Suppression Of RUNX1 Research Articles

In Silico approach: Design an Optimized shRNA against RUNX1 Gene to Target HIV.

Human Immunodeficiency Virus (HIV) is a retrovirus with single-stranded RNA that leads to the challenging disease of acquired immunodeficiency syndrome (AIDS). Combination antiretroviral therapy (cART) can prevent the progression of the disease, but it is not capable of long-term HIV elimination. One of the significant obstacles to treating HIV-1-infected individuals is the creation of latent cell reservoirs early in the infection. Gene-based therapies that utilize RNA interference (RNAi) to silence host or viral gene expression are considered promising therapeutic approaches. It has been demonstrated that RUNX1, a T-cell-specific transcription factor, may significantly affect HIV replication and infection. According to accumulating evidence on the role of interfering RNA techniques in inhibiting gene expression and considering the role of RUNX1 in the replication of HIV-1. In this study, we aim to design shRNAs against RUNX1 that can target the replication of HIV-1. </P> Methods: Several computational methods, including target alignment, similarity search, and secondary structure prediction, have been employed in the design of shRNA against RUNX1. </P> Results: Seven shRNA molecules with the highest efficiency were designed and validated using computational methods to silence the RUNX1 gene. </P> Conclusions: In the present study, we designed shRNA against RUNX1, which can target latent cells infected with HIV. Suppression of RUNX1 by shRNA reactivates HIV in the latent cells and subsequently potentiates the immune response toward identifying accurate virus-infected cells. This process may lead to an effective and efficient reduction of the volume of cell reservoirs infected with HIV.

Abstract 1570: STAT3 inhibition allows for TLR9-induced reprogramming of acute myeloid leukemia into antigen-presenting cells to generate T-cell mediated immune responses

Abstract Signal transducer and activator of transcription factor 3 (STAT3) is commonly activated in acute myeloid leukemia (AML) and known for supporting cancer cell proliferation and survival. Recently, we demonstrated that STAT3 also plays a critical role ensuring AML immune evasion. Intravenous injections of a bi-specific oligonucleotides (CpG-STAT3 decoy inhibitors) blocked STAT3 activity and induced TLR9 signaling in Cbfb/MYH11/Mpl (CMM) AML cells, thereby resulting in immunogenic effects and T cell-mediated immune responses and leukemia regression. To understand the molecular mechanisms of AML differentiation and immunogenicity, we performed global gene expression analysis of leukemic cells treated in vivo using CpG-STAT3 decoy strategy or an inducible STAT3silencing. Transcriptional profiling revealed the upregulation of myeloid cell differentiation related genes, such as Irf8, Cebpa, and Gadd45A with reduction of oncogenic Runx1 and Run1t1 in CMM leukemic cells after CpG-STAT3dODN but not after control treatments. CpG-STAT3dODN treatment also upregulated set of antigen-presentation related genes, such as CIIta, Il12a, and Ifng in CMM AML cells. Importantly, the induction of Irf8 and Cebpa, with the concomitant suppression of Runx1 were found specifically in the subset of differentiated CD11b+ CMM cells but not in the bulk CD11b- leukemic cells. Similar transcriptional effects were triggered in CMM leukemic cells by the combination of inducible STAT3 silencing and TLR9 stimulation in vivo but not by either of these treatments alone. These effects were likely related to epigenetic reprogramming of leukemic cells as indicated by treatment-induced changes in the expression and protein levels of STAT3 regulated DNA methyltransferases, DNMT1 and DNMT3a/b. Furthermore, our studies suggest that STAT3-inhibition/TLR9-stimulation triggers immunogenic effects also in a xenotransplanted human FLT3-ITD MV4-11 leukemia in humanized mice. These effects were augmented by a combination with clinically-relevant demethylating agent (decitabine) and resulted in the differentiation of AML cells into CD11b+HLA-DR+CD86+ antigen-presenting cells (APCs) and increased ratio of CD8+ to regulatory T cells in the bone marrow, thereby reducing leukemia burden. Our results suggest that eliminating STAT3 permits the TLR9-driven reprogramming of AML cells into APCs to unleash T cell-mediated responses against leukemia. Citation Format: Yu-Lin Su, Dongfang Wang, Priyanka Duttagupta, Hiqing Li, Ya-Huei Kuo, Guido Marcucci, Marcin Kortylewski. STAT3 inhibition allows for TLR9-induced reprogramming of acute myeloid leukemia into antigen-presenting cells to generate T-cell mediated immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1570.

Elevated RUNX1 is a prognostic biomarker for human head and neck squamous cell carcinoma.

Runt-related transcription factors regulate many developmental processes such as proliferation and differentiation. In this study, the function of the runt-related transcription factor 1 (RUNX1) was investigated in head and neck squamous cell carcinoma (HNSCC). Our results show that RUNX1 expression was elevated in HNSCC patients, which was greatly correlated with the N stage, tumor size, and American Joint Committee on Cancer stage. Cox proportional hazard models showed that RUNX1 could be used as a prognostic indicator for the overall survival of HNSCC patients (hazard ratio, 5.572; 95% confidence interval, 1.860-9.963; P < 0.001). Moreover, suppression of RUNX1 inhibited HNSCC cell proliferation, migration, and invasion. Using the HNSCC xenograft nude mouse model, we found that the shRUNX1-transfected tumor (sh-RUNX1) was significantly smaller both in size and weight than the control vector-transfected tumor (sh-Control). In conclusion, our results show that the elevated RUNX1 expression was correlated with tumor growth and metastasis in HNSCC, indicating that RUNX1 could be used as a biomarker for tumor recurrence and prognosis.

Activation of Steroidogenesis, Anti-Apoptotic Activity, and Proliferation in Porcine Granulosa Cells by RUNX1 Is Negatively Regulated by H3K27me3 Transcriptional Repression.

H3K27me3 is an epigenetic modification that results in the repression of gene transcription. The transcription factor RUNX1 (the runt-related transcription factor 1) influences granulosa cells’ growth and ovulation. This research uses ELISA, flow cytometry, EDU, ChIP-PCR, WB and qPCR to investigate steroidogenesis, cell apoptosis, and the proliferation effect of RUNX1 in porcine granulosa cells (pGCs) as regulated by H3K27me3. Decreased H3K27me3 stimulates the expression of steroidogenesis-related genes, including CYP11A1, PTGS2, and STAR, as well as prostaglandin. H3K27me3 transcriptionally represses RUNX1 here, whereas RUNX1 acts as an activator of FSHR, CYP11A1, and CYP19A1, promoting the production of androgen, estrogen, and prostaglandin, as well as increasing anti-apoptotic and cell proliferation activity, but decreasing progesterone. Both the complementary recovery of the H3K27me3 antagonist with the siRUNX1 signal, and the H3K27me3 agonist with the RUNX1 signal to maintain RUNX1 lead to the activation of CYP19A1, ER1, HSD17β4, and STAR here. Androgen and prostaglandin are significantly repressed but progesterone is markedly increased with the antagonist and siRUNX1. Prostaglandin is significantly promoted with the agonist and RUNX1. Furthermore, H3K27me3-RUNX1 affects the anti-apoptotic activity and stimulation of proliferation in pGCs. The present work verifies the transcriptional suppression of RUNX1 by H3K27me3 during antral follicular development and maturation, which determines the levels of hormone synthesis and cell apoptosis and proliferation in the pGC microenvironment.

RUNX1-dependent mechanisms in biological control and dysregulation in cancer.

The RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context-dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial-to-mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context-dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.

Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions

AbstractDisrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC–Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML.

MicroRNA-378-mediated suppression of Runx1 alleviates the aggressive phenotype of triple-negative MDA-MB-231 human breast cancer cells.

The Runx1 transcription factor, known for its essential role in normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx1 increases concomitantly with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant downregulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mice and in human breast cancer cell lines MCF7 and triple-negative MDA-MB-231 that represent early- and late-stage diseases, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3' untranslated region (3'UTR) and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion, while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late-stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.

The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation, migration and invasion

Previously, we have identified the RUNX1 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared with primary cultures derived from matched primary (prior to CT) tumors. Here we show that RUNX1 displays a trend of hypomethylation, although not significant, in omental metastases compared with primary EOC tumors. Surprisingly, RUNX1 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. The RUNX1 expression levels were almost identical in primary tumors and omental metastases, suggesting that RUNX1 hypomethylation might have a limited impact on its overexpression in advanced (metastatic) stage of the disease. Knockdown of the RUNX1 expression in EOC cells led to sharp decrease of cell proliferation and induced G1 cell cycle arrest. Moreover, RUNX1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX1 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX1 gene in EOC progression and suggest that RUNX1 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX1 and other members of the RUNX gene family in ovarian tumorigenesis.

Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells

Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2−/− mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4+ CD25high CD127− T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4+CD25− cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.

Suppression of RUNX1 by siRNA in megakaryocytic UT-7/GM cells

RUNX1 is a transcription factor that plays critical roles in hematopoietic proliferation and differentiation. Megakaryocyte is a precursor cell of platelets. Several reports have implied that RUNX1 is important in megakaryocytic differentiation and proliferation. However, the mechanism is not well understood. In this study, we employed a megakaryocytic cell line UT-7/GM and suppressed the RUNX1 gene expression by siRNA. Knocking down of RUNX1 induced the increase of megakaryocyte-specific gene expression and down-regulation of polyploidization. RUNX1 overexpression decreased the PF4 and GPIIb promoter activities. These results suggest that RUNX1 promotes proliferation of megakaryocytic cell line but not megakaryocytic gene expression in UT-7/GM cells.

RUNX1 suppression induces megakaryocytic differentiation of UT-7/GM cells

The transcription factor RUNX1 plays a crucial role in hematopoiesis. RUNX1 regulates both differentiation and proliferation of hematopoietic cells. Several reports have shown that RUNX1 participates in megakaryopoiesis, which is a process that leads to formation of platelets. However, to date, the mechanisms by which this occurs have not been fully elucidated. In the present study, we investigated whether siRNA-mediated depletion of RUNX1 affected megakaryopoiesis of UT-7/GM cells. The depletion of RUNX1 in UT-7/GM cells resulted in up-regulation of the expression of megakaryocytic markers and polyploidization, while cell proliferation was down-regulated. Furthermore, the overexpression of RUNX1 decreased the activity of megakaryocytic gene promoters. These results suggest that RUNX1 down-regulates terminal differentiation of megakaryocytes and promotes proliferation of megakaryocytic progenitors.