C-myb Expression Research Articles

Bisphenol-A alters hematopoiesis through EGFR/ERK signaling to induce myeloblastic condition in zebrafish model

Experimental evidence from the etiology of cancer studies suggests that a correlation between Bisphenol-A (BPA) exposure and alterations in hematopoiesis leads to blood cancer. In our study zebrafish were used to assess the lethality, developmental effect, embryonic apoptosis and changes in transcription factor of hematopoiesis through EGFR/ERK signaling pathways in response to BPA. The in silico interaction of EGFR and BPA was analysed by molecular dynamic simulation. According to our results, BPA induced a significant lethal effect in hatching retardation, reduction in heart rate and teratogenic effects on zebrafish embryos and larvae at three different concentrations 100, 500 and 2500 μg/L. The mortality of adult zebrafish exposed to the acute toxicity of BPA from 5 to 30 mg/L concentrations was determined for 96 h. The peripheral blood cells and vital organs such as kidney, liver and spleen from BPA exposed fish showed predominantly abnormal myeloid blast cells along with severe morphological changes in erythrocytes at sublethal concentration 245 μg/L. The BPA showed the highest binding affinity to zebrafish EGFR with a docking score of −7.5 kcal/mol with an RMSD of 3.0 nm during MD simulation. We found that EGFR/ERK overexpression leads to induce hematopoietic cell proliferation and impaired differentiation, which enhances the myeloid repopulating activity and the accumulation of immature myeloblast cells. BPA also caused a corresponding increase in expression of hematopoietic transcription factor c-MYB and RUNX-1 leading to polychromasia, poikilocytosis, acanthocytes and anisocytosis and promoted myeloblastosis by inhibiting GATA-1 expression. These morphological changes often resulted in the prior condition of acute myeloid leukemia (AML). Comprehensively, our data suggest that BPA can trigger the malignancy of AML cells by alteration of respective hematopoietic transcription factors via EGFR/ERK signaling in the zebrafish model.

C-Myb interferes with inflammatory IL1α-NF-κB pathway in breast cancer cells

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.

Brucine inhibits proliferation of glioblastoma cells by targeting the G-quadruplexes in the c-Myb promoter.

The proto-oncogene c-Myb plays an important role in cell proliferation, and its upregulation affects the development of glioblastomas. G-quadruplexes are secondary DNA or RNA structures that usually form in the promoter region of oncogenes, including c-Myb, and regulate the expression of these genes. The traditional Chinese medicine, brucine, is a ligand of the G-quadruplexes located in the promoter region of c-Myb. The present study investigated the therapeutic effects and mechanism of action of brucine in U87, LN18, and LN229 cells in vitro and in vivo. Our results showed that brucine suppressed the growth of these cells in vitro by arresting the cell cycle and reducing c-Myb expression. Dual-luciferase reporter assays showed that brucine inhibited c-Myb expression by targeting the guanine-rich sequence that forms G-quadruplexes in the c-Myb promoter. Moreover, U87 tumors were suppressed by brucine in a tumor xenograft nude mouse model. Therefore, brucine is potentially effective for treating glioblastomas.

Resveratrol Activates Natural Killer Cells through Akt- and mTORC2-Mediated c-Myb Upregulation.

Natural killer (NK) cells are suitable targets for cancer immunotherapy owing to their potent cytotoxic activity. To maximize the therapeutic efficacy of cancer immunotherapy, adjuvants need to be identified. Resveratrol is a well-studied polyphenol with various potential health benefits, including antitumor effects. We previously found that resveratrol is an NK cell booster, suggesting that it can serve as an adjuvant for cancer immunotherapy. However, the molecular mechanism underlying the activation of NK cells by resveratrol remains unclear. The present study aimed to determine this mechanism. To this end, we investigated relevant pathways in NK cells using Western blot, real-time polymerase chain reaction, pathway inhibitor, protein/DNA array, and cytotoxicity analyses. We confirmed the synergistic effects of resveratrol and interleukin (IL)-2 on enhancing the cytolytic activity of NK cells. Resveratrol activated Akt by regulating Mammalian Target of Rapamycin (mTOR) Complex 2 (mTORC2) via phosphatase and tensin homolog (PTEN) and ribosomal protein S6 kinase beta-1 (S6K1). Moreover, resveratrol-mediated NK cell activation was more dependent on the mTOR pathway than the Akt pathway. Importantly, resveratrol increased the expression of c-Myb, a downstream transcription factor of Akt and mTORC2. Moreover, c-Myb was essential for resveratrol-induced NK cell activation in combination with IL-2. Our results demonstrate that resveratrol activates NK cells through Akt- and mTORC2-mediated c-Myb upregulation.

Determination of the binding of natural products to the human c-myb oncogene promoter G-quadruplex DNA by capillary electrophoresis and electrospray ionization mass spectrometry

The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Recent studies have found that the human oncogene c-myb is overexpressed in cancer tissues such as colorectal cancer. C-myb has become a potential therapeutic target for colorectal cancer, leukemia, and other cancers. A guanine (G)-rich DNA sequence located in the promoter region of c-myb can be spontaneously folded to form an intra-molecular G-quadruplex (G4) with cationic induction. The specific recognition of small molecules can stabilize this G4 folding, thus regulating the transcription and expression of c-myb. In this study, pressure assisted capillary electrophoresis frontier analysis (PACE-FA) combined with electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interactions between the human c-myb promoter G4 and natural product molecules. In PACE-FA, an external pressure (no more than 13.8 kPa) in the same direction of the migration of the analyte was used in capillary electrophoresis frontier analysis (CE-FA), which greatly sped up the analysis while maintaining the accuracy of the results. Meanwhile, the combination of PACE-FA and ESI-MS could rapidly determine the affinity and stoichiometric relationship between binding molecules and targets. First, the intramolecular parallel-stranded G4 formation of the c-myb promoter sequence in the presence of cations (K+, NH4+) was investigated by circular dichroism (CD) and ESI-MS. Then, ESI-MS was used to rapidly screen the natural products for candidate molecules that bound the target G4 DNA. The binding interactions were measured by mixing the c-myb DNA with each natural product separately in a 1:4 molar ratio, and then directly infusing these mixtures into the ESI-MS system. From the ESI-MS spectra, IRa values calculated from the relative intensities of DNA and its complex ions were used to probe the binding affinities of the natural products. This parameter denotes the relative binding affinity for a small molecule with the G4 DNA. Three natural products were identified through the screen, and their binding affinities to G4 DNA were ranked as follows:pseudolaric acid B > scopolamine butylbromide > nuciferine. Considering that both specific and non-specific binding existed in the solution phase, a free solution method using PACE-FA was developed to further test the binding ability of these products to the c-myb promoter G4 DNA. In the PACE-FA experiments, the pre-equilibrated mixture of the c-myb G4 DNA and the selected ligand was injected into the capillary prior to separation. Upon applying voltage and an external pressure (6.9 kPa) to the capillary, different species of analytes in the sample migrated at their own rates due to their different sizes and charges. The results showed that scopolamine butylbromide could bind specifically to target G4 DNA with 1:1 stoichiometry and a binding constant of 1.18×105 L/mol. Nuciferine's binding to G4 DNA showed a linear increasing trend due to nonspecific binding; thus nuciferine is a nonspecific binder. Although pseudolaric acid B showed high affinity for the c-myb G4 DNA and 1:1 and 1:2 G4-bound complex ions were observed in ESI-MS measurements, the PACE-FA results indicated that pseudolaric acid B did not bind to target G4 DNA in free solution. Therefore, scopolamine butylbromide could be the best candidate to regulate the transcription of the c-myb oncogene, and is expected to be a precursor for anticancer drugs. In this work, PACE-FA has allowed for significant improvements to the conventional CE-FA technique. This combination of PACE-FA and ESI-MS not only reduced the time needed for binding analysis, but also improved the accuracy and specificity of the affinity analysis compared to conventional binding approaches. Furthermore, this combination could be used to screen other targeted drug candidates and to evaluate their interaction mechanisms.

Lipopolysaccharide Reverses Hepatic Stellate Cell Activation Through Modulation of cMyb, Small Mothers Against Decapentaplegic, and CCAAT/Enhancer-Binding Protein C/EBP Transcription Factors.

During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFβR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs. We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl4 administration to rats every 3days for 3 or 8weeks, then challenged with LPS (5mg/kg; IP). HSCs were isolated 24hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFβR, transforming growth factor beta receptor 1 (TGFβR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFβR, αSMA, TGFβR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ. In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.

Substitution of Thr572 to Ala in mouse c-Myb attenuates progression of early erythroid differentiation

The expression level of transcription factor c-Myb oscillates during hematopoiesis. Fbw7 promotes ubiquitin-mediated degradation of c-Myb, which is dependent on phosphorylation of Thr572. To investigate the physiological relevance of Fbw7-mediated c-Myb degradation, we generated mutant mice carrying c-Myb-T572A (TA). Homozygous mutant (TA/TA) mice exhibited a reduction in the number of peripheral red blood cells and diminished erythroblasts in bone marrow, presumably as a result of failure during erythroblast differentiation. We found that c-Myb high-expressing cells converged in the Lin−CD71+ fraction, and the expression of c-Myb was higher in TA/TA mice than in wild-type mice. Moreover, TA/TA mice had an increased proportion of the CD71+ subset in Lin− cells. The c-Myb level in the Lin−CD71+ subset showed three peaks, and the individual c-Myb level was positively correlated with that of c-Kit, a marker of undifferentiated cells. Ultimately, the proportion of c-Mybhi subgroup was significantly increased in TA/TA mice compared with wild-type mice. These results indicate that a delay in reduction of c-Myb protein during an early stage of erythroid differentiation creates its obstacle in TA/TA mice. In this study, we showed the T572-dependent downregulation of c-Myb protein is required for proper differentiation in early-stage erythroblasts, suggesting the in vivo significance of Fbw7-mediated c-Myb degradation.

MiR-150 regulates endothelial progenitor cell differentiation via Akt and promotes thrombus resolution

BackgroundDeep venous thrombosis (DVT) constitutes a major global disease burden. Endothelial progenitor cells (EPCs) have been described in association with recanalization of venous thrombus. Furthermore, emerging evidence suggests microRNAs are involved in this progression. The goal of this study was to investigate the influence of miR-150 on the behavior of EPCs and its potential contribution in venous thrombosis resolution.MethodsWe isolated and cultured EPCs from healthy adults. Next, early EPCs or endothelial colony-forming cells (ECFCs or late EPCs) were transfected with miR-150 agomir and antagomir. Gene expression profiles, proliferation, cytokine secretion, and angiogenic capacity of early EPCs and ECFCs were examined. The effects of miR-150 on c-Myb expression and Akt/FOXO1 signaling were also evaluated. Furthermore, a rat model of venous thrombosis was constructed to determine the in vivo function of EPCs.ResultsOur results showed that miR-150 overexpression in early EPCs significantly promoted differentiation to ECFCs and contributed to proliferation and tube formation. However, suppression of miR-150 in late EPCs inhibited proliferation and tube formation. Moreover, we identified that this progression is regulated by inhibition of c-Myb and activation of the Akt/FOXO1 pathway. Our findings also showed that miR-150 led to the enhanced resolution ability of EPCs in a rat venous thrombosis model.ConclusionsIn this study, we present a novel mechanism of miRNA-mediated regulation of EPCs and Akt activation in thrombus resolution.

3115 – GATA2A DETERMINES THE SURVIVAL AND HOMEOSTATIC LINEAGE OUTPUT OF HAEMATOPOIETIC STEM AND PROGENITOR CELLS

The transcription factor Gata2 is key regulator of haematopoietic stem and progenitor cells (HSPCs) during embryonic development of vertebrates. We took advantage of a partial genome duplication in zebrafish that lead to the appearance of two Gata2 genes, Gata2a and Gata2b, to address how Gata2 programmes the haemogenic endothelium (HE) towards the HSPC cell fate. Through deletion of a conserved intronic enhancer (i4 enhancer) in the gata2a locus, we show that reducing gata2a levels in the endothelium leads to decreased expression of gata2b and runx1, thus demonstrating that Gata2a activity is required for HE programming in embryos. By contrast, loss of gata2b impaired cmyb expression in the HSPC population but did not affect HE programming, indicating that Gata2a programmes HE first and Gata2b modulates HSPC programming later. Single cell transcriptional profiling of the adult kidney marrow revealed that gata2a and gata2b are expressed in HSPCs, but in different subsets. Strikingly, homozygous Gata2a mutants are viable and fertile, but show hypocellularity in the marrow niche and neutropenia, resembling GATA2 deficiency syndrome in humans. Gata2a enhancer mutants show fewer HSPCs and a lineage skewing towards the erythroid and B cell fates at the expense of the neutrophilic and monocytic lineages. These differences are already detectable in HSPCs. In addition, myeloid lineages in gata2a enhancer mutants ectopically express B- and erythroid lineage markers and fail to express lineage-specific genes at wild type levels, suggesting that Gata2a activity in HSPCs initiates and maintains lineage commitment in haematopoietic cells.

Regulation of MYB mediated cisplatin resistance of ovarian cancer cells involves miR-21-wnt signaling axis

c-MYB has been reported to be elevated in few cancers, including in ovarian cancer. It influences resistance to cisplatin but the details are not very well understood. The objective of this study was to further evaluate role of c-MYB in ovarian cancer’s cisplatin resistance. To elucidate the underlying mechanism of cisplatin resistance in ovarian cancer, we focused on the epigenetic regulation by miRNAs. Two cell lines, ES2 and OVCAR3, were used as the model systems. C-MYB expression was either up-regulated or silenced and the resulting effect on cisplatin resistance evaluated, along with the mechanistic role of miR-21, through transfections with pre/anti-miRNAs. An in vivo cisplatin resistance model was also employed to verify findings. High c-MYB correlated with increased miR-21. High c-MYB also resulted in induction of EMT and increased resistance against cisplatin which was attenuated by anti-miR-200s. c-MYB decreased β-catenin phosphorylation and thus activated wnt signaling. Silencing of c-MYB resulted in reduced miR-21 levels, reduced EMT, reduced cisplatin IC-50s and increased β-catenin phosphorylation. In an in vivo mice model of cisplatin resistance, c-MYB overexpressing ES2 xenografts were more aggressive than their control counterparts. These c-MYB overexpressing ES xenografts were significantly more resistant to cisplatin but could be sensitized to cisplatin by anti-miR-21. Our results provide a novel mechanism of cisplatin resistance by c-MYB which involves an essential role of miR-21.

RETRACTED: Exosome miR-155 Derived from Gastric Carcinoma Promotes Angiogenesis by Targeting the c-MYB/VEGF Axis of Endothelial Cells

Exosomes, membranous nanovesicles, naturally carry proteins, mRNAs, and microRNAs (miRNAs) and play important roles in tumor pathogenesis. Here we showed that gastric cancer (GC) cell-derived exosomes can function as vehicles to deliver miR-155 to promote angiogenesis in GC. In this study, we first detected that the expression of miR-155 and c-MYB was negatively correlated in GC and that c-MYB was a direct target of miR-155. We next characterized the promotional effect of exosome-delivered miR-155 on angiogenesis and tumor growth in GC. We found that miR-155 could inhibit c-MYB but increase vascular endothelial growth factor (VEGF) expression and promote growth, metastasis, and tube formation of vascular cells, causing the occurrence and development of tumors. We also used a tumor implantation mouse model to show that exosomes containing miR-155 significantly augment the growth rate of the vasculature and tumors in vivo. Our results illustrate the potential mechanism between miR-155 and angiogenesis in GC. These findings contribute to our understanding of the function of miR-155 and exosomes for GC therapy.

Prognostic value of connective tissue growth factor and c-Myb expression in IgA nephropathy and Henoch-Schönlein purpura—A pilot immunohistochemical study

AimAdverse and advanced prognostic signs in IgA nephropathy (IgAN) are interstitial fibrosis and tubular atrophy, but early predictors of bad outcome are still lacking. We investigated expression of connective tissue growth factor (CTGF) and c-Myb in renal biopsies of IgAN and Henoch-Schönlein purpura (HSP), because these gene products are indirectly included in fibrosis and epithelial-mesenchymal transition (EMT). MethodsThe sample included 23 patients and 8 controls who underwent nephrectomy due to renal cancer. The slides cut from the paraffin blocks were prepared for standard indirect immunoflourescence, using antibodies to CTGF and c-Myb. Ten high-power non-overlapping fields were photographed on Olympus IX51 microscope. Average percent of positive tubular cells, as well as number of positive cells per glomerulus were calculated. ResultsThe cytoplasmic tubular CTGF expression was higher in IgAN/HSP than in controls (P < 0.001), whereas no difference was found in glomeruli (P = 0.437). The nuclear c-Myb expresssion in glomeruli and tubules was higher in IgAN/HSP than in controls (P < 0.05). In the follow-up, decline in renal function correlated with glomerular and tubular c-Myb, as well as tubular CTGF expression (all P < 0.05). Conclusion: Our results proposed c-Myb and CTGF as novel, early and sensitive markers of chronic kidney disease and worse renal outcome, but larger series are needed.

TCF-1-Centered Transcriptional Network Drives an Effector versus Exhausted CD8 T Cell-Fate Decision

TCF-1 is a key transcription factor in progenitor exhausted CD8 Tcells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1+Ly108+PD-1+ CD8 Tcell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1Hi effectors while fostering KLRG1Lo Tex precursor cells, and PD-1 stabilized this TCF-1+ Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcation-driving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.

Protective Effect and Mechanisms of New Gelatin on Chemotherapy-Induced Hematopoietic Injury Zebrafish Model.

The aim of the study is to explore the protective effect of new gelatin (NG, Xin'ejiao in China) on hematopoietic injury caused by chemotherapy. Zebrafish, at 48 hours post fertilization (hpf), was treated with different chemotherapeutic drugs to establish the zebrafish hematopoietic damage model with reduced thrombocytes and erythrocytes. The protecting effects of NG on the thrombocytes and erythrocytes were observed, respectively, on zebrafish models. Then, the RT-PCR method was used to detect the change of mRNA level of the hematopoiesis-related cytokines scl1, c-myb, pu.1, GATA1, and runx1 genes. The results showed that 50 μg·mL−1 and 100 μg·mL−1 NG rescued and increased the thrombocytes numbers induced by vinorelbine (NVB) and chloramphenicol (CHL) and the erythrocytes numbers induced by methotrexate (MTX), doxorubicin (ADM), and mechlorethamine hydrochloride (MH) in zebrafish models. Meanwhile, the mRNA expression of scl1, c-myb, and GATA1 genes in the NG treatment group was raised compared with the MTX treatment group. Also, the mRNA expression of pu.1 and Runx1 in the NG treatment group was reduced compared with the MTX treatment group. In consequence, traditional Chinese medicine NG showed a certain degree protective effect on hematopoiesis injury induced by chemotherapy in this study, which may depend on the promotion of erythrocytes proliferation and the regulation of the hematopoietic genes level.

C-Myb promotes growth and metastasis of colorectal cancer through c-fos-induced epithelial-mesenchymal transition.

c‐Myb is a crucial transcription factor that participates in various biological functions; however, its role in colorectal cancer (CRC) remains poorly investigated. We first analyzed the expression and clinical significance of c‐Myb in a retrospective cohort enrolling 132 CRC patients. Then, the CRISPR/Cas9 technique was used to establish c‐Myb gene KO CRC cell lines. Cellular functional assays in vitro and in vivo were used to evaluate the impact of c‐Myb KO in CRC cells. Finally, RNA sequencing was used to investigate the potential oncogenic mechanisms regulated by c‐Myb in CRC progression and related cellular validations were accordingly carried out. As a result, c‐Myb is significantly overexpressed in CRC tissues as compared with adjacent normal tissues. High expression of c‐Myb is positively correlated with lymph node metastasis and poor prognosis. Univariate analysis and multivariate analysis further identify c‐Myb as an independent unfavorable prognostic factor for CRC patients. c‐Myb KO inhibits the proliferation, apoptosis resistance, invasion, metastasis, colony formation and in vivo tumorigenesis of CRC cells. Also, the mechanism investigation indicates that c‐Myb may promote CRC progression by regulating c‐fos. c‐fos overexpression can rescue the inhibitory effect of c‐Myb KO on the malignant characteristics of CRC cells. Finally, we find that c‐Myb KO inhibits the epithelial‐mesenchymal transition (EMT) molecular phenotype in CRC cells, whereas c‐fos overexpression can rescue this inhibitory effect. This study suggests that c‐Myb promotes the malignant progression of CRC through c‐fos‐induced EMT and has the potential to be a promising prognostic biomarker and therapeutic target.

Low infiltration of tumor-associated macrophages in high c-Myb-expressing breast tumors

Tumor-associated macrophages (TAMs) are prominent components of tumor stroma that promotes tumorigenesis. Many soluble factors participate in the deleterious cross-talk between TAMs and transformed cells; however mechanisms how tumors orchestrate their production remain relatively unexplored. c-Myb is a transcription factor recently described as a negative regulator of a specific immune signature involved in breast cancer (BC) metastasis. Here we studied whether c-Myb expression is associated with an increased presence of TAMs in human breast tumors. Tumors with high frequency of c-Myb-positive cells have lower density of CD68-positive macrophages. The negative association is reflected by inverse correlation between MYB and CD68/CD163 markers at the mRNA levels in evaluated cohorts of BC patients from public databases, which was found also within the molecular subtypes. In addition, we identified potential MYB-regulated TAMs recruiting factors that in combination with MYB and CD163 provided a valuable clinical multigene predictor for BC relapse. We propose that identified transcription program running in tumor cells with high MYB expression and preventing macrophage accumulation may open new venues towards TAMs targeting and BC therapy.

LncRNA SNHG10 Facilitates Hepatocarcinogenesis and Metastasis by Modulating Its Homolog SCARNA13 via a Positive Feedback Loop

Understanding the roles of noncoding RNAs (ncRNA) in tumorigenesis and metastasis would establish novel avenues to identify diagnostic and therapeutic targets. Here, we aimed to identify hepatocellular carcinoma (HCC)-specific ncRNA and to investigate their roles in hepatocarcinogenesis and metastasis. RNA-seq of xenografts generated by lung metastasis identified long noncoding RNA small nucleolar RNA host gene 10 (SNHG10) and its homolog SCARNA13 as novel drivers for the development and metastasis of HCC. SNHG10 expression positively correlated with SCARNA13 expression in 64 HCC cases, and high expression of SNHG10 or SCARNA13 was associated with poor overall survival. As SCARNA13 showed significant rise and decline after overexpression and knockdown of SNHG10, respectively, we hypothesized that SNHG10 might act as an upstream regulator of SCARNA13. SNHG10 and SCARNA13 coordinately contributed to the malignant phenotype of HCC cells, where SNHG10 served as a sponge for miR-150-5p and interacted with RPL4 mRNA to increase the expression and activity of c-Myb. Reciprocally, upregulated and hyperactivated c-Myb enhanced SNHG10 and SCARNA13 expression by regulating SNHG10 promoter activity, forming a positive feedback loop and continuously stimulating SCARNA13 expression. SCARNA13 mediated SNHG10-driven HCC cell proliferation, invasion, and migration and facilitated the cell cycle and epithelial-mesenchymal transition of HCC cells by regulating SOX9. Overall, we identified a complex circuitry underlying the concomitant upregulation of SNHG10 and its homolog SCARNA13 in HCC in the process of hepatocarcinogenesis and metastasis. SIGNIFICANCE: These findings unveil the role of a noncoding RNA in carcinogenesis and metastasis of hepatocellular carcinoma.

Abstract 3836: Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML

Abstract Standard AML chemotherapy and JAK inhibitor (ruxolitinib) treatments are ineffective against post-myeloproliferative neoplasm (MPN) sAML. Mutations in JAK2, MPL and calreticulin, and co-epimutations, result in a dysregulated transcriptome, which is responsible for transformation of MPN to sAML and for its therapy-refractoriness. CDK9 is the catalytic subunit of pTEFb that phosphorylates serine 2 in the heptad-repeats of the C-terminal domain of RNA pol II (RNAP2). CDK9 also phosphorylates and inactivates negative transcript elongation factors (NELF and SPT5), which induces promoter-proximal pause-release of RNAP2 to enable productive mRNA transcript elongation. The bromodomain extra-terminal (BET) protein BRD4 recruits pTEFb to the chromatin to promote RNAP2-mediated elongation of transcripts, including those of c-Myc, Bcl-xL, PIM1 and MCL1, important for cell growth and survival of post-MPN sAML cells. Here, we determined the effects of inhibiting BRD4-CDK9-RNAP2 axis in post-MPN sAML cells. Treatment with the CDK9 inhibitor (CDK9i) NVP2 or BAY1143572 (B) dose-dependently induced apoptosis of ruxolitinib-sensitive (Rux-S) sAML cultured cells lines HEL92.1.7 (HEL) and SET2, as well as of patient-derived sAML blast progenitor cells (BPCs), while sparing normal CD34+ progenitor cells (HPCs). CDK9i-induced lethality in sAML cells was associated with attenuation of protein levels of S2-phosphorylated RNAP2, as well as depletion of mRNA and protein levels of c-Myc, PIM1, MCL1, Bcl-xL and XIAP. ATAC-Seq analyses demonstrated that treatment with NVP2 or B caused marked alterations in chromatin accessibility, including large peak losses on the chromatin with binding sites of TFs such as ERG, PU.1, STAT5, c-Myc, and RELA. This was associated with attenuation of mRNA expression of cMyb, c-Myc, RUNX1, LMO2, RELB, NFKB2, c-FLIP, MCL1, CDK6 and Bcl-xL. Following engraftment of luciferase transduced HEL cells into NSG mice, daily treatment with B at 10 mg/kg for 3 wks significantly reduced sAML burden and improved survival of the NSG mice (p &amp;lt; 0.002). Co-treatment of NVP2 or B with ruxolitinib or BCL2/Bcl-xL inhibitor ABT263 synergistically induced apoptosis of ruxolitinib-sensitive sAML cells (CI values of &amp;lt; 1.0). Treatment with NVP2 or B, or with BETi OTX015, also induced apoptosis of ruxolitinib persister/resistant (Rux-P) sAML cells (which demonstrate &amp;gt; 10-fold higher ruxolitinib IC50 values than Rux-S cells). This was associated with attenuated protein levels of p-RNAP2, c-Myc, PIM1, Bcl-xL and XIAP. Co-treatment with OTX015 and NVP2 or B synergistically induced apoptosis of not only HEL and SET2 but also HEL-RuxP and SET-RuxP cells, as well as of patient-derived sAML BPCs (CI values &amp;lt; 1.0). These findings confirm that targeted inhibition of BRD4-CDK9-RNAP2 leads to high level of pre-clinical efficacy against ruxolitinib-sensitive and ruxolitinib-refractory sAML cells. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Joseph D. Khoury, Srdan Verstovsek, Kapil N. Bhalla. Efficacy of CDK9 inhibitor-based combinations as therapy for post-myeloproliferative neoplasm (MPN) secondary (s) AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3836.

Role and mechanism of microRNA-548c-3p/c-Myb in myocardial infarction fibrosis in rats.

To investigate the role of micro ribonucleic acid (miR)-548c-3p in myocardial fibrosis after myocardial infarction (MI), and to explore the possible underlying. The rat model of MI was successfully established in-vivo. MiR-548c-3p was upregulated via lentivirus transfection with miR-548c-3p mimics. Cardiac function of rats was detected via echocardiography. Meanwhile, Sirius red and Masson staining were used to detect the level of fibrosis index in MI model. Subsequently, myocardial fibroblasts were isolated and cultured in vitro. An oxygen-glucose deprivation (OGD) model was established to mimicking the ischemic condition. Furthermore, the relationship between miR-548c-3p and c-Myb was verified, and the levels of fibrosis-related factors (including α-SMA and COL1A1) were measured. In-vivo experiments showed that miR-548c-3p expression in rats was significantly down-regulated at 2 and 4 weeks after MI. Up-regulation of miR-548c-3p significantly improved cardiac function, reduced myocardial fibrosis and inhibited the protein expression of proto-oncogene c-Myb (c-Myb). In vitro experiments revealed that c-Myb was a target gene of miR-548c-3p. In addition, miR-548c-3p could inhibit the expressions of α-SMA and COL1A1 through targeting c-Myb. MiR-548c-3p could improve myocardial fibrosis by targeting c-Myb.

A phase I trial of palbociclib in combination with dexamethasone in relapsed or refractory adult B-cell acute lymphoblastic leukemia (ALL).

TPS7065 Background: c-Myb is a DNA-binding transcription factor that is highly expressed in immature hematopoietic cells. c-Myb and its products are essential in regulating normal hematopoiesis and influencing leukemogenesis. Knockdown of c-Myb causes cell cycle arrest and apoptosis in pre-B-ALL cells. The effects of c-Myb depend on transcriptional regulation of CDK6 and Bcl-2. c-Myb-silenced Ph+ ALL cells exhibit Rb-dependent cell cycle arrest and apoptosis, both of which are rescued by ectopic expression of cyclin D3, CDK6, and Bcl-2 expression. Preclinical studies suggest that the cytotoxic activity of dexamethasone in ALL cells may be due to decreased c-Myb expression and reduced Bcl-2 levels. Thus, the novel combination of palbociclib, a small molecule CDK4/6 inhibitor, and dexamethasone is a logical approach for the treatment of B-cell ALL. Methods: This is a single arm, phase I, dose escalation study with a traditional 3+3 design. Adult patients with relapsed or refractory B-cell ALL are eligible. Patients with Ph+ ALL must be refractory to or intolerant of standard tyrosine kinase inhibitor therapy. Patients receive a 1-week lead-in of palbociclib alone followed by induction with 4 weeks of palbociclib and dexamethasone. If an adequate response is seen, patients move to maintenance therapy, which consists of 1 week of palbociclib plus dexamethasone followed by 3 weeks of palbociclib alone. Treatment continues until disease progression, dose limiting toxicity, or availability of an alternative therapy. The primary endpoints are dose limiting toxicity and maximum tolerated dose of palbociclib and dexamethasone. Correlative studies, which are performed on pretreatment, day +1 and day +8 samples, include RB phosphorylation and FOXM1 expression as measures of palbociclib activity; CD19+ cell gene expression profiling of (1) p21 expression as an indicator of cell cycle activity, (2) S-Phase, Annexin V/Caspase 3 activation as indicators of proliferation and apoptosis and (3) Myb and Bcl-2 expression as indicators of dexamethasone sensitivity. Cohort 1 is currently enrolling. Once a maximum tolerated dose is established, an expansion cohort is planned. Clinical trial information: NCT03472573.