RUNX1T1 Expression Research Articles

Abstract 5589: CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine

Abstract CBFB::MYH11 acute myeloid leukemia (AML) is a subset of Core Binding Factor AML initiated by an oncofusion in which Core Binding Factor Beta (CBFB) is fused to the C-terminus of smooth muscle myosin heavy chain 11 (MYH11). CBFB::MYH11 AML has a high cure rate with conventional anthracycline/cytarabine induction and high-dose cytarabine consolidation in patients under the age of 60, and remains chemosensitive after multiple relapses; the mechanisms underlying chemosensitivity are unknown. TP53 is a key regulator of DNA damage response and apoptosis, and TP53-mutated AML is often chemoresistant. Large sequencing studies have revealed only rare co-occurrence of TP53 mutations with CBFB::MYH11. We initially used CBFB::MYH11 as a negative control for an unrelated study of TP53 function and unexpectedly found striking synergy. While expression of CBFB::MYH11 in WT murine hematopoietic stem and progenitor cells (HSPCs) induced a low-penetrance (~20%), long latency (~6 months) myelomonocytic AML, expression of CBFB::MYH11 in Trp53-/- HSPCs induced AML with 100% penetrance and a median latency of 8 weeks. CBFB::MYH11-induced AMLs arising in Trp53-/- cells were similar by morphology and flow cytometry phenotype. We validated these findings in a second model, using a conditional Trp53flox/flox x Vav1-Cre model in which Trp53 is inactivated in HSPCs, with nearly identical results. Trp53-/- cells transduced with Empty Vector did not develop AML, but did develop T cell lymphoma with a median latency of 16 weeks, similar to prior studies. Trp53 deficiency also cooperated with RUNX1::RUNX1T1, the other Core Binding Factor oncofusion, to induce a high-penetrance (100%) and short latency AML (median latency 10 weeks); consistent with prior data, RUNX1::RUNX1T1 expression alone in Trp53 WT cells was insufficient to induce AML. To determine whether Trp53 influences the chemosensitivity of CBFB::MYH11 expressing cells, we performed cytarabine dose-response experiments. While CBFB::MYH11-transduced WT HSPCs were very sensitive to cytarabine (relative to EV), sensitivity was reduced in CBFB::MYH11-transduced Trp53-/- cells, suggesting that CBFB::MYH11-induced chemosensitivity is, at least in part, mediated by the TP53 pathway. To determine whether CBFB::MYH11 expression results in upregulation of key TP53 pathway genes, we transduced WT HSPCs with EV or CBFB::MYH11, transplanted cells into WT mice, harvested bone marrow at 3 months, and performed RNA-Seq. Key TP53 pathway genes were upregulated, including Bbc3/Puma, Bax, Cdkn1a/p21, and Trp53 (all 2.6-4.6-fold, p≤0.05). These data suggest that the chemosensitivity and the favorable prognosis of CBFB::MYH11 AML may be due to the direct activation of the TP53 pathway by CBFB::MYH11. Similar studies are underway with RUNX1::RUNX1T1-transduced cells to determine whether TP53 is relevant for the chemosensitivity of that CBF AML as well. Citation Format: Ryan B. Day, Julia A. Hickman, Casey D. Katerndahl, Sai Mukund Ramakrishnan, Christopher A. Miller, Timothy J. Ley. CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5589.

Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

Hepatic Runx1t1 improves body fat index after endurance exercise in obese mice

Endurance exercise could attenuate obesity induced by high fat diet (HFD). Thus, the purpose of this study was to explore the crucial targets that play key roles in the improvement of body fat index (BFI) in obese mice by endurance exercise. Firstly, we constructed murine obesity models: High fat diet control (HFD) group, HFD exercise (HFE) group, normal chow diet control (NC) group, and normal chow diet exercise (NE) group. Next, we identified the BFI improvement related genes using differential gene analysis, and investigated these genes’ functional pathways using functional enrichment analysis. The qRT-PCR and western blot assays were used to determine the gene expression and protein expression, respectively. Gene set enrichment analysis was used to explore the potential pathways associated with endurance exercise in obese mice and Mitochondrial respiratory control ratio (RCR) assay was applied to determine the RCR in the liver tissues of mice. We discovered that endurance exercise remarkably reduced the body weights and BFI of HFD-induced obese mice. Runx1t1 was related to the improvement of BFI by endurance exercise in HFD-induced obese mice. Runx1t1 mRNA and protein levels in liver tissues were observably decreased in HFD mice compared to mice in HFE, NC and NE groups. Moreover, Glucagon signaling pathway that was associated with mitochondrial function was significantly activated in HFE mice. The Runx1t1 expression exhibited an observable negative correlation with Acaca in HFD mice. Moreover, the mitochondrial RCR level was significantly increased in HFE mice than that in HFD mice. In HFD-induced obese mice, Runx1t1 was implicated in the improvement of BFI via endurance exercise. Endurance exercise could improve mitochondrial dysfunction in obese mice by activating the Runx1t1.

MiR-210-3p accelerates tumor-relevant cell functions of endometrial carcinoma by repressing RUNX1T1

BackgroundBiological mechanism of miR-210-3p in endometrial carcinoma (EC) remains unclear. Here, our purpose is to study effects of miR-210-3p on malignant progression of EC. MethodsBioinformatics analysis showed miRNA and mRNA are abnormally expressed in EC tissues. Quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) was utilized to compare miR-210-3p mRNA level in EC cells and tissues. qRT-PCR and western blot were used to measure RUNX1T1 and NCAM1 at mRNA and protein levels, and western blot for p-AKT and AKT proteins related to PI3K/AKT signaling pathway. Furthermore, EC cell behaviors were assayed via Cell Counting Kit-8, cell colony formation assay, wound healing, transwell and flow cytometry experiments. Interaction between RUNX1T1 and miR-210-3p was verified through dual-luciferase assay. Immunohistochemistry was used to analyze RUNX1T1 expression in clinical samples ResultsMiR-210-3p was considerably upregulated and RUNX1T1 was significantly under-expressed in EC. Overexpression of miR-210-3p stimulated cell proliferation, migration, invasion, and restrained cell apoptosis in EC. Dual-luciferase assay proved that RUNX1T1 was a target gene of miR-210-3p. The level of RUNX1T1 in EC was downregulated after overexpressing miR-210-3p. Rescue assay showed that overexpression of RUNX1T1 had an inhibitory impact on tumor-relevant cell behaviors, whereas overexpression of miR-210-3p rescued such inhibition. Overexpression of RUNX1T1 reduced p-AKT expression, which was restored with concomitantly overexpressed miR-210-3p. ConclusionIn general, miR-210-3p behaves as an oncogene in EC by down-regulating the expression of RUNX1T1. This study elucidates a new functional mechanism in EC, and indicates miR-210-3p an underlying target.

RUNX1T1, a potential prognostic marker in breast cancer, is co-ordinately expressed with ERα, and regulated by estrogen receptor signalling in breast cancer cells.

RUNX1T1 is extensively studied in the context of AML1-RUNX1T1 fusion protein in acute myeloid leukemia. Little is known about the function of RUNX1T1 itself, although data on its function and regulation have begun to emerge from clinical, and in vitro studies. It is a putative tumor suppressor, whose expression is altered in a variety of solid tumors. Recently, reduced expression of RUNX1T1 in triple-negative breast tumors, and its influence on prognosis was reported. The Kaplan-Meier Plotter online tool was used to study the relationship between RUNX1T1 expression and survival of breast cancer patients. High RUNX1T1 expression was associated with longer overall survival (OS), relapse-free survival (RFS) and distant metastasis free survival (DMFS). RUNX1T1 expression positively and negatively influenced OS of patients with ERα-positive and ERα-negative breast tumors, respectively. It was also associated with prolonged RFS, and DMFS in tamoxifen-treated patients. Expression of RUNX1T1 and ERα mRNA was analyzed in 40 breast tumor samples, and breast cancer cell lines using RT-PCR. TCGA-BRCA data was mined to study the relationship between RUNX1T1 and ERα mRNA expression. ERα-positive breast tumors showed significantly higher RUNX1T1 mRNA expressioncompared to ERα-negative tumors. RUNX1T1 mRNA expression was analyzed by qRT-PCR in MCF-7 or T47D cells, which were treated with 17β-estradiol, or the ERα agonist PPT, alone or in combination with 4-hydroxytamoxifen. Effect of ERα knockdown was also investigated. Results indicate that estrogen downmodulated RUNX1T1 mRNA expression via ERα. Higher expression of RUNX1T1 in breast tumors is associated with favourable prognosis. RUNX1T1 and ERα show co-ordinated expression in breast tumors, and breast cancer cell lines. Estrogen-ERα signalling downmodulates the expression of RUNX1T1 mRNA in ERα-positive breast cancer cells. In-depth investigations on the interaction between RUNX1T1 and ERα are warranted to unravel the role and relevance of RUNX1T1 in breast cancer.

RBM24 exacerbates bladder cancer progression by forming a Runx1t1/TCF4/miR-625-5p feedback loop

RNA–binding motif protein 24 (RBM24) acts as a multifunctional determinant of cell fate, proliferation, apoptosis, and differentiation during development by regulating premRNA splicing and mRNA stability. It is also implicated in carcinogenesis, but the functions of RBM24 in bladder cancer (BC) remain unclear. In the present study, we revealed that RBM24 was upregulated in BC tissues. Importantly, we found that a higher level of RBM24 was correlated with poor prognosis in BC patients. Overexpression of RBM24 promoted BC cell proliferation, while depletion of RBM24 inhibited BC cell proliferation in vivo and in vitro. Mechanistically, RBM24 positively regulated Runx1t1 expression in BC cells by binding to and enhancing Runx1t1 mRNA stability. Furthermore, Runx1t1 in turn promoted RBM24 expression by interacting with the transcription factor TCF4 and suppressing the transcription of miR-625-5p, which directly targets RBM24 and suppresses RBM24 expression. RBM24-regulated BC cell proliferation was moderated via the Runx1t1/TCF4/miR-625-5p feedback loop. These results indicate that the RBM24/Runx1t1/TCF4/miR-625-5p positive feedback loop participates in BC progression. Disruption of this pathway may be a potential therapeutic strategy for BC treatment.

Identification of RUNX1T1 as a potential epigenetic modifier in small-cell lung cancer.

Small-cell lung cancer (SCLC) can be subgrouped into common 'pure' and rare 'combined' SCLC (c-SCLC). c-SCLC features a mixed tumor histology of both SCLC and non-small-cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c-SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c-SCLC, but in only 2 of 88 'pure' SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%-15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c-SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP-seq data in Kasumi-1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

Long non-coding RNA EPB41L4A-AS2 suppresses progression of ovarian cancer by sequestering microRNA-103a to upregulate transcription factor RUNX1T1.

What is the central question of this study? What is the specific mechanism by which EPB41L4A-AS2 exerts a regulatory role in ovarian cancer? What is the main finding and its importance? Overexpressed EPB41L4A-AS2 inhibited cell proliferation, migration and invasion in ovarian cancer by upregulating RUNX1T1 through downregulation of miR-103a. This study provides new insight into the role of EPB41L4A-AS2 in ovarian cancer. Ovarian cancer (OC) is a malignant tumour with a poor prognosis. Emerging evidence has shown that long non-coding RNAs (lncRNAs) are regulators that can be used for prognosis, diagnosis and targeted therapy of cancers. Therefore, our purpose was to investigate the possible regulatory role of the lncRNA EPB41L4A-AS2 in the progression of OC. Initially, EPB41L4A-AS2 expression was determined in OC tissues and matched paracancerous tissues. Then the RNA crosstalk among EPB41L4A-AS2, miR-103a and RUNX1T1 was determined. Subsequently, the expression of EPB41L4A-AS2, miR-103a and RUNX1T1 was up- or downregulated by exogenous transfection in SK-OV-3 cells to investigate their roles in the proliferation, migration, colony formation and invasion of OC cells. Further, the tumour formation ability of nude mice was tested in vivo. EPB41L4A-AS2 was poorly expressed in OC tissues and cells, and microarray data revealed upregulation of miR-103a and downregulation of RUNX1T1 in OC. RUNX1T1 was a target gene of miR-103a and EPB41L4A-AS2 bound to miR-103a. Moreover, EPB41L4A-AS2 increased RUNX1T1 expression by decreasing miR-103a expression. EPB41L4A-AS2-overexpressing SK-OV-3 cells exhibited inhibited proliferation, migration, colony formation and invasion, which was rescued by overexpression of miR-103a or silencing of RUNX1T1. Besides, overexpressed EPB41L4A-AS2 repressed tumour formation in vivo. Altogether, the current study demonstrates that overexpressed EPB41L4A-AS2 can potentially bind to miR-103a to promote the expression of RUNX1T1, thereby inhibiting OC, highlighting the potential of EPB41L4A-AS2 as a target forOC.

Defining the role of RUNX1T1 in "combined" small cell lung cancer.

e20101 Background: The RUNX1T1 gene, also known as ETO, was first identified in the fusion transcript AML1/ETO, generated by the translocation between chromosome 8 and 21, which occurs in 12%-15% of AML. We recently discovered that RUNX1T1 was specifically amplified only in the small cell lung cancer (SCLC) but not the non-small cell lung cancer (NSCLC) component of combined SCLC tumors. Here, we investigated if RUNX1T1 expression plays a role in transforming NSCLC to SCLC in combined SCLC tumor formation. Methods: We examined tumors from 92 of our SCLC patients by targeted exome and CNV analyses. RUNX1T1 mRNA expression was determined in various cancers by analysis of the CCLE and TCGA databases. RUNX1T1 mRNA expression was detected by RNAscope analysis of another 22 patient SCLC tumors. Stable overexpression of RUNX1T1 in NSCLC and SCLC cell lines used commercial RUNX1T1 lentiviral constructs. The resulting stable cell lines were analyzed by RT-qPCR, western blotting, Affymetrix gene array, and E2F-luciferase assays. Results: We found that 2 of 2 patients with combined SCLC demonstrated RUNX1T1 amplification in the SCLC component. Notably, only 2 of the remaining 90 patients with pure SCLC demonstrated RUNX1T1 amplification. RUNX1T1 mRNA is highly expressed in SCLC compared to the majority of other cancers, including NSCLC, using either CCLE or TCGA databases. Western blots confirmed higher RUNX1T1 protein expression in SCLC vs NSCLC cell lines, and RNAscope analysis confirmed greater RUNX1T1 mRNA expression in SCLC vs NSCLC patient tumors. When we stably overexpressed RUNX1T1 in NSCLC cell lines, there was no increase in neuroendocrine protein expression, however we did find an increased sensitivity to etoposide. Bioinformatic analysis of microarray data demonstrated significantly altered E2F pathway activity, and we could show a significant increase in E2F-luciferase activity after RUNX1T1 overexpression. Interestingly, this was associated with decreased p21 expression, which is a putative suppressor of E2F activity. Conclusions: RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. E2F pathway activity is elevated after RUNX1T1 overexpression, consistent with an increased SCLC phenotype.

Abstract 2495: RUNX1T1 is amplified in combined small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a highly aggressive malignancy that expresses neuroendocrine (NE) genes and represents approximately 15% of all lung cancers; the remaining histological subtype is non-small cell lung cancer (NSCLC). Clinically, there are two types of SCLC, termed ‘pure' and ‘combined'. Combined SCLC presents as a mixture of SCLC and NSCLC components within the same tumor mass. We analyzed 88 SCLC patient tumors by targeted exome sequencing and found that four SCLC patients demonstrated RUNX1T1 amplification. Interestingly, among these 88 SCLC patients there were two with combined SCLC and both demonstrated RUNX1T1 amplification only in the small cell component. Neither MYC (8p24) nor FGFR1 (8p11), two genes commonly amplified in SCLC that are nearby the RUNX1T1 locus (8p22), were co-amplified. RUNX1T1, also called ETO or MTG8, was first identified in the oncogenic fusion transcript AML1/ETO in acute myelogenous leukemia (AML) and was previously reported as a transcriptional co-repressor. Based on our observation we hypothesized that: 1) RUNX1T1 may be a specific marker for SCLC and may play a role in determining the SCLC phenotype, including NE expression, and 2) RUNX1T1 may play a role in transforming NSCLC to SCLC in the early stages of combined SCLC tumor formation. First we examined RUNX1T1 mRNA expression among cancer cell lines using the CCLE database and found that RUNX1T1 is highly expressed in SCLC cells compared to other cancer cells, especially NSCLC cells. This was also true in tumors using RNAseq data. Consistent with these mRNA profiles, our western blot results showed higher RUNX1T1 protein expression in SCLC compared to NSCLC cell lines. These findings validate that RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. To investigate a role for RUNX1T1 in determining the SCLC phenotype, we overexpressed RUNX1T1 in SCLC cell lines by lentiviral transduction and observed marked growth suppression in H82, H2171, and H841 cells. Conversely, transient knock down of RUNX1T1 expression in the SCLC cell line H446 using siRNA decreased the expression of two NE proteins (NEUROD1 and TTF1). To explore a potential role of RUNX1T1 in NSCLC to SCLC transformation, we overexpressed RUNX1T1 in three NSCLC cell lines (PC-9, H1650 and A549) but observed no obvious phenotypic changes and no increase in NE gene expression. In conclusion, we have demonstrated that RUNX1T1 expression is upregulated in SCLC compared to NSCLC. Moreover, the results from RUNX1T1 overexpression and knockdown experiments in SCLC cell lines indicates a potential role of RUNX1T1 in regulating the SCLC phenotype and NE expression. Citation Format: Tian He, Karen McColl, Alyssa Savadelis, Yanwen Chen, Gary Wildey, Afshin Dowlati. RUNX1T1 is amplified in combined small cell lung cancer [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 2495.

Runx1t1 regulates the neuronal differentiation of radial glial cells from the rat hippocampus.

The brain has the highest Runx1t1 level relative to the levels in other organs. Runx1t1 might have a regulatory function as a transcriptional corepressor in the differentiation/development of the nervous system. Neurogenesis requires factors that regulate the proliferation of progenitors and activate the neuronal differentiation process. However, the precise role of Runx1t1 in hippocampal neurogenesis is unclear. We knocked down Runx1t1 in hippocampal radial glial cells (RGCs) with Runx1t1-RNA interference using lentiviral vectors. We also used LV-Runx1t1 to induce Runx1t1 overexpression in vitro. We have provided experimental evidence that decreased Runx1t1 expression reduced the neuronal differentiation of RGCs, and increased Runx1t1 expression caused a greater number of RGCs to differentiate into neurons. We have concluded that Runx1t1 could be involved in the process through which RGCs differentiate into neurons.

Abstract 526: Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung

Abstract The amplification of the distal portion of chromosome 3q in lung cancer is a major signature of neoplastic transformation, particularly in the squamous cell carcinoma (SCC) of the lung. A number of potential drivers in the 3q amplicon have been identified and are proposed to contribute to the development of lung SCC. miRNAs have emerged as a new class of small, non-coding RNAs that regulate gene expression and might act as integral parts of the molecular architecture of oncogene and tumor suppressor networks in human cancer, including non-small cell lung cancer (NSCLC). miR 1224-5p, 1248 and 944 are located at 3q amplicon and overexpressed in 50%, 26% and 53% of 30 primary SCCs when compared to normal tissues. The overexpression of these miRNAs was further validated in 237 TCGA lung SCC samples compared with 35 normal tissues (p=0.007, 1.18E-16 and 7.71E-20, respectively). The expression of the three miRNAs was higher in lung SCC compared to lung adenocarcinomas (p=8.19E-05, 1.04E-33 and 3.37E-95, respectively), suggesting unique roles of these miRNAs in the lung SCC development. Using miRNA mRNA target prediction tools (miRANDA and TargetScan) and in the combination with a literature search, we found RUNX1T1 and SMAD4 were candidate tumor suppressor gene target for miRNA-1224-5p and miRNA-1248. Downregulation of RUNX1T1 and SMAD4 were confirmed in TCGA lung SCC dataset. Using real-time RT-PCR, we found that the same three miRNAs 1224-5p, 1248 and 944 are overexpressed in 3q amplified lung SCC cell lines H520 and HCC95. When miRNA inhibitors were used to repress the expression of miR-1224-5p or miR-1248 in H520, we found that RUNX1T1 and SMAD4 protein expression levels were increased, suggesting that these tumor suppressor genes are regulated by miR-1224-5p or miR-1248. Lastly, knockdown of miR-1224-5p or miR-1248 using miRNA inhibitors led to 35-60% growth inhibition in H520 and HCC95 cells. Together, these results indicate potential roles for miR-1245-5p and 1248 in regulating cell proliferation by directly targeting tumor suppressor genes RUNX1T1 or SMAD4 in lung SCC. This work provides, for the first time, evidence for overexpression of miRNAs at the 3q26-29 amplicon that might be implicated in the oncogenesis of lung SCC. The work is supported by the NIH Grant R01 CA102353 to PPM and Lung Cancer Research Foundation grant (2011) to JQ. Note: This abstract was not presented at the meeting. Citation Format: Jun Qian, Xiangming Ji, Yong Zou, Megan Hoeksema, Heidi Chen, Pierre P. Massion. Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 526. doi:10.1158/1538-7445.AM2014-526

Runx1t1 (Runt-Related Transcription Factor 1; Translocated to, 1) Epigenetically Regulates the Proliferation and Nitric Oxide Production of Microglia

BackgroundMicroglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions.Methods and FindingssiRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2.Conclusion/InterpretationIn conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs.

Expression of RUNX1 and RUNX1T1 Correlates with Cytogenetic Subgroups in Pediatric AML: A Report From the Children's Oncology Group

AbstractAbstract 1425 Background:The t(8;21) translocation results in the fusion oncogene RUNX1-RUNX1T1 (also called AML1-ETO) and confers a favorable prognosis in both pediatric and adult AML. Little is known about the expression of RUNX1 and its translocation partner RUNX1T1 in myeloid leukemia. In this study we examined the mRNA expression of both genes in children with newly diagnosed AML and correlated patient clinical features with gene expression. Methods:We isolated mRNA from diagnostic specimens of 206 patients enrolled on COG AAML03P1. RUNX1 and RUNX1T1 mRNA expression was measured using qRT-PCR. GUSB gene served as control for mRNA quality and to standardize expression. Expression was correlated with patient and disease characteristics and clinical outcomes. Results:Relative RUNX1 expression ranged from 0.09 to 11.32 with a median of 1.45. The patient population was divided into quartiles (n=51, 52, 51, 52) with quartile 1 (Q1) having the lowest expression and quartile 4 (Q4) the highest expression. There was no significant difference in sex, age, race, ethnicity, hematologic parameters at diagnosis, or CNS status. Cytogenetic groups t(8;21), inv(16), abnormal 11q23, and monosomy 7 differed significantly in RUNX1 expression. The percentage of patients with t(8;21) decreased for increasing quartiles of RUNX1 expression with 28% of Q1 having t(8;21) compared to 12% of Q2, 8% of Q3 and 0% of Q4 (p=<0.001). Conversely, the percentage of patients with inv(16) increased for increasing quartiles of RUNX1 expression with only 2% of Q1 having inv(16) compared to 12% of Q2, 27% of Q3 and 20% of Q4 (p=0.006). Percentage of patients with abnormal 11q23 decreased for increasing quartiles of RUNX1 expression (Q1=36%, Q2=22%, Q3=15% and Q4=7%, p=0.003). All 4 patients with monosomy 7 had RUNX1 expression levels in Q4 (p=0.005). RUNX1 expression did not differ by FLT3-ITD, CEBPa mutant and NPM mutant. On recent COG studies, low risk includes core binding factor (CBF) patients and CEBPa or NPM mutant, high risk -5/5q-, −7 or FLT3-ITD high AR, and standard risk being all others. There was no difference in distribution of low or standard risk patients. The high risk group had higher RUNX1 expression attributable to the high expression in monosomy 7 (p=0.027). There was no difference across quartiles for rates of CR, 5 yr EFS or OS.Relative RUNX1T1 expression had a wide range from 0 to 139,786 with 69% of patients having no detectable expression. Due to the number of patients with no expression and the logarithmic distribution of expression, we divided the cohort into no expression (“exp=0”, n=143), expression levels >0 to 1 (“exp>0–1”, n=23) and expression >1 (“exp>1”, n=40). Among these 3 groups, there was no difference in sex, age, race, ethnicity, or CNS status. WBC at diagnosis was higher (median 38,000/μL) for exp=0 compared to exp>0–1 (median 14,100/μL) and exp>1 (median 15,800/μL) (p=0.045). RUNX1T1 expression differed significantly in patients with t(8;21) and monosomy 7. Patients with t(8;21) made up a larger proportion of the high RUNX1T1 expression group including 35% (13/37) having exp>1 compared to 0% of patients with exp>0–1 and 7% (10/134) with exp=0 (p=<0.001). Inv(16) patients showed trend toward the lower RUNX1T1 expression group but not significantly (p=0.11). Monosomy 7 patients clustered in the higher expression group accounting for 8% (3/37) of patients with exp>1 compared to 0% with exp>0–1 and 1% (1/134) with exp=0 (p=0.017). Expression did not differ in the molecular groups or risk groupings. Rates for CR, OS and EFS did not differ significantly by RUNX1T1 expression. However, there was a trend toward higher relapse risk among those with higher RUNX1T1 expression in standard risk patients (exp=0, RR=34±14%; exp>0–1, RR=28±33%; exp >1, RR=68%±33; p=0.138). Conclusion:Both RUNX1 and RUNX1T1 expression correlate with cytogenetic subgrouping, particularly in CBF and monosomy 7. Patients with inv(16) and monosomy 7 make up a larger percentage of the high RUNX1 expression patients but t(8;21) patients tend to be in the low expression groups. For RUNX1T1, t(8;21) and monosomy 7 patients had higher percentages in the high expression group, while inv(16) patients showed a trend toward the lower expression group. RUNX1 or RUNX1T1 expression levels alone did not predict OS or EFS, but further study is warranted to understand the role of high RUNX1 and RUNX1T1 expression in the rare high risk pediatric patients with monosomy 7. Disclosures:No relevant conflicts of interest to declare.

Mir193b–365 is essential for brown fat differentiation

Mammals have two principal types of fat. White adipose tissue (WAT) primarily serves to store extra energy as triglycerides, while brown adipose tissue (BAT) is specialized to burn lipids for heat generation and energy expenditure as a defense against cold and obesity 1, 2. Recent studies demonstrate that brown adipocytes arise in vivo from a Myf5-positive, myoblastic progenitor by the action of Prdm16 (PR domain containing 16). Here, we identified a brown fat-enriched miRNA cluster, miR-193b-365, as a key regulator of brown fat development. Blocking miR-193b and/or miR-365 in primary brown preadipocytes dramatically impaired brown adipocyte adipogenesis by enhancing Runx1t1 (runt-related transcription factor 1; translocated to, 1) expression whereas myogenic markers were significantly induced. Forced expression of miR-193b and/or miR-365 in C2C12 myoblasts blocked the entire program of myogenesis, and, in adipogenic condition, miR-193b induced myoblasts to differentiate into brown adipocytes. MiR-193b-365 was upregulated by Prdm16 partially through Pparα. Our results demonstrate that miR-193b-365 serves as an essential regulator for brown fat differentiation, in part by repressing myogenesis.

Aberrant TGFβ/SMAD4 signaling contributes to epigenetic silencing of a putative tumor suppressor, RunX1T1 in ovarian cancer

Aberrant TGFβ signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGF-beta signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (P=0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (P<0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGFβ/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis.

RUNX1T1

Using gene expression profiling on frozen primary pancreatic endocrine tumors (PETs), we discovered RUNX1T1 as a leading candidate progression gene. This study was designed (1) to validate the differential expression of RUNX1T1 protein on independent test sets of metastatic and nonmetastatic PETs and (2) to determine if RUNX1T1 underexpression in primary tumors was predictive of liver metastases. Immunohistochemical expression of RUNX1T1 protein was quantified using Allred scores on archival metastatic (n = 13) and nonmetastatic (n = 24) primary adult PET tissues using custom-designed tissue microarrays. Wilcoxon rank sum/Fisher exact tests and receiver operating characteristic curves were used in the data analysis. Median RUNX1T1 scores were 2 (2-7) and 6 (3-8) in metastatic versus nonmetastatic primaries (P < 0.0001). Eleven of 13 metastatic and 1 of 24 nonmetastatic primaries exhibited RUNX1T1-scores of 4 or less (P < 0.0001). Low RUNX1T1 expression was highly associated with hepatic metastases (P < 0.0001), whereas conventional histological criteria (Ki-67 index, mitotic rate, necrosis) were weakly associated with metastases (P = 0.08-0.15). Considering RUNX1T1 expression (Allred) score of 4 or less to be predictive, the sensitivity to predict hepatic metastases was 85%, with a specificity of 96%. RUNX1T1 protein is underexpressed in well-differentiated metastatic primary PETs relative to nonmetastatic primaries and emerges as a promising novel biomarker for prediction of liver metastases.

Metastasis-Associated Gene Products and Liver Metastases in Pancreatic Endocrine Tumors

Background: Patient outcome in well-differentiated pancreatic endocrine tumors can be difficult to predict based exclusively on pathologic criteria. We recently identified a novel set of 3 metastasis-associated genes by microarray analysis of fresh-frozen pancreatic endocrine neoplasms: Palladin, p21, RUNX1T1. The aim of this analysis was to evaluate the potential for these markers, individually or in combination, to predict liver metastases as an indicator of adverse outcome. Methods: Palladin, p21, and RUNX1T1 immunostains were carried out on a tissue microarray of 39 resected primary pancreatic endocrine neoplasms, 14 of which had hepatic metastases. The Allred score was independently determined by 2 pathologists as the sum of stain intensity (scored 0-3) and % cells stained (scored 0-5). Receiver operating characteristic (ROC) analysis was used to choose the cutpoint in Allred score (high vs low protein expression) to optimize sensitivity and specificity for predicting liver metastases. Results: Nearly all tumors with liver metastases showed high Palladin and p21 expression (Allred score > 3 and > 4, respectively), while expression was lower in the majority of non-metastatic tumors. In contrast, RUNX1T1 expression was low (Allred score < 4) in most tumors with liver metastases, but was higher in all except one of the non-metastatic tumors. Individual test sensitivities for predicting liver metastases were 100% for high Palladin, 93% for high p21 and 85% for low RUNX1T1, while corresponding specificities were 63%, 75%, and 96%. Tumors were correctly classified as being metastatic or not (predictive accuracy) by Palladin, p21, or RUNX1T1 expression in 76%, 76%, and 92% of cases, respectively. If abnormal expression of even one of 3 proteins is considered a positive test (parallel testing), then sensitivity of all 3 together for predicting liver metastases was 100%, specificity 48%, and predictive accuracy 68%. Conclusions: 1) High Palladin, high p21, or low RUNX1T1 expression have good sensitivity and specificity for predicting liver metastases in pancreatic endocrine tumors. 2) Parallel testing with all 3 markers achieved 100% sensitivity but at a cost of reduced specificity. 3) Differential expression of these biomarkers may predict aggressive tumor behavior that warrants more aggressive management.

RUNX1T1

Background: Clinical outcome of patients with well-differentiated pancreatic endocrine tumors (PETs) and carcinomas (PECAs) is often difficult to predict based on conventional pathologic characteristics. We identified a set of candidate progression genes with differential expression in the primary PECAs with liver metastases relative to non-metastatic primary PETs by using gene expression profiling and validated the selected candidates by real-time-PCR. One of the leading progression genes was RUNX1T1. The aim of this analysis was to validate the differential expression of RUNX1T1 at the protein level on independent test sets of PETs and PECAs, and determine if loss of RUNX1T1 expression was predictive of liver metastases in well-differentiated primary PETs. Methods: We determined immunohistochemical expression of RUNX1T1 protein in archival PECAs with liver metastases (N = 13) and non-metastatic PETs (N = 24), using a custom tissue microarray. Allred score (0-8) was used as an independent semi-quantitative measure of RUNX1T1 expression by two pathologists. Results: RUNX1T1 protein was expressed by all 37 primary tumors, however, median RUNX1T1 Allred score was 2 (range 2-7) in metastatic PECAs compared to a median of 6 (range 3-8) in non-metastatic PETs (p < 0.0001). Allred scores of ≤4 were observed in 11 of 13 metastatic PECAs, but in only 1 of 24 non-metastatic PETs (p < 0.0001). If an Allred score of ≤4 was considered predictive, the sensitivity of this test for predicting hepatic metastases was 85%, with a specificity of 96%. Low RUNX1T1 expression was highly associated with the presence of hepatic metastases (p < 0.0001), while conventional histologic criteria (Ki67 proliferation index, mitotic rate, presence of necrosis) were only weakly associated with metastases (p = 0.08-0.15). Conclusions: RUNX1T1 is differentially expressed in metastatic pancreatic endocrine carcinomas relative to non-metastatic pancreatic endocrine tumors and is a promising marker for prediction of liver metastases in well-differentiated, primary pancreatic endocrine neoplasms.

Expression of metastasis-associated gene products and liver metastases in pancreatic endocrine tumors

11089 Background: Outcomes in well-differentiated pancreatic endocrine tumors can be difficult to predict using pathologic criteria. We recently identified a novel set of 3 metastasis-associated genes by microarray: Palladin, p21, RUNX1T1. Our aim was to evaluate the potential for these markers, individually or in combination, to predict liver metastases as an indicator of adverse outcome. Methods: Palladin, p21, and RUNX1T1 immunostains were done on a tissue microarray of 39 resected primary pancreatic endocrine neoplasms, 14 of which had hepatic metastases. The Allred score was determined as the sum of stain intensity (scored 0–3) and % cells stained (scored 0–5). Receiver operating characteristic (ROC) analysis was used to choose the cutpoint in Allred score (high vs low protein expression) to optimize sensitivity and specificity for predicting liver metastases. Results: Nearly all tumors with liver metastases showed high Palladin and p21 levels (Allred score &gt; 3 and &gt; 4, respectively), while protein expression was lower in the majority of non-metastatic tumors. In contrast, RUNX1T1 expression was low (Allred score &lt; 4) in most tumors with liver metastases, but higher in all except one of the non-metastatic tumors. Individual test sensitivities for predicting liver metastases were 100% for high Palladin, 93% for high p21 and 85% for low RUNX1T1, while corresponding specificities were 63%, 75%, and 96%. Tumors were correctly classified as being metastatic or not (predictive accuracy) by Palladin, p21, or RUNX1T1 expression in 76%, 76%, and 92% of cases, respectively. If abnormal expression of even one of 3 proteins is considered a positive test (parallel testing), then sensitivity of all 3 together for predicting liver metastases was 100%, specificity 48%, and predictive accuracy 68%. Conclusions: 1) High Palladin, high p21, or low RUNX1T1 expression have good sensitivity and specificity for predicting liver metastases in pancreatic endocrine tumors. 2) Parallel testing with all 3 markers achieved 100% sensitivity but at a cost of reduced specificity. 3) Differential expression of these biomarkers may predict aggressive tumor behavior that warrants more aggressive management. No significant financial relationships to disclose.