RUNX2 Mutations Research Articles

Mmu-miR-1963 negatively regulates the ameloblast differentiation of LS8 cell line by directly targeting Smoc2 3’UTR

RUNX2 is a key regulator of osteogenic differentiation and odontoblastic differentiation. RUNX2 mutations could cause Cleidocranial dysplasia (CCD; OMIM119600), which is featured by abnormal development of bone and teeth. By using microRNA array, we identified a large number of microRNAs that showed different expression between wild-type Runx2 group and mutant groups. The aim of this study is to find out the effect of mmu-miR-1963, which was downregulated in all mutant Runx2 groups, on the ameloblast differentiation of LS8 cells. qPCR and Western Blot results showed the suppressive effect of mmu-miR-1963 on ameloblast differentiation of LS8 cell line. We further confirmed Smoc2 as one direct target of mmu-miR-1963. For the first time, we showed that mmu-miR-1963 could regulate the ameloblast differentiation of LS8 by targeting Smoc2. This study suggests the suppressive role of mmu-miR-1963 on ameloblast differentiation of LS8 via directly targeting the 3’UTR of Smoc2. We also demonstrated that Smoc2 itself could promote the ameloblast differentiation of LS8 for the first time. Our results indicate a novel explanation to the enamel hypoplasia phenotype in part of CCD patients.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Determination Of RUNX2 Single Nucleotide Polymorphism rs6930053 In Class I, II And III Malocclusions

Introduction: Runt-related transcription factor 2 (RUNX2) plays important roles in osteoblast differentiation, tooth development and chondrocyte maturation; hence its involvement in craniofacial development is paramount. Mutation in RUNX2 is implicated with cleidocranial dysplasia; a bone development disorder, while single nucleotide polymorphism (SNP) in RUNX2 is associated with Class II/2 malocclusion. This study aimed to determine RUNX2 SNP of DNA marker (rs6930053) in malocclusion patients from local population. Materials and Methods: Genomic DNA were extracted from unstimulated saliva of 31 Class I (control samples), 30 Class II and 30 Class III malocclusion patients. Cephalometric measurements were performed prior to saliva samples collection. The DNA was amplified using the specific primers for marker rs6930053 and the genotyping was done by sequencing. Chi-square test was used to determine differences in allele and genotype frequencies (p<0.05). Results: No significant differences were observed in RUNX2 SNP (rs8004560) in Class I and Class III malocclusion. However, there were significant differences between allele (p=0.000) and genotype (p=0.000) frequency within Class II alone; while significant differences was detected only in allele frequency between control and Class II malocclusion (p=0.019). Conclusion(s): There is genetic association between RUNX2 (rs6930053) in Class II malocclusion in our population. Further studies involving larger number of samples and other DNA markers of RUNX2 gene should be developed in order to understand the exact role and mechanism of RUNX2 in different classes of malocclusions.

A novel 18-bp in-frame deletion mutation in RUNX2 causes cleidocranial dysplasia

ObjectivesRunt-related transcription factor 2 (RUNX2) gene is known to cause rare autosomal dominant skeletal disorder Cleidocranial dysplasia (CCD). Here, we explored a novel, large deletion in RUNX2 gene in a Chinese patient with CCD and the function associated with the mutation. DesignGenomic DNA was extracted from the peripheral blood and subjected to do DNA sequencing. Sanger sequencing was used to do mutational analysis of the RUNX2 gene. Function associated with RUNX2 mutation was investigated by performing conservation analysis, secondary structure analysis, subcellular localization study and reporter assay. ResultsWe identified a novel, large deletion mutation involving a c.243-260delGGCGGCTGCGGCGGCGGC mutation in exon 2 of the RUNX2 gene. Conservation and secondary structure analysis revealed that the novel mutation located in QA domain and converted the structure of RUNX2. Subcellular localization analysis revealed that the novel mutant showed the same intracellular localization with the wild type of RUNX2, and both of them localized exclusively in the nucleus. While reporter assay indicated the novel mutant severely impaired the transactivation activities of RUNX2 gene. ConclusionsOur findings demonstrated that the novel c.243-260delGGCGGCTGCGGCGGCGGC mutation resulted in CCD. These results extend the spectrum of RUNX2 mutations in CCD patients and suggest a functional role of the novel mutation in CCD.

A novel gene mutation of Runx2 in cleidocranial dysplasia.

Haploinsufficiency of the runt-related transcription factor 2 (Runx2) gene is widely known to be responsible for cleidocranial dysplasia (CCD). To date, more than 190 mutations in Runx2 gene have been reported to be related to CCD. In this study, a novel mutation of Runx2 gene was observed in a female with CCD. Genomic DNA was extracted from peripheral venous blood of the proband and eleven members of her family. Genetic testing on these twelve people identified a novel missense mutation (c.895 T>C, Y299H) in exon 5 of the RUNX2 gene in the proband. This mutation results in an amino acid change at codon 895 (P.Tyr 299 His.) from a tryptophan codon (TAT) to a histidine codon (CAT). Our finding may further extend the known mutation spectrum of the RUNX2 gene, and facilitate prenatal genetic diagnosis of CCD in the future.

MiR-221-regulated KIT level by wild type or leukemia mutant RUNX1: a determinant of single myeloblast fate decisions that - collectively - drives or hinders granulopoiesis.

RUNX1, a master transcription factor of hematopoiesis, was shown to orchestrate both cell proliferation and differentiation during granulopoiesis by regulating microRNAs (miRs). In this study, taking advantage of the miR-ON reporter system, we monitored first, how the granulocyte colony stimulation factor (GCSF) temporally modulates the concomitant level variation of miR-221 and one of its prototypic targets, the stem cell factor receptor KIT, in single 32DmiR-ON-221 myeloblasts expressing wild type RUNX1. Second, with the same reporter system we assessed how these temporal dynamics are affected by the t(8;21)(q22;q22) acute myelogenous leukemia mutant RUNX1-MTG8 (RM8) in single 32D-RM8miR-ON-221 myeloblasts. Depending on either wild type, or mutant, RUNX1 transcriptional regulation, the cell-context specific miR-221-regulated KIT level translates into differential single cell fate decisions. Collectively, single cell fate choices translate into either initial expansion of undifferentiated myeloblasts followed by terminal granulocyte differentiation, as it happens in normal granulopoiesis, or aggressive growth of undifferentiated myeloblasts, as it happens in RUNX1-MTG8-positive acute myelogenous leukemia. Increasing knowledge of biological changes, due to altered miRNA dynamics, is expected to have relevant translational implications for leukemia detection and treatment.

Runx1 mediates the development of the granular convoluted tubules in the submandibular glands.

The mouse granular convoluted tubules (GCTs), which are only located in the submandibular gland (SMG) are known to develop and maintain their structure in an androgen-dependent manner. We previously demonstrated that the GCTs are involuted by the epithelial deletion of core binding factor β (CBFβ), a transcription factor that physically interacts with any of the Runt-related transcription factor (RUNX) proteins (RUNX1, 2 and 3). This result clearly demonstrates that the Runx /Cbfb signaling pathway is indispensable in the development of the GCTs. However, it is not clear which of the RUNX proteins plays useful role in the development of the GCTs by activating the Runx /Cbfb signaling pathway. Past studies have revealed that the Runx /Cbfb signaling pathway plays important roles in various aspects of development and homeostatic events. Moreover, the Runx genes have different temporospatial requirements depending on the biological situation. In the present study, the GCTs of the SMG showed a remarkable phenotype of, which phenocopied the epithelial deletion of Cbfb, in epithelial-specific Runx1 conditional knock-out (cKO) mice. The results indicate that Runx1 works as a partner of Cbfb during the development of the GCTs. We also discovered that the depletion of Runx1 resulted in the reduced secretion of saliva in male mice. Consistent with this finding, one of the water channels, Aquaporin-5 (AQP5) was mislocalized in the cytoplasm of the Runx1 mutants, suggesting a novel role of Runx1 in the membrane trafficking of AQP5. In summary, the present findings demonstrated that RUNX1 is essential for the development of the GCTs. Furthermore, RUNX1 could also be involved in the membrane trafficking of the AQP5 protein of the acinar cells in the SMG in order to allow for the proper secretion of saliva.

Clinical Observations of AML Expressing Mutant RUNX1 and Pre-Clinical Studies of RUNX1-Targeted Novel Therapy of AML.

7030Background: Runt-related transcription factor 1 (RUNX1) is critically involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 occur in ~10% of AML, especially in older patien...

A novel RUNX2 mutation in exon 8, G462X, in a patient with Cleidocranial Dysplasia.

To identify a novel mutation of Runx2 gene in Cleidocranial Dysplasia (CCD) patients and to characterize the functional consequences of this mutation. The subjects consisted of 12 Korean CCD patients. After oral epithelial cells were collected using a mouthwash technique, genomic DNA was extracted. Screening for Runx2 mutation was performed using direct sequencing of polymerase chain reaction (PCR) products for exons 1-8. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transacting activity, cyclohexamide chase assay for Runx2 protein stability, real-time PCR for mRNA level of Runx2 downstream bone marker genes, and alkaline phosphatase (ALP) staining assay in mesenchymal stem cells for osteoblast differentiation. Of the 12 patients, seven showed Runx2 mutations reported previously and four showed no mutation. A novel mutation, G462X in exon 8, which was located in the C-terminus of proline/serine/threonine-rich (PST) domain, was found in one patient. In the luciferase assay, Runx2 transacting activity was decreased in Runx2-G462X transfected cells. In the cyclohexamide chase assay, Runx2-G462X mutation reduced the stability of Runx2 protein. Expression of the bone marker genes (osteocalcin, ALP, Type I collagen αI, matrix metalloproteinase-13, bone sialoprotein, and osteopontin) decreased in G462X-transfected cells. In the ALP staining assay, osteoblast differentiation was reduced in Runx2-G462X overexpressed cell. The G462X mutation might reduce the Runx2 transacting activity, lower the protein stability, downgrade the expression of bone marker genes, and eventually diminish osteoblast differentiation in CCD patients.

Clinical Outcomes and Co-Occurring Mutations in Patients with RUNX1-Mutated Acute Myeloid Leukemia.

(1) Runt-related transcription factor 1 (RUNX1) mutations in acute myeloid leukemia (AML) are often associated with worse prognosis. We assessed co-occurring mutations, response to therapy, and clinical outcomes in patients with and without mutant RUNX1 (mRUNX1); (2) We analyzed 328 AML patients, including 177 patients younger than 65 years who received intensive chemotherapy and 151 patients >65 years who received hypomethylating agents. RUNX1 and co-existing mutations were identified using next-generation sequencing; (3) RUNX1 mutations were identified in 5.1% of younger patients and 15.9% of older patients, and were significantly associated with increasing age (p = 0.01) as well as intermediate-risk cytogenetics including normal karyotype (p = 0.02) in the elderly cohort, and with lower lactate dehydrogenase (LDH; p = 0.02) and higher platelet count (p = 0.012) overall. Identified co-occurring mutations were primarily ASXL1 mutations in older patients and RAS mutations in younger patients; FLT3-ITD and IDH1/2 co-mutations were also frequent. Younger mRUNX1 AML patients treated with intensive chemotherapy experienced inferior treatment outcomes. In older patients with AML treated with hypomethylating agent (HMA) therapy, response and survival was independent of RUNX1 status. Older mRUNX1 patients with prior myelodysplastic syndrome or myeloproliferative neoplasms (MDS/MPN) had particularly dismal outcome. Future studies should focus on the prognostic implications of RUNX1 mutations relative to other co-occurring mutations, and the potential role of hypomethylating agents for this molecularly-defined group.

Analysis of novel RUNX2 mutations in Chinese patients with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant inheritable skeletal disorder characterized by cranial dysplasia, clavicle hypoplasia and dental abnormalities. This disease is mainly caused by heterozygous mutations in RUNX2, a gene that encodes an osteoblast-specific transcription factor. In the present study, mutational analyses of RUNX2 gene were performed on four unrelated Chinese patients with CCD. Four different RUNX2 mutations were detected in these patients, including one nonsense mutation (c.199C>T p.Q67X) and three missense mutations (c.338T>G p.L113R, c.557G>C p.R186T and c.673C>T p.R225W). Among them, two mutations (c.199C>T p.Q67X and c.557G>C p.R186T) were novel and the other two had been reported in previous literatures. Except for Q67X mutation located in the Q/A domain, other three mutations were clustered within the highly conserved Runt domain. Green fluorescent protein (GFP) and RUNX2 fusion protein analyses in vitro showed that nuclear accumulation of RUNX2 protein was disturbed by Q67X mutation, while the other two mutations (c.338T>G p.L113R and c.557G>C p.R186T) had no effects on the subcellular distribution of RUNX2. Luciferase reporter assay demonstrated that all the three novel RUNX2 mutations significantly reduced the transactivation activity of RUNX2 on osteocalcin promoter. Our findings enrich the evidence of molecular genetics that the mutations of RUNX2 gene are responsible for CCD.

Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of p53-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63.

Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant p53 stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant p53-depleted MiaPaCa-2 cells. Surprisingly, RUNX2 silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant p53 or RUNX2 potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by p63 depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of p53-mutated pancreatic cancer cells.

Abstract LB-081: Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation

Abstract RUNX1 is a transcription factor involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 (mtRUNX1) have been documented in de novo and secondary AML (10%), MDS (~10%) and CMML (up to 37%). Germ-line, mono-allelic mutations and deletions in RUNX1 cause the highly-penetrant (~40%) autosomal dominant, Familial Platelet Disorder with a propensity to Myeloid Malignancy (FPD-MM). The majority of mtRUNX1 are missense mutations, large deletions or truncation-mutations in the DNA-binding ‘RUNT’ homology domain, or in the C-terminal transactivation domain. These mutations behave mostly as loss of function mutations, associated with relative resistance to standard chemotherapy and an unfavorable prognosis in AML. This highlights an unmet need to develop and test novel targeted therapies for AML due to germ-line or somatic mtRUNX1. In the present studies, we demonstrate that shRNA-mediated knockdown of mutant and wild-type RUNX1 repressed its target genes MYC, PU.1 and MPO (myeloperoxidase), as well as inhibited growth and induced apoptosis of AML cells expressing mtRUNX1 (OCI-AML5 and Mono-Mac-1). Ex vivo depletion of RUNX1 abrogated the leukemia initiating potential of OCI-AML5 cells. After engraftment, tetracycline-inducible shRNA-mediated in vivo knockdown of RUNX1, as compared to the non-induced controls, enhanced survival of the immune-depleted (NSG) mice engrafted with OCI-AML5 cells. RUNX1 transcription is driven by the BET protein BRD4-occupied super enhancer in the first intron of the RUNX1 gene. A heat map of Hi-C interaction scores within the RUNX1 TAD (topology-associated domain) showed triangle-shaped regions of high interaction, with CTCF binding sites defining the TAD boundaries, anchoring a loop that separates the RUNX1 TAD from other TADs. Consistent with this, our findings show that, shRNA-mediated depletion of BRD4 or treatment with BET protein (BETP) inhibitor (BETi) OTX015 reduced BRD4 occupancy at the enhancer and promoter of RUNX1. This was associated with depletion of RUNX1 and its target-gene expressions and apoptosis of cultured and patient-derived (PD), primary AML blast progenitor cells (BPCs). Additionally, treatment of NSG mice engrafted with luciferase-transduced OCI-AML5 cells with OTX015 (50 mg/kg/day X 5 days, for 3 weeks) reduced the AML burden and significantly improved their survival (p < 0.01). Co-treatment with the BETi and BCL2-antagonist venetoclax or CDK4/6 antagonist palbociclib or decitabine synergistically induced apoptosis of OCI-AML5 and PD AML BPCs (combination index values of < 1.0). Results describing the in vivo efficacy of BETi and BETP-PROTAC (proteolysis targeting chimera), alone and in combinations against AML expressing mtRUNX1 will be updated at the AACR meetings. Collectively, these findings highlight novel and effective, mechanistically-targeted, single agent or combination, BETP antagonist-based therapy of AML expressing mtRUNX1. Citation Format: Christopher P. Mill, Courtney DiNardo, Warren C. Fiskus, Dyana T. Saenz, Baohua Sun, Agnieszka J. Nowak, Misun Kim, Mark Routbort, Koichi Takahashi, Kapil N. Bhalla. Novel and effective RUNX1-targeted therapy for AML expressing RUNX1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-081. doi:10.1158/1538-7445.AM2017-LB-081

Novel Mutation of the RUNX2 Gene in Patients with Cleidocranial Dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder linked to mutations in the Runt-related transcription factor 2, encoded by the RUNX2 gene, which is essential for osteoblast differentiation and skeletal development. Here, we describe a novel nonsense mutation (c.532C>T; p.Q178X) in RUNX2 identified in 3 affected members of a Polish family with CCD. The localization and transcriptional transactivation studies show that the mutated form of the protein has altered the subcellular localization and significantly decreased transactivation properties, respectively. Consequently, our data show that the c.532C>T mutation generates a defective RUNX2 protein and is genetically linked to the CCD phenotype.

Characterization of Runx2 phosphorylation sites required for TGF-β1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells.

Transforming growth factor-beta1 (TGF-β1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase-13 (MMP-13) expression in osteoblastic cells. MMP-13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF-β1-mediated stimulation of MMP-13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF-β1-stimulation of MMP-13 expression via Runx2 in osteoblastic cells was elucidated. TGF-β1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106-01) and human (MG-63) osteoblastic cells. Pretreatment with okadaic acid, a serine-threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF-β1-mediated stimulation of MMP-13 mRNA expression decreased. Nano-ESI/LC/MS analysis identified that TGF-β1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF-β1-mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF-β1 stimulated MMP-13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF-β1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP-13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone-related diseases.

Clinical observations of AML expressing mutant RUNX1 and pre-clinical studies of RUNX1-targeted novel therapy of AML.

7030 Background: Runt-related transcription factor 1 ( RUNX1) is critically involved in normal and malignant hematopoiesis. Somatic mutations in RUNX1 occur in ~10% of AML, especially in older patients with history of radiation or antecedent hematologic disorder. Presence of mRUNX1 is reported to confer relative resistance to therapy and poorer prognosis in AML, and there are no mRUNX1-targeted or specific therapies available. Methods: We retrospectively analyzed outcomes of 94 mRUNX1 and 444 wild-type RUNX1 AML patients treated at our institution from 9/2013 to 12/2016. We also determined the pre-clinical efficacy of a targeted therapy against cultured and primary AML cells expressing mRUNX1. Results: 67% of mRUNX1 patients were > 65 years of age. Co-occurring mutations with mRUNX1 were ASXL1 (33%), N/KRAS (20%), FLT3 (20%), IDH2 (18%), IDH1 (13%) and TET2 (10%). In patients > 65 years treated with hypomethylating agent-based therapy, the presence or absence of mRUNX1 did not impact response rate (42% vs 46% CR/CRp, p = 0.67), median event-free survival (3.4 vs 4.7 mo, p = 0.82) or overall survival (11.5 vs 9.3 mo, p = 0.97). In mRUNX1 expressing AML OCI-AML5 and MonoMac1 cells, knockdown of RUNX1 by shRNA repressed its targets, e.g., MYC and PU.1, inhibited growth and induced apoptosis. Ex vivo knockdown of RUNX1 abrogated in vivo leukemia initiation by OCI-AML5 cells. After engraftment, inducible shRNA-mediated in vivo knockdown of RUNX1 restored survival of immune-depleted NSG mice engrafted with OCI-AML5 cells. RUNX1 transcription is driven by a super enhancer occupied by the bromodomain extraterminal protein (BETP), BRD4. Accordingly, BRD4 knockdown by shRNA or treatment with the BRD4-inhibitor OTX015 depleted RUNX1 and its targets, and induced apoptosis of AML cells. Treatment of OCI-AML5 cell-engrafted NSG mice with OTX015 (50 mg/Kg/day X 5, for 3 wks) reduced AML burden and improved survival (p < 0.01). Co-treatment with the BETi and BCL2 inhibitor venetoclax or CDK4/6 antagonist palbociclib or decitabine synergistically induced apoptosis of OCI-AML5 and primary AML blasts. Conclusions: These findings highlight a novel, promising, BETP antagonist-based therapy of AML expressing mtRUNX1.

Functional analysis of novel RUNX2 mutations in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal disorder caused by mutation of runt-related transcription factor 2 (RUNX2) gene. The purpose of this study was to explore novel RUNX2 mutations in seven individuals with CCD and investigate the function of the mutant RUNX2 proteins. DNA samples were prepared from the peripheral blood of the CCD individuals, and then subjected to DNA sequencing. Conservation and secondary structure analysis were performed based on RUNX2 sequencing results. pEGFP-C1 plasmids containing GFP-tagged wild-type RUNX2 and three novel RUNX2 mutations expression cassettes were constructed, and then transfected into HEK293T cells. Cell fluorescence, luciferase assay and western blotting were used to analyse the subcellular distribution and function of the mutant RUNX2 proteins. Three novel mutations (R193G, 258fs, Y400X) were found in the seven CCD patients. Conservation and structure analysis show one novel mutation (R193G) in Runt domain and two novel mutations (258fs and Y400X) in PST domain of RUNX2. Western blotting confirmed that the 258fs and Y400X mutations produced truncated proteins. Fluorescence detection showed that the three novel mutants localised exclusively in the nucleus. However, luciferase assay indicated all mutants severely impaired the transactivation activities of RUNX2 on osteocalcin promoter. Our results broaden the spectrum of RUNX2 mutations in CCD individuals and demonstrated that loss of function in RUNX2 is responsible for CCD.

MAP Kinase-Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic Modification of Chromatin During Osteoblast Differentiation.

RUNX2, an essential transcription factor for osteoblast differentiation and bone formation is activated by ERK/MAP kinase-dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2-binding regions of Bglap2 and Ibsp. Growth of MC3T3-E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin-bound P-ERK, P-RUNX2, p300, and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di-methylation, another gene activation-associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono-, di-, and tri-methylation in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation-dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2-deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non-phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin-associated P-ERK, p300, and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P-RUNX2-dependent process involving epigenetic modifications of specific promoter regions. J. Cell. Physiol. 232: 2427-2435, 2017. © 2016 Wiley Periodicals, Inc.

A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia

BackgroundHaploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations.MethodsGenomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies.ResultsWe identified 2 complex heterozygous mutations involving a c.398–399 insACAGCAGCAGCAGCA insertion and a c.411–412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus.ConclusionsOur findings demonstrated that the novel c.398–399insACAGCAGCAGCAGCA mutation occurred alongside the c.411–412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.

A limb-girdle myopathy phenotype of RUNX2 mutation in a patient with cleidocranial dysplasia: a case study and literature review

BackgroundCleidocranial dysplasia (CCD) is a rare hereditary disorder that arises from heterozygous loss of function mutations in the runt-related transcription factor 2 (RUNX2) gene. As RUNX2 is mainly expressed in osteoblasts, CCD typically affects the skeletal and dental systems. Few studies have investigated RUNX2 mutation effects on non-skeletal systems. Here, we describe limb-girdle myopathy, an uncommon phenotype of CCD, in a patient with a heterozygous missense mutation (p.R225Q) in the RUNX2 gene.Case presentationA 58 year-old man presented with progressive back pain and six months of weakness in the proximal parts of all four limbs. Physical examinations showed that he was short in stature (height, 164.4 cm; weight, 79.1 kg) with a dysmorphic face, including hypertelorism, midface hypoplasia, and chin protrusion. At a young age, he had received orthodontic surgery, due to dental abnormalities. Neurological examinations revealed sloping shoulders, weakness, and atrophy in the proximal areas of the arms, shoulder girdle muscles, and legs. The deep tendon reflex and sensory system were normal. Radiological examinations revealed mild scoliosis, shortened clavicles, and a depressed skull bone, which were consistent with a clinical diagnosis of CCD. Electromyography (EMG) studies showed myogenic polyphasic waves in the deltoid, biceps brachii, and rectus femoris muscles. Instead, the EMG findings were normal in the first dorsal interosseous, tibialis anterior and facial muscles. The EMG findings were compatible with a limb-girdle pattern with facial sparing. The patient’s family history showed his father and eldest daughter with similar dysmorphic faces, skeletal disorders and proximal upper extremity weakness. We sequenced the RUNX2 gene and discovered a heterozygous missense mutation (c.G674A, p.R225Q), which altered the C-terminal end of the RUNX2 protein. This mutation was predicted to inactivate the protein and might affect its interactions with other proteins. This mutation co-segregated with the disease phenotypes in the family.ConclusionsWe described limb-girdle myopathy in a patient with CCD that carried a heterozygous RUNX2 missense mutation. This uncommon phenotype expanded the phenotypic spectrum of the RUNX2 p.R225Q mutation. The role of RUNX2 in myogenic development merits future studies. Our findings remind clinicians that myopathic patients with myopathies combined with facial dysmorphism and shortened clavicles should consider the diagnosis of CCD.