Overexpression of transcription factor TBX5 inhibits the activation of YAP1-TEAD1 pathway to promote ferroptosis in lung cancer cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for more than 80 % of lung cancer (LC) cases, making it the primary cause of cancer-related mortality worldwide. T-box transcription factor 5 (TBX5) is an important regulator of embryonic and organ development and plays a key role in cancer development. Here, our objective was to investigate the involvement of TBX5 in ferroptosis within LC cells and the underlying mechanisms. METHODS: First, TBX5 expression was examined in human LC cells. Next, overexpression of TBX5 and Yes1-associated transcriptional regulator (YAP1) and knockdown of TEA domain 1 (TEAD1) were performed in A549 and NCI-H1703 cells. The proliferation ability of A549 and NCI-H1703 cells, GSH, MDA, ROS, and Fe2+ levels were measured. Co-immunoprecipitation (Co-IP) was performed to verify whether TBX5 protein could bind YAP1. Then TBX5, YAP1, TEAD1, GPX4, p53, FTH1, SLC7A11 and PTGS2 protein levels were assessed. Finally, we verified the effect of TBX5 on ferroptosis in LC cells in vivo. RESULTS: TBX5 expression was down-regulated in LC cells, especially in A549 and NCI-H1703 cells. Overexpression of TBX5 significantly decreased proliferation ability of A549 and NCI-H1703 cells, downregulated GPX4 and GSH levels, and upregulated MDA, ROS, and Fe2+ levels. Co-IP verified that TBX5 protein could bind YAP1. Moreover, oe-YAP1 promoted proliferation ability of A549 and NCI-H1703 cells transfected with Lv-TBX5, upregulated GPX4 and GSH levels and downregulated MDA, ROS, and Fe2+ levels. Additionally, oe-YAP1 promoted FTH1 and SLC7A11 levels and inhibited p53 and PTGS2 levels in A549 and NCI-H1703 cells transfected with Lv-TBX5. However, transfection with si-TEAD1 further reversed these effects. In vivo experiments further validated that TBX5 promoted ferroptosis in LC cells. CONCLUSIONS: TBX5 inhibited the activation of YAP1-TEAD1 pathway to promote ferroptosis in LC cells.

Proteostatic reactivation of the developmental transcription factor TBX3 drives BRAF/MAPK-mediated tumorigenesis.

MAPK pathway-driven tumorigenesis, often induced by BRAFV600E, relies on epithelial dedifferentiation. However, how lineage differentiation events are reprogrammed remains unexplored. Here, we demonstrate that proteostatic reactivation of developmental factor, TBX3, accounts for BRAF/MAPK-mediated dedifferentiation and tumorigenesis. During embryonic development, BRAF/MAPK upregulates USP15 to stabilize TBX3, which orchestrates organogenesis by restraining differentiation. The USP15-TBX3 axis is reactivated during tumorigenesis, and Usp15 knockout prohibits BRAFV600E-driven tumor development in a Tbx3-dependent manner. Deleting Tbx3 or Usp15 leads to tumor redifferentiation, which parallels their overdifferentiation tendency during development, exemplified by disrupted thyroid folliculogenesis and elevated differentiation factors such as Tpo, Nis, Tg. The clinical relevance is highlighted in that both USP15 and TBX3 highly correlates with BRAFV600E signature and poor tumor prognosis. Thus, USP15 stabilized TBX3 represents a critical proteostatic mechanism downstream of BRAF/MAPK-directed developmental homeostasis and pathological transformation, supporting that tumorigenesis largely relies on epithelial dedifferentiation achieved via embryonic regulatory program reinitiation.

Unraveling molecular advancements in chordoma tumorigenesis and treatment response: a review of scientific discoveries and clinical implications.

Chordomas are tumors thought to originate from notochordal remnants that occur in midline structures from the cloves of the skull base to the sacrum. In adults, the most common location is the sacrum, followed by the clivus and then mobile spine, while in children a clival origin is most common. Most chordomas are slow growing. Clinical presentation of chordomas tend to occur late, with local invasion and large size often complicating surgical intervention. Radiation therapy with protons has been proven to be an effective adjuvant therapy. Unfortunately, few adjuvant systemic treatments have demonstrated significant effectiveness, and chordomas tend to recur despite intensive multimodal care. However, insight into the molecular underpinnings of chordomas may guide novel therapeutic approaches including selection for immune and molecular therapies, individualized prognostication of outcomes, and real-time noninvasive assessment of disease burden and evolution. At the genomic level, elevated levels of brachyury stemming from duplications and mutations resulting in altered transcriptional regulation may introduce druggable targets for new surgical adjuncts. Transcriptome and epigenome profiling have revealed promoter- and enhancer-dependent mechanisms of protein regulation, which may influence therapeutic response and long-term disease history. Continued scientific and clinical advancements may offer further opportunities for treatment of chordomas. Single-cell transcriptome profiling has further provided insight into the heterogeneous molecular pathways contributing to chordoma propagation. New technologies such as spatial transcriptomics and emerging biochemical analytes such as cell-free DNA have further augmented the surgeon-clinician's armamentarium by facilitating detailed characterization of intra- and intertumoral biology while also demonstrating promise for point-of-care tumor quantitation and assessment. Recent and ongoing clinical trials highlight accelerating interest to translate laboratory breakthroughs in chordoma biology and immunology into clinical care. In this review, the authors dissect the landmark studies exploring the molecular pathogenesis of chordoma. Incorporating this into an outline of ongoing clinical trials and discussion of emerging technologies, the authors aimed to summarize recent advancements in understanding chordoma pathogenesis and how neurosurgical care of chordomas may be augmented by improvements in adjunctive treatments.

Exhaustive identification of genome-wide binding events of transcriptional regulators.

Genome-wide binding assays aspire to map the complete binding pattern of gene regulators. Common practice relies on replication-duplicates or triplicates-and high stringency statistics to favor false negatives over false positives. Here we show that duplicates and triplicates of CUT&RUN are not sufficient to discover the entire activity of transcriptional regulators. We introduce ICEBERG (Increased Capture of Enrichment By Exhaustive Replicate aGgregation), a pipeline that harnesses large numbers of CUT&RUN replicates to discover the full set of binding events and chart the line between false positives and false negatives. We employed ICEBERG to map the full set of H3K4me3-marked regions, the targets of the co-factor ß-catenin, and those of the transcription factor TBX3, in human colorectal cancer cells. The ICEBERG datasets allow benchmarking of individual replicates, comparing the performance of peak calling and replication approaches, and expose the arbitrary nature of strategies to identify reproducible peaks. Instead of a static view of genomic targets, ICEBERG establishes a spectrum of detection probabilities across the genome for a given factor, underlying the intrinsic dynamicity of its mechanism of action, and permitting to distinguish frequent from rare regulation events. Finally, ICEBERG discovered instances, undetectable with other approaches, that underlie novel mechanisms of colorectal cancer progression.

Zebrafish as a model to investigate a biallelic gain-of-function variant in MSGN1, associated with a novel skeletal dysplasia syndrome.

BACKGROUND/OBJECTIVES: Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos. RESULTS: The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix-Loop-Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function. CONCLUSION: In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.

Role of TBX20 Truncating Variants in Dilated Cardiomyopathy and Left Ventricular Noncompaction.

BACKGROUND: Less than 40% of patients with dilated cardiomyopathy (DCM) have a pathogenic/likely pathogenic genetic variant identified. TBX20 has been linked to congenital heart defects; although an association with left ventricular noncompaction (LVNC) and DCM has been proposed, it is still considered a gene with limited evidence for these phenotypes. This study sought to investigate the association between the TBX20 truncating variant (TBX20tv) and DCM/LVNC. METHODS: TBX20 was sequenced by next-generation sequencing in 7463 unrelated probands with a diagnosis of DCM or LVNC, 22 773 probands of an internal comparison group (hypertrophic cardiomyopathy, channelopathies, or aortic diseases), and 124 098 external controls (individuals from the gnomAD database). Enrichment of TBX20tv in DCM/LVNC was calculated, cosegregation was determined in selected families, and clinical characteristics and outcomes were analyzed in carriers. RESULTS: TBX20tv was enriched in DCM/LVNC (24/7463; 0.32%) compared with internal (1/22 773; 0.004%) and external comparison groups (4/124 098; 0.003%), with odds ratios of 73.23 (95% CI, 9.90-541.45; P<0.0001) and 99.76 (95% CI, 34.60-287.62; P<0.0001), respectively. TBX20tv was cosegregated with DCM/LVNC phenotype in 21 families for a combined logarythm of the odds score of 4.53 (strong linkage). Among 57 individuals with TBX20tv (49.1% men; mean age, 35.9±20.8 years), 41 (71.9%) exhibited DCM/LVNC, of whom 14 (34.1%) had also congenital heart defects. After a median follow-up of 6.9 (95% CI, 25-75:3.6-14.5) years, 9.7% of patients with DCM/LVNC had end-stage heart failure events and 4.8% experienced malignant ventricular arrhythmias. CONCLUSIONS: TBX20tv is associated with DCM/LVNC; congenital heart defect is also present in around one-third of cases. TBX20tv-associated DCM/LVNC is characterized by a nonaggressive phenotype, with a low incidence of major cardiovascular events. TBX20 should be considered a definitive gene for DCM and LVNC and routinely included in genetic testing panels for these phenotypes.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

TBX15 and SDHB expression changes in colorectal cancer serve as potential prognostic biomarkers.

Alterations in the expression of certain genes could be associated with both patient mortality rates and drug resistance. This study aimed to identify genes in colorectal cancer (CRC) that potentially serve as hub genes influencing patient survival rates. RNA-Seq data were downloaded from the cancer genome atlas database, and differential expression analysis was performed between tumors and healthy controls. Through the utilization of univariate and multivariate Cox regression analyses, in combination with the MCODE clustering module, the genes whose expression changes were related to survival rate and the hub genes related to them were identified. The mortality risk model was computed using the hub genes. CRC samples and the RT-qPCR method were utilized to confirm the outcomes. PharmacoGx data were employed to link the expression of potential genes to medication resistance and sensitivity. The results revealed the discovery of seven hub genes, which emerged as independent prognostic markers. These included HOXC6, HOXC13, HOXC8, and TBX15, which were associated with poor prognosis and overexpression, as well as SDHB, COX5A, and UQCRC1, linked to favorable prognosis and downregulation. Applying the risk model developed with the mentioned genes revealed a markedly higher incidence of deceased patients in the high-risk group compared to the low-risk group. RT-qPCR results indicated a decrease in SDHB expression and an elevation in TBX15 levels in cancer samples relative to adjacent healthy tissue. Also, PharmacoGx data indicated that the expression level of SDHB was correlated with drug sensitivity to Crizotinib and Dovitinib. Our findings highlight the potential association between alterations in the expression of genes such as HOXC6, HOXC13, HOXC8, TBX15, SDHB, COX5A, and UQCRC1 and increased mortality rates in CRC patients. As revealed by the PPI network, these genes exhibited the most connections with other genes linked to survival.

Computed tomographic findings in TBX4 mutation: a common cause of severe pulmonary artery hypertension in children.

BACKGROUND: Mutations in the T-Box 4 (TBX4) gene are a lesser-known cause of heritable pulmonary arterial hypertension (PAH). Patients with heritable PAH typically have worse outcomes when compared with patients with idiopathic PAH, yet little is known about the phenotypical presentation of this mutation. OBJECTIVE: This article reviews the pattern of chest CT findings in pediatric patients with PAH and TBX4 mutations and compares their radiographic presentation with those of age-matched patients with PAH but without TBX4 mutations. MATERIALS AND METHODS: A retrospective chart review of the pulmonary arterial hypertension database was performed. Pediatric patients with PAH-confirmed TBX4 mutations and an available high CT were included. Fifteen (9 females) patients met the inclusion criteria. Fourteen (8 females) age-matched controls with diagnosed PAH but without TBX4 mutations were also evaluated. The median age at diagnosis was 7.4 years (range: 0.1-16.4 years). Demographic information and clinical outcomes were collected. CTs of the chest were reviewed for multiple airway, parenchymal, and structural abnormalities (16 imaging findings in total). Chi-square tests were used to compare the prevalence of each imaging finding in the TBX4 cohort compared to the control group. RESULTS: Patients with TBX-4 mutations had increased presence of peripheral or subpleural irregularity (73% vs 0%, P < 0.01), cystic lucencies (67% vs 7%, P < 0.01), and linear or reticular opacity (53% vs 0%, P < 0.01) compared to the control group. Ground glass opacities, bronchiectasis, and centrilobular nodules were not significantly different between the two patient groups (P > 0.05). CONCLUSION: TBX4 mutations have distinct imaging phenotypes in pediatric patients with PAH. Compared to patients without this mutation, patients with TBX-4 genes typically present with peripheral or subpleural irregularity, cystic lucencies, and linear or reticular opacity.

A novel non-recurrent CNV deletion involving TBX4 and leaving TBX2 intact causes congenital alveolar dysplasia.

Congenital alveolar dysplasia (CAD) belongs to rare lethal lung developmental disorders (LLDDs) in neonates, manifesting with acute respiratory failure and pulmonary arterial hypertension refractory to treatment. The majority of CAD cases have been associated with copy-number variant (CNV) deletions at 17q23.1q23.2 or 5p12. Most CNV deletions at 17q23.1q23.2 were recurrent and encompassed two closely located genes, TBX4 and TBX2. In a few CAD cases, intragenic frameshifting deletions or single-nucleotide variants (SNVs) involved TBX4 but not TBX2. Here, we describe a male neonate who died at 27 days of life from acute respiratory failure caused by lung growth arrest along the spectrum of CAD confirmed by histopathological assessment. Trio-based genome sequencing revealed in the proband a novel non-recurrent ~1.07 Mb heterozygous CNV deletion at 17q23.2, encompassing TBX4 that arose de novo on the paternal chromosome. This is the first report of a larger-sized CNV deletion in a CAD patient involving TBX4 and leaving TBX2 intact. Our results, together with previous reports, indicate that perturbations of TBX4, rather than TBX2, cause severe lung phenotypes in humans.

Abnormal activation of the Wnt3a/ß-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

BACKGROUND: T-box transcription factor 3(TBX3) is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM's pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). METHODS AND RESULTS: We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (ICR) mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/ß-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/ß-catenin pathway as well as Anti-proliferation cell nuclear antigen( PCNA) and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/ß-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. CONCLUSION: These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/ß-catenin pathway. The activation of the Wnt3a/ß-catenin pathway in ADM promoted TBX3 expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM.

Phase II Trial of Nivolumab in Metastatic Rare Cancer with dMMR or MSI-H and Relation with Immune Phenotypic Analysis (the ROCK Trial).

PURPOSE: Mismatch repair deficiency (dMMR)/microsatellite instability-high (MSI-H) are positive predictive markers for immune checkpoint inhibitors. However, data on the activity of nivolumab in advanced dMMR/MSI-H rare cancers and more accurate biomarkers are worth exploring. PATIENTS AND METHODS: We conducted a multicenter phase II, open-label, single-arm clinical trial to explore the effectiveness and safety of nivolumab monotherapy in patients with advanced rare cancers with dMMR/MSI-H, in parallel with immune phenotype analysis, to explore new biomarkers. A Bayesian adaptive design was applied. Characterization of peripheral blood mononuclear cells (PBMC) was characterized by multicolor flow cytometric analysis and CyTOF using samples collected before and after the intervention. The dMMR was identified by the complete loss of MLH1/MSH2/MSH6/PMS2. RESULTS: From May 2018 to March 2021, 242 patients were screened, and 11 patients were enrolled, of whom 10 were included in the full analysis. Median follow-up was 24.7 months (interquartile range, 12.4-31.5). Objective response rate was 60% [95% confidence interval (CI), 26.2-87.8] by central assessment and 70% (95% CI, 34.8-93.3) by local investigators. Median progression-free survival was 10.1 months (95% CI, 0.9-11.1). No treatment-related adverse events of grade 3 or higher were observed. Patients with a tumor mutation burden of ≥10/Mb showed a 100% response rate (95% CI, 47.8-100). Responders had increased T-bet+ PD-1+ CD4+ T cells in PBMC compared with nonresponders (P < 0.05). CONCLUSIONS: The trial met its primary endpoint with nivolumab, demonstrating clinical benefit in advanced dMMR/MSI-H rare solid cancers. Besides, the proportion of T-bet+ PD-1+ CD4+ T-cells may serve as a novel predictive biomarker.

Homeostatic PD-1 signaling restrains EOMES-dependent oligoclonal expansion of liver-resident CD8 T cells.

The co-inhibitory programmed death (PD)-1 signaling pathway plays a major role in the context of tumor-specific T cell responses. Conversely, it also contributes to the maintenance of peripheral tolerance, as patients receiving anti-PD-1 treatment are prone to developing immune-related adverse events. Yet, the physiological role of the PD-1/PDL-1 axis in T cell homeostasis is still poorly understood. Herein, we show that under steady-state conditions, the absence of PD-1 signaling led to a preferential expansion of CD8+ T cells in the liver. These cells exhibit an oligoclonal T cell receptor (TCR) repertoire and a terminally differentiated exhaustion profile. The transcription factor EOMES is required for the clonal expansion and acquisition of this differentiation program. Finally, single-cell transcriptomics coupled with TCR repertoire analysis support the notion that these cells arise locally from liver-resident memory CD8+ T cells. Overall, we show a role for PD-1 signaling in liver memory T cell homeostasis.

CircTBX5 knockdown modulates the miR-558/MyD88 axis to alleviate IL-1ß-induced inflammation, apoptosis and extracellular matrix degradation in chondrocytes via inactivating the NF-κB signaling.

BACKGROUND: It has been widely shared that the dysregulation of circular RNA (circRNA) may contribute to the progression of osteoarthritis (OA). OA is characterized by persistent chondrocyte injury. We aimed to clarify the role of circTBX5 in IL-1ß-induced chondrocyte injury. METHODS: The expression of circTBX5, miR-558 and MyD88 mRNA was measured using quantitative real-time PCR (qPCR). Cell viability, proliferation and apoptosis were assessed by CCK-8, EdU or flow cytometry assay. The protein levels of extracellular matrix (ECM)-associated markers, MyD88, IkBα, p65 and phosphorylated IkBα were measured by western blot. The release of inflammatory factors was assessed by ELISA. The targets of circTBX5 were screened by RIP and pull-down assay. The putative binding between miR-558 and circTBX5 or MyD88 was validated by dual-luciferase reporter assay. RESULTS: CircTBX5 and MyD88 were enhanced, while miR-558 was downregulated in OA cartilage tissues and IL-1ß-treated C28/I2 cells. IL-1ß induced C28/I2 cell injury by impairing cell viability and proliferation and promoting cell apoptosis, ECM degradation and inflammatory response, while circTBX5 knockdown alleviated IL-1ß induced injury. CircTBX5 bound to miR-558 to regulate IL-1ß induced cell injury. In addition, MyD88 was a target of miR-558, and circTBX5 targeted miR-558 to positively regulate MyD88 expression. MiR-558 enrichment attenuated IL-1ß induced injury by sequestering MyD88 expression. Moreover, circTBX5 knockdown weakened the activity of NF-κB signaling, while miR-558 inhibition or MyD88 overexpression recovered the activity of NF-κB signaling. CONCLUSION: CircTBX5 knockdown modulated the miR-558/MyD88 axis to alleviate IL-1ß induced chondrocyte apoptosis, ECM degradation and inflammation via inactivating the NF-кB signaling pathway.

T-BET and EOMES sustain mature human NK cell identity and antitumor function.

Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.

A rare case of isolated right atrial enlargement and TBX5 mutation associated with Holt-Oram syndrome.

Holt-Oram syndrome or atriodigital dysplasia is commonly associated with cardiac malformations, most often with defects of the muscular septum. We describe the case of a fetus referred for fetal cardiology evaluation in the setting of right atrial enlargement without tricuspid valve abnormalities with small muscular VSDs, and without other significant cardiac lesions. On serial fetal echocardiograms, isolated right atrial enlargement was persistent as was relative fetal bradycardia without apparent AV block or other signs of abnormal conduction. Limb or other anatomic abnormalities were also not visualized on prenatal scans. A postnatal diagnosis of Holt-Oram Syndrome was made. In the setting of isolated right atrial enlargement, we suggest a comprehensive sonographic search for upper limb abnormalities as well as genetic evaluation.

The overexpression and clinical significance of TBX15 in human gliomas.

T-box transcription factor 15 (TBX15) is upregulated in a variety of tumors and has been reported to promote uncontrolled proliferation of tumor cells and induce tumor cells to avoid apoptosis, thus accelerating the malignant transformation of malignant tumors. However, the prognostic value of TBX15 in glioma and its relationship with immune infiltration remain unknown. In this study, we intended to explore the prognostic value of TBX15 and its link to glioma immune infiltration and examine TBX15 expression in pan-cancer using RNAseq data in TPM format from TCGA and GTEx. TBX15 mRNA and protein expressions in glioma cells and adjacent normal tissue were detected and compared by RT-qPCR and Western blot. The effect of TBX15 on survival was assessed by Kaplan-Meier Method. The correlation between TBX15 upregulation and the clinicopathological characteristics of glioma patients was assessed by using TCGA databases, and the relationship between TBX15 and other genes in glioma was evaluated by using TCGA data. The top 300 genes most significantly associated with TBX15 were selected to establish a PPI network through the STRING database. The relationship between TBX15 mRNA expression and immune cell infiltration was explored by using ssGSEA and the TIMER Database. It was found that TBX15 mRNA expression in glioma tissues was significantly higher than that in the adjacent normal tissues, and this difference was most obvious in high-grade gliomas. TBX15 expression was increased in human gliomas and associated with worse clinicopathological characteristics and poorer survival prognosis in glioma patients. In addition, elevated TBX15 expression was linked to a collection of genes involved in immunosuppression. In conclusion, TBX15 played an important role in immune cell infiltration in glioma and may prove to be a predictor of the prognosis in glioma patients.

Intracellular mechanics and TBX3 expression jointly dictate the spreading mode of melanoma cells in 3D environments.

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma.

miR-4732-3p prevents lung cancer progression via inhibition of the TBX15/TNFSF11 axis.

Aim: Possible roles of miRNAs in cancer treatment have been highly studied. This study aimed to elucidate the role of miR-4732-3p in lung cancer. Methods: Bioinformatics analysis was conducted to predict miR-4732-3p-related mRNA targets in lung cancer. Following interaction determination between miR-4732-3p and TBX15 as well as between TBX15 and TNFSF11, their in vitro and in vivo roles were assayed. Results: miR-4732-3p negatively targeted TBX15, which upregulated TNFSF11 by enhancing the activity of the TNFSF11 promoter. Overexpression of miR-4732-3p or silencing of TBX15 or TNFSF11 inhibited the malignant phenotype of lung cancer cells and reduced tumorigenicity in vivo. Conclusion: Overall, this study highlighted the inhibitory role of miR-4732-3p in lung cancer progression through the TBX15/TNFSF11 axis.

This study describes the role of miR-4732-3p in lung cancer. The authors conducted bioinformatics analysis to predict miR-4732-3p-related mRNA targets in lung cancer. Then they analyzed the potential interaction between miR-4732-3p and TBX15 and between TBX15 and TNFSF11. To evaluate their effects on the progression of lung cancer, the authors performed in vitro and in vivo assays. They discovered that miR-4732-3p negatively targeted TBX15, which upregulated TNFSF11 by enhancing the activity of TNFSF11 promoter. Overexpression of miR-4732-3p or silencing of TBX15 or TNFSF11 inhibited the malignant phenotype of lung cancer cells and reduced tumorigenicity in vivo. This study, which has identified potential for the miR-4732-3p/TBX15/TNFSF11 axis as an antioncogenic tool, opens the possibility for better monitoring of lung cancer.

Identification of bi-allelic LFNG variants in three patients and further clinical and molecular refinement of spondylocostal dysostosis 3.

Spondylocostal dysostosis (SCD), a condition characterized by multiple segmentation defects of the vertebrae and rib malformations, is caused by bi-allelic variants in one of the genes involved in the Notch signaling pathway that tunes the "segmentation clock" of somitogenesis: DLL3, HES7, LFNG, MESP2, RIPPLY2, and TBX6. To date, seven individuals with LFNG variants have been reported in the literature. In this study we describe two newborns and one fetus with SCD, who were found by trio-based exome sequencing (trio-ES) to carry homozygous (c.822-5C>T) or compound heterozygous (c.[863dup];[1063G>A]) and (c.[521G>T];[890T>G]) variants in LFNG. Notably, the c.822-5C>T change, affecting the polypyrimidine tract of intron 5, is the first non-coding variant reported in LFNG. This study further refines the clinical and molecular features of spondylocostal dysostosis 3 and adds to the numerous investigations supporting the usefulness of trio-ES approach in prenatal and neonatal settings.