Helicase Activity Research Articles

Inhibition of CDC7 with Sgr-2921 in AML Models Results in Enhanced DNA Damage and Anti-Leukemic Activity As Monotherapy and in Combination with Standard of Care Agents

Introduction: CDC7 is a protein kinase that is active during the S-phase of the cell cycle. CDC7 plays a critical role in the replication stress response by phosphorylation and activation of the BRCA1 and Cohesin complexes, which protect and restart stalled replication forks. In addition, by maintaining DNA helicase activity during replication fork stalling, CDC7 establishes a platform for ATR checkpoint activation. Acute myeloid leukemia (AML) is typically a rapidly proliferating cancer and is characterized by high replication stress and DNA damage. Agents that target replication stress and DNA damage response pathways represent novel therapeutic opportunities in AML. Here we show that inhibition of CDC7 in AML models results in potent anti-tumor activity by amplifying replication stress and DNA damage. We show that CDC7 inhibition has synergistic anti-proliferative effects when combined with hypomethylating agents and a BCL2 inhibitor. We previously discovered that CDC7 activity drives resistance to FLT3 inhibition (Cancer Cell. 2021 Jul 12;39(7):999-1014.e8), and now show that inhibition of CDC7 overcomes resistance to a variety of FLT3 inhibitor resistant cell lines. CDC7 inhibition is efficacious in multiple AML models resistant to standard of care agents, suggesting that CDC7 inhibitor combinations may be efficacious in the clinic. Results: We identified a potent and selective CDC7 inhibitor, SGR-2921, using large-scale physics-based computational modeling technology. In a panel of ~300 cancer cell lines, AML cell lines were on average the most sensitive to SGR-2921, and AML patient samples were highly sensitive to CDC7 inhibition ex vivo. In vivo, SGR-2921 showed strong anti-tumor growth activity in MV-4-11 and Molm-16 AML xenograft models at tolerated doses. In combination with hypomethylating agents, SGR-2921 increased the level of replication stress, DNA damage and apoptosis markers in vitro. Combination of SGR-2921 with venetoclax (BCL2 inhibitor) showed synergy on anti-tumor activity both in vitro and in vivo. SGR-2921 was highly efficacious in AML cell lines resistant to FLT3 inhibitors, hypomethylating agents and venetoclax, and in multi-agent resistant cell lines. Co-treatment with SGR-2921 partially restored sensitivity to FLT3 inhibition in FLT3 resistance AML cell lines. Conclusions: SGR-2921, a novel, potent CDC7 small molecule inhibitor, demonstrates strong anti-proliferative activity both in vitro in AML cell models and in vivo in AML xenograft models. SGR-2921 showed synergistic inhibitory effects on cell-proliferation and tumor growth in combination with standard of care agents, and was anti-proliferative in AML cell lines and patient samples resistant to standard of care agents. Together, these data show that SGR-2921-mediated CDC7 inhibition is an attractive novel treatment opportunity in AML, with a potential utility in patients with relapsed and refractory AML.

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase.

Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay.

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machineryand the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work;K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue.

Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue.

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism.

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lackstatistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5'-, but not 3'-ends of biotinylated DNA.

LBMON272 Chromatoid Bodies In The Regulation Of Spermatogenesis: Novel Role Of Grth

Abstract More than 5 million couples in US are affected by infertility. Male infertility is a major cause (&amp;gt;30%), and understanding spermatogenesis is important to address the problem. During early spermatogenesis, mRNAs are actively transcribed, transported and stored transiently in chromatoid bodies (CBs). CBs are membrane-less perinuclear organelles which serve as storehouses of mRNAs until later stages of spermatogenesis. Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) plays critical role in spermatogenesis. In addition to its inherent helicase activity, GRTH transports mRNAs from nucleus to cytoplasm and from there to CBs for storage until translation. In Germ cells, there are two species of GRTH, the 56 kDa non-phospho and 61 kDa phospho (pGRTH) forms. Our early studies revealed a missense mutation (R242H) of GRTH in Japanese azoospermic men which resulted in the lack of pGRTH in in vitro studies. GRTH knock-in (KI) mice with the human mutant GRTH gene (R242H), show loss of the phospho-species from cytoplasm and CBs with preservation of the non-phospho form in the cytoplasm, Nucleus and CBs. GRTH-KI mice are sterile, lack elongated spermatids and spermatozoa with spermatogenic arrest at step 8 of round spermatids (RS). CBs isolated from GRTH KI mice are smaller, highly condensed, lack pGRTH and RNA-Seq analysis of isolated mRNA (from CBs) revealed differential abundance of mRNAs related to spermatid differentiation. Integrative analysis of small RNA and mRNA expression will provide cohesive outlook of molecular mechanisms of gene regulation during spermatogenesis. Hence, we proceeded with small RNAseq analysis which reveal differential expression (Padj&amp;lt;0. 05) of piRNAs and miRNAs. In total 226706 piRNAs upregulated and 166567 piRNAs downregulated in the round spermatids. Several miRNAs like miR32, miR7, miR184, miR335, miR140, miR141, miR1981, miR202, miR880, miR669c were downregulated. These are involved in the positive regulation of spermatid differentiation, sperm motility, mRNA processing and decay. The miRNAs, miR150, miR196a-2, miR7b, miR652, miR146, miR10b, miR379, miR122a, miR27a, miR127 and miR328 were upregulated, which negatively regulate the spermatid differentiation. Gene ontology analysis revealed that the majority of downregulated miRNAs belong to regulation of gene expression. Majority of upregulated miRNAs belong to miRNA mediated suppression of translation mostly associated with spermiogenesis and CBs. The identified miRNAs regulate essential genes like Tnp2, TSSK6, Pabpc1, Ppp1cc and UPF2 etc. These indicates that the CBs mediate highly specialized mRNA metabolism process and serve as a processing center of essential mRNAs. These results demonstrate the importance of GRTH and phospho-GRTH in the regulation of CB and their role in mRNA storage and stability of germ cell specific mRNAs during spermiogenesis and healthy sperm formation. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Inhibition of transcription of VP2 by mutations in the DNA binding domains of mink enteritis virus NS1 protein

The NS1 protein of mink enteritis virus (MEV) is a multidomain and multifunctional protein that plays a critical role in viral replication, with predicted nuclease, helicase and transactivation activities. The nuclease and helicase domains of NS1 protein are involved in interaction with viral DNA. Herein, potential amino acids critical for DNA binding in the MEV NS1 were mutated, all of which resulted in a termination of viral production from an infectious MEV clone. Although E121, H129/131, Y212 and K470/472 mutants retained their P38 and 5’UTR transactivation activity, K196/197 and K406 mutations eliminated this. Interestingly, VP2 protein was produced following transfection of F81 cells with pMEV-NS1-196K2G (K196G and K197G) and pMEV-NS1-K406G when pNS1 was co-transfected in trans, indicating that the substitutions did not affect the integrity of the DNA sequence that bound to NS1 protein but inhibited the biological properties of NS1 protein itself. The ability of NS1 protein to interact with SP1 was inhibited by both 196K2G and K406G substitutions, while 196K2G resulted in failure to bind to the DNA-binding sites in the P38 promoter, and the oligomerization of K406G was inhibited. All of these could explain the transcriptional repression.

The Vaccinia Virus DNA Helicase Structure from Combined Single-Particle Cryo-Electron Microscopy and AlphaFold2 Prediction.

Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323–785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.

MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair

Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD). We show MutL-DNA binding is undetectable in physiological conditions. Instead, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone, likely enabling diffusion-mediated wrapping of the remaining MutL domains around the DNA. The resulting MutL sliding clamp enhances MutH endonuclease and UvrD helicase activities on the DNA, which also engage the PCC during strand-specific incision/excision. These MutS clamp-loader progressions are significantly different from the replication clamp-loaders that attach the polymerase processivity factors β-clamp/PCNA to DNA, highlighting the breadth of mechanisms for stably linking crucial genome maintenance proteins onto DNA.

Overexpressed Histocompatibility Minor 13 was Associated with Liver Hepatocellular Carcinoma Progression and Prognosis.

Among primary liver carcinoma cases, the proportion of liver hepatocellular carcinoma (LIHC) cases is 75%–85%. Current treatments for LIHC include chemotherapy, surgical excision, and liver transplantation, which are effective for early LIHC treatment. Nevertheless, the early symptoms of liver carcinoma are atypical, so a large proportion of LIHC patients are diagnosed at an advanced stage. Histocompatibility minor 13 (HM13), located in the endoplasmic reticulum, is responsible for catalysing the hydrolysis of some signal peptides after cleavage from the precursor protein. Here, we studied the role of HM13 in LIHC development through bioinformatics analysis. Database analysis showed that HM13 was of great significance for LIHC tumorigenesis. Compared to normal liver tissues, HM13 expression was increased to a greater extent in LIHC tissues. After analysis of Kaplan‒Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA) datasets, we discovered that highly expressed HM13 exhibited an association with shorter overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS). We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to analyse HM13-related genes, and the data indicated that these genes obviously participated in rRNA processing, ribosome biogenesis, spliceosome, Huntington's disease, and ATP-dependent helicase activity. The Cell Counting Kit-8 (CCK-8) assay and Transwell assay showed that reducing HM13 expression hindered LIHC cell proliferation, migration, and invasion. In conclusion, these findings indicate that HM13 is a biomarker and is related to the poor prognosis of LIHC. Our results are conducive to discovering new targets for LIHC treatment.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an ‘active helicase,’ capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Identification of DDX5 as an indispensable activator of the glucocorticoid receptor in adipocyte differentiation.

The expression of CCAAT/enhancer-binding protein (C/EBP) family members and peroxisome proliferator-activated receptor γ (PPAR γ) is essential for the differentiation of pre-adipocyte 3T3-L1 cells into mature adipocytes induced by a combined stimulation with dexamethasone, 3-isobutyl-1-methylxanthine and insulin (DMI). We herein demonstrated that the RNA helicase DDX5, the expression of which was induced by DMI, played an important role in the adipocyte differentiation of 3T3-L1 cells. The DMI-induced accumulation of lipid droplets and expression of adipocyte markers in 3T3-L1 cells were significantly inhibited by the knockdown of DDX5. The knockdown of DDX5 interfered with the expressional induction of C/EBPδ, which was the first to be induced in the transcription factor cascade, and inhibited the subsequent expression of the other transcription factors, C/EBPβ, PPARγ and C/EBPα. DDX5 interacted with the glucocorticoid receptor (GR), which induced the expression of C/EBPδ. The knockdown of DDX5 failed to induce the nuclear translocation of GR, suggesting the essential role of DDX5 in the early stage of adipocyte differentiation. Furthermore, the reconstitution of DDX5, but not the DDX5 mutant (K144N) lacking RNA helicase activity, restored DMI-induced GR activation and adipocyte differentiation in 3T3-L1 cells in which DDX5 was knocked down, confirming that the RNA helicase activity of DDX5 is essential for adipogenesis. Collectively, these results revealed for the first time that DDX5 is necessary for GR activation and plays an essential role in early adipocyte differentiation.

The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

Hrq1/RECQL4 regulation is critical for preventing aberrant recombination during DNA intrastrand crosslink repair and is upregulated in breast cancer.

Human RECQL4 is a member of the RecQ family of DNA helicases and functions during DNA replication and repair. RECQL4 mutations are associated with developmental defects and cancer. Although RECQL4 mutations lead to disease, RECQL4 overexpression is also observed in cancer, including breast and prostate. Thus, tight regulation of RECQL4 protein levels is crucial for genome stability. Because mammalian RECQL4 is essential, how cells regulate RECQL4 protein levels is largely unknown. Utilizing budding yeast, we investigated the RECQL4 homolog, HRQ1, during DNA crosslink repair. We find that Hrq1 functions in the error-free template switching pathway to mediate DNA intrastrand crosslink repair. Although Hrq1 mediates repair of cisplatin-induced lesions, it is paradoxically degraded by the proteasome following cisplatin treatment. By identifying the targeted lysine residues, we show that preventing Hrq1 degradation results in increased recombination and mutagenesis. Like yeast, human RECQL4 is similarly degraded upon exposure to crosslinking agents. Furthermore, over-expression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. Using bioinformatic analysis, we observe that RECQL4 overexpression correlates with increased recombination and mutations. Overall, our study uncovers a role for Hrq1/RECQL4 in DNA intrastrand crosslink repair and provides further insight how misregulation of RECQL4 can promote genomic instability, a cancer hallmark.

WRN promotes bone development and growth by unwinding SHOX-G-quadruplexes via its helicase activity in Werner Syndrome

Werner Syndrome (WS) is an autosomal recessive disorder characterized by premature aging due to mutations of the WRN gene. A classical sign in WS patients is short stature, but the underlying mechanisms are not well understood. Here we report that WRN is indispensable for chondrogenesis, which is the engine driving the elongation of bones and determines height. Zebrafish lacking wrn exhibit impairment of bone growth and have shorter body stature. We pinpoint the function of WRN to its helicase domain. We identify short-stature homeobox (SHOX) as a crucial and direct target of WRN and find that the WRN helicase core regulates the transcriptional expression of SHOX via unwinding G-quadruplexes. Consistent with this, shox−/− zebrafish exhibit impaired bone growth, while genetic overexpression of SHOX or shox expression rescues the bone developmental deficiency induced in WRN/wrn-null mutants both in vitro and in vivo. Collectively, we have identified a previously unknown function of WRN in regulating bone development and growth through the transcriptional regulation of SHOX via the WRN helicase domain, thus illuminating a possible approach for new therapeutic strategies.

Revealing the DNA Unwinding Activity and Mechanism of Fork Reversal by RecG While Exposed to Variants of Stalled Replication-fork at Single-Molecular Resolution

RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. The elemental mechanism of the helicase activity of RecG with several non-homologous stalled fork structures resembling intermediates formed during the process of DNA repair has been investigated in the present study to capture the dynamic stages of genetic rearrangement. The functional characterization has been exemplified through quantifying the response of the substrate in terms of their molecular heterogeneity and dynamical response by employing single-molecule fluorescence methods. An elevated processivity of RecG is observed for the stalled fork where progression of lagging daughter strand is ahead as compared to that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. RecG is found to adopt an asymmetric mode of locomotion to unwind the lagging daughter strand for facilitating formation of Holliday junction that acts as a suitable intermediate for recombinational repair pathway. Our results emphasize the mechanism adopted by RecG during its ‘sliding back’ mode along the lagging daughter strand to be ‘active translocation and passive unwinding’. This also provide clues as to how this helicase decides and controls the mode of translocation along the DNA to unwind.

A single-molecule approach to unravel the molecular mechanism of the action of Deinococcus radiodurans RecD2 and its interaction with SSB and RecA in DNA repair

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. They are responsible for resolving double-stranded DNA (dsDNA) into single-strands, which is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions to the organism's unusually high tolerance to gamma radiation and hydrogen peroxide. Although the results from X-ray Crystallography studies have revealed the structural characteristics of the protein, direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5′ terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

Unique role of DDX41, a DEAD-box type RNA helicase, in hematopoiesis and leukemogenesis.

In myeloid malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), patient selection and therapeutic strategies are increasingly based on tumor-specific genetic mutations. Among these, mutations in DDX41, which encodes a DEAD-box type RNA helicase, are present in approximately 2–5% of AML and MDS patients; this disease subtype exhibits a distinctive disease phenotype characterized by late age of onset, tendency toward cytopenia in the peripheral blood and bone marrow, a relatively favorable prognosis, and a high frequency of normal karyotypes. Typically, individuals with a loss-of-function germline DDX41 variant in one allele later acquire the p.R525H mutation in the other allele before overt disease manifestation, suggesting that the progressive decrease in DDX41 expression and/or function is involved in myeloid leukemogenesis.RNA helicases play roles in many processes involving RNA metabolism by altering RNA structure and RNA-protein interactions through ATP-dependent helicase activity. A single RNA helicase can play multiple cellular roles, making it difficult to elucidate the mechanisms by which mutations in DDX41 are involved in leukemogenesis. Nevertheless, multiple DDX41 functions have been associated with disease development. The enzyme has been implicated in the regulation of RNA splicing, nucleic acid sensing in the cytoplasm, R-loop resolution, and snoRNA processing.Most of the mutated RNA splicing-related factors in MDS are involved in the recognition and determination of 3’ splice sites (SS), although their individual roles are distinct. On the other hand, DDX41 is likely incorporated into the C complex of the spliceosome, which may define a distinctive disease phenotype. This review summarizes the current understanding of how DDX41 is involved in this unique myeloid malignancy.

1708P SMARCA4/SMARCB1 alterations detected through tissue comprehensive genomic profiling (CGP): A single-center study of prevalence and clinical characteristics

SMARCA4 (also known as BRG1) and SMARCB1 are proteins with transcription-regulation through helicase and ATPase activity in the SWI/SNF chromatin-remodelling complex. Alterations in these genes have been described as prognostic and related to tumor differentiation and metastasis. Inactivation has a role in the development, early dedifferentiation and metastasis of up to 20% solid tumors.

Tideglusib Inhibits Pif1 Helicase of Bacteroides sp. via an Irreversible and Cys-380-Dependent Mechanism.

Pif1 helicase plays multiple roles in maintaining genome stability, which is an attractive therapeutic target for helicase-related diseases, while small molecules targeting Pif1 are not yet available. In this study, we performed a fluorescence polarization-based high-throughput screening and identified that an FDA-approved drug, Tideglusib (TD), could inhibit the DNA-binding activity (IC50 = 6.2 ± 0.4 μM) and ATPase and helicase activity (IC50 = 2–4 μM) of Bacteroides sp. Pif1 (BaPif1), which was also confirmed with human Pif1. In addition, the TD analogue TDZD-8 displayed similar inhibitory effects on Pif1 activities. Notably, TD irreversibly inhibited BaPif1 and severely induced BaPif1 aggregation. Furthermore, inhibition of BaPif1 by TD was significantly attenuated in the presence of dithiothreitol, indicating that TD could be a thiol-reactive compound. We also identified that Cys-380 of BaPif1 is critical for the inhibition by TD, suggesting that TD inhibits BaPif1 via an irreversible and Cys-380-dependent mechanism.