MYC Gene Expression Research Articles

Antiproliferative and cytotoxic effects of grape pomace and grape seed extracts on colorectal cancer cell lines.

Grape pomace is the source of bioactive compounds (anthocyanins, flavonols, flavan‐3‐ols, and stilbenes) which exhibit antiproliferative actions on cell cultures. We have investigated the antitumoral effects of grape pomace and grape seed extracts on colon cancer cells (Caco‐2, HT‐29) and fibroblasts. Crude extracts prepared from white and red pomace, and grape seeds, reduced the viability and proliferation of Caco‐2. HT‐29 cells were resistant to these actions. Purified extracts were then prepared from the same sources and compared with the LDH test; again, all three extracts were active and purified extract from grape seed was the most potent and specific on Caco‐2 cells. HT‐29 cells were more sensitive to these purified extracts. The biological activity resided almost exclusively in the flavonol and flavan‐3‐ols subfractions, rather than the anthocyanin subfraction. Preliminary results on the mechanisms involved in these effects revealed downregulation of Myc gene expression in HT‐29 and upregulation of Ptg2 in Caco‐2 cells.

PB1868 B‐CELL PROLYMPHOCYTIC LEUKEMIA (B‐PLL) AND PROLYMPHOCYTOID MANTLE CELL LYMPHOMA (PMCL) (MORE THAN 55% OF PROLYMPHOCYTES) ARE CLOSED BUT DISTINCT ENTITIES. ON BEHALF GFCH AND FILO GROUPS

Background:B‐PLL is a rare entity that was debated. It is now recognized in the WHO classification and defined by the presence of prolymphocytes in peripheral blood (PB) exceeding 55% of lymphoid cells, different from transformed chronic lymphocytic leukemia (CLL) and cases with translocation t(11;14) that are classified as MCL. We previously showed that B‐PLL have a high frequency of MYC activation by translocation [t(MYC)] or gain, and frequent del17p, patients with both abnormalities (abs) having the worst prognosis. It has been suggested that B‐PLL should be considered as a subgroup of MCL.Aims:In order to evaluate the relationship between B‐PLL and MCL, we compared cytogenetics and molecular data.Methods:We studied 35 B‐PLL and 4 pMCL (≥55% of prolymphocytes in PB) (multicentric study by the Groupe Francophone de Cytogénétique Hématologique and the FILO group), 24 MCL and 16 CLL as control, using karyotype (K), FISH, whole‐exome sequencing (WES), IGHV and RNA‐sequencing. All MCL had a t(11;14), while the presence of a CCND1, CCND2 or CCND3 rearrangement was excluded in B‐PLL cases.Results:A morphological review, performed by 3 independent experts, did not show any distinctive features between B‐PLL and pMCL. In B‐PLL, we found a complex K (CK) (≥3 abs) in 71%, and a highly complex K (HCK ≥ 5) in 44%. The most frequent chromosomal abs were: MYC abs (n = 27, 77%): t(MYC) (n = 22, 63%) or MYC gain (n = 5, 14%) and del17p (n = 13, 37%). Among the 19/24 MCL with an informative K, we found CK in 13 (68%), including 11 HCK (58%) and del11q (ATM) in 3 (16%). By FISH, we found 9 (38%) del17p and 7 (29%) MYC abs (6 gains and 1 t(MYC)). All 4 pMCL had a CK, including 3 HCK, 2 had a t(MYC), 1 a MYC gain, 2 a del17p and 2 a del11q (Figure A). Using WES on 23 B‐PLL, the most frequent mutated genes were TP53 (n = 12, 52%), MYC, BCOR (n = 5, 22% each), SETD2, MYD88 (n = 4, 17% each), SF3B1 and CHD2 (n = 3, 13% each). WES performed on 3 pMCL showed 2 cases ATMmut, and 1 CCND1mut/TP53mut (Figure B). Overall, ATM inactivation was present in 1/35 (2.9%) B‐PLL vs 3/4 (75%) pMCL (p = .001). The majority of B‐PLL used a VH3 or VH4 gene (17/19, 89%), but none of the 4 pMCL (VH1, n = 3, VH2, n = 1). The level of somatic hypermutation was higher in B‐PLL: 16/19 (84%) had ≤98% of homology vs 1/4 pMCL (p = .04). Principal component analysis of gene expression data in 12 B‐PLL, 3 pMCL and 16 CLL analyzed by RNA‐Seq showed that B‐PLL and pMCL clustered together, distinctively from CLL (Figure C). However, 386 genes were differently expressed between B‐PLL and pMCL, with enrichment in cell cycle genes in pMCL, including CCND1 and CCND2. The level of SOX11 expression was significantly lower in B‐PLL than in pMCL (p = .02). Comparing B‐PLL and CLL, GSEA analysis revealed enrichment in MYC signaling pathway, and cell metabolism signatures in B‐PLL.Summary/Conclusion:In conclusion, our results show that B‐PLL have a genomic profile distinct from MCL and CLL, with frequent t(MYC), TP53, MYC and BCOR mutations. However, B‐PLL have similarities with the rare subgroup of pMCL: morphology, high frequency of MYC abs and gene expression profiles related to cell cycle and proliferation. Nevertheless, we highlighted distinctive features in addition to t(11;14): IGHV repertoire and mutation level, SOX11 expression and frequency of ATM abs. Our data advocate that pMCL could be an aggressive form of MCL, which have to be definitely distinguished from B‐PLL with a routine test, the karyotype.image

FGF2 induces breast cancer growth through ligand-independent activation and recruitment of ERα and PRBΔ4 isoform to MYC regulatory sequences.

Progression to hormone-independent growth leading to endocrine therapy resistance occurs in a high proportion of patients with estrogen receptor alpha (ERα) and progesterone receptors (PR) positive breast cancer. We and others have previously shown that estrogen- and progestin-induced tumor growth requires ERα and PR interaction at their target genes. Here, we show that fibroblast growth factor 2 (FGF2)-induces cell proliferation and tumor growth through hormone-independent ERα and PR activation and their interaction at the MYC enhancer and proximal promoter. MYC inhibitors, antiestrogens or antiprogestins reverted FGF2-induced effects. LC-MS/MS identified 700 canonical proteins recruited to MYC regulatory sequences after FGF2 stimulation, 397 of which required active ERα (ERα-dependent). We identified ERα-dependent proteins regulating transcription that, after FGF2 treatment, were recruited to the enhancer as well as proteins involved in transcription initiation that were recruited to the proximal promoter. Also, among the ERα-dependent and independent proteins detected at both sites, PR isoforms A and B as well as the novel protein product PRBΔ4 were found. PRBΔ4 lacks the hormone-binding domain and was able to induce reporter gene expression from estrogen-regulated elements and to increase cell proliferation when cells were stimulated with FGF2 but not by progestins. Analysis of the Cancer Genome Atlas data set revealed that PRBΔ4 expression is associated with worse overall survival in luminal breast cancer patients. This discovery provides a new mechanism by which growth factor signaling can engage nonclassical hormone receptor isoforms such as PRBΔ4, which interacts with growth-factor activated ERα and PR to stimulate MYC gene expression and hence progression to endocrine resistance.

The ERH gene regulates migration and invasion in 5637 and T24 bladder cancer cells

BackgroundThis study aimed to determine whether the enhancer of the rudimentary homolog (ERH) gene regulates cell migration and invasion in human bladder urothelial carcinoma (BUC) T24 cells and the underlying mechanism.MethodsFirst, we knocked down ERH in BUC T24 and 5637 cells by shRNA and then used wound healing cell scratch migration assays, transwell cell migration assays, transwell cell invasion chamber experiments and nude mouse tail vein transfer assays to determine the migration and invasion ability after ERH was knocked down. Moreover, we used gene expression profiling chip analysis and further functional experiments to explore the possible mechanism through which ERH knockdown downregulated metastasis ability in T24 cells.ResultsWound healing cell scratch migration assays, transwell cell migration assays, transwell cell invasion chamber experiments and nude mouse tail vein transfer assays all showed that the metastasis ability was significantly inhibited in human BUC T24 and 5637 cells with ERH knockdown. A gene expression profiling chip analysis in T24 cells showed that the MYC gene may be an important downstream target of the ERH gene, and the functional experiments showed that MYC is a functional target of ERH in BUC T24 cells.ConclusionERH knockdown could inhibit the metastasis of BUC T24 cells in vitro and in vivo. This study further explored the mechanism of the ERH gene in the metastasis of the T24 human bladder cancer cell line and found that ERH may regulate MYC gene expression. The results of this research provide a basis for the clinical application of ERH as a potential target for BUC treatment.

Association of Overexpressed MYC Gene with Altered PHACTR3 and E2F4 Genes Contributes to Non-small Cell Lung Carcinoma Pathogenesis.

SummaryBackgroundC-Myc is one of the major cellular oncogenes overexpressed in non-small cell lung carcinoma (NSCLC). Its deregulated expression is necessary but not sufficient for malignant transformation. We evaluated expression of MYC gene in NSCLC patients and its association with alterations in the genes previously identified to be related to NSCLC pathogenesis, PHACTR3 and E2F4.MethodsWe analyzed MYC gene expression by qRT-PCR in 30 NSCLC patients’ samples and paired normal lung tissue. MYC expression was further statistically evaluated in relation to histopathological parameters, PHACTR3 and E2F4 gene alterations and survival. Alterations in aforementioned genes were previously detected and identified based on AP-PCR profiles of paired normal and tumor DNA samples, selection of DNA bands with altered mobility in tumor samples and their characterization by the reamplification, cloning and sequencing.ResultsMYC expression was significantly increased in NSCLC samples and its overexpression significantly associated with squamous cell carcinoma subtype. Most importantly, MYC overexpression significantly coincided with mutations in PHACTR3 and E2F4 genes, in group of all patients and in squamous cell carcinoma subtype. Moreover, patients with jointly overexpressed MYC and altered PHACTR3 or E2F4 showed trend of shorter survival.ConclusionsOverall, MYC is frequently overexpressed in NSCLC and it is associated with mutated PHACTR3 gene, as well as mutated E2F4 gene. These joint gene alterations could be considered as potential molecular markers of NSCLC and its specific subtypes.

The Crohn's disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells.

Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that arises from chronic inflammation in the gastrointestinal tract. Genome-wide association studies (GWAS) have identified over 200 single nucleotide polymorphisms (SNPs) that are associated with a predisposition for developing IBD. For the majority, the causal variant and target genes affected are unknown. Here, we investigated the CD-associated SNP rs6651252 that maps to a gene desert region on chromosome 8. We demonstrate that rs6651252 resides within a Wnt responsive DNA enhancer element (WRE) and that the disease associated allele augments binding of the TCF7L2 transcription factor to this region. Using CRISPR/Cas9 directed gene editing and epigenetic modulation, we find that the rs6651252 enhancer regulates expression of the c-MYC proto-oncogene (MYC). Furthermore, we found MYC transcript levels are elevated in patient-derived colonic segments harboring the disease-associated allele in comparison to those containing the ancestral allele. These results suggest that Wnt/MYC signaling contributes to CD pathogenesis and that patients harboring the disease-associated allele may benefit from therapies that target MYC or MYC-regulated genes.

Induction of GNMT by 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside through proteasome-independent MYC downregulation in hepatocellular carcinoma

Glycine-N-methyl transferase (GNMT) a tumor suppressor for hepatocellular carcinoma (HCC) plays a crucial role in liver homeostasis. Its expression is downregulated in almost all the tumor tissues of HCC while the mechanism of this downregulation is not yet fully understood. Recently, we identified 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranoside (PGG) as a GNMT promoter enhancer compound in HCC. In this study, we aimed to delineate the mechanism by which PGG enhances GNMT expression and to investigate its effect on GNMT suppression in HCC. Microarray and pathway enrichment analysis revealed that MYC was a major target of PGG. PGG suppressed MYC mRNA and protein expression in Huh7 and Hep G2 cells in a dose- and time-dependent fashion. Furthermore, MYC expression was also reduced in xenograft tumors in PGG treated mice. Moreover, shRNA-mediated knocked-down or pharmacological inhibition of MYC resulted in a significant induction of GNMT promoter activity and endogenous GNMT mRNA expression in Huh7 cells. In contrast, overexpression of MYC significantly inhibited GNMT promoter activity and endogenous GNMT protein expression. In addition, antibodies against MYC effectively precipitated the human GNMT promoter in a chromatin immunoprecipitation assay. Lastly, GNMT expression was negatively correlated with MYC expression in human HCC samples. Interestingly, PGG not only inhibited MYC gene expression but also promoted MYC protein degradation through proteasome-independent pathways. This work reveals a novel anticancer mechanism of PGG via downregulation of MYC expression and establishes a therapeutic rationale for treatment of MYC overexpressing cancers using PGG. Our data also provide a novel mechanistic understanding of GNMT regulation through MYC in the pathogenesis of HCC.

Effect of Helicobacter pylori, black tea and sodium bicarbonate on iron metabolism and MDCK cell survival

Background: Iron deficiency anemia is the most common nutritional disorder in the world. Diet and Helicobacter pylori infection are among the main causes of this disorder. Objective: In this study, the effect of black tea extract and sodium bicarbonate with Helicobacter pylori on the genes involved in iron absorption and storage, as well as cell proliferation, were studied. Methods: Simultaneous cultivation of MDCK and Helicobacter pylori cell lines was performed at concentrations of 10, 20, 40 and 80 μg/ml of tea extract and 30, 40, 60 and 100 mM sodium bicarbonate at 24 and 48 hours. The effect of treatment on cell survival was investigated by trypan blue staining and expression of MYC, TFRC, FTH1, IRP2, IRP1, and NDRG1 genes by real-time PCR and analyzed by ANOVA and independent T-test. Findings: There was no significant change in the expression of the genes involved in iron metabolism under the influence of tea, sodium bicarbonate and Helicobacter pylori treatment in MDCK cell line. Upregulation MYC gene expression was observed in the presence of Helicobacter pylori after 24 hours treatment with tea extract, and sodium bicarbonate, and in the absence of Helicobacter pylori upregulation with tea extract after 48 hours (P<0.05). Also upregulation NDRG1 gene expression was seen after tea extract treatment of cells with or without Helicobacter pylori in both 24 and 48 h (P<0.05). Conclusion: Sodium bicarbonate and tea each one alone didn’t not influence iron status. This study suggests that reduction of tea intake could be served as a risk prevention strategy.

Genetic modifiers of the BRD4-NUT dependency of NUT midline carcinoma uncovers a synergism between BETis and CDK4/6is.

Bromodomain and extraterminal (BET) domain inhibitors (BETis) show efficacy on NUT midline carcinoma (NMC). However, not all NMC patients respond, and responders eventually develop resistance and relapse. Using CRISPR and ORF expression screens, we systematically examined the ability of cancer drivers to mediate resistance of NMC to BETis and uncovered six general classes/pathways mediating resistance. Among these, we showed that RRAS2 attenuated the effect of JQ1 in part by sustaining ERK pathway function during BRD4 inhibition. Furthermore, overexpression of Kruppel-like factor 4 (KLF4), mediated BETi resistance in NMC cells through restoration of the E2F and MYC gene expression program. Finally, we found that expression of cyclin D1 or an oncogenic cyclin D3 mutant or RB1 loss protected NMC cells from BETi-induced cell cycle arrest. Consistent with these findings, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors showed synergistic effects with BETis on NMC in vitro as well as in vivo, thereby establishing a potential two-drug therapy for NMC.

Abstract 5790: TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models

Abstract BET (bromodomain and extra-terminal) proteins bind to acetylated lysine residues on chromatin and participate in the regulation of gene transcription. Inhibition of BET protein binding to chromatin with small molecules selectively suppresses the transcription of a set of oncogenes, including MYC and BCL-2. TG-1601 is a novel, selective and potent small molecule inhibitor of BET bromodomains. TG-1601 binds to the first and second bromodomains (BD1, BD2) of the BET protein family, BRD2, BRD3, BRD4, and BRDT, with Kd values ranging from 0.5 nM to 9.1 nM. MYC protein expression is strongly inhibited in the MV4-11 cancer cell line with an EC50 of 5 nM, with GI50 comprised between 15 nM and 85 nM in a variety of leukemia and myeloma cancer cell lines, indicating potent inhibition of cell proliferation. Time course and dose-response studies conducted in mice bearing subcutaneous MV4-11 xenografts showed that MYC protein was undetectable 3 hours following a single 25 mg/kg oral dose, with a TG-1601 tumor concentration of 6uM achieved. Interestingly, at 24h post-dose, while TG-1601 is cleared from the tumor, MYC protein level remains below 40% of its initial level, indicating a long-lasting effect pharmacodynamic of TG-1601, potentially attributable to enhanced binding affinity compared to earlier generation molecules. In agreement with this long-lasting effect, efficacy studies in MV4-11 tumor-bearing mice, dosed with a 20 mg/kg/day PO regimen interrupted by increasing drug holiday periods, showed that drug holidays of 2, 3 and 4 days per week only modestly affected efficacy (3%, 15% and 12% TGI respectively), suggesting discontinuous dosing of TG-1601 in clinic may not significantly impact efficacy. Anti-cancer agents have been shown - in vitro or in vivo - to synergize with BET inhibitors. Here we show that TG-1601 and anti PD-1 antibody demonstrated synergistic anti-tumor activity when combined in the B16F10 model, an aggressive syngeneic model of melanoma. TG-1601 inhibition of MYC, CCR-2 and IL1RN gene expression was tested in a whole blood ex-vivo experiment, and the genes were validated as pharmacodynamic markers to monitor TG-1601 activity in clinic. In conclusion, TG-1601 is a novel BET inhibitor with remarkably strong affinity and a potent ability to inhibit MYC expression and cell growth, with favorable pharmacokinetic properties supporting clinical development. Its properties in vivo provide an opportunity for the rational development of TG-1601 as an anti-cancer agent, taken alone or in combination with other small molecules or antibodies. IND enabling studies are underway, with clinical evaluation expected to commence in the first half of 2018. Citation Format: Emmanuel Normant, Leonid Gorelik, Rama Shmeis, Henry Le, Robert Nisch, Karen TenHuisen, Teja Turpuseema, James Oliviero, Hari P. Miskin, Peter Sportelli, Michael S. Weiss. TG-1601 is a novel BET inhibitor with strong binding affinity and long-lasting effect in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5790.

Clinicopathological Features and Prognostic Factors of DLBCL

To analyze the clinicopathological features and prognostic factors of patients with diffuse large B-cell lymphoma(DLBCL). Ninety-four cases of DLBCL followed up were selected in Fujian Tumor Hospital. The immunohistochemistry method was used to detect the protein expressions of BCL-2 BCL-6, MYC, CD10 and MUM-1, the gene abnormalities of MYC and BCL-2 were analyzed by fluorescence in situ hybridization, and the clinical pathological features and the related factors affecting prognosis in the patients with DLBCL were analyzed. The protein positive rates of BCL-2, BCL-6, MYC, CD10 and MUM-1 in 94 patients were 75.53% (71/94), 58.51% (55/94), 52.13% (49/94), 15.96% (15/94) and 34.04% (32/94) respectively. The detection rate of MYC gene abnormality was 20.93% (9/43) and the detection rate of BCL-2 gene abnormality was 44% (22/50); 2 kinds of gene abnormalities were of multiple copies, and 2 cases (2.13%) were abnormal in MYC and BCL-2 genes simultaneously. The median survival time of 3 years in 94 patients was 21.79 months (2-36 months), and the overall survival rates of 1 and 3 years were 82.98% and 64.89% respectively. Single factor analysis revealed that the high ECOG score (≥ 2), high international prognostic index (IPI) classification, positive expression of BCL-6 protein, and MYC and BCL-2 gene simultaneously abnormal were the risk factors influencing the prognosis (all P<0.05). COX regression analysis showed that IPI classification, ECOG score and treatment methods were independent factors influencing the prognosis (all P<0.05). IPI classification, ECOG score and treatment methods have greater impacts on the prognosis of patients with DLBCL. Chemotherapy combined with radiotherapy or surgical treatment can significantly improve the prognosis of patients.

Inhibition of Glycolysis in Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate.

We have shown previously that dietary administration of phenethyl isothiocyanate (PEITC), a small molecule from edible cruciferous vegetables, significantly decreases the incidence of poorly differentiated prostate cancer in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without any side effects. In this study, we investigated the role of c-Myc-regulated glycolysis in prostate cancer chemoprevention by PEITC. Exposure of LNCaP (androgen-responsive) and 22Rv1 (castration-resistant) human prostate cancer cells to PEITC resulted in suppression of expression as well as transcriptional activity of c-Myc. Prostate cancer cell growth inhibition by PEITC was significantly attenuated by stable overexpression of c-Myc. Analysis of the RNA-Seq data from The Cancer Genome Atlas indicated a significant positive association between Myc expression and gene expression of many glycolysis-related genes, including hexokinase II and lactate dehydrogenase A Expression of these enzyme proteins and lactate levels were decreased upon PEITC treatment in prostate cancer cells, and these effects were significantly attenuated by ectopic expression of c-Myc. A normal prostate stromal cell line (PrSC) was resistant to lactic acid suppression by PEITC treatment. Prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate and pyruvate levels. However, a 1-week intervention with 10 mg PEITC (orally, 4 times/day) was not sufficient to decrease lactate levels in the serum of human subjects. These results indicated that although prostate cancer prevention by PEITC in TRAMP mice was associated with suppression of glycolysis, longer than 1-week intervention might be necessary to observe such an effect in human subjects. Cancer Prev Res; 11(6); 337-46. ©2018 AACR.

Transcriptional Dysregulation of MYC Reveals Common Enhancer-Docking Mechanism

SUMMARYTranscriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.

НОВАЯ КОРОТКАЯ ФОРМА СЕКУРИНА СТИМУЛИРУЕТ ЭКСПРЕССИЮ ЦИКЛИНА D3 И АНГИОГЕННЫХ ФАКТОРОВ VEGFA И FGF2, НО НЕ ВЛИЯЕТ НА ЭКСПРЕССИЮ ТРАНСКРИПЦИОННОГО ФАКТОРА MYC, "Молекулярная биология"

Pituitary tumor-transforming gene-1 (PTTG1) encodes securin, a multifunctional protein involved in development of various types of cancer. Securin participates in the regulation of sister chromatids separation and the expression of multiple genes involved in the control of the cell cycle, metabolism, and angiogenesis. In several human cell lines, we have found a novel short isoform of securin mRNA, which does not contain exons 3 and 4. After the translation of this new mRNA, a shortened protein is produced that, like the full-size form, is able to activate the transcription of cyclin D3 gene (CCND3), which controls the G1/S transition and angiogenesis factors VEGFA (vascular endothelial growth factor), and FGF2 (fibroblast growth factor 2) in HEK293 cells. However, unlike the full-size protein, the short isoform of PTTG1 does not affect the MYC gene expression because it lacks the DNA-binding domain, which is needed for its interactions with the MYC promoter. Furthermore, the short form of securin does not influence the expression of MYC transcriptional targets, such as TP53 and IL-8. Thus, we found a novel isoform of securin which is able to activate a more restricted repertoire of genes compared to the full-size protein.

A bioclinical prognostic model using MYC and BCL2 predicts outcome in relapsed/refractory diffuse large B-cell lymphoma

The objective of this study was to create a bioclinical model, based on clinical and molecular predictors of event-free and overall survival for relapsed/refractory diffuse large B-cell lymphoma patients treated on the Canadian Cancer Trials Group (CCTG) LY12 prospective study. In 91 cases, sufficient histologic material was available to create tissue microarrays and perform immunohistochemistry staining for CD10, BCL6, MUM1/IRF4, FOXP1, LMO2, BCL2, MYC, P53 and phosphoSTAT3 (pySTAT3) expression. Sixty-seven cases had material sufficient for fluorescent in situ hybridization (FISH) for MYC and BCL2. In addition, 97 formalin-fixed, paraffin-embedded tissue samples underwent digital gene expression profiling (GEP) to evaluate BCL2, MYC, P53, and STAT3 expression, and to determine cell-of-origin (COO) using the Lymph2Cx assay. No method of determining COO predicted event-free survival (EFS) or overall survival (OS). Factors independently associated with survival outcomes in multivariate analysis included primary refractory disease, elevated serum lactate dehydrogenase (LDH) at relapse, and MYC or BCL2 protein or gene expression. A bioclinical score using these four factors predicted outcome with 3-year EFS for cases with 0–1 vs. 2–4 factors of 55% vs. 16% (P<0.0001), respectively, assessing MYC and BCL2 by immunohistochemistry, 46% vs. 5% (P<0.0001) assessing MYC and BCL2 messenger ribonucleic acid (mRNA) by digital gene expression, and 42% vs. 21% (P=0.079) assessing MYC and BCL2 by FISH. This proposed bioclinical model should be further studied and validated in other datasets, but may discriminate relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients who could benefit from conventional salvage therapy from others who require novel approaches. The LY12 study; clinicaltrials.gov Identifier: 00078949.

P1.15-005 Relationship Between MYC Family Status and Sensitivity to Aurora Kinase Inhibitors in Neuroendocrine and Other Lung Cancer Cell Lines

Recently, it has been reported that MYC-driven SCLC cell lines exhibit synthetic lethality with Aurora kinase (AURK) inhibitors. The aims of this study are to evaluate sensitivities to neuroendocrine (NE) lung cancer cell lines with or without any MYC family overexpression and/or amplification using representative AURK inhibitors and to investigate the associations between drug sensitivities, MYC family status and NE differentiation. We screened a panel of 62 cell lines including 33 SCLC, 18 other NE, and 11 NSCLC, with various MYC family status for growth inhibition upon AURK inhibitors. MYC family copy number, gene expression, and protein status were examined by quantitative real-time PCR, microarray and Western blotting. Relative cell growth was analyzed by MTS assay, and selective AURK A and B inhibitors, MLN8237 (Alisertib) and AZD1152 (Barasertib), respectively, were used in this study. Cell lines with IC50 values <0.1 μM were defined as sensitive. Drug effects on cell cycle and morphology were determined by flow cytometry and examination of formalin-fixed paraffin embedded cell pellets. Of 31 NE cell lines with MYC family overexpression/amplification, 21 (68%) and 18 (58%) were sensitive to Alisertib and Barasertib, respectively. All 20 NE (and most NSCLC) cell lines lacking MYC family overexpression/amplification were resistant to AURK inhibitors. There was an excellent concordance (86%) between response to the two AURK inhibitors. Both MYC family overexpression and amplification were predictive of sensitivity; however, some overexpressing lines had normal copy number. Cell cycle analysis showed a mitotic arrest in some sensitive cell lines with treatments of these drugs. Representative sensitive cell lines showed cell ballooning and bizarre multinucleated polyploid cells, which indicated cell cycle arrest in G2/M. MYC family overexpression/amplification in NE lung cancer cell lines confers sensitivity to AURK inhibitors in approximately 70% of cases. Lack of MYC family overexpression/amplification was always associated with drug resistance. MYC family overexpression may occur in the absence of gene amplification, and, thus, overexpression is a better predictor of sensitivity than amplification. We are currently investigating the mechanism of resistance in overexpressing NE cell lines and determining whether NE differentiation plays a role in drug sensitivity.

Genetic causes of resistance to entinostat in luminal breast cancer model systems.

1065 Background: Based on promising phase II data, the histone deacetylase inhibitor Entinostat (ENT) is in phase III trials for patients with metastatic ER-positive breast cancer. Predictors of sensitivity and resistance, however, remain unknown. Methods: Luminal cell lines SKBR3 (ER-/HER2+), BT474 (ER+/HER2+) and MCF7 (ER+/HER2-) were treated with or without ENT at their IC50 doses and their gene expression profiles determined. In addition, a total of 27 MMTV-Neu mouse tumors (luminal) were untreated (N = 8), or treated with ENT at 12 mg/kg for 3 weeks (N = 5), 6 weeks (N = 6), or until progression after complete response (N = 8). We investigated their gene expression profiles by microarray and copy number (CN) by arrayCGH, and utilized the Dawnrank analysis, a network-based bioinformatics tool that integrates DNA and RNA data to identify driver genes, to find predictors of resistance to ENT. Results: Supervised analysis of gene expression data coming from the 3 treated cell lines showed significant upregulation of multiple MYC gene signatures. Therefore, we constitutively overexpressed MYC using lentiviral MYC shRNA in SKBR3 and MCF7, and MYC overexpression made cell lines more resistant to ENT. In MMTV-Neu mice, both MYC gene mRNA and gene signatures were downregulated while cells responded to ENT, and became upregulated when the tumors progressed. aCGH CN analysis revealed that a large portion of mouse Chromosome 4 had DNA CN loss and low gene expression in tumors that progressed while on ENT. Within this region, JUN was computationally identified to be a top driver gene associated with resistance. JUN was next knocked down using lentiviral JUN shRNA in BT474 and T47D, and JUN knock-down repeatedly made cell lines more resistant to ENT. MYC gene-expression was also upregulated in JUN-knockdown BT474 and T47D. Finally, JUN CN loss was found in 22% (132/588) of luminal tumors in The Genome Cancer Atlas breast cancer, and all the MYC signature scores were significantly higher in JUN-deleted TCGA samples. Conclusions: ENT was an effective drug for all of our luminal models, both in vitro and in vivo. Using these models, we selected for resistant variants and identified MYC signature expression, and JUN CN deletion as being associated with resistance.

MYC Protein-positive Diffuse Large B-Cell Lymphoma Features an Activated B-Cell Receptor Signal Pathway.

Components of the B-cell receptor (BCR) signaling pathway represent promising therapeutic targets in diffuse large B-cell lymphoma (DLBCL) and other B-cell malignancies. MYC, a transcriptional factor and oncoprotein, is overexpressed in a fraction of DLBCL and indicates poor prognosis and aggressive clinical course when treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). However, BCR signaling status in MYC-positive DLBCL cases and the potential efficacy of BCR signal inhibitors in treating this aggressive disease are unknown. To further elucidate the BCR signaling pathway in MYC-positive DLBCL, we analyzed the levels of BCR-associated genes according to MYC gene status, detected phosphorylated protein with primary DLBCL samples, and estimated the patient survival with MYC expression. In addition, we manipulated MYC gene expression and tested its effects on BCR signaling in vitro. We found that CD19, SYK, and BLK were highly expressed in DLBCL with MYC gene overexpression. MYC-positive DLBCL had higher levels of pSYK and pBLK, but only pSYK level correlated with patient survival. The in vitro studies demonstrated that overexpression of the MYC gene augmented BCR signaling, whereas MYC gene knockdown attenuated BCR signaling. Thus, MYC protein-positive DLBCL features highly activated BCR signaling and may represent a potential candidate for BCR inhibitor therapy.

A Myc Activity Signature Predicts Poor Clinical Outcomes in Myc-Associated Cancers.

Myc transcriptional activity is frequently deregulated in human cancers, but a Myc-driven gene signature with prognostic ability across multiple tumor types remains lacking. Here, we selected 18 Myc-regulated genes from published studies of Myc family targets in epithelial ovarian cancer (EOC) and neuroblastoma. A Myc family activity score derived from the 18 genes was correlated to MYC/MYCN/MYCL1 expression in a panel of 35 cancer cell lines. The prognostic ability of this signature was evaluated in neuroblastoma, medulloblastoma, diffuse large B-cell lymphoma (DLBCL), and EOC microarray gene expression datasets using Kaplan-Meier and multivariate Cox regression analyses and was further validated in 42 primary neuroblastomas using qPCR. Cell lines with high MYC, MYCN, and/or MYCL1 gene expression exhibited elevated expression of the signature genes. Survival analysis showed that the signature was associated with poor outcome independently of well-defined prognostic factors in neuroblastoma, breast cancer, DLBCL, and medulloblastoma. In EOC, the 18-gene Myc activity signature was capable of identifying a group of patients with poor prognosis in a "high-MYCN" molecular subtype but not in the overall cohort. The predictive ability of this signature was reproduced using qPCR analysis of an independent cohort of neuroblastomas, including a subset of tumors without MYCN amplification. These data reveal an 18-gene Myc activity signature that is highly predictive of poor prognosis in diverse Myc-associated malignancies and suggest its potential clinical application in the identification of Myc-driven tumors that might respond to Myc-targeted therapies. Cancer Res; 77(4); 971-81. ©2016 AACR.

B-Cell Receptor Driven MALT1 Activity Regulates MYC Signaling in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor prognosis. Recent studies revealed the importance of constitutive B-cell receptor (BCR) signaling in maintaining survival of a subset of MCLs. MALT1 is an essential component of the CARD11-BCL10-MALT1 (CBM) complex that links BCR signaling to the nuclear factor kappa-B (NF-κB) pathway. Additionally, MALT1 functions as a protease that cleaves various substrates to promote proliferation and survival. However, its role in the molecular pathogenesis of MCL is unknown.To elucidate the functional role of MALT1 in the biology of MCL, we determined its proteolytic activity in primary MCL cells and in MCL derived cell lines. A large fraction of MCLs displayed constitutive activity of MALT1. This MALT1 activity is driven by constitutive BCR signaling, as we were able to show that RNA interference-mediated knockdown of central components of the BCR cascade abolished MALT1 activity.To gain insights into the functional significance of MALT1 in MCL, we knocked down its expression by different MALT1 shRNAs. Transduction of these shRNAs induced cytotoxicity in models that are characterized by constitutive MALT1 activity, whereas no effect on survival was observable in MCLs without MALT1 activation. To determine if this MALT1 addiction translates into an in vivosetting, we knocked down MALT1 in mouse MCL models and detected a significant inhibition of tumor growth. This indicates that MALT1-activated MCLs are dependent on the function of MALT1. These results were confirmed as pharmacologic inhibition of MALT1 significantly reduced cell viability in MALT1-activated MCLs, implying that MALT1 inhibition might represent a promising therapeutic strategy for MCL patients.To understand which biologic processes are regulated by MALT1 in MCL, we profiled gene expression changes at different time points following MALT1 inhibition. An unbiased gene set enrichment analysis identified various previously described MYC gene expression signatures to be among the top downregulated signatures, suggesting that MALT1 regulates MYC expression. MYC downregulation following MALT1 inhibition or MALT1 knockdown was confirmed at the protein level and various analyses revealed that MALT1 regulates MYC expression posttranslationally by preventing its proteasomal degradation. These results were further confirmed in primary mouse splenocytes, indicating that this novel molecular mechanism of regulating MYC expression is not restricted to MCL.To confirm that MYC is indeed expressed in primary MCLs, we determined MYC expression in 234 primary MCL samples by immunohistochemistry. These analyses revealed that 75 samples (32.1%) displayed an intermediate and 55 samples (23.5%) a high MYC positivity, suggesting that MYC is expressed in a substantial number of primary MCLs. As common alterations such as MYC high-levelamplifications and translocations determined by FISH occurred extremely rarely in our primary MCL samples (1% of samples), it is conceivable that BCR-driven MALT1 signaling is the predominant mechanism of MYC upregulation in MCL.In summary, we report for the first time that a substantial fraction of MCLs is addicted to constitutive MALT1 signaling. Thus, MCLs can be divided based on their MALT1 activation status into two distinct subgroups. We further identified a novel regulatory mechanism of MYC expression by MALT1. Thus, our study provides a strong mechanistic rationale to investigate the therapeutic efficacy in targeting the MALT1-MYC axis in MCL patients. DisclosuresTrneny:Janssen Research & Development: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Dreyling:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau.