HIVEP3 as a potential prognostic factor promotes the development of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common malignancy worldwide. Human immune deficiency virus type 1 enhancer-binding protein 3 (HIVEP3) was verified to play a vital role in types of cancers. However, the functional role of HIVEP3 in AML was rarely reported. In this study, CCK-8, colony formation assay, flow cytometry, and Trans-well chamber experiments were applied for detecting cell proliferation, apoptosis, and invasion in AML cells. The expression of proteins related to TGF-ß/Smad signaling pathway was determined by western blot. Our data showed that the expression level of HIVEP3 was closely related to the risk classification and prognosis of AML patients. Moreover, HIVEP3 was highly expressed in AML patients and cells. Knockdown of HIVEP3 significantly repressed cell proliferation invasion, and enhanced cell apoptosis in HL-60 and THP-1 cells. In addition, HIVEP3 donwreglation could inhibit the TGF-ß/Smad signaling pathway. TGF-ß overexpression could reverse the inhibition effects of HIVEP3 knockdown on AML development and the TGF-ß/Smad signaling pathway. These findings indicated that HIVEP3 contributed to the progression of AML via regulating the TGF-ß/Smad signaling pathway and had a prognostic value for AML.

Nuclear factor kappa B activation appears weaker in schizophrenia patients with high brain cytokines than in non-schizophrenic controls with high brain cytokines.

BACKGROUND: High inflammation status despite an absence of known infection characterizes a subpopulation of people with schizophrenia who suffer from more severe cognitive deficits, less cortical grey matter, and worse neuropathology. Transcripts encoding factors upstream of nuclear factor kappa B (NF-κB), a major transcriptional activator for the synthesis of pro-inflammatory cytokines, are increased in the frontal cortex in schizophrenia compared to controls. However, the extent to which these changes are disease-specific, restricted to those with schizophrenia and high-neuroinflammatory status, or caused by loss of a key NF-κB inhibitor (HIVEP2) found in schizophrenia brain, has not been tested. METHODS: Post-mortem prefrontal cortex samples were assessed in 141 human brains (69 controls and 72 schizophrenia) and 13 brains of wild-type mice and mice lacking HIVEP2 (6 wild-type, 7 knockout mice). Gene expression of pro-inflammatory cytokines and acute phase protein SERPINA3 was used to categorize high and low neuroinflammation biotype groups in human samples via cluster analysis. Expression of 18 canonical and non-canonical NF-κB pathway genes was assessed by qPCR in human and mouse tissue. RESULTS: In humans, we found non-canonical upstream activators of NF-κB were generally elevated in individuals with neuroinflammation regardless of diagnosis, supporting NF-κB activation in both controls and people with schizophrenia when cytokine mRNAs are high. However, high neuroinflammation schizophrenia patients had weaker (or absent) transcriptional increases of several canonical upstream activators of NF-κB as compared to the high neuroinflammation controls. HIVEP2 mRNA reduction was specific to patients with schizophrenia who also had high neuroinflammatory status, and we also found decreases in NF-κB transcripts typically induced by activated microglia in mice lacking HIVEP2. CONCLUSIONS: Collectively, our results show that high cortical expression of pro-inflammatory cytokines and low cortical expression of HIVEP2 in a subset of people with schizophrenia is associated with a relatively weak NF-κB transcriptional signature compared to non-schizophrenic controls with high cytokine expression. We speculate that this comparatively milder NF-κB induction may reflect schizophrenia-specific suppression possibly related to HIVEP2 deficiency in the cortex.

Regional, cellular and species difference of two key neuroinflammatory genes implicated in schizophrenia.

The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.

Expanding the phenotype of intellectual disability caused by HIVEP2 variants.

De novo pathogenic variants in the human immunodeficiency virus enhancer type I binding protein 2 (HIVEP2) gene, a large transcription factor predominantly expressed in the brain have previously been associated with intellectual disability (ID) and dysmorphic features in nine patients. We describe the phenotype and genotype of two additional patients with novel de novo pathogenic HIVEP2 variants, who have previously unreported features, including hyperphagia and Angelman-like features. Exome sequencing was utilized in the investigation of the patients who had previously incurred a rigorous genetic workup for their neurodevelopmental delay, and in whom no genetic cause had been detected. Information pertaining to phenotype and genotype for new patients was collated along with data from previous reports, showing that the phenotypic spectrum of patients with HIVEP2 variants is broader than first noted. Additional characteristics are: an increased body mass index; and features of Angelman-like syndromes including: ID, limited speech, post-natal microcephaly, and hypotonia. Dysmorphic features vary between patients. As yet, no clear association between the type of gene aberration and phenotype can be concluded. HIVEP2-related ID needs to be considered in the differential diagnosis of patients with Angelman-like phenotypes and hyperphagia, and whole-exome sequencing should be considered in the genetic diagnostic armamentarium for patients with ID of inconclusive etiology.

Mutations in HIVEP2 are associated with developmental delay, intellectual disability, and dysmorphic features.

Human immunodeficiency virus type I enhancer binding protein 2 (HIVEP2) has been previously associated with intellectual disability and developmental delay in three patients. Here, we describe six patients with developmental delay, intellectual disability, and dysmorphic features with de novo likely gene-damaging variants in HIVEP2 identified by whole-exome sequencing (WES). HIVEP2 encodes a large transcription factor that regulates various neurodevelopmental pathways. Our findings provide further evidence that pathogenic variants in HIVEP2 lead to intellectual disabilities and developmental delay.

Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

WITHDRAWN: Modulation of TLR3, TLR4 and TLR7 mediated IFN-ß, Rantes and TNFα production by HIVEP1.

The manuscript was withdrawn by the author.

Parental gonadossomatic mosaicism in HIVEP2-related intellectual disability and impact on genetic counseling-case report.

Intellectual development disorder, autosomal dominant 43 (MRD43) is an autosomal dominant disorder caused by heterozygous mutations in the HIVEP2 gene. In this report, we describe a case of a 4-year-old boy with global development delay, hypotonia, and dysmorphic features, in whom the finding of a heterozygous nonsense pathogenic variant in exon 5 of HIVEP2 [c.2827C>T p. (Arg943*)] through WES established a MRD43 diagnosis. Our patient's phenotype overlaps with other MRD43 descriptions in the literature. Unlike previously reported cases, where the condition was almost invariably de novo, the healthy mother in this case presented mosaicism for the pathogenic variant. Thus, the recurrence risk increased significantly from 1% to up to 50%. The description of a variant inherited for MDR43 is singular in the literature and this description highlights the importance of parental studies for accurate genetic counseling, particularly for family planning.

HIVEP3 inhibits fate decision of CD8+ invariant NKT cells after positive selection.

CD8+ invariant natural killer T (iNKT) cells are functionally different from other iNKT cells and are enriched in human but not in mouse. To date, their developmental pathway and molecular basis for fate decision remain unclear. Here, we report enrichment of CD8+ iNKT cells in neonatal mice due to their more rapid maturation kinetics than CD8- iNKT cells. Along developmental trajectories, CD8+ and CD8- iNKT cells separate at stage 0, following stage 0 double-positive iNKT cells, and differ in HIVEP3 expression. HIVEP3 is lowly expressed in stage 0 CD8+ iNKT cells and negatively controls their development, whereas it is highly expressed in stage 0 CD8- iNKT cells and positively controls their development. Despite no effect on IFN-γ, HIVEP3 inhibits granzyme B but promotes interleukin-4 production in CD8+ iNKT cells. Together, we reveal that, as a negative regulator for CD8+ iNKT fate decision, low expression of HIVEP3 in stage 0 CD8+ iNKT cells favors their development and T helper 1-biased cytokine responses as well as high cytotoxicity.

Expanding the Phenotypic Spectrum of HIVEP2-Related Intellectual Disability: Description of Two Portuguese Patients and Review of the Literature.

Introduction: Pathogenic variants in HIVEP2 have been associated with a neurodevelopmental disorder mainly characterized by intellectual disability, severe language impairment, and motor developmental delay. Since its first description in 2016, only 15 patients have been described in the literature. Methods: Here, we report 2 additional unrelated Portuguese children presenting intellectual disability and motor delay in whom de novo nonsense pathogenic variants in HIVEP2 have been identified by next-generation sequencing analysis. Results: In patient 1, the variant c.2827C>T, p.(Arg943*) was detected, whereas patient 2 carried the variant c.6667C>T, p.(Arg2223*). Interestingly, patient 1 presented with a rapid growth of the occipitofrontal diameter in the first months of life due to external hydrocephalus, a feature that, as far as we know, has never been reported in patients with HIVEP2 pathogenic variants. Conclusion: This report expands the phenotypic spectrum of this rare syndrome and provides deeper insights by comparing the clinical features of our patients with previously reported affected individuals.

Novel HIVEP1-ALK fusion in a patient with lung adenocarcinoma demonstrating sensitivity to alectinib: a case report.

Background: Anaplastic lymphoma kinase (ALK) fusion is an important oncogenic driver in non-small cell lung cancer (NSCLC). Reports on the intergenic region (IGR) as an ALK fusion partner are rare. Here, we report the case of a patient with advanced NSCLC harboring a human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1)-ALK fusion that responded effectively to alectinib. Case Description: A 60-year-old non-smoking male was referred with a 3-month history of productive cough secondary to lung adenocarcinoma metastatic to mediastinal lymph nodes, brain, liver, and bone (T2N3M1c, stage IVB). Next-generation sequencing identified an IGR (upstream HIVEP1-) ALK fusion, and immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results were consistent with an ALK-positive tumor. The patient was subsequently started on alectinib, with no obvious adverse reaction. After 1 month of therapy, the patient achieved significantly remission of the clinical symptoms and had led to an ongoing partial response (PR) lasting >33 months. Conclusions: Our experience highlights the efficacy of alectinib in a patient with HIVEP1-ALK fusion positive NSCLC with multiple metastases including brain disease, and the need for multiple genetic testing methods to verify the oncogenicity of ALK fusions prior to treatment. It could provide useful guidance for the treatment of similar cases in the future.

HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis.

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

Novel HIVEP2 Variant p.Q1248* is Associated with Developmental Delay: A Case Report.

In this report, we describe a 5-year-old boy with global developmental delay who presented for medical genetic evaluation. We performed whole exome sequencing that revealed the involvement of a heterogenous variant p.Gln1248Ter (CAG > TAG): c.3742 C > T inherited de novo in exon 5 of HIVEP2 (human immunodeficiency virus type I enhancer binding protein 2; NM_006734.3). The gene variant p.Q1248* is interpreted to be associated as a cause of the intellectual disability. We review pathomechanisms of HIVEP2 and discuss the reasoning behind the pathogenicity of this novel variant. To the best of our knowledge, this the first reported case that demonstrates the p.Q1248* variant as pathogenic.

Novel HIVEP2 Variants in Patients with Intellectual Disability.

Intellectual disability (ID) occurs in approximately 1% of the population. Over the last years, broad sequencing approaches such as whole exome sequencing (WES) substantially contributed to the definition of the molecular defects underlying nonsyndromic ID. Pathogenic variants in HIVEP2, which encodes the human immunodeficiency virus type I enhancer binding protein 2, have recently been reported as a cause of ID, developmental delay, behavioral disorders, and dysmorphic features. HIVEP2 serves as a transcriptional factor regulating NF-ĸB and diverse genes that are essential in neural development. To date, only 8 patients with pathogenic de novo nonsense or frameshift variants and 1 patient with a pathogenic missense variant in HIVEP2 have been reported. By WES, we identified 2 novel truncating HIVEP2 variants, c.6609_6616delTGAGGGTC (p.Glu2204*) and c.6667C>T (p.Arg2223*), in 2 young adults presenting with developmental delay and mild ID without any dysmorphic features, systemic malformations, or behavioral issues.

Combined overexpression of HIVEP3 and SOX9 predicts unfavorable biochemical recurrence-free survival in patients with prostate cancer.

BACKGROUND: To clarify the involvement of HIVEP3 and SOX9 coexpression in prostate cancer (PCa). METHODS: A small interfering RNA was used to knockdown SOX9 expression in a PCa cell line and to analyze the effects of SOX9 inhibition on the expression of HIVEP3 in vitro. Then, HIVEP3 and SOX9 expression patterns in the human PCa tissues were detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis and immunohistochemistry. RESULTS: We found that the downregulation of SOX9 could inhibit the expression of HIVEP3 in the PCa cells in vitro. In addition, both HIVEP3 and SOX9 messenger RNA expression levels in the PCa tissues were significantly higher than those in the noncancerous prostate tissues (P=0.006 and P<0.001, respectively). Moreover, the immunohistochemical staining scores of HIVEP3 in the PCa tissues with PSA failure were significantly higher than those without (P=0.042); the increased SOX9 protein expression was more frequently found in the PCa tissues with a high Gleason score (P=0.045) and a high clinical stage (P=0.012). The tumors showing the HIVEP3-high/SOX9-high expression more frequently had PSA failure (P=0.024). When the patients with an HIVEP3 overexpression combined with the SOX9 overexpression, this group had a worse biochemical recurrence-free survival (P<0.001). Furthermore, the multivariate analysis showed that the HIVEP3/SOX9 coexpression was an independent predictor of an unfavorable biochemical recurrence-free survival. CONCLUSION: Our data offer the convincing evidence for the first time that a combined analysis of HIVEP3 and SOX9 may help to predict the tumor progression and prognosis of PCa patients. In particular, the overexpression of HIVEP3 in PCa might partly explain the poor prognosis of patients with an upregulation of SOX9.