MYC-driven Cancer Cells Research Articles

Abstract A167: Cellular fitness of MYC-driven cancer cells to genetic and pharmacologic perturbations in normoxia, hypoxia and 3D culture

Abstract MYC and hypoxia gene clusters are two most commonly recurring transcriptional programs in a heterogenous tumor. Understanding the cellular fitness of MYC-driven cancer cells in normoxia and hypoxia may lead to identification of cancer cell vulnerabilities. Using genome-wide CRISPR screenings of MYC-driven liver cancer cells cultured in 21% and 1% oxygen in monolayer, and 21% oxygen in 3D spheroid over 4 weeks, we not only identify many targetable essential genes under all three conditions but also context-specific fitness genes and pathways. In parallel, we perform drug screening under three conditions. We find that cells respond to 125 FDA approved oncology drugs in different ways in normoxia, hypoxia and 3D. Our resource study highlights unique epigenetic and metabolic dependency of MYC-driven cancer cells in different tumor environments. Citation Format: Jun Yang. Cellular fitness of MYC-driven cancer cells to genetic and pharmacologic perturbations in normoxia, hypoxia and 3D culture [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A167.

Abstract B010: Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. The complex genetic makeup and heterogeneity of its microenvironment make PDAC difficult to treat. Desmoplasia, a major feature of PDAC’s TME, are dense fibrotic and inflammatory stromal regions. Desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs) because the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. The ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6𝛼 (ATF6𝛼). We hypothesize that ER stress responses are tumor promoting in PDAC and used as a survival strategy under uFA deprivation. In addition, previous studies showed that MYC-driven cancer cells are highly reliant on the ER stress response pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” have distinct dependencies on ER stress response pathways, with the basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Our results suggest that opposite to cancer cell promoting, ER stress responses mediated by IRE1𝛼, PERK and ATF6𝛼 are cytotoxic in vitro under uFA deprived conditions. By analyzing the published bulk RNA-seq from patient samples, we found that the basal-like PDAC subtype is enriched with an ER stress gene signature. To avoid contamination from other cell populations, we are currently analyzing a published scRNA-seq dataset from 15 independent patient samples. We will investigate whether the ER stress signature is also enhanced in basal-like cells. Basal-like PDAC cells are usually more mesenchymal; we therefore created isogenic “epithelial” and “mesenchymal” lines to mimic PDAC subtypes. However, we did not see signaling preferences between the lines. One caveat is that the basal-like subtype have many more features than being mesenchymal, and our isogenic system might not be a faithful modeling of the subtypes. We are studying whether hypoxic PDAC cells experience ER stress in vivo due to a deprivation in uFAs using multiplex fluorescent imaging, stimulated Raman microscopy and/or mass spectrometry. We are evaluating the role of ER stress responses in vivo and see whether inhibiting ER stress response alone or in combination with other reagents gives rise to potential therapeutic opportunities. This project will provide a better understanding of the role ER stress responses play under uFA deprivation and will also enable us to further understand PDAC subtypes in terms of how they utilize ER stress responses. Citation Format: Yanqing (Christine) Jiang, Xu Han, Mai Wang, Raymond Ng, Pamela Burgess-Jones, John Tobias, Nathan Coffey, Andrew Hu, Brian Keith, Celeste Simon. Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B010.

A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth

BackgroundChromosome translocations involving mixed lineage leukemia 1 (MLL1) cause acute leukemia in most infants and 5–10% children/adults with dismal clinical outcomes. Most frequent MLL1-fusion partners AF4/AFF4, AF9/ENL and ELL, together with CDK9/cyclin-T1, constitute super elongation complexes (SEC), which promote aberrant gene transcription, oncogenesis and maintenance of MLL1-rearranged (MLL1-r) leukemia. Notably, ENL, but not its paralog AF9, is essential for MLL1-r leukemia (and several other cancers) and therefore a drug target. Moreover, recurrent ENL mutations are found in Wilms tumor, the most common pediatric kidney cancer, and play critical roles in oncogenesis.MethodsProteolysis-Targeting Chimera (PROTAC) molecules were designed and synthesized to degrade ENL. Biological activities of these compounds were characterized in cell and mouse models of MLL1-r leukemia and other cancers.ResultsCompound 1 efficiently degraded ENL with DC50 of 37 nM and almost depleted it at ~ 500 nM in blood and solid tumor cells. AF9 (as well as other proteins in SEC) was not significantly decreased. Compound 1-mediated ENL reduction significantly suppressed malignant gene signatures, selectively inhibited cell proliferation of MLL1-r leukemia and Myc-driven cancer cells with EC50s as low as 320 nM, and induced cell differentiation and apoptosis. It exhibited significant antitumor activity in a mouse model of MLL1-r leukemia. Compound 1 can also degrade a mutant ENL in Wilms tumor and suppress its mediated gene transcription.ConclusionCompound 1 is a novel chemical probe for cellular and in vivo studies of ENL (including its oncogenic mutants) and a lead compound for further anticancer drug development.

Abstract PO-027: Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of only 10%. A major feature of PDAC tumor microenvironment (TME) is desmoplasia, which are dense fibrotic and inflammatory stromal regions constituting the PDAC TME. This desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a significant deficiency in unsaturated fatty acids (UFAs) because the de novo synthesis of UFAs requires molecular oxygen. Our data indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger a subsequent unfolded protein response (UPR) under tumor-like stress, which can be mitigated by UFAs. The UPR regulates an intricate signaling network mediated by three major stress sensors: Inositol-requiring enzyme 1a (IRE1a), PKR-like ER kinase (PERK) and activating transcription factor 6a (ATF6a). UPR output can be either cytoprotective or cytotoxic depending on tumor types and the severity of stress. Our data suggest that B-I09, a selective IRE1 RNase inhibitor, impairs PDAC cell viability under stress conditions, indicating a pro-survival role for IRE1a. Previous studies showed that MYC-driven cancer cells are highly reliant on UPR pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” may have distinct dependencies on UPR pathways, with the Basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Overall, we hypothesize that IRE1a, PERK and/or ATF6a play(s) an oncogenic role in PDAC tumorigenesis due to lipid saturation abnormalities, and the Basal-like PDAC subtype is more sensitive to inhibition of UPR pathway(s). We are evaluating the role of IRE1a, PERK and ATF6a using pharmacologic inhibition and genetic knockdown in vitro and in vivo under UFA deprived conditions. We will also evaluate PDAC intracellular and interstitial fluid lipidomics using LC-MS. In addition, we will assess whether hypoxic PDAC cells experience ER stress in vivo due to UFA deprivation using Stimulated Raman scattering (SRS) microscopy. To explore the subtype-specific dependency on the UPR pathways, we are querying a UPR gene signature in different tumor cell clusters using single cell RNA-seq datasets of patient samples. In addition, we will employ an isogenic system for PDAC subtypes and examine subtype-specific responses to UPR inhibition in vitro and in vivo. Our goal is to better understand UPR pathways in PDAC cells as a survival mechanism in response to the “lipotoxicity” induced by ER membrane saturation, with a consideration of intratumoral heterogeneity of PDAC cell subtype. Citation Format: Yanqing (Christine) Jiang, Xu Han, Michelle Burrows, Carson Poltorack, Celeste Simon, Brian Keith. Investigating unfolded protein responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-027.

Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth.

Chromosome translocations involving mixed lineage leukemia (MLL) gene cause acute leukemia with a poor prognosis. MLL is frequently fused with transcription cofactors AF4 (~35%), AF9 (25%) or its paralog ENL (10%). The AHD domain of AF9/ENL binds to AF4, its paralog AFF4, or histone-H3 lysine-79 (H3K79) methyltransferase DOT1L. Formation of AF9/ENL/AF4/AFF4-containing super elongation complexes (SEC) and the catalytic activity of DOT1L are essential for MLL-rearranged leukemia. Protein-protein interactions (PPI) between AF9/ENL and DOT1L/AF4/AFF4 are therefore a potential drug target.Methods: Compound screening followed by medicinal chemistry was used to find inhibitors of such PPIs, which were examined for their biological activities against MLL-rearranged leukemia and other cancer cells.Results: Compound-1 was identified to be a novel small-molecule inhibitor of the AF9/ENL-DOT1L/AF4/AFF4 interaction with IC50s of 0.9-3.5 µM. Pharmacological inhibition of the PPIs significantly reduced SEC and DOT1L-mediated H3K79 methylation in the leukemia cells. Gene profiling shows compound-1 significantly suppressed the gene signatures related to onco-MLL, DOT1L, HoxA9 and Myc. It selectively inhibited proliferation of onco-MLL- or Myc-driven cancer cells and induced cell differentiation and apoptosis. Compound-1 exhibited strong antitumor activity in a mouse model of MLL-rearranged leukemia.Conclusions: The AF9/ENL-DOT1L/AF4/AFF4 interactions are validated to be an anticancer target and compound-1 is a useful in vivo probe for biological studies as well as a pharmacological lead for further drug development.

Discovery and Structure-Based Optimization of Potent and Selective WD Repeat Domain 5 (WDR5) Inhibitors Containing a Dihydroisoquinolinone Bicyclic Core.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.

MP99-18 ROLE OF LONG NON-CODING RNA PVT1 IN REGULATING MYC IN HUMAN CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2017MP99-18 ROLE OF LONG NON-CODING RNA PVT1 IN REGULATING MYC IN HUMAN CANCER Kojiro Tashiro, Yuen-Yi Tseng, Badrinath Konety, and Anindya Bagchi Kojiro TashiroKojiro Tashiro More articles by this author , Yuen-Yi TsengYuen-Yi Tseng More articles by this author , Badrinath KonetyBadrinath Konety More articles by this author , and Anindya BagchiAnindya Bagchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3104AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Though gain of 8q24.21 is a common mutation in human cancers, its functional annotation is limited to studying myelocytomatosis (MYC), the prominent oncogene in the amplicon. However, MYC is co-gained with an adjacent long non-coding RNA gene plasmacytoma variant translocation 1 (PVT1), CCDC26 and gasdermin C (Gsdmc). Whether copy number gain of one or more of these genes drives neoplasia is unknown. METHODS We developed chromosome engineered mouse strains with an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. These mice were crossed with transgenic mice harboring rat Neu was introduced to test the change in the latency in mammary tumor development. We also used genetically engineered human cancer cell lines to identify the molecular basis of co-operation between MYC and PVT1 in human cancer. RESULTS Only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc developed adenocarcinomas, suggesting that may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the tumors. Ablation of PVT1 decreased MYC protein levels, suggesting a PVT1-dependence of MYC protein in MYC amplified cancer cells. These data suggests that PVT1 can potentiate MYC in human cancers. CRISPR-cas9 mediated deletion of PVT1 in HCT116 impaired tumor formation in xenografts, and significantly reduced MYC levels. Additionally, we have identified the putative functional domain of PVT1 which confers its oncogenic potential. We have recently discovered that the exon 2 of PVT1 can undergo ‘backsplicing’ and form circular RNA of 410 bases (CircPVT1). This CircPVT1 is more abundant in MYC-driven cancer cell lines. Though PVT1 is known as a long non-coding RNA, we found that CircPVT1 can form an open reading frame (ORF) of 104 amino acids, which we have annotated as PVT1 encoded protein upon circularization (PEPc). While CRISPR-Cas9 mediated deletion of PVT1 exon 2 results into decreased MYC levels and reduced transformation potential in MYC-driven cancer cells, PEPc can independently augment MYC when added exogenously to LNCaP cells, and increase their transformation potential. CONCLUSIONS These results suggest that PEPc may play a key role in boosting MYC levels in metastatic prostate cancers. We propose that the dependence of high levels of MYC on PVT1 provides a much-needed therapeutic window against MYC protein, known to be refractory to small molecule inhibition. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1327-e1328 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Kojiro Tashiro More articles by this author Yuen-Yi Tseng More articles by this author Badrinath Konety More articles by this author Anindya Bagchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract PL02-02: Transcriptional regulation by the MAX-like protein MLX is required for adaptive metabolic responses in development and tumorigenesis

Abstract The MYC transcription factor, together with its obligate dimerization partner, MAX, regulates widespread gene expression and is implicated in the etiology of a broad spectrum of cancers. MYC and MAX are members of the MAX-MLX superfamily of bHLHZ transcription factors (which includes MYC, MAX, MXD, MLX, and MONDO/ChREBP). Members of this highly conserved family respond to environmental cues to modulate gene expression programs and control cellular fate. We recently showed that metabolic reprogramming and survival in MYC-driven cancer cells is coordinately regulated by MYC-MAX and MONDOA-MLX. While knockdown of MONDOA or MLX affected multiple metabolic pathways in MYC-induced tumors, we found that decreased fatty acid synthesis primarily accounted for the loss of viability (Carroll et al. Cancer Cell 27:271, 2015) To explore the physiological role of the MLX branch of the MAX-MLX network we generated mice with targeted deletion of Mlx. Mlx null mice appeared to develop normally; however, we found that Mlx null males are infertile, with strikingly diminished sperm production, viability, and motility. MondoA knockout mice exhibited a similar phenotype. By employing tissue-specific Cre-mediated deletion of Mlx, we uncovered metabolic defects in both Sertoli cells as well as germ cells, including loss of lipogenic enzymes and altered lipid content. Moreover, we found that human male germ cell tumors (MGCTs) overexpress MAX-MLX network proteins and undergo apoptosis upon loss of MONDOA/MLX. Importantly, the same metabolic pathways shown to be crucial for survival of neuroblastoma are also required for survival of MGCTs. Furthermore. we have uncovered a critical role for MLX in myokine production required for myoblast fusion in vitro. Moreover, mice bearing homozygous deletion of Mlx fail to regenerate muscle following cardiotoxin treatment (Hunt et al. Genes Dev. 29:2475, 2015). Taken together, our studies suggest a critical role for the MLX arm of the MAX-MLX network in adaptive metabolic responses in tumorigenesis and development. Funding: NIH grants CA57138 (to RNE) and GM055668 (to DEA); ALSAC of St. Jude Children's Research Hospital, the Ellison Medical Foundation, the Glenn Foundation for Medical Research, and the American Federation for Aging Research (AFAR) (to FD). Citation Format: Patrick A. Carroll, Pei-Feng Cheng, Charles H. Muller, Liam Hunt, Fabio Demontis, Don E. Ayer, Robert N. Eisenman. Transcriptional regulation by the MAX-like protein MLX is required for adaptive metabolic responses in development and tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr PL02-02.

Abstract 1139: Translational control of MYC protein stability as a target of small molecules

Abstract It is well established that MYC family proteins drive tumor growth and sustain the malignant phenotype. However, MYC family proteins have gained the reputation of being “un-druggable” by small molecule inhibitors. This is because MYC proteins lack intrinsic enzymatic activities or appropriate molecular grooves to which small molecules may bind and inhibit their function. Nonetheless, previous studies suggest that MYC family proteins (MYC, MYCN, MYCL) can be “targetable” indirectly by accelerating their turnover rate at different biochemical pathways that regulate the steady state level of the proteins. These include transcriptional-inhibition of the corresponding genes and acceleration of their proteasome-dependent degradation. The goal of this study is to gain a better understanding of how the MYC family protein turnover is regulated, which in turn might help develop future new drugs against “MYC family-driven” cancers. We have used neuroblastoma, a childhood cancer, as a disease model and identified several small molecules that rapidly destabilize MYC and MYCN proteins in MYC-driven neuroblastoma cells. The small molecules include FCCP, OSU-03012 and Salinomycin. Preliminary data suggest that the common target of these compounds is mitochondria. Recently, we have also found Halofuginone, an FDA approved orphan drug for Scleroderma treatment exhibits a similar MYC-destabilizing activity. At submicromolar concentrations and less than one hour of drug-treatment, Halofuginone rapidly causes significant down-regulation of MYC proteins in neuroblastoma cells. Halofuginone is known to activate the eIF2α pathway, which controls translation. Activation of the eIF2α pathway is initiated by the activation of eIF2a kinases (GCN2, HRI, PERK, PKR) via autophosphorylation by various stress-related stimuli such as reactive oxygen species and misfolded proteins. The activated eIF2α kinases then inactivates eIF2α by phosphorylation, which subsequently halts translation globally. Interestingly, FCCP, OSU-03012 and Salinomycin treatments also caused eIF2α phosphorylation. These observations suggest that activation of the eIF2α pathway results in translational block of the MYC family protein expression in tumor cells. Because half-lives of MYC family proteins are very short, a sudden attenuation in protein translation can cause rapid down-regulation of the proteins. Hence, activation of the eIF2α pathway by small molecules may prove effective for rapid down-regulation of MYC family proteins in MYC-driven cancer cells. Citation Format: Naohiko Ikegaki, Luqman Baloch, Christopher Chu, Joshua Lomahan, Jamie Harris, Bill Chiu. Translational control of MYC protein stability as a target of small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1139.

A Time for MYC: Metabolism and Therapy.

The MYC oncogene is frequently deregulated in human cancers, whereas the proto-oncogene is exquisitely, tightly regulated in normal cells. Deregulated MYC drives transcriptional imbalance, thereby altering metabolism and disrupting the circadian Bmal1-Clock E-box-dependent transcriptional circuitry. Sustained oncogenic MYC expression drives a constitutive growth program with mammalian target of rapamycin (mTOR) activation that renders cells dependent on nutrients, such that glucose or glutamine deprivation could trigger cell death and key enzymes such as lactate dehydrogenase A (LDHA) and glutaminase (GLS) amenable for targeting in cancers. Further, MYC-mediated suppression of the circadian clock is surmised to suspend the inhibitory effect of Bmal1-Clock on metabolism, allowing for MYC-driven cancer cells to reach a higher state of anabolic metabolism. Hence, metabolic therapy could be deployed, particularly at specific times of the day, to diminish side effects to normal tissues while maintaining antitumor efficacy.

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.

The spliceosome is a therapeutic vulnerability in MYC-driven cancer.

c-MYC (MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its pro-tumorigenic functions have been attributed to its ability to regulate gene expression programs1–3. Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts4–7. While such increases in RNA and protein production may endow cancer cells with pro-tumor hallmarks, this elevation in synthesis may also generate new or heightened burden on MYC-driven cancer cells to properly process these macromolecules8. Herein, we discover the spliceosome as a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (SF3B1, U2AF1, and others) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total pre-mRNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Importantly, genetic or pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.

Abstract 2953: Rev-erbα modulates Myc-driven cancer cell growth and altered metabolism

Abstract Circadian rhythms are regulated by feedback loops comprising a network of factors that regulate Clock-associated genes. Chronotherapy seeks to take advantage of altered circadian rhythms in some cancers to better time administration of treatments to increase efficacy and reduce toxicity. While many cancers have perturbed expression of core circadian rhythm genes, the molecular basis underlying these perturbations and their functional implications in oncogenesis are still poorly understood, and so it is impossible to predict which cancers have altered circadian rhythms and would best benefit from chronotherapy. We have observed in cancer cell models of osteosarcoma, hepatocellular carcinoma, and neuroblastoma that the c-Myc and N-Myc oncogenic transcription factors disrupt oscillation of the circadian clock by specifically upregulating the circadian rhythm gene and nuclear hormone receptor NR1D1 (Rev-erbα). Interestingly, while Rev-erbα has not been previously recognized as an oncogene, data from The Cancer Genome Atlas revealed that it is amplified in many forms of human cancer, and we also observed that Rev-erbα was upregulated in primary human neuroblastoma and associated with poor prognosis. Therefore, we hypothesized that Rev-erbα is a novel oncogene downstream of Myc and is important for cancer cell growth. Here we show that Rev-erbα is specifically essential for the growth of Myc-driven hepatocellular carcinoma cells, as the related protein Rev-erbβ did not strongly influence growth. While knockdown of Rev-erbα expression by siRNA slowed growth, it did not cause cell death or canonical cell cycle arrest. Rev-erbα modulates circadian rhythm by downregulating the central circadian regulatory protein Bmal1, but this pathway did not play a central role in Rev-erbα control of cell growth. Additionally, while Rev-erbα has a well-described role in heme metabolism and subsequent support of mitochondria respiration, this pathway was not directly altered in Myc-driven liver cancer cells. Rather, knockdown of Rev-erbα was associated with decreased glycolytic activity characterized by a decrease in intracellular lactate and extracellular lactate production as well as an increase in certain glycolytic intermediates. In addition to these glycolytic changes, the maximum respiratory capacity of cells lacking Rev-erbα increased, as measured by oxygen consumption. These data suggest a novel role for Rev-erbα in promoting the growth of cancer cells through modulation of glucose metabolism and a shift towards increased respiration, and imply that cancers with upregulated Myc and Rev-erbα may be good candidates for chronotherapy. We thank the following funding sources: NIH R01CA57341, LLS 6106-14. Citation Format: Brian J. Altman, Annie Hsieh, Arvin M. Gouw, Zachary E. Stine, Anand Venkataraman, David I. Bellovin, Sharon J. Diskin, Wenyun Lu, Sisi Zhang, Dean W. Felsher, John M. Maris, Mitchell A. Lazar, Joshua D. Rabinowitz, John B. Hogenesch, Chi V. Dang. Rev-erbα modulates Myc-driven cancer cell growth and altered metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2014-2953

Therapeutic Targeting of Myc-Reprogrammed Cancer Cell Metabolism

Studies from many laboratories document that the MYC oncogene produces a pleiotropic transcription factor, Myc, which influences genes driven by all three RNA polymerases to orchestrate nutrient import with biomass accumulation for cell division. Myc has been shown to activate genes involved in glycolysis, glutaminolysis, and mitochondrial biogenesis to provide ATP and anabolic substrates for cell mass accumulation. Myc stimulates ribosome biogenesis and orchestrates the energetic demand for biomass accumulation through its regulation of glucose and glutamine import and metabolism. When normal cells are deprived of nutrients, endogenous MYC expression diminishes and cells withdraw from the cell cycle. However, ectopic MYC-driven cancer cells are locked in a state of deregulated biomass accumulation, which renders them addicted to glucose and glutamine. This addictive state can be exploited for cancer therapy, because nutrient deprivation kills Myc-driven cells and inhibition of the Myc targets, lactate dehydrogenase A or glutaminase, diminishes tumor xenograft growth in vivo.