RUNX Family Transcription Factor Research Articles

PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice

BackgroundInduction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE−/−) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE−/− mouse model of atherosclerosis and under the influence of a high-fat diet.MethodsFor this purpose, PACAP−/−/ApoE−/−, PAC1−/−/ApoE−/−, and ApoE−/− mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified.ResultsApoE−/− mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1−/−/ApoE−/− mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE−/− mice. In contrast, PACAP deficiency in ApoE−/− mice did not affect the chondrogenic signature under either diet.ConclusionsOur data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Loss of SLC27A5 Activates Hepatic Stellate Cells and Promotes Liver Fibrosis via Unconjugated Cholic Acid.

Although the dysregulation of bile acid (BA) composition has been associated with fibrosis progression, its precise roles in liver fibrosis is poorly understood. This study demonstrates that solute carrier family 27 member 5 (SLC27A5), an enzyme involved in BAs metabolism, is substantially downregulated in the liver tissues of patients with cirrhosis and fibrosis mouse models. The downregulation of SLC27A5 depends on RUNX family transcription factor 2 (RUNX2), which serves as a transcriptional repressor. The findings reveal that experimental SLC27A5 knockout (Slc27a5-/- ) mice display spontaneous liver fibrosis after 24months. The loss of SLC27A5 aggravates liver fibrosis induced by carbon tetrachloride (CCI4 ) and thioacetamide (TAA). Mechanistically, SLC27A5 deficiency results in the accumulation of unconjugated BA, particularly cholic acid (CA), in the liver. This accumulation leads to the activation of hepatic stellate cells (HSCs) by upregulated expression of early growth response protein 3 (EGR3). The re-expression of hepatic SLC27A5 by an adeno-associated virus or the reduction of CA levels in the liver using A4250, an apical sodium-dependent bile acid transporter (ASBT) inhibitor, ameliorates liver fibrosis in Slc27a5-/- mice. In conclusion, SLC27A5 deficiency in mice drives hepatic fibrosis through CA-induced activation of HSCs, highlighting its significant implications for liver fibrosis treatment.

Aberrant SUMO2/3 modification of RUNX1 upon SENP1 inhibition is linked to the development of diabetic retinopathy in mice

Our previous report established that RUNX family transcription factor 1 (RUNX1) promotes proliferation of mouse retinal microvascular endothelial cells (mRMECs) and exacerbates diabetic retinopathy (DR). However, the mechanism behind the upregulation of RUNX1 remains unclear. This study aims to investigate the possible correlation between histone SUMOylation and RUNX1 in DR, as well as the involved molecules. A mouse model of diabetes was induced by streptozotocin (STZ). These mice had increased retinal thickness and elevated production of inflammatory cytokines. Additionally, they showed elevated levels of SUMO1 and SUMO2/3, but reduced levels of SUMO specific peptidase 1 (SENP1) in retinal tissues. Co-immunoprecipitation and Western blot assays revealed that the RUNX1 protein was primarily modified by SUMO2/3, and SENP1 inhibited SUMO2/3 modification, thereby reducing RUNX1 expression. Overexpression of SENP1 alleviated symptoms in mice and alleviated inflammation. In vitro experiments demonstrated that the SENP1 overexpression suppressed the proliferation, migration, and angiogenesis of high-glucose-induced mRMECs. However, further overexpression of RUNX1 counteracted the alleviating effects of SENP1 both in vivo and in vitro. In conclusion, this study demonstrates that the downregulation of SENP1 in DR leads to SUMO2/3-dependent activation of RUNX1. This activation promotes proliferation of mRMECs and exacerbates DR symptoms in mice.

MicroRNA-584-5p/RUNX family transcription factor 2 axis mediates hypoxia-induced osteogenic differentiation of periosteal stem cells.

The hypoxic environment during bone healing is important in regulating the differentiation of periosteal stem cells (PSCs) into osteoblasts or chondrocytes; however, the underlying mechanisms remain unclear. To determine the effect of hypoxia on PSCs, and the expression of microRNA-584-5p (miR-584-5p) and RUNX family transcription factor 2 (RUNX2) in PSCs was modulated to explore the impact of the miR-584-5p/RUNX2 axis on hypoxia-induced osteogenic differentiation of PSCs. In this study, we isolated primary mouse PSCs and stimulated them with hypoxia, and the characteristics and functional genes related to PSC osteogenic differentiation were assessed. Constructs expressing miR-584-5p and RUNX2 were established to determine PSC osteogenic differentiation. Hypoxic stimulation induced PSC osteogenic differentiation and significantly increased calcified nodules, intracellular calcium ion levels, and alkaline phosphatase (ALP) activity in PSCs. Osteogenic differentiation-related factors such as RUNX2, bone morphogenetic protein 2, hypoxia-inducible factor 1-alpha, and ALP were upregulated; in contrast, miR-584-5p was downregulated in these cells. Furthermore, upregulation of miR-584-5p significantly inhibited RUNX2 expression and hypoxia-induced PSC osteogenic differentiation. RUNX2 was the target gene of miR-584-5p, antagonizing miR-584-5p inhibition in hypoxia-induced PSC osteogenic differentiation. Our study showed that the interaction of miR-584-5p and RUNX2 could mediate PSC osteogenic differentiation induced by hypoxia.

RUNX1 predicts poor prognosis and correlates with tumor progression in clear cell renal carcinoma

BackgroundRunt-related transcription factor 1 (RUNX1), also called acute myeloid leukaemia 1, is a member of RUNX family of transcription factors. This family is composed of evolutionarily conserved transcription factors that function as critical lineage determinants in various tissues, however its function in cancer development and clinical significance in RCC are still unknown. MethodsWe used paraffin-embedded tumor tissues from 100 patients and fresh-harvested and paired adjacent normal renal tissues from 15 RCC patients who underwent primary surgical resection in Xijing Hospital between 2018 and 2022. The expression level of RUNX1 was evaluated by immunohistochemistry and Western Blot. RUNX1 promoted tumor cells proliferation, migration and invasion were verified by CCK-8, wound-healing and transwell assays. Finally, we constructed a xenografts model of the 786-O cell lines to observe the effect of RUNX1 on tumorigenesis in vivo. ResultsTCGA database showed higher RUNX1 expression levels in KIRC (kidney renal clear cell carcinoma). In overall survival analysis, RCC patients with higher RUNX1 expression level would have a shorter survival period than those with lower expression. Similarly, immunohistochemical results of our cohort also showed that RUNX1 was over-expression in cancer tissues than in corresponding non-cancer tissues. We also proved this result at protein level by western-blot. Meanwhile, prognostic and OS analyses of our cohort showed that the RUNX1 expression level was an individual prognostic factor in RCC patients. CCK-8, wound-healing and transwell assays proved that the overexpression of RUNX1 in Caki-1 cells promoted the proliferation, migration and invasion of the cells. Knocking down RUNX1 in 786-O cells inhibited the proliferation, migration and invasion of cells. The experimental results of xenografts model in nude mice showed that the knockdown of RUNX1 in 786-O cells slowed down the growth of tumor. ConclusionRUNX1 is a poor prognostic factor of clear cell renal carcinoma, which may provide a novel therapeutic target for ccRCC.

The combined treatment of insulin and naringin improves bone properties in rats with type 1 diabetes mellitus.

We have studied the effects of individual and combined treatment of insulin (I) and naringin (NAR) on the bone structure and biomechanical properties of femurs from streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into five groups: (1) controls, (2) STZ-induced diabetic rats, (3) STZ-induced diabetic rats treated with I, (4) STZ-induced diabetic rats treated with NAR, and (5) STZ-induced diabetic rats treated with I+NAR. Bone mineral density (BMD), bone histomorphometry, biomechanical testing, and bone biomarker expressions were accomplished in femur of all animals, as well as serum biochemical analyses. The combined treatment of I+NAR increased the body weight and the femur BMD from STZ-induced diabetic rats. The bone biomechanical properties and the bone morphology of the femurs from STZ-induced diabetic rats were also improved by the combined treatment. The increased number of osteoclasts in STZ-induced diabetic rats was partially prevented by I, NAR, or I+NAR. NAR or I+NAR completely blocked the decrease in the number of osteocalcin (+) cells in the femur from STZ-induced diabetic rats. RUNX family transcription factor 2 immunostaining was much lower in STZ-induced diabetic rats than in control animals; the combination of I+NAR totally blocked this effect. The combined treatment not only ameliorated bone quality and function, but also normalized the variables related to glucose metabolism. Therefore, the combination of I+NAR might be a better therapeutic strategy than the individual I or NAR administration to reduce bone complications in diabetic patients.

DAP1 regulates osteoblast autophagy via the ATG16L1–LC3 axis in Graves’ disease-induced osteoporosis

ObjectiveThis study aimed to uncover a critical protein and its mechanisms in modulating autophagy in Graves’ disease (GD)-induced osteoporosis (OP).MethodsWe discovered the target protein, death-associated protein 1 (DAP1), using bone proteomics analysis. Furthermore, genetic overexpression and knockdown (KD) of DAP1 in bone and MC3T3-E1 cells revealed DAP1 effects on autophagy and osteogenic markers, and autophagic vacuoles in cells were detected using transmission electron microscopy and the microtubule-associated protein 1 light chain 3 alpha (MAP1LC3/LC3) dual fluorescence system. An autophagy polymerase chain reaction (PCR) array kit was used to identify the key molecules associated with DAP1-regulated autophagy.ResultsDAP1 levels were significantly higher in the bone tissue of GD mice and MC3T3-E1 cells treated with triiodothyronine (T3). DAP1 overexpression reduced LC3 lipidation, autophagic vacuoles, RUNX family transcription factor 2 (RUNX2), and osteocalcin (OCN) expression in MC3T3-E1 cells, whereas DAP1 KD reversed these changes. In vivo experiments revealed that GD mice with DAP1 KD had greater bone mass than control mice. DAP1-overexpressing (OE) cells had lower levels of phosphorylated autophagy-related 16-like 1 (ATG16L1) and LC3 lipidation, whereas DAP1-KD cells had higher levels.ConclusionsDAP1 was found to be a critical regulator of autophagy homeostasis in GD mouse bone tissue and T3-treated osteoblasts because it negatively regulated autophagy and osteogenesis in osteoblasts via the ATG16L1–LC3 axis.

Effect of RUNX1/FOXP3 axis on apoptosis of T and B lymphocytes and immunosuppression in sepsis.

Lymphocyte apoptosis is a latent factor for immunosuppression in sepsis. Forkhead box protein P3 (FOXP3) can interact with RUNX family transcription factor 1 (RUNX1) in regulatory T cells. Our research was to probe whether RUNX1/FOXP3 axis affects immunosuppression in the process of sepsis by modulating T and B lymphocyte apoptosis. We constructed sepsis model in mice and mouse CD4+ T and CD19+ B lymphocytes. RUNX1 and FOXP3 expressions and apoptosis in cells were assessed by western blot, quantitative real-time PCR, and flow cytometer. Inflammation of serum and pathological damage was assessed by ELISA and H&E staining. Relationship between RUNX1 and FOXP3 was assessed by co-immunoprecipitation. The findings showed that RUNX1 ameliorated the survival rate, pathological damage, and decreased inflammation-related factors, and inhibited apoptosis of CD4+ T and CD19+ B cells in cecal ligation and puncture mice. Furthermore, RUNX1 up-regulated the viability and down-regulated apoptotic rate with the changed expressions of apoptosis-related molecules in lipopolysaccharide (LPS)-mediated CD4+ T and CD19+ B cells. Additionally, FOXP3 interacted with RUNX1, and its silencing decreased RUNX1 expression and reversed the inhibitory effect of RUNX1 on apoptosis of LPS-mediated CD4+ T and CD19+ B cells. In summary, the RUNX1/FOXP3 axis alleviated immunosuppression in sepsis progression by weakening T and B lymphocyte apoptosis.

Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats

To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.

YTHDC2 inhibits rat bone mesenchymal stem cells osteogenic differentiation by accelerating RUNX2 mRNA degradation via m6A methylation

As the most abundant internal mRNA modification, N6-methyladenosine (m6A) RNA methylation has been found to influence many biological events including bone mesenchymal stem cells (BMSCs) osteogenic differentiation. YTH N6-methyladenosine RNA binding protein C2 （YTHDC2） is an m6A reading protein with the ability to mediate the decay of combined methylated mRNA, however its role in BMSCs osteogenic differentiation remains unknown. In this study, we first found an increase of RUNX family transcription factor 2 （RUNX2） expression and a decrease of YTHDC2 expression during the process of BMSCs osteogenic differentiation. Furthermore, we transfected BMSCs with YTHDC2 interference fragment, resulting in an increased content of RUNX2 mRNA and protein inside BMSCs. Finally, through RNA Immunoprecipitation experiments, we confirmed that YTHDC2 protein can bind to RUNX2 mRNA and accelerate its decomposition. Moreover, the immunofluorescence staining also showed a negative correlation between YTHDC2 and RUNX2. In conclusion, during BMSCs osteogenic differentiation, YTHDC2 protein showed decreased expression, resulting in a higher level of RUNX2 (mRNA and protein) expression inside cells, indicating YTHDC2 as a promising molecular target for the regulation of BMSCs osteogenic differentiation.

Incorporation of synthetic water-soluble curcumin polymeric drug within calcium phosphate cements for bone defect repairing.

Modified macroporous structures and active osteogenic substances are necessary to overcome the limited bone regeneration capacity and low degradability of self-curing calcium phosphate cement (CPC). Curcumin (CUR), which possesses strong osteogenic activity and poor aqueous solubility/bioavailability, esterifies the side chains in hyaluronic acid (HA) to form a water-soluble CUR-HA macromolecule. In this study, we incorporated the CUR-HA and glucose microparticles (GMPs) into the CPC powder to fabricate the CUR-HA/GMP/CPC composite, which not only retained the good injectability and mechanical strength of bone cements, but also significantly increased the cement porosity and sustained release property of CUR-HA in vitro. CUR-HA incorporation greatly improved the differentiation ability of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by activating the RUNX family transcription factor 2/fibroblast growth factor 18 (RUNX2/FGF18) signaling pathway, increasing the expression of osteocalcin and enhancing the alkaline phosphatase activity. In addition, in vivo implantation of CUR-HA/GMP/CPC into femoral condyle defects dramatically accelerated the degradation rate of cement and boosted local vascularization and osteopontin protein expression, and consequently promoted rapid bone regeneration. Therefore, macroporous CPC based composite cement with CUR-HA shows a remarkable ability to repair bone defects and is a promising translational application of modified CPC in clinical practice.

Impact of supraphysiologic MDM2 expression on chromatin networks and therapeutic responses in sarcoma

Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents.

RING finger gene 180 inhibits osteosarcoma progression through regulating chromobox homolog 4 ubiquitination

ABSTRACT Osteosarcoma (OS) is still the most common malignant bone tumor whose etiology remains largely unclear. Here, we aimed to investigate the role of a novel E3 ubiquitin ligase RING finger gene 180 (RNF180) in OS progression. RNF180 was significantly down-regulated in both OS tissues and cell lines. We up-regulated RNF180 using over-expression vector and knocked down RNF180 using specific short hairpin RNAs in OS cell lines. RNF180 over-expression inhibited the viability and proliferation yet promoted apoptosis in OS cells, while RNF180 knockdown showed the opposite effects. RNF180 also suppressed tumor growth and lung metastasis in mouse model, accompanied with elevated E-cadherin level and decreased ki-67 level. Besides, chromobox homolog 4 (CBX4) was predicted as a substrate of RNF180. RNF180 and CBX4 were both localized mainly in nucleus and their interaction was validated. RNF180 aggravated the decline of CBX4 level after cycloheximide treatment. RNF180 also promoted the ubiquitination of CBX4 in OS cells. Furthermore, CBX4 was significantly up-regulated in OS tissues. RNF180 also up-regulated Kruppel like factor 6 (KLF6) yet down-regulated RUNX family transcription factor 2 (Runx2) in OS, which served as downstream targets of CBX4. In addition, RNF180 inhibited migration, invasion and epithelial-mesenchymal transition (EMT) in OS cells, which were partially abolished by CBX4 over-expression. In conclusion, our findings demonstrated that RNF180 inhibits OS development via regulating CBX4 ubiquitination, and RNF180-CBX4 axis is a potential therapeutic target for OS treatment.

SOX9 Inhibits the Progression of Osteonecrosis of the Femoral Head via the Activation of the Wnt/Beta-Catenin Pathway

Background: In this study, we aimed to explore whether the SRY-box transcription factor 9 (SOX9) can play protective roles against the occurrence and development of osteonecrosis of the femoral head (ONFH) by regulating the proliferation, apoptosis, and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) via the Wnt/β-catenin pathway. Methods: We used 1600 mg of glucocorticoid (GC) to induce hBMSCs to establish an ONFH cell model and performed various experiments. Reverse transcription-quantitative polymerase chain reaction and western blotting assays were used to determine the expression levels of SOX9 and osteoblast markers, such as the RUNX family transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osterix, Wnt3a, and β-catenin. An ALP detection kit was used to measure the ALP activity. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to determine the cell viability. Results: GC treatment decreased the expression levels of RUNX2, ALP, and osterix, suppressed ALP activity, and inhibited SOX9 expression. SOX9 overexpression promoted GC-induced cell proliferation and decreased cell apoptosis. Additionally, hBMSCs were transfected with SOX9-small interfering RNA in GC treatment, and SOX9 knockdown was found to suppress the osteogenic differentiation of cells and decrease their viability. Conclusion: Our results revealed that SOX9 is related to the Wnt/β-catenin pathway in ONFH. Moreover, SOX9 participated in ONFH development by activating the Wnt/β-catenin pathway.

The RUNX Family of Proteins, DNA Repair, and Cancer.

The RUNX family of transcription factors, including RUNX1, RUNX2, and RUNX3, are key regulators of development and can function as either tumor suppressors or oncogenes in cancer. Emerging evidence suggests that the dysregulation of RUNX genes can promote genomic instability in both leukemia and solid cancers by impairing DNA repair mechanisms. RUNX proteins control the cellular response to DNA damage by regulating the p53, Fanconi anemia, and oxidative stress repair pathways through transcriptional or non-transcriptional mechanisms. This review highlights the importance of RUNX-dependent DNA repair regulation in human cancers.

Abstract 3280: Vrd induction therapy drives memory like NK cell development in newly diagnosed multiple myeloma patients

Abstract VRd induction therapy is a common approach to treat newly diagnosed multiple myeloma (MM) patients. Although in vitro studies of bortezomib (Velcade, V), lenalidomide (Revlimid, R), and dexamethasone (d) reveal mechanisms of these individual drugs, VRd therapy induced immune cell phenotype changes in vivo are unknown. We have utilized multimodal single cell methods to elucidate novel mechanisms of induction therapy response in human immune cells over time as a longitudinal study: pre-treatment (pretrt), after 2-cycles of VRd therapy (post-induction; pi) and after the end of 4-6 additional cycles of VRd therapy (end induction; ei). Protein measurements in plasma demonstrate significantly increased levels of IL-15 and IL-18 only at pi compared to pretrt but IL-2 and IFN-γ are progressively reduced from pretrt to ei. Both flow cytometry and single cell RNA -seq reveal that the proportion of NK cells (NK) significantly increased in PBMC only at pi compared to pretrt. NK subsets with strong dexamethasone effect emerge at pi; In contrast, memory like NK subsets (CD56dimCD16+NKG2A+CD94+) become the major NK in PBMC and bone marrow (BM) at ei. In addition, single cell ATAC-seq suggests that RUNX-family transcription factors may mediate lenalidomide to up-regulate NK cytotoxic gene expression during VRd therapy. In summary, comprehensive immune profiling of newly diagnosed MM patients suggests that transient increase of IL-15 and IL-18 may mediate the generation of memory like NK cells, which may play important roles in controlling MM growth and/or reducing risk of infection during VRd induction therapy. Citation Format: Tao Peng, Emma Kuan, Anthony Cicalo, Qiuyu Gong, Troy Torgerson, Xiaojun Li. Vrd induction therapy drives memory like NK cell development in newly diagnosed multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3280.

CircRBM23 regulates the switch between osteogenesis and adipogenesis of mesenchymal stem cells via sponging miR-338-3p.

The disruption of the differentiation between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) in bone marrow contributes to the adipocytes accumulation and bone loss, which leads to the development of osteoporosis (OP). The circular RNA (circRNA), circRBM23, was generated from the RNA binding motif protein 23 (RBM23) gene. It was reported that circRBM23 was downregulated in OP patients, but it remains unknown whether its downregulation is involved in the lineage switch of MSCs. We aimed to explore the role and mechanism of circRBM23 in regulating the switch between osteogenic and adipogenic differentiation of MSCs. The expression and function of circRBM23 in vitro was detected by qRT-PCR, alizarin red staining, and oil Red O staining. The interactions between circRBM23 and microRNA-338-3p (miR-338-3p) were analyzed by RNA pull-down assay, FISH, and dual-luciferase reporter assay. MSCs treated with lentivirus overexpression of circRBM23 was applied for both in vitro and in vivo experiments. CircRBM23 was expressed at lower levels in OP patients. Besides, circRBM23 was upregulated during osteogenesis and downregulated during adipogenesis of MSCs. CircRBM23 could promote the osteogenic differentiation, but inhibit the adipogenic differentiation of MSCs. Mechanistically, circRBM23 acted as a sponge for microRNA-338-3p (miR-338-3p) to enhance the expression of RUNX family transcription factor 2 (RUNX2). Our research indicates that circRBM23 could promote the switch from adipogenic to osteogenic differentiation of MSCs via sponging miR-338-3p. It might improve the understanding of the lineage switch of MSCs and provide a potential target for diagnosing and treating OP.

In vitro efficacy of whole-grain finger millet (Eleusine coracana) extract on primary rat osteoblasts

To improve the primary osteoblast cells of neonatal rats for mineralization, Finger millet grain (Eleusine coracana) extract was employed. In vitro digested extract (IVD) and citric acid buffered extract (CAB) were evaluated for cell proliferation, regeneration and matrix mineralization. The cytotoxic effect and the osteogenic differentiation were examined using MTT (3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, Giemsa stain and Von-Kossa respectively. Cell progression and osteogenic differentiation markers like Osteocalcin (OCN), Osteopontin (OPN), collagen type I (Col-I), collagen type II (Col-II) and collagen type III (Col-III), RUNX family transcription factor 2 (Runx2), Matrix metallo peptidase 9 (MMP-9) and peroxisome proliferator-activated receptor (PPARG) expression were made using reverse transcriptase-polymerase chain reaction. Exposure of different concentrations of the extract (100-800μg/ml of CAB and 25-400 μg/ml of IVD) on cells showed the characteristic morphology change in osteoblastic cells including the increase in cell number. In a dose-dependent way (p&lt;0.005), osteoblast matrix mineralization revealed that CAB stimulates osteoblasts. In light of these findings, finger millet (CAB extract) could be used for osteoporosis prevention and bone rebuilding.

Management of patients with germline predisposition to haematological malignancies considered for allogeneic blood and marrow transplantation: Best practice consensus guidelines from the UK Cancer Genetics Group (UKCGG), CanGene-CanVar, NHS England Genomic Laboratory Hub (GLH) Haematological Malignancies Working Group and the British Society of Blood and Marrow Transplantation and cellular therapy (BSBMTCT).

Germline predisposition to haematological cancers is increasingly being recognised. Widespread adoption of high-throughput and whole genome sequencing is identifying large numbers of causative germline mutations. Constitutional pathogenic variants in six genes (DEAD-box helicase 41 [DDX41], ETS variant transcription factor 6 [ETV6], CCAAT enhancer binding protein alpha [CEBPA], RUNX family transcription factor 1 [RUNX1], ankyrin repeat domain containing 26 [ANKRD26] and GATA binding protein 2 [GATA2]) are particularly significant in increasing the risk of haematological cancers, with variants in some of these genes also associated with non-malignant syndromic features. Allogeneic blood and marrow transplantation (BMT) is central to management in many haematological cancers. Identification of germline variants may have implications for the patient and potential family donors. Beyond selection of an appropriate haematopoietic stem cell donor there may be sensitive issues surrounding identification and counselling of hitherto asymptomatic relatives. If BMT is needed, there is frequently a clinical urgency that demands a rapid integrated multidisciplinary approach to testing and decision making involving haematologists in collaboration with Clinical and Laboratory Geneticists. Here, we present best practice consensus guidelines arrived at following a meeting convened by the UK Cancer Genetics Group (UKCGG), the Cancer Research UK (CRUK) funded CanGene-CanVar research programme (CGCV), NHS England Genomic Laboratory Hub (GLH) Haematological Oncology Malignancies Working Group and the British Society of Blood and Marrow Transplantation and Cellular Therapy (BSBMTCT).

Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability

BackgroundSolid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to...