Differentiation of cancer stem cells into erythroblasts in the presence of CoCl2

Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1α changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34+ and c-kit+ was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity.

FLT3 Inhibitor Quizartinib Facilitates Proliferation and CXCL12-Induced Chemotaxis in Quizartinib Resistant FLT3/ITD + Hematopoietic Cells By Increasing RUNX1

RUNX1 generally functions as a tumor suppressor in the hematopoietic system. However, RUNX1 expression is significantly elevated in human AML cells with FLT3/ITD mutations, promotes leukemogenesis induced by FLT3/ITD (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD + cells to type-II FLT3 inhibitor quizartinib (Hirade et al IJH 2016). We previously reported that RUNX1 expression is higher in CXCR4-low FLT3/ITD + cells compared to Cxcr4-high FLT3/ITD + cells, even though Cxcr4 expression is trans-activated by RUNX1. This difference in RUNX1 expression level was associated with divergent response to CXCL12 in FLT3/ITD + cells harboring different CXCR4 expression levels that were exposed to quizartinib (Fukuda S. et al. ASH 2019). Our data also demonstrated that RUNX1 expression is down-regulated following withdrawal of quizartinib in FLT3/ITD + cells that became refractory to quizartinib (Hirade et al. IJH 2016), suggesting that RUNX1 expression may be up-regulated by quizartinib in FLT3/ITD + cells. Since RUNX1 regulates proliferation of FLT3/ITD + AML cells, the present study investigated association between RUNX1 expression levels and proliferation of quizartinib resistant FLT3/ITD + cells that are exposed to quizartinib. In the sensitive FLT3/ITD + Ba/F3 cells, RUNX1 protein expression was transiently up-regulated but eventually down-regulated by 5 nM quizartinib, coincident with decline in the viable cells. In contrast, RUNX1 expression was up-regulated by quizartinib and remained elevated in the resistant FLT3/ITD + Ba/F3 cells. Since RUNX1 enhances proliferation of FLT3/ITD + cells, we next examined whether proliferation FLT3/ITD + cells that acquired resistance to quizartinib is facilitated by quizaritinib as a result from quizartinib-mediated up-regulation of RUNX1, using the Cxcr4-low and Cxcr4-high FLT3/ITD + cells that acquired resistance to quizartinib. Although CXCL12 barely enhanced the proliferation of refractory FLT3/ITD + Ba/F3 cells, 5 nM quizartinib significantly increased the proliferation of both Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to quizartinib compared to those without quizartinib. This increase in the proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells coincided with the elevation in RUNX1 and CXCR4 protein expression. Moreover, the resistant Cxcr4-low FLT3/ITD + Ba/F3 cells proliferated significantly faster than Cxcr4-high FLT3/ITD + cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Likewise, type-I FLT3 inhibitor gilteritinib significantly enhanced proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to gilteritinib. Knocking down Runx1 using shRNAs significantly decreased the enhanced proliferation induced by quizartinib in refractory FLT3/ITD + Ba/F3 cells, coincident with reduction in CXCR4 expression. Since CXCR4 expression level was elevated by quizartinib in the FLT3/ITD + cells refractory to quizartinib, we next examined CXCL12-induced migration in quizartinib-resistant FLT3/ITD + cells following exposure to quzartinib. Pre-incubating the quizartinib resistant Cxcr4-low or Cxcr4-high FLT3/ITD + Ba/F3 cells with 5 nM quizartinib for 72 hours significantly enhanced their migration to 100 ng/ml of Cxcl12 compared to those without quizartinib, coincident with elevation in RUNX1 levels. Surprisingly, migration of CXCR4-low FLT3/ITD + cells to CXCL12 was significantly elevated compared to CXCR4-high cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Silencing Runx1 using shRNAs significantly decreased migration to CXCL12 in refractory Cxcr4-low FLT3/ITD + Ba/F3 cells. These data indicate that the FLT3 inhibitor itself can facilitate the proliferation and migration to CXCL12 in FLT3/ITD + cells that are refractory to FLT3 inhibitors by up-regulating RUNX1. The results implicate that FLT3 inhibitors may worsen the disease progression in the patients that became refractory to FLT3 inhibitors by facilitating proliferation and migration to CXCL12 of the resistant FLT3/ITD + AML cells. In this regard, targeting RUNX1 may represent additional strategy to eradicate resistant FLT3/ITD + AML cells, in which their proliferation and migration are supported by FLT3 inhibitors. Disclosures No relevant conflicts of interest to declare.

RUNX1 Expression Can be Complementary to RUNX1 Mutation in MDS Prognostication

RUNX1 is a member of the core-binding factor family of transcription factors and is imperative for establishing definitive haematopoiesis. Mutated RUNX1 is an adverse risk factor for myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Meanwhile, high expression of RUNX1 is correlated with dismal prognosis in cytogenetically normal AML patients and critical for maintaining leukemic stem cells across AML genetic subgroups. However, the clinical relevancy of RUNX1 expression in MDS patients remains elusive. This study aimed to investigate the prognostic and biologic impacts of RUNX1 expression in MDS patients. We recruited 341 primary MDS patients who had enough bone marrow (BM) samples for RNA and next-generation sequencing. We first examined the difference in RUNX1 expression among patients with wild RUNX1 and N-terminal or C-terminal RUNX1 mutation. Among the 341 patients, 54 (15.8%) harboured RUNX1 mutation, 15 (27.8%) in N-terminal and 39 (72.2%), C-terminal. Patients with C-terminal RUNX1 mutant had higher RUNX1 expression than those with N-terminal RUNX1 mutant or wild RUNX1 (p<0.001 ). Patients were then divided into two groups with higher- and lower-RUNX1 expression (median as cut-off). Higher RUNX1 expression was closely associated with lower platelet counts, higher blast percentages in the BM and peripheral blood and complex karyotypes at diagnosis. Patients with higher-RUNX1 expression were more frequently categorized into higher-risk groups based on the revised international prognosis scoring system (IPSS-R). Higher RUNX1 expression was intimately associated with ASXL1, NPM1, RUNX1, SRSF2, STAG2, TET2, TP53, and ZRSR2 mutations, whereas lower RUNX1 was associated with SF3B1 mutation. Regarding survival, we first examined the impact of RUNX1 mutation on survival in this cohort. As expected, patients with mutated RUNX1 had significantly inferior leukaemia-free survival (LFS) and overall survival (OS) than those with unmutated RUNX1 (p=0.007, and p=0.008, respectively). We then explored the effects of RUNX1 expression on patients' survival. Patients with higher RUNX1 expression had significantly inferior LFS and OS than those with lower expression (both p<0.001, Figure 1a and 1b). We further interrogated RUNX1 expression and mutation statuses for risk stratification. The higher-RUNX1 group consistently had shorter LFS and OS than the lower-RUNX1 group no matter RUNX1 was mutated or not (Figure 1c-1f). Subgroups analysis revealed the same findings in IPSS-R lower-risk (very low, low, and intermediate-risk) and IPSS-R higher-risk (high and very high risk) subgroups (all p<0.05). Moreover, time-dependent ROC curves indicated that RUNX1 expression had better predictive power for LFS and OS than RUNX1 mutation. In multivariate analysis, higher RUNX1 expression appeared as an independent adverse risk factor for LFS and OS irrespective of age, IPSS-R, and mutations in ASXL1, EZH2, RUNX1, SF3B1, SRSF2, STAG2, TET2, and TP53 (Table). The prognostic significance of RUNX1 expression was further validated in two external public cohorts, GSE 114922 and GSE15061. Patients with higher-RUNX1 expression consistently had significantly inferior survival than those with lower-RUNX1 expression (Figure 2). Bioinformatic analysis revealed that higher-RUNX1 patients had more robust IL-17 and MAPK signallings but exhausted antioxidant activities and antimicrobial humoral responses. In summary, we present the characteristics and prognosis of MDS patients with various RUNX1 expressions and propose that RUNX1 expression can complement RUNX1 mutation in MDS prognostication, wherein patients with wild RUNX1 but high expression may need more proactive treatment. Figure 1 Figure 1. Disclosures Tien: AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Chou: Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; IQVIA: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Bristol Myers Squibb: Honoraria, Research Funding; Kirin: Honoraria, Research Funding.

Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH

Background: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). Methods: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. Results: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. Conclusions: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.

Identification of Regulatory circRNAs Involved in the Pathogenesis of Acute Myocardial Infarction

BackgroundAcute myocardial infarction (AMI) has high morbidity and mortality worldwide. However, the pathogenesis of AMI is still unclear, and the impact of circular RNAs (circRNAs) on AMI has rarely been recognized and needs to be explored.Materials and MethodsThe circRNA array was applied to investigate the expression level of circRNAs in the blood samples of coronary arteries of three AMI patients and three normal persons. Principal component analysis (PCA) and unsupervised clustering analysis were performed to reveal the distinguished expression patterns of circRNAs. The miRNA expression profiles of AMI patients were identified from a public dataset from the Gene Expression Omnibus (GEO) database (GSE31568). The miRNA binding sites on the circRNAs were predicted by miRanda. The miRNA enrichment analysis and annotation tool were used to explore the pathways that the dysregulated circRNAs may participate in.ResultsIn total, 142 differentially expressed circRNAs, including 89 upregulated and 53 downregulated in AMI samples, were identified by the differential expression analysis. AMI patients had quite different circRNA expression profiles to those of normal controls. Functional characterization revealed that circRNAs that had the potential to regulate miRNAs were mainly involved in seven pathways, such as the Runt-related transcription factor-1 (RUNX1) expression and activity-related pathway. Specifically, hsa_circRNA_001654, hsa_circRNA_091761, hsa_circRNA_405624, and hsa_circRNA_406698 were predicted to sponge four miRNAs including hsa-miR-491-3p, hsa-miR-646, hsa-miR-603, and hsa-miR-922, thereby regulating RUNX1 expression or activity.ConclusionWe identified dysregulated blood circRNAs in the coronary arteries of AMI patients and predicted that four upregulated circRNAs were involved in the regulation of RUNX1 expression or activity through sponging four miRNAs.

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.

TET2/IDH1/2/WT1 and NPM1 Mutations Influence the RUNX1 Expression Correlations in Acute Myeloid Leukemia

Background and objectives: Mutational analysis has led to a better understanding of acute myeloid leukemia (AML) biology and to an improvement in clinical management. Some of the most important mutations that affect AML biology are represented by mutations in genes related to methylation, more specifically: TET2, IDH1, IDH2 and WT1. Because it has been shown in numerous studies that mutations in these genes lead to similar expression profiles and phenotypes in AML, we decided to assess if mutations in any of those genes interact with other genes important for AML. Materials and Methods: We downloaded the clinical data, mutational profile and expression profile from the TCGA LAML dataset via cBioPortal. Data were analyzed using classical statistical methods and functional enrichment analysis software represented by STRING and GOrilla. Results: The first step we took was to assess the 196 AML cases that had a mutational profile available and observe the mutations that overlapped with TET2/IDH1/2/WT1 mutations. We observed that RUNX1 mutations significantly overlap with TET2/IDH1/2/WT1 mutations. Because of this, we decided to further investigate the role of RUNX1 mutations in modulating the level of RUNX1 mRNA and observed that RUNX1 mutant cases presented higher levels of RUNX1 mRNA. Because there were only 16 cases of RUNX1 mutant samples and that mutations in this gene determined a change in mRNA expression, we further observed the correlation between RUNX1 and other mRNAs in subgroups regarding the presence of hypermethylating mutations and NPM1. Here, we observed that both TET2/IDH1/2/WT1 and NPM1 mutations increase the number of genes negatively correlated with RUNX1 and that these genes were significantly linked to myeloid activation. Conclusions: In the current study, we have shown that NPM1 and TET2/IDH1/2/WT1 mutations increase the number of negative correlations of RUNX1 with other transcripts involved in myeloid differentiation.

Fzr1 Preserves Hematopoietic Stem Cell Quiescence through Inhibiting Runx1

Regulated blood production is achieved through the hierarchical organization of dormant hematopoietic stem cell (HSC) subsets that differ in self-renewal potential and division frequency, with long-term (LT)-HSCs dividing the least. FZR1, as a regulatory subunit of anaphase promoting complex/cyclosome (APC), is a master regulator of cell cycle, but little is known with regard to its role in HSC quiescence. Thus, we examined the function of Fzr1 in HSCs during steady-state hematopoiesis and under stress. We demonstrate that Fzr1 deletion led to perturbed hematopoiesis with an approximately 2-fold decrease in HSC pool size, as consequence of Fzr1 loss driving HSC from quiescence into rapid cycling (~70.1% to ~28.4%), elevating proliferation (~4.58% to ~18.5%) and apoptosis (~0.23% to ~2.86%) in HSC. As shown by serial bone marrow transplantation and competitive repopulation assays, self-renewal capacity and regenerative capacity were impaired, but differentiation of HSCs was not affected post Fzr1 deletion. Mechanistically, Fzr1 loss led to upregulation of Runx1 expression, in line with the protein expression, the ubiquitin sites of the gene was also obviouly decreased, knockdown Runx1 can rescue the Fzr1-deficient phenotype in HSC. Together, our data therefore support that Fzr1 acts a positive regulator of HSC quiescence and self-renewal capacity through inhibiting Runx1 expression. Disclosures No relevant conflicts of interest to declare.

87 Transcription factor RUNX1 activates OPN to promote tumor progression via MAPK signaling in head and neck cancer

BackgroundTumor progression and metastasis are still major burdens for head and neck squamous cell carcinoma (HNSCC) and are associated with eventual resistance to prevailing therapies. Complex molecular transcription and downstream signaling pathways have been implicated in the development, progression, invasion, metastasis, and treatment resistance of HNSCC. Runt-related transcription factor 1 (RUNX1) are involved in aggressive phenotypes in several cancers, while the molecular role of RUNX1 underlying cancer progression and metastasis of HNSCC remains largely unknown.MethodsRUNX1 expression levels in HNSCC cells and tissues were detected by quantitative real-time PCR (qPCR), Western blotting and immunohistochemistry (IHC). In vitro and in vivo assays were performed to investigate the function of RUNX1 in the metastatic phenotype and the tumorigenic capability of HNSCC cells. Luciferase reporter and chromatin immunoprecipitation (ChIP)-qPCR assays were performed to determine the underlying mechanism of RUNX1-mediated HNSCC aggressiveness.ResultsIn our study, RUNX1 expression was increased with disease progression in patients with HNSCC (figure 1). The silencing of RUNX1 significantly decelerated the malignant progression of HNSCC cells, reduced Osteopontin (OPN) expression in vitro, and weakened the tumorigenicity of HNSCC cells in vivo (figure 2). Moreover, we demonstrated that RUNX1 activated the MAPK signaling by directly binding to the promoter of OPN in tumor progression and metastasis of HNSCC (figure 3).Abstract 87 Figure 1RUNX1 expression in cancer progression of HNSCC. (A) Representative images of RUNX1 immunohistochemical staining between normal tissues and HNSCC tissues (scale bar 20μm). Insets (bottom) are lower magnification (15×) images of respective cores to show a more global view of individual samples. (B) The RUNX1 mRNA expression in tumor versus normal tissues from the TCGA database, which contains 31 normal samples and 91 HNSCC samples. (C) Immunoblotting analysis of RUNX1 expression in 3 pairs of HNSCC and non-tumoral laryngeal tissues. (D) Quantitative and statistical analysis of the immunoblotting analysis. *P<0.05, **P<0.01Abstract 87 Figure 2Effect of RUNX1 on progression and the interrelationship between RUNX1 and OPN in HNSCC. (A) The migration ability of FaDu and SCC-9 cells transfected as above were assessed by wound-healing assay. Representative images were obtained at 0h and 24h (upper, magnification 40×) and quantified (bottom). (B) The migration and invasion ability detected by transwell assays. Representative images of FaDu and SCC-9 cells from migration and invasion assays experiment were obtained at 24h (upper, magnification 12×) and quantified (bottom). (C) Correlation analysis was performed between RUNX1 expression and OPN expression in HNSCC tissues (n = 29) and (D) in TCGA HNSCC database (n = 91). All P values are shown in the graphs. (E) Levels of nucleus OPN mRNA and (F) protein in the FaDu cells transfected with lentiviral vector encoding shRUNX1 or scrambled control were determined by real-time RT-PCR and immunoblotting analysis. (G) The predicted OPN promoter sequence bound by RUNX1 and their ChIP-PCR primers. (H) The binding of RUNX1 to predicted OPN promoter binding region was confirmed in FaDu using ChIP-qPCR and ChIP-PCR. IgG was used as the control. (I) Relative OPN activity was detected by luciferase assay in 293T cells co-transfected with RUNX1 and luciferase reporter. **P<0.01, ****P<0.0001Abstract 87 Figure 3RUNX1-mediated HNSCC cell metastasis in MAPK pathway via stimulating OPN. (A) The migration ability of FaDu cells transfected as above were assessed by wound-healing assay. Representative images were obtained at 0h and 24h (magnification 40×). (B) The migration and invasion ability detected by transwell assays. Representative images of FaDu cells from migration and invasion assays experiment were obtained at 24h (magnification 12×). (C) Immunoblotting analysis for protein markers expression levels of the MAPK pathway in FaDu cells transfected as above. (D) The graph of tumor growth/volumes curve at the indicated time intervals (left). Tumor weights were quantified at the end of the experiment (right). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001ConclusionsOur results may provide new insight into the mechanisms underlying the role of RUNX1 in tumor progression and metastasis and reveal the potential therapeutic target in HNSCC.Ethics ApprovalThe study was approved by the Ethics Board of BenQ Medical Center, the Affiliated BenQ Hospital of Nanjing Medical University.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.