High Levels Of MYC Research Articles

Mitochondrial AKAP1 supports mTOR pathway and tumor growth

Mitochondria are the powerhouses of energy production and the sites where metabolic pathway and survival signals integrate and focus, promoting adaptive responses to hormone stimulation and nutrient availability. Increasing evidence suggests that mitochondrial bioenergetics, metabolism and signaling are linked to tumorigenesis. AKAP1 scaffolding protein integrates cAMP and src signaling on mitochondria, regulating organelle biogenesis, oxidative metabolism and cell survival. Here, we provide evidence that AKAP1 is a transcriptional target of Myc and supports the growth of cancer cells. We identify Sestrin2, a leucine sensor and inhibitor of the mammalian target of rapamycin (mTOR), as a novel component of the complex assembled by AKAP1 on mitochondria. Downregulation of AKAP1 impaired mTOR pathway and inhibited glioblastoma growth. Both effects were reversed by concomitant depletion of AKAP1 and sestrin2. High levels of AKAP1 were found in a wide variety of high-grade cancer tissues. In lung cancer, AKAP1 expression correlates with high levels of Myc, mTOR phosphorylation and reduced patient survival. Collectively, these data disclose a previously unrecognized role of AKAP1 in mTOR pathway regulation and cancer growth. AKAP1/mTOR signal integration on mitochondria may provide a new target for cancer therapy.

The MYC mRNA 3'-UTR couples RNA polymerase II function to glutamine and ribonucleotide levels.

Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.

Therapeutic Targeting of PIM Protein Kinases in a Subset of T-Cell Acute Lymphoblastic Leukemia

New approaches are needed to treat and cure T-cell acute lymphoblastic leukemia (T-ALL) patients who are currently failing to achieve a remission with chemotherapy. While very intensive chemotherapy regimens result in remission in many T-ALL patients, early T-cell precursor (ETP)-ALL, defined by the absence of CD4, CD8, and CD1a and frequent expression of one or more myeloid markers, is associated with a higher rate of relapse and induction failure with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. The PIM serine/threonine protein kinase has been identified as a potential novel target for the development of new treatment regimens for the cure of hematopoietic malignancies.To explore the potential association of elevated PIM1 expression with ETP-ALL, we analyzed a publically available St. Judes gene expression dataset GSE28703. Results demonstrated that PIM1 is highly expressed in 75% of patients with ETP-ALL and in 13% of non-ETP ALL. PIM1 overexpression in immature or ETP-ALL is further validated using two other publically available independent cohorts: E-MEXP-313 and GSE62156.Based on this observation, we then tested the ability of pan-PIM inhibitors (AZD1208/LGB-321) on the growth of 6 human T-ALL cell lines. qRT-PCR and western blot analysis confirmed, PIM inhibitor sensitive (XTT assay: IC50 < 1 micromolar) cell lines (H-SB2, DU.528, and KOPT-K1) had elevated levels of PIM1 (>9 folds, p<0.01), activated JAK/STAT pathway and lower levels of MYC. Sensitive cells (H-SB2 and DU.528), had an immature or ETP-ALL phenotype (CD4-/CD8- and TdT-). Insensitive CUTLL1, SUP-T1, and HPB-ALL (XTT assay: IC50 > 10 micromolar) cell lines express significantly low PIM1, activated NOTCH mutations, activated AKT signaling, and higher levels of MYC. Immunophenotype of all the PIM inhibitor insensitive cells lines was consistent with a more mature T-ALL phenotype (CD4+/CD8+ and TdT+). When we selected NOTCH-mutant mature T-ALL cells for resistance to gamma secretase inhibitors (SUP-T1-Persister cells) they developed elevated PIM1 kinase levels and were sensitive to the growth inhibitory activities of PIM inhibition. Thus, PIM appears to be a potential target for chemotherapeutic treatment intervention in T-ALL and especially ETP-ALL tumor cells.While PIM kinase inhibitors block ETP-ALL growth they do not induce cell death. The observation that treatment with PIM inhibitors activated ERK and STAT phosphorylation, along with the knowledge that T-ALL cells have increased levels of SRC family tyrosine kinases suggested that adding a broadly active tyrosine kinase inhibitor could enhance the killing of these tumor cells. We have made the novel observation that the combination of Ponatinib, a tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia, with a PIM kinase inhibitor is synergistically lethal to ETP-ALL cells (CI < 1.0). These agents synergize to induce PARP and Caspase cleavage, sufficient to drive apoptosis. In murine models, following engraftment of NOD/scid/IL-2Rγ null (NSG) mice with H-SB2-luc cell xenografts, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly blocked leukemia growth as evidenced by optical scanning for luciferase producing cells (Figure 1, p<0.05), and reduced the number of leukemic cells (hCD45 staining) in the peripheral blood and bone marrow. This dual therapy significantly prolonged the survival (p<0.005) of the treated mice. This outcome was obtained at one-tenth of normal dose of Ponatinib used in animals, suggesting that in the humans the side effects induced by this agent could be avoided by using this combination therapy.Taken together, these findings highlight the important role for PIM protein kinase activity in T-ALL and especially in the ETP-ALL, phenotype. Tumor xenograft experiments provide strong preclinical rationale for a novel treatment strategy of combining PIM and tyrosine kinase inhibitors for treatment of patients with PIM overexpressing T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting the interaction of HDAC2 and MYC in Group 3 medulloblastoma

Patients with high-risk subgroup medulloblastoma (MB) show poor overall survival rates despite aggressive treatment while survivors suffer from important treatment-associated morbidity. Particularly poor outcome is observed in patients with tumors of the molecular subgroup Group 3 with an amplification and high levels of the transcription factor MYC. We have previously shown that Group 3 MB cell lines with MYC amplification are highly susceptible to pan- and class I-histone deacetylase (HDAC) inhibitor treatment. We here explore the molecular interaction of the well known oncogenic driver MYC and class I HDAC2. Six established MB cell lines were used for experiments in cell culture, including MYC amplified cell lines. The functional interaction between MYC and HDAC2 was investigated by enzymatic inhibition of class I HDACs using class I-selective HDAC inhibitors (HDACi) and by siRNA-mediated knockdown of HDAC2. Observed protein degradation was rescued with proteasome inhibition using small molecule proteasome inhibitor MG132. The direct interaction of HDAC2 and MYC was analyzed in co-immunoprecipitation and mass spectrometry studies. The pharmacological inhibition of class I HDACs including HDAC2 as well as artificial HDAC2 protein depletion lead to reduced MYC protein levels, most likely due to increased proteasomal degradation. MYC and HDAC2 are localized in a protein complex together with other potential drug targets. Our data suggest that class I HDAC inhibition leads to destabilization of MYC protein depriving the cells from one of their main oncogenic drivers. The MYC/HDAC2 multi-protein complex represents an attractive target for novel combinatorial treatment approaches. Funding: MYCNet (BMBF Germany), Verein fur krebskranke Kinder Odenwald e.V.

Abstract B121: Oncogenic MYC contributes to tumor super-enhancers via eRNA amplification

Abstract Super-enhancers (SEs) are large clusters of enhancers that control transcription of genes that define cell identity, and they are frequently acquired by cancer cells to drive key oncogenes. Indeed, the prominent oncogene MYC recurrently acquires SEs in many types of cancer, which may explain its overexpression in a broad array of cancers. Elevated levels of MYC promote transcriptional elongation at promoters of all active genes, thereby amplifying RNA output of the cellular gene expression program. High levels of MYC also lead to increased binding of MYC at enhancers, but the significance of enhancer invasion by MYC is unclear. Here, we propose a model that at tumor cell SEs MYC amplifies the production of enhancer RNAs (eRNAs), which in turn facilitate maintenance of SEs by stabilizing transcription factor and co-factor occupancy. We found that in Burkitt's lymphoma cells SEs depend on high levels of MYC and eRNAs, and that MYC is required for elevated levels of eRNA transcription. We demonstrate that eRNAs directly interact with RNA-binding transcription factors at SEs, and that perturbation of eRNA affects SE signal. Elevated MYC thus contributes to the dynamic formation and maintenance of SEs through amplification of eRNAs. Our results may identify novel RNA targets for new therapies for Burkitt's lymphoma, as well as other cancers. New ways to modulate the immune response may also be revealed by our studies. Citation Format: Yang Eric Guo, Michael Seyffert, Brian Abraham, Xiong Ji, Richard Young. Oncogenic MYC contributes to tumor super-enhancers via eRNA amplification [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B121.

OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

Myc Expression Drives Aberrant Lipid Metabolism in Lung Cancer.

MYC-mediated pathogenesis in lung cancer continues to attract interest for new therapeutic strategies. In this study, we describe a transgenic mouse model of KRAS-driven lung adenocarcinoma that affords reversible activation of MYC, used here as a tool for lipidomic profiling of MYC-dependent lung tumors formed in this model. Advanced mass spectrometric imaging and surface analysis techniques were used to characterize the spatial and temporal changes in lipid composition in lung tissue. We found that normal lung tissue was characterized predominantly by saturated phosphatidylcholines and phosphatidylglycerols, which are major lipid components of pulmonary surfactant. In contrast, tumor tissues displayed an increase in phosphatidylinositols and arachidonate-containing phospholipids that can serve as signaling precursors. Deactivating MYC resulted in a rapid and dramatic decrease in arachidonic acid and its eicosanoid metabolites. In tumors with high levels of MYC, we found an increase in cytosolic phospholipase A2 (cPLA2) activity with a preferential release of membrane-bound arachidonic acid, stimulating the lipoxygenase (LOX) and COX pathways also amplified by MYC at the level of gene expression. Deactivating MYC lowered cPLA2 activity along with COX2 and 5-LOX mRNA levels. Notably, inhibiting the COX/5-LOX pathways in vivo reduced tumor burden in a manner associated with reduced cell proliferation. Taken together, our results show how MYC drives the production of specific eicosanoids critical for lung cancer cell survival and proliferation, with possible implications for the use of COX and LOX pathway inhibitors for lung cancer therapy. Cancer Res; 76(16); 4608-18. ©2016 AACR.

Abstract 1257: A MYC-Aurka protein complex represents an actionable target in p53 altered liver cancer

Abstract Myc oncoproteins are causally involved in the genesis of a large fraction of human tumors. Three closely related Myc proteins, c-myc, N-myc and L-myc, have been implicated in cancer, whereas c-myc was identified as an important oncogenic driver in frequent solid tumors.Applying transposon-based mosaic liver cancer mouse models and direct in vivo shRNA screening technology, we found that bypassing a latent p19Arf- and Aurka mediated G2/M cell cycle arrest is required for development of p53 altered liver carcinomas with activated Ras/MAPK signalling. The resulting tumors depend on high MYC levels for survival. A direct interaction of Aurka and MYC was identified in p53 altered liver cancer cells, which could be efficiently disrupted by conformation changing Aurka inhibitors, resulting in MYC degradation and cell death specifically of p53 altered human and murine hepatoma cells. Treatment studies in mouse models of p53 deficient therapy resistant liver cancer revealed marked therapeutic efficacy of conformation changing Aurka inhibitors, thus suggesting a new therapeutic strategy against this major lethal cancer. Citation Format: Dauch Daniel, Ramona Rudalska, Giacomo Cossa, Jean Charles Nault, Sandrine Imbeaud, Nisar P. Malek, Thomas Longerich, Stefan Laufer, Antti Poso, Jessica Zucman-Rossi, Martin Eilers, Lars Zender. A MYC-Aurka protein complex represents an actionable target in p53 altered liver cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1257.

Abstract 3703: MDM2 regulation of MYC and MYCN in pediatric neural cancers

Abstract Background MYC and MYCN have critical roles in a wide range of cancers and can be deregulated through diverse transcriptional and post-transcriptional mechanisms. Recent studies have shown that the high-level MYCN expression needed to drive retinoblastoma and MYCN-amplified neuroblastoma proliferation depends upon efficient MYCN translation mediated by highly expressed MDM2 and acting via a p53-independent mechanism. Here, we assessed MDM2-mediated regulation of MYC as well as MYCN in other pediatric neural tumors, specifically in Group 3 medulloblastoma and in MYC-overexpressing neuroblastoma. We also assessed MDM2-mediated regulation of MYCN in small cell lung cancer (SCLC), which has genetic and morphologic features in common with retinoblastoma. Methods MDM2, MYCN, and MYC expression were defined in Group 3 medulloblastoma lines D283, D425, D341; in MYC-overexpressing neuroblastoma lines CHLA 255 and SY-5Y; and in p53-wild type and p53-mutant SCLC lines H69 and H526 by western blotting. Cells were infected with shMDM2 and scrambled control lentivirus and effects on MDM2, MYC, and MYCN protein expression defined using western blot and effects on cell growth defined using Cell Titer-Glo. In neuroblastoma cells, MDM2 knockdown effects were defined with or without p53 co-knockdown. Results Group 3 medulloblastoma lines D283, D341, D425 highly expressed MYC but not MYCN. MDM2 knockdown did not decrease MYC expression in these cells. MDM2 knockdown decreased MYC expression In CHLA 255 and SY5Y neuroblastoma cells, along with p53 induction and cell death, and MYC down-regulation and cell death were mitigated by co-knockdown of p53. MDM2 knockdown decreased MYCN expression and impaired viability in p53-wild type H69 SCLC cells and in p53-mutant H526 SCLC cells. Conclusions MDM2 is not needed to sustain high-level MYC expression in three Group 3 medulloblastoma cell lines. MDM2 sustains high-level MYC in two MYC-overexpressing neuroblastoma cell lines via a p53-dependent mechanism distinct from the p53-independent MYCN regulation in MYCN-amplified neuroblastomas. In SCLC, MDM2 sustains high-level MYCN expression and viability in a p53-independent manner, similar to MDM2 effects in retinoblastoma and in MYCN-amplified neuroblastoma. These findings suggest that MDM2 selectively sustains MYCN but not MYC expression through a p53-independent mechanism and raise the possibility that cis-acting elements specific to MYCN-RNA mediate MDM2 regulation. Citation Format: Hung N. Tran, Donglai Qi, David Cobrinik. MDM2 regulation of MYC and MYCN in pediatric neural cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3703.

The epigenetic modifier JMJD6 is amplified in mammary tumors and cooperates with c-Myc to enhance cellular transformation, tumor progression, and metastasis.

BackgroundOncogene overexpression in primary cells often triggers the induction of a cellular safeguard response promoting senescence or apoptosis. Secondary cooperating genetic events are generally required for oncogene-induced tumorigenesis to overcome these biologic obstacles. We employed comparative genomic hybridization for eight genetically engineered mouse models of mammary cancer to identify loci that might harbor genes that enhance oncogene-induced tumorigenesis.ResultsUnlike many other mammary tumor models, the MMTV-Myc tumors displayed few copy number variants except for amplification of distal mouse chromosome 11 in 80 % of the tumors (syntenic to human 17q23-qter often amplified in human breast cancer). Analyses of candidate genes located in this region identified JMJD6 as an epigenetic regulatory gene that cooperates with Myc to enhance tumorigenesis. It suppresses Myc-induced apoptosis under varying stress conditions through inhibition of p19ARF messenger RNA (mRNA) and protein, leading to reduced levels of p53. JMJD6 binds to the p19ARF promoter and exerts its inhibitory function through demethylation of H4R3me2a. JMJD6 overexpression in MMTV-Myc cell lines increases tumor burden, induces EMT, and greatly enhances tumor metastasis. Importantly, we demonstrate that co-expression of high levels of JMJD6 and Myc is associated with poor prognosis for human ER+ breast cancer patients.ConclusionsA novel epigenetic mechanism has been identified for how JMJD6 cooperates with Myc during oncogenic transformation. Combined high expression of Myc and JMJD6 confers a more aggressive phenotype in mouse and human tumors. Given the pleiotropic pro-tumorigenic activities of JMJD6, it may be useful as a prognostic factor and a therapeutic target for Myc-driven mammary tumorigenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0205-6) contains supplementary material, which is available to authorized users.

Strategically targeting MYC in cancer.

MYC is a major driver of cancer cell growth and mediates a transcriptional program spanning cell growth, the cell cycle, metabolism, and cell survival. Many efforts have been made to deliberately target MYC for cancer therapy. A variety of compounds have been generated to inhibit MYC function or stability, either directly or indirectly. The most direct inhibitors target the interaction between MYC and MAX, which is required for DNA binding. Unfortunately, these compounds do not have the desired pharmacokinetics and pharmacodynamics for in vivo application. Recent studies report the indirect inhibition of MYC through the development of two compounds, JQ1 and THZ1, which target factors involved in unique stages of transcription. These compounds appear to have significant therapeutic value for cancers with high levels of MYC, although some effects are MYC-independent. These approaches serve as a foundation for developing novel compounds to pharmacologically target MYC-driven cancers.

Optimal Management of Double-Hit Lymphoma.

Although the majority of patients with diffuse large B-cell lymphoma (DLBCL) can be cured by using standard immunochemotherapy, a sizeable proportion of patients cannot. Therefore, it is critical to be able to identify these patients up-front by looking at various clinical and biologic characteristics. DLBCL is a heterogeneous disease with clinical, morphologic, and genetically distinct subtypes, and it is important to understand how these various factors impact treatment outcome as a prerequisite for the development of effective and specific treatment strategies for clinically and molecularly defined subtypes.Whereas the role of clinical characteristics, such as those comprising the International Prognostic Index score, in predicting outcome has been understood for some time, many recent studies demonstrate a prognostic role for individual tumor biomarkers. Biomarkers are thus becoming more andmore of an important consideration in first-line optimal therapy selection. Most DLBCL cases can be divided into one of two molecular subtypes—a germinal center B-cell subtype or an activated B-cell (ABC) subtype—on the basis of mechanisms of oncogenic activation.Outcomes for patients with the ABC subtype are significantly inferior after standard therapy than are outcomes for those with the germinal center B-cell subtype; therefore, there is a need todevelop novel strategies for theABC subtype. It has beenhelpful that anumberof recently developed agents have shown selective activity within the ABC subtype, and randomized clinical trials to test someof these agents in the up-front setting are in progress. Role of MYC and BCL2 Whereas discovering the cell of origin has been helpful in the development of therapeutic agents, there has been much interest and focus in the literature on the roles ofMYC and BCL2 in DLBCL. Several retrospective studies have investigated these roles, and it has been shown that after standard CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or R-CHOP(rituximabplusCHOP) therapy, cases that harbor a MYC rearrangement, which is usually detected by fluorescent in situ hybridization, or both aMYC and a BCL2 rearrangement (double-hit lymphomas [DHLs]) have inferior outcomes. In patients with DHL, BCL2 is typically the translocation partner withMYC but it can sometimes be BCL6, or there can be three rearrangements with BCL2 and BCL6 (triple-hit lymphoma). In addition, by using immunohistochemistry to assess expression ofMYCandBCL2 inDLBCL,various groups have demonstrated that approximately 20% to 25% of newly diagnosed patients with DLBCL have high levels of MYC and BCL2 protein expression, without translocations in the majority of cases. These cases have been called double-expresser lymphomas, and patients do not respond well to treatment with standard R-CHOP therapy. An important question, therefore, is how should these patients be managed?

MicroRNA-206 impairs c-Myc-driven cancer in a synthetic lethal manner by directly inhibiting MAP3K13.

c-Myc (Myc) is one of the most frequently dysregulated oncogenic transcription factors in human cancer. By functionally screening a microRNA (miR) library, we identified miR-206 as being a synthetic lethal in Myc over-expressing human cancer cells. miR-206 inhibited MAP3K13, which resulted in Myc protein de-stabilization, and an inhibition of anchorage-independent growth and in vivo tumorigenesis by Myc over-expressing human cancer cells. Eliminating MAP3K13 by shRNA recapitulated the effects caused by miR-206, thus supporting the idea that miR-206's effect on Myc was mediated through MAP3K13. Conversely, enforced expression of MAP3K13 stabilized Myc by promoting its N-terminal phosphorylation and enhancing its transcriptional activity. Gene expression analyses of breast cancers expressing high levels of Myc indicated that low miR-206 expression and high MAP3K13 expression correlated with poor patient survival. The critical link between miR-206 and MAP3K13 in the development of Myc over-expressing human cancers suggests potential points of therapeutic intervention for this molecular sub-category.

MYC and integrins interplay in colorectal cancer.

MYC and integrins interplay in colorectal cancer.

MYC, Metabolic Synthetic Lethality, and Cancer.

The MYC oncogene plays a pivotal role in the development and progression of human cancers. It encodes a transcription factor that has broad reaching effects on many cellular functions, most importantly in driving cell growth through regulation of genes involved in ribosome biogenesis, metabolism, and cell cycle. Upon binding DNA with its partner MAX, MYC recruits factors that release paused RNA polymerases to drive transcription and amplify gene expression. At physiologic levels of MYC, occupancy of high-affinity DNA-binding sites drives 'house-keeping' metabolic genes and those involved in ribosome and mitochondrial biogenesis for biomass accumulation. At high oncogenic levels of MYC, invasion of low-affinity sites and enhancer sequences alter the transcriptome and cause metabolic imbalances, which activates stress response and checkpoints such as p53. Loss of checkpoints unleashes MYC's full oncogenic potential to couple metabolism with neoplastic cell growth and division. Cells that overexpress MYC, however, are vulnerable to metabolic perturbations that provide potential new avenues for cancer therapy.

Clinical features, tumor biology, and prognosis associated with MYC rearrangement and Myc overexpression in diffuse large B-cell lymphoma patients treated with rituximab-CHOP

MYC dysregulation, including MYC gene rearrangement and Myc protein overexpression, is of increasing clinical importance in diffuse large B-cell lymphoma (DLBCL). However, the roles of MYC and the relative importance of rearrangement vs overexpression remain to be refined. Gaining knowledge about the tumor biology associated with MYC dysregulation is important to understand the roles of MYC and MYC-associated biology in lymphomagenesis. In this study, we determined MYC rearrangement status (n=344) and Myc expression (n=535) in a well-characterized DLBCL cohort, individually assessed the clinical and pathobiological features of patients with MYC rearrangement and Myc protein overexpression, and analyzed the prognosis and gene expression profiling signatures associated with these MYC abnormalities in germinal center B-cell-like and activated B-cell-like DLBCL. Our results showed that the prognostic importance of MYC rearrangement vs Myc overexpression is significantly different in germinal center B-cell-like vs activated B-cell-like DLBCL. In germinal center B-cell-like DLBCL, MYC-rearranged germinal center B-cell-like DLBCL patients with Myc overexpression significantly contributed to the clinical, biological, and prognostic characteristics of the overall Myc-overexpressing germinal center B-cell-like DLBCL group. In contrast, in activated B-cell-like DLBCL, the occurrence, clinical and biological features, and prognosis of Myc overexpression were independent of MYC rearrangement. High Myc levels and Myc-independent mechanisms, either tumor cell intrinsic or related to tumor microenvironment, conferred significantly worse survival to MYC-rearranged germinal center B-cell-like DLBCL patients, even among MychighBcl-2high DLBCL patients. This study provides new insight into the tumor biology and prognostic effects associated with MYC dysregulation and suggest that detection of both MYC translocations and evaluation of Myc and Bcl-2 expression is necessary to predict the prognosis of DLBCL patients.

Abstract B01: An allelic series of deregulated c-Myc expression interrogates the oncogenicity of distinct Myc levels in lung adenocarcinoma

Abstract A plethora of transgenic mouse models have found that c-Myc induces tumors inefficiently and requires cooperating mutations. Acute activation of high levels of Myc induces cellular proliferation, tempered by intrinsic tumor suppression via apoptosis. We hypothesized that low, near physiological, levels of deregulated Myc that fail to invoke tumor suppression pathways, would more effectively initiate sporadic tumors. We previously described the Rosa26lslMycER (R26MER) allele in which deregulated c-Myc is expressed at an endogenous-like level following expression of Cre-recombinase. This c-Myc is fused to a modified estrogen receptor, such that Myc function is dependent upon administration of tamoxifen. We have now generated a Rosa26CAGlslMycER (R26(C)MER) allele, in which the addition of the CAG enhancer has increased the amount of MycER driven by this locus 10-fold. Genetic combination of these alleles generates for the first time mice with tightly controlled and defined levels of switchable Myc protein. Lung cancer is the leading cause of cancer death worldwide and is commonly associated with elevated Myc levels. To determine the role of deregulated Myc expression in lung adenocarcinoma, intranasally administered Adenoviral Cre-recombinase was used to induce sporadic c-MycER expression at different levels in lung epithelial cells, and tamoxifen diet was used to activate c-MycER function. Whereas acute low levels of Myc induced proliferation, high levels of Myc induced both proliferation and apoptosis. Contrary to our hypothesis that Myc-induced apoptosis would delay tumorigenesis, mice with high Myc expression exhibited shorter tumor latency and decreased survival compared to mice with lower levels of expression. The observation that acute Myc activation stimulates p53 activity suggests that the Arf-p53 pathway would be selected against during tumorigenesis. We observed that the hyperplastic clusters of cells observed following Myc activation in R26(C)MER mice were enhanced in mice lacking functional Arf protein. However at later time points their number gradually decreased and the clusters remained small, suggesting that further mutations are necessary for tumorigenesis. Of the tumors that develop following long-term Myc activation, 40% carried activating mutations in Kras. We therefore crossed our R26(C)MER mice to the KrasLSL-G12D allele to investigate the mechanism of this oncogene cooperation. Endogenous levels of oncogenic Kras cooperated dramatically with high levels of Myc, inducing rapid growth of lesions in spite of apparent activation of the p53 tumor suppressor pathway and frequent apoptosis. We then hypothesized that Kras provided increased survival via the Akt pathway to counter Myc-induced apoptosis, as has been previously suggested. Indeed, we found that inhibition of PI3K through treatment with PF-04691502 increased the amount of apoptosis in R26(C)MER/(C)MER;KrasLSL-G12D/+ mice. Importantly, a more dramatic effect was observed when the same lesions were treated with a MEK inhibitor, PD 0325901. Despite having no effect on apoptosis, treatment with PD 0325901 significantly reduced proliferation in these tumors, clearing early lesions within a week of treatment, presumably by allowing Myc-induced apoptosis to overcome the residual proliferation. Consistent with this notion, MEK inhibition of human lung cancer cell lines is more effective at inducing apoptosis in the presence of higher levels of Myc protein. These data suggest that MEK inhibition may have a potent therapeutic role in lung adenocarcinomas that express high levels of Myc. Citation Format: Deborah L. Burkhart, Catherine H. Wilson, Megan Bywater, Roderik Kortlever, Jinyang Li, Mark J. Arends, Trevor D. Littlewood, Gerard I. Evan. An allelic series of deregulated c-Myc expression interrogates the oncogenicity of distinct Myc levels in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B01.

Abstract A23: Identification of KJ-Pyr-9 as a potent MYC inhibitor

Abstract We have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. KJ-Pyr-9 functions as an inhibitor of MYC in biochemical assays, in cells and in xenograft experiments. In vitro, KJ-Pyr-9 binds to MYC as well as MYC-MAX dimers and can disrupt binding of MYC-MAX dimers with E-box DNA. In cells, MYC-MAX dimerization can be disrupted in cells as demonstrated by protein fragment complementation assays. KJ-Pyr-9 disrupts the transcriptional targets of MYC. The P493-6 cell line has tetracycline regulatable MYC expression. For this cell line, the transcriptional targets of MYC can be readily observed upon the addition of 0.1 ug/mL doxycycline. Both doxycycline and KJ-Pyr-9 negatively regulate MYC transcriptional signatures as determined by GSEA. The growth of a variety of different MYC-dependent cell lines is inhibited, including MYC-translocated Burkitt's lymphoma cell lines expressing high levels of MYC as well as solid tumor cell lines expressing moderate amounts of MYC. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. Acknowledgements: This work was supported by the National Cancer Institute under award number R01 CA078230 (PKV), The Skaggs Institute for Chemical Biology (KDJ) and by the Austrian Science Fund (FWF) grant P23652 (KB). This is manuscript 25089 of The Scripps Research Institute. Citation Format: Jonathan R. Hart, Klaus Bister, Kim D. Janda, Peter K. Vogt. Identification of KJ-Pyr-9 as a potent MYC inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A23.

Abstract A27: Epigenomic and transcriptional analyses in a Tet-Myc driven mouse model of liver cancer

Abstract The c-myc proto-oncogene is frequently overexpressed in human hepatoblastoma due to gene amplification or constitutively active Wnt signaling. High Myc levels characterize an aggressive hepatoblastoma subtype, reproduced in mice with a liver-specific tetracyclin-regulated c-myc transgene (1, 2). Here, we used an analogous mouse model (tetO-MYC/Lap-tTA; ref. 3) to study the epigenomic and transcriptional mechanisms underlying tumor progression and maintenance. Tet-Myc induction in utero resulted in rapid and fully penetrant tumorigenesis by the age of six weeks. Pre-tumoral changes were detectable already at a late embryonic stage (E18.5) with increased liver size and hepatoblast fraction. As previously described (2), tumors remained dependent on the constitutive expression of the transgene. To study transcriptional changes during tumor progression and regression, we established RNA-seq profiles in control hepatoblasts, pre-tumoral Tet-Myc hepatoblasts and tumor nodules, in the latter before and after short-term inactivation of Tet-Myc. In parallel, we used ChIP-seq to profile the genome-wide distribution of Myc, RNAPII and several histone marks (H3K4me1, H3K4me3, H3K27ac), yielding comprehensive maps of Myc binding to regulatory elements (promoters and enhancers). This unique dataset is providing the basis for mechanistic studies addressing how Myc deregulates its target genes during liver tumorigenesis: comparison of differentially expressed mRNAs and Myc-binding profiles enables us to discriminate between directly and potentially indirectly regulated genes, for which we are currently investigating the underlying molecular basis. Furthermore, our genome-wide data are allowing us to unravel potentially druggable genes and pathways regulated by Myc during hepatoblastoma progression and maintenance. Our progress in characterizing this model will be presented at the meeting.

Abstract 965: Identification of KJ-Pyr-9 as a potent MYC inhibitor

Abstract We have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. KJ-Pyr-9 functions as an inhibitor of MYC in biochemical assays, in cells and in xenograft experiments. In vitro, KJ-Pyr-9 binds to MYC as well as MYC-MAX dimers and can disrupt binding of MYC-MAX dimers with E-box DNA. In cells, MYC-MAX dimerization can be disrupted in cells as demonstrated by protein fragment complementation assays. KJ-PYR-9 disrupts the transcriptional targets of MYC. The P493-6 cell line has tetracycline regulatable MYC expression. For this cell line, the transcriptional targets of MYC can be readily observed upon the addition of 0.1 ug/mL doxycycline. Both doxycycline and KJ-PYR-9 negatively regulate MYC transcriptional signatures as determined by GSEA. The growth of a variety of different MYC-dependent cell lines are inhibited, including MYC translocated Burkitt's lymphoma cell lines expressing high levels of MYC as well as solid tumor cell lines expressing moderate amounts of MYC. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells. Citation Format: Jonathan R. Hart, Klaus Bister, Kim D. Janda, Peter K. Vogt. Identification of KJ-Pyr-9 as a potent MYC inhibitor. [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 965. doi:10.1158/1538-7445.AM2015-965