MYC-driven Tumors Research Articles

Abstract B34: PIM kinase as a novel therapeutic target for triple-negative breast cancer

Abstract The greatest clinical challenge in treating breast cancer occurs in those patients whose tumors lack expression of the estrogen and progesterone receptors and that of the HER2 oncoprotein. No targeted therapeutic strategies currently exist against this aggressive type of “triple negative” breast cancer (TNBC) due to lack of validated targets. We previously found that MYC mRNA, protein, and its signaling were disproportionally elevated in TN compared to receptor positive (RP) breast cancer. We sought to take advantage of the unique molecular feature found in this tumor type to identify potent and effective treatment strategies. Since MYC is an oncogenic transcription factor, rationally designed small molecule inhibitors that can directly inhibit its activity are not available for clinical use. An alternative approach to selectively kill MYC-driven tumors is to inhibit those proteins that are indispensable for the viability of such tumors, but are not essential in non-tumorigenic cells. This form of “indirect” treatment strategy has become known as the “synthetic-lethal” approach. To identify novel targets that are readily druggable for treating MYC-driven TNBC, we conducted a kinome MYC synthetic lethal shRNA screen in non-immortalized human mammary epithelial cells expressing a 4-hydroxytamoxifen (TAM)-activatable MycER transgene (HMEC-MycER). Of 600 human kinases targeted by 2,000 individual shRNA clones, 9 kinases were identified as hits as they were essential specifically for the MYC-activated HMEC cells. Among these hits, we focused on PIM1, a non-essential kinase, the knock-down of which had the greatest efficacy in causing cell death in the MYC-activated cells and had minimum inhibitory effect on the growth of the control cells. We determined that PIM1 expression was elevated in TN tumors and was associated with poor prognosis specifically in patients with hormone receptor-negative tumors. Small molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in a patient-derived tumor xenograft (PDX) mouse model by inhibiting oncogenic transcriptional activity of MYC while simultaneously restoring the function of the endogenous cell cycle inhibitor p27. Thus, our findings warrant clinical evaluation of small molecule PIM kinase inhibitors in patients with TN tumors that exhibit elevated MYC expression. Note: This abstract was not presented at the conference. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Sanjeev Balakrishnan, Kai Kessenbrock, Devon A. Lawson, Roman Camarda, Brittany N. Anderton, Alexey V. Bazarov, Henok Eyob, Julia Rohrberg, Paul Yaswen, Michael T. McManus, Hope S. Rugo, Zena Werb, Andrei Goga. PIM kinase as a novel therapeutic target for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B34.

Myc-Directed Suppression of Autophagy Provides Therapeutic Vulnerabilities Targeting Amino Acid Homeostasis

Metabolic reprogramming is a hallmark of cancer. MYC oncoproteins control many aspects of this response, by inducing the expression of genes involved in mitochondrial biogenesis, glycolysis, glutaminolysis and amino acid transport. This coordinated response allows cancer cells to meet the demands for macromolecules and energy necessary to sustain the anabolic state.Normal cells adapt to nutrient-limiting conditions, such as amino acid (AA) starvation, by activating the autophagy-lysosomal pathway that is necessary for the maintenance of amino acid pools and for providing other building blocks (e.g., ATP) that are needed for cell survival. Surprisingly, our ex vivo and in vivo studies of premalignant and neoplastic MYC-expressing B cells of Eμ-Myc transgenic mice, and of human MYC-driven B cell lymphoma (e.g., Burkitt lymphoma), revealed that MYC suppresses the catabolic autophagy-lysosomal pathway, and that, accordingly, Myc-expressing premalignant and neoplastic B cells are exquisitely sensitive to AA starvation. For example, analyses of the effects of low (6%) versus high (20%) protein diets revealed that limiting AA pools in vivo selectively reduces the numbers of circulating premalignant Eμ-Myc B220+ B cells without affecting B cell numbers in wild type littermate mice. Thus, MYC-driven tumor cells are unable to sufficiently adapt to a state of nutrient deprivation (Figure 1).Expression analyses revealed that this MYC suppresses the autophagy-lysosomal system by transcriptionally repressing genes that encode regulators and components of this pathway, and that this response is a hallmark of human malignancies with MYC involvement. Further, suppressing these genes has functional consequences, where MYC provokes marked reductions in autophagic flux that lead to marked increases in the levels of cargo such as p62/Sequestrin that are normally degraded by this pathway.A master regulator of autophagy and lysosomal biogenesis is TFEB that, like MYC, functions as a basic helix-loop-helix leucine zipper transcription factor and shares a similar DNA recognition sequence. Our studies suggest that MYC blocks TFEB function at three levels. First, MYC can directly repress TFEB transcription. Second, MYC can directly repress TFEB transcription targets by competing with TFEB for binding to the promoter-regulatory regions of autophagy-lysosome gene targets. Third, MYC-expressing B cells have activated mTORC1, which phosphorylates TFEB and blocks its nuclear localization. Notably, forced reactivation of the autophagy-lysosomal pathway via inducible expression of a of a constitutively active (mTORC1-resistant and nuclear) form of TFEB (TFEBS211A) disables the malignant state, where TFEBS211A triggers cell cycle arrest and senescence of both mouse and human MYC-driven lymphomas ex vivo, and compromises tumorigenic potential in vivo. Thus, TFEB acts as tumor suppressor for MYC-driven malignancies.We hypothesized that MYC-driven tumor cells compensate for the reductions in the autophagy pathway and maintain AA homeostasis by activating compensatory mechanisms, including AA transport and the proteasome. In support of this notion, the expression of AA transporters and components of the proteasome, and AA transport and proteasome activity, are markedly augmented in premalignant and neoplastic MYC-expressing B cells. Accordingly, MYC-expressing B cells are exquisitely sensitive to treatment with proteasome inhibitors.Collectively, these findings suggest that MYC drives the anabolic state by suppressing the catabolic autophagy-lysosomal pathway, and that to maintain AA pools MYC-driven cancer cells up-regulate AA transport and the proteasome. This scenario provides attractive opportunities for combination therapies that should disable MYC-driven malignancies, including protein-restricted diets and proteasome and TORC1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A101: Comprehensive genomic profile analyses of small cell lung cancer patient-derived xenografts for pharmacogenomics

Abstract Small Cell Lung Cancers (SCLC) are fast growing tumors with frequent neuroendocrine differentiation. They represent 10-15% of lung cancers and arise in heavy smokers. Most SCLC cancer patients respond well to chemotherapy initially but rapidly develop resistance and die within a few months. Thus, the identification of alternative therapeutic approaches is urgently needed to improve patient's prognosis. Since SCLC is rarely treated by surgery, the number of SCLC models developed for pharmacogenomic research is very limited, however we have established 6 patient-derived xenograft (PDX) models from primary and metastasis SCLC. Complementary to histology, growth characteristic and response to SOCs, here we present comprehensive genomic analyses of these models, revealing similarity with patient tumors and druggable genes and pathways. First we confirmed that the SCLC PDX models retained the morphology characteristics of SCLC tumors and all were heterogeneous in stroma and vasculature content. The median volume doubling time of SCLC-PDX was 7.4 days, which was faster than adenoma (9.6 days) or squamous (9.5 days) NSCLC and slower than large cell NSCLC-PDX (5.7 days). In vivo analyses demonstrated that the SCLC-PDX responded to the SOCs irinotecan and cyclophosphamide but were relatively resistant to cisplatin. Whole exome sequencing, RNAseq and SNP6.0 microarray data revealed mutations in 1542 genes (bi-allelic mutations occurring in 328 of these) as well as larger chromosomal alterations including high copy number gains in 33 genes (of the 33 gene copy gains found, 28 occurred in a model with amplification of chromosome 1), 3 homozygous gene deletions and >20 gene fusions. Genes involved in cell cycle/apoptosis, ECM and focal adhesion via PI3K/AKT were frequently altered, as identified using KEGG database resources and as in patient tumors, recurrent mutations were seen in TP53, RB1 and SPDYE5. Furthermore, similar to SCLC data reported by the TCGA, we saw alterations of the NOTCH signaling pathway in some models, with mutations in NOTCH 1, 2 or 4 genes as well as downstream signaling molecules (CTBP2, KAT2A, DTX2). A search for drug targetable gene alterations identified mutations in BRAF, ERBB2, ALK, mTOR and JAK3 genes while EGFR, KRAS, PI3KCA and PTEN genes were not affected in these models. We also discovered gene fusions such as IP6K1-TRAIP or TM9SF4 fused with the oncogene SRC. At the gene expression level (RNAseq and Affymetrix U133 2Plus), 5 out of 6 SCLC-PDX expressed high levels of neuroendocrine markers characteristic of SCLC such as ASCL1, DLK1, GRP or CHGA, and had gene signatures characteristic of high proliferation and expression of stem cell markers such as LGR5. Analysis of the model with amplification of chromosome 1 confirmed that gene copy number gains were frequently associated with increased expression. This included the MYC family member MCL1, suggesting MYC-driven tumors and sensitivity toward compounds such as aurora kinase inhibitors. In conclusion, we show here SCLC-PDX closely resemble to patient tumors for histology and genomic profiles resulting in fast growing models of various sensitivity toward chemotherapeutics. Numerous drug targetable alterations have been revealed by comprehensive genomic profile analysis. These models therefore represent useful tools for pharmacogenomics investigations. Citation Format: Vincent Vuaroqueaux, Anne-Lise Peille, Bruno Zeitouni, Vanina Fiebig, Heinz-Herbert Fiebig. Comprehensive genomic profile analyses of small cell lung cancer patient-derived xenografts for pharmacogenomics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A101.

Monocytes promote liver carcinogenesis in an oncogene-specific manner

The leukocyte composition of tumors is heterogeneous, as is the involvement of each leukocyte subset in promoting or restraining tumorigenesis. This heterogeneity reflects the tissue of origin, tumor stage, and the functional state of leukocyte activation, but its biological roots remain poorly understood. Since tumorigenesis is driven by various genetic events, we assessed the role of driver genes in shaping the profiles and the roles of leukocytes in tumorigenesis. Mouse liver tumors were induced by hepatic overexpression of either MYC or the combination of myristoylated AKT and NRAS(V12) oncogenes via hydrodynamic transfection. A comparative, flow cytometry- and histology-based immunophenotyping of liver-infiltrating leukocytes was performed at various stages of liver tumorigenesis. The roles of the most abundant leukocyte subsets in tumorigenesis were addressed by immunodepletion. The contribution of liver injury was assessed by comparing the injury-inducing hydrodynamic transfection model to a model in which MYC is an inducible transgene. Myristoylated AKT and NRAS(V12) promoted a marked recruitment of CD11b(+)Ly6G(hi)Ly6C(int) neutrophils and CD11b(+)Ly6G(-)Ly6C(hi) monocytes to the liver, but their immunodepletion did not alter tumorigenesis. In contrast, despite minimal invasion by monocytes/neutrophils during MYC-driven tumorigenesis, immunodepletion of these cells reduced MYC tumor burden and extended survival. MYC-driven tumor initiation was augmented specifically by Ly6C+ monocytes and their ability to promote liver injury. Our results demonstrate that leukocyte profiles do not necessarily predict their involvement in tumorigenesis, the functional role of leukocytes can be shaped by oncogenes, and that monocyte-dependent tissue injury selectively cooperates with MYC during tumorigenesis.

The spliceosome, a potential Achilles heel of MYC-driven tumors.

Alterations in RNA splicing are frequent in human tumors. Two recent studies of lymphoma and breast cancer have identified components of the spliceosome — the core splicing machinery — that are essential for malignant transformation driven by the transcription factor MYC. These findings provide a direct link between MYC and RNA splicing deregulation, and raise the exciting possibility of targeting spliceosome components in MYC-driven tumors.

Increased metastasis with loss of E2F2 in Myc-driven tumors.

In human breast cancer, mortality is associated with metastasis to distant sites. Therefore, it is critical to elucidate the biological mechanisms that underlie tumor progression and metastasis. Using signaling pathway signatures we previously predicted a role for E2F transcription factors in Myc induced tumors. To test this role we interbred MMTV-Myc transgenic mice with E2F knockouts. Surprisingly, we observed that the loss of E2F2 sharply increased the percentage of lung metastasis in MMTV-Myc transgenic mice. Examining the gene expression profile from these tumors, we identified genetic components that were potentially involved in mediating metastasis. These genes were filtered to uncover the genes involved in metastasis that also impacted distant metastasis free survival in human breast cancer. In order to elucidate the mechanism by which E2F2 loss enhanced metastasis we generated knockdowns of E2F2 in MDA-MB-231 cells and observed increased migration in vitro and increased lung colonization in vivo. We then examined genes that were differentially regulated between tumors from MMTV-Myc, MMTV-Myc E2F2-/-, and lung metastases samples and identified PTPRD. To test the role of PTPRD in E2F2-mediated breast cancer metastasis, we generated a knockdown of PTPRD in MDA-MB-231 cells. We noted that decreased levels of PTPRD resulted in decreased migration in vitro and decreased lung colonization in vivo. Taken together, these data indicate that E2F2 loss results in increased metastasis in breast cancer, potentially functioning through a PTPRD dependent mechanism.

Abstract A01: MYC regulates glutathione depletion and glutamine utilization via miR-18a in vivo

Abstract Recent research suggests that MYC activation in tumor cells leads to a unique metabolic phenotype that is characterized by glutamine addiction and TCA cycle activation. We wish to identify novel metabolic pathways that are altered in MYC-driven cancer in vivo. To that end, we have performed microarray-based transcriptomics and mass-spectrometry-based metabolomics profiling of tumors from a murine model of MYC-driven liver cancer. Using Fisher's exact test, we have identified glutathione metabolism as the most significantly enriched pathway across the two datasets. Notably, both the reduced and oxidized isoforms of glutathione, a major cellular antioxidant, are depleted in tumors relative to naïve control livers. We find that the rate-limiting enzyme of glutathione synthesis, Gclc, is attenuated by a MYC-regulated microRNA miR-18a. Loss of glutathione production is concomitant with increased glutamine conversion into TCA cycle metabolites in vivo. Despite loss of glutathione and another antioxidant, NADPH, our MYC-driven tumors do not exhibit markers of elevated oxidative stress. Instead, they appear to upregulate a host of alternative antioxidant pathways including Vitamin C and thioredoxins. In summary, while it is well established that MYC-driven tumors have increased dependence on glutamine resulting in increased glutamate production, whether MYC also regulates glutamate utilization decisions remains poorly understood. Here we show that MYC can regulate glutamate flux by attenuating glutathione production, shifting glutamate utilization towards the TCA cycle. Citation Format: Brittany Anderton, Asha Balakrishnan, Sanjeev Balakrishnan, Lionel Lim, Rebecca Kohnz, Dan Nomura, Andrei Goga. MYC regulates glutathione depletion and glutamine utilization via miR-18a in vivo. [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 A01.

Abstract B30: Regulation of the core pre-mRNA splicing machinery by MYC and PRMT5 is essential to sustain lymphomagenesis

Abstract Deregulated expression of the MYC transcription factor occurs in a majority of human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Over-expressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here, we show that during lymphomagenesis in Eµ-myc transgenic mice, MYC directly up-regulates the transcription of the core snRNP assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins. This coordinated regulatory effect is critical for snRNP biogenesis, effective mRNA splicing, cell survival and proliferation. Indeed, Prmt5 haplo-insufficiency significantly delays lymphoma onset in Eµ-myc transgenic mice and the acute deletion of PRMT5 in established lymphomas enhances the survival of tumor-bearing animals. In human lymphomas and leukemias, the expression of core snRNP genes also correlates with that of MYC, and PRMT5 knockdown results in aberrant pre-mRNA splicing and loss of viability. In both human or mouse lymphomas cells, MYC knockdown also reduces the expression of core snRNP components, causing aberrant pre-mRNA splicing. Finally, we identify pre-mRNAs that are particularly sensitive to perturbation of the MYC-PRMT5 axis, resulting in either intron retention or exon skipping. Using antisense oligonucleotides (ASOs), we mimic these splicing defects and demonstrate their individual contribution to the anti-proliferative/apoptotic phenotype observed in Eµ-myc B-cells. Thus, coordinate regulation of the core snRNP assembly machinery and PRMT5 is critical for the maintenance of normal pre-mRNA splicing and cell survival upon oncogenic activation of c-myc, pointing to PRMT5 methyl-transferase activity as a potential therapeutic target in MYC-driven tumors. Citation Format: Cheryl Koh, Marco Bezzi, Diana Low, WeiXia Ang, ShunXie Teo, Florence Gay, Muthafar Al Haddawi, SooYong Tan, Motomi Osato, Arianna Sabo', KengBoon Wee, Bruno Amati, Ernesto Guccione. Regulation of the core pre-mRNA splicing machinery by MYC and PRMT5 is essential to sustain lymphomagenesis. [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 B30.

Abstract B33: Transient upregulation of Myc with GSK3-β inhibitors in B-cell lymphomas enhances p53-independent apoptotic responses to chemotherapy

Abstract The Myc proto-oncogene is known to regulate p53-dependent apoptosis through the Myc -> ARF -| MDM2 -| p53 pathway. We had previously shown that this pathway could be exploited to increase sensitivity to anti-cancer drugs. Specifically, small increases in Myc levels obtained by inhibiting the Myc-targeting microRNA-34a resulted in enhanced apoptotic response of B-cells to bortezomib (Sotillo et al, Oncogene 2010). However, many Myc-driven tumors are deficient in p53 activity. Thus, we sought to determine whether transient up-regulation of Myc also increases cell death in response to chemotherapeutic drugs in p53-deficient backgrounds. We chose to study this in human Burkitt's lymphoma cell lines and murine B-lymphoid cells isolated from bone marrows of p53ER™ knock-in mice and subsequently transduced with a Myc-expressing retrovirus (Amaravadi et al, J Clin Inv 2007, Yu et al, Blood 2007). This latter system allowed us to distinguish between p53-dependent (cells treated with tamoxifen) and independent (no tamoxifen added) cell death. We also considered that Myc expression is regulated not only transcriptionally, most notably by the bromodomain containing protein 4 (BRD4), but also post-transcriptionally by the glycogen synthase kinase 3β (GSK3-β). GSK3-β phosphorylates Myc on its Thr58 residue, targeting it for proteasomal degradation, and inhibition of GSK3-β activity results in stabilization of Myc protein (Chung et al, J Clin Inv 2012). Using the GSK3-β inhibitor CHIR 99021, we transiently increased Myc protein levels prior to treating B-lymphoma cells with chemotherapeutic drugs. Remarkably, pharmacological stabilization of Myc strongly enhanced p53-independent doxorubicin-induced apoptosis in the p53ER™/Myc system as well as in Burkitt's lymphoma cell lines with inactive p53. This enhancement was accompanied by cleavage of caspase 8 with concomitant de-regulation of the death receptor pathway genes, indicating engagement of the extrinsic apoptotic pathway. In contrast, we did not observe any significant differences in the intrinsic pro-apoptotic genes (BAX, BAK, BIM). To determine whether chemosensitization was mediated by Myc or other GSK3-β targets, we used a BRD4 inhibitor (iBET-151) at concentrations that were sufficient to prevent the CHIR 99021-mediated increase in Myc levels but didn't grossly affect cell viability. Inhibition of Myc with iBET-151 abrogated the increase in apoptosis brought about by CHIR 99021 and doxorubicin, suggesting that Myc is key to chemosensitization by CHIR 99021. Our results suggest that Myc stabilization could be a viable adjuvant therapy, even for tumors with p53 loss or MDM2 amplification. Citation Format: Elena Sotillo-Piñeiro, Colleen T. Harrington, Daniel Soto De Jesus, Andrei Thomas-Tikhonenko. Transient upregulation of Myc with GSK3-β inhibitors in B-cell lymphomas enhances p53-independent apoptotic responses to chemotherapy. [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 B33.

Abstract A17: Identification and characterization of small molecules targeting MYC-cofactor interactions

Abstract Our aim is to develop specific inhibitors targeting the interaction between the oncoprotein/transcription factor MYC and its cofactors. MYC-family genes (MYC, MYCN, MYCL) are involved in over half of all cancers, including many childhood cancers such as neuroblastoma, medulloblastoma, leukemia and lymphoma, and is often strongly associated with poor prognosis. This emphasizes the need to identify clinically relevant MYC inhibitors. It regulates numerous genes involved in cell growth, metabolism, apoptosis, senescence and other processes. MYC is functionally dependent on interaction with the dimerization partner MAX and with several cofactors involved in different aspects of transcription regulation. Targeting such interactions with low Mw compounds is a plausible way to inhibit the transcriptional function of MYC. We have established several techniques to visualize MYC-cofactor interactions in living (BiFC, split Gaussia Luc) or fixed (isPLA) cells to screen small Mw compound libraries for MYC/cofactor-interaction inhibitors. In MYC/MAX BiFC and Gaussia Luc screens, 15 compounds were identified that directly or indirectly inhibit MYC/MAX interactions in cells. Several of these compounds including analogues are potent inhibitors of MYC/MYCN-dependent proliferation and transformation in neuroblastoma, leukemia/lymphoma and other cancer cells by inducing apoptosis or senescence. One of these, #2, and analogues completely inhibit MYC/MAX interactions in vitro by binding MYC. Another compound, 2XU, inhibit MYC/MAX interactions in sub-nanomolar concentration. We are evaluating the biological activities of these molecules further in different MYC-driven cell culture systems, and are elucidating their mechanisms of action by X-ray crystallography, FRET and SPR. In vivo studies with some of these molecules in mouse tumor models is ongoing. Furthermore, extended screens for finding molecules targeting MYC and other cofactor interactions, such as E3-ligases, are ongoing. The MYC inhibitors identified here have the potential to become important tools in the studies of MYC function in cells and in vivo as well as lead molecules for drug development for treatment of MYC-driven tumors. Citation Format: Alina Castell, Qinzi Yan, Karin Ridderstråle, Lars-Gunnar Larsson. Identification and characterization of small molecules targeting MYC-cofactor interactions. [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 A17.

Abstract PR12: Selective targeting of MYC mRNA by chemically stabilized antisense oligonucleotides

Abstract Despite its well-characterized properties as a proto-oncogene in numerous cancers, direct potent and selective inhibition of MYC or its protein product c-Myc with cell-permeable synthetic agents remains a significant challenge. Systemic inhibition of c-Myc activity in mice by expression of dominant negative MYC transgenes has been shown to dramatically decrease tumor burden with apparently tolerable side effects (Soucek et al, Nature 2008). In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC at the transcriptional, translational, and post-translational level. Here we describe the application of synthetic antisense oligonucleotides designed to target specific sequences of the MYC mRNA (MYCASOs). We have incorporated several chemical features into MYCASOs, including the addition of locked nucleic acid (LNA) bases at the 5' and 3' ends, phosphorothioate linkers, and internal DNA bases in order to increase mRNA target affinity and cleavage, cellular permeability, stability, and systemic distribution in vivo. Treatment of MYC¬-expressing cancer cells with MYCASOs leads to a potent decrease in MYC mRNA and c-Myc protein levels. Cleaved MYC mRNA in MYCASO-treated cells is detected with a sensitive 5' rapid amplification of cDNA ends (RACE) assay. MYCASO-treatment of cancer cell lines leads to significant inhibition of cellular proliferation and induces apoptosis in c-Myc-dependent myeloma and leukemia cell lines while decreasing c-Myc-mediated gene expression. In vivo administration of MYCASOs simply dissolved in saline was performed in mice by tail vein injection. MYCASOs are well tolerated up to 25 mg per kg on a twice-weekly dosing regimen. In a MYC-induced model of hepatocellular carcinoma, MYCASO treatment leads to detectable cleavage of the MYC transcript, decreases c-Myc levels within tumors, and significantly decreases tumor burden. MYCASOs represent a new chemical tool for in vitro and in vivo modulation of c-Myc activity, and promising therapeutic agents for MYC-driven tumors. Citation Format: Christopher J. Ott, Raj Bandaru, Taylor Gill, Junyan Tao, Xin Chen, Yixian Zhang, James E. Bradner. Selective targeting of MYC mRNA by chemically stabilized antisense oligonucleotides. [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 PR12.

Abstract PR13: miR-17-92 mediates MYC oncogene addiction

Abstract The MYC oncogene is frequently overexpressed in human cancers. MYC can transcriptionally and translationally regulate the expression of thousands of genes. However, it was unclear which specific genes are responsible for MYC to maintain a neoplastic state. The microRNA cluster miR-17-92 is a major MYC target gene known to regulate proliferation, survival, and angiogenesis, which are several of the key phenotypes associated with MYC oncogene addiction. The resemblance of biological functions between MYC and miR-17-92 thus evoked the hypothesis that miR-17-92 is causally responsible for at least part of the mechanism by which MYC maintains a neoplastic state. We have found that miR-17-92 regulates multiple histone modifiers, such as Sin3b, Hbp1, Suv420h1, and Btg1, as well as the apoptosis regulator Bim, to maintain autonomous proliferation, survival, and self-renewal of MYC-driven tumors. Conversely, MYC inactivation downregulates the expression of miR-17-92 and results in the loss of neoplastic features as a consequence of restoration of senescence, apoptosis, and differentiation. Thus, the expression of miR-17-92 can dictate the cellular fates of MYC-driven tumors between survival versus apoptosis and proliferation versus senescence. Our findings provide a mechanistic insight into why tumors are dependent on or addicted to MYC. Citation Format: Yulin Li, Peter S. Choi, Stephanie C. Casey, David L. Dill, Dean W. Felsher. miR-17-92 mediates MYC oncogene addiction. [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 PR13.

Abstract IA16: Transcriptional regulation by physiological and oncogenic Myc levels

Abstract Myc proteins bind globally to open promoters of all three RNA polymerases and in some instances globally enhance transcription. Surprisingly, Myc-driven tumor cells are characterized by specific gene expression profiles, which are defined by both activation and repression of specific target genes and which hold considerable prognostic value and biological information about tumor physiology. One mechanism that contributes to this specificity is promoter affinity and functionally distinct classes of Myc target genes vary widely in their apparent affinity to Myc. As a result, physiological and supraphysiological/oncogenic levels of Myc regulate different gene expression programs. Furthermore, oncogenic levels of Myc engage the zinc finger protein, Miz1, to repress transcription and I will briefly review what we know about the mechanism of repression. I will discuss two examples how different levels of Myc sensitize cells towards apoptosis and regulate the self-renewing potential of adult stem cells. Citation Format: Bjoern von Eyss, Elmar Wolf, Katrin Wiese, Martin Eilers. Transcriptional regulation by physiological and oncogenic Myc levels. [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 IA16.

Abstract A40: CD30: A therapeutic target of MYC-driven cancer

Abstract The MYC oncogene induces a multitude of human malignancies and has been well characterized as a critical driver of select lymphoma subtypes. In MYC-amplified tumor progression, there is a robust dependence on MYC activity; however, strategies developed to modulate MYC function in cancer have yielded little success. Patients harboring these MYC-driven tumors would benefit from a directed therapy that circumvents explicit molecular targeting of MYC expression. Our preliminary data from an unbiased expression-profiling screen identified CD30 as a primary downstream target of the MYC oncoprotein. CD30 is a member of the tumor necrosis factor superfamily and resides on the extracellular membrane; it is highly expressed in many lymphomas while acutely restricted in normal cells. Within the last few decades, CD30 has emerged as a diagnostic marker and therapeutic target of many lymphoproliferative disorders and diseases. The biological function of CD30 is pleiotropic: CD30 stimulation is reportedly linked to cell cycle arrest, apoptosis, and proliferation through activation of the pro-survival transcription factor, NF-kB. However, the underlying mechanisms that regulate CD30 expression remain unclear. CD30-driven oncogenic pathways phenocopy those initiated by MYC, suggesting CD30's potential status as an effector of MYC-driven tumorigenic events. Here we report that CD30 is regulated at the transcriptional level by MYC. Through a genetic and biochemical approach, we define CD30 induction in a MYC-dependent manner across a variety of human cell lines of both lymphomagenic and non-lymphomagenic origin. Furthermore, in a murine lymphoma model where tumor formation is driven by a conditional allele of MYC, CD30 transcript levels are tightly correlated with MYC activity. Ultimately, CD30 induction offers a novel therapeutic avenue by which to target MYC-driven oncogenesis and has the potential to reshape detection and treatment of MYC-regulated cancer. Citation Format: Victoria Gennaro, Xiao-yong Zhang, Lauren DeSalle, Duonan Yu, Andrei Thomas-Tikhonenko, Steven McMahon. CD30: A therapeutic target of MYC-driven cancer. [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 A40.

Abstract 2892: A MYC-driven medulloblastoma model derived from human induced pluripotent stem cells

Abstract Brain tumors are the most common cause of childhood oncological death, and medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current MB treatments yield five-year survival rates of 60-70%, but usually result in significant neurological, intellectual and physical disabilities. Recent gene expression studies have identified four MB subgroups, many of which have unique clinical and histopathological features. Patients with Group 3 MB are more likely to have aggressive and invasive tumors with large cell/anaplastic histology, and have the worst prognosis. Group 3 MB is characterized by amplification and overexpression oncogenic transcription factor MYC, herein referred to as MYC-driven MB. Modeling MYC-driven MB is critical for developing and testing potential therapies for this highly aggressive MB. Recently, murine MYC-driven MB models have been developed using mouse neural stem cells (NSCs) or neuronal precursor cells (NPCs). But human MB models derived from individual-specific cells are still lacking. Human induced pluripotent stem cells (iPSCs) can be differentiated into various types of cells and hold great promise for developing individual-specific disease models. It is valuable to develop MB models using human iPSCs from both MB patients and unaffected persons. In comparison with mouse-cell-derived MB models, human-iPSC-derived MB models will provide a unique and high-impact platform not only for personalized drug discovery but also for studying the role of individual's distinctive genetic background in carcinogen sensitivity and MB susceptibility. Transcription factor Atoh1 governs the development of cerebellar granule neurons and is essential for MB formation. Here, we induced Atoh1 in human iPSCs to differentiate these cells into NPCs. We further infected these Atoh1-induced NPCs with lentiviruses encoding a stabilized form of MYC (MYCT58A) and dominant-negative p53 (DNp53). These NPCs generated aggressive tumors after being transplanted into mouse cerebellum. NPCs infected by DNp53 alone did not form tumors after 90 days. These MYC-driven tumors were comprised of poorly differentiated, medium to large size cells which showed nuclear molding, prominent nucleoli and numerous mitotic (Ki67+) and apoptotic (Cleaved-Caspase-3+) cells. These tumors also expressed early neuronal lineage marker (β-tubulin III). All these features closely mimic human Group 3 MB. Moreover, we also established neurosphere cultures from these MYC-driven tumors to enrich cancer stem-like cells that have the capability for long-term self-renewal and tumor initiation upon serial transplantations. In summary, we established a novel human-iPSC-drived cancer model for modeling MYC-driven MB. Our results support the feasibility to recapitulate human cancers using progenies derived from human iPSCs. The iPSC-derived MB model we established will facilitate mechanistic studies and drug testing for human aggressive MB. Citation Format: Jonathan Sagal, Charles G. Eberhart, Mingyao Ying. A MYC-driven medulloblastoma model derived from human induced pluripotent stem cells. [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 2892. doi:10.1158/1538-7445.AM2015-2892

S6 Kinase is essential for MYC-dependent rDNA transcription in Drosophila

Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, we performed a transient expression screen for known oncogenic signalling pathways in Drosophila. Strikingly, from the broad range of pathways tested, we found that ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, we demonstrated that one of the mechanism(s) by which MYC and S6K cooperate is through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

The AURKA/TPX2 axis drives colon tumorigenesis cooperatively with MYC

The MYC oncogene has long been established as a central driver in many types of human cancers including colorectal cancer. However, the realization of MYC-targeting therapies remains elusive; as a result, synthetic lethal therapeutic approaches are alternatively being explored. A synthetic lethal therapeutic approach aims to kill MYC-driven tumors by targeting a certain co-regulator on the MYC pathway. We analyzed copy number and expression profiles from 130 colorectal cancer tumors together with publicly available datasets to identify co-regulators on the MYC pathway. Candidates were functionally tested by in vitro assays using colorectal cancer and normal fibroblast cell lines. Additionally, survival analyses were carried out on another 159 colorectal cancer patients and public datasets. Our in silico screening identified two MYC co-regulator candidates, AURKA and TPX2, which are interacting mitotic regulators located on chromosome 20q. We found the two candidates showed frequent co-amplification with the MYC locus while expression levels of MYC and the two genes were positively correlated with those of MYC downstream target genes across multiple cancer types. In vitro, the aberrant expression of MYC, AURKA and TPX2 resulted in more aggressive anchorage-independent growth in normal fibroblast cells. Furthermore, knockdown of AURKA or TPX2, or treatment with an AURKA-specific inhibitor effectively suppressed the proliferation of MYC-expressing colorectal cancer cells. Additionally, combined high expression of MYC, AURKA and TPX2 proved to be a poor prognostic indicator of colorectal cancer patient survival. Through bioinformatic analyses and experiments, we proposed TPX2 and AURKA as novel co-regulators on the MYC pathway. Inhibiting the AURKA/TPX2 axis would be a novel synthetic lethal therapeutic approach for MYC-driven cancers.

Deficiency for the cysteine protease cathepsin L impairs Myc-induced tumorigenesis in a mouse model of pancreatic neuroendocrine cancer.

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycERTAM;Bcl-xL model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycERTAM;Bcl-xL tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.

Interaction with WDR5 Promotes Target Gene Recognition and Tumorigenesis by MYC

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.

Gene expression profiling of MYC-driven tumor signatures in porcine liver stem cells by transcriptome sequencing.

To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells. In this study, we have used an immortal porcine liver stem cell line, PICM-19, to study the role of c-MYC in hepatocarcinogenesis. PICM-19 cells were converted into cancer cells (PICM-19-CSCs) by overexpressing human MYC. To identify MYC-driven differential gene expression, transcriptome sequencing was carried out by RNA sequencing, and genes identified by this method were validated using real-time PCR. In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice. Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells (PICM-19-CSCs). By using comparative bioinformatics analyses, we have determined that > 1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs. Gene ontology analysis further showed that the MYC-induced, altered gene expression was primarily associated with various cellular processes, such as metabolism, cell adhesion, growth and proliferation, cell cycle, inflammation and tumorigenesis. Interestingly, six genes expressed by PICM-19 cells (CDO1, C22orf39, DKK2, ENPEP, GPX6, SRPX2) were completely silenced after MYC-induction in PICM-19-CSCs, suggesting that the absence of these genes may be critical for inducing tumorigenesis. MYC-driven genes may serve as promising candidates for the development of hepatocellular carcinoma therapeutics that would not have deleterious effects on other cell types in the liver.