DDX3X Research Articles

PRRSV hijacks DDX3X protein and induces ferroptosis to facilitate viral replication

Porcine reproductive and respiratory syndrome virus (PRRSV) is a severe disease with substantial economic consequences for the swine industry. The DEAD-box helicase 3 (DDX3X) is an RNA helicase that plays a crucial role in regulating RNA metabolism, immunological response, and even RNA virus infection. However, it is unclear whether it contributes to PRRSV infection. Recent studies have found that the expression of DDX3X considerably increases in Marc-145 cells when infected with live PRRSV strains Ch-1R and SD16; however, it was observed that inactivated viruses did not lead to any changes. By using the RK-33 inhibitor or DDX3X-specific siRNAs to reduce DDX3X expression, there was a significant decrease in the production of PRRSV progenies. In contrast, the overexpression of DDX3X in host cells substantially increased the proliferation of PRRSV. A combination of transcriptomics and metabolomics investigations revealed that in PRRSV-infected cells, DDX3X gene silencing severely affected biological processes such as ferroptosis, the FoxO signalling pathway, and glutathione metabolism. The subsequent transmission electron microscopy (TEM) imaging displayed the typical ferroptosis features in PRRSV-infected cells, such as mitochondrial shrinkage, reduction or disappearance of mitochondrial cristae, and cytoplasmic membrane rupture. Conversely, the mitochondrial morphology was unchanged in DDX3X-inhibited cells. Furthermore, silencing of the DDX3X gene changed the expression of ferroptosis-related genes and inhibited the virus proliferation, while the drug-induced ferroptosis inversely promoted PRRSV replication. In summary, these results present an updated perspective of how PRRSV infection uses DDX3X for self-replication, potentially leading to ferroptosis via various mechanisms that promote PRRSV replication.

Abstract P33: Novel Reporter Cell Models for Efficient Interrogation of Helicase Activity in Cancer-associated DDX3X Mutations

Abstract DDX3X is an ATP-dependent RNA helicase involved in diverse cellular processes. Somatic mutations in the DDX3X gene exhibit oncogenic or tumor suppressive activities depending on cancer types. However, helicase modulating roles of specific DDX3X mutations remain elusive mainly due to lack of an appropriate cell culture model system. Here, we report our development of novel transgenic cell lines capable of interrogating DDX3X helicase activity. To evaluate the helicase activity of the DDX3X mutations in question, we designed a reporter plasmid that measures the luciferase signal corresponding to the helicase activity. Using 8 different point mutations in the DDX3X that we identified earlier in relapsed/refractory patients with diffuse large B cell lymphoma (DLBCL), we show that these DDX3X mutants displayed defective helicase activity. DDX3X mutant knock-in DLBCL cells also displayed abolished helicase unwinding capabilities, validating our reporter cell models. We were able to extend the application of our reporter cells to interrogate Y chromosome-linked male paralog DDX3Y. In male lymphoma patients, ectopic DDX3Y expression can compensate for DDX3X loss to restore global protein synthesis. We narrowed down DDX3Y helicase and ATPase domains to be critical for cellular translation, ruling out DDX3Y involvement in ribosomal elongation. Unexpectedly, DDX3Y helicase is less efficient in RNA unwinding as compared to DDX3X revealing subtle differences despite having a high level of homology. The developed DDX3X reporter cells can be applied widely to evaluate the effects of DDX3X mutations in diseases as well as study the biology of DDX3X and DDX3Y. Citation Format: Zhi Sheng Poh, James Tan Chia Wei, Brandon Han Siang Wong, Kottaiswamy Amuthavalli, Suat Hoon Tan, Nicholas Francis Grigoropoulos, Navin Kumar Verma. Novel Reporter Cell Models for Efficient Interrogation of Helicase Activity in Cancer-associated DDX3X Mutations [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P33.

MiR-181a-5p targets DDX3X to inhibit the progression of osteoarthritis via NF-ΚB signaling pathway

Osteoarthritis (OA) is the most common age-related joint disease, characterized by chronic inflammation, progressive articular cartilage destruction and subchondral osteosclerosis. More and more evidence showed that microRNAs (miRNAs) play a key role in various diseases, but the specific mechanism of miRNAs in OA is not clear. The purpose of this study was to investigate the expression level and role of miR-181a-5p in OA and its related mechanism. Here we identified the key gene DEAD-box RNA helicase 3X (DDX3X) in the OA dataset by bioinformatics analysis. At the same time, miRNAs targeting DDX3X were screened, and miR-181a-5p was selected as the next research object. Then we used different concentrations of interleukin-1 beta (IL-1β)-induced in vitro model of arthritis, and found that IL-1β can stimulate cells to release nitric oxide. The expression levels of miR-181a-5p and DDX3X in mouse chondrocyte cell line ATDC5 induced by IL-1β at a concentration of 10ug/mL were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IL-1β induced a decrease in the expression of miR-181a-5p and an increase in the expression of DDX3X in ATDC5 cells. mimic miR-181a-5p or inhibitor miR-181a-5p were transfected into ATDC5 cells, and the levels of inflammatory mediators in the cells were detected by enzyme-linked immunosorbent assay, and the results showed that miR-181a-5p could reduce the release of tumor necrosis factor-α, IL-1β, IL-6 and inducible nitric oxide nitric oxide synthase in a cellular model of arthritis. Luciferase reporter assays confirmed that the miR-181a-5p binding site was in the DDX3X gene 3′-untranslated region (3′-UTR), and DDX3X was negatively regulated by miR-181a-5p. Rescue assays confirmed that miR-181a-5p reduced the expression of DDX3X by targeting the 3′-UTR region of DDX3X, thereby reducing the release of inflammatory factors. Finally, in this paper, western blot was used to detect the mechanism of miR-181a-5p regulating OA. The results showed that interfering with the expression of miR-181a-5p could up-regulate the expression of DDX3X protein, increase the expression of nuclear factor- kappaB (NF-κB) related proteins, and reduce the inflammatory response of OA, thereby increasing the secretion of the matrix proteinases MMP-3 and MMP-13. Taken together, the results of the study suggested that miR-181a-5p may be a promising therapeutic target for the treatment of human OA.

Characteristics and prognosis of rrDLBCL with TP53 mutations and a high-risk subgroup represented by the co-mutations of DDX3X-TP53.

TP53 mutations have a prognostic significance in relapsed and refractory diffuse large B-cell lymphoma (rrDLBCL) patients, and their treatment still faces a great challenge. This study aimed to evaluate the prognosis of patients with TP53 mutations (TP53mut) in the context of CAR-T therapy (Chimeric antigen receptor T-cell therapy) as well as explore the heterogeneity in their cohort and identify the possible risk factors. A retrospective study was conducted to investigate the clinical characteristics of rrDLBCL patients with TP53 mutations and their prognostic factors, receiving CAR-T therapy. And the expression level of TP53 and DDX3X, which was an important co-mutation of TP53 revealed in the cohort, were explored in public databases and cell lines. The median overall survival time of 40 patients with TP53 mutations was 24.5months, while their median progression-free survival time after CAR-T was 6.8months. There were no significant differences in the ORR (objective remission rate, X2 =3.0498, p > 0.05) and PFS (after CAR-T therapy) between the patients with wild-type and mutated TP53 genes after CAR-T therapy, while the OS of patients with TP53 mutations was significantly worse (p < 0.01). In patients with TP53 mutations, the performance status (ECOG score) was identified as the most important prognostic factor, while the efficacies of induction and salvage treatments were also correlated with the prognosis. Among molecular indicators, the co-mutations of Chr-17 and those located on the exon 5 of the TP53 gene showed a tendency for a worse prognosis. Moreover, the patients with TP53-DDX3X co-mutations were identified as a subgroup with an extremely bad prognosis. The expression levels of DDX3X and TP53 were explored in a public database and the cell lines with their co-mutations, which indicated that inhibiting the DDX3X gene could affect the proliferation of rrDLBCL cells and the expression of TP53. This study indicated rrDLBCL patients with TP53 mutations was still the group of poor prognosis in the CAR-T therapy era. CAR-T therapy can benefit some TP53mut patients, and the performance status (ECOG) might help predict their prognosis. The study also revealed a subgroup of TP53-DDX3X co-mutations in rrDLBCL, which showed a strong clinical significance.

The RNA helicase DDX3 and its role in c-MYC driven germinal center-derived B-cell lymphoma.

DDX3X is an RNA helicase with many functions in RNA metabolism such as mRNA translation, alternative pre-mRNA splicing and mRNA stability, but also plays a role as a regulator of transcription as well as in the Wnt/beta-catenin- and Nf-κB signaling pathways. The gene encoding DDX3X is located on the X-chromosome, but escapes X-inactivation. Hence females have two active copies and males only one. However, the Y chromosome contains the gene for the male DDX3 homologue, called DDX3Y, which has a very high sequence similarity and functional redundancy with DDX3X, but shows a more restricted protein expression pattern than DDX3X. High throughput sequencing of germinal center (GC)-derived B-cell malignancies such as Burkitt Lymphoma (BL) and Diffuse large B-cell lymphoma (DLBCL) samples showed a high frequency of loss-of-function (LOF) mutations in the DDX3X gene revealing several features that distinguish this gene from others. First, DDX3X mutations occur with high frequency particularly in those GC-derived B-cell lymphomas that also show translocations of the c-MYC proto-oncogene, which occurs in almost all BL and a subset of DLBCL. Second, DDX3X LOF mutations occur almost exclusively in males and is very rarely found in females. Third, mutations in the male homologue DDX3Y have never been found in any type of malignancy. Studies with human primary GC B cells from male donors showed that a loss of DDX3X function helps the initial process of B-cell lymphomagenesis by buffering the proteotoxic stress induced by c-MYC activation. However, full lymphomagenesis requires DDX3 activity since an upregulation of DDX3Y expression is invariably found in GC derived B-cell lymphoma with DDX3X LOF mutation. Other studies with male transgenic mice that lack Ddx3x, but constitutively express activated c-Myc transgenes in B cells and are therefore prone to develop B-cell malignancies, also showed upregulation of the DDX3Y protein expression during the process of lymphomagenesis. Since DDX3Y is not expressed in normal human cells, these data suggest that DDX3Y may represent a new cancer cell specific target to develop adjuvant therapies for male patients with BL and DLBCL and LOF mutations in the DDX3X gene.

A novel de novo DDX3X missense variant in a female with brachycephaly and intellectual disability: a case report

BackgroundDe novo pathogenic variants in the DDX3X gene are reported to account for 1–3% of unexplained intellectual disability (ID) in females, leading to the rare disease known as DDX3X syndrome (MRXSSB, OMIM #300958). Besides ID, these patients manifest a variable clinical presentation, which includes neurological and behavioral defects, and abnormal brain MRIs.Case presentationWe report a 10-year-old girl affected by delayed psychomotor development, delayed myelination, and polymicrogyria (PMG). We identified a novel de novo missense mutation in the DDX3X gene (c.625C > G) by whole exome sequencing (WES). The DDX3X gene encodes a DEAD-box ATP-dependent RNA-helicase broadly implicated in gene expression through regulation of mRNA metabolism. The identified mutation is located just upstream the helicase domain and is suggested to impair the protein activity, thus resulting in the altered translation of DDX3X-dependent mRNAs. The proband, presenting with the typical PMG phenotype related to the syndrome, does not show other clinical signs frequently reported in presence of missense DDX3X mutations that are associated with a most severe clinical presentation. In addition, she has brachycephaly, never described in female DDX3X patients, and macroglossia, that has never been associated with the syndrome.ConclusionsThis case expands the knowledge of DDX3X pathogenic variants and the associated DDX3X syndrome phenotypic spectrum.

975. Why Sex Make a Difference in HIV Clinical Course? Bioinformatics Analysis of Differential Expressed Gene in Females and Males with HIV Disease

BackgroundHuman immunodeficiency virus (HIV) disease progression are different among genders, in which women usually progress to acquired immunodeficiency syndrome (AIDS) faster than men. The mechanisms resulting in the gender biases of HIV progression are unclear. We conducted a bioinformatics analysis of differentially expressed genes (DEGs) in women and men with HIV disease to understand the sex-based differences in HIV pathogenesis.MethodsWe obtained microarray data from the Gene Expression Omnibus (GEO) database using our pre-defined search strategy and analyzed data using the GEO2R platform. The t-test was done to compare DEGs between females and males with HIV diseases. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was implemented to systematically extract biological features and processes of retrieving DEGs via gene ontology (GO) analysis. A Systemic search was performed to evaluate each DEG function and its possible association with HIV.ResultsOne gene expression profiling data were retrieved: GSE 140713, composed of 40 males and 10 females with HIV1 infected samples. A GEO2R analysis yielded 19 DEGs (Table 1). The GO analysis result was demonstrated in Tables 2 and 3. Following a systemic search, we found two DEGs, which have previous studies reported an association with HIV: DDX3X (20 studies) and PDS5 (1 study). We proposed DDX3X (t 5.3, p 0.0037) is responsible for gender inequalities of HIV progression because of: 1. DDX3X is needed in the HIV1 life cycle. 2. Several studies confirmed a positive correlation between DDX3X expression and HIV1 replication. 3. Our study found an up-regulated DDX3X expression in women corresponded to the fact that women progress to AIDS faster than men. 4. Our GO analysis showed female up-regulated genes were enriched in positive regulation of the gene expression pathway, which can be explained by DDX3X and its underlying mechanism.Table 1: DEGs in women and men with HIV1 disease Table 2: GO functional enrichment pathway analyses of overall retrieving DEGs Table 3: GO functional enrichment pathway analyses of down- and up-regulated clusters of DEGs ConclusionAberrant DDX3X expression may contribute to sex-based differences in HIV disease. Drugs modifying DDX3X gene expression will be beneficial in the treatment of HIV especially resolving the HIV drug resistance problem because current anti-HIV drugs target viral components posed the risk of viral mutation.Disclosures All Authors: No reported disclosures

Clinical and genetic characteristics of X-linked mental retardation 102 type caused by novel mutations in the DDX3X gene (OMIM:300958)

Introduction. X-linked mental retardation 102 type caused by novel mutations in the DDX3X gene is one of the most common monogenic variants of intellectual deficiency in women.Purpose of the study. Description of the clinical and genetic characteristics of Russian female patients with type 102 mental retardation due to newly identified mutations.Materials and methods. The diagnosis of mental retardation of type 102 was established on the basis of the features of clinical manifestations and the detection of the mutations in the DDX3X gene as a result of exome sequencing and subsequent confirmation of the identified variants of Sanger sequencing.Results. A description is given of the clinical and genetic characteristics of two female patients with type 102 X-linked mental retardation due newly to identified mutations p.1703C&gt; G and c.113A&gt; G (NM_001193416) in the DDX3X gene in the heterozygous state. New features of the phenotype are described. The mechanism of the appearance of clinical and genetic correlations is suggested, which can be used as a prognostic marker of the development of the disease.Conclusion. Clinical and genetic characteristics of two patients with mutations in the DDX3X gene that violate the amino acid sequence of different protein regions with different severity of clinical manifestations are described. The results obtained may testify in favor of the existence of a dependence of the severity of the phenotype on the localization and nature of mutations in the gene and determine the relevance of further research aimed at searching for clinical and genetic correlations.

The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma

The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5' UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential invivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas.

Whole-Genome Genomics Correlates of Response to Anti-PD1 Therapy in Relapsed/Refractory Natural Killer/T Cell Lymphoma

The genetic landscape of Natural killer/T-cell nasal-type lymphoma (NKTL) has been recently unraveled by discoveries describing recurring mutations altering the JAK-STAT pathway, epigenetic modifiers, the DDX3X gene and genetic predisposition in the HLA-DPB1 gene but none has employed whole-genome sequencing (WGS).Whole-genome sequencing was performed for 11 pairs of tumor-blood samples to study the association between somatic mutations and response to pembrolizumab. Interestingly, recurrent PD-L1 SRs were validated in four of the seven complete responders (CR) cases. JAK3-activating (p.A573V) mutations were also validated in another two pembrolizumab-treated patients who have achieved CR. Lastly, we also found a homozygous 3 bp insertion (p.Q131_H132insQ) in the ARID1B gene, a chromatin remodeler gene and a subunit in the SWI/SNF complex in the last remaining CR case. A recent study has also reported PBRM1-deficient and ARID2-deficient tumors correlated with better response to anti-PD1/PD-L1 therapy renal cell carcinoma. There seems to be a relationship between truncating alterations in the subunits of the SWI/SNF complex and response to PD1/PD-L1 therapy. However, the exact mechanisms behind these associations remain to be elucidated for NKTL.Analysis of the WGS data from the four remaining progressive disease (PD) patients' tumors did not reveal similar alterations in the PD-L1 and JAK3 genes. A careful inspection was also carried out on the genes associated with major histocompatibility complex and interferon gamma pathways, which are known to associate with resistance to immune checkpoint blockade in melanoma, but no further mutation in these groups of genes was found in our cohort. Furthermore, a TP53 (p.W14X) stop-gain mutation, a hallmark tumor suppressor gene, was detected in a patient who had progressive disease after given pembrolizumab.We went on to check if PD-L1 IHC staining could explain the response of these NKTL patients to pembrolizumab. In this study, tumors were stained and assessed for PD-L1 positivity by the same pathologist. All cases, except two cases, have greater than 20% of tumor cells stained positive for PD-L1. Interesting, both cases are CR. In addition, all four PD cases were strongly stained for PD-L1 with an average of 69% PD-L1 positive cells (range, 50% - 90%) but their outcomes were dismal. This suggests that there could be a companion biomarker that could be added to PD-L1 IHC positivity for better predictive power of response to PD1 blockade therapy.Here we report retrospectively, for the first time, the genomic mutational profiles of anti-PD1 blockade in 11 relapsed/refractory NKTL patients using WGS data, which provide proof-of-concept data that the response to anti-PD1 is relevant and correlates with recurrent PD-L1 and JAK3 genomic alterations in this malignancy. DisclosuresNo relevant conflicts of interest to declare.

DDX3X Biomarker Correlates with Poor Survival in Human Gliomas.

Primary high-grade gliomas possess invasive growth and lead to unfavorable survival outcome. The investigation of biomarkers for prediction of survival outcome in patients with gliomas is important for clinical assessment. The DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked (DDX3X) controls tumor migration, proliferation, and progression. However, the role of DDX3X in defining the pathological grading and survival outcome in patients with human gliomas is not yet clarified. We analyzed the DDX3X gene expression, WHO pathological grading, and overall survival from de-linked data. Further validation was done using quantitative RT-PCR of cDNA from normal brain and glioma, and immunohistochemical (IHC) staining of tissue microarray. Statistical analysis of GEO datasets showed that DDX3X mRNA expression demonstrated statistically higher in WHO grade IV (n = 81) than in non-tumor controls (n = 23, p = 1.13 × 10−10). Moreover, DDX3X level was also higher in WHO grade III (n = 19) than in non-tumor controls (p = 2.43 × 10−5). Kaplan–Meier survival analysis showed poor survival in patients with high DDX3X mRNA levels (n = 24) than in those with low DDX3X expression (n = 53) (median survival, 115 vs. 58 weeks, p = 0.0009, by log-rank test, hazard ratio: 0.3507, 95% CI: 0.1893–0.6496). Furthermore, DDX3X mRNA expression and protein production significantly increased in glioma cells compared with normal brain tissue examined by quantitative RT-PCR, and Western blot. IHC staining showed highly staining of high-grade glioma in comparison with normal brain tissue. Taken together, DDX3X expression level positively correlates with WHO pathologic grading and poor survival outcome, indicating that DDX3X is a valuable biomarker in human gliomas.

DDX3X, the X homologue of AZFa gene DDX3Y, expresses a complex pattern of transcript variants only in the male germ line.

DDX3X, the functional X homologue of the major AZFa gene, DDX3Y, belongs to the highly conserved PL10-subfamily of DEAD-box RNA helicase genes which are functionally conserved from yeast to man. They are mainly involved in cell cycle control and translation initiation control of gene transcripts with long 5'UTR extensions containing complex secondary structures. Interestingly, in humans both gene copies were found to be expressed at different phases of human spermatogenesis. Whereas DDX3Y transcripts are translated only in premeiotic male germ cells, the DDX3X protein is expressed only in postmeiotic spermatids. In this study, we found that the major class of DDX3X transcripts in human testis become activated first after meiosis and at a specific core promoter not active in somatic tissues and not present upstream of the DDX3Y homologue. Two alternative 5'UTR transcript lengths are subsequently produced by an additional testis-specific 5'UTR splicing event. Both transcripts are mainly processed for polyadenylation in their proximal 3'UTR. A minor transcript class starting at the same male germ line-specific core promoter produces primary transcripts with an extremely long 3'UTR (∼ 17 kb), which is subsequently spliced at distinct sites resulting in six short 3'UTR splice variants (I-VI). Comparative analyses of the DDX3X transcripts in mouse and primates revealed that this complex pattern of male germ line-specific transcript variants first evolved in primates. Our data thus suggest complex translational control mechanism(s) for the human DDX3X gene locus functioning only in the male germ line and resulting in expression of its protein only in the postmeiotic spermatids.

Advances in childhood cancers

Advances in childhood cancers