Box RNA Helicase Family Research Articles

Helicase-independent function of RIG-I against murine gammaherpesvirus 68 via blocking the nuclear translocation of viral proteins

Innate immunity is the first line of defense against viral pathogens. Retinoic Acid-Inducible Gene 1 (RIG-I) is a pattern recognition receptor that recognizes virus-associated double-stranded RNA and initiates the interferon responses. Besides signal transduction, RIG-I exerts direct antiviral functions to displace viral proteins on dsRNA via its Helicase activity. Nevertheless, this effector-like activity of RIG-I against herpesviruses remains largely unexplored. It has been previously reported that herpesviruses deamidate RIG-I, resulting in the abolishment of its Helicase activity and signal transduction. In this study, we discovered that RIG-I possessed signaling-independent antiviral activities against murine gamma herpesviruses 68 (γHV68, murid herpesvirus 4). Importantly, a Helicase-dead mutant of RIG-I (K270A) demonstrated comparable inhibition on herpesviruses lytic replication, indicating that this antiviral activity is Helicase-independent. Mechanistically, RIG-I bound the Replication and Transcription Activator (RTA) and diminished its nuclear localization to repress viral transcription. We further demonstrated that RIG-I blocked the nuclear translocation of ORF21 (Thymidine Kinase), ORF75c (vGAT), both of which form a nuclear complex with RTA and RNA polymerase II (Pol II) to facilitate viral transcription. Moreover, RIG-I retained ORF59 (DNA processivity factor) in the cytoplasm to repress viral DNA replication. Altogether, we illuminated a previously unidentified, Helicase-independent effector-like function of RIG-I against γHV68, representing an exquisite host strategy to counteract viral manipulations on innate immune signaling. ImportanceRetinoic acid-inducible gene I (RIG-I), a member of DExD/H box RNA helicase family, functions as a key pattern recognition receptor (PRR) responsible for the detection of intracellular double-stranded RNA (dsRNA) from virus-infected cells and induction of type I interferon (IFN) responses. Nevertheless, our understanding of the helicase-independent effector-like activity of RIG-I against virus infection, especially herpesvirus infection, remains largely unknown. Herein, by deploying murine gamma herpesviruses 68 (γHV68) as a model system, we demonstrated that RIG-I possessed an interferon and helicase-independent antiviral activity against γHV68 via blocking the nuclear trafficking of viral proteins, which concomitantly repressed the viral early transcription and genome replication thereof. Our work illuminates a previously unidentified antiviral strategy of RIG-I against herpesvirus infection.

DDX43 mRNA expression and protein levels in relation to clinicopathological profile of breast cancer.

Breast cancer (BC) is the most often diagnosed cancer in women globally. Cancer cells appear to rely heavily on RNA helicases. DDX43 is one of DEAD- box RNA helicase family members. But, the relationship between clinicopathological, prognostic significance in different BC subtypes and DDX43 expression remains unclear. Therefore, the purpose of this study was to assess the clinicopathological significance of DDX43 protein and mRNA expression in different BC subtypes. A total of 80 females newly diagnosed with BC and 20 control females that were age-matched were recruited for this study. DDX43 protein levels were measured by ELISA technique. We used a real-time polymerase chain reaction quantification (real-time PCR) to measure the levels of DDX43 mRNA expression. Levels of DDX43 protein and mRNA expression within BC patients had been compared to those of control subjects and correlated with clinicopathological data. The mean normalized serum levels of DDX43 protein were slightly higher in control than in both benign and malignant groups, but this result was non-significant. The mean normalized level of DDX43 mRNA expression was higher in the control than in both benign and malignant cases, although the results were not statistically significant and marginally significant, respectively. Moreover, the mean normalized level of DDX43 mRNA expression was significantly higher in benign than in malignant cases. In malignant cases, low DDX43 protein expression was linked to higher nuclear grade and invasive duct carcinoma (IDC), whereas high mRNA expression was linked to the aggressive types of breast cancer such as TNBC, higher tumor and nuclear grades. This study explored the potential of using blood DDX43 mRNA expression or protein levels, or both in clinical settings as a marker of disease progression in human breast cancer. DDX43 mRNA expression proposes a less invasive method for discriminating benign from malignant BC.

2B and 3C Proteins of Senecavirus A Antagonize the Antiviral Activity of DDX21 via the Caspase-Dependent Degradation of DDX21.

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently discovered picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. It can replicate efficiently in cells, but the molecular mechanism remains poorly understood. This study determined the host’s differentially expressed proteins (DEPs) during SVA infection using dimethyl labeling based on quantitative proteomics. Among the DE proteins, DDX21, a member of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase (DDX) family, was downregulated and demonstrated inhibiting SVA replication by overexpression and knockdown experiment. To antagonize this antiviral effect of DDX21, SVA infection induces the degradation of DDX21 by 2B and 3C proteins. The Co-IP results showed that 2B and 3C did not interact with DDX21, suggesting that the degradation of DDX21 did not depend on their interaction. Moreover, the 3C protein protease activity was necessary for the degradation of DDX21. Furthermore, our study revealed that the degradation of DDX21 by 2B and 3C proteins of SVA was achieved through the caspase pathway. These findings suggest that DDX21 was an effective antiviral factor for suppressing SVA infection and that SVA antagonized its antiviral effect by degrading DDX21, which will be useful to guide further studies into the mechanism of mutual regulation between SVA and the host.

Genera specific distribution of DEAD-box RNA helicases in cyanobacteria.

Although RNA helicases are essentially ubiquitous and perform roles in all stages of RNA metabolism, phylogenetic analysis of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase family in a single phylum has not been performed. Here, we performed a phylogenetic analysis on DEAD-box helicases from all currently available cyanobacterial genomes, comprising a total of 362 helicase protein sequences from 280 strains. DEAD-box helicases belonging to three distinct clades were observed. Two clades, the CsdA (cold shock DEAD-box A)-like and RhlE (RNA helicase E)-like helicases, cluster with the homologous proteins from Escherichia coli . The third clade, the CrhR (cyanobacterial RNA helicase Redox)-like helicases, is unique to cyanobacteria and characterized by a conserved sequence motif in the C-terminal extension. Restricted distribution is observed across cyanobacterial diversity with respect to both helicase type and strain. CrhR-like and CsdA-like helicases essentially never occur together, while RhlE always occurs with either a CrhR-like or CsdA-like helicase. CrhR-like and RhlE-like proteins occurred in filamentous cyanobacteria of the orders Nostocales , Oscillatoriales and Synechococcales . Similarly, CsdA- and RhlE-like proteins are restricted to unicellular cyanobacteria of the genera Cyanobium and Synechococcus . In addition, the unexpected occurrence of RhlE in two Synechococcus strains suggests recent acquisition and evolutionary divergence. This study, therefore, raises physiological and evolutionary questions as to why DEAD-box RNA helicases encoded in cyanobacterial lineages display restricted distributions, suggesting niches that require either CrhR or CsdA RNA helicase activity but not both. Extensive conservation of gene synteny surrounding the previously described rimO–crhR operon is also observed, indicating a role in the maintenance of photosynthesis. The analysis provides insights into the evolution, origin and dissemination of sequences within a single gene family to yield divergent functional roles.

The DDX39B/FUT3/TGF\u03b2R-I axis promotes tumor metastasis and EMT in colorectal cancer

DDX39B is a member of the DEAD box (DDX) RNA helicase family required for nearly all cellular RNA metabolic processes. The exact role and potential molecular mechanism of DDX39B in the progression of human colorectal cancer (CRC) remain to be investigated. In the present study, we demonstrate that DDX39B expression is higher in CRC tissues than in adjacent normal tissues. Gain- and loss-of-function assays revealed that DDX39B facilitates CRC metastasis in vivo and in vitro. Mechanistically, RNA-sequencing (RNA-seq) and RNA-binding protein immunoprecipitation-sequencing (RIP-seq) showed that DDX39B binds directly to the FUT3 pre-mRNA and upregulates FUT3 expression. Splicing experiments in vitro using a Minigene assay confirmed that DDX39B promotes FUT3 pre-mRNA splicing. A nuclear and cytoplasmic RNA separation assay indicates that DDX39B enhances the mRNA export of FUT3. Upregulation of FUT3 accelerates the fucosylation of TGFβR-I, which activates the TGFβ signaling pathway and eventually drives the epithelial–mesenchymal transition (EMT) program and contributes to CRC progression. These findings not only provide new insight into the role of DDX39B in mRNA splicing and export as well as in tumorigenesis, but also shed light on the effects of aberrant fucosylation on CRC progression.

The Inherited MDS Gene DDX41 Is Required for Ribosome Biogenesis and Cell Viability

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders in which myeloid cell differentiation is impaired, causing blood lineage cytopenias and potentially leading to acute myeloid leukemia (AML) through malignant transformation. MDS occurs in adults with a median age of 71 years, and is associated with multiple cytogenetic and genetic abnormalities in the diseased HSC. Younger patients can also have MDS as a result of underlying congenital diseases or secondary effects from cancer therapy. It has recently been discovered that some families with high rates of MDS incidence bear heterozygous inherited mutations in DDX41, a member of the DEAD box RNA helicase family of genes. These patients typically have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general population. Inherited DDX41 mutations are always heterozygous and are typically frame-shift mutations, indicating they are likely loss of function. Approximately half of MDS patients with inherited DDX41 mutations acquire a second-hit, often R525H, in the healthy DDX41 allele in their disease clones. This mutation is also observed in 1-2% of de novo AML patients, suggesting it causes gain of function or dominant negative activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor and as an RNA splicing regulator, but its role in the pathogenesis of MDS remains unknown. We set out to model DDX41 mutations by generating conditional DDX41 knockout and R525H-knock-in mice. Combining these alleles and crossing to Rosa-Cre-ERT expressing mice allowed for tamoxifen-inducible acquisition of knockout (KO), heterozygous (HET), heterozygous knock-in (KI/+) and knock-in alone (KI/-) HSPC. The KO and KI/- HSPC were incapable of engrafting into recipient mice and underwent rapid cell cycle arrest and apoptosis, indicating that Ddx41 is required for HSPC cell viability and that the R525H mutation causes loss of the required function. In contrast, the HET and KI/+ HSPC survived and proliferated normally in culture and successfully engrafted irradiated recipient mouse bone marrow. HET and KI/+ transplanted mice had increased numbers of LSK cells, and subset of mice developed a myeloid malignancy, resembling the human disease. To determine the function of DDX41 that is critical for hematopoiesis, we performed a tandem-pulldown followed by mass spectrometry analysis to identify relevant DDX41 interacting proteins in human AML cells. We found that DDX41 interacts with multiple proteins in the small ribosomal subunit, including RPS3 and RPS14. Consistent with disruption of the assembly and function of the small ribosomal subunit, KO and KI/-HSPC exhibited rapid and robust impairment of global protein translation. Polysome profiling indicated an increase in monosomes and a decrease in polysomes in KO cells, consistent with an inability of ribosomes to initiate translation and move along the mRNA. To determine the role of the translation defect in the cell growth deficiency of DDX41-deficient cells, we treated WT, HET, and KO HSPC with the translation inhibitor puromycin and determined that KO cells were relatively more sensitive to translation inhibition, indicating that DDX41-deficient cells are specifically sensitive to further reduction in protein translation. This data supports the conclusion that the cell lethality caused by DDX41 loss is related to ribosome dysfunction. Mechanistically, we find that DDX41-deficient cells have stalled ribosomal RNA (rRNA) processing, characterized by increased unprocessed rRNA and decreased processed rRNA intermediates. In conclusion, we identify a novel function of DDX41 in regulating rRNA processing and ribosome formation that is essential for the survival and proliferation of HSPC. The loss of DDX41 may contribute to MDS as a result of impaired ribosome function, as has been previously reported in patients bearing mutations in other ribosome regulators. Disclosures Starczynowski: Kurome Therapeutics: Consultancy.

MON-310 Oncogenic Roles of Tripartite Motif 25 in Endometrial Cancer

The tripartite motif (TRIM) family proteins are known for their structure typically composed of integrated three components of RING, B-box and coiled-coil domains. Some of the TRIM proteins have been paid attention as they contribute to the development and progression of cancers. We previously identified that TRIM25/Efp promotes breast cancer proliferation by exerting its E3 ubiquitin ligase function to degrade a cell cycle checkpoint 14-3-3σ. TRIM25 also plays a critical role in antiviral immunity and interferon production by interacting with the DExD/H box RNA helicase family proteins. During the antiviral signaling, TRIM25 is involved in the activation of transcription factors such as nuclear factor κB (NF-κB). In the present study, we investigated whether TRIM25 contributes to the pathophysiology of endometrial cancer, one of the major gynecological tumors with an increasing incidence especially among postmenopausal women. In both estrogen receptor (ER)-positive and -negative endometrial cancer cells, the knockdown of TRIM25 substantially repressed the cell proliferation and migration. As expected, TRIM25 knockdown in endometrial cancer cells increased 14-3-3σ protein levels and decreased the cell population at proliferative stage. Moreover, TRIM25-specific siRNAs could successfully suppress the in vivo tumor growth of endometrial cancer cells in both subcutaneous and orthotopic xenograft models. Microarray analysis showed that genes involved in NF-κB-dependent pathway were particularly downregulated in endometrial cancer treated with TRIM25-specific siRNAs. Stable expression of TRIM25 substantially promoted the proliferation of endometrial cancer cells. Taken together, TRIM25 plays an oncogenic role in endometrial cancer by modulating 14-3-3σ protein degradation and NF-κB pathway. TRIM25 and its interacting factors would be potential diagnostic and therapeutic targets for clinical management of endometrial cancer.

DEAD box protein DDX1 promotes colorectal tumorigenesis through transcriptional activation of the LGR5 gene.

DDX1, a member of the DEAD box RNA helicase family, plays a critical role in testicular tumors. However, it remains to be clarified whether DDX1 is involved in other types of malignant tumors such as colorectal cancer. We disrupted the DDX1 gene in a human colorectal cancer cell line LoVo using the CRISPR/Cas9‐mediated gene‐targeting system. DDX1‐KO LoVo cells exhibited a much slower growth rate, produced fewer colonies in soft agar medium, and generated smaller solid tumors in nude mice than parental LoVo cells. Such phenotypes of the DDX1‐KO cells were mostly reversed by exogenous expression of DDX1. These results indicate that DDX1 is required for tumorigenicity of colorectal cancer cells. In the DDX1‐KO cells, the cancer stem cell marker genes LGR5, CD133, ALDH1 and SOX2 were markedly suppressed. Among them, expression of LGR5, which is essential for tumorigenicity of colorectal cancer cells, was restored in the DDX1‐transfected DDX1‐KO cells. Consistently, the DDX1‐KO cells lost sphere‐forming capacity in a DDX1‐dependent fashion. Reporter and chromatin immunoprecipitation assays revealed that DDX1 directly bound to the −1837 to −1662 region of the enhancer/promoter region of the human LGR5 gene and enhanced its transcription in LoVo cells. Repression of LGR5 by DDX1 knockdown was observed in 2 other human colorectal cancer cell lines, Colo320 and SW837. These results suggest that LGR5 is a critical effector of DDX1 in colorectal cancer cells. The DDX1‐LGR5 axis could be a new drug target for this type of malignant cancer.

Abstract 2802: Discovery of the first selective and cell-active eIF4A3 inhibitors

Abstract Background: Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the Asp-Glu-Ala-Asp (DEAD) box RNA helicase family. There are three subtypes of eIF4A, eIF4A1, 2, and 3. eIF4A1 and eIF4A2 are required for translation initiation as their name suggest, however eIF4A3 is functionally distinct and one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD), which is the surveillance mechanism that recognizes mRNAs containing premature termination codons to prevent the accumulation of truncated proteins. It is known that eIF4A3 is an ATP-dependent RNA clamp that can serve as a nucleation center to recruit other EJC components, and siRNA-mediated knockdown of eIF4A3 leads to a defect in NMD. In order to investigate the functions of eIF4A3 further and evaluate the therapeutic potential, we conducted a search for molecular probes of eIF4A3. Methods: Using an RNA dependent ATPase assay as a guide, intensive structure activity relationship (SAR) study and chemical optimization of high-throughput screening hit were conducted. Thereafter, NMD inhibitory activity, selectivity, helicase inhibitory activity, and physicochemical properties of optimized compounds were confirmed. Additionally, direct binding of inhibitors to eIF4A3 and their binding mode were analyzed using biophysical methods. Results: Optimized compounds showed high selectivity for eIF4A3 and exhibited significant NMD inhibitory activity in HEK293T cells. The drastic difference in eIF4A3 inhibitory activity between eutomers and distomer revealed the importance of stereochemistry at the 3-position of the piperazine ring for eIF4A3 inhibition. The lack of significant NMD inhibition by distomers suggests that NMD inhibition of eutomers is mediated by eIF4A3 inhibition. A surface plasmon resonance (SPR) biosensing assay and hydrogen/deuterium exchange mass spectrometry (HDX MS) provided strong evidence for the direct binding of the inhibitors to eIF4A3 and implications for their binding site. Conclusion: We discovered the first selective eIF4A3 inhibitors exhibiting cellular NMD inhibitory activity. These novel inhibitors not only represent novel molecular probes for investigation of RNA biology but also could be promising lead compounds for drug discovery research.

DDX49 is an RNA helicase that affects translation by regulating mRNA export and the levels of pre-ribosomal RNA.

Among the proteins predicted to be a part of the DExD box RNA helicase family, the functions of DDX49 are unknown. Here, we characterize the enzymatic activities and functions of DDX49 by comparing its properties with the well-studied RNA helicase, DDX39B. We find that DDX49 exhibits a robust ATPase and RNA helicase activity, significantly higher than that of DDX39B. DDX49 is required for the efficient export of poly (A)+ RNA from nucleus in a splicing-independent manner. Furthermore, DDX49 is a resident protein of nucleolus and regulates the steady state levels of pre-ribosomal RNA by regulating its transcription and stability. These dual functions of regulating mRNA export and pre-ribosomal RNA levels enable DDX49 to modulate global translation. Phenotypically, DDX49 promotes proliferation and colony forming potential of cells. Strikingly, DDX49 is significantly elevated in diverse cancer types suggesting that the increased abundance of DDX49 has a role in oncogenic transformation of cells. Taken together, this study shows the physiological role of DDX49 in regulating distinct steps of mRNA and pre-ribosomal RNA metabolism and hence translation and potential pathological role of its dysregulation, especially in cancers.

Comparative proteomic analysis provides insight into the biological role of protein phosphatase inhibitor-2 from Arabidopsis

Protein phosphatase inhibitor-2 (PPI-2) is a conserved eukaryotic effector protein that inhibits type one protein phosphatases (TOPP). A transfer-DNA knockdown of AtPPI-2 resulted in stunted growth in both vegetative and reproductive phases of Arabidopsis development. At the cellular level, AtPPI-2 knockdown had 35 to 40% smaller cells in developing roots and leaves. This developmental phenotype was rescued by transgenic expression of the AtPPI-2 cDNA behind a constitutive promoter. Comparative proteomics of developing leaves of wild type (WT) and AtPPI-2 mutant revealed reduced levels of proteins associated with chloroplast development, ribosome biogenesis, transport, and cell cycle regulation processes. Decreased abundance of several ribosomal proteins, a DEAD box RNA helicase family protein (AtRH3), Clp protease (ClpP3) and proteins associated with cell division suggests a bottleneck in chloroplast ribosomal biogenesis and cell cycle regulation in AtPPI-2 mutant plants. In contrast, eight out of nine Arabidopsis TOPP isoforms were increased at the transcript level in AtPPI-2 leaves compared to WT. A protein-protein interaction network revealed that >75% of the differentially accumulated proteins have at least secondary and/or tertiary connections with AtPPI-2. Collectively, these data reveal a potential basis for the growth defects of AtPPI-2 and support the presumed role of AtPPI-2 as a master regulator for TOPPs, which regulate diverse growth and developmental processes. Biological significanceComparative label-free proteomics was used to characterize an AtPPI-2T-DNA knockdown mutant. The complex, reduced growth phenotype supports the notion that AtPPI-2 is a global regulator of TOPPs, and possibly other proteins. Comparative proteomics revealed a range of differences in protein abundance from various cellular processes such as chloroplast development, ribosome biogenesis, and transporter activity in the AtPPI-2 mutant relative to WT Arabidopsis. Collectively the results of proteomic analysis and the protein-protein network suggest that AtPPI-2 is involved in a wide range of biological processes either directly or indirectly including plastid biogenesis, translational mechanisms, and cell cycle regulation. The proposed protein interaction network comprises a testable model underlying changes in protein abundance in the AtPPI-2 mutant, and provides a better framework for future studies.

X chromosome gene Ddx3x is involved in B-cell development

Abstract Ddx3x, located on the X chromosome, belongs to the DEAD box RNA helicase family. Ddx3x is previously implicated in RNA metabolism, cell cycle control, apoptosis, tumorigeneses and viral infections. In the process of exploring the role of ddx3x in the female predominance of systemic lupus erythematosus, we found abnormalities in B-cell development within conditional ddx3x-deficient mice. Breeding of ddx3x-floxed mice with Vav1-Cre mice led to deletion of ddx3x in hematopoietic cells (Vav1ddx3x). Ddx3x expression was reduced 80% in the bone marrow (BM) of Vav1ddx3x mice. Hematopoietic deficiency of ddx3x is associated with a reduced proportion and absolute number of B cells, starting at the small pre-B cell stage, and then subsequently immature B cells and mature B cells in bone marrow, relative to wild-type mice. In spleen, the frequency and number of follicular B cells (FOB) were significantly decreased whereas the numbers of marginal zone, germinal center and plasma-blast B cells were sustained. Mixed Vav1ddx3x and wild-type bone marrow chimera mice demonstrated a B-cell intrinsic effect. Surprisingly, despite fewer B cells in the BM and spleen, serum IgM, IgA and IgG titers were elevated in Vav1ddx3x mice, suggesting ddx3x may affect B-cell function as well. In conclusion, we showed that ddx3x deficiency affects B-cell development resulting in a loss of B cells in bone marrow and the periphery and an increase in serum immunoglobulin levels.

DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation

DDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation. DDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.

Prognostic Impact of Molecular Mutations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) on Allogeneic Hematopoietic Cell Transplant (HCT) Outcomes: Adverse Impact of TET2 Mutations

▪AML and MDS are heterogeneous myeloid neoplasms with variable biologic and clinical outcomes. Although allogeneic HCT is the only potentially curative therapy for high risk AML and MDS, survival after transplant remains poor, and identifying who benefits is challenging.We hypothesized that next-generation sequencing (NGS) mutational analyses can predict outcome in MDS and AML patients undergoing allogeneic HCT. We performed multi-amplicon targeted pre-HCT NGS using a somatic panel of the 60 most commonly mutated genes in myeloid neoplasias as previously determined by whole exome sequencing, on 123 patients with AML (N=64, 52%) and MDS (N=59, 48%) who subsequently underwent HCT. Median age at transplant was 53 years (range, 20-73). 21 (17%) patients had complex karyotype, 10 (8%) with monosomy 7, 48 (39%) normal, and 48 (39%) with other or unknown cytogenetic abnormalities. 45 (37%) patients were in a complete remission (CR) prior to transplant, while 78 (63%) were in less than a CR; with CR as defined by International Working Group criteria for MDS, or <5% blasts for AML. The majority of patients received myeloablative conditioning (N=83, 68%), and 40 (33%) received a reduced-intensity preparative regimen. Donor source was matched sibling (N=52, 42%), matched unrelated (N=56, 46%), cord-blood (N=12, 10%), and haplo-identical (N=3, 2%). Median follow up was 35 months (range 5-178). Mutations were analyzed individually and by molecular pathway. 88 (72%) patients had at least one mutation, most frequently in STAG2 (10.2%), TET2 (9.8%), ASXL1 (8.1%), and RUNX1 (8.1%). TP53 mutations were more common in MDS patients compared to AML (10% versus 1.6%, P=0.05). NRAS (P=0.019) and TP53 (P=0.022)mutations were more commonly associated with complex karyotype. Mutations in BCOR (P=0.048) and TP53 (P=0.047)were associated with less than CR, while TET2 (P=0.03)mutations were associated with CR prior to HCT. In univariable analyses, the presence of complex karyotype was associated with shorter overall (OS) and relapse-free survival (RFS) (hazard ratio [HR] 2.4; P=0.002 and HR 3.1; P<0.001). Mutations in TET2 (HR 2.1; P=0.042) and EZH2 (HR 2.3; P=0.048), or presence of any mutation in the histone modification pathway (ASXL1, EZH2, KDM6A, SUZ12); (HR 1.7; P=0.039) was associated with poor OS. The presence of any mutation in the DEAD box RNA-helicase family genes (DHX29, DDX54, DDX41) was associated with poor RFS (HR 3.1; P=0.009). Nothing except complex karyotype was specifically associated with higher relapse. Unlike in previous reports, TP53 mutations were not found to be significantly associated with poor OS or RFS, though these cases (N=7) were limited. In multivariable analyses, adjusting for clinical variables, complex karyotype remained significantly associated with poor OS (HR 2.7; P<0.001) and RFS (HR 3.9; P<0.001). TET2 also remained independently associated with poor OS (HR 2.4; P=0.022). Presence of any of the DNA methylation mutations (TET2, DNMT3A, IDH1, IDH2) was associated with poor RFS (HR 1.7; P=0.05). 3-year OS was 23% in patients with a complex karyotype versus 48% in patients without (P=0.002); and 14% in patients with a TET2 mutation and 46% without (P=0.042) (Figure 1).Molecular abnormalities are important variables in determining outcome after allogeneic HCT. We demonstrate that TET2 mutations in AML and MDS predict for poor survival after HCT. Ongoing serial mutational analyses in an extended cohort of patients will enhance our understanding of the role of NGS in informing care decisions for patients undergoing allogeneic HCT for AML and MDS. [Display omitted] DisclosuresMajhail:Gamida Cell Ltd.: Consultancy; Anthem Inc.: Consultancy. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Ketorolac salt is a newly discovered DDX3 inhibitor to treat oral cancer

DDX3 belongs to DEAD box RNA helicase family and is involved in the progression of several types of cancer. In this work, we employed a High Throughput Virtual screening approach to identify bioactive compounds against DDX3 from ZINC natural database. Ketorolac salt was selected based on its binding free energy less than or equals to −5 Kcal/mol with reference to existing synthetic DDX3 inhibitors and strong hydrogen bond interactions as similar to crystallized DDX3 protein (2I4I). The anti-cancer activity of Ketorolac salt against DDX3 was tested using oral squamous cell carcinoma (OSCC) cell lines. This compound significantly down regulated the expression of DDX3 in human OSCC line (H357) and the half maximal growth inhibitory concentration (IC50) of Ketorolac salt in H357 cell line is 2.6 µM. Ketorolac salt also inhibited the ATP hydrolysis by directly interacting with DDX3. More importantly, we observed decreased number of neoplastic tongue lesions and reduced lesion severity in Ketorolac salt treated groups in a carcinogen induced tongue tumor mouse model. Taken together, our result demonstrates that Ketorolac salt is a newly discovered bioactive compound against DDX3 and this compound can be used as an ideal drug candidate to treat DDX3 associated oral cancer.

Abstract 2747: EWS-FLI1 reduces RNA Helicase A activity.

Abstract Background: EWS-FLI1 requires the protein binding of RNA Helicase A (RHA) to enable oncogenic transformation. RHA is a DExH box RNA helicase family member. RHA has critical roles modulating transcription, splicing and translation. RHA also functions as a scaffolding protein in multi-protein complexes including BRCA1, DICER, EGFR, POL2R, TOPO2. Our previous work demonstrated that YK-4-279 inhibited the protein-protein interaction of RHA, with EWS-FLI1 and led to cellular apoptosis. The biochemical mechanism remained cryptic, so we investigated the effect of EWS-FLI1 upon the helicase activity of RHA. Methods: Full-length purified recombinant RHA from insect cells. Infrared-labeled double stranded RNA was used as a substrate to measure helicase activity of RHA. We included full-length recombinant EWS-FLI1 in to the helicase reaction to test the effect of EWS-FLI1. Results: RHA unwinds double stranded RNA in the presence of ATP with Km value of 5.322 nM. We also showed that RHA manifests reannealing activity of single stranded RNA to double stranded RNA in the absence of ATP. Unlike ADP, ATP and additionally the non-hyrolysable gamma-S-ATP prevent reannealing activity of RHA. A molecular modeling of RHA structure predicted the surface exposure of the region (between 823-832aa), which EWS-FLI1 interacts with. The function of this region is not well described but presumed to support helicase activity. EWS-FLI1 interferes RNA helicase activity of RHA in a dose dependent manner. Moreover, the reduction of RHA protein level exerted cytotoxic effect on Ewing Sarcoma cells. Decreased RHA protein level changed EWS-FLI1 and its target gene expression profile. IGFBP3, GRK5, p21, and PRR3 gene expression levels increased massively. RPLA1 and NR0B1 mRNA levels were also increased by moderately in these cells. The change in the profile may contribute to apoptosis. Conclusions: The purified recombinant RHA is very active in vitro helicase assays. We demonstrated that RHA's helicase activity might depend on ATP binding but not ATP hydrolysis. The protein partnering as well as posttranslational modification of RHA might modulate the helicase and/or reannealing activity of RHA, which may augment oncogenesis of EWS-FLI1. This is the first time that a protein partner reduces the helicase activity of RHA. Further experiments are ongoing that investigate whether YK-4-279 blocking of EWS-FLI1 binding to RHA will restore the helicase activity of RHA as part of it contribution to cell death. Citation Format: Hayriye Verda Erkizan, Kamal Sajwan, Maksymilian Chruszcz, Jeffrey Schneider, Sarah E. Gamble, Aykut Uren, Wladek Minor, Radhakrishnan Padmanabhan, Jeffrey Toretsky. EWS-FLI1 reduces RNA Helicase A activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2747. doi:10.1158/1538-7445.AM2013-2747

Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis.

Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

From unwinding to clamping — the DEAD box RNA helicase family

RNA helicases of the DEAD box family are present in all eukaryotic cells and in many bacteria and Archaea. These highly conserved enzymes are required for RNA metabolism from transcription to degradation and are therefore important players in gene expression. DEAD box proteins use ATP to unwind short duplex RNA in an unusual fashion and remodel RNA-protein complexes, but they can also function as ATP-dependent RNA clamps to provide nucleation centres that establish larger RNA-protein complexes. Structural, mechanistic and molecular biological studies have started to reveal how these conserved proteins can perform such diverse functions and how accessory proteins have a central role in their regulation.

Oncogenic role of DDX3 in breast cancer biogenesis

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.

Intracellular characterization of DDX39, a novel growth-associated RNA helicase

DDX39 belongs to the DEAD box RNA helicase family and is overexpressed in human lung squamous cell carcinoma. In this study, in order to seek the biological relevance of DDX39, we conducted its intracellular characterization. When expressed in 293 cells, DDX39 undergoes heavy ubiquitylation and the stability of DDX39 is regulated via a ubiqutin–proteasome pathway. DDX39 tethers ALY, an essential mRNA export factor, in vivo, confirming the role of DDX39 in the RNA splicing/export process. Co-immunoprecipitation and mass spectrometry analyses detected CIP29, a recently discovered growth and cell cycle-related factor, as a main DDX39-interacting protein. CIP29 binds RNA on its own and enhances RNA unwinding activity of DDX39. Thus, CIP29 physically and functionally associates with DDX39, suggesting their cooperation in the RNA metabolism. Extension of the search for the protein–protein interactions encompassing DDX39 identified FUS/TLS, a nucleic acid binding protein participating in both transcription and splicing, as a CIP29-interacting protein. The connections comprising ALY, DDX39, CIP29 and FUS/TLS may be an integral part of transcription, splicing and RNA export. We simultaneously examined the properties of DDX39-S, a C-terminally truncated variant of DDX39 stemmed from alternative splicing, to understand its biological significance.