CCAAT/enhancer-binding Protein Alpha Expression Research Articles

A comprehensive analysis of CEBPA on prognosis and function in uterine corpus endometrial carcinoma

Uterine corpus endometrial carcinoma (UCEC) is one of the most common tumours of the female reproductive system. CCAAT enhancer-binding protein alpha (CEBPA) is a member of the transcription factor family involved in regulating processes such as cell proliferation, differentiation, metabolism, and the immune response. However, the role of CEBPA in UCEC has not been clarified. Here, we performed a comprehensive analysis to explore the expression level, prognostic value, immune infiltration and biological function of CEBPA in UCEC. In this study, we found that CEBPA expression was upregulated and associated with poor prognosis in UCEC patients. KEGG and GO analyses revealed that the genes positively correlated with CEBPA were enriched primarily in immune regulation and oxidative phosphorylation. Immune infiltration analysis revealed that CEBPA is strongly correlated with immune cell infiltration in UCEC. RT-qPCR indicated that CEBPA may regulate the OXPHOS level in Ishikawa cells. CCK-8, cell cycle, Transwell and scratch wound healing assays revealed that CEBPA promoted Ishikawa cell proliferation, invasion and migration. In addition, PPI and survival analyses suggested that CEBPG may be a potential target of CEBPA in UCEC. These results demonstrated that CEBPA may be a potential therapeutic target in UCEC.

MAFB-mediated CEBPA regulated human urothelium growth through Wnt/β-catenin signaling pathway

MAFB is essential for regulating male-type urethral differentiation, and especially, its variation can contribute to hypospadias in mice. However, the potential mechanism is still unclear. Here we observed that the basic leucine zipper (bZIP) transcription factor MAFB and CCAAT/enhancer-binding protein alpha (CEBPA) could promote human urothelium SV-HUC-1 growth. Moreover, MAFB and CEBPA expression were reduced in the prepuce tissues of hypospadias patients. Based on transcriptome sequencing analysis and Western blot, MAFB knockdown was found to suppress CEBPA protein expression and repress Wnt/β-catenin signaling in urothelium cells. Meanwhile, we observed blocked cell-cycle progression from the G1 to the S phase, inhibited cell proliferation, and activated apoptosis. Furthermore, MAFB could facilitate CEBPA transcription and regulate the proliferation of urothelium. The above results indicated that MAFB-mediated inhibition of urothelial SV-HUC-1 growth resulted from inhibiting the Wnt/β-catenin signaling pathway by down-regulating CEBPA. Our findings provide new insight into the understanding of genes associated with hypospadias and the pathogenic mechanism of this disorder.

Targeting CEBPA to restore cellular identity and tissue homeostasis in pulmonary fibrosis.

Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency-mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.

CEBPA Restrains the Malignant Progression of Breast Cancer by Prompting the Transcription of SOCS2.

The suppressor of cytokine signaling 2 (SOCS2) has been identified to act as a tumor suppressor in breast cancer (BC) progression. However, the action of SOCS2 in macrophage polarization in BC cells has not been reported yet. The qRT-PCR and western blotting were adopted for detecting the levels of mRNAs and proteins. The macrophage M2 polarization was analyzed by flow cytometry. Analyses of cell oncogenic phenotypes and tumor growth were conducted using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, scratch, Transwell, tube formation assays in vitro, and tumor xenograft assay in vivo, respectively. The interaction between CEBPA (CCAAT Enhancer Binding Protein Alpha) and SOCS2 was confirmed using bioinformatics analysis and dual-luciferase reporter assay. SOCS2 was lowly expressed in BC tissues and cells. Functionally, overexpression of SOCS2 inhibited macrophage M2 polarization, and impaired BC cell proliferation, angiogenesis, and metastasis. Mechanistically, CEBPA bound to the promoter region of SOCS2, and promoted its transcription. A low CEBPA expression was observed in BC tissues and cells. Forced expression of CEBPA also suppressed macrophage M2 polarization, BC cell proliferation, angiogenesis, and metastasis. Moreover, the anticancer effects mediated by CEBPA were abolished by SOCS2 knockdown. In addition, CEBPA overexpression impeded BC growth in nude mice by regulating SOCS2. CEBPA suppressed macrophage M2 polarization, BC cell proliferation, angiogenesis, and metastasis by promoting SOCS2 transcription in a targeted manner.

Targeting the nuclear long noncoding transcript LSP1P5 abrogates extracellular matrix deposition by trans-upregulating CEBPA in keloids

Keloids are characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix (ECM) and are a major global healthcare burden among cutaneous diseases. However, the function of long noncoding RNA (lncRNA)-mediated ECM remodeling during the pathogenesis of keloids is still unclear. Herein, we identified a long noncoding transcript, namely, lymphocyte-specific protein 1 pseudogene 5 (LSP1P5), that modulates ECM component deposition in keloids. First, high-throughput transcriptome analysis showed that LSP1P5 was selectively upregulated in keloids and correlated with more severe disease in a clinical keloid cohort. Therapeutically, the attenuation of LSP1P5 significantly decreased the expression of ECM markers (COL1, COL3 and FN1) both in vitro and in vivo. Intriguingly, an antifibrotic gene, CCAAT enhancer binding protein alpha (CEBPA), is a functional downstream candidate of LSP1P5. Mechanistically, LSP1P5 represses CEBPA expression by hijacking Suppressor of Zeste 12 to the promoter of CEBPA, thereby enhancing the polycomb repressive complex 2-mediated H3K27me3 and changing the chromosomal opening status of CEBPA. Taken together, these findings indicate that targeting LSP1P5 abrogates fibrosis in keloids through epigenetic regulation of CEBPA, revealing a novel antifibrotic therapeutic strategy that bridges our current understanding of lncRNA regulation, histone modification and ECM remodeling in keloids.

Transfer of mRNA Encoding CEBPA in Lipid Nanoparticles to CEBPA Mutated Leukemia Cell Line KO52 and Primary AML Reduces Cell Growth and Induces Myeloid Differentiation

There are known constitutional mutations associated with bone marrow failure and progression to malignancy, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). For the majority of the defects associated with clonal hematopoiesis and progression to MDS/AML, there are no specific treatments. We sought to determine if messenger RNA (mRNA) transfer can restore expression of the normal proteins, modulate abnormal proliferation and differentiation, and enhance response to chemotherapy and targeted agents. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor involved in myeloid differentiation that is mutated in ~7-20% of patients with AML (Su L et al., Frontier Oncol. 2022). A germline alteration was identified in 4.5% of AML patients with CEBPA mutation (Ho PA et al., Blood 2009). While it was originally believed that biallelic mutations of CEBPA confer favorable prognosis, more recently it was realized that a single basic region leucine zipper (bZIP) mutation in the C terminal region suffices to confer favorable risk, as categorized by ELN2022. There are currently no targeted therapies for mutated CEBPA, and no known specific therapy for patients with constitutional or acquired CEBPA mutations. Although most patients with mutated CEBPA achieve initial CR, there is a moderate relapse rate (30-50%). The objective of this study was to determine if restoration of normal CEBPA mRNA would result in expression of normal CEBPA protein in AML cell lines (e.g.-U937), as well as an AML cell line with mutated CEBPA (KO52), and in primary AML blasts, including those with mutated CEBPA. We tested whether expression of normal CEBPA would lead to phenotypic changes including overcoming differentiation block, induce apoptosis, and eliminate leukemic blasts. There are normally two translation initiation sites, leading to production of the CEBPA-p42 or CEBPA-p30 proteins. U937 has high level expression of normal CEBPA-p42, while the KO52 cell line has two CEBPA mutations, p.E50* and p.L324P, the former leading to decrease in CEBPA-p30. To study the consequences of mRNA transfer, we performed immunofluorescence, multicolor flow cytometry (MFC), and Western blot (WB) analysis for non-transfected control cells, GreenLantern mRNA lipid nanoparticle (LNP) transfected control cells, and cells transfected with two types of CEBPA mRNA LNP (#1 and #2). We observed transfection efficiencies of 90-97% in cell lines and 20-95% in primary cells. Figure 1A shows the mean fluorescence intensity (MFI) of Alexa Fluor® 647-labeled monoclonal antibody to the C-terminus of CEBPA. Wild-type CEBPA-p42 was expressed in U937 and KO52 cell lines at 2-4 times control levels by WB quantitation. With two additions of LNP at 0h and 48h, the induced CEBPA protein maintained stable expression up to 5 days. Immunofluorescence demonstrated staining of CEBPA in the nucleus, co-localizing with nuclear DAPI fluorescence. To evaluate the functional consequences of CEBPA introduction, we analyzed morphology by Wright/Giemsa staining, proliferation by cell counting, apoptosis by Annexin V staining, cell cycle using BrdU labeling assay, and immunophenotype with stem and lineage markers including CD45, CD34, CD38, CD123, CD117, HLA-DR, CD13, CD33, CD16, CD11b, CD64, CD14. Strikingly, the enhanced expression of CEBPA was associated with decrease in proliferation and fraction in S phase, and increase in apoptosis and myeloid differentiation, suggesting induction of differentiation that could have therapeutic implications similar to ATRA in acute promyelocytic leukemia. The myeloid differentiation was evident by MFC showing decreased expression of primitive markers CD34, CD117 and HLA-DR, with increased expression of CD64 (Figure 1B), CD38, and CD11b. With this method we have also transfected primary AML patient blasts including a sample that harbored 4 CEBPA mutations. We observed 95% transfection efficiency and >50% decrease in cell number relative to controls with increased myeloid differentiation. Studies of the combination of CEBPA mRNA LNP transfection with targeted inhibitors to the other mutations present in this sample are in progress. Our findings reveal a novel therapeutic strategy using CEBPA mRNA LNP applicable to inherited or acquired disorders of CEBPA. This approach of restoration of normal mRNA could have potential for therapy of many pre-malignant and malignant disorders.

CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA.

Enhancer RNAs (eRNA) are noncoding transcripts produced from active enhancers and have potential gene regulatory function. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor generally involved in metabolism, cell cycle inhibition, hematopoiesis, adipogenesis, hepatogenesis, and is associated with tumorigenesis. In this study, we demonstrate that an enhancer-associated long noncoding RNA (elncRNA), transcribed from an enhancer located 9 kb downstream from the transcriptional start site (TSS) of CEBPA, positively regulates the expression of CEBPA. As a result, we named this elncRNA “CEBPA regulatory elncRNA downstream 9 kb” or “CRED9.” CRED9 expression level positively correlates with CEBPA mRNA expression across multiple cell lines as detected by RT droplet digital PCR. Knockdown of CRED9 resulted in a reduction of CEBPA mRNA expression in Hep3B cells. Additionally, CRED9 knockdown in Hep3B and HepG2 cells resulted in lower CEBPA protein expression. We also found that knockdown of CRED9 in Hep3B cells caused a 57.8% reduction in H3K27ac levels at the +9 kb CEBPA enhancer. H3K27ac has previously been described as a marker of active enhancers. Taken together, the evidence presented here supports a previously proposed model whereby, in some contexts, eRNA transcripts are necessary to amplify and maintain H3K27ac levels at a given enhancer. Ultimately, this study adds to the growing body of evidence that elncRNA transcripts have important roles in enhancer function and gene regulation.

Dysregulation of CCAAT/enhancer binding protein-alpha (CEBPA) expression in the bone marrow of acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by accumulation of different types of mutations commonly the CCAAT/enhancer binding protein-alpha (CEBPA). However, the dysregulations of CEBPA expression in AML is still a debatable issue. The aim of the current study was to assess CEBPA gene expression in bone marrow (BM) aspiration specimens of 91 AML patients, compared to 20 control donors of bone marrow transplantation (BMT), using RT-PCR. Data were correlated with patients’ clinico-pathological features, response to treatment, progression-free survival (PFS), and overall survival (OS) rates.ResultsThere was overexpression of CEBPA gene in AML patients compared to normal control [1.7 (0.04–25.6) versus 0.17 (0–4.78), respectively, P < 0.001]. Upregulation of CEBPA expression associated significantly with increased BM hypercellularity, total leucocyte counts, peripheral blood blast cell count, and poor PFS (P < 0.001, 0.002, 0.001, and 0.013, respectively). There was no significant association between CEBPA expression and any other relevant clinico-pathological features or OS rates (P = 0.610) of the patients. ROC analysis for biological relevance of CEBPA expression with AML showed that sensitivity and specificity of CEBPA expression at a cut-off value of 0.28 are 92.3% and 78.6%, respectively (P < 0.001). All patients who had CEBPA overexpression and mutant FLT3 showed BM hypercellularity, adverse cytogenetic risk, increased TLC, and PB blast cells count (P = 0.007, P < 0.001, 0.016, and 0.002, respectively).ConclusionCEBPA overexpression could be used as a genetic biological marker for AML diagnosis, as well as a poor prognostic factor for disease progression. It has no impact on OS rates of the patients.

Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression

BackgroundFibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated.MethodsLung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-β1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression.ResultsWe found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and αSMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states.ConclusionsCEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.

Expression level of CEBPA gene in acute lymphoblastic leukemia individuals

Currently, acute lymphoblastic leukemia (ALL) has an overall survival of nearly 80% when it occurs in children, however cure rates among adults are far reduced. Leukemogenesis can be driven up by a slight change in the expression or function of certain transcription factors. CCAAT Enhancer Binding Protein Alpha (CEBPA) is a transcription factor with role in cell cycle regulation, granulocytic differentiation and more. Some studies suggest its oncogenic function. The potential role of CEBPA as an oncogene in ALL development has not been completely elucidated so far. Therefore, the aim of the present study was to evaluate mRNA level of CEBPA gene in 60 adult patients diagnosed with ALL. Quantitative analysis was performed by qPCR reaction. Analysis revealed that men tended to have higher and more variable CEBPA expression levels (P = 0.032). No associations for other parameters (ALL subtype, age, leukocytosis, blast percentage, Philadelphia chromosome presence, CD10 marker presence) were found. When comparing the results of CEBPA expression with patients suffering from acute myeloid leukemia, ALL cases showed statistically significant lower levels of CEBPA (P < 0.0000). It may seem that CEBPA expression level itself has potentially no effect on arising and progression of acute lymphoblastic leukemia, although it is a matter that needs further investigation.

Expression analysis of CEBPA and its antisense RNA revealed their dysregulation in peripheral blood of coronary artery disease patients

Coronary artery disease (CAD) is a major cause of mortality. It has been associated with several genetic and lifestyle factors. CCAAT enhancer binding protein alpha (CEBPA) encodes a protein with regulatory function on the expression of genes participating in cell cycle control as well as in body weight homeostasis. A non-coding transcript namely adipogenic differentiation induced noncoding RNA (ADINR) has been shown to be transcribed from the upstream region of the CEBPA gene. In the current study, we compared expression of CEBPA and ADINR between CAD patients and healthy subjects. ADINR expression was significantly lower in CAD cases compared with healthy subjects. However, expression of CEBPA was significantly higher in cases compared with controls. A significant correlation was detected between expression of CEBPA and FBS in female subjects (r = 0.50, P < 0.0001). Notably, expression of ADINR was inversely correlated with FBS (r = −0.64, P < 0.0001) and with HDL (r = −0.30, P < 0.0001) in male subjects. Moreover, expression of CEBPA was correlated with FBS in male subjects (r = 0.29, P < 0.0001). The diagnostic power values of ADINR and CEBPA in differentiating between CAD patients and normal subjects were 93% and 80% respectively. Consequently, CEBPA and ADINR are possibly involved in the pathophysiology of CAD and are putative biomarkers for this disorder.

Abstract 3211: MTL-CEBPA combined with radiofrequency ablation and immunotherapy enhances immunological anti-tumour response in an HCC mouse model

Abstract The transcription factor CEBPA (CCAAT/enhancer-binding protein alpha) is recognised for its antiproliferative effects. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA and amongst other effects causes and immunomodulatory effect on peripheral granulocytes. Radiofrequency ablation (RFA) is standard treatment for some tumour types such as liver cancer and induces modulation of both innate and adaptive immune systems. To investigate any synergistic effect of MTL-CEBPA with RFA and immune checkpoint inhibition we initiated a reverse translation experiment, where syngeneic BNL hepatocellular carcinoma tumour cells were injected in the two opposite flanks of immunocompetent BALB/c mice (n=8 in each group). Treatments for hepatoma bearing mice included: 1) RFA on one flank (day 0), 2) Anti-PD-1 inhibition immunotherapy (RMP1-14 antibody, BioXCell, West Lebanon, NH, USA at 200 μg IV/mouse/dose on days 0, 2 &amp; 5) and 3) MTL-CEBPA (3mg/kg IV/mouse/dose on days 0, 2 &amp; 5) as well as combinations of all 3 interventions. We found that tumour control on the opposite flank was augmented by addition of RFA and most significant in the triple combination group (RFA + anti-PD1 + MTL-CEBPA) in which 2/8 animals showed a complete response and 5/8 a partial response. This was also the only group that showed a statistically significant increase in CD8+ (Cytotoxic) as well as CD49b+/CD45+ (Natural Killer) tumour infiltrating lymphocytes. These data suggest a clinical role for combination treatment with checkpoint blockade, RFA and MTL-CEBPA through synergistic priming of the immune tumour response, enabling RFA to have a pronounced anti-tumour abscopal effect. Citation Format: Mikael H. Sodergren, Kai-Wen Huang, Vikash Reebye, Cheng-Ean Chee, Dimitris Zacharoulis, Robert Habib, David Blakey, John Rossi, Nagy Habib. MTL-CEBPA combined with radiofrequency ablation and immunotherapy enhances immunological anti-tumour response in an HCC mouse model [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 3211.

Loss of CCAAT-enhancer-binding protein alpha (CEBPA) is linked to poor prognosis in PTEN deleted and TMPRSS2:ERG fusion type prostate cancers.

The transcription factor CCAAT-enhancer-binding protein alpha (CEBPA) is a crucial regulator of cell proliferation and differentiation. Expression levels of CEBPA have been suggested to be prognostic in various tumor types. Here, we analyzed the immunohistochemical expression of CEBPA in a tissue microarray containing more than 17 000 prostate cancer specimens with annotated clinical and molecular data including for example TMPRSS2:ERG fusion and PTEN deletion status. Normal prostate glands showed moderate to strong CEBPA staining, while CEBPA expression was frequently reduced (40%) or lost (30%) in prostate cancers. Absence of detectable CEBPA expression was markedly more frequent in ERG negative (45%) as compared to ERG positive cancers (20%, P < 0.0001). Reduced CEBPA expression was linked to unfavorable phenotype (P < 0.0001) and poor prognosis (P = 0.0008). Subgroup analyses revealed, that the prognostic value of CEBPA loss was entirely driven by tumors carrying both TMPRSS2:ERG fusions and PTEN deletions. In this subgroup, CEBPA loss was tightly linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), positive nodal stage (0.0003), and early biochemical recurrence (P = 0.0007), while these associations were absent or markedly diminished in tumors with normal PTEN copy numbers and/or absence of ERG fusion. CEBPA is down regulated in about one third of prostate cancers, but the clinical impact of CEBPA loss is strictly limited to the subset of about 10% prostate cancers carrying both ERG fusion and deletions of the PTEN tumor suppressor. Our findings challenge the concept that prognostic molecular markers may be generally applicable to all prostate cancers.

PIWIL3/OIP5-AS1/miR-367-3p/CEBPA feedback loop regulates the biological behavior of glioma cells.

Rationale: PIWI-interacting RNAs (piRNAs), a class of newly discovered small RNA molecules that function by binding to the Argonaute protein family (i.e., the PIWIL protein subfamily), and long noncoding RNAs (lncRNA) are implicated in several cancers. However, the detailed roles of ncRNAs in glioma remain unclear.Methods: The expression of PIWIL3, piR-30188, OIP5-AS1, miR-367, CEBPA and TRAF4 were measured in glioma tissues and cells. The role of PIWIL3/OIP5-AS1/miR-367-3p/CEBPA feedback loop was evaluated in cell and animal models. The association of the above molecules was analyzed.Results: Over-expression of PIWIL3, piR-30188 and miR-367-3p or knockdown of OIP5-AS1 resulted in inhibition of glioma cells progression. Binding sites between piR-30188 and OIP5-AS1 as well as between OIP5-AS1 and miR-367-3p were confirmed by RNA immunoprecipitation and luciferase assays. OIP5-AS1 knockdown or miR-367-3p over-expression contributed to a decrease in CEBPA (CCAAT/enhancer binding protein alpha) protein. Furthermore, CEBPA was detected as a target of miR-367-3p and played an oncogenic role in glioma. Treatment with CEBPA and miR-367-3p resulted in the modulation of downstream TRAF4 (TNF receptor-associated factor 4). PIWIL3 was also a target of CEBPA, forming a positive feedback loop in the growth regulation of glioma cells. Significantly, knockdown of OIP5-AS1 combined with over-expression of PIWIL3 and miR-367-3p resulted in tumor regression and extended survival in vivo.Conclusion: These results identified a novel molecular pathway in glioma cells that may provide a potential innovative approach for tumor therapy.

Abstract 5347: SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells

Abstract Although differentiation-inducing agents have significantly improved the management of acute promyelocytic leukemia, no significant progress has been made in the treatment of other acute myeloid leukemias (AML). Numerous proteins involved in tumor development have so-called allosteric disulfide bonds amenable to modifications affecting protein structure and function. We have developed SK053, a small molecule and mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI). The aim of our studies was to evaluate anti-leukemic activity of SK053 in human AML cells. To validate if SK053 targets PDI, a binding assay and an insulin turbidimetric activity assay were used. Cytostatic/cytotoxic effects in HL60, NB4, KG-1 and MOLM14 cells as well as in primary AML cells were assessed with trypan blue exclusion. Differentiation of AML cells was studied with May-Grünwald-Giemsa staining, nitro blue tetrazolium reduction assay and flow cytometry analysis of CD11b, CD14 and CD15 levels and by RNA sequencing, qRT-PCR and western blotting (WB). We observed covalent binding of SK053 to PDI and inhibition of its enzymatic activity with IC50 of 10 μM. Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), a transcription factor crucial for neutrophils maturation, we evaluated the potential of SK053 to induce differentiation and cytostatic/cytotoxic effects in human AML cells. SK053 exerts significant cytostatic/cytotoxic activity in human AML cells (HL60, NB4, KG-1 and MOLM14), and induces differentiation of AML blasts into more mature myeloid cells. Incubation of AML cells with SK053 induced expression of CEBPA and hexokinase 3 mRNA in quantitative RT-PCR and increased amount of CEBPA protein in nuclear fraction measured in WB. Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes as well as for histone proteins. Expression of other genes of mature myeloid lineage such as adhesion molecules (collagen type XV, fibronectin I, MAC-1), hydrolytic enzymes (carboxypeptidase, proteinase 3, CA12 anhydrase, ADAM19 metalloprotease), proteoglycan 2 (core of eosinophilic granules) and PGLYRP3 (peptidoglycan recognition protein 3) was significantly up-regulated. The GeneOntology analysis done with the RNAseq results revealed enrichment of gene transcripts regulating myeloid cells differentiation. SK053 exerts potent anti-leukemic activity and induces differentiation of numerous types of human AML cells. Targeting allosteric disulfide bonds with small molecule inhibitors presents a promising therapeutic strategy in AML. Citation Format: Dominika Nowis, Justyna Chlebowska, Pawel Gaj, Michal Lazniewski, Malgorzata Firczuk, Karolina Furs, Radoslaw Sadowski, Pawel Leszczynski, Piotr Stawinski, Szymon Klossowski, Ryszard Ostaszewski, Krzysztof Giannopoulos, Rafal Ploski, Dariusz Plewczynski, Jakub Golab. SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2015-5347

CEBPA methylation and mutation in myelodysplastic syndrome.

Aberrant methylation of CCAAT/enhancer-binding protein alpha (CEBPA) promoter has been observed in acute myeloid leukemia. However, little is known about CEBPA promoter in myelodysplastic syndrome (MDS). The purpose of this study was to investigate the alteration of CEBPA promoter in MDS patients and further determine the association with CEBPA expression and mutation. CEBPA promoter was significantly methylated in 105 MDS patients compared to 22 controls (median 0.016 vs. 0.000) (P < 0.0001). Receiver operating characteristic curve analysis discriminated all patients or cytogenetically normal patients from normal controls. Three cases (3 %) were identified with single-mutated CEBPA and one (1 %) with double-mutated CEBPA. CEBPA methylation and mutation occurred mutually exclusive. No significant correlation was found between CEBPA expression and methylation (P = 0.586). Our findings indicate that CEBPA methylation is a common event in MDS, but could not act as a prognostic biomarker for MDS patients.

SK053 An Inhibitor Of Enzymes Involved In Allosteric Disulfide Bonds Formation Induces Differentiation Of Human AML Cells

Introduction of differentiation-inducing agents [all-trans retinoic acid (ATRA) and arsenic trioxide] to the treatment of acute promyelocytic leukemia (APL) was a remarkable therapeutic breakthrough resulting in cure rates exceeding 80%. However, there is no such significant progress in the treatment of other acute myeloid leukemia (AML) types. Thus search for new agents exerting anti-leukemic effects by targeting novel and unique cellular mechanisms is of utmost clinical importance. Numerous human proteins involved in tumor formation, such as C-terminal SRC kinase (CSK) or tissue factor, contain allosteric disulfide bonds that are cleaved by oxidoreductases or by thiol-disulfide exchange. Such disulfide modifications participate in post-translational protein control and affect protein function. Targeting of allosteric disulfide bonds is a novel promising strategy in cancer therapy (Hogg P, Nat Rev Cancer 2013). We have recently developed SK053, a small molecule inhibitor of thioredoxin/thioredoxin reductase system, that showed anti-tumor effects both in vitro and in murine tumor models (Klossowski S, Muchowicz A et al., J Med Chem 2012). Our ongoing studies revealed that SK053 is not a target-specific, but mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as protein disulfide isomerase (PDI). Its biotinylated form (SK231) precipitated PDI from human AML NB4 cells, and mass spectrometry analysis revealed that SK053 covalently binds to PDI. In a turbidimetric assay of insulin disulfide reduction SK053 inhibits the enzymatic activity of PDI with IC50 of 10 µM (Fig. 1B). Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), one of the transcription factors crucial for normal neutrophils maturation (Haefliger S et al., Blood 2011), we set out to evaluate the activity of SK053 in human AML cells to see whether it can induce differentiation and cytostatic/cytotoxic effects against these cells. We observed that SK053 exerts significant cytostatic/cytotoxic activity in various types of human AML cells (HL60, NB4, KG-1 and MOLM14, Fig. 1A), and induces differentiation of AML blasts into more mature myeloid cells as evidenced morphologically in May-Grünwald-Giemsa staining, nitro blue tetrazolium (NBT) reduction assay as well as by increased expression of cell membrane differentiation markers (CD11b, CD14 and CD15), measured with flow cytometry. Moreover, incubation of AML cells with SK053 induces expression of CEBPA and hexokinase 3 (HK3) mRNA in quantitative RT-PCR (Fig. 1C) and increases amount of CEBPA protein in nuclear fraction measured with immunoblotting (Fig. 1D). Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. In summary, SK053 targets PDI and thioredoxin/thioredoxin reductase system, has significant anti-leukemic activity and induces differentiation of various types of human AML cells. Thus, targeting of enzymes involved in allosteric disulfide bonds formation with small molecule inhibitors presents a novel and promising therapeutic strategy in acute myeloid leukemia. [Display omitted] The research was supported by the Ministry of Science and Higher Education [IP2011038971 (DN)], National Science Centre Poland [NN405127640 (AM)] and a grant from the European Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION. Disclosures:No relevant conflicts of interest to declare.

Detection ofCEBPADouble Mutants in Acute Myeloid Leukemia Using a Custom Gene Expression Array

Double (bi-allelic) mutations in the gene encoding the CCAAT/enhancer-binding protein-alpha (CEBPA) transcription factor have a favorable prognostic impact in acute myeloid leukemia (AML). Double mutations in CEBPA can be detected using various techniques, but it is a notoriously difficult gene to sequence due to its high GC-content. Here we developed a two-step gene expression classifier for accurate and standardized detection of CEBPA double mutations. The key feature of the two-step classifier is that it explicitly removes cases with low CEBPA expression, thereby excluding CEBPA hypermethylated cases that have similar gene expression profiles as a CEBPA double mutant, which would result in false-positive predictions. In the second step, we have developed a 55 gene signature to identity the true CEBPA double-mutation cases. This two-step classifier was tested on a cohort of 505 unselected AML cases, including 26 CEBPA double mutants, 12 CEBPA single mutants, and seven CEBPA promoter hypermethylated cases, on which its performance was estimated by a double-loop cross-validation protocol. The two-step classifier achieves a sensitivity of 96.2% (95% confidence interval [CI] 81.1 to 99.3) and specificity of 100.0% (95% CI 99.2 to 100.0). There are no false-positive detections. This two-step CEBPA double-mutation classifier has been incorporated on a microarray platform that can simultaneously detect other relevant molecular biomarkers, which allows for a standardized comprehensive diagnostic assay. In conclusion, gene expression profiling provides a reliable method for CEBPA double-mutation detection in patients with AML for clinical use.

Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia

Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia

Yin Yang-1 suppresses differentiation of hepatocellular carcinoma cells through the downregulation of CCAAT/enhancer-binding protein alpha

As a member of the GLI-Kruppel family of transcriptional factors, Yin Yang-1 (YY1) functions as an oncogene in various types of cancers. However, the role of YY1 in hepatocellular carcinogenesis remains unknown. In this report, we investigated the relevance of YY1 to hepatocellular carcinoma (HCC) development. We found that YY1 was upregulated in HCC cell lines. Ectopic YY1 expression promoted the growth of non-tumor liver cells that expressed low level of YY1. In contrast, YY1 depletion inhibited the growth of HCC cells which was accompanied with distinct morphological changes. Moreover, the phenotypic changes induced by YY1 depletion were attributed to cellular differentiation rather than cellular senescence. CCAAT/enhancer-binding protein alpha (CEBPA) which was important to regulate differentiation of hepatocytes was found as the direct target downregulated by YY1. Restoration of CEBPA in YY1-expressing HCC cells induced cellular differentiation and growth inhibition while knockdown of CEBPA expression in non-tumor liver cells promoted cell growth. In summary, our study demonstrated that YY1 could promote hepatocellular carcinogenesis and inhibit cellular differentiation through the downregulation of CEBPA expression.