Myc-related Genes Research Articles

Additional copies of 1q negatively impact the outcome of multiple myeloma patients and induce transcriptomic deregulation in malignant plasma cells

Additional copies of chromosome 1 long arm (1q) are frequently found in multiple myeloma (MM) and predict high-risk disease. Available data suggest a different outcome and biology of patients with amplification (Amp1q, ≥4 copies of 1q) vs. gain (Gain1q, 3 copies of 1q) of 1q. We evaluated the impact of Amp1q/Gain1q on the outcome of newly diagnosed MM patients enrolled in the FORTE trial (NCT02203643). Among 400 patients with available 1q data, 52 (13%) had Amp1q and 129 (32%) Gain1q. After a median follow-up of 62 months, median progression-free survival (PFS) was 21.2 months in the Amp1q group, 54.9 months in Gain1q, and not reached (NR) in Normal 1q. PFS was significantly hampered by the presence of Amp1q (HR 3.34 vs. Normal 1q, P < 0.0001; HR 1.99 vs. Gain1q, P = 0.0008). Patients with Gain1q had also a significantly shorter PFS compared with Normal 1q (HR 1.68, P = 0.0031). Concomitant poor prognostic factors or the failure to achieve MRD negativity predicted a median PFS < 12 months in Amp1q patients. Carfilzomib–lenalidomide–dexamethasone plus autologous stem cell transplantation treatment improved the adverse effect of Gain1q but not Amp1q. Transcriptomic data showed that additional 1q copies were associated with deregulation in apoptosis signaling, p38 MAPK signaling, and Myc-related genes.

Machine Learning Developed a MYC Expression Feature-Based Signature for Predicting Prognosis and Chemoresistance in Pancreatic Adenocarcinoma.

MYC has been identified to profoundly influence a wide range of pathologic processes in cancers. However, the prognostic value of MYC-related genes in pancreatic adenocarcinoma (PAAD) remains unclarified. Gene expression data and clinical information of PAAD patients were obtained from The Cancer Genome Atlas (TCGA) database (training set). Validation sets included GSE57495, GSE62452, and ICGC-PACA databases. LASSO regression analysis was used to develop a risk signature for survival prediction. Single-cell sequencing data from GSE154778 and CRA001160 datasets were analyzed. Functional studies were conducted using siRNA targeting RHOF and ITGB6 in PANC-1 cells. High MYC expression was found to be significantly associated with a poor prognosis in patients with PAAD. Additionally, we identified seven genes (ADGRG6, LINC00941, RHOF, SERPINB5, INSYN2B, ITGB6, and DEPDC1) that exhibited a strong correlation with both MYC expression and patient survival. They were then utilized to establish a risk model (MYCsig), which showed robust predictive ability. Furthermore, MYCsig demonstrated a positive correlation with the expression of HLA genes and immune checkpoints, as well as the chemotherapy response of PAAD. RHOF and ITGB6, expressed mainly in malignant cells, were identified as key oncogenes regulating chemosensitivity through EMT. Downregulation of RHOF and ITGB6 reduced cell proliferation and invasion in PANC-1 cells. The developed MYCsig demonstrates its potential in enhancing the management of patients with PAAD by facilitating risk assessment and predicting response to adjuvant chemotherapy. Additionally, our study identifies RHOF and ITGB6 as novel oncogenes linked to EMT and chemoresistance in PAAD.

Identification and validation of functional roles for three MYC-associated genes in hepatocellular carcinoma

BackgroundAberrations in MYC underlie a large proportion of liver hepatocellular carcinoma (LIHC) cases; however, MYC is difficult to target because of its undruggable structure. We aimed to uncover MYC-associated molecular targets to provide new strategies for LIHC treatment. MethodsLIHC transcriptome datasets and clinical information were obtained from The Cancer Genome Atlas. A series of bioinformatics analyses were performed for 370 patients who were stratified based on the median MYC expression level (high-MYC group and low-MYC group). Correlation analysis was performed to determine relationships between the expression of key MYC-associated genes and prognosis, DNA promotor methylation, and immune cell infiltration. Gene ontology and Kyoto Encyclopedia of Genes and Genomes Pathway enrichment analyses were performed to elucidate the functions of these genes in LIHC. Their expression and functions in LIHC were further verified using transgenic mice overexpressing c-Myc under control of the hepatocyte-specific promoter (Alb-Cre). ResultsAURKB, CCNB2, and CDKN3 were overexpressed in LIHC patients with high MYC expression and were associated with poor prognosis. Upregulation of these 3 genes was significantly correlated with hypomethylated promoter status, advanced T stage, metastasis, and immune cell infiltration in LIHC patients. Functional enrichment analyses indicated that these genes participate in the “p53 signaling pathway” and “cell cycle”. Furthermore, RT-PCR and IHC analysis revealed that their mRNA and protein expression levels were upregulated in an Alb-Cre;cMYClsl/- mouse model. Drugs that target these 3 MYC-related genes were identified. ConclusionTaken together, our results identify biomarkers of potential utility for managing liver cancer therapy owing to their significance in tumorigenesis, proliferation, and tumor immunity.

Computational Gene Expression and Network Analysis of Myc Reveal Insights into Its Diagnostic and Prognostic Role in Subtypes of Renal Cancer.

In this study, we analysed the Myc expression in the pan-kidney cohort (KIPAN) and kidney renal clear cell carcinoma (KIRC) in human tumour tissues compared to normal tissues. Myc is overexpressed and associated with poor overall survival (OS) in the KIPAN and KIRC. It shows that Myc plays a crucial role in the growth and maintenance of these malignancies. Additionally, we explored coexpressed genes, gene-set enrichment analysis of coexpressed genes, proteins and regulatory partners directly linked with Myc in KIPAN and KIRC and their role in cancer-specific events. Pathway enrichment analysis concluded that Myc-related genes are involved in many cancer-related pathways. Furthermore, we studied that among KIPAN, mutant forms of tumour suppressor genes have a poor prognosis and are associated with higher Myc expression but not in KIRC. This paper also investigates the correlation between Myc expression and promoter methylation, tumour-infiltrating lymphocytes, and the interaction of Myc with drugs. Our study indicates that Myc can be used as a diagnostic and prognostic biomarker in patients with KIPAN and KIRC with diverse clinical and pathological characteristics.

The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

ObjectivesChronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts.MethodsUsing qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features.ResultsSignificantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT.ConclusionIn conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

L-myc Gene Expression in Canine Fetal Fibroblasts Promotes Self-Renewal Capacity but Not Tumor Formation.

Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.

Differential Contributions of MYCs to Insect Defense Reveals Flavonoids Alleviating Growth Inhibition Caused by Wounding in Arabidopsis.

In Arabidopsis, basic helix–loop–helix transcription factors (TFs) MYC2, MYC3, and MYC4 are involved in many biological processes, such as defense against insects. We found that despite functional redundancy, MYC-related mutants displayed different resistance to cotton bollworm (Helicoverpa armigera). To screen out the most likely genes involved in defense against insects, we analyzed the correlation of gene expression with cotton bollworm resistance in wild-type (WT) and MYC-related mutants. In total, the expression of 94 genes in untreated plants and 545 genes in wounded plants were strongly correlated with insect resistance, and these genes were defined as MGAIs (MYC-related genes against insects). MYC3 had the greatest impact on the total expression of MGAIs. Gene ontology (GO) analysis revealed that besides the biosynthesis pathway of glucosinolates (GLSs), MGAIs, which are well-known defense compounds, were also enriched in flavonoid biosynthesis. Moreover, MYC3 dominantly affected the gene expression of flavonoid biosynthesis. Weighted gene co-expression network analysis (WGCNA) revealed that AAE18, which is involved in activating auxin precursor 2,4-dichlorophenoxybutyric acid (2,4-DB) and two other auxin response genes, was highly co-expressed with flavonoid biosynthesis genes. With wounding treatment, the WT plants exhibited better growth performance than chalcone synthase (CHS), which was defective in flavonoid biosynthesis. The data demonstrated dominant contributions of MYC3 to cotton bollworm resistance and imply that flavonoids might alleviate the growth inhibition caused by wounding in Arabidopsis.

Identification of Gene Expression Signatures in Leukemia Stem Cells and Minimal Residual Disease Following Treatment of Adverse Risk Acute Myeloid Leukemia

Identification of Gene Expression Signatures in Leukemia Stem Cells and Minimal Residual Disease Following Treatment of Adverse Risk Acute Myeloid Leukemia

Effects of Polymorphisms in Myc-Related Genes on Bleeding Complications in Patients with Stable Warfarin Responses.

Objectives This study aimed to identify the possible effects of Myc and 8q24 polymorphisms on bleeding complications in patients who maintained international normalized ratio (INR) of 2.0-3.0 with warfarin therapy after cardiac valve replacement. Methods Twenty-five single nucleotide polymorphisms were analyzed, including VKORC1, CYP2C9, Myc, and 8q24. Univariate and multivariate analyses were conducted to evaluate the associations between genetic polymorphisms and bleeding complications. Attributable risk and the number needed to genotype (NNG) were also calculated to evaluate the potential clinical value of genotyping. Results We included 142 patients, among whom 21 experienced bleeding complications. Multivariate models showed that patients carrying the CC genotype of rs6983561 and the A allele of rs13281615 at 8q24 had 27.6- and 10.0-fold higher bleeding complications, compared with patients with the A allele and the GG genotype, respectively. For rs6983561, the attributable risk and NNG were 96.4% and 36.8, respectively, whereas, for rs13281615, the attributable risk and NNG were 90.0% and 8.3, respectively. Atrial fibrillation was associated with a 5.5-fold increased risk of bleeding complications. The AUROC value was 0.761 (95% CI 0.659-0.863, p<0.001), and the Hosmer–Lemeshow test showed that the fitness of the multivariate analysis model was satisfactory (χ2=0.846; 3 degrees of freedom; p=0.838). Conclusions Bleeding complications during warfarin therapy were associated with 8q24 polymorphisms and atrial fibrillation in patients with mechanical heart valves.

Role of CXCL12-Expressing Mesenchymal Stromal Cell Niches in Maintaining Treatment-Resistant Leukemia Stem Cells

Chronic myeloid leukemia (CML) results from hematopoietic stem cell (HSC) transformation by the BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKI), although effective in inducing remissions in CML, fail to eradicate leukemia stem cells (LSC) which persist as source of relapse. LSC resistance to TKI-treatment occurs through kinase-independent mechanisms, which include alterations in intrinsic cell-regulatory mechanisms as well as signals from the bone marrow (BM) microenvironment that support LSC persistence. HSC have been shown to be regulated by C-X-C motif chemokine ligand 12 (CXCL12)-expressing bone marrow niches, but the nature and regulatory role of BM niches for LSC remains poorly understood.Here, we used CXCL12-GFP mice and CXCL12f/fmice crossed with Cre lines targeting specific CXCL12-expressing cells to investigate the contribution of CXCL12-expressing populations to LSC regulation.We found that targeted deletion of CXCL12 from Prx1+ mesenchymal stromal cells (MSC) reduced normal HSC numbers. In contrast, deletion of CXCL12 from Prx1+ MSC in the setting of CML, resulted in increased leukocytosis, neutrophilia, BM cellularity and LSC numbers, and reduced survival, compared to control CML mice. CXCL12 deletion from Prx1+ MSC was found to enhance LSC cycling. Despite increased cycling, the expanded LSC from these mice maintained their in vivo repopulating capacity.To evaluate the effect of CXCL12 deletion on LSC and MSC distribution, we performed 3D imaging of BM volumes from Prx1-Cre mice crossed with tdTomato reporter mice. CML development resulted in formation of large pathological tissue niches harboring an abnormally high density of MSCs as well as c-Kit+ leukemia progenitor cells. However, these MSC and leukemic progenitor clusters were not observed in Prx1-Cre+CXCL12fl/fl mice, indicating that formation of tissue structures with colocalized leukemic progenitors is dependent on CXCL12 expression in MSC.We performed gene expression analysis of LSC from Prx1-Cre+CXCL12f/f and Cre-negative mice. Gene expression analysis revealed enrichment of cell cycling and MYC related genes in CML LSC from Prx1-Cre+CXCL12f/f mice, and downregulation of Polycomb Repressive Complex 2 (PRC2) target genes, indicating increased PRC2 activity. We confirmed that EZH2 expression and H3K27 trimethylation were increased in LSC from Prx1-Cre+CXCL12f/f mice. Treatment with the EZH2 inhibitor, GSK 343, resulted in significant reduction in WBC, neutrophils, BM cellularity and LSC in Prx1-Cre+CXCL12f/f mice, but not control CML mice. These results support a role for increased PRC2 activity in LSC expansion in mice with CXCL12 deletion from Prx1+ MSC.We evaluated the effect of CXCL12 deletion from Prx1+ MSC on LSC sensitivity to treatment with the TKI Nilotinib. Treatment of Cre-negative CML mice with Nilotinib reduced WBC counts, spleen cellularity, and splenic LSC numbers, but did not reduce BM cellularity or LSC numbers. On the other hand, Nilotinib treatment significantly reduced BM cellularity and LSC numbers, and enhanced survival, in Prx1-Cre+CXCL12f/f CMLmice. Transplantation of BM from TKI-treated Prx1-Cre+CXCL12f/f mice to irradiated normal recipients resulted in significantly reduced long-term donor engraftment and donor LSC numbers compared to vehicle-treated mice. These results indicate that CXCL12 deletion from Prx1+ MSC leads to enhanced sensitivity of CML LSC to elimination by TKI treatment.In conclusion, our studies show that deletion of CXCL12 from Prx1+ MSC niches leads to loss of MSC clustering and colocalization with leukemic progenitors, and to loss of quiescence and expansion of LSC, dependent on enhanced PRC2 activity. CXCL12 deletion from MSC also increases TKI-mediated targeting of resistant, quiescent, self-renewing CML LSC. Strategies to inhibit CXCL12-mediated niche interactions represent a promising approach for LSC depletion to enhance opportunities for cures in CML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5517: The correlation of AIMP2-DX2 expression and oncogenic signaling pathways in lymphoma

Abstract Introduction: ARS-interacting multifunctional protein 2 (AIMP2) has been known to play an important role in the regulation of cell fate, and acts as a tumor-suppressive agent. Unlike normal AIMP2, AIMP2-DX2, a splicing variant of exon 2 deletion, promotes carcinogenic stress. We recently discovered that AIMP2-DX2 is an important poor prognostic factor in acute myeloid leukemia. Likewise, we conjectured AIMP2-DX2 would have a potential role in lymphoid hematologic malignancy. The aim of this study is to investigate the biologic role of AIMP2-DX2 in lymphoma. Materials and Methods: We studied the composition of genetically categorized variation in lymphoma using 38 diffuse large B cell lymphomas (DLBC) and 56 germinal center B cell lymphoma (MALY) of RNA sequencing data shared by International Cancer Genome Consortium (ICGC). The Differentially Expressed Gene (DEG) analysis method was applied to the RNA data aligned to the human reference, and the different expression pattern in each group was confirmed by nonbiased clustering. We observed whether clustering identifies certain subset of high AIMP2-DX2 level tumors. The Gene Set Enrichment Analysis (GSEA) was performed to confirm the statistical differences or similarities in the number of genes in each subgroup with adjusted p-value &amp;lt;0.05 to identify the representative pathway. Malignant lymphoma-based NAMALWA and HS-sultan cell lines were used and expression level of DX2 was quantitatively analyzed by quantitative real-time PCR. The relationship between AIMP2-DX2 and oncogenic signaling pathway components was verified with Western blotting. Results: As a result of DEG using unbiased clustering, we could successfully cluster patients into two groups in both DLBC and MALY. Interestingly, unbiased clustering of tumors resulted in a distinct cluster composed of AIMP2-DX2 high-expressing tumors. This phenomenon was observed both in DLBCL and MALY. GSEA analysis revealed that, in DLBC, the expression of JAK-STAT, MAPK, KRAS, and MYC-related genes was increased while the expression of the spliceosome-related genes was reduced as DX2 expression increased. MALY also showed high expression of JAK-STAT, MAPK and KRAS-associated genes and high expression of WNT signaling pathway in high AIMP2-DX2 samples. Using cell lines, we confirmed the expression of AKT MEK, and ERK correlates in malignant lymphoma cell line with expression of AIMP2-DX2 than in cell line with low AIMP2-DX2 expression. Conclusion: Thus, our data suggest that differences in expression of DX2 cause lymphomagenesis by affecting other oncogenic signaling pathway, such as JAK-STAT, MAPK and KRAS signaling pathways are related to AIMP2 DX2-induced lymphoid hematologic malignancy. Citation Format: Yungyeong Park, Heejoo Han, Seulki Song, Dongchan Kim, Youngil Koh, Sung-Soo Yoon. The correlation of AIMP2-DX2 expression and oncogenic signaling pathways in lymphoma [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 5517.

NOTCH1 MUTATED CHRONIC LYMPHOCYTIC LEUKEMIA CELLS ARE CHARACTERIZED BY a MYC‐RELATED OVEREXPRESSION OF NUCLEOPHOSMIN‐1 AND RIBOSOME ASSOCIATED COMPONENTS

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

Antimyeloma activity of bromodomain inhibitors on the human myeloma cell line U266 by downregulation of MYCL.

Bromodomain and extraterminal protein (BET) inhibitors suppress the expression of c-MYC. U266, a human myeloma cell line, expresses the MYCL gene, but not the c-MYC gene. Our aim was to analyse the antimyeloma activity of BET inhibitors on U266 cells. Two BET inhibitors, I-BET151 and JQ1, were tested. U266 cell proliferation decreased to 61.5 and 54.0% of the control after incubation with 500 nmol/l I-BET151 for 72 and 96 h and to 53.5 and 56.4% of control after incubation with 500 nmol/l JQ1 for 72 and 96 h by MTS tetrazolium, respectively. BET inhibitors induced cell cycle arrest at the G1 phase in U266 cells, but did not induce apoptosis by flow cytometry. According to Gene Set Enrichment Analysis, MYC-related genes were significantly downregulated in U266 cells treated with I-BET151 similar to KMS11 cells that expressed c-MYC. The MYCL1 was expressed in U266 cells, whereas c-MYC and MYCN were not by quantitative real-time reverse-transcription-PCR. Incubation with I-BET151 induced downregulation of MYCL1 in U266 cells. BET inhibitors decreased the cell proliferation in U266 cells with overexpression of MYCL less than those without overexpression of MYCL. BET inhibitors induce G1 arrest without apoptosis and interfere with the proliferation of U266 myeloma cells, which express MYCL, but not c-MYC. BET inhibitors might be active in cancers that express MYCL, but not c-MYC.

Characterization of Leukemia-Inducing Genes Using a Proto-Oncogene/Homeobox Gene Retroviral Human cDNA Library in a Mouse In Vivo Model.

The purpose of this research is to develop a method to screen a large number of potential driver mutations of acute myeloid leukemia (AML) using a retroviral cDNA library and murine bone marrow transduction-transplantation system. As a proof-of-concept, murine bone marrow (BM) cells were transduced with a retroviral cDNA library encoding well-characterized oncogenes and homeobox genes, and the virus-transduced cells were transplanted into lethally irradiated mice. The proto-oncogenes responsible for leukemia initiation were identified by PCR amplification of cDNA inserts from genomic DNA isolated from leukemic cells. In an initial screen of ten leukemic mice, the MYC proto-oncogene was detected in all the leukemic mice. Of ten leukemic mice, 3 (30%) had MYC as the only transgene, and seven mice (70%) had additional proto-oncogene inserts. We repeated the same experiment after removing MYC-related genes from the library to characterize additional leukemia-inducing gene combinations. Our second screen using the MYC-deleted proto-oncogene library confirmed MEIS1and the HOX family as cooperating oncogenes in leukemia pathogenesis. The model system we introduced in this study will be valuable in functionally screening novel combinations of genes for leukemogenic potential in vivo, and the system will help in the discovery of new targets for leukemia therapy.

Anthocyanin production by over-expression of grape transcription factor gene VlmybA2 in transgenic tobacco and Arabidopsis

An myb-related transcription factor gene of the anthocyanin biosynthetic pathway, VlmybA2, from the Kyoho grape (Vitis labruscana) was introduced into tobacco and Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. The 35S:VlmybA2-induced anthocyanin production was prominent in transformed tobacco calli, and the regenerated tobacco plants were completely purple. Except for the color, the transgenic plants were apparently not different from the control plants. During plant growth in pots, the purple color was not uniformly distributed but appeared as patches in the leaves, whereas the flowers showed intense pigmentation. In Arabidopsis, T1 transformants showing two prominent phenotypes: completely purple seedlings and seedlings with green leaves and purple roots. The partially purple seedlings grown in pots produced fertile and viable seeds of two distinguishable colors, purple and brown. VlmybA2 alone, without the aid of other myc-related genes, could induce complete pigmentation in tobacco and Arabidopsis, indicating its potential over other previously used myb- and myc-related genes.

Expression and Mutation Analysis of the Myc Antagonist Gene Mad1 in Acute Leukemia.

Objectives: Overexpression of Myc genes has been observed in many solid tumors and hematological malignancies, and Myc proteins are believed to act as transcription factors. In the past decade, several Myc-related genes have been identified, and the interaction mechanisms between these genes have been clarified. Members of Myc superfamily proteins belong to four different families: the Max family of common dimerization partners (including Max and MLx), the Myc family of proto-oncoproteins (including c-, N-, and L-Myc), the Myc antagonists including Mad family (including Mad1 and Mxi1) and Rox (Mnt). Mad1 gene maps to human chromosome 2p13 and encodes a 221-amino acid protein. Mad1 protein was shown to counteract the effects of Myc proteins and to inhibit the proliferation and tumorigenicity of glioblastoma and astrocytoma cells, and therefore was considered to be a kind of tumor suppressor. All Myc super-family members share a highly conserved structural domain, basic helix-loop-helix leucine zipper domain (bHLHZip). This bHLHzip domain is essential for these proteins to function during molecular switching from proliferation to differentiation. To function in vivo, Myc and Mad1 must heterodimerize with Max through the HLHZip motif. Then through the b (basic) motif, the heterodimers bind with CACGTG (E-box) containing DNA to activate or inactivate transcription. The present study is thus designed to examine the expression of Mad1 gene and to search for mutations in bHLHzip encoding domain.Methods: we screened bone marrow mononuclear cells (BMMNC) from 26 patients with AL and peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, using reverse transcription- polymerase chain reaction, single strand conformation polymorphism analysis and sequencing.Results: Mad1 gene was expressed in all samples. Two polymorphisms of Mad1 were found in BMMNC from AL patients and PBMNC from health volunteers. A point mutation was detected at a very important position, a substitution of A397G inducing Lys78Glu in two patients. This might have significant implication for the function of Mad1, as Mad1 will heterodimerize with Max at this very point. Mutation at this point could thus disrupt proper intermolecular alignment between Mad1 and Max. The two patients with Mad1 gene mutation had poor clinical outcomes, patient No.9 never achieved CR and died 2 months after diagnosis, while patient No.11 got CR, after two courses chemotherapy, but relapsed quickly and died 3 months after diagnosis.Conclusion: Mad1 gene expressed and displayed mutations in acute leukemia patients. It would be interesting to know whether these mutations influence either the heterodimerizing or DNA binding potentials of Mad1 protein, by performing functional analysis of the mutant protein.

New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes

The basic helix-loop-helix (bHLH) proteins represent an evolutionary conserved class of transcription factors that are known to play important roles in cell determination and differentiation during animal embryonic development. Following an exhaustive search of the complete Drosophila genome sequence using a PSI-BLAST strategy, we identified 19 new genes, bringing the total number of bHLH-encoding genes in the Drosophila genome to 56. These new genes belong to various subfamilies of bHLH transcription factors, such as the Daughterless, Hairy-Enhancer of split, bHLH-PAS or bHLHZip subfamilies. The embryonic expression pattern of each of these new genes has been analyzed by in situ hybridization. By looking for closely structurally related motifs, we found two genes that represent likely orthologues of vertebrate Mnt and Mlx. Together with previous reports, our data suggest that, similar to networks involved in neurogenesis and myogenesis, the network of Myc-related genes has been globally conserved throughout evolution.

L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer.

Altered structure and regulation of the c-myc proto-oncogene have been associated with a variety of human tumours and derivative cell lines, including Burkitt's lymphoma, promyelocytic leukaemia and small cell lung cancer (SCLC). The N-myc gene, first detected by its homology to the second exon of the c-myc gene, is amplified and/or expressed in tumours or cell lines derived from neuroblastoma, retinoblastoma and SCLC. Here we describe a third myc-related gene (L-myc) cloned from SCLC DNA with homology to a small region of both the c-myc and N-myc genes. Human genomic DNA shows an EcoRI restriction fragment length polymorphism (RFLP) of L-myc defined by two alleles (10.0- and 6.6-kilobase (kb) EcoRI fragments), neither associated disproportionately with SCLC. Mouse and hamster DNAs exhibit a 12-kb EcoRI L-myc homologue, which indicates conservation of the gene in mammals. Gene mapping studies assign L-myc to human chromosome region 1p32, a location distinct from that of either c-myc or N-myc but associated with cytogenetic abnormalities in certain human tumours. This L-myc sequence is amplified 10-20-fold in four SCLC cell line DNAs and in one SCLC tumour specimen taken directly from a patient. Either the 10.0- or 6.6-kb allele can be amplified and in heterozygotes only one of the two alleles was amplified in any SCLC genome. SCLC cell lines with amplified L-myc sequences express L-myc-derived transcripts not seen in SCLC with amplified c-myc or N-myc genes. In addition, some SCLCs without amplification also express L-myc-related transcripts. Together, these findings suggest an enlarging role for myc-related genes in human lung cancer and provide evidence for the concept of a myc family of proto-oncogenes.