Myc Oncoprotein Research Articles

MYC Protein Inhibits Transcription of the MicroRNA Cluster MC-let-7a-1∼let-7d via Noncanonical E-box

The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

Mechanistic insight into Myc stabilization in breast cancer involving aberrant Axin1 expression

High expression of the oncoprotein Myc has been linked to poor outcome in human tumors. Although MYC gene amplification and translocations have been observed, this can explain Myc overexpression in only a subset of human tumors. Myc expression is in part controlled by its protein stability, which can be regulated by phosphorylation at threonine 58 (T58) and serine 62 (S62). We now report that Myc protein stability is increased in a number of breast cancer cell lines and this correlates with increased phosphorylation at S62 and decreased phosphorylation at T58. Moreover, we find this same shift in phosphorylation in primary breast cancers. The signaling cascade that controls phosphorylation at T58 and S62 is coordinated by the scaffold protein Axin1. We therefore examined Axin1 in breast cancer and report decreased AXIN1 expression and a shift in the ratio of expression of two naturally occurring AXIN1 splice variants. We demonstrate that this contributes to increased Myc protein stability, altered phosphorylation at S62 and T58, and increased oncogenic activity of Myc in breast cancer. Thus, our results reveal an important mode of Myc activation in human breast cancer and a mechanism contributing to Myc deregulation involving unique insight into inactivation of the Axin1 tumor suppressor in breast cancer.

Abstract 617: Small molecule that simultaneously inhibits Myc oncoprotein and activates HIF1A

Abstract Aberrant expression of Myc oncoproteins is a major causal factor in a large proportion of human cancer. In particular, MYCN oncogene amplification is one of the most powerful prognostic markers identified in the childhood cancer neuroblastoma and, as such, represents a valuable therapeutic target for the development of novel treatment approaches. A diverse chemical library of small molecules was screened using a cell-based assay to identify inhibitors of MYCN. Among a number of molecules identified as potential Myc inhibitors, M606 was found to reduce protein expression of MYCN and its downstream target genes in the MYCN-amplified neuroblastoma cell line BE(2)-C. A similar effect was also observed in a number of c-Myc over-expressing tumor cell lines. Analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc.) indicated that this small molecule inhibited Myc mediated transcription in a dose-dependent manner. Interestingly, hypoxia inducible factor 1 alpha (HIF1A) was induced ∼100 fold. Consistently, we observed HIF1A protein accumulation and nuclear translocation post-M606 treatment under normoxic conditions accompanied by activation of transcription of the HIF1A target, VEGF. siRNA-mediated knockdown of c-Myc or HIF1A in HepG2 cells followed by M606 treatment demonstrated that downregulation of c-Myc and upregulation of HIF1A by M606 are two independent events. Furthermore, inhibition of HIF1A prolyl hydroxylases by dimethyloxalylglycine (DMOG) resulted in downregulation of c-Myc protein independent of any HIFA upregulation. These results suggest that the same molecular mechanism, targeted by M606 and presumably involving inhibition of prolyl hydroxylases, can affect both HIF1A and Myc but in opposite ways. Further development of this compound may be useful in the treatment of cancers in which Myc oncoproteins are overexpressed as well as in the treatment or prevention of ischemic injury since recent studies suggest HIF1A activation may protect against ischemia-reperfusion injury. 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 617. doi:10.1158/1538-7445.AM2011-617

TGFβ1 enhances MAD1 expression and stimulates promoter-bound Pol II phosphorylation: basic functions of C/EBP, SP and SMAD3 transcription factors

BackgroundThe MAD1 protein, a member of the MYC/MAX/MAD network of transcriptional regulators, controls cell proliferation, differentiation and apoptosis. MAD1 functions as a transcriptional repressor, one direct target gene being the tumor suppressor PTEN. Repression of this gene is critical to mediate the anti-apoptotic function of MAD1. Under certain conditions it also antagonizes the functions of the oncoprotein MYC. Previous studies have demonstrated that MAD1 expression is controlled by different cytokines and growth factors. Moreover we have recently demonstrated that the MAD1 promoter is controlled by the cytokine granulocyte colony-stimulating factor (G-CSF) through the activation of STAT3, MAP kinases and C/EBP transcription factors.ResultsWe observed that in addition to G-CSF, the cytokine transforming growth factor β (TGFβ1) rapidly induced the expression of MAD1 mRNA and protein in promyelocytic tumor cells. Moreover we found that C/EBP and SP transcription factors cooperated in regulating the expression of MAD1. This cooperativity was dependent on the respective binding sites in the proximal promoter, with the CCAAT boxes being bound by C/EBPα/β heterodimers. Both C/EBP and SP transcription factors bound constitutively to DNA without obvious changes in response to TGFβ1. In addition SMAD3 stimulated the MAD1 reporter, cooperated with C/EBPα and was bound to the core promoter region. Thus SMAD3 appears to be a potential link between TGFβ1 signaling and C/EBP regulated promoter activity. Moreover TGFβ1 stimulated the phosphorylation of polymerase II at serine 2 and its progression into the gene body, consistent with enhanced processivity.ConclusionsOur findings suggest that C/EBP and SP factors provide a platform of transcription factors near the core promoter of the MAD1 gene that participate in mediating signal transduction events emanating from different cytokine receptors. SMAD3, a target of TGFβ1 signaling, appears to be functionally relevant. We suggest that a key event induced by TGFβ1 at the MAD1 promoter is the recruitment or activation of cofactors, possibly in complex with C/EBP, SP, and SMAD3 transcriptional regulators, that control polymerase activity.

The MK5/PRAK Kinase and Myc Form a Negative Feedback Loop that Is Disrupted during Colorectal Tumorigenesis

Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNAscreening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.

Ubiquitylation of the amino terminus of Myc by SCFβ-TrCP antagonizes SCFFbw7-mediated turnover

The SCFFbw7 ubiquitin ligase mediates growth-factor-regulated turnover of the Myc oncoprotein. Here we show that SCFβ-TrCP binds to Myc by means of a characteristic phosphodegron and ubiquitylates Myc; this results in enhanced Myc stability. SCFFbw7 and SCFβ-TrCP can exert these differential effects through polyubiquitylation of the amino terminus of Myc. Whereas SCFFbw7 with the Cdc34 ubiquitin-conjugating enzyme specifically requires lysine 48 (K48) of ubiquitin, SCFβ-TrCP uses the UbcH5 ubiquitin-conjugating enzyme to form heterotypic polyubiquitin chains on Myc. Ubiquitylation of Myc by SCFβ-TrCP is required for Myc-dependent acceleration of cell cycle progression after release from an arrest in S phase. Therefore, alternative ubiquitylation events at the N terminus can lead to the ubiquitylation-dependent stabilization of Myc.

Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

Transcriptional upregulation of histone deacetylase 2 promotes Myc-induced oncogenic effects

Myc oncoproteins and histone deacetylases (HDACs) modulate gene transcription and enhance cancer cell proliferation, and HDAC inhibitors are among the most promising new classes of anticancer drugs. Here, we show that N-Myc and c-Myc upregulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells, respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Cyclin G2 (CCNG2) was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. 5-bromo-2'-deoxyuridine incorporation assays showed that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc- and HDAC2-induced cell proliferation. Dual crosslinking chromatin immunoprecipitation assay demonstrated that N-Myc acted as a transrepressor by recruiting the HDAC2 protein to Sp1-binding sites at the CCNG2 gene core promoter. Moreover, HDAC2 was upregulated, and CCNG2 downregulated, in pre-cancerous and neuroblastoma tissues from N-Myc transgenic mice, and c-Myc overexpression correlated with upregulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of upregulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers.

Myc-Nick: A Cytoplasmic Cleavage Product of Myc that Promotes α-Tubulin Acetylation and Cell Differentiation

The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation.

MicroRNA Biogenesis Is Required for Myc-Induced B-Cell Lymphoma Development and Survival

Many tumor cells express globally reduced levels of microRNAs (miRNA), suggesting that decreased miRNA expression in premalignant cells contributes to their tumorigenic phenotype. In support of this, Dicer, an RNase III-like enzyme that controls the maturation of miRNA, was recently shown to function as a haploinsufficient tumor suppressor in nonhematopoietic cells. Because the Myc oncoprotein, a critical inducer of B-cell lymphomas, was reported to suppress the expression of multiple miRNAs in lymphoma cells, it was presumed that a deficiency of Dicer and subsequent loss of miRNA maturation would accelerate Myc-induced lymphoma development. We report here that, surprisingly, a haploinsufficiency of Dicer in B cells failed to promote B-cell malignancy or accelerate Myc-induced B-cell lymphomagenesis in mice. Moreover, deletion of Dicer in B cells of CD19-cre(+)/Emicro-myc mice significantly inhibited lymphomagenesis, and all lymphomas that did arise in these mice lacked functional Cre expression and retained at least one functional Dicer allele. Uncharacteristically, the lymphomas that frequently developed in the CD19-cre(+)/Dicer(fl/fl)/Emicro-myc mice were of very early precursor B-cell origin, a stage of B-cell development prior to Cre expression. Therefore, loss of Dicer function was not advantageous for lymphomagenesis, but rather, Dicer ablation was strongly selected against during Myc-induced B-cell lymphoma development. Moreover, deletion of Dicer in established B-cell lymphomas resulted in apoptosis, revealing that Dicer is required for B-cell lymphoma survival. Thus, Dicer does not function as a haploinsufficient tumor suppressor in B cells and is required for B-cell lymphoma development and survival.

Myc suppression of Nfkb2 accelerates lymphomagenesis

BackgroundDeregulated c-Myc expression is a hallmark of several human cancers where it promotes proliferation and an aggressive tumour phenotype. Myc overexpression is associated with reduced activity of Rel/NF-κB, transcription factors that control the immune response, cell survival, and transformation, and that are frequently altered in cancer. The Rel/NF-κB family member NFKB2 is altered by chromosomal translocations or deletions in lymphoid malignancies and deletion of the C-terminal ankyrin domain of NF-κB2 augments lymphocyte proliferation.MethodsPrecancerous Eμ-Myc-transgenic B cells, Eμ-Myc lymphomas and human Burkitt lymphoma samples were assessed for Nfkb2 expression. The contribution of Nfkb2 to Myc-driven apoptosis, proliferation, and lymphomagenesis was tested genetically in vivo.ResultsHere we report that the Myc oncoprotein suppresses Nfkb2 expression in vitro in primary mouse fibroblasts and B cells, and in vivo in the Eμ-Myc transgenic mouse model of human Burkitt lymphoma (BL). NFKB2 suppression by Myc was also confirmed in primary human BL. Promoter-reporter assays indicate that Myc-mediated suppression of Nfkb2 occurs at the level of transcription. The contribution of Nfkb2 to Myc-driven lymphomagenesis was tested in vivo, where Nfkb2 loss was shown to accelerate lymphoma development in Eμ-Myc transgenic mice, by impairing Myc's apoptotic response.ConclusionsNfkb2 is suppressed by c-Myc and harnesses Myc-driven lymphomagenesis. These data thus link Myc-driven lymphomagenesis to the non-canonical NF-κB pathway.

Transcriptional Repression: The Dark Side of Myc

The (c-)Myc oncoprotein and its cousins, the N-Myc and L-Myc proteins, show all hallmarks of transcriptional activator proteins: Myc carries a carboxy-terminal DNA binding domain, which mediates sequence-specific binding to DNA. At its amino-terminus, Myc carries a transcriptional regulatory domain that strongly activates transcription when fused to an ectopic DNA binding domain; moreover, the strength of activation of different members of the Myc family correlates with their ability to transform rodent cells. Furthermore, activation of conditional alleles of Myc, either tetracycline or estrogen inducible, upregulates expression of a large number of genes, both in tissue culture and in transgenic animals. Indeed, many of these genes have essential roles in cell proliferation, cell growth, and metabolism; two of them, odc, encoding ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, and rpl24, encoding a constituent of the large ribosomal subunit, are haploinsufficient for Myc-induced lymphomagenesis but not for normal development, arguing very strongly that upregulation of both genes is critical for Myc-dependent tumor formation. Undoubtedly, therefore, Myc exerts part of its biological activities via transcriptional upregulation of a large number of target genes. One of the key issues in the field is whether there are additional biochemical activities of the Myc protein and, if so, whether and how they contribute to Myc biology. This review summarizes evidence demonstrating that Myc has the ability to repress transcription and that this may be an important function during oncogenic transformation.

Myc: The Beauty and the Beast

The iconic history of the Myc oncoprotein encompasses 3 decades of intense scientific discovery. There is no question that Myc has been a pioneer, advancing insight into the molecular basis of cancer as well as functioning as a critical control center for several diverse biological processes and regulatory mechanisms. This narrative chronicles the journey and milestones that have defined the understanding of Myc, and it provides an opportunity to consider future directions in this challenging yet rewarding field.

Emerging Concepts in the Analysis of Transcriptional Targets of the MYC Oncoprotein: Are the Targets Targetable?

Activation of the MYC oncoprotein is among the most ubiquitous events in human cancer. MYC functions in part as a sequence-specific regulator of transcription. Although early searches for direct downstream target genes that explain MYC's potent biological activity were met with enthusiasm, the postgenomic decade has brought the realization that MYC regulates the transcription of not just a manageably small handful of target genes but instead up to 15% of all active loci. As the dust has begun to settle, two important concepts have emerged that reignite hope that understanding MYC's downstream targets might still prove valuable for defining critical nodes for therapeutic intervention in cancer patients. First, it is now clear that MYC target genes are not a random sampling of the cellular transcriptome but instead fall into specific, critical biochemical pathways such as metabolism, chromatin structure, and protein translation. In retrospect, we should not have been surprised to discover that MYC rewires cell physiology in a manner designed to provide the tumor cell with greater biosynthetic properties. However, the specific details that have emerged from these studies are likely to guide the development of new clinical tools and strategies. This raises the second concept that instills renewed optimism regarding MYC target genes. It is now clear that not all MYC target genes are of equal functional relevance. Thus, it may be possible to discern, from among the thousands of potential MYC target genes, those whose inhibition will truly debilitate the tumor cell. In short, targeting the targets may ultimately be a realistic approach after all.

Widespread Genomic Instability Mediated by a Pathway Involving Glycoprotein Ibα and Aurora B Kinase

c-Myc (Myc) oncoprotein induction of genomic instability (GI) contributes to its initial transforming function and subsequent tumor cell evolution. We describe here a pathway by which Myc, via its target protein glycoprotein Ibalpha (GpIb alpha), mediates GI. Proteomic profiling revealed that the serine/threonine kinase Aurora B is down-regulated by GpIb alpha in p53-deficient primary human fibroblasts. The phenotypes of Aurora B deficiency are strikingly reminiscent of Myc or GpIb alpha overexpression and include double-stranded DNA breaks, altered nuclear size and morphology, chromatin bridges, cleavage furrow regression, and tetraploidy. During mitosis, GpIb alpha and Aurora B redistribute to the cleavage furrow along with other cleavage furrow proteins. GpIb alpha overexpression at levels comparable with those seen in some tumor cells causes the dispersal of these proteins but not Aurora B, resulting in furrow regression and cytokinesis failure. Aurora B normalization redirects the mislocalized furrow proteins to their proper location, corrects the cleavage furrow abnormalities, and restores genomic stability. Aurora B thus appears necessary for a previously unrecognized function in guiding and positioning a number of key proteins, including GpIb alpha to the cleavage furrow. These findings underscore the importance of maintaining a delicate balance among cleavage furrow-associated proteins during mitosis. Suppression of Aurora B via GpIb alpha provides a unifying and mechanistic explanation for several types of Myc-mediated GI.

Opposing Effects of Two Tissue Transglutaminase Protein Isoforms in Neuroblastoma Cell Differentiation

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.

Evaluating the Antitumor Activity of MLN9708 in a Disseminated Mouse Model of Double Transgenic iMyc Ca/Bcl-XL Plasma Cell Malignancy.

Abstract 3835Poster Board III-771 IntroductionThe first generation proteasome inhibitor VELCADE® (bortezomib) is indicated for the treatment of patients with multiple myeloma (MM), a form of plasma cell malignancy (PCM). MLN9708 is our novel proteasome inhibitor that selectively and reversibly binds to, and potently inhibits the b5 site of the 20s proteasome in preclinical studies. We have recently demonstrated that MLN9708 significantly prolongs tumor-free survival of double transgenic iMycCa/Bcl-XL mice, a genetically-engineered mouse model of de novo PCM. Here we describe the in vivo evaluation of cell lines derived from double transgenic iMycCa/Bcl-XL mice and the antitumor activity of MLN9708 in a disseminated mouse model of iMycCa/Bcl-XL PCM. MaterialsMLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Double transgenic iMycCa/Bcl-XL mice develop de novo PCM, in which neoplastic plasma cell development is driven by the targeted expression of the oncoprotein Myc and anti-apoptotic Bcl-XL (J. Clin. Invest. 113:1763-1773, 2004). DP54 and DP42 are plasma cell tumor cell lines isolated from the bone marrow and lymph nodes, respectively, of syngeneic mice previously inoculated with iMycCa/Bcl-XL tumors (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 and DP42 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and have gene expression profiles very similar to human MM. MethodsCell viability studies were performed to determine the antiproliferative effects of MLN2238 in DP54 and DP42 cells in vitro. To evaluate DP54 and DP42 cells in vivo, these cells were aseptically inoculated into the tail vein of NOD-SCID mice. Progressions of the resultant PCM were monitored and tumor burdens were evaluated by magnetic resonance imaging (MRI), ex vivo mCT imaging, and histopathology. Mouse plasma samples were collected at the end of the studies and levels of immunoglobulin were assessed. To establish a preclinical disseminated mouse model of iMycCa/Bcl-XL PCM, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically inoculated into the tail vein of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated with vehicle, bortezomib (at 0.7mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (at 11 mg/kg IV BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. ResultsIn vitro, both DP54 and DP42 cells were sensitive to MLN2238 treatment (LD50 values of 14 and 25 nM, respectively). In vivo, NOD-SCID mice rapidly succumbed to PCM after being inoculated with DP54 and DP42 cells (25 and 14 days post-inoculation, respectively), where the disease was accompanied by marked elevation of plasma immunoglobulins. MRI scans revealed the presence of multiple lesions and several abnormalities were found including: cranial deformation, bowel distortion, splenomegaly and renal edema. Tumor infiltrates, ranging from minor to extensive, were identified in multiple organ compartments (brain<kidney<liver<lymph nodes<spleen<bone marrow) by histopathological analysis. Ex vivo mCT imaging has also revealed signs of bone erosion in the cranial sagittal sutures. Dissemination of DP54-Luc cells after tail vein inoculations was detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were identified in the spleen, kidneys, liver, intestine, lymph nodes, spinal bone and cranium. To assess the antitumor activity of MLN2238, an efficacy study was performed using the DP54-Luc disseminated model. Tumor burden (bioluminescence), skeletal malformation (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. ConclusionThe DP54-Luc disseminated mouse model of double transgenic iMycCa/Bcl-XL PCM recapitulated several key features of human MM and provided real-time assessment of novel MM therapy preclinically. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures:Cao:Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Li:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Silverman:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.

Gene Regulation and Epigenetic Remodeling in Murine Embryonic Stem Cells by c-Myc

BackgroundThe Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells.Methodology/Principal FindingsTo elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc null E7 embryos demonstrates a 70–80% decrease in H3-K4me3, with little change in H3-K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation.Conclusions/SignificanceWe show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation.

TGF-β enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity

Inhibition of tumor growth factor (TGF)-β-mediated cell cycle exit is considered an important tumorigenic function of Myc oncoproteins. Here we found that TGF-β1 enforced G 1 cell cycle arrest and cellular senescence in human U-937 myeloid tumor cells ectopically expressing v-Myc, which contains a stabilizing mutation frequently found in lymphomas. This correlated with induced expression of the Myc antagonist Mad1, resulting in replacement of Myc for Mad1 at target promoters, reduced histone acetylation and strong repression of Myc-driven transcription. The latter was partially reversed by histone deacetylase (HDAC) inhibitors, consistent with involvement of Mad1. Importantly, knockdown of MAD1 expression prevented TGF-β1-induced senescence, underscoring that Mad1 is a crucial component of this process. Enforced Mad1 expression sensitized U-937-myc cells to TGF-β and restored phorbol ester-induced cell cycle exit, but could not alone induce G 1 arrest, suggesting that Mad1 is required but not sufficient for cellular senescence. Our results thus demonstrate that TGF-β can override Myc activity despite a stabilizing cancer mutation and induce senescence in myeloid tumor cells, at least in part by induction of Mad1. TGF-β-induced senescence, or signals mimicking this pathway, could therefore potentially be explored as a therapeutic principle for treating hematopoietic and other tumors with deregulated MYC expression.

DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-15a and miR-16-1

The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.