RUNX1 Protein Research Articles

Erythropoietin regulates energy metabolism through EPO-EpoR-RUNX1 axis

Erythropoietin (EPO) plays a key role in energy metabolism, with EPO receptor (EpoR) expression in white adipose tissue (WAT) mediating its metabolic activity. Here, we show that male mice lacking EpoR in adipose tissue exhibit increased fat mass and susceptibility to diet-induced obesity. Our findings indicate that EpoR is present in WAT, brown adipose tissue, and skeletal muscle. Elevated EPO in male mice improves glucose tolerance and insulin sensitivity while reducing expression of lipogenic-associated genes in WAT, which is linked to an increase in transcription factor RUNX1 that directly inhibits lipogenic genes expression. EPO treatment in wild-type male mice decreases fat mass and lipogenic gene expression and increase in RUNX1 protein in adipose tissue which is not observed in adipose tissue EpoR ablation mice. EPO treatment decreases WAT ubiquitin ligase FBXW7 expression and increases RUNX1 stability, providing evidence that EPO regulates energy metabolism in male mice through the EPO-EpoR-RUNX1 axis.

RUNX1, FUS, and ELAVL1-induced circPTPN22 promote gastric cancer cell proliferation, migration, and invasion through miR-6788-5p/PAK1 axis-mediated autophagy

BackgroundAn increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated.MethodsWe used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2′-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22’s influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status.ResultsUpregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC.ConclusionUnder the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.

The roles and mechanisms of APOL1 in the development of colorectal cancer.

Research has demonstrated that apolipoprotein L1 (APOL1) has a role in the emergence and progression of a number of malignant cancers. It is unclear, however, how APOL1 functions in colorectal cancer (CRC). In this study, we examined the possible molecular processes underlying APOL1's biological role in CRC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to identify APOL1 expression in patients with CRC and the cell line of cancer tissue. Following transfection of human colon carcinoma cells (HCT116) and human colon adenocarcinoma cells (SW1116) with sh-APOL1, the effects of APOL1 on the biological behavior of CRC cell lines were examined. In nude mice, the effect of APOL1 on tumor growth was noted. The protein interaction between APOL1 and RUNX1 was detected via coimmunoprecipitation. The expression of relevant proteins and cell biological behaviors were examined to confirm the APOL1-RUNX1 pathway in CRC cell lines. The CRC tissues and cells exhibited elevated expression of APOL1. HCT116 and SW1116 cells' proliferation, migration, and invasion were suppressed by sh-APOL1, and sh-APOL1 also increased the expression of E-cadherin and decreased the expression of RUNX1, cyclin D1, β-catenin, N-cadherin, and vimentin. APOL1 bound to the RUNX1 protein and regulated its protein levels. The counteractive effect of sh-APOL1 epithelial-mesenchymal transition (EMT), proliferation, migration, and invasion of CRC cells was counteracted by the overexpression of RUNX1. By silencing APOL1, the Wnt-β-catenin pathway was able to restrain EMT and regulate the biological behavior processes in CRC cells. APOL1 has potential as a diagnostic biomarker for CRC. By preventing the Wnt-β-catenin pathway from being activated, the sh-APOL1-binding protein RUNX1 inhibited the EMT and biological behavior of CRC cells.

RUNX1 regulates MCM2/CDC20 to promote COAD progression modified by deubiquitination of USP31

Colon adenocarcinoma (COAD) is the second leading cause of cancer death, and there is still a lack of diagnostic biomarkers and therapeutic targets. In this study, bioinformatics analysis of the TCGA database was used to obtain RUNX1, a gene with prognostic value in COAD. RUNX1 plays an important role in many malignancies, and its molecular regulatory mechanisms in COAD remain to be fully understood. To explore the physiological role of RUNX1, we performed functional analyses, such as CCK-8, colony formation and migration assays. In addition, we investigated the underlying mechanisms using transcriptome sequencing and chromatin immunoprecipitation assays. RUNX1 is highly expressed in COAD patients and significantly correlates with survival. Silencing of RUNX1 significantly slowed down the proliferation and migratory capacity of COAD cells. Furthermore, we demonstrate that CDC20 and MCM2 may be target genes of RUNX1, and that RUNX1 may be physically linked to the deubiquitinating enzyme USP31, which mediates the upregulation of RUNX1 protein to promote transcriptional function. Our results may provide new insights into the mechanism of action of RUNX1 in COAD and reveal potential therapeutic targets for this disease.

#1995 Runt-related Transcription Factor 1 (RUNX1) is a mediator of acute kidney injury

Abstract Background and Aims A better understanding of the pathogenesis of acute kidney injury (AKI) may lead to new therapeutic approaches. Kidney transcriptomics analyses of murine folic acid-induced AKI (FA-AKI) identified Runx1 as the most upregulated RUNX family gene. RUNX1 is a key regulator of hematopoiesis, associated to leukemias and other non-immunological cancers and modulates myoblast proliferation during muscle regeneration and cardiac remodelling after myocardial infarction. In kidney diseases, RUNX1 is overexpressed in polycystic kidney disease and kidney cancer and favours fibrosis. Method We examined the expression of RUNX1 in folic acid-AKI (FA-AKI), in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI) and in human AKI. For this, female 12- to 14-week-old C57BL/6J wild type (WT) mice, received a single intraperitoneal (i.p) injection of acid folic (250 mg/kg), LPS (5 mg/kg) or vehicle. In cultured murine tubular MCT cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. The chemical inhibitor of RUNX1 Ro5-3335 and a specific small interfering RNA (siRNA) against RUNX1 were used in cultured cells to explore the role of RUNX1 in TWEAK and LPS-induced inflammation. Ro5-3335 was also used in murine AKI (FA-AKI and CS-AKI) to test its potential as a therapeutic target. In all experimental models, plasma samples were collected to assess renal function. Kidneys were collected for RNA (RT-PCR), protein (Western blot, ELISA, immunohistochemistry) and histopathologic studies. Results Kidney transcriptomics identified Runx1 as the most upregulated Runx gene in FA-AKI at 24 h and the most overactive transcription factor in upstream regulator analysis. RUNX1 overexpression in FA-AKI was validated at mRNA and protein levels and localized mainly to tubular cell nuclei. CS-AKI also upregulated kidney RUNX1 in nuclei. In a transcriptomics array of murine tubular cells stimulated with TWEAK for 6 h, Runx1 was upregulated compared with non-stimulated cells, while Runx2 and Runx3 were not. RT-PCR confirmed an early upregulation of Runx1 (3 h) followed by a progressive return to baseline by 24 h. TWEAK also increased total and nuclear RUNX1 protein levels from 6 h onwards. Since kidney RUNX1 is also increased in CS-AKI, we tested whether LPS regulated RUNX1 in cultured tubular cells. LPS transiently increased Runx1 mRNA expression and it also increased whole cell and nuclear RUNX1 protein levels. Mechanistically, RUNX1 bound to the Il-6 gene promoter in response to TWEAK and LPS, and RUNX1 targeting with the chemical inhibitor Ro5-3335 or a specific siRNA prevented the TWEAK- and LPS-induced upregulation of IL-6. NFκB1 p50 pathway is involved in the RUNX1/IL-6 axis since the specific p50 inhibitor SN50 decreased the nuclear accumulation of RUNX1 and IL-6 expression in TWEAK- or LPS-stimulated MCT cells. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. Moreover, in FA-AKI, Ro5-3335 also prevented the expression of profibrotic genes. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factors such as Yap1 and p53 as key targets of Ro5-3335 in CS-AKI. Conclusion RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI.

Expression pattern of Runt-related transcription factor (RUNX) family members and the role of RUNX1 during kidney development

Runt-related transcription factor (RUNX) family members play critical roles in the development of multiple organs. Mammalian RUNX family members, consisting of RUNX1, RUNX2, and RUNX3, have distinct tissue-specific expression and function. In this study, we examined the spatiotemporal expression patterns of RUNX family members in developing kidneys and analyzed the role of RUNX1 during kidney development. In the developing mouse kidney, RUNX1 protein was strongly expressed in the ureteric bud (UB) tip and weakly expressed in the distal segment of the renal vesicle (RV), comma-shaped body (CSB), and S-shaped body (SSB). In contrast, RUNX2 protein was restricted to the stroma, and RUNX3 protein was only expressed in immune cells. We also analyzed the expression of RUNX family members in the cynomolgus monkey kidney. We found that expression patterns of RUNX2 and RUNX3 were conserved between rodents and primates, whereas RUNX1 was only expressed in the UB tip, not in the RV, CSB, or SSB of cynomolgus monkeys, suggesting a species differences. We further evaluated the roles of RUNX1 using two different conditional knockout mice: Runx1f/f:HoxB7-Cre and Runx1f/f:R26-CreERT2 and found no abnormalities in the kidney. Our findings showed that RUNX1, which is mainly expressed in the UB tip, is not essential for kidney development.

Gypenoside A Protects Human Myocardial Cells from Ischemia/Reperfusion Injury via the circ_0010729/miR-370-3p/RUNX1 Axis.

Ischemia/reperfusion (I/R) injury is one of the major causes of cardiovascular disease. Gypenoside A (GP), the main active component of Gynostemma pentaphyllum, alleviates myocardial I/R injury. Circular RNAs (circRNAs) and microRNAs (miRNAs) are involved in the I/R injury. We explored the protective effect of GP on human cardiomyocytes (HCMs) via the circ_0010729/miR-370-3p/RUNX1 axis. Overexpression of circ_0010729 abolished the effects of GP on HMC, such as suppression of apoptosis and increase in cell viability and proliferation. Overexpression of miR-370-3p reversed the effect of circ_0010729 overexpression, resulting in the stimulation of HMC viability and proliferation and inhibition of apoptosis. The knockdown of miR-370-3p suppressed the effects of GP in HCMs. RUNX1 silencing counteracted the effect of miR-370-3p knockdown and maintained GP-induced suppression of apoptosis and stimulation of HMC viability and proliferation. The levels of RUNX1 mRNA and protein were reduced in cells expressing miR-370-3p. In conclusion, this study confirmed that GP alleviated the I/R injury of myocardial cell via the circ_0010729/miR-370-3p/RUNX1 axis.

MYCT1 inhibits hematopoiesis in diffuse large B-cell lymphoma by suppressing RUNX1 transcription

BackgroundThe abnormality of chromosomal karyotype is one factor causing poor prognosis of lymphoma. In the analysis of abnormal karyotype of lymphoma patients, three smallest overlap regions were found, in which MYCT1 was located. MYCT1 is the first tumor suppressor gene cloned by our research team, but its studies relating to the occurrence and development of lymphoma have not been reported.MethodsR banding analyses were employed to screen the abnormality of chromosomal karyotype in clinical specimen and MYCT1 over-expression cell lines. FISH was to monitor MYCT1 copy number aberration. RT-PCR and Western blot were to detect the mRNA and protein levels of the MYCT1 and RUNX1 genes, respectively. The MYCT1 and RUNX1 protein levels in clinical specimen were evaluated by immunohistochemical DAB staining. The interaction between MYCT1 and MAX proteins was identified via Co-IP and IF. The binding of MAX on the promoter of the RUNX1 gene was detected by ChIP and Dual-luciferase reporter assay, respectively. Flow cytometry and CCK-8 assay were to explore the effects of MYCT1 and RUNX1 on the cell cycle and proliferation, respectively.ResultsMYCT1 was located in one of three smallest overlap regions of diffuse large B-cell lymphoma, it altered chromosomal instability of diffuse large B-cell lymphoma cells. MYCT1 negatively correlated with RUNX1 in lymphoma tissues of the patients. MAX directly promoted the RUNX1 gene transcription by binding to its promoter region. MYCT1 may represses RUNX1 transcription by binding MAX in diffuse large B-cell lymphoma cells. MYCT1 binding to MAX probably suppressed RUNX1 transcription, leading to the inhibition of proliferation and cell cycle of the diffuse large B-cell lymphoma cells.ConclusionThis study finds that there is a MYCT1-MAX-RUNX1 signaling pathway in diffuse large B-cell lymphoma. And the study provides clues and basis for the in-depth studies of MYCT1 in the diagnosis, treatment and prognosis of lymphoma.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Rps19 and Rpl5 Play Distinct Roles in hematopoietic Stem Cell maintenance and Erythroid Differentiation

Mutations in ribosomal proteins (RPs) lead to ribosomopathies. Mechanistic basis underlying the failure of erythropoiesis due to mutations in various RPs is yet to be fully defined. Lack of relevant murine models of ribosomapathies has hindered significant progress. To fill this gap, we generated Rps19 and Rpl5 floxed mice using CRISPR/Cas9 technologies and characterized their hematologic phenotype following crossing them to a Vav-iCremice. Vav-iCre;Rps19 fl/+mice are born at expected Mendelian rates with severe macrocytic anemia and reticulocytopenia. These mice die by post-natal day 10 (P10), due to bone marrow failure and absence of splenic stress erythropoiesis. Analysis of fetal erythropoiesis revealed a 40% reduction in the total number of erythroid cells in Vav-iCre;Rps19 fl/+, however erythroblasts at all stages of terminal erythroid differentiation were found. Intriguingly, studies of hematopoietic stem and progenitor cell s demonstrated a progressive exhaustion of the stem cell compartment. scRNAseq studies of Vav-iCre;Rps19 fl/+ fetal livers demonstrated increased expression of RUNX1, a transcription factor implicated in the regulation of ribosome biogenesis. Accordingly, we observed that hematopoietic progenitors of Vav-iCre,Rps19 fl/+had enhanced global protein synthesis along with increased RUNX1 protein, despite ribosomal protein haploinsufficiency demonstrated by polysome profiling. In marked contrast, Vav-iCre;Rpl5 fl/+ mice presented with a more severe phenotype and died perinatally from anemia. Further analysis revealed a failure of erythropoiesis at erythroid progenitor stages in Vav-iCre;Rpl5 fl/+. In striking contrast with the Rps19 model, Vav-iCre;Rpl5 fl/+ mice had an expansion of the hematopoietic stem and progenitor cell compartment that also had an increased protein translation rate . This increased translation occurred in the context of normal expression levels of RUNX1. Interestingly Vav-iCre;Rpl5 fl/+ micehad reduced mTORC1 activity and a dramatic reduction in the expression of other ribosomal proteins. Consistent with previous studies about the pivotal role of mTORC1 in erythropoiesis Vav-iCre,Raptor fl/fl(mTORC1 conditional knockout) had reduced expressions of RPs in fetal liver. In summary, we report different requirements for Rps19 and Rpl5 during fetal hematopoiesis in regulating erythroid progenitor biology. While Rps19 plays an essential role at the HSC level to maintain the generation of erythroid progenitors, Rpl5 is critical once progenitors are committed towards an erythroid fate. Along with the findings that translation is increased in RP haploinsufficient models, our study unravels potential compensatory mechanisms during fetal hematopoiesis.

Age-related promoter-switch regulates Runx1 expression in adult rat hearts

BackgroundRunt-related transcription factor-1 (RUNX1), a key member of the core-binding factor family of transcription factors, has emerged as a novel therapeutic target for cardiovascular disease. There is an urgent need to fully understand the expression pattern of Runx1 in the heart and the mechanisms by which it is controlled under normal conditions and in response to disease. The expression of Runx1 is regulated at the transcriptional level by two promoters designated P1 and P2. Alternative usage of these two promoters creates differential mRNA transcripts diversified in distribution and translational potential. While the significance of P1/P2 promoter-switch in the transcriptional control of Runx1 has been highlighted in the embryogenic process, very little is known about the level of P1- and P2-specific transcripts in adult hearts, and the underlying mechanisms controlling the promoter-switch.MethodsTo amplify P1/P2 specific sequences in the heart, we used two different sense primers complementary to either P1 or P2 5'-regions to monitor the expression of P1/P2 transcripts. DNA methylation levels were assessed at the Runx1 promoter regions. Rats were grouped by age.ResultsThe expression levels of both P1- and P2-derived Runx1 transcripts were decreased in older rats when compared with that in young adults, paralleled with an age-dependent decline in Runx1 protein level. Furthermore, older rats demonstrated a higher degree of DNA methylation at Runx1 promoter regions. Alternative promoter usage was observed in hearts with increased age, as reflected by altered P1:P2 mRNA ratio.ConclusionOur data demonstrate that the expression of Runx1 in the heart is age-dependent and underscore the importance of gene methylation in the promoter-mediated transcriptional control of Runx1, thereby providing new insights to the role of epigenetic regulation in the heart.

The E3 ligase DTX2 inhibits RUNX1 function by binding its C-terminus and prevents the growth of RUNX1-dependent leukemia cells.

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome-independent ubiquitination, is poorly understood. We previously identified DTX2 as a RUNX1-interacting E3 ligase using a cell-free AlphaScreen assay. In this study, we examined whether DTX2 is involved in the regulation of RUNX1 using in vitro and ex vivo analyses. DTX2 bound to RUNX1 and other RUNX family members RUNX2 and RUNX3 through their C-terminal region. DTX2-induced RUNX1 ubiquitination did not result in RUNX1 protein degradation. Instead, we found that the acetylation of RUNX1, which is known to enhance the transcriptional activity of RUNX1, was inhibited in the presence of DTX2. Concomitantly, DTX2 reduced the RUNX1-induced activation of an MCSFR luciferase reporter. We also found that DTX2 induced RUNX1 cytoplasmic mislocalization. Moreover, DTX2 overexpression showed a substantial growth-inhibitory effect in RUNX1-dependent leukemia cell lines. Thus, our findings indicate a novel aspect of the ubiquitination and acetylation of RUNX1 that is modulated by DTX2 in a proteosome-independent manner.

Silver Nanoparticles Promote the Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Modulating the O-GlcNAcylation of RUNX1

Human BMSCs are multifunctional progenitor cells that can transferred into different functional cells. Silver nanoparticles (AgNPs) has been demonstrated to modulate the osteogenesis of stem cells. However, effects of AgNPs on hBMSC functions and their potential modulatory mechanism remain elusive. Our study aimed to thoroughly analyze the role of AgNPs in mediating hBMSCs osteogenic differentiation. In this study, we found that hBMSCs viability as well as osteogenic differentiation were significantly promoted when treated with 4 μg/ml AgNPs. Aberrant O-GlcNAcylation can affect multiple biological processes of human eukaryotes, including osteogenic differentiation of hBMSCs. Here, we demonstrated that AgNPs could increase the O-GlcNAcylation level to exert their functions. Further mechanistic investigation revealed that AgNPs enhanced the protein level of runt-related transcription Factor 1 (RUNX1) by O-glycosylating it at the T219 site to stabilize the RUNX1 protein. Finally, we determined that silencing RUNX1 abrogated AgNPs function on the viability and hBMSCs osteogenic differentiation. In conclusion, this study reported a novel mechanism by which AgNPs promoted hBMSCs osteogenic differentiation.

USP10 deubiquitinates RUNX1 and promotes proneural-to-mesenchymal transition in glioblastoma

The mesenchymal (MES) subtype of glioblastoma (GBM) is a highly aggressive, malignant and proliferative cancer that is resistant to chemotherapy. Runt-related transcription factor 1 (RUNX1) was shown to support MES GBM, however, its underlying mechanisms are unclear. Here, we identified USP10 as a deubiquitinating enzyme that regulates RUNX1 stabilization and is mainly expressed in MES GBM. Overexpression of USP10 upregulated RUNX1 and induced proneural-to-mesenchymal transition (PMT), thus maintaining MES properties in GBM. Conversely, USP10 knockdown inhibited RUNX1 and resulted in the loss of MES properties. USP10 was shown to interact with RUNX1, with RUNX1 being stabilized upon deubiquitylation. Moreover, we found that USP10 inhibitor Spautin-1 induced RUNX1 degradation and inhibited MES properties in vitro and in vivo. Furthermore, USP10 was strongly correlated with RUNX1 expression in samples of different subtypes of human GBM and had prognostic value for GBM patients. We identified USP10 as a key deubiquitinase for RUNX1 protein stabilization. USP10 maintains MES properties of GBM, and promotes PMT of GBM cells. Our study indicates that the USP10/RUNX1 axis may be a potential target for novel GBM treatments.

Downregulation of RUNX1-activated osteopontin facilitates burn wound healing by activating the MAPK pathways.

Burn wounds require intervention to ensure timely progression to reduce morbidity and mortality. The migrative and proliferative capabilities of keratinocytes are impaired in wounds. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix (ECM), allowing epithelial cells to migrate. As reported, osteopontin can regulate cell migration, cell adhesion, and ECM invasion in endothelial and epithelial cells, and its expression is significantly increased in chronic wounds. Therefore, this study investigates the biological functions of osteopontin and its related mechanisms involved in burn wounds. We established cellular and animal models of burn injury. Levels of osteopontin, RUNX1, MMPs, collagen I, CK19, PCNA and pathway-associated proteins were measured by RT-qPCR, western blotting and immunofluorescence staining. Cell viability and migration were examined by CCK-8 and wound scratch assays. Histological changes were analyzed by hematoxylin and eosin staining and Masson's trichrome staining. For in vitro analysis, osteopontin silencing facilitated the growth and migration of HaCaT cells and promoted ECM degradation in HaCaT cells. Mechanistically, RUNX1 bound to osteopontin promoter, and RUNX1 upregulation attenuated the promoting efficacy of osteopontin silencing on cell growth and migration and ECM degradation. Additionally, RUNX1-activated osteopontin inactivated the MAPK signaling pathway. For in vivo analysis, osteopontin depletion facilitated burn wound healing by promoting re-epithelialization and ECM degradation. In conclusion, RUNX1 activates the osteopontin expression at the transcriptional level and osteopontin depletion facilitates the recovery of burn wounds by promoting the migration of keratinocytes and re-epithelization and ECM degradation by activating the MAPK pathway.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

Mechanistic Study of Wee1 Kinase Inhibition with AZD1775 Exposes Drug Targetable Vulnerabilities in Acute B-Lymphoblastic Leukemia

Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side effects of chemotherapy. Despite a high response rate to induction chemotherapy in acute lymphoblastic leukemia (ALL) there is an urgent need to identify effective combination therapy to overcome treatment resistance that associates with disease relapse. ALL cells are acutely highly sensitive to the WEE1 inhibitor AZD1775 that compromises the G2/M-phase cell cycle checkpoint. Here, we show that AZD1775 selectively inhibits recovery of proliferation in KMT2A/MLL-rearranged (MLL-r) ALL, a group of leukemias that respond poorly to current chemotherapy and have dismal prognosis compared to other leukemia subgroups. This vulnerability could be distinguished by elevated WEE1 expression in MLL-r ALL and through biochemical analysis of the resulting mitotic catastrophe upon WEE1 inhibition as assessed by PARP cleavage and flow cytometric analysis of DNA metabolism. Extensive genomics profiling of the drug response by joint single-cell transcriptome and chromatin accessibility profiling revealed that AZD1775 led to a strong p53-driven gene regulatory response resulting in induction of apoptosis and senescence and disrupts a crucial hematopoietic TF-network involving RUNX1, MYC and GATA2. We further demonstrate that RUNX1 protein was degradaded in a CDK-dependent manner. Leukemia relapse may arise from rare cells that escape treatment through genetic mutations, transcriptional reprogramming or signaling rewiring. We here demonstrate that upon the pre-mature mitosis entry resulting from AZD1775 treatment, a rapid transcriptional reprogramming is triggered involving activation of NfKB and transcription factors regulating lipid metabolism, BCL6 and pre-BCR signaling that resulted in a small surviving cell population. Single-cell characterization of the AZD1775 response in a MLL-r patient-derived xenograft (PDX) model supported the in vivo relevance of this drug resistance mechanism. Informed by the genomic profiles, sequential administration of AZD1775 with inhibitors of pre-BCR signaling or fatty acid synthesis was designed to target the resistant cell state and this approach effectively prevented cell survival and recovery of ALL cells. Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell survival and cell fate regulation in response to drugs targeting cell cycle regulation and provide a rationale for combination therapies with low toxicity drugs such as dasatinib or fatostatin to counteract drug resistance that occurs through cell state switching.

NEXT-GENERATION GENOMIC MONITORING REVEAL A NOVEL RUNX1 PATHOGENIC MUTATION ASSOCIATED WITH TRISOMY 4 AS A SOLE ABNORMALITY IN A YOUNG ADULT WITH ACUTE MYELOID LEUKEMIA

Acute Myeloid Leukemia (AML) with a single trisomy show particular clinical, morphological, genomic, and immunophenotypic features. Trisomy 4 was first described in 1986 as the sole chromosomal abnormality in AML. However, due to its rarity (< 1%), there are not enough data concerning its clinical impact. Here we report clinical, cyto-molecular, and genomic data of a young adult with AML and a rare trisomy 4 associated with a novel RUNX1 pathogenic mutation. A 19-year-old man was referred to Hospital dos Servidores do Estado, Rio de Janeiro, Brazil. He presented a history of 30 days of anemia, fever, weight loss, thrombocytopenia, and mucocutaneous bleeding. Physical examination revealed hepatomegaly (15 cm) and splenomegaly (14 cm). Laboratory data included hemoglobin level of 6.9 g/dL, WBC of 350 × 10 9 /L, and platelet count of 8 × 10 9 /L. The LDH was 1650 U/L. Flow cytometry analysis revealed 83.4% of blasts showing myeloid morphology that expressed CD34, CD117, CD13, CD7, CD38, and HLA-DR, compatible with FAB AML-M0 with minimal differentiation/undifferentiated. The patient was treated under a high-risk AML protocol. On day 14 of treatment, he developed visceral septicemia and died due to septic shock. We performed G-banding and FISH experiments. NGS was performed on a targeted approach. G-Banding revealed the karyotype 47,XY,+4. FISH with a centromeric probe for chromosome 4 confirmed trisomy 4. Molecular biology tests were positive for FLT3/ITD and negative for NPM1 mutations. NGS results revealed a novel RUNX1 missense pathogenic mutation. Trisomy 4 is a rare chromosomal abnormality in AML, accounting for 1 < % of cases. Although its prognostic relevance has been a frequent topic of discussion in the literature, its relation to prognosis remains to be elucidated. Chilton and coworkers (2016) described 35 patients with trisomy 4 as an isolated abnormality and observed only two patients (6%) in the 16‒25 age group. Patients with trisomy 4 have a poor prognosis when associated with mutations in KIT, mapped at position 4q12, which may influence the role of trisomy 4 in leukemogenesis. The patient in the present study belongs to the same age group as the patients in the abovementioned study. However, to our knowledge, it is the first case with trisomy 4 as a sole abnormality presenting FAB AML-M0. Our patient had a poor prognosis, including hyperleukocytosis. In addition, we characterized a missense RUNX1 mutation not yet described in the literature. RUNX1 is a transcription factor part of a complex that provides stability to the RUNX1 protein, which plays a pivotal role in generating hematopoietic stem cells and their differentiation. Loss of RUNX1 function has been shown to impair cell differentiation, often resulting in leukemogenesis. Besides, RUNX1 mutations are known to cause thrombocytopenia and familial platelet disorder. Here we present a rare AML-M0 case with trisomy 4 as a sole abnormality and highlight its association with a novel RUNX1 mutation. Although, the prognostic implications remain to be elucidated. In particular, when to consider stem cell transplantation as curative therapy of choice in RUNX1 mutated individuals to maximize benefit and minimize risk.

Transcription Factors Runx1 and Runx3 Suppress Keratin Expression in Undifferentiated Keratinocytes.

The Runt-related transcription factor (Runx) family has been suggested to play roles in stem cell regulation, tissue development, and oncogenesis in various tissues/organs. In this study, we investigated the possible functions of Runx1 and Runx3 in keratinocyte differentiation. Both Runx1 and Runx3 proteins were detected in primary cultures of mouse keratinocytes. Proteins were localized in the nuclei of undifferentiated keratinocytes but translocated to the cytoplasm of differentiated cells. The siRNA-mediated inhibition of Runx1 and Runx3 expression increased expression of keratin 1 and keratin 10, which are early differentiation markers of keratinocytes. In contrast, overexpression of Runx1 and Runx3 suppressed keratin 1 and keratin 10 expression. Endogenous Runx1 and Runx3 proteins were associated with the promoter sequences of keratin 1 and keratin 10 genes in undifferentiated but not differentiated keratinocytes. In mouse skin, the inhibition of Runx1 and Runx3 expression by keratinocyte-specific gene targeting increased the ratios of keratin 1- and keratin 10-positive cells in the basal layer of the epidermis. On the other hand, inhibition of Runx1 and Runx3 expression did not alter the proliferation capacity of cultured or epidermal keratinocytes. These results suggest that Runx1 and Runx3 likely function to directly inhibit differentiation-induced expression of keratin 1 and keratin 10 genes but are not involved in the regulation of keratinocyte proliferation.

In-silico probing of AML related RUNX1 cancer-associated missense mutations: Predicted relationships to DNA binding and drug interactions.

The molecular consequences of cancer associated mutations in Acute myeloid leukemia (AML) linked factors are not very well understood. Here, we interrogated the COSMIC database for missense mutations associated with the RUNX1 protein, that is frequently mis-regulated in AML, where we sought to identify recurrently mutated positions at the DNA-interacting interface. Indeed, six of the mutated residues, out of a total 417 residues examined within the DNA binding domain, evidenced reduced DNA association in in silico predictions. Further, given the prominence of RUNX1’s compromised function in AML, we asked the question if the mutations themselves might alter RUNX1’s interaction (off-target) with known FDA-approved drug molecules, including three currently used in treating AML. We identified several AML-associated mutations in RUNX1 that were calculated to enhance RUNX1’s interaction with specific drugs. Specifically, we retrieved data from the COSMIC database for cancer-associated mutations of RUNX1 by using R package “data.table” and “ggplot2” modules. In the presence of DNA and/or drug, we used docking scores and energetics of the complexes as tools to evaluate predicted interaction strengths with RUNX1. For example, we performed predictions of drug binding pockets involving Enasidenib, Giltertinib, and Midostaurin (AML associated), as well as ten different published cancer associated drug compounds. Docking of wild type RUNX1 with these 13 different cancer-associated drugs indicates that wild-type RUNX1 has a lower efficiency of binding while RUNX1 mutants R142K, D171N, R174Q, P176H, and R177Q suggested higher affinity of drug association. Literature evidence support our prediction and suggests the mutation R174Q affects RUNX1 DNA binding and could lead to compromised function. We conclude that specific RUNX1 mutations that lessen DNA binding facilitate the binding of a number of tested drug molecules. Further, we propose that molecular modeling and docking studies for RUNX1 in the presence of DNA and/or drugs enables evaluation of the potential impact of RUNX1 cancer associated mutations in AML.