the possibility of exploiting this consequence of Opa1 reduction as a means of selectively eliminating tumor cells that display Myc over-expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1114. doi:1538-7445.AM2012-1114

The miR-17∼92 family regulates the response to Toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes

Chronic lymphocytic leukemia (CLL) cells from clinically aggressive cases have a greater capacity to respond to external microenvironmental stimuli, including those transduced through Toll-like-receptor-9 (TLR9). Concomitant microRNA and gene expression profiling in purified CLL cells (n=17) expressing either unmutated (UM) or mutated (M) IGHV genes selected microRNAs from the miR-17∼92 family as significantly upregulated and in part responsible for modifications in the gene expression profile of UM CLL cells stimulated with the TLR9 agonist CpG. Notably, the stable and sustained upregulation of miR-17∼92 microRNAs by CpG was preceded by a transient induction of the proto-oncogene MYC. The enforced expression of miR-17, a major member from this family, reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected cells from serum-free-induced apoptosis (P ≤ 0.05). Consistently, transfection with miR-17∼92 family antagomiRs reduced Bromo-deoxy-uridine incorporation in CpG-stimulated UM CLL cells. Finally, miR-17 expression levels, evaluated in 83 CLL samples, were significantly higher in UM (P=0.03) and ZAP-70(high) (P=0.02) cases. Altogether, these data reveal a role for microRNAs of the miR-17∼92 family in regulating pro-survival and growth-promoting responses of CLL cells to TLR9 triggering. Overall, targeting of this pathway may represent a novel therapeutic option for management of aggressive CLL.

Suppression of Ras/Mapk pathway signaling inhibits Myc-induced lymphomagenesis

Although the Myc transcription factor has been shown necessary for the oncogenic function of Ras, the contribution of Ras pathway signaling to the oncogenic function of Myc remains unresolved. We report the novel findings that Myc alone induced Ras/Mapk pathway signaling, and increased signaling following growth factor stimulation. Deletion of the scaffold protein kinase suppressor of Ras 1 (Ksr1) attenuated signaling through the Ras/Mapk pathway, including activation following Myc induction. B cells that lacked Ksr1 exhibited reduced proliferation and increased cytokine deprivation-induced apoptosis. Overexpression of Myc rescued the proliferation defect of Ksr1-null B cells, but loss of Ksr1 increased sensitivity of B cells to Myc-induced apoptosis. Notably, there was a significant delay in lymphoma development in Ksr1-null mice overexpressing Myc in B cells (Eμ-myc transgenic mice). There was an elevated frequency of p53 inactivation, indicative of increased selective pressure to bypass the p53 tumor suppressor pathway, in Ksr1-null Eμ-myc lymphomas. Therefore, loss of Ksr1 inhibits Ras/Mapk pathway signaling leading to increased Myc-induced B-cell apoptosis, and this results in reduced B-cell transformation and lymphoma development. Our data indicate that suppression of Myc-induced Ras/Mapk pathway signaling significantly impairs Myc oncogenic function. These results fill a significant gap in knowledge about Myc and should open new avenues of therapeutic intervention for Myc-overexpressing malignancies.

Oncogenic Splicing Factor SRSF1 Is a Critical Transcriptional Target of MYC

The SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here, we show that SRSF1 is a direct target of the transcription factor oncoprotein MYC. These two oncogenes are significantly coexpressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two noncanonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC's oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.

Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic lymphocytic leukemia cells

AbstractAlthough long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.

Regulation of c-Myc Protein Abundance by a Protein Phosphatase 2A-Glycogen Synthase Kinase 3 -Negative Feedback Pathway

Regulation of Myc protein abundance is critical for normal cell growth as evidenced by the fact that deregulated Myc expression is a hallmark of many cancers. One of several important mechanisms that control Myc levels involves its phosphorylation-dependent proteolysis. Previous studies have shown that phosphorylation of threonine 58 by glycogen synthase kinase 3β (GSK3β) within the conserved Myc Box I sequence results in binding by the ubiquitin ligase Fbw7-SCF complex, followed by ubiquitination and proteasome-mediated degradation of Myc. Here, we show that induction of Myc in several cell types correlates with loss of the inhibitory serine 9 phosphorylation of GSK3β and its increased kinase activity. The Myc-induced decrease in serine 9 phosphorylation is blocked by okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We therefore examined components of PP2A complexes and found that, among the regulatory B56 subunits, only the promoter of the ppp2r5d gene, encoding the B56δ isoform, is directly bound and transcriptionally activated by Myc in an E-box-dependent manner. Furthermore, we find that B56δ associates with both GSK3β and Myc, resulting in phosphorylation of Myc threonine 58, the well-established signal for ubiquitination and degradation. Furthermore, overexpression, or siRNA-mediated knockdown, of B56δ respectively results in accelerated, or retarded, rates of Myc degradation. Together, our data indicate that Myc limits its own abundance through a negative feedback pathway involving PP2A and GSK3β.

Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model

SUMMARYMyc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifen-inducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.

Early induction of c-Myc is associated with neuronal cell death

Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases.

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

BackgroundGenetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways.ResultsOur biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia.ConclusionsOur study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.

Down-regulation of BLIMP1α by the EBV oncogene, LMP-1, disrupts the plasma cell differentiation program and prevents viral replication in B cells: implications for the pathogenesis of EBV-associated B-cell lymphomas

AbstractAn important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.

Romo1 is a negative-feedback regulator of Myc

Degradation of Myc protein is mediated by E3 ubiquitin ligases, including SCF(Fbw7) and SCF(Skp2), but much remains unknown about the mechanism of S-phase kinase-associated protein (Skp2)-mediated Myc degradation. In the present study, we show that upregulated Myc protein, which triggers the G1-S phase progression in response to growth-stimulatory signals, induces reactive oxygen species modulator 1 (Romo1) expression. Romo1 subsequently triggers Skp2-mediated ubiquitylation and degradation of Myc by a mechanism not previously reported in normal lung fibroblasts. We also show that reactive oxygen species (ROS) derived from steady-state Romo1 expression are necessary for cell cycle entry of quiescent cells. From this study, we suggest that the generation of ROS mediated by pre-existing Romo1 protein is required for Myc induction. Meanwhile, Romo1 expression induced by Myc during G1 phase stimulates Skp2-mediated Myc degradation in a negative-feedback mechanism.

Abstract 1259: c-Myc influences mitochondrial structure and function by regulating fusion and fission

Abstract Background: c-Myc (Myc) is a global transcription factor that controls the expression of a large part of the human genome. The numerous Myc target genes function in a variety of normal cellular processes whose collective expression of these processes is referred to as the “Myc phenotype”. One such cellular process is the regulation of mitochondrial biogenesis. Objectives: To understand the specific influence that Myc has on mitochondrial structure and function. We hypothesize that Myc is involved in the regulation of both the production and destruction of mitochondria, and that the ability of Myc to maintain this balance has an impact on cellular transformation. Design/Method: Various cell lines that allow for the manipulation of Myc levels have been used. These include cells that have been engineered to have inducible Myc over-expression as well as inducible expression of Myc shRNA. These strains were used in a series of assays that measured different aspects of the Myc effect on mitochondrial structure and function. Most notably, metabolic pathways were measured by an extracellular flux analyzer and electron and confocal microscopy was performed to examine the structure of the mitochondria in detail. Results: The induction of Myc results in dramatic increases in overall mitochondrial mass, as well as increasing individual mitochondrial size and cristae density. This increase in recognizable mitochondria correlates to increases in oxidative phosphorylation and decreased mitochondrial loss through mitophagy. Finally, Myc levels correlate to increased mitochondrial inter-connectivity. This appears to be related to the ability of Myc to influence the expression of the mitochondrial fusion protein, Opa1 and the fission protein, DLP1. Conclusions Reached: The changes in mitochondrial structure and function are directly influenced by the expression of Myc. There appear to be multiple pathways involved in maintaining a specific mitochondrial mass, which emphasizes the importance of this organelle in cellular homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1259. doi:10.1158/1538-7445.AM2011-1259

SKP2 Oncogene Is a Direct MYC Target Gene and MYC Down-regulates p27KIP1 through SKP2 in Human Leukemia Cells

SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.

Myc-dependent Mitochondrial Generation of Acetyl-CoA Contributes to Fatty Acid Biosynthesis and Histone Acetylation during Cell Cycle Entry

Cell reprogramming from a quiescent to proliferative state requires coordinate activation of multiple -omic networks. These networks activate histones, increase cellular bioenergetics and the synthesis of macromolecules required for cell proliferation. However, mechanisms that coordinate the regulation of these interconnected networks are not fully understood. The oncogene c-Myc (Myc) activates cellular metabolism and global chromatin remodeling. Here we tested for an interconnection between Myc regulation of metabolism and acetylation of histones. Using [(13)C(6)]glucose and a combination of GC/MS and LC/ESI tandem mass spectrometry, we determined the fractional incorporation of (13)C-labeled 2-carbon fragments into the fatty acid palmitate, and acetyl-lysines at the N-terminal tail of histone H4 in myc(-/-) and myc(+/+) Rat1A fibroblasts. Our data demonstrate that Myc increases mitochondrial synthesis of acetyl-CoA, as the de novo synthesis of (13)C-labeled palmitate was increased 2-fold in Myc-expressing cells. Additionally, Myc induced a forty percent increase in (13)C-labeled acetyl-CoA on H4-K16. This is linked to the capacity of Myc to increase mitochondrial production of acetyl-CoA, as we show that mitochondria provide 50% of the acetyl groups on H4-K16. These data point to a key role for Myc in directing the interconnection of -omic networks, and in particular, epigenetic modification of proteins in response to proliferative signals.

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.