Role Of MYC Research Articles

MYC-dependent recruitment of RUNX1 and GATA2 on the SET oncogene promoter enhances PP2A inactivation in acute myeloid leukemia.

The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the SET gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates SET expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of SET. These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of SET and MYC, RUNX1, and GATA2 in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to SET overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of SET expression in AML.

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC) compared to estrogen, progesterone and human epidermal growth factor 2 receptor-positive (RP) breast tumors1,2. We and others have shown that MYC alters metabolism during tumorigenesis3,4. However, the role of MYC in TNBC metabolism remains largely unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for MYC-overexpressing (MO) TNBC and may thus identify novel therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. A lipid metabolism gene signature was identified in patients with TNBC from The Cancer Genome Atlas (TCGA) database and multiple other clinical datasets, implicating FAO as a dysregulated pathway critical for TNBC metabolism. We find that MO-TNBC displays increased bioenergetic reliance upon fatty acid oxidation (FAO), and that pharmacologic inhibition of FAO catastrophically decreases energy metabolism of MO-TNBC, blocks growth of a MYC-driven transgenic TNBC model and that of MO-TNBC patient-derived xenografts. Our results demonstrate that inhibition of FAO is a novel therapeutic strategy against MO-TNBC.

MYC is not detected in highly proliferating normal spermatogonia but is coupled with CIP2A in testicular cancers.

High MYC expression is linked to proliferative activity in most normal tissues and in cancer. MYC also supports self-renewal and proliferation of many types of tissue progenitor cells. Cancerous inhibitor of PP2A (CIP2A) promotes MYC phosphorylation and activity during intestinal crypt regeneration in vivo and in various cancers. CIP2A also supports male germ cell proliferation in vivo. However, the role of MYC in normal germ cell proliferation and spermatogonial progenitor self-renewal is currently unclear. Here, we demonstrate that male germ cells are CIP2A-positive but lack detectable levels of MYC protein; whereas MYC is highly expressed in Leydig cells and peritubular myoid cells contributing thereby to the testicular stem cell niche. On the other hand, MYC was co-expressed with CIP2A in testicular cancers. These results demonstrate that CIP2A and MYC are spatially uncoupled in the regulation of spermatogenesis, but functional relationship between these two human oncoproteins is established during testicular cancer transformation. We propose that further analysis of mechanisms of MYC silencing in spermatogonial progenitors may reveal novel fundamental information relevant to understanding of MYC expression in cancer.

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?

Abstract IA21: Targeting amplified MYCN through CDK7 inhibition in high-risk neuroblastoma

Abstract Neuroblastoma, a tumor of the peripheral nervous system, is the most common extracranial malignancy of childhood. It often presents with widely disseminated disease, leading to survival rates that seldom exceed 50% regardless of treatment strategy. Amplification of the MYCN oncogene, which occurs in over half of high-risk cases is the most unequivocal marker of aggressive disease, automatically stratifying patients to more intensive therapy. However this target has proved elusive for a number of reasons, which include the lack of a druggable domain and the absence of an unequivocal MYCN target-gene signature. The identification of the oncogenic role of MYC as a universal amplifier of transcription in MYC-overexpressing cancer cells provides a new paradigm for understanding how this oncogene promotes the cancer state and suggests a novel focus - RNA polymerase II (Pol II) - for the development of targeted therapy. The transcription cycle of RNA polymerase II is governed by a group of cyclin-dependent kinases, with critical roles in transcriptional initiation, pause release and elongation. We chose to investigate CDK7, because of its dual roles in transcriptional regulation and cell cycle progression, the latter, through its CDK-activating kinase function. By using a novel, highly selective, covalent inhibitor of CDK7 that binds to a unique cysteine residue outside its ATP-binding site, we demonstrated striking activity specifically against MYCN-overexpressing neuroblastoma cells that was accompanied by a global decrease in Pol II-mediated transcription. Selective inhibition of CDK7 not only depleted short-lived transcripts involved in cell cycle progression and apoptosis regulation, but importantly, also downregulated MYCN, itself a short-lived target. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without introducing discernible toxicity. We demonstrated that as with deregulated MYC, amplified MYCN is a global transcriptional regulator that can induce massive activation of genes involved in multiple cellular processes, and that CDK7 disruption led to inhibition of MYCN-dependent transcriptional amplification. Another general feature of deregulated MYCN that seemed highly relevant to its effective targeting was its own transcriptional regulation by super-enhancers (SEs). The selective effect of THZ1 was associated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known lineage-specifying genes in neuroblastoma. This study provides a compelling rationale for targeting CDK7 to block MYCN-mediated global transcription in high-risk neuroblastoma. The challenge now is to exploit the exquisite sensitivity of SEs to transcriptional perturbation, to ensure more durable control over the progression of MYCN-driven tumor cells. Citation Format: Rani George. Targeting amplified MYCN through CDK7 inhibition in high-risk neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA21.

Abstract PD3-05: Inhibition of fatty-acid oxidation as a therapy for triple-negative breast cancer

Abstract Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC) compared to estrogen, progesterone and/or human epidermal growth factor 2 receptor-positive (RP) breast tumors. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains largely unexplored. We hypothesized that pharmacologic inhibition of MYC-driven metabolic pathways may serve as a therapeutic strategy for this clinically challenging subtype of breast cancer. Using an unbiased metabolomics approach, we identified fatty acid oxidation intermediates as dramatically up-regulated in MYC-driven models of TNBC. A lipid metabolism gene signature was identified in patients with TNBC in the TCGA and multiple other clinical datasets, implicating fatty acid oxidation as a deregulated pathway critical for TNBC metabolism. We find that MYC-overexpressing TNBC, including transgenic models and patient-derived xenografts (PDX), display increased bioenergetic reliance upon fatty-acid oxidation (FAO). Pharmacologic inhibition of FAO catastrophically decreases energy metabolism of MYC over-expressing breast cancer, blocks growth of a MYC-driven transgenic TNBC model and MYC-overexpressing patient-derived xenografts. In vivo inhibition of FAO induced proliferation arrest and increased cell death in PDX models of TNBC. Our results demonstrate that inhibition of FAO is a novel therapeutic strategy against TNBCs that over-express MYC. Citation Format: Goga A, Camarda R, Zhou AY, Kohnz RA, Balakrishnan S, Anderton B, Mahieu C, Eyob H, Krings G, Nomura DK. Inhibition of fatty-acid oxidation as a therapy for triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-05.

Abstract P5-08-50: Associations of MYC protein expression and gene status with breast cancer subtypes and outcome in patients treated with anthracycline-based adjuvant chemotherapy

Abstract Background-Aim: Breast cancer is a heterogeneous disease and despite recent scientific progress there is still need for the identification of biomarkers associated with risk for relapse, as well as for markers identifying patients who will benefit from specific treatments. The aim of the present study was to investigate the role of MYC, as a clinically meaningful biomarker, in the outcome of breast cancer subtypes. Patients and Methods: We have pooled the patients and the respective breast carcinomas from two randomized anthracycline-based adjuvant phase III trials, consecutively conducted by the Hellenic Cooperative Oncology Group (HE10/97 and HE10/00). The HE10/97 trial included a non-paclitaxel arm. Tissue microarrays were constructed from 1,060 formalin-fixed paraffin-embedded tumor tissue samples that were collected retrospectively in the first and prospectively in the second trial. MYC was evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 986 cases. Results: In total 61.0% of the cases showed positive cytoplasmic MYC immunostaining, while 26.5% showed positive nuclear staining. 65-80% of the patients were characterized as non-amplified or loss/normal-low gain in all FISH cut-offs examined. A weak association was observed between FISH and nuclear protein expression of MYC. High histological grade was associated with MYC protein overexpression and gene amplification. In terms of disease-free survival (DFS), low (2.5-5 copies) and high (≥5 copies) gain of MYC was of adverse prognostic value compared to loss/normal (<2.5 copies) MYC (HR=1.50, 95% CI 1.13-1.98, Wald's p=0.004 and HR=1.45, 95% CI 1.07-1.97, p=0.016, respectively). Comparable results were observed for overall survival (OS) (HR=1.51, 95% CI 1.09-2.08, p=0.013 and HR=1.65, 95% CI 1.17-2.33, p=0.005, respectively). The comparison of neoplasms with CEP8 ratio ≥1.3 and polysomy 8 for MYC versus all others resulted in worse survival prognosis (HR=1.44, 95% CI 1.13-1.83, p=0.004), while tumors with nuclear protein overexpression were associated with better DFS (HR=0.77, 95% CI 0.60-0.99, p=0.039) and OS (HR=0.73, 95% CI 0.55-0.98, p=0.034). In HER2-enriched patients, MYC amplification was found to be an adverse prognostic factor for DFS (HR=2.11, 95% CI 1.09-4.07, p=0.026) and OS (HR=2.41, 95% CI 1.12-5.15, p=0.024). Treatment with paclitaxel was found to differentiate the effect of MYC: CEP8 ratio ≥1.3 and polysomy 8 in terms of DFS and OS in our total cohort. Among patients with CEP8 ratio ≥1.3 and polysomy 8, those treated with paclitaxel performed significantly better than those not treated, while among patients not treated with paclitaxel, those with CEP8 ratio ≥1.3 and polysomy 8 performed much worse than those with CEP8 ratio <1.3 or no polysomy 8. Conclusions: Our data suggest that MYC has prognostic and predictive value in patients with breast cancer. MYC amplification and MYC protein overexpression are detected in breast cancer patients and are of adverse prognostic value for DFS and OS. Polysomy 8 is also associated with worse prognosis. Treatment with paclitaxel in the adjuvant setting benefits breast cancer patients with MYC:CEP8 ratio ≥1.3 and polysomy 8. Citation Format: Batistatou A, Razis E, Bobos M, Tsolaki E, Timotheadou E, Alexopoulou Z, Goussia A, Gogas H, Koutras A, Karina M, Pentheroudakis G, Efstratiou I, Petraki K, Sotiropoulou M, Pavlakis K, Koletsa T, Kotoula V, Fountzilas G. Associations of MYC protein expression and gene status with breast cancer subtypes and outcome in patients treated with anthracycline-based adjuvant chemotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-50.

Abstract B25: Novel hepatic phenotypes caused by conditional c-Myc deletion

Abstract The transcription factor c-Myc (hereafter Myc) is among the most frequently deregulated oncoproteins. Inhibition of Myc triggers proliferative arrest of transformed cells and promotes tumor regression and/or apoptosis. Myc is also developmentally necessary and myc-/- embryos die at E9.5-10.5. However, Myc's role in the maintenance of specific tissues has been shown to be of variable importance and necessity. For example, several studies of Myc's role in promoting liver regeneration following partial hepatectomy (PH) have given conflicting results, although all show Myc to be generally dispensable for this function. We have used a conditional murine knockout (KO) model of Myc to study its role in liver regeneration. By employing a mycfl/fl;Alb-Cre+ model in which loss of Myc occurs perinatally, we studied non-oncogenic liver proliferation and metabolism in the absence of Myc signaling. We employed basic metabolic benchmarks of liver function including measurements of triglyceride levels, oxidative phosphorylation, and TCA cycle and electron transport chain function. At the molecular level, RNAseq was performed on isolated hepatocytes and the mitochondrial proteome was evaluated by both differential and unbiased mass spectrometry. At the time of active Myc excision, myc-/- mice had a significantly lower liver: body weight ratios relative to myc+/+ controls. However, this was reversed in older mice and was associated with the hepatic accumulation of neutral lipids, cholesterol and increased fatty acid β-oxidation in myc-/- mice. RNAseq on hepatocytes and Ingenuity Pathway analyses showed differences in 105 transcripts (q<0.05), the major pathways encoding ribosomal proteins, members of the cytochrome p450 family and enzymes involved in cholesterol and bile metabolism. These findings correlated with abnormalities in fatty acid and sterol metabolism and storage in liver samples. PH may provide an insufficiently sustained proliferative challenge to allow adequate evaluation of Myc's potential role in liver regeneration. We therefore utilized a mouse model of hereditary tyrosinemia in which knockout of the gene encoding fumarylacetoacetate hydrolase (FAH) leads to hepatocellular death that can be rescued by the infusion of fah+/+ hepatocytes, which expand and eventually replace the fah-/- recipient hepatocytes. FAH-deficient animals could be rescued equally well by both myc+/+ and myc-/- hepatocytes. However, livers from the latter group showed excessive neutral lipid accumulation and fibrosis, reminiscent of non-alcoholic steatohepatitis (NASH). Taken together, our results provide unequivocal evidence that Myc is dispensable for long-term hepatic regeneration but is necessary to maintain proper lipid and steroid metabolism. In Myc's absence the excessive accumulation of these intermediates predisposes to the development of a relatively mild pathology mimicking non-alcoholic fatty liver disease, which under the duress of chronic proliferation, progresses to a more severe NASH-like picture of end-stage liver disease. Our studies thus reveal a heretofore unappreciated role for Myc in hepatic metabolic homeostasis. Citation Format: Lia R. Edmunds, Lokendra Sharma, Peter Anthony Otero, Sonia D'Souza, James M. Dolezal, Xuemei Zeng, Ying Ding, Fei Ding, Megan E. Beck, Lisa E. Kratz, Jerry Vockley, Eric Goetzman, Donald Scott, Nathan Yates, Andrew W. Duncan, Edward V. Prochownik. Novel hepatic phenotypes caused by conditional c-Myc deletion. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B25.

Modeling the CNS Tropism of Diffuse Large B-Cell Lymphomas in Vivo

Introduction: Central nervous system (CNS) manifestation accounts for dismal prognosis in patients diagnosed with systemic diffuse large B-cell lymphoma (DLBCL). Myc rearrangement, deletions encompassing the CDKN2A (a.k.a. INK4a/ARF) or ATM gene loci as well as NF-kB-hyperactivating mutations are frequently detected in primary and secondary CNS-tropic lymphoma, however, investigations that functionally link these lesions to CNS involvement in adequate in vivo model systems are missing.Methods: We generated primary Eµ-myc transgenic mouse lymphomas with and without distinct naturally occurring NF-kB mutations (within genes encoding for MyD88, CD79B, A20, IkBζ, IkBε or BIRC3) or deletions at the INK4a/ARF and ATM loci by retroviral gene transfer and crossbreeding to the respective knockout strains. A subset of the lymphomas was subjected to gene expression profiling and whole-exome sequencing (WES). Wild-type recipient mice propagated with lymphoma cells via tail vein injection were monitored for systemic lymphoma development, the time at which the brain was isolated and examined regarding lymphoma infiltration.Results: Underlining Myc's role as a putative co-driver of CNS involvement, we found in about 40% of primary Eµ-myc lymphomas (with no additional exogenous lesions) meningeal lymphoma manifestations, and transplantation of the same individual lymphomas into numerous recipients reproduced the CNS lymphoma status. Gene set enrichment analysis of genome-wide transcriptome profiles indicated NF-kB hyperactivation in the CNS-tropic lymphoma group, suggesting that constitutive NF-kB signaling may promote CNS-prone pathogenesis in vivo. Transplantation of Eµ-myc transgenic hematopoietic stem cells expressing a variety of NF-kB-activating mutants in myeloablated recipient mice resulted in a significant acceleration of Eµ-myc -driven lymphomagenesis, with some, but not all of these mutants conferring a CNS-tropic lymphoma phenotype. Global NF-kB suppression in CNS-tropic Eµ-myc lymphomas via the NF-kB-antagonizing IkBΔN super-repressor did not fully abrogate lymphoma infiltration of the brain, suggesting that additional factor(s) must contribute. Accordingly, targeted ablation of the INK4a/ARF and ATM loci robustly enhanced CNS tropism of Eµ-myc lymphomas.Conclusions: The Eµ-myc mouse lymphoma model is well-suited to genetically dissect and rebuild components of DLBCL-like CNS tropism. We identified CDKN2A or ATM deletions as critical determinants of CNS tropism in vivo. Our systematic analyses of different NF-kB mutants - so far rather recognized as functionally interchangeable - indicated that only distinct NF-kB mutants contribute to CNS tropism in B-cell lymphomas. WES data, results from compound genotypes (e.g. combining ATM deletions with an NF-kB-activating mutation), and the impact of the host's cellular immune status will be reported at the meeting. Our findings underscore the need for functional analyses of oncogenic network contexts, and provide important insights into candidate target lesions for personalized CNS-directed therapies in DLBCL patients in the future. DisclosuresNo relevant conflicts of interest to declare.

Abstract IA03: Role of MYC in germinal-center development and in lymphomagenesis

Abstract Germinal centers (GCs) are transient structures that form in second¬ary lymphoid organs where B cells are selected on the basis of their ability to produce high-affinity antibodies. B cells in the GCs are also the cells of origin for most B cell lymphomas, including Burkitt Lymphoma carrying chromosomal translocations involving c-Myc. After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. We have shown that the GC reaction requires a biphasic regulation of expression of c-Myc that involves: 1) its transient induction during early GC commitment; 2) its repression by Bcl-6 in highly proliferating DZ B cells; 3) its re-induction in B cells selected for reentry into the DZ. Elimination of c-MYC expression during GC formation results in GC absence, while its inhibition in formed GCs leads to GC collapse. Thus c-Myc has essential roles in early induction of GCs and in their maintenance, by eliminating the LZ-to DZ recirculation. These results suggest an unusual, independent of cell proliferation role of c-Myc in GC development and has implications for the role of c-Myc in lymphomagenesis. By overriding the physiological control of c-Myc expression at critical checkpoints in the GC reaction (entry into the GC and reentry into the DZ), translocations involving immunoglobu¬lin loci and c-MYC may allow B cells to bypass affinity-based selection signals and impose a continuous reentry phenotype that, by perturb¬ing normal GC dynamics, may contribute to lymphomagenesis. Note: This abstract was not presented at the conference. Citation Format: Riccardo Dalla-Favera. Role of MYC in germinal-center development and in 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 IA03.

Prognostication of High-Grade Diffuse Large B-Cell Lymphoma and the Role of MYC, BCL2, and Phosphohistone H3

Prognostication of High-Grade Diffuse Large B-Cell Lymphoma and the Role of MYC, BCL2, and Phosphohistone H3

Abstract A03: Integrative analyses reveal distinct metabolic dysregulation in MYC-driven, basal breast cancer

Abstract Dysregulated metabolism is a hallmark of all human cancers. The observation that tumors display unique changes in metabolism led to the hypothesis that tumors may be dependent upon an altered metabolic state. c-MYC (MYC) is a proto-oncogene and transcription factor activated in many cancers that regulates multiple metabolic pathways. Our lab and others have shown that MYC is activated in a large proportion of basal-like breast cancer, the most aggressive subtype of human breast cancer. However, the role of MYC in basal breast cancer metabolism is largely unknown. No efficacious MYC inhibitor is available for clinical use, however, an alternative treatment strategy is to target cellular pathways required in the context of MYC activation. We hypothesize that basal-like breast cancer harbors distinct, MYC-driven metabolic dysregulation, and that this dysregulation can be targeted therapeutically to induce MYC-dependent synthetic lethality. Utilizing in vitro and in vivo models of MYC-driven and basal breast cancer, including patient-derived cell lines and patient-derived xenograft mouse tumor models, we have performed metabolomic and transcriptomic analyses. We investigate MYC-dependent metabolic dysregulation via integrated analyses of these datasets, and find distinct changes that could reveal essential metabolic pathways for MYC-driven, basal tumorigenesis. Citation Format: Roman Camarda, Sanjeev Balakrishnan, Alicia Y. Zhou, Brittany Anderton, Henok Eyob, Rebecca Kohnz, Daniel K. Nomura, Andrei Goga. Integrative analyses reveal distinct metabolic dysregulation in MYC-driven, basal breast 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 A03.

Abstract IA04: Neuroblastoma as a paradigm of MYC(N) driven cancers

Abstract Neuroblastoma is an enigmatic childhood cancer with phenotypes ranging from spontaneous regression of seemingly advanced disease to relentless metastatic progression. MYCN amplification was defined as a powerful predictor of metastatic potential and acquisition of therapy resistance in the 1980's, and arguably was the first genomic biomarker utilized in routine clinical practice to stratify therapeutic intensity. While there have been incremental improvements in cure rates over the last three decades, these have come at the cost of a highly complex and intensive chemoradiotherapeutic regimen that leaves survivors with significant long-term morbidity. Here we will discuss our ongoing efforts to define the genetic basis of neuroblastoma, and recent insight into how heritable DNA variation influences neuroblastoma susceptibility and clinical phenotypes, including MYCN amplification. We will also discuss the major oncogenic drivers of high-risk neuroblastoma beyond MYCN, including recent insights into the role of MYC in the MYCN-non-amplified high-risk cases, as well as the major role of the ALK oncogene, often in cooperation with MYCN. Finally, we will discuss recent efforts to leverage this information therapeutically, with a focus on how therapeutically targetable oncogenic pathways evolve under the selective pressure of chemoradiotherapy. Finally, we will discuss nascent efforts to deliver on the holy grail of neuroblastoma translational research—targeting the MYCN oncoprotein itself, discussing opportunities and challenges. Citation Format: John M. Maris. Neuroblastoma as a paradigm of MYC(N) driven cancers. [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 IA04.

Abstract IA24: MYC in germinal center B cell physiology and pathology

Abstract Germinal centers (GC)s are sites of intense B-cell proliferation, central for T-cell dependent antibody responses. Although much is known of GC B-cell physiology, the mechanisms of cellular proliferation have remained elusive. Specifically, the role of MYC, a key cell cycle regulator, in GC B-cell proliferation has been questioned. We have identified MYC positive B-cell subpopulations in both immature and mature GCs, and shown through genetic ablation of Myc at these stages that they play indispensable roles in GC formation and maintenance, respectively. The identification of functionally critical MYC-expressing GC B-cell subsets has important implications for B-cell lymphomagenesis, which mostly originates from GC cells and frequently involves MYC deregulation through chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the MYC positive GC subpopulations may represent B-cells with a particularly high risk for malignant transformation. Citation Format: Dinis Pedro Calado. MYC in germinal center B cell physiology and pathology. [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 IA24.

Abstract IA20: Reduced expression of MYC increases longevity and enhances healthspan

Abstract MYC is a highly pleiotropic transcription factor whose deregulation promotes cancer. We find that Myc haploinsufficient (Myc+/–) mice exhibit increased lifespan. They show resistance to several age-associated pathologies, including osteoporosis, cardiac fibrosis and immunosenescence. They also appear to be more active, with a higher metabolic rate and healthier lipid metabolism. Transcriptomic analysis reveals a gene expression signature enriched for metabolic and immune processes. The ancestral role of MYC as a regulator of ribosome biogenesis is reflected in reduced protein translation, which is inversely correlated with longevity. We also observe changes in nutrient and energy sensing pathways, including reduced serum IGF-1, increased AMPK activity, and decreased AKT, TOR and S6K activities. In contrast to observations in other longevity models, Myc+/– mice do not show improvements in stress management pathways. Our findings indicate that MYC activity has a significant impact on longevity and multiple aspects of mammalian healthspan. Citation Format: John M. Sedivy. Reduced expression of MYC increases longevity and enhances healthspan. [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 IA20.

Abstract IA27: Inhibition of MYC transcriptional signaling

Abstract MYC is a master regulator of stem cell state, embryogenesis, tissue homeostasis, and aging. As in health, in disease MYC figures prominently. Decades of biological research have identified a central role for MYC in the pathophysiology of cancer, inflammation and heart disease. The centrality of MYC to such a vast breadth of disease biology has attracted significant attention to the historic challenge of developing inhibitors of MYC. This presentation will discuss therapeutic strategies towards the development of inhibitors of MYC-dependent transcriptional signaling and the elusive goal of developing potent, direct-acting inhibitors of MYC. Citation Format: James E. Bradner. Inhibition of MYC transcriptional signaling. [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 IA27.

Abstract A31: Elucidating mechanisms that cooperate with the therapeutic inhibition of RNA polymerase I to treat MYC-driven hematological malignancies

Abstract The MYC oncoprotein and transcription factor has a well-described role in harnessing and driving ribosome biogenesis (RiBi) via its regulation of all three RNA polymerases, and it is proposed that this is a crucial output of MYC's malignant function. Thus, the potential for therapeutic intervention in a broad range of MYC-driven cancers may converge on targeting the RiBi program, potentially as monotherapy or, more likely, as a basis for rational combination therapies. Indeed, small molecule (CX-5461) inhibition of RNA Polymerase I (Pol I), a major MYC target, selectively kills MYC-driven lymphoma in vivo via activation of a p53-dependent nucleolar stress response. While CX-5461 potently inhibits ribosome biogenesis in a MYC-driven B-cell lymphoma via p53-dependent apoptosis, it is unclear whether all malignancies will respond similarly. Furthermore, as we predict resistance to CX-5461 and tumor relapse from our in vivo mouse studies, the need to identify pathways responsible for mediating resistance to Pol I inhibition in the background of oncogenic MYC is necessary for future development of rational combination therapies. To address these key questions, we have undertaken multiple approaches. Firstly, we are utilizing model systems of acute myeloid leukemia (AML) to take both an unbiased and a direct approach to understanding the role of MYC in mediating CX-5461 sensitivity. Secondly, to identify factors conferring acquired resistance to CX-5461 we have used the well-characterized Eµ-Myc mouse model of MYC-mediated lymphoma where, following a period of disease remission, prolonged dosing of CX-5461 in mice bearing lymphomas results in eventual relapse due to acquired drug resistance. Thus, we compared whole-exome sequencing data between isogenic sensitive and CX-5461 resistant B-cell lymphomas to identify additional mutations conferring drug resistance. Finally, we are using the Eµ-Myc model to investigate the regulation of rDNA transcription itself during cancer progression. Compared to wild type B-cells, malignant MYC over-expressing B-cells have hyper-activated Pol I and increased rDNA transcription. This increase in transcription is associated with a dramatic increase in the number of transcriptionally active rDNA repeats, indicating changes in chromatin structure during transformation. In order to assess epigenetic changes at the rDNA loci and to identify long distance rDNA interactions occurring during malignant progression, we have used ChIP-seq and circularized chromosome conformation capture sequencing (4C-seq) at all cell stages of malignancy in vivo (wild type, pre-malignant and malignant Eµ-Myc B-cells). We demonstrate that transition from premalignancy to malignancy is associated with robust changes in rDNA chromatin as well as reorganization of rDNA-genome interactions with a significant increase in these interactions being detected in malignant cells. Our data provide evidence for MYC-driven activation of rDNA in cancer progression beyond ribosome biogenesis and offer novel insights into the spatial and transcriptional dynamics of the rDNA-associated genome during malignant transformation. We will present our latest data on all fronts aimed at understanding the critical role that MYC plays in therapeutic inhibition of Pol I transcription. Citation Format: Gretchen Poortinga, Jeannine Diesch, Elaine Sanij, Nadine Hein, Jirawas Sornkum, Donald Cameron, Megan J. Bywater, Ricky Johnstone, Denis Drygin, Sean O'Brien, Richard B. Pearson, Grant A. McArthur, Ross D. Hannan. Elucidating mechanisms that cooperate with the therapeutic inhibition of RNA polymerase I to treat MYC-driven hematological malignancies. [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 A31.

Abstract A44: Selective transcriptional regulation by Myc in growth control and tumorigenesis

Abstract Myc binds DNA with a preference for the E-box consensus CACGTG. In vivo, however, DNA recognition is primarily determined by chromatin context, preceding sequence-specific DNA binding. Myc preferentially associates with active/poised promoters and, to a lesser extent, distal enhancer elements. When expressed at high levels, Myc targets virtually all active promoters and enhancers in the genome, a phenomenon termed “invasion”, which contributed to the concept that Myc acts as a general amplifier of transcriptional activity (1, 2). We recently presented data that support a different scenario (3). While Myc has the potential to interact with all active/poised regulatory elements in the genome, this does not systematically lead to a productive regulatory interaction: instead, promoter/enhancer invasion by Myc most likely reflects the detection of low-affinity interactions (protein-protein or protein-DNA) that allow the transcription factor to target the relevant genomic regions, preceding local scanning of the DNA sequence for high-affinity binding sites (4). Most importantly, when productively engaged by Myc, target genes can be either induced or repressed, as shown in different cell types (3, 5). Our data also show that the global increase in mRNA copies per cell (or amplification), observable either during tumor progression or upon mitogenic stimulation of normal cells, can occur in the absence of overt promoter/enhancer invasion by Myc. RNA amplification is thus indirect, most likely attributable to changes in physiological features that impact on global RNA production and turnover (e.g. cell size, energy metabolism, etc..). Consistent with this view, many Myc-regulated genes have - or may have - central roles in these physiological processes. Putting together all available data, the emerging unifying model supports a central role for Myc in the control of cellular physiology and metabolism, including - albeit indirectly - global RNA production and turnover. An important challenge lying ahead is the full characterization of the Myc-regulated genes and pathways that mediate these biological effects and - by the same token - endow Myc with such pervasive oncogenic potential. 1. Lin CY, et al. (2012) Transcriptional amplification in tumor cells with elevated c-Myc. Cell 151:56-67. 2. Nie Z, et al. (2012) c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell 151:68-79. 3. Sabo A, et al. (2014) Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis. Nature 511:488-492. 4. Sabo A & Amati B (2014) Genome Recognition by MYC. Cold Spring Harb Perspect Med 4. 5. Walz S, et al. (2014) Activation and repression by oncogenic MYC shape tumour-specific gene expression profiles. Nature 511:483-487. Citation Format: Arianna Sabò, Theresia K. Kress, Mattia Pelizzola, Stefano de Pretis, Marcin Gorski, Alessandra Tesi, Marco J. Morelli, Pranami Bora, Mirko Doni, Alessandro Verrecchia, Claudia Tonelli, Giovanni Fagà, Valerio Bianchi, Alberto Ronchi, Diana Low, Heiko Muller, Ernesto Guccione, Stefano Campaner, Bruno Amati. Selective transcriptional regulation by Myc in growth control and tumorigenesis. [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 A44.

A Role for MYC in Lithium-Stimulated Repair of the Colonic Epithelium After DSS-Induced Damage in Mice.

Chronic inflammation disrupts the colonic epithelial layer in patients afflicted by ulcerative colitis (UC). The use of inhibitors of glycogen synthase kinase three beta (GSK3β) has proven efficacious to mitigate disease symptoms in rodent models of UC by reducing the pro-inflammatory response. Less is known about whether these inhibitors promote colonic regeneration by stimulating proliferation of colonic epithelial cells. We investigated whether delivery of the GSK3β inhibitor, lithium chloride (LiCl), during the recovery period from acute DSS-induced colitis in mice promoted colonic regeneration and ameliorated disease symptoms. We also tested whether the c-MYC transcription factor (MYC) was involved in this response. Acute colitis was induced by administration of 2.5% dextran sodium sulfate (DSS) to wild-type C57BL/6 mice for 5days. During the recovery period, mice received a daily intraperitoneal (IP) injection of LiCl or 1X PBS as a control. Mice were weighed, colon lengths measured, disease activity index (DAI) scores were assessed, and histological analyses were performed on colonic sections. We analyzed transcripts and proteins in purified preparations of the colonic epithelium. We delivered the MYC inhibitor 10058-F4 via IP injection to assess the role of MYC in colonic regeneration. Lithium treatments promoted recovery from acute DSS-induced damage by increasing expression of Myc transcripts, MYC proteins, and expression of a subset of Wnt/MYC target genes in the colonic epithelium. Inhibiting MYC function with 10058-F4 blunted the lithium response. By inducing Myc expression in the colonic epithelium, lithium promotes colonic regeneration after DSS-induced colitis. Therefore, the use of lithium may be of therapeutic value to manage individuals afflicted by UC.

Abstract 2023: MYC amplification and overexpression in metastatic anaplastic thyroid cancer dictates response to therapy

Abstract Anaplastic thyroid cancer (ATC) is a very aggressive and lethal cancer with few months of survival from diagnosis. Although less than 2% of all thyroid carcinomas are ATC, it accounts for up to 40% of thyroid carcinoma deaths. TRP53 inactivating mutations and PI3K activation through loss of PTEN, or PIK3CA mutations and amplification are common in ATC. In fact, targeted deletion of Pten and Trp53 in the mouse thyroid results in ATC development. Using this mouse model, we found that Myc is amplified in 38% (11/29) of [Pten,Trp53]thyr-/− ATCs with distant metastasis, but only in 10% (3/30) non-metastatic ATCs. Conversely Myc was not amplified in any of 15 metastatic follicular thyroid carcinomas developed by Ptenthyr-/− mice. Analysis of human ATCs showed Myc amplification in 17% (3/18) tumors and protein overexpression in 25% of samples in a tissue microarray. Myc amplification has been shown to be associated with resistance to PI3K targeted therapies in different tumor types. We evaluated the role of Myc in ATC growth and response to therapy using cell lines derived from our [Pten,p53]thyr-/− mouse model. Myc-amplified cell lines grew faster than cell lines from tumors without Myc amplification both in full medium and under serum deprivation. Myc knockdown resulted in a dramatic decrease in growth rate, especially under serum starvations, suggesting that tumors with Myc amplification are less dependent on growth factors present in the serum. Myc knockdown in Myc-amplified cells completely impaired tumor formation in syngeneic mice. To evaluate the effect of Myc amplification on drug sensitivity, we screened a panel of drugs on cultured cells. Myc knockdown sensitized ATC cells to PLK1 inhibition with BI6727, PI3K inhibition with BKM120, as well as to doxorubicin and etoposide. Conversely, Myc-amplified cells were more sensitive to carboplatin. Our results show that Myc amplification is a biomarker for more aggressive and resistant ATCs and that MYC-targeting strategies could be employed to sensitize these tumors to conventional and targeted therapy. Citation Format: Emrullah Yilmaz, Marika A. Russo, Arturo Orlacchio, Toni Forde, Antonio Di Cristofano. MYC amplification and overexpression in metastatic anaplastic thyroid cancer dictates response to therapy. [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 2023. doi:10.1158/1538-7445.AM2015-2023