Elevated Levels Of c-Myc Research Articles

Identification of antimycin A as a c-Myc degradation accelerator via high-throughput screening

c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.

IL-6 promotes MYC-induced B cell lymphomagenesis independent of STAT3.

The inflammatory cytokine IL-6 is known to play a causal role in the promotion of cancer, although the underlying mechanisms remain to be completely understood. Interplay between endogenous and environmental cues determines the fate of cancer development. The Eμ-myc transgenic mouse expresses elevated levels of c-Myc in the B cell lineage and develops B cell lymphomas with associated mutations in p53 or other genes linked to apoptosis. We generated Eμ-myc mice that either lacked the IL-6 gene, or lacked the STAT3 gene specifically in B cells to determine the role of the IL-6/JAK/STAT3 pathway in tumor development. Using the Eμ-myc lymphoma mouse model, we demonstrate that IL-6 is a critical tumor promoter during early stages of B cell lymphomagenesis. IL-6 is shown to inhibit the expression of tumor suppressors, notably BIM and PTEN, and this may contribute to advancing MYC-driven B cell tumorigenesis. Several miRNAs known to target BIM and PTEN are upregulated by IL-6 and likely lead to the stable suppression of pro-apoptotic pathways early during the tumorigenic process. STAT3, a classical downstream effector of IL-6, appears dispensable for Eμ-myc driven lymphomagenesis. We conclude that the growth-promoting and anti-apoptotic mechanisms activated by IL-6 are critically involved in Eμ-myc driven tumor initiation and progression, but the B cell intrinsic expression of STAT3 is not required.

Targeting PRMT5 to Circumvent Acquired Ibrutinib Resistance in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by genetic dysregulation of cyclin D1 and activation of signaling pathways driving uncontrolled MCL cell proliferation and survival. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK), a downstream target of the B-cell receptor (BCR) pathway. While ibrutinib exhibits significant single-agent therapeutic activity in patients with relapsed/refractory MCL, the vast majority of MCL patients on ibrutinib progress with aggressive disease and short survival (3-8 mo). Although ~80% of chronic lymphocytic leukemia patients with acquired ibrutinib resistance have mutations in BTK and PLCγ2, this is uncommon in MCL suggesting alternative mechanisms driving this resistant phenotype. Understanding drug-resistance mechanisms and developing effective therapies for ibrutinib resistant (IR) MCL are urgently needed. The major type II protein arginine methyltransferase enzyme, PRMT5, catalyzes symmetric dimethylation of arginine residues on histone tails (H3R8 and H4R3) and other proteins. PRMT5 regulates a vast array of biologic functions including RNA processing, DNA damage response, signal transduction, and gene expression. Amplified PRMT5 activity drives the expression and activity of key oncogenes (MYC, CYCLIND1, NOTCH1) while silencing expression and activity of tumor suppressors (ST7, RBL2, and p53). Our group has shown PRMT5 is overexpressed and dysregulated in MCL and strategies aimed at selectively targeting PRMT5 show anti-tumor activity in preclinical lymphoma models. Here we describe the development of a novel patient derived xenograft (PDX) of IR-MCL and explore PRMT5 inhibition as an alternative therapeutic option to circumvent IR. Peripheral blood mononuclear cells from a 75 yo male patient diagnosed with acquired classic IR-MCL were engrafted intravenously into NSG mice. After 5 passages, all mice engrafted with 107 MCL cells developed histologically confirmed MCL infiltrating kidney, lymph nodes, bone marrow, spleen and peripheral blood. Circulating human CD5+/CD19+ cells were detectable and quantifiable by flow cytometry by day 21 post-engraftment. Karyotype analysis confirmed the hallmark t(11;14)(q13;q32) of MCL while retaining nearly all cytogenetic abnormalities present in the patient's primary tumor including a deletion of chromosome 9, associated with deletion of MTAP, a therapeutic vulnerability for PRMT5-targeted therapy. Whole exome sequencing confirmed genomic stability with successive passages. Ex vivo cytotoxicity assays and protein pathway analysis further confirmed resistance to ibrutinib (IC50 >1 µM) with maintained hyper-phosphorylation of AKT (Ser473) and ERK (Thr202/Tyr204). Western blot analysis showed elevated levels of c-MYC, CYCLIND1, BCL2, and pERK. After validation of circulating disease at day 25 post engraftment, mice were treated with either a novel small molecule inhibitor of PRMT5 (PRT382, 10 mg/kg orally 4 days on 3 days off) or ibrutinib (75 mg/kg administered in drinking water, n=5 mice per treatment group). Treatment of this PDX model with PRT382 resulted in significantly decreased disease burden and improved median survival compared to control animals from 48 to 83 days, respectively (p=0.0045). We found no significant difference in survival (p= 0.6540) or circulating disease burden with ibrutinib therapy compared to control group. The full BTK occupancy of ibrutinib treated mice was validated using fluorescence resonance energy transfer-based assay. Ex vivo PDX MCL cells from PRT382-treated mice showed loss of symmetric dimethyl arginine with preservation of asymmetric dimethyl arginine levels, reduced H4(Sme2)R3 epigenetic marks, and elevated levels of BCL2, MYC, and pAKT/pERK. We developed a cell line (SEFA) allowing for in vitro mechanistic studies. We are currently investigating potential mechanisms responsible for circumventing IR-MCL by integrating genome-wide changes to chromatin accessibility and whole transcriptome analysis. This IR-MCL PDX mouse model serves as a useful tool to investigate mechanisms of drug resistance, provides a platform to explore novel pre-clinical therapeutic strategies to circumvent IR and demonstrates the therapeutic activity of PRMT5 targeted therapy in this aggressive disease. Disclosures Byrd: Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Genentech: Research Funding; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau. Vaddi:Prelude Therapeutics: Employment. Scherle:Prelude Therapeutics: Employment. Baiocchi:Prelude: Consultancy.

Heterogeneous nuclear ribonucleoprotein M promotes the progression of breast cancer by regulating the axin/β-catenin signaling pathway.

Despite significant progress in the treatment of breast cancer due to advances in surgery, cytotoxic agents, and endocrine therapy, the prognosis for patients has not improved much. Accumulated evidence indicates that heterogeneous nuclear ribonucleoprotein M (hnRNPM) and Wnt/β-catenin function as tumor oncogenes in the progression of many cancers. The present study aimed to explore whether HnRNPM/β-catenin signaling molecules might serve as a genetic target for breast cancer treatment. To shed light on this issue, quantitative real-time polymerase chain reaction (qRT-PCR) detection, Western blotting, and immunohistochemical staining were performed. The hnRNPM is expressed at a much higher level in breast cancer tissues and cell lines than in noncancerous tissues and cell lines. In vitro studies revealed that overexpressed hnRNPM promoted cell proliferation and colony formation but inhibited cell apoptosis. In vivo results demonstrated that upregulation of hnRNPM dramatically increased breast cancer xenograft tumor growth. Western blotting and immunofluorescence studies revealed that hnRNPM markedly activated the Wnt/β-catenin pathway and catalyzed its translocation from the cytoplasm to the nucleus by targeting axin, a negative regulator of Wnt/β-catenin signaling in MCF-7 and KPL-4 cells. Elevated levels of c-Myc and cyclin D1 were observed when MCF-7 and KPL-4 cells were transfected with a hnRNPM vector. These findings indicate that the hnRNPM/axin/β-catenin signaling pathway acts as an oncogenic promoter in the progression of breast cancer, suggesting that hnRNPM may be a potential target for the treatment of this disease.

MiR-182 promotes prostate cancer progression through activating Wnt/β-catenin signal pathway

Although prostate cancer can be surgical excised and effectively treated by androgen-deprivation therapy, radiotherapy, or chemotherapy, management of patients with advanced or drug-resistance prostate cancer stills remains a big trouble. Accumulated evidence indicated that miR-182 and Wnt/β-catenin function as tumor oncogene in the progression of a variety of tumors. However, little is known about how miR-182 regulates β-catenin signal molecular and impacts on the tumorigenesis of human prostate cancer. In this study, employing the analyses of qRT-PCR, we found that prostate cancer tissues expressed much more miR-182 than non-cancer tissues did. In vitro studies revealed that overexpression of miR-182 promoted cell proliferation, colony formation, migration, invasion and inhibited cell apoptosis; in vivo results demonstrated that silencing of miR-182 mediated by inhibitor dramatically reduced prostate cancer xenograft tumor growth. Importantly, through western blotting analysis, we identified that miR-182 dramatically activated Wnt/β-catenin pathway by targeting multiple negative regulators of Wnt/β-catenin signaling, including GSK-3β, APC, CK1 and Axin. Besides, we observed the elevated levels of c-myc and Cyclin D1 when PC-3 and LNCap cells were up-regulated miR-182. Our findings indicate that miR-182 acts as one of oncogenic factor in the progression of prostate cancer by recruiting a mechanism of aberrant activation of Wnt/β-catenin signaling.

Therapeutic aspects of c-MYC signaling in inflammatory and cancerous colonic diseases.

Colonic inflammation is required to heal infections, wounds, and maintain tissue homeostasis. As the seventh hallmark of cancer, however, it may affect all phases of tumor development, including tumor initiation, promotion, invasion and metastatic dissemination, and also evasion immune surveillance. Inflammation acts as a cellular stressor and may trigger DNA damage or genetic instability, and, further, chronic inflammation can provoke genetic mutations and epigenetic mechanisms that promote malignant cell transformation. Both sporadical and colitis-associated colorectal carcinogenesis are multi-step, complex processes arising from the uncontrolled proliferation and spreading of malignantly transformed cell clones with the obvious ability to evade the host’s protective immunity. In cells upon DNA damage several proto-oncogenes, including c-MYC are activated in parelell with the inactivation of tumor suppressor genes. The target genes of the c-MYC protein participate in different cellular functions, including cell cycle, survival, protein synthesis, cell adhesion, and micro-RNA expression. The transcriptional program regulated by c-MYC is context dependent, therefore the final cellular response to elevated c-MYC levels may range from increased proliferation to augmented apoptosis. Considering physiological intestinal homeostasis, c-MYC displays a fundamental role in the regulation of cell proliferation and crypt cell number. However, c-MYC gene is frequently deregulated in inflammation, and overexpressed in both sporadic and colitis-associated colon adenocarcinomas. Recent results demonstrated that endogenous c-MYC is essential for efficient induction of p53-dependent apoptosis following DNA damage, but c-MYC function is also involved in and regulated by autophagy-related mechanisms, while its expression is affected by DNA-methylation, or histone acetylation. Molecules directly targeting c-MYC, or agents acting on other genes involved in the c-MYC pathway could be selected for combined regiments. However, due to its context-dependent cellular function, it is clinically essential to consider which cytotoxic drugs are used in combination with c-MYC targeted agents in various tissues. Increasing our knowledge about MYC-dependent pathways might provide direction to novel anti-inflammatory and colorectal cancer therapies.

PP2A Inhibition By CIP2A or SETBP1 Leads to Elevated Levels of AKT S473 Which Can be Used As a Biomarker of Outcome in Acute Myeloid Leukaemia

Many cells utilise reversible phosphorylation as a mechanism of post-translational modification for activating and deactivating key regulatory molecules involved in cell signalling. Many malignancies are characterised by overactive kinases e.g. BCR-ABL or FLT-3 in myeloid malignancy, or ERK or ErbB2 in solid tumours. A major phosphatase working in opposition to kinases is protein phosphatase 2A (PP2A) but why the cellular phosphatases such as PP2A simply do not counteract the overactive kinase activity is unclear. PP2A activity is regulated by inhibitor proteins SET, (which is stabilised by a binding protein SETBP1) and cancerous inhibitor of PP2A (CIP2A). In CML, we have shown that high levels of CIP2A at diagnosis is a prospective biomarker for future progression to blast crisis. High levels of CIP2A in other malignancies have also been reported to confer a poor prognosis. In CML, high CIP2A levels lead to the stabilisation of c-Myc and elevation of E2F1, as well as PP2A inactivation. In AML there are data suggesting that high SETBP1 levels may confer a poor outcome in AML, by PP2A inhibition via increased action of SET, though little is known of CIP2A in AML. The aim of the study was to investigate if CIP2A or network related proteins could predict clinical outcome in AML patients.CIP2A and network related proteins were investigated in 119 AML patient samples collected in the UK MRC/NCRI trials AML15, 16 and 17. These were stratified into intermediate or adverse risk, based on cytogenetics as previously defined (Grimwade et al, Blood. 1998: 92; 2322-33). Protein levels were studied in diagnostic mono-nuclear cell samples by flow cytometry. The diagnostic CIP2A protein level (mean fluorescence intensity (MFI)) for all patients was calculated. This range was 0-13.69, the interquartile range was 1.47-5.11, the median was 3 and the mean was 3.5. High CIP2A patients are defined as those patients with a CIP2A level greater than or equal to 3.In all AML samples studied high CIP2A was associated with inactive PP2A as shown by high levels of PP2AY307 (p=0.05), as well as elevated levels of c-Myc p=<0.0001 and E2F1 p=<0.0001, a similar finding to what we have observed in CML. We also found that AKTS473 and STAT5 were also elevated in high CIP2A AML patients (p=0.002 and p=0.006 respectively). Unlike CML, we found in AML patients high CIP2A was associated with high levels of SET (p=<0.0001) and SETBP1 (p=0.01).Younger intermediate AML patients had significantly higher diagnostic CIP2A levels than adverse risk patients (p=0.004). In the intermediate risk group, overall survival was dominated by the survival from relapse. For younger intermediate risk patients with high and low CIP2A levels, median survival from relapse (censored at transplant) was 56 days and 303 days respectively. Multivariate analysis adjusted for FLT3-ITD showed that a high CIP2A level predicted inferior survival from relapse (p=0.04, hazard ratio 4.02). Interestingly, high diagnostic CIP2A was strongly associated with the presence of a FLT3-ITD mutation (p=0.03).In adverse risk patients SETBP1 levels were elevated but CIP2A levels were lower. SETBP1 acts via SET as an alternative inhibitor of PP2A. In younger patients with adverse cytogenetics, high SETBP1 levels predict for an inferior overall survival (p=0.02). For all 119 AML patients those with detectable SETBP1 protein levels at diagnosis had an inferior overall survival (p=0.01). High SETBP1 was also associated with secondary AML.In univariate analysis, high levels of AKTphosphorylation at seine 473 (which is inversely correlated with PP2A activity)were associated with poor survival in all patients. Multivariate analysis of known predictors of outcome (age, cytogenetics, white count, secondary leukaemia, FLT3-ITD) together with CIP2A and related proteins (PP2AY307, SET, SETBP1, c-Myc E2F1, AKTS473, STAT5, and E2F1) was used to derive a prognostic model for survival. This ranked high levels of AKTS473 as the third most important predictor of outcome after age and cytogenetics. Our data suggest that AKTS473 levels are elevated as a result of PP2A inactivation (p=>0.0001) caused by high level of PP2A inhibitors, either CIP2A (p=0.003) or SETBP1 (p=0.03).In summary, AKT phosphorylation at S473 is a biomarker of PP2A inhibition by either CIP2A (intermediate risk) or SETBP1 (adverse risk patients). The diagnostic AKTS473 level appears to be a novel biomarker for survival in AML. DisclosuresNo relevant conflicts of interest to declare.

ID: 42: IL-6/STAT3 Pathway in B Cell Gammaherpesvirus Latency and Myc-Driven B Cell Lymphoma

STAT3 is at the molecular crossroads of innate and adaptive immunity, and in B cells it responds to many cytokines including IL-6, IL-10, and IL-21. We investigated the role of STAT3 in two animal models of disease, the establishment of gammaherpesvirus latency in B cells, and the promotion of B cell lymphomas by the c-myc proto-oncogene. Murine gammaherpesvirus, MHV68, was used to evaluate the requirement of STAT3 for establishment of viral latency in B cells. We infected mice that lacked expression of STAT3 specifically in B cells by both intranasal and intraperitoneal routes. Establishment of viral latency in these animals was substantially impaired compared to controls, as assayed by colonization of the spleen and persistent infection of germinal center immunoglobulin class-switched B cells. There were no dramatic changes in total or virus-specific IgG titers in the absence of B cell STAT3. Our findings recognize STAT3 as a mediator of gammaherpesvirus latency in B cells. The E μ -myc transgenic mouse is a model of Burkitt’s B cell lymphoma in which elevated levels of c-Myc are expressed in the B cell lineage. We generated E μ -myc mice that either lacked the IL-6 gene, or lacked the STAT3 gene specifically in B cells to determine the role of the IL-6/STAT3 pathway in tumor development. We found that IL-6 promotes the development of E μ -myc B cell lymphomas, but the B cell intrinsic expression of STAT3 does not contribute to the development of E μ -myc lymphomas. Therefore, an IL-6-dependent signal pathway distinct from STAT3 cooperates with E μ -myc in oncogenesis.

Abstract 2292: The role of immature colon carcinoma transcript 1 during c-myc deregulation in fast-onset mouse plasmacytoma

Abstract Murine plasmacytoma (mPCT) models human blood cancers that involve the deregulation of c-myc by a translocation that juxtaposes c-myc to one of the immunoglobin loci. The fast-developing mPCT does not depend on c-myc translocation; rather infection with v-abl/c-myc retrovirus produces elevated c-myc levels. Chromosome 11 subcytoband E2 is always (100%) found duplicated compared to slow-developing mPCT (7.1%). The gene, immature colon carcinoma transcript1 (ict1) is located within 11E2 and overexpressed as a result of 11E2 duplication. The effect of ict1 overexpression during c-myc deregulation may contribute to the aggressive development of mPCT. Here we look at the tumorigenic potential of ict1 in vitro. The ict1 gene has been cloned into an Ecdysone inducible vector and transiently transfected (Nucleofection) into mouse PreB cells that also have inducible c-myc expression activated by 4-hydroxytamoxifen. This allows us to look at overexpression of ict1 along with c-myc simultaneously or each separately. Immunofluorescence was used to confirm overexpression of the target genes. Proliferation and apoptosis levels were determined for each condition by Edu and TUNEL assay respectively. Metaphase spreads were prepared before and after each experiment. We have confirmed by immunofluorescence overexpression of ict1 using the inducible vector system. A study in these cell lines shows that ict1 overexpression increases cell proliferation levels, and may reduce apoptosis compared to cells that were not induced for ict1/c-myc expression. Metaphase spreads show normal diploid chromosome number of 40 (at time 0, and every 24 hours). Further genetic analysis to identify any chromosomal abnormalities that may account for these changes will be performed (SKY). We have identified a potential role of ict1 overexpression in vitro. In order to further explore these results we will try to restore proliferation and apoptotic levels relative to a normal PreB cell phenotype in plasmacytoma cells in vitro in the MOPC 460D cell line, and ex vivo in plasmacytoma cells isolated from PCT-carrying mice. 11E2 is syntenic to human chromosome 17q25, which has chromosomal abnormalities in many other neoplasms, such as acute and chronic myeloid leukemia, neuroblastoma, breast, ovarian, thyroid cancers. If we can identify the role of overexpression of ict1 during c-myc deregulation in fast-onset mPCT, we can gain insight into the aggressiveness of the disease progression and determine if ict1 could lead to a new therapeutic target for aggressive blood cancers with c-myc deregulation. Citation Format: Amy K. Dahl, Ruedee Sakulratchata, Sabine Mai. The role of immature colon carcinoma transcript 1 during c-myc deregulation in fast-onset mouse plasmacytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2015-2292

The E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to suppress innate variant TFH cell development, thymocyte expansion, and lymphomagenesis.

It is now well established that the E and Id protein axis regulates multiple steps in lymphocyte development. However, it remains unknown how E and Id proteins mechanistically enforce and maintain the naïve T-cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate variant follicular helper T (TFH) cells. Innate variant TFH cells required major histocompatibility complex (MHC) class I-like signaling and were associated with germinal center B cells. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of a TFH transcription signature. We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion. We found that mice depleted for Id2 and Id3 expression developed colitis and αβ T-cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc, whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities to genetic deficiencies associated with Burkitt lymphoma. We propose that, in response to antigen receptor and/or cytokine signaling, the E-Id protein axis modulates the activities of the PI3K-AKT-mTORC1-Hif1a and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.

Registered report: transcriptional amplification in tumor cells with elevated c-Myc.

The Lin et al. (2012), published in Cell in 2012. The experiments that will be replicated are those reported in Figures 3E and 3F. In these experiments, elevated levels of c-Myc in the P493-6 cell model of Burkitt's lymphoma results in an increase of the total level of RNA using UV/VIS spectrophotometry (Figure 3E; Lin et al., 2012) and on the mRNA levels/cell for a large set of genes using digital gene expression technology (Figure 3F; Lin et al., 2012). The Reproducibility Project: Cancer Biology is a collaboration between the eLife.

Abstract 3519: The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs

Abstract Several clinical studies have shown a correlation between elevated c-Myc levels and a better response to chemotherapy. Although elevated c-Myc might indirectly cause such chemosensitivity, it is possible that a direct effect is occurring due to underlying molecular mechanisms. The purpose of the current study was to determine whether c-Myc overexpression could acutely cause chemosensitivity. The c-Myc protein is known to activate DNA replication origins, but the mechanism for this is unknown. Origins are regulated by the loading of MCM2-7 hexamers, which form the core of the helicase that starts the initiation of replication. Mammalian cells load more MCM complexes than are necessary to complete one round of replication. The excess MCM complexes function as backups to allow recovery of replication when forks stall during drug treatment. Consistent with this, we found that loss of Mcm4 or Mcm7 sensitizes PDAC cell lines to 5-FU and Gemcitabine, two S phase stress drugs that are used to treat PDAC patients but are not highly effective on their own. Similar to depletion of backup MCM complexes via loss of Mcm4 or Mcm7, elevated c-Myc expression over-activates backup MCMs, leaving fewer available to recover from drug treatment. Cells containing overexpressed c-Myc display increased chemosensitivity to Gemcitabine that is not seen in cells containing normal c-Myc levels or a non-functional c-Myc mutant. These results demonstrate that reduction in backup MCM complexes, either by direct downregulation or by oncogene driven over-activation (via c-Myc), render cells sensitive to chemotherapy drugs. These results suggest that development of drugs that target MCM functionality may be useful in combination with S-phase stress drugs currently used in the clinic. These results also demonstrate that c-Myc has the ability to acutely cause chemosensitivity and c-Myc elevated tumors may respond better to chemotherapy due to the c-Myc effects on MCM complexes. Citation Format: Victoria Bryant, Mark G. Alexandrow. The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2014-3519

Abstract 3090: Targeting glutamine metabolism as a therapeutic strategy in MYC-driven medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Currently, treatment consists of surgical resection, chemotherapy, craniospinal radiation, and is associated with high morbidity. Of the four molecular subgroups of MB, group 3 tumors are have the worst clinical prognosis and are associated with elevated c-MYC levels. Because MYC over-expression can lead to increased glutamine metabolism and glutamine addiction in other cancer types, we hypothesized that MYC-driven MB would exhibit increased glutamine metabolism and be sensitive to inhibitors of glutamine metabolism. In MB cell lines, MYC expression positively correlates with increased expression of enzymes involved in glutamine metabolism as measured by western blot. To investigate the hypothesis that inhibition of glutamine metabolism would preferentially target MYC driven MB, we tested the effects of two glutamine analogs—acivicin and 6-diazo-5-L-norleucine (DON)—that inhibit glutaminolytic enzymes. Acivicin and DON significantly decreased the growth of the high-MYC MB cell lines D425 and D283 as measured by MTS assay (D425 p&amp;lt;0.05 vehicle vs acivicin, D283 p&amp;lt;0.05 vehicle vs 50uM acivicin; D425 p&amp;lt;0.05 vehicle vs DON, D283 p&amp;lt;0.05 vehicle vs DON). Treatment with acivicin and DON also decreased proliferation as measured by BrdU incorporation. Apoptosis increased upon treatment with acivicin and DON treatment as determined by cleaved caspase 3 immunofluorescence positivity and an increased percentage of cells in the sub2N population as measured by flow cytometry. In neurosphere models of MYC and non-MYC driven MB, MYC-expressing cells showed a significant decrease in proliferation after acivicin treatment as measured by BrdU incorporation (p&amp;lt;0.005), while the proliferation of MYC-negative cells was unaffected. MB neurosphere models expressing MYC also showed an increase in apoptosis as measured by cleaved caspase 3 immunofluorescence positivity (p&amp;lt;0.003) and an increased percentage of cells in the sub2N population. Fewer mice receiving acivicin treatment following D425 xenograft developed flank tumors than untreated mice (40% treated mice vs 100% untreated mice). Treated mice also had smaller tumors than untreated mice (80.9mm3 in treated mice vs 273.8mm3 in untreated mice). Knocking down the expression of the glutaminolytic enzyme glutaminase using a short hairpin resulted in a significant decrease in growth in D425 (p&amp;lt;0.05 scramble control vs GLS hairpin) and D283 (p&amp;lt;0.05 scramble control vs GLS hairpins). Our laboratory is currently conducting in vivo orthotopic xenograft experiments using a MB cell line where GLS expression has been knocked-down using a short hairpin, and we predict that GLS knock-down will prolong survival. Taken together, our data suggest that glutamine metabolism may be a therapeutic target in MYC-driven MB and that glutamine analogs may be useful therapeutic agents. Citation Format: Allison Rose Hanaford, Charles G. Eberhart, Eric H. Raabe. Targeting glutamine metabolism as a therapeutic strategy in MYC-driven medulloblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3090. doi:10.1158/1538-7445.AM2014-3090

Oncogenic activation of glypican-3 by c-Myc in human hepatocellular carcinoma

Glypican-3 (GPC3) is a heparan sulfate proteoglycan that has an important role in cell growth and differentiation, and its function in tumorigenesis is tissue-dependent. In hepatocellular carcinoma (HCC), the overexpression of GPC3 has been demonstrated to be a reliable diagnostic indicator. However, the mechanisms that regulate the expression and function of GPC3 remain unclear. The oncoprotein c-Myc is a transcription factor that plays a significant role in more than 50% of human tumors. We report here that GPC3 is a transcriptional target of c-Myc and that the expression of c-Myc is also regulated by GPC3, thus forming a positive feedback signaling loop. We found that the overexpression of c-Myc could induce GPC3 promoter-dependent luciferase activity in luciferase reporter experiments. Furthermore, mutational analysis identified c-Myc-binding sites within the GPC3 promoter. The exogenous overexpression of c-Myc increased the endogenous messenger RNA (mRNA) and protein levels of GPC3. Chromatin immunoprecipitation experiments revealed the binding of c-Myc to the endogenous GPC3 promoter, indicating that c-Myc can directly transcriptionally activate GPC3. Interestingly, GPC3 can also elevate c-Myc expression. Overexpression of GPC3 increased c-Myc protein levels, whereas the knockdown of GPC3 reduced c-Myc expression levels. Lastly, the elevated levels of c-Myc correlate with the overexpression of GPC3 in human HCC samples. These data provide new mechanistic insight into the roles of GPC3 and of c-Myc in the development of HCC.

Abstract 4189: Excess c-Myc loads tissue-specific enhancers in tumor cells

Abstract Excessive expression of c-Myc occurs frequently in human cancers, where high levels are associated with tumor aggression and poor clinical outcome. It is well established that the c-Myc protein forms a heterodimer with Max, binds to E-box sequences typically located near the core promoter elements of target genes involved in cellular proliferation, and alters expression of these genes. It is not yet clear, however, how altering the levels of c-Myc affects its activity across the genome to enhance the oncogenic state of cells. We show here that c-Myc occupies occupies enhancer elements only in tumor cells with elevated levels of c-Myc. The enhancers that are occupied are often tumor-specific and associated with the cell-of-origin of the tumor. These and additional results provide insights into a key mechanism by which c-Myc overexpression alters the transcriptional state of cancer cells to enforce their tumorigenic state and malignant potential. 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 4189. doi:1538-7445.AM2012-4189

Role of Pirh2 in Mediating the Regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2−/− mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.

Modeling ductal carcinoma in situ: a HER2–Notch3 collaboration enables luminal filling

A large fraction of ductal carcinoma in situ (DCIS), a non-invasive precursor lesion of invasive breast cancer, overexpresses the HER2/neu oncogene. The ducts of DCIS are abnormally filled with cells that evade apoptosis, but the underlying mechanisms remain incompletely understood. We overexpressed HER2 in mammary epithelial cells and observed growth factor-independent proliferation. When grown in extracellular matrix as 3- dimensional spheroids, control cells developed a hollow lumen, but HER2-overexpressing cells populated the lumen by evading apoptosis. We demonstrate that HER2 overexpression in this cellular model of DCIS drives transcriptional up-regulation of multiple components of the Notch survival pathway. Importantly, luminal filling required up-regulation of a signaling pathway comprising Notch3, its cleaved intracellular domain (NICD) and the transcriptional regulator HES1, resulting in elevated levels of c-MYC and Cyclin D1. In line with HER2- Notch3 collaboration, drugs intercepting either arm reverted the DCIS-like phenotype. In addition, we report up-regulation of Notch3 in hyperplastic lesions of HER2 transgenic animals, as well as an association between HER2 levels and expression levels of components of the Notch pathway in tumor specimens of breast cancer patients. Therefore, it is conceivable that the integration of the Notch and HER2 signaling pathways contributes to the pathophysiology of DCIS.

2‐D DIGE profiling of hepatocellular carcinoma tissues identified isoforms of far upstream binding protein (FUBP) as novel candidates in liver carcinogenesis

Hepatocellular carcinoma (HCC) is a major cause of cancer worldwide and is often characterized by aggressive tumour behaviour and poor prognosis. One of the major etiologies is hepatitis B or C virus (HBV or HCV) infections. In order to better comprehend the molecular mechanisms involved in HCC progression, we performed a systematic analysis on moderately and poorly differentiated human HCC tissues using 2-D DIGE coupled to MALDI-TOF/TOF MS. A total of 52 and 26 proteins were found to be dysregulated in moderately and poorly differentiated HCC tissues, respectively. For the first time, the over-expression of a novel protein family, far upstream binding proteins (FUBPs) was identified in both stages of HCC and confirmed by western blots. FUBPs are of particular interest due to their transcriptional activity on the oncogene, c-myc. It has generally been accepted that c-myc plays an important role in HCC progression but its exact activators remain poorly understood. Interestingly, we also observed elevated c-myc levels in the tissues used in this study by western blot analysis. We therefore propose that the FUBP family of proteins may be one of the possible upstream players that are involved in modulating the c-myc levels in HCC tumorigenesis.

MYC‐induced sensitivity of human malignant melanoma to proteasome inhibitors – a KaMYCaze effect

MYC‐induced sensitivity of human malignant melanoma to proteasome inhibitors – a KaMYCaze effect

Modulation in the activity of lactate dehydrogenase and level of c-Myc and c-Fos by modified base queuine in cancer

Cancer is characterized by uncontrolled cell growth, which results from unlimited proliferation and disturbs various cellular activities. Queuine is a highly modified base analogue of guanine found at first anti-codon position of specific tRNAs i.e. tRNATyr, tRNAHis, tRNAAsp and tRNAAsn. These tRNAs are known as Q-family of tRNA. The tRNAs of Q-family are completely modified to Q-tRNAs in terminally differentiated somatic cells, however hypomodification of Q-tRNA is closely associated with cell proliferation and malignancy. Queuosine modification of tRNAs may be essential for normal development, differentiation and cellular functions. Physiological role of queuine remains ill defined but direct or indirect evidences suggest that queuine or Q-tRNA participates in many cellular functions such as regulation of cell proliferation, control of glycolytic metabolism, alteration in expression of proto-oncogenes, modulation of signal transduction pathways but the mechanism is not well known. Increase in LDH-A expression regulated by c-myc is well documented in a variety of tumor cells. Over expression of proto-oncogenes cause deregulated cellular responses which may lead to development of cancer. The cellular proto-oncogenes like c-myc and c-fos have important role in cell growth, proliferation and differentiation. The present study is aimed to investigate queuine mediated modulation in the activity of lactate dehydrogenase and expression of proto-oncogenes like c-myc and c-fos in T-cell lymphoma (DLAT) induced cancerous mouse. The results indicate that elevated lactate dehydrogenase activity is brought down by queuine treatments and the elevated levels of c-myc and c-fos in DLAT cancerous mouse are down regulated, suggesting that queuine inhibits anaerobic metabolism and cell proliferation.