Regulation Of C-myc Transcription Research Articles

Selinexor Synergizes CX4945 on Anti-Tumor Effect By Targeting XPO1/Ikros/c-Myc Signaling in T-Cell Acute Lymphoblastic Leukemia

Background Acute lymphocytic leukemia (ALL) is an aggressive hematologic malignancy with poor outcomes. The IKZF1 (Ikaros) gene plays a crucial role in normal hematopoiesis as well as acts as a leukemia suppressor in ALL. Casein kinase II (CK2) inhibitor, CX4945, has shown potential function in restoring Ikaros on transcriptional repression of target genes in ALL. Exportin-1 (XPO1) is a critical mediator of nuclear export, which is essential for regulating the cell cycle and proliferation of normal and malignant tissues. Increased expression of XPO1 has been observed in various types of cancer, including ALL. In this study, we investigated the combined effects of Selinexor and CX4945 in T-cell ALL (T-ALL) and explored the underlying mechanisms. Methods Cell Counting Kit-8(CCK-8) was used for cell viability assay, and Annexin-V/PI staining following flow cytometry analysis for apoptosis. qPCR was used for gene expression in CEM and Molt4 T-ALL cell lines. CalcuSyn analysis was performed for a synergistic effect. Results Treatment of CX4945 or Selinexor significantly led to cell proliferation arrest of CEM and Molt4 cells in a dose-dependent manner. The combination of Selinexor with CX4945 significantly enhanced the cell proliferation arrest of CEM and Molt4 cells versus the single drug controls, and CalcuSyn analysis exhibited a synergistic effect of the two drugs in the cells (Fig.1A, B). The combination also significantly increased the apoptotic cells compared to the single-drug controls (Fig.1C). c-Myc oncogene plays an essential role in human cancers. Our results showed that c-Myc mRNA level was significantly higher expressed in the cohort of T-ALL patients compared to that of normal bone marrow controls (Fig.1D). To gain insight into the underlying mechanism of the synergy, we observed the effect of the drugs on the expression of c-Myc. Results showed that the combination of Selinexor with CX4945 significantly down-regulated the expression of c-Myc compared to the single-drug controls in CEM and Molt4 cells (Fig.1E). Moreover, the combination of Selinexor with CX4945 significantly enhanced the cell growth arrest in primary cells from T-ALL patients compared to single drug controls (Fig.1F). Taken together, our data indicated the synergistic effect of Selinexor with CX4945 on cell proliferation arrest and apoptosis by down-regulation of c-Myc expression. We previously reported that Ikaros directly regulates c-Myc transcription in ALL and CX4945 suppresses c-Myc expression in a Ikaros-dependent manner. Also, c-Myc is the cargo of XPO1. Thus, our results in this study reveal a model that treatment with XPO1 inhibitor, Selinexor prevents the nuclear transport of c-Myc and leads to its nuclear accumulation and down-regulation of c-Myc expression. The combination of Selinexor and CX4945 showed the synergistic anti-tumor effect by suppressing c-Myc expression in both transcriptional and post-transcriptional levels. The mechanism model is summarized in Fig.1G. ConclusionsThe combination of Selinexor and CX4945 has synergistic effects on cell proliferation arrest and apoptosis in T-ALL by targeting the XPO1/IKROS/c-Myc signaling. Our results also provide experimental evidence for the new combination of Selinexor and CX4945 as a new potential therapeutic option for T-ALL patients.

New Dibenzoquinoxalines Inhibit Triple-Negative Breast Cancer Growth by Dual Targeting of Topoisomerase 1 and the c-MYC G-Quadruplex.

As c-MYC is one of the central players in triple-negative breast cancer (TNBC) oncogenesis, inhibiting c-MYC expression would be an effective anticancer strategy. Transcription-induced negative supercoiling is crucial in the regulation of c-MYC transcription, which facilitates the formation of a G4 structure in NHE III1 that can silence the transcription. However, topoisomerase 1 (Topo1) can dissipate this negative supercoiling, leading to continuous activation of c-MYC transcription. Thus, dual ligands targeting both Topo1 and c-MYC G4 appear to be significant in cancer therapy. In this study, a series of new dibenzoquinoxaline derivatives were designed, synthesized, and evaluated for both Topo1 and c-MYC inhibition. Among them, 5 was identified as the most promising dual ligand, which could effectively inhibit Topo1 activity and strongly stabilize c-MYC G4, thereby inhibiting cancer cell growth. Accordingly, this work suggests that this dual-targeting strategy may be effective in cancer therapy.

Non-duplex G-Quadruplex DNA Structure: A Developing Story from Predicted Sequences to DNA Structure-Dependent Epigenetics and Beyond.

The story of the non-duplex DNA form known as the G-quadruplex (G4) has traversed a winding path. From initial skepticism followed by debate to a surge in interest, the G4 story intertwines many threads. Starting with computational predictions of a gene regulatory role, which now include epigenetic functions, our group was involved in many of these advances along with many other laboratories. Following a brief background, set in the latter half of the last century when the concept of the G4 as a structure took ground, here we account the developments. This is through a lens that though focused on our groups' research presents work from many other groups that played significant roles. Together these provide a broad perspective to the G4 story. Initially we were intrigued on seeing potential G4 (pG4)-forming sequences, then known to be found primarily at the telomeres and immunoglobin switch regions, occurring throughout the genome and being particularly prevalent in promoters of bacteria. We further observed that pG4s were not only prevalent but also conserved through evolution in promoters of human, chimpanzee, mouse and rat genomes. This was between 2005 and 2007. Encouraged by these partly and partly in response to the view held by many that genome-wide presence of G4s were genomic "accidents", the focus shifted to seeking experimental evidence.In the next year, 2008, two independent findings showed promise. First, on treating human cancer cells with G4-binding ligands, we observed widespread change in gene expression. Second, our search for the missing G4-specific transcription factor, without which, importantly, G4s in promoters posed only half the story, yielded results. We determined how NM23-H2 (also known as NME2 or NDPK-B) interacts with G4s and how interaction of NM23-H2 with a G4 in the promoter of the oncogene c-myc was important for regulation of c-myc transcription. NM23-H2, and subsequently many other similar factors discovered by multiple groups, is possibly giving shape to what might be the "G4-transcriptome". Later, a close look at NM23-H2-G4 interaction in regulation of the human reverse transcriptase gene (hTERT) revealed the role of G4s in local epigenetic modifications. Meanwhile work from others showed how G4s impact histone modifications following replication. Together these show the intrinsic role of DNA sequence, through formation of DNA structure, in epigenetics.More recent work, however, was waiting to reveal aspects that tend to bring forth a completely new understanding of G4s. We observed that the telomere-repeat-binding-factor-2 (TRF2), known canonically to be telomere-associated, binds extensively outside telomeres throughout the genome. Moreover, a large fraction of the non-telomeric TRF2 sites comprise G4s. Second, the extent of non-telomeric TRF2 binding at promoters was dependent on telomere length. Thereby TRF2-induced epigenetic gene regulation was telomere-dependent. Together these implicate underlying connections that show signs of addressing an intriguing unanswered question that takes us back to the beginning: Why are G4s prevalent in two distinct regions, the telomeres and gene promoters?

Transcriptional positive cofactor 4 promotes breast cancer proliferation and metastasis through c-Myc mediated Warburg effect

BackgroundThe human positive cofactor 4 (PC4) is initially identified as a transcriptional cofactor and has an important role in embryonic development and malignant transformation. However, the clinical significance and the molecular mechanisms of PC4 in breast cancer development and progression are still unknown.MethodsWe investigated PC4 expression in 114 cases of primary breast cancer and matched normal breast tissue specimens, and studied the impact of PC4 expression as well as the molecular mechanisms of this altered expression on breast cancer growth and metastasis both in vitro and in vivo.ResultsPC4 was significantly upregulated in breast cancer and high PC4 expression was positively correlated with metastasis and poor prognosis of patients. Gene set enrichment analysis (GSEA) demonstrated that the gene sets of cell proliferation and Epithelial-Mesenchymal Transition (EMT) were positively correlated with elevated PC4 expression. Consistently, loss of PC4 markedly inhibited the growth and metastasis of breast cancer both in vitro and in vivo. Mechanistically, PC4 exerted its oncogenic functions by directly binding to c-Myc promoters and inducing Warburg effect.ConclusionsOur study reveals for the first time that PC4 promotes breast cancer progression by directly regulating c-Myc transcription to promote Warburg effect, implying a novel therapeutic target for breast cancer.

Loss of FOXP3 and TSC1 Accelerates Prostate Cancer Progression through Synergistic Transcriptional and Posttranslational Regulation of c-MYC.

Although c-MYC and mTOR are frequently activated proteins in prostate cancer, any interaction between the two is largely untested. Here, we characterize the functional cross-talk between FOXP3-c-MYC and TSC1-mTOR signaling during tumor progression. Deletion of Tsc1 in mouse embryonic fibroblasts (MEF) decreased phosphorylation of c-MYC at threonine 58 (pT58) and increased phosphorylation at serine 62 (pS62), an observation validated in prostate cancer cells. Conversely, inhibition of mTOR increased pT58 but decreased pS62. Loss of both FOXP3 and TSC1 in prostate cancer cells synergistically enhanced c-MYC expression via regulation of c-Myc transcription and protein phosphorylation. This crosstalk between FOXP3 and TSC1 appeared to be mediated by both the mTOR-4EBP1-c-MYC and FOXP3-c-MYC pathways. In mice, Tsc1 and Foxp3 double deletions in the prostate led to prostate carcinomas at an early age; this did not occur in these mice with an added c-Myc deletion. In addition, we observed synergistic antitumor effects of cotreating mice with inhibitors of mTOR and c-MYC in prostate cancer cells and in Foxp3 and Tsc1 double-mutant mice. In human prostate cancer, loss of nuclear FOXP3 is often accompanied by low expression of TSC1. Because loss of FOXP3 transcriptionally induces c-Myc expression and loss of TSC1 activates mTOR signaling, these data suggest cross-talk between FOXP3-c-MYC and TSC1-mTOR signaling that converges on c-MYC to regulate tumor progression. Coadministration of c-MYC and mTOR inhibitors may overcome the resistance to mTOR inhibition commonly observed in prostate cancer cells. SIGNIFICANCE: These results establish the principle of a synergistic action of TSC1 and FOXP3 during prostate cancer progression and provide new therapeutic targets for patients who have prostate cancer with two signaling defects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1413/F1.large.jpg.

Abstract 1474: Identifying novel chromatin binding sites of nucleolin protein in cancer cells

Abstract Nucleolin is an important regulator of cell proliferation and has been reported to play a role in c-Myc transcriptional regulation. Elevated expression levels of nucleolin have been shown in a number of malignant cells, but how nucleolin contributes to cancer development and progression remains unknown. Recently, G-quadruplexes (G4s) have been shown in vivo to form in G-rich sequences with biological importance. Nucleolin has been reported to bind to G-quadruplexes for its biological functions. In particular, G4s have been shown to form in the MYC promoter and inhibit MYC transcription; nucleolin was found to display a remarkable binding affinity to the c-Myc promoter G4 over its best-known RNA target. CX-3543, the first-in-class G-quadruplex-targeting small molecule, was shown to induce nucleolin redistribution from the cell nucleolus to the cell nucleoplasm and apoptosis in cancer cells, which was suggested to act through the c-Myc promoter G-quadruplex. Therefore, unbiased analysis of the genomic binding sites of nucleolin in human chromatin is pivotal in understanding nucleolin's biological functions. Using chromatin immunoprecipitation (ChIP) assay, we demonstrated that nucleolin binds to the c-Myc NHE III1 region in vivo. Using an anti-nucleolin antibody, the isolated DNA from the immunoprecipitated DNA-protein complexes from HeLa cells yielded numerous c-Myc promoter sequences that were determined by real-time PCR, suggesting the direct interaction between nucleolin and the c-Myc promoter G4-forming region in vivo. G4-interactive ligands have been shown to facilitate the formation of G4 structures in cells. To elucidate the association of nucleolin with G-quadruplexes in vivo, we monitored cellular localization of nucleolin before and after G4 ligand treatments and observed a rapid cellular translocation of nucleolin from the cell nucleolus to the cell nucleoplasm. Using ChIP coupled with next-generation sequencing analysis (ChIP-seq), we identified several binding sites of nucleolin in human chromatin. The determined chromatin binding sites of nucleolin will provide insights into the biological functions of nucleolin and its relationship with G-quadruplexes in cancer cells. Citation Format: Guanhui Wu, Buket Onel, Danzhou Yang. Identifying novel chromatin binding sites of nucleolin protein in cancer cells [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 1474.

Abstract 1445: Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract Colorectal Cancer (CRC) is one of the leading causes of mortality in the US, being the third most common cancer among both men and women. While an exact cause of CRC has not yet been elucidated, the existence of a clear heritable genetic component has been established. With the recent surge of data from Genome-Wide Association Studies, a handful of CRC-associated loci have been identified, each containing numerous Single Nucleotide Polymorphisms (SNPs). We hypothesize that among the ~25 loci associated with increased risk for CRC, there are driver SNPs functioning to contribute to initiation or progression of CRC development. It is crucial, in order to enhance our understanding CRC tumorigenesis, to extract these functional variants from the pool of associated SNPs and validate their causative significance within the cell. To model these functional SNPs, we have developed a novel “scarless” isolation pipeline for selecting isogenic clones with the desired single-base edit without selection markers following CRISPR-Cas9 mediated HDR. With this pipeline, we have successfully modeled our first selected SNP, rs6983267, from the 8q24 risk locus in HCT-116 cells, producing the heterozygous genotype (G/T) from the parental homozygous risk (G/G) genotype. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in CRC pathogenesis. In line with previous studies, we observe a slight decrease in c-Myc expression in our heterozygous clones compared to wildtype homozygous risk. Moreover, we observe a preferential binding of the transcription factor in control of c-Myc transcriptional regulation, TCF7L2, to the risk allele over the reference allele while binding of proximal factors such as CTCF show no allelic preference. Interestingly, we also see significant changes in expression of target genes of the WNT signaling pathway. Dysregulation of WNT is a hallmark characteristic of CRC. These changes indicate there is indeed an effect of the risk allele of rs6983267 on overall WNT activity in CRC cells due to increased binding of transcription factor TCF7L2. This novel SNP editing pipeline provides a method to study precise effects of single base changes located anywhere in the genome. Utilizing it to model other risk SNPs will enable the enhancement of our current understanding of GWAS-identified risk loci in CRC pathogenesis at the molecular level. Citation Format: Nicole Coggins, Jacob Stultz, David Segal, Luis Carvajal-Carmona. Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1445. doi:10.1158/1538-7445.AM2017-1445

Nac1 promotes self-renewal of embryonic stem cells through direct transcriptional regulation of c-Myc.

The pluripotency transcriptional network in embryonic stem cells (ESCs) is composed of distinct functional units including the core and Myc units. It is hoped that dissection of the cellular functions and interconnections of network factors will aid our understanding of ESC and cancer biology. Proteomic and genomic approaches have identified Nac1 as a member of the core pluripotency network. However, previous studies have predominantly focused on the role of Nac1 in psychomotor stimulant response and cancer pathogenesis. In this study, we report that Nac1 is a self-renewal promoting factor, but is not required for maintaining pluripotency of ESCs. Loss of function of Nac1 in ESCs results in a reduced proliferation rate and an enhanced differentiation propensity. Nac1 overexpression promotes ESC proliferation and delays ESC differentiation in the absence of leukemia inhibitory factor (LIF). Furthermore, we demonstrated that Nac1 directly binds to the c-Myc promoter and regulates c-Myc transcription. The study also revealed that the function of Nac1 in promoting ESC self-renewal appears to be partially mediated by c-Myc. These findings establish a functional link between the core and c-Myc-centered networks and provide new insights into mechanisms of stemness regulation in ESCs and cancer.

Design, Synthesis, and Evaluation of Isaindigotone Derivatives To Downregulate c-myc Transcription via Disrupting the Interaction of NM23-H2 with G-Quadruplex.

Transcriptional control of c-myc oncogene is an important strategy for antitumor drug design. G-quadruplexes in the promoter region have been proven to be the transcriptional down-regulator of this gene. The transcriptional factor NM23-H2 can reactivate c-myc transcription by unwinding the G-quadruplex structure. Thus, down-regulation of c-myc transcription via disrupting G-quadruplex-NM23-H2 interaction might be a potential approach for cancer therapy. Here, a series of new isaindigotone derivatives were designed and synthesized based on our previous study. The abilities of these derivatives on interacting with G-quadruplexes or NM23-H2, and disrupting G-quadruplex-NM23-H2 interaction were evaluated. Among these derivatives, 19d and 22d showed remarkable abilities on disrupting G-quadruplex-NM23-H2 interaction. They exhibited significant effects on c-myc-relating processes in SiHa cells, including inhibiting the transcription and translation, inhibiting cellular proliferation, inducing apoptosis, and regulating cell cycle. Our findings provided the basis for the anticancer strategy based on c-myc transcriptional regulation via small molecules disrupting G-quadruplex-protein interaction.

Abstract 406: Enhancer templated RNAs as predictors of therapeutic response to epigenetic therapy

Abstract Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we utilized a CRISPR loss of function screen and identified a number of essential genes, including the Bromodomain and Extraterminal (BET) protein, BRD4. We find BRD4 is critical for colon cancer proliferation and its loss leads to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers, characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct super-enhancer in CIMP(+) colon cancers that regulates cMYC transcription. We find that the CCAT1 long non-coding RNA (lncRNA) is transcribed from this super-enhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose CCAT1 as a clinically tractable biomarker for identifying patients likely to benefit from BET inhibitors. Citation Format: Ron Firestein, Mark McCleland, Kathryn Mesh, Florian Gnad. Enhancer templated RNAs as predictors of therapeutic response to epigenetic therapy. [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 406.

A reciprocal regulatory circuit between CD44 and FGFR2 via c-myc controls gastric cancer cell growth.

Despite their suggested importance, the mechanistic roles of FGFR2 and gastric cancer stem cell (GCSC) marker CD44 remain unclear. We investigated cross talk between CD44 and FGFR2. FGFR2 and CD44 positively regulate each other's expression. While FGFR2 suppresses c-Myc transcription, CD44 activates it. c-Myc in turn augments FGFR2 transcription. CD44 knockdown (KD) depleted FGFR2 and other GCSC markers, decreased c-Myc and Sox2 expression, and suppressed tumor growth, whereas CD44 activation led to FGFR2 induction. FGFR2 KD decreased most GCSC marker expression, including CD44, but increased c-Myc and Sox2 expression and attenuated tumor growth. FGFR2 kinase inhibitor and FGFR2 neutralizing antibody decreased the CD44+/hi GCSC fraction. Conversely, FGFR2 overexpression increased CD44 and accelerated tumor growth in mice. FGFR2 was co-expressed and colocalized diffusively with CD44, EpCAM, and LGR5. In contrast, phospho-FGFR2 colocalized densely with CD44, forming an aggregated signaling complex that was prevented by FGFR2 inhibition. The c-Myc KD depleted FGFR2 but not CD44. Similarly to CD44+/hi phenotypes, sorted FGFR+/hi cells had larger volumes, formed more tumor spheres, grew faster in vivo with bigger tumor mass, and expressed more CD44, EpCAM, and HER2. These findings suggest that FGFR2+/hi cells have stemness properties. Moreover, in situ FGFR2 expression in patient-derived gastric cancer tissue correlated with tumorigenic potential in a xenograft model. In conclusion, CD44 and FGFR2 maintain stemness in gastric cancer by differentially regulating c-Myc transcription.

Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex.

CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer.

Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.

SMARCAL1 Negatively Regulates C-Myc Transcription By Altering The Conformation Of The Promoter Region.

SMARCAL1, a member of the SWI2/SNF2 protein family, stabilizes replication forks during DNA damage. In this manuscript, we provide the first evidence that SMARCAL1 is also a transcriptional co-regulator modulating the expression of c-Myc, a transcription factor that regulates 10–15% genes in the human genome. BRG1, SMARCAL1 and RNAPII were found localized onto the c-myc promoter. When HeLa cells were serum starved, the occupancy of SMARCAL1 on the c-myc promoter increased while that of BRG1 and RNAPII decreased correlating with repression of c-myc transcription. Using Active DNA-dependent ATPase A Domain (ADAAD), the bovine homolog of SMARCAL1, we show that the protein can hydrolyze ATP using a specific region upstream of the CT element of the c-myc promoter as a DNA effector. The energy, thereby, released is harnessed to alter the conformation of the promoter DNA. We propose that SMARCAL1 negatively regulates c-myc transcription by altering the conformation of its promoter region during differentiation.

Abstract A11: Biophysical characterization of the b-HLH-LZ of deltaMax

Abstract Myc proteins (N-, L- and c-Myc) are transcriptional regulators with a broad spectrum of target genes. While essential for development, cell growth and proliferation as well as apoptosis, they play a major role in cancer onset and progression when deregulated. Myc protein functions depend on their heterodimerization with Max, another transcriptional regulator capable of forming a homodimer and antagonizing c-Myc transcriptional regulation. Recent studies have shown that c-Myc can control the alternative splicing of Max's pre-messenger RNA to produce a c-terminal truncated isoform called deltaMax. The ratio of deltaMax/WT-Max (p21) in cancer cells correlates with increased malignancy. In addition to producing a shorter gene product, the alternative splicing (exon-inclusion) generates a change in sequence in the LZ. The LZ domain is an integral and crucial part of the b-HLH-LZ motif of Max controlling its homodimerization and specific heterodimerization with c-Myc as well as binding to the E-box sequences found in gene promoters and enhancers. Whereas it has been demonstrated that deltaMax increases c-Myc transcriptional activities, the underlying mechanisms have not been elucidated yet. Here, we provide original insights into such mechanisms. First, we show that contrary to the WT motif, the b-HLH-LZ of deltaMax does not homodimerize. In addition, while the b-HLH-LZ of deltaMax can still heterodimerize with the b-HLH-LZ of c-Myc, it can no longer compete with the binding of the heterodimer to E-box sequences. Taken together, our results demonstrate that deltaMax can maximize the amount of c-Myc bound target gene promoters and enhancers and explain how deltaMax and the ratio of deltaMax/WT-Max (p21) in cancer cells can increase c-Myc oncogenic activities. Citation Format: Loïka Maltais, Cynthia Tremblay, Mikaël Bédard, Martin Montagne, Pierre Lavigne. Biophysical characterization of the b-HLH-LZ of deltaMax. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A11.

Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer

Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studies indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis.

Abstract 2607: G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies

Abstract Introduction: Many cancer-related pro-oncogenes have quadruplex forming oligonucleotide sequences in their promoter regions. These genes include, amongst others, c-Myc, Bcl-2, K-ras, or HIF-1a which are individually or together over-expressed in the vast majority of neoplasms. For instance, c-Myc, a key regulator of cell cycle and proliferation, is commonly over-expressed in both hematological and solid cancers. The c-myc promoter region contains a guanine-rich sequence (PU27), capable of forming four-stranded quadruplex DNA structures, which negatively regulates c-myc transcription. Also, Bcl-2, is commonly over-expressed in a variety of hematological malignancies and the Bcl-2 quadruplex-forming sequence is located upstream of the P1 promoter of the Bcl-2 gene; it is also implicated in negative regulation of Bcl-2 transcription. Treatment of cancer cells over-expressing c-Myc and / or Bcl-2 with oligonucleotides encoding the c-Myc (PU27) and Bcl-2 (Bcl-2-GRO) guanine-rich sequences, located in their respective promoter regions, selectively inhibits c-Myc or Bcl-2 gene expressions, cancer cells proliferation and induces cell death. Methods: Four cancer cell lines obtained from ATCC were used in these studies (Raji, HL-60, U937, A549). Cell Titer-Glo© Assay was used for the cell proliferation experiments. Cell cycle analysis was determined in the HL-60 cell line using fixed doses of PU27 and Bcl-2-GRO and time dependent FACS analysis. Internalization studies were analyzed by FACS analysis using FAM labeled PU27 and DY647 labeled Bcl-2-GRO. In vivo tumor growth inhibition studies were done in females NOD/SCID mice (6 animals per group); 5x106 cells in a Matrigel mixture were implanted in the flank of the animals; tumors developed and treatments with PU27 and Bcl-2-GRO at different dose levels alone and in combination were performed while body weights were monitored during the course of the study. Results: Treating the Raji, HL-60, U937, or A549 cancer cell lines with PU27 or Bcl-2-GRO causes inhibition of proliferation with IC50 between 200 nM and 5mM after 144 hours depending on expression of the target pro-oncogene. When treating the same cell lines using a combination of these two sequences, a significant synergy is observed, leading to more than 100 fold increase in potency. The same synergistic effect was seen when PU27 was combined with paclitaxel, leading to picomolar IC50. Internalization of these oligonucleotides was very rapid and quantitative. Localization studies show that PU27 and Bcl-2-GRO are localized in the nucleus. Cell cycle results showed a significantly increase in G0-G1 cell cycle arrest for the combination. In vivo xenograft tumor growth inhibition studies confirmed the in vitro synergistic results with 60 to 80% tumor growth inhibition while treatments were well tolerated (no body weight loss). Citation Format: Gilles H. Tapolsky, Kara Sedoris, Shelia Thomas, Francine Rezzoug, Donald M. Miller. G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies. [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 2607. doi:10.1158/1538-7445.AM2014-2607

Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

The transcription factors Runx1 and c-Myc have individually been shown to regulate important gene targets as well as to collaborate in oncogenesis. However, it is unknown whether there is a regulatory relationship between the two genes. In this study, we investigated the transcriptional regulation of endogenous c-Myc by Runx1 in the human T cell line Jurkat and murine primary hematopoietic cells. Endogenous Runx1 binds to multiple sites in the c-Myc locus upstream of the c-Myc transcriptional start site. Cells transduced with a C-terminally truncated Runx1 (Runx1.d190), which lacks important cofactor interaction sites and can block C-terminal-dependent functions of all Runx transcription factors, showed increased transcription of c-Myc. In order to monitor c-Myc expression in response to early and transiently-acting Runx1.d190, we generated a cell membrane-permeable TAT-Runx1.d190 fusion protein. Murine splenocytes treated with TAT-Runx1.d190 showed an increase in the transcription of c-Myc within 2 hours, peaking at 4 hours post-treatment and declining thereafter. This effect is dependent on the ability of Runx1.d190 to bind to DNA. The increase in c-Myc transcripts is correlated with increased c-Myc protein levels. Collectively, these data show that Runx1 directly regulates c-Myc transcription in a C-terminal- and DNA-binding-dependent manner.

Mechanistic studies for the role of cellular nucleic-acid-binding protein (CNBP) in regulation of c-myc transcription

BackgroundGuanine-rich sequence of c-myc nuclease hypersensitive element (NHE) III1 is known to fold in G-quadruplex and subsequently serves as a transcriptional silencer. Cellular nucleic-acid-binding protein (CNBP), a highly conserved zinc-finger protein with multiple biological functions, could bind to c-myc NHE III1 region, specifically to the single strand G-rich sequence. MethodsIn the present study, a variety of methods, including cloning, expression and purification of protein, EMSA, CD, FRET, Ch-IP, RNA interference, luciferase reporter assay, SPR, co-immunoprecipitation, and co-transfection, were applied to investigate the mechanism for the role of CNBP in regulating c-myc transcription. ResultsWe found that human CNBP specifically bound to the G-rich sequence of c-myc NHE III1 region both in vitro and in cellulo, and subsequently promoted the formation of G-quadruplex. CNBP could induce a transient decrease followed by an increase in c-myc transcription in vivo. The interaction of CNBP with NM23-H2 was responsible for the increase of c-myc transcription. ConclusionsBased on above experimental results, a new mechanism, involving G-quadruplex related CNBP/NM23-H2 interaction, for the regulation of c-myc transcription was proposed. General significanceThese findings indicated that the regulation of c-myc transcription through NHE III1 region might be governed by mechanisms involving complex protein–protein interactions, and suggested a new possibility of CNBP as a potential anti-cancer target based on CNBP's biological function in c-myc transcription.

Androgen Receptor Promotes Ligand-Independent Prostate Cancer Progression through c-Myc Upregulation

The androgen receptor (AR) is the principal therapeutic target in prostate cancer. For the past 70 years, androgen deprivation therapy (ADT) has been the major therapeutic focus. However, some patients do not benefit, and those tumors that do initially respond to ADT eventually progress. One recently described mechanism of such an effect is growth and survival-promoting effects of the AR that are exerted independently of the AR ligands, testosterone and dihydrotestosterone. However, specific ligand-independent AR target genes that account for this effect were not well characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer growth, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-growth effects of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer – AR and c-Myc – and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control.