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NFκBp50 and NFκBp65 induce MYB elongation via the MYB SLR polyU. (A) Top panel: The 5´ genomic structure of MYB and the CAT reporter constructs is depicted. MYB ΔSLR polyU CAT has a 76 bp deletion of the SLR sequence up to the 19 nucleotide polyU stretch. MYB SLR ΔpolyU CAT contains a deletion of the 19 nucleotide polyU stretch. Bottom left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter and 0.5 μg of pcDNA NFκBp50. Bottom right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.5 μg of pcDNA NFκBp65. (B) Left panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB ΔSLR polyU CAT or MYB SLR ΔpolyU CAT reporters and 0.25 μg of pcDNA NFκBp50-p65; Right panel: Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT or MYB Promoter CAT reporters and 0.25 μg of pcDNA NFκBp50-p65. (C) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT, MYB SLR 3L mutation polyU CAT, MYB SLR 5C mutation polyU CAT or MYB SLR 23C mutation polyU CAT reporters with 0.25 μg of pcDNA NFκBp50-p65. (D) Transactivation studies in 293 cells using 2 μg of the MYB SLR polyU CAT reporter with 0.25 μg of pcDNA NFκBp50-p65, 0.25 μg of pcDNA NFκBp50 K148A-p65 or 1 μg of pcDNA NFκBp50 K146-148A-p65. Error bars represent mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.
Source publication
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homolog...
Citations
... MYB transcription elongation arrest is relieved by ESR1 in ER + breast cancer cells 43 and by NF-κB factors in colon cancer cells through interaction with P-TEFb. 44 Pbx1, Pbx2, Meis1, and Hoxa9 bind the alternative MYB promoter located in intron 1 in mouse hematopoietic cells. 18 In our study, we found evidence of positive regulation of MYB exon 1 expression by methylation of the conventional promoter at chr6:135,502,378 (GRCh37). ...
Therapy-resistant disease is a major cause of death in patients with acute lymphoblastic leukemia (ALL). Activation of the MYB oncogene is associated with ALL and leads to uncontrolled neoplastic cell proliferation and blocked differentiation. Here, we used RNA-seq to study the clinical significance of MYB expression and MYB alternative promoter (TSS2) usage in 133 pediatric ALLs. RNA-seq revealed that all cases analyzed overexpressed MYB and demonstrated MYB TSS2 activity. qPCR analyses confirmed the expression of the alternative MYB promoter also in seven ALL cell lines. Notably, high MYB TSS2 activity was significantly associated with relapse (p = 0.007). Moreover, cases with high MYB TSS2 usage showed evidence of therapy-resistant disease with increased expression of ABC multidrug resistance transporter genes (e.g., ABCA2, ABCB5, and ABCC10) and enzymes catalyzing drug degradation (e.g., CYP1A2, CYP2C9, and CYP3A5). Elevated MYB TSS2 activity was further associated with augmented KRAS signaling (p < 0.05) and decreased methylation of the conventional MYB promoter (p < 0.01). Taken together, our results suggest that MYB alternative promoter usage is a novel potential prognostic biomarker for relapse and therapy resistance in pediatric ALL.
... NF-κB-PTEFb forms a multiprotein complex, which is the main driving force of transcription. [42][43][44] Then, we speculated that the formation of the transcription complex of NF-κB-PTEFb controls the transcriptional regulation of c-Myb. Further experiments found that the CDK9 protein level was significantly decreased by 4-IPP treatment. ...
Background:
As an inflammatory factor and oncogenic driver protein, the pleiotropic cytokine macrophage migration inhibitory factor (MIF) plays a crucial role in the osteosarcoma microenvironment. Although 4-iodo-6-phenylpyrimidine (4-IPP) can inactivate MIF biological functions, its anti-osteosarcoma effect and molecular mechanisms have not been investigated. In this study, we identified the MIF inhibitor 4-IPP as a specific double-effector drug for osteosarcoma with both anti-tumour and anti-osteoclastogenic functions.
Methods:
The anti-cancer effects of 4-IPP were evaluated by wound healing assay, cell cycle analysis, colony formation assay, CCK-8 assay, apoptosis analysis, and Transwell migration/invasion assays. Through the application of a luciferase reporter, chromatin immunoprecipitation assays, and immunofluorescence and coimmunoprecipitation analyses, the transcriptional regulation of the NF-κB/P-TEFb complex on c-Myb- and STUB1-mediated proteasome-dependent MIF protein degradation was confirmed. The effect of 4-IPP on tumour growth and metastasis was assessed using an HOS-derived tail vein metastasis model and subcutaneous and orthotopic xenograft tumour models.
Results:
In vitro, 4-IPP significantly reduced the proliferation and metastasis of osteosarcoma cells by suppressing the NF-κB pathway. 4-IPP hindered the binding between MIF and CD74 as well as p65. Moreover, 4-IPP inhibited MIF to interrupt the formation of downstream NF-κB/P-TEFb complexes, leading to the down-regulation of c-Myb transcription. Interestingly, the implementation of 4-IPP can mediate small molecule-induced MIF protein proteasomal degradation via the STUB1 E3 ligand. However, 4-IPP still interrupted MIF-mediated communication between osteosarcoma cells and osteoclasts, thus promoting osteoclastogenesis. Remarkably, 4-IPP strongly reduced HOS-derived xenograft osteosarcoma tumourigenesis and metastasis in an in vivo mouse model.
Conclusions:
Our findings demonstrate that the small molecule 4-IPP targeting the MIF protein exerts an anti-osteosarcoma effect by simultaneously inactivating the biological functions of MIF and promoting its proteasomal degradation. Direct destabilization of the MIF protein with 4-IPP may be a promising therapeutic strategy for treating osteosarcoma.
... The link between NF-κB signaling and c-Myb expression has been described for colorectal cancer, where the NF-κB proteins trigger transcription of Myb [50] . To test whether expression of Myb itself could be stimulated by NF-κB in our experimental settings, we treated 4T1 cells with the NF-κB-specific inhibitor JSH-23 [51] . ...
The transcription factor c-Myb can be involved in the activation of many genes with protumorigenic function; however, its role in breast cancer (BC) development is still under discussion. c-Myb is considered as a tumor-promoting factor in the early phases of BC, on the other hand, its expression in BC patients relates to a good prognosis. Previously, we have shown that c-Myb controls the capacity of BC cells to form spontaneous lung metastasis. Reduced seeding of BC cells to the lungs is linked to high expression of c-Myb and a decline in expression of a specific set of inflammatory genes. Here, we unraveled a c-Myb-IL1α-NF-κB signaling axis that takes place in tumor cells. We report that an overexpression of c-Myb interfered with the activity of NF-κB in several BC cell lines. We identified IL1α to be essential for this interference since it was abrogated in the IL1α-deficient cells. Overexpression of IL1α, as well as addition of recombinant IL1α protein, activated NF-κB signaling and restored expression of the inflammatory signature genes suppressed by c-Myb. The endogenous levels of c-Myb negatively correlated with IL1α on both transcriptional and protein levels across BC cell lines. We concluded that inhibition of IL1α expression by c-Myb reduces NF-κB activity and disconnects the inflammatory circuit, a potentially targetable mechanism to mimic the antimetastatic effect of c-Myb with therapeutic perspective.
... In view of the findings above showing the importance of CDK9 and its recruitment by MLL-AF9 in the regulation of MYB expression, we next investigated how and where within the MYB gene CDK9 inhibition was acting. In particular we asked whether the mechanism in MLL-AF9 leukaemia cells was the same as that we reported in breast and colon cancers, in which transcriptional elongation is blocked at the SL-dT motif in the first intron [15][16][17][18] by CDK9 inhibitors. Accordingly, we used the previously-described 17 RT-PCR primer pairs (Fig. 3a) to detect exonic and intronic Myb transcripts upstream and downstream of the SL-dT region, and primers from exon 9 to measure mature transcript levels. ...
... Having confirmed that MYB is a direct target of MLL-AF9, we examined the mechanism of MYB regulation and the roles of known, key mediators of the transcriptional regulatory activity of MLL-fusions. As mentioned in the Introduction, it is well established that in many systems MYB expression is regulated by a transcriptional elongation block within the first intron [15][16][17][18] . Furthermore, overcoming this block to allow continued transcription requires CDK9 activity and the resultant phosphorylation of the Pol II CTD at Ser2 residues 17,30 . ...
... We then asked whether MYB regulation by MLL-fusions involved overcoming an elongation block in the first intron and specifically, at the SL-dT motif required for this block in several other cell systems, as discussed above [15][16][17][18] . We did this through two approaches: examining the levels and distribution of Pol II and its key CTD-phosphorylated forms across the Myb gene in the DOX-regulated MLL-AF9 cell line, and measuring transcription across various regions along the length of the gene. ...
Acute leukaemias express high levels of MYB which are required for the initiation and maintenance of the disease. Inhibition of MYB expression or activity has been shown to suppress MLL-fusion oncoprotein-induced acute myeloid leukaemias (AML), which are among the most aggressive forms of AML, and indeed MYB transcription has been reported to be regulated by the MLL-AF9 oncoprotein. This highlights the importance of understanding the mechanism of MYB transcriptional regulation in these leukaemias. Here we have demonstrated that the MLL-AF9 fusion protein regulates MYB transcription directly at the promoter region, in part by recruiting the transcriptional regulator kinase CDK9, and CDK9 inhibition effectively suppresses MYB expression as well as cell proliferation. However, MYB regulation by MLL-AF9 does not require H3K79 methylation mediated by the methyltransferase DOT1L, which has also been shown to be a key mediator of MLL-AF9 leukemogenicity. The identification of specific, essential and druggable transcriptional regulators may enable effective targeting of MYB expression, which in turn could potentially lead to new therapeutic approaches for acute myeloid leukaemia with MLL-AF9.
... The MYB promoter, upstream of exon 1, is G-C rich and responds to a variety of stimuli [12,13]. In some tissues, a secondary regulatory mechanism involving a transcriptional pause site in the first intron is also important [14][15][16][17]. For example, estrogen receptor-regulated RNA polymerase stalling controls MYB expression in some types of breast cancer [12]. ...
Adenoid cystic carcinoma (ACC) is an aggressive salivary gland tumor that frequently displays perineural invasion and is often associated with translocations or overexpression of the MYB oncogene. Detailed analyses of MYB transcripts from ACC patient samples revealed that ACC tumors utilize an alternative MYB promoter, which is rarely used in normal cells or other tumor types. The alternative promoter transcripts produce N-terminally truncated Myb proteins lacking a highly conserved and phosphorylated domain, which includes the pS11 epitope that is frequently used to detect Myb proteins. In RNA-seq assays, Myb isoforms lacking the N-terminal domain displayed unique transcriptional activities, regulating many genes differently than full-length Myb. Thus, a regulatory pathway unique to ACC activates the alternative MYB promoter, leading to the production of a truncated Myb protein with altered transcriptional activities. This could provide new therapeutic opportunities for ACC patients.
... A literature search revealed that NFκB transcription factors regulate MYB expression by various mechanisms. [34][35][36][37] Consistent with a reported NFκB response element (NFκB-RE) in the MYB promoter situated at -278 to -256 bp upstream of the transcriptional start site, 37 tumor necrosis factor-α treatment, or p65/RELA transfection activated a MYB (-687-+204) promoter reporter 38 ( Figure 5A). Given that clofazimine downregulated PRDX1 promoter luc in HEK-293 cells ( Figure 2L) and that p65 is endogenously expressed in HEK-293 cells, 39 we investigated whether clofazimine regulates p65 expression. ...
Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.
... As one of the important transcription factors, c-Myb is frequently elevated in a number of carcinomas including acute myelogenous leukemia [9,10], salivary adenoid cystic carcinoma [11], breast cancer [12], and CRC [9]. This gene participates in a wide variety of biological life processes including cell proliferation, cell cycle progression, and apoptosis, indicating that the malignancy maintenance of CRC cells is c-Myb dependent [13][14][15]. Moreover, recent studies discovered the mediation of chronic inflammatory reaction by c-Myb in the gut [13][14][15]. ...
... This gene participates in a wide variety of biological life processes including cell proliferation, cell cycle progression, and apoptosis, indicating that the malignancy maintenance of CRC cells is c-Myb dependent [13][14][15]. Moreover, recent studies discovered the mediation of chronic inflammatory reaction by c-Myb in the gut [13][14][15]. These findings help us understand why antiinflammatory therapy is useful in the CRC treatment. ...
Background: One of our previous studies have demonstrated that the cancer suppressor miR-150 regulated the progression of colorectal cancer (CRC) by down-regulating v-myb avian myeloblastosis viral oncogene homolog (c-Myb). The purpose of present study was to evaluate the prognostic value of the expression of c-Myb and its effector, prostaglandin-endoperoxide synthase 2 (COX-2) in patients with CRC.
Methods: We used tissue microarrays (containing 202 CRC tissues and matched adjacent normal tissues) and conducted immunohistochemical analysis and western blotting analysis (containing 3 CRC tissues and matched adjacent normal tissues) to detect the expression of c-Myb and COX-2.
Results: Compared with the adjacent nontumorous tissues, both the expression levels of c-Myb and COX-2 were higher in the cancer tissues. A statistically significant correlation was found between the expression of c-Myb and COX-2. Elevated c-Myb and COX-2 were associated with more advanced tumor invasion and poorer overall survival by univariate analysis. Higher expression levels of both c-Myb and COX-2 were significantly associated with shorter overall survival for stage II and stage III patients with 5-Fu based chemotherapy. Multivariate analysis identified the lymph node involvement, distant metastatic spread and the elevated c-Myb and COX-2 as independent factors of poor prognosis for CRC.
Conclusions: In conclusion, the overexpression of both c-Myb and COX-2 would be of prognostic screening value in patients with CRC.
... Therefore, it was hypothesized in CRC, that tissue specific transcription factors and associated co-factors which include P-TEFb complex, may be recruited on this site to drive the transcription. Recent studies of Pereira et al. (53) showed that the p50 subunit of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) binds to the nascent stem-loop RNA synthesized by transcription of the 72 bp regulatory region. NF-κB p50 subunit forms a heterodimer with p65 subunit of NF-κB which is required to drive the transcription. ...
Basal transcription factors have never been considered as a priority target in the field of drug discovery. However, their unparalleled roles in decoding the genetic information in response to the appropriate signal and their association with the disease progression are very well-established phenomena. Instead of considering transcription factors as such a target, in this review, we discuss about the potential of the regulatory mechanisms that control their gene expression. Based on our recent understanding about the critical roles of c-MYB at the cellular and molecular level in several types of cancers, we discuss here how MLL-fusion protein centred SEC in leukaemia, ligand-estrogen receptor (ER) complex in breast cancer (BC) and NF-κB and associated factors in colorectal cancer regulate the transcription of this gene. We further discuss plausible strategies, specific to each cancer type, to target those bona fide activators/co-activators, which control the regulation of this gene and therefore to shed fresh light in targeting the transcriptional regulation as a novel approach to the future drug discovery in cancer.
... However, there is a CA/TA microsatellite (8443 bp from the exon 1/intron 1 junction) followed by a predicted hairpin (at 9057 bp). Both elements, microsatellites and stem-loops, have been shown to be sufficient to terminate transcription in some eukaryotic genes 39,40 . CA expansions that are not bound to the protective hnRNP L 41 lead to cleavage of pre-mRNA upstream of the expansion 42 . ...
Huntington's disease is caused by a CAG repeat expansion in exon 1 of the HTT gene. We have previously shown that exon 1 HTT does not always splice to exon 2 producing a small transcript (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The mechanisms by which this incomplete splicing occurs are unknown. Here, we have generated a minigene system that recapitulates the CAG repeat-length dependence of HTTexon1 production, and has allowed us to define the regions of intron 1 necessary for incomplete splicing. We show that manipulation of the expression levels of the splicing factor SRSF6, predicted to bind CAG repeats, modulates this aberrant splicing event and also demonstrate that RNA polymerase II transcription speed regulates the levels of HTTexon1 production. Understanding the mechanisms by which this pathogenic exon 1 HTT is generated may provide the basis for the development of strategies to prevent its production.
... Another approach aiming at suppression of the MYB protein expression proposes to target protein complexes that relieve the elongation blockade imposed by the poly-T motifs located in the first intron of MYB (see above). Deregulation of the interaction between these complexes (involving NF-kB and c-Jun) [88,89] and the intronic region of MYB might constitute an attractive therapeutic strategy [40]. As MYB activity is regulated by an interplay with various partner proteins such as CBP/ p300 co-activator, disruption of their physical associations might pose as another promising approach to reduce MYB activity in cancer. ...
Adenoid cystic carcinoma (ACC), the second most common salivary gland malignancy, is notorious for poor prognosis, which reflects the propensity of ACC to progress to clinically advanced metastatic disease. Due to high long-term mortality and lack of effective systemic treatment, the slow-growing but aggressive ACC poses a particular challenge in head and neck oncology. Despite the advancements in cancer genomics, up until recently relatively few genetic alterations critical to the ACC development have been recognized. Although the specific chromosomal translocations resulting in MYB-NFIB fusions provide insight into the ACC pathogenesis and represent attractive diagnostic and therapeutic targets, their clinical significance is unclear, and a substantial subset of ACCs do not harbor the MYB-NFIB translocation. Strategies based on detection of newly described genetic events (such as MYB activating super-enhancer translocations and alterations affecting another member of MYB transcription factor family-MYBL1) offer new hope for improved risk assessment, therapeutic intervention and tumor surveillance. However, the impact of these approaches is still limited by an incomplete understanding of the ACC biology, and the manner by which these alterations initiate and drive ACC remains to be delineated. This manuscript summarizes the current status of gene fusions and other driver genetic alterations in ACC pathogenesis and discusses new therapeutic strategies stemming from the current research.
