First-in-human, phase I study of PF-06647263, an anti-EFNA4 calicheamicin antibody-drug conjugate, in patients with advanced solid tumors.

PF-06647263, a novel antibody-drug conjugate consisting of an anti-EFNA4 antibody linked to a calicheamicin payload, has shown potent antitumor activity in human xenograft tumor models, including triple-negative breast cancer (TNBC). In the dose-escalation part 1 of this multicenter, open-label, phase I study (NCT02078752), successive cohorts of patients (n, 48) with advanced solid tumors and no available standard therapy received PF-06647263 every 3 weeks (Q3W) or every week (QW), following a modified toxicity probability interval (mTPI) method (initial dosing: 0.015 mg/kg Q3W). Primary objective in part 1 was to estimate the maximum tolerated dose (MTD) and select the recommended phase 2 dose (RP2D). In part 2 (dose-expansion cohort), 12 patients with pretreated, metastatic TNBC received PF-06647263 at the RP2D to further evaluate tumor response and overall safety. PF-06647263 QW administration (n, 23) was better tolerated than the Q3W regimen (n, 25) with only 1 DLT reported (thrombocytopenia). The most common AEs with the QW regimen (fatigue, nausea, vomiting, mucosal inflammation, thrombocytopenia, and diarrhea) were mostly mild to moderate in severity. The MTD was not estimated. PF-06647263 exposures increased in a dose-related manner across the doses evaluated. The RP2D was determined to be 0.015 mg/kg QW. Six (10%) patients achieved a confirmed partial response and 22 (36.7%) patients had stable disease. No correlations were observed between tumor responses and EFNA4 expression levels. Study findings showed manageable safety and favorable PK for PF-06647263 administered QW at the RP2D, with preliminary evidence of limited antitumor activity in patients with TNBC and ovarian cancer.

Molecular Analysis of Ephrin A4 and Ephrin B1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis (CS) has a prevalence of approximately 1 in every 2000 live births and is characterized by the premature fusion of one or more cranial sutures. Failure to maintain the cell lineage boundary at the coronal suture is thought to be involved in the pathology of some forms of CS. The Ephrin family of receptor tyrosine kinases consists of membrane-bound receptors and ligands that control cell patterning and the formation of developmental boundaries. Mutations in the ephrin A4 (EFNA4) and ephrin B1 (EFNB1) ligands have been linked to nonsyndromic CS and craniofrontonasal syndrome, respectively, in patient samples. We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if EFNA4 or EFNB1 could be the loci of the causal mutation in this unique animal model. Sequencing of EFNA4 and EFNB1 was performed using templates obtained from wild-type (n = 4) and craniosynostotic (n = 4) rabbits. No structural coding errors were identified in either gene. A single-nucleotide transversion was identified in one wild-type rabbit within the third intron of EFNA4. These data indicate that the causal locus for heritable CS in this rabbit model is not located within the structural coding regions of either EFNA4 or EFNB1.

Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/ß-catenin axis and activating dCK.

Background: Previously, our investigations have underscored the potential of hyperthermia to improve the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC). Nonetheless, the precise underlying mechanisms remain elusive. Methods: We engineered two GEM-resistant PC cell lines (BxPC-3/GEM and PANC-1/GEM) and treated them with GEM alongside hyperthermia. The impact of hyperthermia on the therapeutic potency of GEM was ascertained through MTT assay, assessment of the concentration of its active metabolite dFdCTP, and evaluation of deoxycytidine kinase (dCK) activity. Lentivirus-mediated dCK silencing was further employed to validate its involvement in mediating the GEM-sensitizing effect of hyperthermia. The mechanism underlying hyperthermia-mediated dCK activation was explored using bioinformatics analyses. The interplay between hyperthermia and the ephrin A4 (EFNA4)/ß-catenin/dCK axis was investigated, and their roles in GEM resistance was further explored via the establishment of xenograft tumor models in nude mice. Results: Hyperthermia restored dCK expression in GEM-resistant cell lines, concurrently enhancing GEM sensitivity and fostering DNA damage and cell death. These observed effects were negated by dCK silencing. Regarding the mechanism, hyperthermia activated dCK by downregulating EFNA4 expression and mitigating ß-catenin activation. Overexpression of EFNA4 activated the ß-catenin while suppressing dCK, thus diminishing cellular GEM sensitivity-a phenomenon remediated by the ß-catenin antagonist MSAB. Consistently, in vivo, hyperthermia augmented the therapeutic efficacy of GEM on xenograft tumors through modulation of the ephrin A4/ß-catenin/dCK axis. Conclusion: This study delineates the role of hyperthermia in enhancing GEM sensitivity of PC cells, primarily mediated through the suppression of the EFNA4/ß-catenin axis and activation of dCK.

The EFNA4 gene is a potential prognostic biomarker in pancreatic cancer: a bioinformatics analysis.

Background: Pancreatic cancer is a highly aggressive malignancy with poor prognosis, and there is an urgent need to understand its molecular mechanisms for early diagnosis and treatment. Despite surgical resection being the only effective treatment, most patients are diagnosed at an advanced stage, missing the optimal window for therapy. Identifying novel biomarkers is crucial for prognostic assessment, treatment planning, and early intervention. Ephrin A4 (EFNA4), a member of the receptor tyrosine kinase family, is involved in vascular and epithelial development via regulation of cell migration and rejection. However, the role of EFNA4 in pancreatic cancer has not been reported. Therefore, our study aimed to clarify the role of EFNA4 in pancreatic cancer through bioinformatics analysis and vitro experiments. Methods: The expression of EFNA4 and its potential value as a diagnostic and prognostic biomarker in pancreatic cancer was analyzed using data from The Cancer Genome Atlas (TCGA) and the Gene Expression Profiling Interactive Analysis (GEPIA) database. According to the expression level of EFNA4, patients were divided into high expression group and low expression group, and the correlation between overall survival (OS) and disease-free survival (DFS) with different expression levels of EFNA4 and clinical parameters were analyzed. Subsequently, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect EFNA4 expression. The proliferation, invasion, and cloning ability of the cells were detected via Cell Counting Kit 8 (CCK8), Transwell, and plate cloning assays, respectively. Results: EFNA4 is highly expressed in pancreatic cancer, and upregulation of EFNA4 is associated with poor prognosis. In this study, EFNA4 expression was correlated with T stage and TNM (tumor-node-metastasis) stage of pancreatic cancer, and the median survival time and progression-free survival (PFS) were worse in those with high EFNA4 expression (394 days) than in those with low expression (525 days) [hazard ratio (HR): 1.47, 95% confidence interval (CI): 1.00-2.16, P=0.047]. In addition, EFNA4 was also found to be involved in the regulation of signal pathways such as cell adhesion, cyclic AMP, insulin secretion, pancreatic secretion, and protein digestion and absorption. In vitro experiments demonstrated that EFNA4 knockdown significantly inhibited the proliferation, cloning ability, and invasiveness of the PANC-1 and SW1990 pancreatic cancer cell lines. Conclusions: The abnormal expression of EFNA4 in pancreatic cancer is associated with poor prognosis. Knockout of EFNA4 gene could significantly inhibit the proliferation and invasion of pancreatic cancer cells. Therefore, EFNA4 may be one of the molecular targets for poor prognosis of patients with pancreatic cancer.

Ephrin-A4 Ligand (EFNA4) Predicts Poor Prognosis of Hepatocellular Carcinoma and Promotes Tumor Proliferation.

Background/Aims: Primary hepatocellular carcinoma (HCC) is one of the most lethal tumor diseases in the world. Receptor tyrosine kinases (RTKs) are thought to play a vital role in HCC and Ephrin-A4 ligand (EFNA4) is a membrane-bound molecule that can activate RTKs through erythropoietin-producing hepatocellular (Eph) receptors. However, the specific role of EFNA4 remains unknown. The aim of our study was to explore the prognostic value of EFNA4 expression in HCC. Methods: Bioinformatics analyses were conducted to probe the expression levels and prognostic value of EFNA4 in HCC. The quantitative real-time polymerase chain reaction, immunohistochemical and western blot were used to confirm the expression of EFNA4 in paired clinical specimens of HCC. Colony formation assay was used to confirm the proliferation of tumor cell. Results: The expression of EFNA4 is generally elevated in various cancers. Especially, EFNA4 was upregulated in tumor tissue and associated with clinical stage in HCC patients. HCC patients with lower levels of EFNA4 possessed better survival and progression-free survival times. Colony formation assay indicated that the overexpression of EFNA4 promoted tumor cell proliferation. Conclusion: These results demonstrated that EFNA4 played as an oncogenic gene and a prognostic biomarker for HCC patients.

TBX3 and EFNA4 Variant in a Family with Ulnar-Mammary Syndrome and Sagittal Craniosynostosis.

Ulnar-mammary syndrome (UMS) is a rare, autosomal dominant disorder characterized by anomalies affecting the limbs, apocrine glands, dentition, and genital development. This syndrome is caused by haploinsufficiency in the T-Box3 gene (TBX3), with considerable variability in the clinical phenotype being observed even within families. We describe a one-year-old female with unilateral, postaxial polydactyly, and bilateral fifth fingernail duplication. Next-generation sequencing revealed a novel, likely pathogenic, variant predicted to affect the canonical splice site in intron 3 of the TBX3 gene (c.804 + 1G > A, IVS3 + 1G > A). This variant was inherited from the proband's father who was also diagnosed with UMS with the additional clinical finding of congenital, sagittal craniosynostosis. Subsequent whole genome analysis in the proband's father detected a variant in the EFNA4 gene (c.178C > T, p.His60Tyr), which has only been reported to be associated with sagittal craniosynostosis in one patient prior to this report but reported in other cranial suture synostosis. The findings in this family extend the genotypic spectrum of UMS, as well as the phenotypic spectrum of EFNA4-related craniosynostosis.

Oncogenic EFNA4 Amplification Promotes Lung Adenocarcinoma Lymph Node Metastasis.

Lymph nodes metastases are common in patients with lung cancer. Additionally, those patients are often at a higher risk for death from lung tumor than those with tumor-free lymph nodes. Somatic DNA alterations are key drivers of cancer, and copy number alterations (CNAs) are major types of DNA alteration that promote lung cancer progression. Here, we performed genome-wide DNA copy number analysis, and identified a novel lung-cancer-metastasis-related gene, EFNA4. The EFNA4 genome locus was significantly amplified, and EFNA4 mRNA expression was significantly up-regulated in lung cancer compared with normal lung tissue, and also in lung cancer with lymph node metastases compared with lung cancer without metastasis. EFNA4 encodes Ephrin A4, which is the ligand for Eph receptors. The function of EFNA4 in human lung cancer remains largely unknown. Through cell line experiments we showed that EFNA4 overexpression contributes to lung tumor cells growth, migration and adhesion. Conversely, EFNA4 knockdown or knockout led to the growth suppression of cells and tumor xenografts in mice. Lung cancer patients with EFNA4 overexpression have poor prognosis. Together, by elucidating a new layer of the role of EFNA4 in tumor proliferation and migration, our study demonstrates a better understanding of the function of the significantly amplified and overexpressed gene EFNA4 in lung tumor metastasis, and suggests EFNA4 as a potential target in metastatic lung cancer therapy.

EFNA4 promotes cell proliferation and tumor metastasis in hepatocellular carcinoma through a PIK3R2/GSK3ß/ß-catenin positive feedback loop.

Rapid tumor progression, metastasis, and diagnosis in advanced stages of disease are the main reasons for the short survival time and high mortality rate of patients with hepatocellular carcinoma (HCC). Ephrin A4 (EFNA4), the ligand of the EPH family, participates in the development of blood vessels and epithelium by regulating cell migration and rejection. In our study, based on bioinformatics analyses, we found that EFNA4 was highly expressed and led to poor prognosis in patients with HCC. We demonstrated that overexpression of EFNA4 significantly promoted HCC cell proliferation and migration in vivo or in vitro. In addition, knockdown of EFNA4 inhibited the proliferation and migration of HCC cells. Furthermore, EFNA4 was found to directly interact with EPHA2 and promote its phosphorylation at Ser897, followed by recruitment of phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) and activation of the glycogen synthase kinase-3beta (GSK3ß)/ß-catenin signaling pathway. Moreover, overexpression of ß-catenin further promoted the expression of PIK3R2, which formed a positive feedback loop. The results revealed that abnormal expression of EFNA4 is the main switch of the PIK3R2/GSK3ß/ß-catenin loop that influenced the proliferation and migration of HCC cells and suggest that EFNA4 is a potential prognostic marker and a prospective therapeutic target in patients with HCC.