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Malignant rhabdoid tumors (MRTs) are rare, aggressive pediatric solid tumors, characterized by a 22q11 deletion that inactivates the SMARCB1 gene. Outcomes remain poor despite multimodality treatment. MRTs are among the most genomically stable cancers and lack therapeutically targetable genetic mutations. We utilized metaVIPER, an extension of the Virtual Inference of Protein-activity by Enriched Regulon (VIPER) algorithm, to computationally infer activated druggable proteins in the tumor of an eight month old patient and then expanded the analysis to TCGA and TARGET cohorts.In vitrostudies were performed on a panel of MRT and atypical teratoid/rhabdoid tumor cell lines. Two patient-derived xenograft (PDX) mouse models of MRT were used forin vivoefficacy studies. MetaVIPER analysis from the patient’s tumor identified significantly high inferred activity of nuclear export protein Exportin-1 (XPO1). Expanded metaVIPER analysis of TCGA and TARGET cohorts revealed consistent elevations in XPO1 inferred activity in MRTs compared to other cancer types. All MRT cell lines demonstrated baseline activation of XPO1. MRT cell lines demonstratedin vitrosensitivity to the XPO1 inhibitor, selinexor which led to cell cycle arrest and induction of apoptosis. Targeted inhibition of XPO1 in patient-derived xenograft models of MRT using selinexor resulted in abrogation of tumor growth. Selinexor demonstrates efficacy in preclinical models of MRT. These results support investigation of selinexor in a phase II study in children with MRT and illustrate the importance of an N-of-1 approach in driving discovery beyond the single patient.Statement of Translational RelevanceWe describe the patient-driven discovery of XPO1 activation as a non-genetically encoded vulnerability in MRTs. The application of metaVIPER analysis to tumors lacking actionable oncogenic alterations represents a novel approach for identifying potential therapeutic targets and biomarkers of response. Our preclinical validation of selinexor confirms XPO1 inhibition as a promising therapeutic strategy for the treatment of MRT.

Glioblastoma heterogeneity and plasticity remain controversial, with proposed subtypes representing the average of highly heterogeneous admixtures of independent transcriptional states. Single-cell, protein-activity-based analysis allowed full quantification of >6,000 regulatory and signaling proteins, thus providing a previously unattainable single-cell characterization level. This helped identify four novel, molecularly distinct subtypes that successfully harmonize across multiple GBM datasets, including previously published bulk and single-cell profiles and single cell profiles from seven orthotopic PDX models, representative of prior subtype diversity. GBM is thus characterized by the plastic coexistence of single cells in two mutually-exclusive developmental lineages, with additional stratification provided by their proliferative potential. Consistently, all previous subtypes could be recapitulated by single-cell mixtures drawn from newly identified states. Critically, drug sensitivity was predicted and validated as highly state-dependent, both in single-cell assays from patient-derived explants and in PDX models, suggesting that successful treatment requires combinations of multiple drugs targeting these distinct tumor states.SignificanceWe propose a new, 4-subtype GBM classification, which harmonizes across bulk and single-cell datasets. Single-cell mixtures from these subtypes effectively recapitulate all prior classifications, suggesting that the latter are a byproduct of GBM heterogeneity. Finally, we predict single-cell level activity of three clinically-relevant drugs, and validate them in patient-derived explant.

CD70 (CD27L) is a member of the tumor necrosis factor superfamily transiently expressed on activated adaptive and innate immune cells. Engagement of CD70 to the co-stimulatory receptor CD27, which is present on adaptive immune cells, promotes antigen-specific T cell responses. Both CD70 and CD27 have been reported to be aberrantly expressed on AML and MDS blasts, but are absent on normal hematopoietic progenitor cells, making them appealing therapeutic targets. CD70-CD27 interactions, working through auto- and/or paracrine signaling, can induce a stem-like signature and stimulate the proliferation of leukemic blasts. In addition, the CD70-CD27 interaction drives inflammatory cytokine production from both CD27 and CD70 pathways, resulting in cytokine production from leukemic blasts and the myeloid cells. Inflammatory signaling is a critical component of the crosstalk between hematopoietic stem and progenitor cells and their microenvironment during infection or hematopoietic stress. However, chronic inflammation can suppress normal hematopoietic stem cell function and eventually lead to bone marrow failure. Similarly, preleukemic and leukemic cell' interactions with the bone marrow microenvironment can induce secretion of pro-inflammatory factors, which in turn, could suppress normal blood cell formation. Therefore, blockade of the CD70-CD27 inflammatory signal pathway could help restore normal hematopoiesis, limit immune evasion, and increase the immune directed responses in MDS and AML. SEA-CD70 is a humanized, nonfucosylated monoclonal antibody targeting CD70. SEA-CD70 retains the ability to block CD27 mediated signaling and binds significantly better than the fucosylated parent to the FcγRIIIa receptor. SEA-CD70 mediates potent target killing through Fc-dependent antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). SEA-CD70 can also disrupt CD70-CD27 signaling and could block cellular proliferation and survival of blast cells in vitro. SEA-CD70 may also inhibit the inflammatory signals originating from the interaction between the tumor microenvironment and leukemic blasts. In vivo, SEA-CD70 delayed tumor growth and increased survival in subcutaneous AML xenograft mouse tumor models both as a single agent and when combined with standard of care (SOC) agents. Overall, our data highlight the therapeutic potential of SEA-CD70 for the treatment of AML and MDS patients. Disclosures Diolaiti: Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Haass:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Ulrich:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Chen:Seattlke Genetics:Current Employment, Current equity holder in publicly-traded company.Ho:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Gardai:Seattle genetics:Current Employment, Current equity holder in publicly-traded company.

Summary There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.

PI3K/AKT and NOTCH1 signaling pathways are frequently dysregulated in T-cell acute lymphoblastic leukemias (T-ALL). Although we have shown that the combined activities of the class I PI3K isoforms p110γ and p110δ play a major role in the development and progression of PTEN-null T-ALL, it has yet to be determined whether their contribution to leukemogenic programing is unique from that associated with NOTCH1 activation. Using an Lmo2-driven mouse model of T-ALL in which both the PI3K/AKT and NOTCH1 pathways are aberrantly upregulated, we now demonstrate that the combined activities of PI3Kγ/δ have both overlapping and distinct roles from NOTCH1 in generating T-ALL disease signature and in promoting tumor cell growth. Treatment of diseased animals with either a dual PI3Kγ/δ or a γ-secretase inhibitor reduced tumor burden, prolonged survival, and induced proapoptotic pathways. Consistent with their similar biological effects, both inhibitors downregulated genes involved in cMYC-dependent metabolism in gene set enrichment analyses. Furthermore, overexpression of cMYC in mice or T-ALL cell lines conferred resistance to both inhibitors, suggesting a point of pathway convergence. Of note, interrogation of transcriptional regulators and analysis of mitochondrial function showed that PI3Kγ/δ activity played a greater role in supporting the disease signature and critical bioenergetic pathways. Results provide insight into the interrelationship between T-ALL oncogenic networks and the therapeutic efficacy of dual PI3Kγ/δ inhibition in the context of NOTCH1 and cMYC signaling. Mol Cancer Ther; 16(10); 2069–82. ©2017 AACR.

Pleomorphic xanthoastrocytoma (PXA) is a World Health Organization (WHO) Grade II glioma occurring primarily in children and young adults. Most PXAs harbor the known activating mutation BRAF V600E. We report a case of locally recurrent PXA with anaplastic features in a 10-yr-old female. The PXA was negative by immunohistochemical (IHC) staining for BRAF V600E mutation. Whole-exome and transcriptome sequencing of the tumor confirmed the absence of BRAF V600E, but identified copy-number alterations (including loss of the tumor suppressor CDKN2A) and a novel TMEM106B-BRAF fusion. Based on similar BRAF fusion proteins, this novel fusion is predicted to result in activation of BRAF signaling. Demonstration of positive IHC for phospho-ERK1/2 and phospho-MEK1/2 supported this prediction, and implicated MEK inhibitors as a potential therapeutic strategy.

The biological complexity of NGF action is achieved by binding two distinct neurotrophin receptors, TrkA and p75(NTR). While several reports have provided lines of evidence on the interaction between TrkA and p75(NTR) at the plasma membrane, much fewer data are available on the consequence of such an interaction in terms of intracellular signaling. In this study, we have focused on how p75(NTR) may affect TrkA downstream signaling with respect to neuronal differentiation. Here, we have shown that cooperation between p75(NTR) and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite outgrowth is concomitant with a selective enhancement of the AP-1 activity and the transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be involved in the differentiation process. Collectively, our results unveil a functional link between the specific expression profile of neurotrophin receptors in neuronal cells and the NGF-mediated regulation of the differentiation process possibly through a persistent ERKs activation and the selective control of the AP-1 activity. The biological complexity of NGF action is achieved by binding two distinct neurotrophin receptors, TrkA and p75(NTR). While several reports have provided lines of evidence on the interaction between TrkA and p75(NTR) at the plasma membrane, much fewer data are available on the consequence of such an interaction in terms of intracellular signaling. In this study, we have focused on how p75(NTR) may affect TrkA downstream signaling with respect to neuronal differentiation. Here, we have shown that cooperation between p75(NTR) and TrkA results in an increased NGF-mediated TrkA autophosphorylation, leads to a sustained activation of ERK1/2 and accelerates neurite outgrowth. Interestingly, neurite outgrowth is concomitant with a selective enhancement of the AP-1 activity and the transcriptional activation of genes such as GAP-43 and p21(CIP/WAF), known to be involved in the differentiation process. Collectively, our results unveil a functional link between the specific expression profile of neurotrophin receptors in neuronal cells and the NGF-mediated regulation of the differentiation process possibly through a persistent ERKs activation and the selective control of the AP-1 activity.

NUTM1-rearranged tumors are defined by the presence of a gene fusion between NUTM1 and various gene partners and typically follow a clinically aggressive disease course with poor outcomes despite conventional multimodality therapy. NUTM1-rearranged tumors display histologic features of a poorly differentiated carcinoma with areas of focal squamous differentiation and typically express the BRD4–NUTM1 fusion gene defining a distinct clinicopathologic entity—NUT carcinoma (NC). NCs with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high-grade spindle cell sarcoma harboring a novel MGA–NUTM1 fusion. Whole-genome sequencing identified the presence of complex rearrangements resulting in a MGA–NUTM1 fusion gene in the absence of other significant somatic mutations. Genetic rearrangement was confirmed by fluorescence in situ hybridization, and expression of the fusion gene product was confirmed by transcriptomic analysis. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1–22) and NUTM1 (exons 3–8). Histopathologically, both cases were high-grade spindle cell sarcomas without specific differentiation markers. In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA–NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high-grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.

The amplification of the gene MYCN (V-myc myelocytomatosis viral-valeted oncogene, neuroblastoma derived) has been a well-documented indicator for poor prognosis in neuroblastoma, a childhood cancer. Unfortunately, there has been limited success in understanding MYCN functionality in the landscape of neuroblastoma and more importantly, given that MYCN has been deemed "undruggable," the need to potentially illuminate key opportunities that indirectly target MYCN is of great interest. To this end, this work employs an emerging quantitative technique from network science, namely network curvature, to quantify the biological robustness of MYCN and its surrounding neighborhood. In particular, when amplified in Stage IV cancer, MYCN exhibits higher curvature (more robust) than those samples with under expressed MYCN levels. When examining the surrounding neighborhood, the above argument still holds for network curvature, but is lost when only analyzing differential expression - a common technique amongst oncologists and computational/molecular biologists. This finding points to the problem (and possible solution) of drug targeting in the context of complexity and indirect cell signaling affects that have often been obfuscated through traditional techniques.

Cancer cells exhibit altered metabolic requirements compared to their normal counterparts. This metabolic reprogramming is mediated by activation of oncogenes such as MYC. Here, we summarize our recent findings demonstrating a metabolic dependency of deregulated MYC on MLXIP-MLX, critical components of the nutrient-sensing arm of the extended MYC transcriptional network.

CENP-C is a fundamental component of the inner kinetochore plate and contributes to the formation of functional centromeres in eukaryotic organisms. Recruitment of CENP-C to kinetochore requires other centromere proteins, particularly CENP-A, CENP-H, and CENP-I. However, how CENP-C is correctly localized at the kinetochore is not clearly determined, mainly due to the functional variety of its domains, which hints at a complex recruitment mechanism. Here, by both immunofluorescent labeling and chromatin/immunoprecipitation we could show that human CENP-C contains two distinct domains, one in the central region, between amino acids 426 and 537, and the second one in the carboxyl terminal region, between amino acids 638 and 943, which are both capable of localizing at centromeres and binding α-satellite DNA. The presence of two domains that iterate the same function despite being significantly different in their amino acid sequence and structure suggests that CENP-C may target the centromere by establishing multiple contacts with both the DNA and protein constituents of the kinetochore. © 2002 Elsevier Science (USA). All rights reserved.

The transcription factor MYC and its related family members MYCN and MYCL have been implicated in the etiology of a wide spectrum of human cancers. Compared to other oncoproteins, such as RAS or SRC, MYC is unique because its protein coding region is rarely mutated. Instead, MYC's oncogenic properties are unleashed by regulatory mutations leading to unconstrained high levels of expression. Under both normal and pathological conditions MYC regulates multiple aspects of cellular physiology including proliferation, differentiation, apoptosis, growth and metabolism by controlling the expression of thousands of genes. How a single transcription factor exerts such broad effects remains a fascinating puzzle. Notably, MYC is part of a network of bHLHLZ proteins centered on the MYC heterodimeric partner MAX and its counterpart, the MAX-like protein MLX. This network includes MXD1-4, MNT, MGA, MONDOA and MONDOB proteins. With some exceptions, MXD proteins have been functionally linked to cell cycle arrest and differentiation, while MONDO proteins control cellular metabolism. Although the temporal expression patterns of many of these proteins can differ markedly they are frequently expressed simultaneously in the same cellular context, and potentially bind to the same, or similar DNA consensus sequence. Here we review the activities and interactions among these proteins and propose that the broad spectrum of phenotypes elicited by MYC deregulation is intimately connected to the functions and regulation of the other network members. Furthermore, we provide a meta-analysis of TCGA data suggesting that the coordinate regulation of the network is important in MYC driven tumorigenesis. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

Background: The proto-oncogene MYC encodes a bHLHLZ transcription factor that binds to its obligate DNA-binding partner, MAX, leading to heterodimer occupancy of E-Box motifs within the genome. MYC is normally mitogen-dependent and tightly regulated where it plays a role in transcriptionally coordinating metabolic sufficiency for cell cycle progression, regulating both proliferation and viability. MYC is deregulated in the majority of human cancers. Importantly, MYC exists within an extended family of related bHLHLZ transcription factors including the MYC antagonizing MAX dimerization (MXD) proteins, MYC paralogs, such as MYCN, as well as MAX-like protein X (MLX), and its nutrient-sensing heterodimerization partners MondoA and carbohydrate response element binding protein (ChREBP). We recently defined a role for the extended MAX/MLX network in the regulation of metabolism and survival of cancer cells specifically with deregulated MYC or MYCN, in which MYC-MAX or MYCN-MAX heterodimers coordinately regulate transcription of important metabolic targets with MondoA-MLX to facilitate tumor cell survival. Targeting a number of these downstream effectors is sufficient to kill cancer cells with deregulated MYC. Methods: Expanding on our initial findings we have used molecular and biochemical approaches to demonstrate non-redundant roles for both MondoA-MLX and ChREBP-MLX in the transcriptional regulation of metabolism and survival of human cancer cells with deregulated MYC, including liver and colon cancer cells. We have also characterized the metabolic and biological phenotypes associated with targeted deletion of Mlx in the mouse, combining in vivo and ex vivo analysis with both whole-body deletion and tissue-specific ablation to unmask novel functions of Mlx and its binding partners. Results: Our results demonstrate that MLX and its binding partners regulate pathways associated with mitochondrial function as well as glucose and lipid metabolism in a non-redundant, cell-type-dependent manner. The presence of MLX stabilizes MondoA and ChREBP, and deletion of Mlx in the mouse leads to decreased levels of MondoA and Chrebp proteins in vivo. Whole-body deletion of Mlx in the mouse is well tolerated but leads to male infertility and teratozoospermia. This sterility phenotype is recapitulated by specific deletion of Mlx in the Sertoli cells of the testis, a major metabolic cell type within the tissue and the central regulator of multiple steps associated with germ cell fate, including production and/or transport of critical metabolites required by the germ cells. This indicates both cell-autonomous (germ cell) and non-cell-autonomous (Sertoli cell) regulation of survival and metabolism in spermatogenesis mediated by Mlx. Importantly, human patients with teratozoospermia exhibit diminished expression of MondoA, MLX and their metabolic targets. Moreover, overexpression of MYCN, MondoA, MLX and their metabolic targets occurs in patients with germ cell-derived tumors, such as seminomas. Conclusions: Transcriptional coordination between the mitogen- and nutrient-responsive arms of the MAX/MLX network exhibits context-dependent lethality. MondoA and Chrebp exhibit non-redundant functions in both cancer cells and normal mouse tissue, and inactivation of Mlx phenocopies loss of both factors, suggesting that targeting Mlx can inhibit both MondoA and Chrebp transcriptional activity and subsequent metabolic output. Metabolic regulation by Mlx impacts normal testicular tissue homeostasis and differentiation in the mouse, and its deregulation is associated with human diseases such as loss of function associated with infertility and gain of function associated with germ cell malignancy. While deletion of Myc, Mycn or Max is embryonic lethal in the mouse, deletion of MondoA, Chrebp or Mlx is well tolerated, indicating that targeting this arm of the extended MYC network, or their critical downstream targets, could be of therapeutic value in metabolic syndrome, fertility and cancer. Citation Format: Patrick A. Carroll, Daniel Diolaiti, Pei-Feng Cheng, Haiwei Gu, Danijel Djukovic, Daniel Raftery, Donald E. Ayer, Charles H. Muller, Robert N. Eisenman. Transcriptional regulation of metabolism by MLX and its binding partners is essential for tumor cell survival and spermatogenesis. [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 PR12.

The origin of metazoans required the evolution of mechanisms for maintaining differentiated cell types within a multicellular individual, in part through spatially differentiated patterns of gene transcription. The unicellular ancestor of metazoans was presumably capable of regulating gene expression temporally in response to changing environmental conditions, and spatial cell differentiation in metazoans may represent a co-option of preexisting regulatory mechanisms. Myc is a critical regulator of cell growth, proliferation, and death that is found in all metazoans but absent in other multicellular lineages, including fungi and plants. Homologs of Myc and its binding partner, Max, exist in two of the closest living relatives of animals, the choanoflagellate Monosiga brevicollis (Mb) and Capsaspora owczarzaki, a unicellular opisthokont that is closely related to metazoans and choanoflagellates. We find that Myc and Max from M. brevicollis heterodimerize and bind to both canonical and noncanonical E-boxes, the DNA-binding sites through which metazoan Myc proteins act. Moreover, in M. brevicollis, MbMyc protein can be detected in nuclear and flagellar regions. Like metazoan Max proteins, MbMax can form homodimers that bind to E-boxes. However, cross-species dimerization between Mb and human Myc and Max proteins was not observed, suggesting that the binding interface has diverged. Our results reveal that the Myc/Max network arose before the divergence of the choanoflagellate and metazoan lineages. Furthermore, core features of metazoan Myc function, including heterodimerization with Max, binding to E-box sequences in DNA, and localization to the nucleus, predate the origin of metazoans.

Resistance to chemotherapeutic agents remains one of the major impediments to a successful treatment of chronic myeloid leukemia (CML). Misregulation of the activity of a specific group of ATP-binding cassette transporters (ABC) is responsible for reducing the intracellular concentration of drugs in leukemic cells. Moreover, a consistent body of evidence also suggests that ABC transporters play a role in cancer progression beyond the efflux of cytotoxic drugs. Despite a large number of studies that investigated the function of the ABC transporters, little is known about the transcriptional regulation of the ABC genes. Here, we present data showing that the oncoprotein c-MYC is a direct transcriptional regulator of a large set of ABC transporters in CML. Furthermore, molecular analysis carried out in CD34+ hematopoietic cell precursors of 21 CML patients reveals that the overexpression of ABC transporters driven by c-MYC is a peculiar characteristic of the CD34+ population in CML and was not found either in the population of mononuclear cells from which they had been purified nor in CD34+ cells isolated from healthy donors. Finally, we describe how the methylation state of CpG islands may regulate the access of c-MYC to ABCG2 gene promoter, a well-studied gene associated with multidrug resistance in CML, hence, affecting its expression. Taken together, our findings support a model in which c-MYC–driven transcriptional events, combined with epigenetic mechanisms, direct and regulate the expression of ABC genes with possible implications in tumor malignancy and drug efflux in CML. Mol Cancer Res; 9(8); 1054–66. ©2011 AACR.

The histone variant H2A.Z has been implicated in the regulation of gene expression, and in plants antagonizes DNA methylation. Here, we ask whether a similar relationship exists in mammals, using a mouse B-cell lymphoma model, where chromatin states can be monitored during tumorigenesis. Using native chromatin immunoprecipitation with microarray hybridization (ChIP-chip), we found a progressive depletion of H2A.Z around transcriptional start sites (TSSs) during MYC-induced transformation of pre-B cells and, subsequently, during lymphomagenesis. In addition, we found that H2A.Z and DNA methylation are generally anticorrelated around TSSs in both wild-type and MYC-transformed cells, as expected for the opposite effects of these chromatin features on promoter competence. Depletion of H2A.Z over TSSs both in cells that are induced to proliferate and in cells that are developing into a tumor suggests that progressive loss of H2A.Z during tumorigenesis results from the advancing disease state. These changes were accompanied by increases in chromatin salt solubility. Surprisingly, ∼30% of all genes showed a redistribution of H2A.Z from around TSSs to bodies of active genes during the transition from MYC-transformed to tumor cells, with DNA methylation lost from gene bodies where H2A.Z levels increased. No such redistributions were observed during MYC-induced transformation of wild-type pre-B cells. The documented role of H2A.Z in regulating transcription suggests that 30% of genes have the potential to be aberrantly expressed during tumorigenesis. Our results imply that antagonism between H2A.Z deposition and DNA methylation is a conserved feature of eukaryotic genes, and that transcription-coupled H2A.Z changes may play a role in cancer initiation and progression.

Increased expression of specific ATP-binding cassette (ABC) transporters is known to mediate the efflux of chemotherapeutic agents from cancer cells. Therefore, establishing how ABC transporter genes are controlled at their transcription level may help provide insight into the role of these multifaceted transporters in the malignant phenotype. We have investigated ABC transporter gene expression in a large neuroblastoma data set of 251 tumor samples. Clustering analysis demonstrated a strong association between differential ABC gene expression patterns in tumor samples and amplification of the MYCN oncogene, suggesting a correlation with MYCN function. Using expression profiling and chromatin immunoprecipitation studies, we show that MYCN oncoprotein coordinately regulates transcription of specific ABC transporter genes, by acting as either an activator or a repressor. Finally, we extend these notions to c-MYC showing that it can also regulate the same set of ABC transporter genes in other tumor cells through similar dynamics. Overall our findings provide insight into MYC-driven molecular mechanisms that contribute to coordinate transcriptional regulation of a large set of ABC transporter genes, thus affecting global drug efflux.

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from it's action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repressionin vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activityin vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.

N-Myc is a transcription factor that forms heterodimers with the protein Max and binds gene promoters by recognizing a DNA sequence, CACGTG, called E-box. The identification of N-myc target genes is an important step for understanding N-Myc biological functions in both physiological and pathological contexts. In this study, we describe the identification of N-Myc-responsive genes through chromatin immunoprecipitation and methylation-sensitive restriction analysis. Results show that N-Myc is a direct regulator of several identified genes, and that methylation of the CpG dinucleotide within the E-box prevents the access of N-Myc to gene promoters in vivo . Furthermore, methylation profile of the E-box within the promoters of EGFR and CASP8 , two genes directly controlled by Myc, is cell type-specific, suggesting that differential E-box methylation may contribute to generating unique patterns of Myc-dependent transcription. This study illuminates a central role of DNA methylation in controlling N-Myc occupancy at gene promoters and modulating its transcriptional activity in cancer cells.

In many tumors, the increased biosynthesis and growth required for tumor progression is achieved by metabolic reprogramming associated with the activation of specific oncogenes. This can manifest itself as a shift to aerobic glycolysis coupled with increased anabolic metabolism or altered catabolism. This is especially relevant to tumors with activated Myc, in which Myc-Max heterodimers are transcriptional mediators of biosynthesis and growth. Work from many labs has implicated Myc in multiple aspects of the shift to increased glucose and glutamine uptake and stimulation of mitochondrial biogenesis and activity. Importantly, Myc-Max does not function alone, but within the context of an extended transcriptional network comprised of the Max-binding, Myc-antagonizing Mxd proteins as well as the Max-like protein, Mlx, and its heterodimerization partners MondoA and ChREBP (also known as MondoB) In order to understand how functional interactions and dependencies within the network influence Myc's role in normal and neoplastic cell behavior, we examined cell growth after siRNA mediated knockdown (KD) of different Max and Mlx network members in a panel of c-Myc and N-myc inducible cell lines. Results showed a synthetic lethal interaction between high level of Myc expression and loss of MondoA, or its heterodimeric binding partner Mlx. This synthetic effect was observed both in tissue culture and in mouse xenografts. By employing transcriptomic and metabolomic analyses we show that the MondoA-Mlx arm of the extended Max-Mlx network is required for deregulated Myc to drive metabolic reprograming and maintain survival and growth of these tumor cell. We further show that a subset of Myc-induced genes involved in metabolism require MondoA for full expression. Interestingly, knockdown of a number of these genes alone, which control a variety of metabolic pathways, can recapitulate the synthetic lethal interaction with deregulated Myc. Taken together, these data suggest that both Myc-Max and MondoA-Mlx complexes coordinately regulate the transcriptional activity of a group of genes critical for Myc's ability to reprogram tumor metabolism. The integration of Myc and MondoA functions may serve to link Myc to nutrient sensing and to augment metabolic flexibility within the evolving tumor. PC and DD contributed equally to this work. This research supported by NIH/NCI grant R37CA57138. Citation Format: Patrick A. Carroll, Daniel Diolaiti, Lisa McFerrin, Michelle Hulrich, Pei Feng Cheng, Haiwei Gu, Danijel Djukovic, Daniel Raftery, Robert N. Eisenman. Synthetic lethal interactions between members of the extended MYC network during metabolic reprograming in MYC-driven cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B30.

In this study, we report that the human p14(ARF) associates in vivo with the N-Myc and inhibits N-Myc mediated transcriptional activation. We have determined that the region (aa 140-300) encompassing the N-Myc BoxIII is required for efficient interaction in vivo. Furthermore, we demonstrate that in the SK-N-BE neuroblastoma cell line p14(ARF) over-expression delocalized N-Myc from the nucleoplasm into nucleoli and that N-Myc regions required for interaction with p14(ARF) are also important for nucleoli co-localization. Finally, we determine that the N-terminal region of the p14(ARF) protein is involved in binding to c-Myc and N-Myc proteins.

SummaryDeregulated Myc transcriptionally reprograms cell metabolism to promote neoplasia. Here we show that oncogenic Myc requires the Myc superfamily member MondoA, a nutrient-sensing transcription factor, for tumorigenesis. Knockdown of MondoA, or its dimerization partner Mlx, blocks Myc-induced reprogramming of multiple metabolic pathways, resulting in apoptosis. Identification and knockdown of genes coregulated by Myc and MondoA have allowed us to define metabolic functions required by deregulated Myc and demonstrate a critical role for lipid biosynthesis in survival of Myc-driven cancer. Furthermore, overexpression of a subset of Myc and MondoA coregulated genes correlates with poor outcome of patients with diverse cancers. Coregulation of cancer metabolism by Myc and MondoA provides the potential for therapeutics aimed at inhibiting MondoA and its target genes.