Interaction Of RUNX1 Research Articles

RUNX1 interacts with lncRNA SMANTIS to regulate monocytic cell functions

Monocytes, the circulating macrophage precursors, contribute to diseases like atherosclerosis and asthma. Long non-coding RNAs (lncRNAs) have been shown to modulate the phenotype and inflammatory capacity of monocytes. We previously discovered the lncRNA SMANTIS, which contributes to cellular phenotype expression by controlling BRG1 in mesenchymal cells. Here, we report that SMANTIS is particularly highly expressed in monocytes and lost during differentiation into macrophages. Moreover, different types of myeloid leukemia presented specific SMANTIS expression patterns. Interaction studies revealed that SMANTIS binds RUNX1, a transcription factor frequently mutated in AML, primarily through its Alu-element on the RUNT domain. RNA-seq after CRISPR/Cas9-mediated deletion of SMANTIS or RUNX1 revealed an association with cell adhesion and both limited the monocyte adhesion to endothelial cells. Mechanistically, SMANTIS KO reduced RUNX1 genomic binding and altered the interaction of RUNX1 with EP300 and CBFB. Collectively, SMANTIS interacts with RUNX1 and attenuates monocyte adhesion, which might limit monocyte vascular egress.

RUNX1-Survivin Axis Is a Novel Therapeutic Target for Malignant Rhabdoid Tumors.

Malignant rhabdoid tumor (MRT) is a highly aggressive pediatric malignancy with no effective therapy. Therefore, it is necessary to identify a target for the development of novel molecule-targeting therapeutic agents. In this study, we report the importance of the runt-related transcription factor 1 (RUNX1) and RUNX1-Baculoviral IAP (inhibitor of apoptosis) Repeat-Containing 5 (BIRC5/survivin) axis in the proliferation of MRT cells, as it can be used as an ideal target for anti-tumor strategies. The mechanism of this reaction can be explained by the interaction of RUNX1 with the RUNX1-binding DNA sequence located in the survivin promoter and its positive regulation. Specific knockdown of RUNX1 led to decreased expression of survivin, which subsequently suppressed the proliferation of MRT cells in vitro and in vivo. We also found that our novel RUNX inhibitor, Chb-M, which switches off RUNX1 using alkylating agent-conjugated pyrrole-imidazole polyamides designed to specifically bind to consensus RUNX-binding sequences (5'-TGTGGT-3'), inhibited survivin expression in vivo. Taken together, we identified a novel interaction between RUNX1 and survivin in MRT. Therefore the negative regulation of RUNX1 activity may be a novel strategy for MRT treatment.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

SUMMARYThe transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Inhibition of Core Binding Factor Transcriptional Complex Provides a Novel Pharmacological Target for Osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor in canines and humans. However, there have been no new successful therapies that improve long‐term survival in several decades. This indicates the need for novel therapeutic targets for treating OS. Core binding factor is a heterodimeric transcriptional complex, comprised of proteins runt‐related transcription factor 2 (RUNX2) and core‐binding factor subunit Beta (CBFβ), that promotes transcription of genes related to bone formation, osteoblast differentiation, and bone mineralization. RUNX2 protein expression is deregulated in OS tumors which contributes to chemoresistance, increased proliferation, and defective differentiation, suggesting it could be a therapeutic target in OS. This study utilizes small molecule allosteric inhibitor of CBFβ, AI‐14‐91, to target the interaction of RUNX2 and CBFβ, thereby reducing RUNX2 binding to target genes, in order to evaluate the CBFβ and RUNX2 interaction as a pharmacological target in OS. We hypothesized that AI‐14‐91 will reduce the malignant phenotype of human OS cell lines by disrupting the RUNX2‐CBFβ interaction.In vitro assays with compound AI‐14‐91 were performed with a panel of unique human OS cell lines (LM7, MG‐63, Saos‐2, SJSA‐1, and U2OS). This panel contains unique genetic mutations in key tumor‐related genes such as p53, MDM2, Rb1, or CDKN2A. RUNX2 knockout (KO) and CBFβ KO cell lines were also generated from the parental U2OS by CRISPR/Cas9 editing, which was confirmed via sequencing and western blot. Concentration‐ and time‐dependent proliferation assays were performed with all cell lines, demonstrating AI‐14‐91 IC50 values ranging from 13‐25 µM across the panel. Clonogenic assays showed AI‐14‐91 treatment significantly reduced colony formation in the majority of cell lines. Flow cytometry revealed cell cycle progression was altered in the parental U2OS and the RUNX2 KO and CBFβ KO cell lines with a significant decrease of cell number in G0/G1 phase and an increase in S phase upon 48 exposure to 20 µM AI‐14‐91. Apoptotic and necrotic cell populations in parental U2OS and KO lines were quantified by flow cytometry bivariate analysis with Annexin V/PI co‐stain. Batch 3’ Tag‐Seq RNAseq analysis was performed on parental U2OS cells exposed to 5 or 20 µM AI‐14‐91 for 6 or 48 hours and on RUNX2 KO and CBFβ KO cell lines. Differential gene expression (DGE) and enrichment analysis was performed to evaluate pertinent transcriptional pathways altered by inhibitor treatment, and to compare the effects of inhibitor treatment with knockout of putative inhibitor targets.Treatment with AI‐14‐91 has shown desirable antitumor effects in human OS cell lines that may be independent of the absence of proteins RUNX2 or CBFβ. Further analysis into DGE pathways combined with ChIP assays will help delineate mechanisms of action and identify additional potential targets for combination treatments. Orthotopic mouse models utilizing the metastasizing LM7 cell line with provide further information on the potential clinical utility of targeting the RUNX2‐CBFβ interaction in OS.

MiR-21 regulates immunosuppression mediated by myeloid-derived suppressor cells by impairing RUNX1-YAP interaction in lung cancer

BackgroundMyeloid-derived suppressor cells (MDSCs) are known suppressors of antitumor immunity and contribute to immunosuppressive microenvironment during tumor development including lung cancer. Accumulating evidence shows microRNAs (miRNAs) affect tumor-expanded MDSC accumulation and function in tumor microenvironment and favor solid tumor growth. Herein, we aim to characterize the role of miR-21 in regulating the accumulation and activity of MDSCs in lung cancer.MethodsThe proportions of MDSCs, T helper cells (Th), and cytotoxic T lymphocytes (CTL) were evaluated by flow cytometric analyses of peripheral blood and tumor tissues collected from Lewis lung-cancer-bearing mice. T cell proliferation assay was performed in CD4+ or CD8+ T cells cocultured with MDSCs. MDSC apoptosis was examined by flow cytometric analysis. The levels of IL-10, TGF-β, and GM-CSF in mouse serum were determined by ELISA. miR-21 targeting RUNX1 and RUNX1 interaction with YAP were evaluated by RIP, dual-luciferase reporter gene, and ChIP assays.ResultsMiR-21 inhibition by its antagomir reduced the proportion of MDSCs, increased the proportion of Th and CTL in peripheral blood and tumor tissues of Lewis lung-cancer-bearing mice, protected Th and CTL from the suppression of MDSCs, increased apoptosis of MDSCs, but reduced IL-10, TGF-β and GM-CSF levels in mouse serum. RUNX1 could transcriptionally inhibit the YAP expression, whereas miR-21 targeting RUNX1 led to elevated YAP expression levels. Mechanistic investigation showed that miR-21 maintained MDSC accumulation in tumor microenvironment and promoted immunosuppressive ability of MDSCs in Lewis lung-cancer-bearing mice by down-regulating RUNX1and up-regulating YAP.ConclusionsTaken together, the study provides evidence that targeting miR-21 in MDSCs may be developed as an immunotherapeutic approach to combat lung cancer development.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

The Runx1/Notch1 Signaling Pathway Participates in M1/M2 Microglia Polarization in a Mouse Model of Temporal Lobe Epilepsy and in BV-2 Cells.

Microglial activation and phenotypic shift play vital roles in many neurological diseases. Runt-related transcription factor-1 (Runx1), which is localized on microglia, inhibits amoeboid microglial proliferation. Preliminary data have indicated that the interaction of Runx1 with the Notch1 pathway affects the hemogenic endothelial cell shift. However, little is known about the effect of Runx1 and the Notch1 signaling pathway on the phenotypic shift of microglia during neuroinflammation, especially in temporal lobe epilepsy (TLE). A mouse model of TLE induced by pilocarpine and the murine microglia cell line BV-2 were used in this study. The proportion of microglia was analyzed using flow cytometry. Western blot (WB) analysis and quantitative real-time polymerase chain reaction were used to analyze protein and gene transcript levels, respectively. Immunohistochemistry was used to show the distribution of Runx1. In the present study, we first found that in a male mouse model of TLE induced by pilocarpine, flow cytometry revealed a time-dependent M2-to-M1 microglial transition after status epilepticus. The dynamic expression patterns of Runx1 and the downstream Notch1/Jagged1/Hes5 signaling pathway molecules in the epileptic hippocampus were determined. Next, Runx1 knockdown by small interfering RNA in BV-2 cells strongly promoted an M2-to-M1 microglial phenotype shift and inhibited Notch1/Jagged1/Hes5 pathway expression. In conclusion, Runx1 may play a critical role in the M2-to-M1 microglial phenotype shift via the Notch1 signaling pathway during epileptogenesis in a TLE mouse model and in BV-2 cells.

E2A-PBX1 functions as a coactivator for RUNX1 in acute lymphoblastic leukemia

AbstractE2A, a basic helix-loop-helix transcription factor, plays a crucial role in determining tissue-specific cell fate, including differentiation of B-cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. Although previous studies have shown the oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1–enforced cistrome, the E2A-PBX1 interactome, and related mechanisms underlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)–positive pre-B ALL cells and show that, compared with normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci cobound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetylation). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Transcriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1–mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

LAPTM5 is transactivated by RUNX2 and involved in RANKL trafficking in osteoblastic cells.

The present study aimed to investigate the role of lysosomal-associated transmembrane protein 5 (LAPTM5) in osteoclast differentiation induced by osteoblasts. The results demonstrated that the expression levels of LAPTM5 were downregulated following runt-related transcription factor 2 (RUNX2) silencing and upregulated following RUNX2 overexpression in ST2 cells. Chromatin immunoprecipitation analysis identified the binding of RUNX2 to the LAPTM5 promoter at the −1176 to −1171 position. Dual-luciferase reporter assays confirmed that RUNX2 directly activated the LAPTM5 gene. The concentration of receptor activator of nuclear factor-κB ligand (RANKL) protein in the cytoplasm and in the media was significantly increased following LAPTM5 knockdown. LAPTM5 silencing in ST2 cells enhanced osteoclastic differentiation of co-cultured RAW264.7 cells. The present study indicated that expression of LAPTM5 was regulated by the interaction of RUNX2 with its promoter region and that LAPTM5 was involved in the trafficking of RANKL. These findings suggested a possible coupling mechanism between osteogenesis and osteoclastogenesis in which RUNX2 may be involved in osteoclast differentiation through the regulation of the lysosome-associated genes that modulate RANKL expression.

Abstract 3357: Genetic regulation of RUNX2 induce apoptotic cell death through regulating the expression of SOX2 in CRPC-NE cells

Abstract Although runt-related transcription factor 2 (RUNX2) is well known for its bone-specific transcriptional regulator in highly metastatic prostate cancer, there is little knowledge about its role of RUNX2 in castration-resistant neuroendocrine prostate cancer (it is named CRPC-NE). It is well known that expression of Androgen Receptor (AR) in CRPC-NE cases is lower than other types and AR suppresses RUNX2 expression by binding it and abrogating its recruitment to DNA. This fact suggests that in CRPC-NE, low expression of AR, the addiction of RUNX2 is relatively higher than that of other prostate cancer types. Extensive analysis of the clinical datasets revealed that RUNX2 is one of the most consistently up-regulated genes in CRPC-NE and the overexpression of SOX2 confers an accelerated disease progression and shortened overall survival periods to CRPC-NE patients. We also found that the silencing RUNX2 induced apoptotic cell death in CRPC-NE cells independent on p53, but through transcriptionally down-regulating SOX2 expressions. Mechanistically, ChIP-qPCR assay and luciferase reporter experiments confirmed that RUNX2 positively regulates SOX2 expression by binding to the consensus RUNX2 binding sequence (5'-TGTGGT-3') located in the proximal promoter region of SOX2. Short hairpin RNA (shRNA)-mediated knockdown of RUNX2 in the CRPC-NE cell line PC-3 induced the decreased expression of SOX2 and the increased expression of cleaved form of caspase-3 and PARP in a p53 independent manner. Silencing of SOX2 in PC-3 cells suppressed the proliferation of these cancer cells and induced apoptosis, which phenotypes were rescued by restoring SOX2 in the cells. These data indicates that inhibition of SOX2-apoptosis axis via RUNX2 could be a better therapeutic choice in CRPC-NE. Lastly, we examined the efficacy of our novel molecule Chlorambucil-conjugated Pyrrole Imidazole Polyamide (we named it as Chb-M'), which specifically binds to the consensus RUNX2 binding sequence and inhibits RUNX2 target gene. Surprisingly, Chb-M' had tremendous inhibitory effect on PC-3 cells (IC50 value at 620 nM) through effective SOX2 inhibition. Additionally, it was exceptionally well-tolerated in mice and exerted excellent efficacy against xenograft mice models of CRPC-NE. Taken together, our work identified a novel interaction of RUNX2 and SOX2-apoptosis axis, offering a new strategy for the management of poor-prognostic advanced stage CRPC-NE cancer patients. Citation Format: Yuki Noguchi, Natsuki Wariishi, Shiina Iwai, Gengo Kashiwazaki, Junichi Taniguchi, Toshikazu Bando, Masaya Baba, Souichi Adachi, Hiroshi Sugiyama, Yasuhiko Kamikubo. Genetic regulation of RUNX2 induce apoptotic cell death through regulating the expression of SOX2 in CRPC-NE cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3357.

RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells

The dual function of runt-related transcriptional factor 1 (RUNX1) as an oncogene or oncosuppressor has been extensively studied in various malignancies, yet its role in gastric cancer remains elusive. Up-regulation of the ErbB2/HER2 signaling pathway is frequently-encountered in gastric cancer and contributes to the maintenance of these cancer cells. This signaling cascade is partly mediated by son of sevenless homolog (SOS) family, which function as adaptor proteins in the RTK cascades. Herein we report that RUNX1 regulates the ErbB2/HER2 signaling pathway in gastric cancer cells through transactivating SOS1 expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, RUNX1 interacts with the RUNX1 binding DNA sequence located in SOS1 promoter and positively regulates it. Knockdown of RUNX1 led to the decreased expression of SOS1 as well as dephosphorylation of ErbB2/HER2, subsequently suppressed the proliferation of gastric cancer cells. We also found that our novel RUNX inhibitor (Chb-M’) consistently led to the deactivation of the ErbB2/HER2 signaling pathway and was effective against several gastric cancer cell lines. Taken together, our work identified a novel interaction of RUNX1 and the ErbB2/HER2 signaling pathway in gastric cancer, which can potentially be exploited in the management of this malignancy.

Abstract 1530: RUNX1 positively regulates ErbB2/HER2 signaling pathway through modulating the expression of SOS1 in gastric cancer cells

Abstract Although runt-related transcription factor 1 (RUNX1) is well known for its context-dependent oncogenic properties in various malignancies, its role in gastric cancers has been poorly defined. Up-regulation of receptor tyrosine kinase (RTK) ErbB2/HER2 signaling pathway is encountered in the vast majority of gastric cancer cases and contributes to the initiation and maintenance of these cancer cells. This signaling cascade is partly yet arbitrary mediated by son of sevenless gene 1 (SOS1), which functions as an adaptor protein in RTK cascades. Herein we report that RUNX1 regulates ErbB2/HER2 signaling pathway in gastric cancer cells through transcriptionally regulating SOS1 expressions, rendering itself an ideal target in anticancer strategies. Mechanistically, RUNX1 interacts with the consensus RUNX1 binding sequence (5’-TGTGGT-3’) located in the proximal promoter region of SOS1 and positively regulates it. Short hairpin RNA (shRNA)-mediated knockdown of RUNX1 in the gastric cancer cell line MKN45 led to the decreased expression of SOS1 and of phosphorylated form of ErbB2/HER2 as well as the deactivation of its downstream targets such as AKT and ERK. RUNX1 knockdown subsequently induced cell cycle arrest at G0/G1 phase and successive apoptotic cell death in MKN45 cells. Silencing of HER2 or SOS1 in MKN45 cells unalterably suppressed the proliferation of these cancer cells, highlighting the importance of this ErbB2/HER2 signaling cascade in the maintenance of gastric cancer cells. We also found that SOS1 is one of the most consistently up-regulated genes in RUNX1-high expressing primary gastric cancer cells derived from previously reported human clinical samples. These data collectively indicates that inhibition of RUNX1 could be a legitimate therapeutic choice in the management of gastric cancers. Lastly, we examined the efficacy of a novel small molecule specifically binds and inhibits RUNX1 (we named it as CM). Intriguingly, CM was exceptionally effective against MKN45 cells (IC50 value at 403.5 nM). Besides, CM was well-tolerated in mice and fabulously suppressed the growth of xenotransplanted MKN45 cells in immunodeficient mice in vivo. Taken together, our work identified a novel interaction of RUNX1 and ErbB2/HER2 signaling pathway, paving a new way for the management of dismal-prognostic advanced stage HER2-positive gastric cancer patients. Citation Format: Yoshihide Mitsuda, Ken Morita, Shintaro Maeda, Kensho Suzuki, Gengo Kashiwazaki, Junichi Taniguchi, Toshikazu Bando, Hiroshi Sugiyama, Souichi Adachi, Yasuhiko Kamikubo. RUNX1 positively regulates ErbB2/HER2 signaling pathway through modulating the expression of SOS1 in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1530. doi:10.1158/1538-7445.AM2017-1530

Paradoxical Enhancement of Leukemogenesis in Acute Myeloid Leukemia Cells with Moderately Attenuated RUNX1 Expressions

Although Runt-related transcription factor 1 (RUNX1), a member of RUNX transcription family, is known for its oncogenic role in the development of acute myeloid leukemia (AML), evidence from other groups support the oncosuppressive property of RUNX1 in leukemia cells, casting a question over the bidirectional function of RUNX1 and it is currently highly controversial. Here we report that the dual function of RUNX1 possibly arise from the total level of RUNX family expressions.To examine the precise mechanism of RUNX1 expression in leukemogenesis, we first prepared several tetracycline-inducible short hairpin RNAs (shRNAs) which could attenuate the expressions of RUNX1 at different levels in AML cells (MV4-11 and MOLM-13 cells). Intriguingly, while AML cells transduced with shRNAs which could down-regulate RUNX1 expression below 10% at protein level (sh_Rx1_profound) deteriorated the proliferation speed of AML cells, AML cells transduced with shRNAs which could moderately down-regulate RUNX1 expression to 70% at protein level (sh_Rx1_moderate) paradoxically promoted the cell cycle progression and doubled the growth rate of AML cells. Besides, RUNX1-moderately expressing AML patient cohort exhibited the worse outcome compared to RUNX1-high or RUNX1-low expressing cohorts (n = 187), indicating an underlying mechanism that confer growth advantage to AML cells with moderately inhibited RUNX1 expressions.To further investigate the correspondent gene in this paradoxical enhancement of oncogenesis in sh_Rx1_moderate-transduced AML cells, we performed comprehensive gene expression array and extracted genes that are highly up-regulated in RUNX1 moderate inhibition and down-regulated in AML cells transduced with sh_Rx1_profound. We hereafter focused on the top-listed gene glutathione S-transferase alpha 2 (GSTA2) and addressed the interaction of RUNX1 and GSTA2 and their functions in AML cells. Real time quantitative PCR (RT-qPCR) and immunoblotting revealed that the expression of GSTA2 was actually up-regulated in sh_Rx1_moderate-transduced AML cells and down-regulated in AML cells transduced with sh_Rx1_profound.Interestingly, equivalent level of compensatory up-regulation of RUNX2 and RUNX3 were observed in sh_Rx1_moderate- and sh_Rx1_profound-transduced AML cells, creating an absolute gap in the expression of total amount of RUNX (RUNX1 + RUNX2 + RUNX3), which was confirmed by RT-qPCR (total amount of RUNX expressions were estimated by primers amplifying the specific sequence common to all RUNX family members). Luciferase reporter assay of GSTA2 promoter and chromatin immunoprecipitation (ChIP) assay in the proximal promoter region of GSTA2 gene proved the association of RUNX family members with this genomic region. These results indicated that total amount of RUNX family expressions modulate the expression of GSTA2 in AML cells, which might results in a paradoxical outbursts of RUNX1 moderately-inhibited AML cells.Since GSTA2 catabolizes and scavenges free radicals such as hydrogen peroxide (H2O2), and decreased intracellular free radicals promote acceleration of cell cycle progression, we next measured the intracellular accumulation of H2O2 in RUNX1 inhibited AML cells. As we have expected, intracellular amount of H2O2 was decreased in sh_Rx1_moderate-transduced AML cells and increased in AML cells transduced with sh_Rx1_profound. Additive transduction of sh_RNAs targeting GSTA2 to AML cells with sh_Rx1_moderate reverted the proliferation speed to the control level, underpinning that growth advantage of moderate RUNX1 inhibition could be attributed to the GSTA2 overexpressions.Taken together, these findings indicate that moderately attenuated RUNX1 expressions paradoxically enhance leukemogenesis in AML cells through intracellular environmental change via GSTA2, which could be a novel therapeutic target in anti-leukemia strategy. DisclosuresNo relevant conflicts of interest to declare.

PCTP (Phosphatidylcholine Transfer Protein) is Regulated By RUNX1 in Platelets/Megakaryocytes and is Associated with Adverse Cardiovascular Events

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5’ upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p < 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value < 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors.Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. DisclosuresNo relevant conflicts of interest to declare.

Transcription Factor RUNX1 Regulates Pctp (Phosphatidylcholine Transfer Protein) in Platelets: A Potential Role in Regulating Platelet Function

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a critical hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, numerous platelet function defects, and increased leukemia risk. We have previously described a patient with a heterozygous RUNX1 nonsense mutation in the conserved runt domain necessary for DNA binding (Sun et al Blood 2004;103;948-54). The patient's platelet abnormalities included impaired aggregation and secretion in response to multiple agonists, granule deficiency, and decreased phosphorylation of myosin light chain and pleckstrin, activation of GPIIb-IIIa, 12-HETE production, and platelet protein kinase C-θ. Transcript expression profiling of patient platelets (Sun et al, J Thromb Haemost 2007;5:146-54) showed numerous genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP is a member of the START (Steroidogenic Acute Regulatory Protein-Related Transfer) domain superfamily and is responsible for the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Several findings indicate that PCTP is important for platelet function. PC is the main fraction of platelet phospholipids and source of arachidonic acid (AA) upon activation. Release of free AA from PC is the rate-limiting step in thromboxane production. PC is also a substrate for phospholipase D, which yields phosphatidic acid that can generate the second messenger diacylglycerol. Importantly, platelet PCTP has recently been demonstrated to have a major role in the racial differences in platelet responses - increased PCTP expression has been linked to the higher platelet aggregation and calcium mobilization induced by thrombin receptor protease-activated receptor 4 (PAR4) in blacks as compared to whites (Edelstein et al Nat Med 2013;19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to 995 bp of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding sites 1 (-232/-227) and 2 (-345/-340), at site 3 (-519/-514), and encompassing sites 4 and 5 (-861/-856, -884/-879). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-232/-227) and both sites 4 and 5 (-861/-856, -884/-879). To assess the effect of modulating RUNX1 expression on PCTP expression, PMA-treated HEL cells were transfected with RUNX1 overexpression plasmid or siRNA. PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown, suggesting that PCTP is regulated by RUNX1.Conclusions: Our results provide evidence that PCTP is a direct transcriptional target of and regulated by RUNX1, and a cogent molecular mechanism for downregulation of platelet PCTP in our patient with RUNX1 haplodeficiency. Regulation of PCTP by RUNX1 may be relevant to the platelet dysfunction in RUNX1 haplodeficiency as well as to racial differences in platelet responses linked to the differential platelet expression of PCTP. DisclosuresNo relevant conflicts of interest to declare.

RUNX2 and the PI3K/AKT axis reciprocal activation as a driving force for tumor progression.

From the first reported role of the transcription factor RUNX2 in osteoblast and chondrocyte differentiation and migration to its involvement in promigratory/proinvasive behavior of breast, prostate, and thyroid cancer cells, osteosarcoma, or melanoma cells, RUNX2 currently emerges as a key player in metastasis. In this review, we address the interaction of RUNX2 with the PI3K/AKT signaling pathway, one of the critical axes controlling cancer growth and metastasis. AKT, either by directly phosphorylating/activating RUNX2 or phosphorylating/inactivating regulators of RUNX2 stability or activity, contributes to RUNX2 transcriptional activity. Reciprocally, the activation of the PI3K/AKT pathway by RUNX2 regulation of its different components has been described in non-transformed and transformed cells. This mutual activation in the context of cancer cells exhibiting constitutive AKT activation and high levels of RUNX2 might constitute a major driving force in tumor progression and aggressiveness.

Structural basis of Ets1 activation by Runx1

Runx1 is required for definitive hematopoiesis and is well-known for its frequent chromosomal translocations and point mutations in leukemia. Runx1 regulates a variety of genes via Ets1 activation on an Ets1•Runx1 composite DNA sequence. The structural basis of such regulation remains unresolved. To address this problem, we determined the crystal structure of the ternary complex containing Runx11-242 and Ets1296-441 bound to T cell receptor alpha (TCRα) enhancer DNA. In the crystal, an Ets1-interacting domain of Runx1 is bound to the Ets1 DNA-binding domain and displaced an entire autoinhibitory module of Ets1, revealing a novel mechanism of Ets1 activation. The DNA binding and transcriptional studies with a variety of structure-guided Runx1 mutants confirmed a critical role of direct Ets1•Runx1 interaction in Ets1 activation. More importantly, the discovered mechanism provides a plausible explanation for how the Ets1•Runx1 interaction effectively activates not only a wild-type Ets1, but also a highly inhibited phosphorylated form of Ets1.

Genomic occupancy of Runx2 with global expression profiling identifies a novel dimension to control of osteoblastogenesis

BackgroundOsteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed.ResultsBy performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation - proliferation, matrix deposition and mineralization - we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions.ConclusionsOur data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis.

The RUNX1-Regulated Cebpa +37 Kb Enhancer Directs Human CD4 Transgene Expression To Long-Term Hematopoietic Stem Cells and Preferentially To Myeloid Compared With Lymphoid Or Erythroid Progenitors

We found that adult mice lacking RUNX1 have increased monopoiesis relative to granulopoiesis correlated with reduce Cebpa RNA expression in myeloid progenitors and that RUNX1 binds and activates Cebpa reporters via two sites in the Cebpa promoter and four conserved sites in a 450 bp +37 kb enhancer (Guo et al, Blood, 2012). This enhancer interacts with p300 and contains the H3K4me1 enhancer mark in myeloid cells. Previously reported ChIP-Seq data had demonstrated interaction of RUNX1 with the +37 kb enhancer, along with GATA2, SCL, Gfi-1b, MEIS1, Fli-1, ERG, PU.1, Lyl1, and LMO2. We have now generated transgenic C57BL/6 mice in which the Cebpa +37 kb enhancer positioned upstream of its 845 bp promoter (-720 to +125) directs expression of cytoplasmically truncated human CD4 (hCD4). Two founders with germ line transmission have been identified – both have detectable hCD4 RNA in marrow, at approximately 10-fold increased levels in one line compared to the other, with hCD4 expression detectable by FACS only in the higher expressing line. Analysis of 4-7 mice reveals that hCD4 expression is detected only in a small subset of mature hematopoietic cells, present in 6% of Mac-1+Gr-1+ neutrophils, 31% of Mac-1+Gr-1- monocytes, 4% of CD3+ T cells, 3% of CD19+ or 7% of B220+ B-cells, and 5% of Ter119+ erythroid cells. 6.5+/-1.9% of total marrow mononuclear cells and 13+/-4% of Lin- marrow cells express hCD4. At the progenitor level, hCD4 was evident in 56+/-7% of CMP, 70+/-5% of GMP, 36+/-9% of CLP, and only 1.5+/-1.0% of MEP. There was>100-fold enrichment of myeloid CFU but a 2-fold decrease in B-lymphoid colonies comparing hCD4+ versus hCD4- fractions of total marrow. Staining LSK cells for CD34 and FcγR demonstrated that 91+/-10% of multi-potent progenitors (MPP), 61+/-8% of ST-HSC, and 17+/-8% of LT-HSC express hCD4. Similarly, staining for the SLAM markers demonstrated that 19+/-9% of LSK-CD48-CD150+ LT-HSC express hCD4. Mean fluorescence index of hCD4 staining was about 2-fold higher in LSK, MPP, ST-HSC, CMP, and GMP compared with CLP and LT-HSC and about 3-fold higher than mature blood myeloid cells. 2E6 hCD4+ or hCD4- CD45.2 marrow cells were transplanted into lethally irradiated CD45.1 syngeneic recipients. 5/5 mice receiving hCD4- cells died at 2 weeks, reflecting lack of even progenitor engraftment, whereas 5/5 mice receiving hCD4+ cells were alive at 20 weeks, reflecting expression of the transgene in functional LT-HSC, with secondary transplanted mice viable at 4 week to date. The marrow cells of surviving mice were almost exclusively CD45.2, with hCD4+ expression in Lin- or Lin-c-kit+ marrow cells similar to that of donor mice. Although our second founder does not allow hCD4 detection by FACS, analysis of hCD4 RNA in marrow stem and progenitor subsets for this line is in progress, as is competitive transplantation of hCD4- versus hCD4+ marrow cells from the higher-expressing line. Murine E13.5 fetal liver hematopoietic stem and progenitor cells express hCD4 in a pattern similar to adult marrow. In zebrafish, the murine Cebpa +37 kb enhancer directs ZsGreen expression specifically to the caudal hematopoietic territory (CHT), analogous to the murine fetal liver, at 72 hour post-fertilization. Murine Cebpa +37 kb enhancer:ZsGreen transgene expression, lacking the Cebpa promoter, overlaps in the CHT with that of murine Runx1 +23 kb enhancer:mCherry in compound transgenic fish. The Cebpa and Runx1 enhancers direct similar tdTomato expression from a Sleeping Beauty vector in the K562 cell line, and these vectors are being compared for expression in hESC derived hematopoietic cells. In summary, the Cebpa gene contains an enhancer active in both LT-HSC and myeloid progenitors. Further characterization of its regulation may provide insights into the development of these hematopoietic stem and progenitor subsets and into how Cebpa expression is impaired in myelodysplastic syndromes or in acute myeloid leukemia. In addition, the Cebpa enhancer may prove useful in efforts to optimize LT-HSC formation from hESC. Disclosures:Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

RUNX1 Regulates PLDN (Pallidin) Expression In Platelets: A Potential Mechanism For Dense Granule Deficiency In RUNX1 Haplodeficiency

Transcription factor RUNX1 plays a major role in hematopoiesis. Haplodeficiency of RUNX1 is associated with familial thrombocytopenia, impaired platelet function and megakaryopoiesis, and predisposition to acute myeloid leukemia. Platelet abnormalities include impaired aggregation, secretion, protein phosphorylation, and activation of GPIIb-IIIa on platelet activation. Dense granule deficiency, either alone or in combination with alpha granule deficiency, has been reported in patients with RUNX1 haplodeficiency, but the mechanisms involved are unknown. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood 87:1368-76, 1996) associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood 103: 948-54, 2004). The platelets showed impaired aggregation, dense granule secretion, phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-θ. Platelet mRNA expression profiling showed downregulation of several genes, including myosin light chain 9 (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRKB), and 12-lipoxygenase (ALOX12) in the patient compared to 6 normal subjects (Sun et al, J. Thromb Haemost. 5: 146-54, 2007). (We have shown that these genes are direct transcriptional targets of RUNX1.) In addition, PLDN, which encodes for protein pallidin, was four-fold down-regulated in platelets (fold change 0.239, p=0.029). In mouse model pallid, knock out of pldn leads to dense granule deficiency. Recent reports document mutations in PLDN in human subjects with platelet dense granule deficiency and the Hermansky-Pudlak syndrome. Pallidin is one of 8 subnunits that constitute the BLOC-1 (biogenesis of lysosome-related organelles complex-1), which plays a major role in granule/vesicle biogenesis. PLDN has two known human transcripts; PLDN1 is expressed ubiquitously, with exception of brain, while PLDN2 is expressed in brain, testes and leukocytes. We have addressed that PLDN is a direct transcriptional target of RUNX1 and hypothesize that its decreased expression constitutes a mechanism for the platelet dense granule deficiency in patients with RUNX1 haplodeficiency.We validated the decreased expression of PLDN on platelet profiling by quantitative real-time PCR - PLDN1 mRNA expression was decreased by 16-fold in the patient relative to normal subjects. We studied the regulation of PLDN1 by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. In silico analyses revealed the presence of 6 RUNX1 consensus binding sites in 2288bp of PLDN1 5' upstream region from ATG. To determine endogenous interaction of RUNX1 with PLDN promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. There was RUNX1 binding to PLDN chromatin at regions encompassing the putative RUNX1 binding site 1 (-184 to -179 bp) and site 3 (-1370/-1365 bp). We performed electrophoretic mobility shift assay (EMSA) using probes with RUNX1 motifs and PMA-treated HEL cell nuclear extracts. With 30–34 bp probes encompassing site 1 (-184 to -179 bp) and site 3 (-1370 to -1365 bp), protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. To study the functional effect of the binding sites, the wild type PLDN1 promoter construct –2288/-2 bp containing the putative RUNX1 sites or mutant constructs with each site individually mutated were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Mutation of RUNX1 sites 1 and 3 individually caused 60-70% reduction in promoter activity indicating that these sites were functional. Studies on the other RUNX1 consensus sites in PLDN promoter are underway. ConclusionsOur results provide the first evidence that PLDN gene is transcriptionally regulated by and is a direct target of RUNX1. These studies provide a cogent mechanism for the PLDN transcript downregulation observed in the patient platelets. More importantly, they provide a mechanism for the dense granule deficiency and impaired vesicle formation associated with RUNX1 haplodeficiency. Disclosures:No relevant conflicts of interest to declare.