The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity.

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

IGF2BP3 regulates the expression of RRM2 and promotes the progression of rheumatoid arthritis via RRM2/Akt/MMP-9 pathway.

BACKGROUND: Rheumatoid arthritis (RA) is a common inflammatory and autoimmune disease. Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) is a crucial and a rate-limiting enzyme responsible for deoxynucleotide triphosphate(dNTP) production. We have found a high expression level of RRM2 in patients with RA, but the molecular mechanism of its action remains unclear. METHODS: We analyzed the expression of hub genes in RA using GSE77298 datasets downloaded from Gene Expression Omnibus database. RRM2 and insulin-like growth factor-2 messenger ribonucleic acid (mRNA)-binding protein 3 (IGF2BP3) gene knockdown was achieved by infection with lentiviruses. The expression of RRM2, IGF2BP3, matrix metalloproteinase (MMP)-1, and MMP-9 were detected via western blotting assay. Cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MeRIP-qRT-PCR was performed to test the interaction of IGF2BP3 and RRM2 mRNA via m6A modification. Cell proliferation was determined by clone formation assay. Migration and invasion assays were performed using transwell Boyden chamber. RESULTS: RRM2 and IGF2BP3 were highly expressed in clinical specimens and tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1ß-stimulated synovial cells. RRM2 and IGF2BP3 knockdown inhibited the proliferation, migration, and invasion of MH7A cells. The inhibitory effects of IGF2BP3 knockdown were effectively reversed by simultaneously overexpressing RRM2 in MH7A cells. By analyzing N6-methyladenosine (m6A)2Target database, five m6A regulatory target binding sites for IGF2BP3 were identified in RRM2 mRNA, suggesting a direct relationship between IGF2BP3 and RRM2 mRNA. Additionally, in RRM2 small hairpin (sh)RNA lentivirus-infected cells, the levels of phosphorylated Akt and MMP-9 were significantly decreased compared with control shRNA lentivirus-infected cells. CONCLUSION: The present study demonstrated that RRM2 promoted the Akt phosphorylation leading to high expression of MMP-9 to promote the migration and invasive capacities of MH7A cells. Overall, IGF2BP promotes the expression of RRM2, and regulates the migration and invasion of MH7A cells via Akt/MMP-9 pathway to promote RA progression.

Physical interactions between specifically regulated subpopulations of the MCM and RNR complexes prevent genetic instability.

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.

RRM2 Is a CTNNB1 Transport Regulator Promoting Colon Cancer Progression.

BACKGROUND/AIM: The cytoplasmic retention and stabilization of CTNNB1 (ß-catenin) in response to Wnt is well documented in playing a role in tumor growth. Here, through the utilization of a multiplex siRNA library screening strategy, we investigated the modulation of CTNNB1 function in tumor cell progression by ribonucleoside-diphosphate reductase subunit M2 (RRM2). MATERIALS AND METHODS: We conducted a multiplex siRNA screening assay to identify targets involved in CTNNB1 nuclear translocation. In order to examine the effect of inhibition of RRM2, selected from the siRNA screening results, we performed RRM2 knockdown and assayed for colon cancer cell viability, sphere formation assay, and invasion assay. The interaction of RRM2 with CTNNB1 and its impact on oncogenesis was examined using immunoprecipitation, immunoblotting, immunocytochemistry, and RT-qPCR. RESULTS: After a series of screening and filtration steps, we identified 26 genes that were potentially involved in CTNNB1 nuclear translocation. All candidate genes were validated in various cell lines. The results revealed that siRNA-mediated knockdown of RRM2 reduces the nuclear translocation of CTNNB1. This reduction was accompanied by a decrease in cell count, resulting in a suppressive effect on tumor cell growth. CONCLUSION: High throughput siRNA screening is an attractive strategy for identifying gene functions in cancers and the interaction between RRM2 and CTNNB1 is an attractive drug target for regulating RRM2-CTNNB1-related pathways in cancers.

Ribonucleotide reductase inhibition improves the symptoms of a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2, in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aß1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200 ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200 ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment.

Reversible promoter demethylation of PDGFD confers gemcitabine resistance through STAT3 activation and RRM1 upregulation.

Drug resistance is a major problem in cancer treatment with traditional or targeted therapeutics. Gemcitabine is approved for several human cancers and the first line treatment for locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, gemcitabine resistance frequently occurs and is a major problem in successful treatments of these cancers and the mechanism of gemcitabine resistance remains largely unknown. In this study, we identified 65 genes that had reversible methylation changes in their promoters in gemcitabine resistant PDAC cells using whole genome Reduced Representation Bisulfite Sequencing analyses. One of these genes, PDGFD, was further studied in detail for its reversible epigenetic regulation in expression and shown to contribute to gemcitabine resistance in vitro and in vivo via stimulating STAT3 signaling in both autocrine and paracrine manners to upregulate RRM1 expression. Analyses of TCGA datasets showed that PDGFD positively associates with poor outcome of PDAC patients. Together, we conclude that the reversible epigenetic upregulation plays an important role in gemcitabine resistance development and targeting PDGFD signaling alleviates gemcitabine resistance for PDAC treatment.

A missense mutation in RRM1 contributes to animal tameness.

The increased tameness to reduce avoidance of human in wild animals has been long proposed as the key step of animal domestication. The tameness is a complex behavior trait and largely determined by genetic factors. However, the underlying genetic mutations remain vague and how they influence the animal behaviors is yet to be explored. Behavior tests of a wild-domestic hybrid goat population indicate the locus under strongest artificial selection during domestication may exert a huge effect on the flight distance. Within this locus, only one missense mutation RRM1I241V which was present in the early domestic goat ~6500 years ago. Genome editing of RRM1I241V in mice showed increased tameness and sociability and reduced anxiety. These behavioral changes induced by RRM1I241V were modulated by the alternation of activity of glutamatergic synapse and some other synapse-related pathways. This study established a link between RRM1I241V and tameness, demonstrating that the complex behavioral change can be achieved by mutations under strong selection during animal domestication.

Ribonucleotide reductase subunit switching in hepatoblastoma drug response and relapse.

Prognosis of children with high-risk hepatoblastoma (HB), the most common pediatric liver cancer, remains poor. In this study, we found ribonucleotide reductase (RNR) subunit M2 (RRM2) was one of the key genes supporting cell proliferation in high-risk HB. While standard chemotherapies could effectively suppress RRM2 in HB cells, they induced a significant upregulation of the other RNR M2 subunit, RRM2B. Computational analysis revealed distinct signaling networks RRM2 and RRM2B were involved in HB patient tumors, with RRM2 supporting cell proliferation and RRM2B participating heavily in stress response pathways. Indeed, RRM2B upregulation in chemotherapy-treated HB cells promoted cell survival and subsequent relapse, during which RRM2B was gradually replaced back by RRM2. Combining an RRM2 inhibitor with chemotherapy showed an effective delaying of HB tumor relapse in vivo. Overall, our study revealed the distinct roles of the two RNR M2 subunits and their dynamic switching during HB cell proliferation and stress response.

RRM1 is mediated by histone acetylation through gemcitabine resistance and contributes to invasiveness and ECM remodeling in pancreatic cancer.

The invasiveness of pancreatic cancer and its resistance to anticancer drugs define its malignant potential, and are considered to affect the peritumoral microenvironment. Cancer cells with resistance to gemcitabine exposed to external signals induced by anticancer drugs may enhance their malignant transformation. Ribonucleotide reductase large subunit M1 (RRM1), an enzyme in the DNA synthesis pathway, is upregulated during gemcitabine resistance, and its expression is associated with worse prognosis for pancreatic cancer. However, the biological function of RRM1 is unclear. In the present study, it was demonstrated that histone acetylation is involved in the regulatory mechanism related to the acquisition of gemcitabine resistance and subsequent RRM1 upregulation. The current in vitro study indicated that RRM1 expression is critical for the migratory and invasive potential of pancreatic cancer cells. Furthermore, a comprehensive RNA sequencing analysis showed that activated RRM1 induced marked changes in the expression levels of extracellular matrix­related genes, including N­cadherin, tenascin­C and COL11A. RRM1 activation also promoted extracellular matrix remodeling and mesenchymal features, which enhanced the migratory invasiveness and malignant potential of pancreatic cancer cells. The present results demonstrated that RRM1 has a critical role in the biological gene program that regulates the extracellular matrix, which promotes the aggressive malignant phenotype of pancreatic cancer.

Prognostic and Immunological Potential of Ribonucleotide Reductase Subunits in Liver Cancer.

Background: Ribonucleotide reductase (RR) consists of two subunits, the large subunit RRM1 and the small subunit (RRM2 or RRM2B), which is essential for DNA replication. Dysregulations of RR were implicated in multiple types of cancer. However, the abnormal expressions and biologic functions of RR subunits in liver cancer remain to be elucidated. Methods: TCGA, HCCDB, CCLE, HPA, cBioPortal, and GeneMANIA were utilized to perform bioinformatics analysis of RR subunits in the liver cancer. GO, KEGG, and GSEA were used for enrichment analysis. Results: The expressions of RRM1, RRM2, and RRM2B were remarkably upregulated among liver cancer tissue both in mRNA and protein levels. High expression of RRM1 and RRM2 was notably associated with high tumor grade, high stage, short overall survival, and disease-specific survival. Enrichment analyses indicated that RRM1 and RRM2 were related to DNA replication, cell cycle, regulation of nuclear division, DNA repair, and DNA recombination. Correlation analysis indicated that RRM1 and RRM2 were significantly associated with several subsets of immune cell, including Th2 cells, cytotoxic cells, and neutrophils. RRM2B expression was positively associated with immune score and stromal score. Chemosensitivity analysis revealed that sensitivity of nelarabine was positively associated with high expressions of RRM1 and RRM2. The sensitivity of rapamycin was positively associated with high expressions of RRM2B. Conclusion: Our findings demonstrated high expression profiles of RR subunits in liver cancer, which may provide novel insights for predicting the poor prognosis and increased chemosensitivity of liver cancer in clinic.

Comprehensive bioinformatics analysis of ribonucleoside diphosphate reductase subunit M2(RRM2) gene correlates with prognosis and tumor immunotherapy in pan-cancer.

Ribonucleotide reductase (RNR) small subunit M2 (RRM2) levels are known to regulate the activity of RNR, a rate-limiting enzyme in the synthesis of deoxyribonucleotide triphosphates (dNTPs) and essential for both DNA replication and repair. The high expression of RRM2 enhances the proliferation of cancer cells, thereby implicating its role as an anti-cancer agent. However, little research has been performed on its role in the prognosis of different types of cancers. This pan-cancer study aimed to evaluate the effect of high expression of RRM2 the tumor prognosis based on clinical information collected from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases. We found RRM2 gene was highly expressed in 30 types of cancers. And we performed a pan-cancer analysis of the genetic alteration status and methylation of RRM2. Results indicated that RRM2 existed hypermethylation, associated with m6A, m1A, and m5C related genes. Subsequently, we explored the microRNAs (miRNA), long non-coding RNAs (lncRNA), and the transcription factors responsible for the high expression of RRM2 in cancer cells. Results indicated that has-miR-125b-5p and has-miR-30a-5p regulated the expression of RRM2 along with transcription factors, such as CBFB, E2F1, and FOXM. Besides, we established the competing endogenous RNA (ceRNA) diagram of lncRNAs-miRNAs-circular RNAs (circRNA) involved in the regulation of RRM2 expression. Meanwhile, our study demonstrated that high-RRM2 levels correlated with patients' worse prognosis survival and immunotherapy effects through the consensus clustering and risk scores analysis. Finally, we found RRM2 regulated the resistance of immune checkpoint inhibitors through the PI3K-AKT single pathways. Collectively, our findings elucidated that high expression of RRM2 correlates with prognosis and tumor immunotherapy in pan-cancer. Moreover, these findings may provide insights for further investigation of the RRM2 gene as a biomarker in predicting immunotherapy's response and therapeutic target.

ARID1A promotes chemosensitivity to gemcitabine in pancreatic cancer through epigenetic silencing of RRM2.

Pancreatic cancer is one of the most common malignancies with very poor prognosis due to its broad resistance to chemotherapy. ARID1A, a subunit of SWI/SNF complex, is involved in pancreatic carcinogenesis through epigenetic silencing of oncogenes. In this study, we aimed to explore whether ARID1A was implicated in the gemcitabine resistance in pancreatic cancer patients via regulating RRM2. We examined the effect of ARID1A depletion on the gemcitabine sensitivity in pancreatic cancer cells and explored the role of RRM2 in ARID1A-mediated pancreatic cancer cells chemosensitivity to gemcitabine. We found that Knockout of ARID1A led to gemcitabine resistance in pancreatic cancer cells, effect of which could be reversed by RRM2, a gemcitabine resistance related gene. ARID1A decreased the transcription of RRM2, and directly bound to the promoter of RRM2. Moreover, expression of RRM2 was negatively correlated with ARID1A in pancreatic cancer tissues. Thus, ARID1A-mediated RRM2 epigenetic suppression is crucial for enhancement of pancreatic cancer chemosensitivity to gemcitabine, and ARID1A could be used as a biomarker to guide the gemcitabine chemotherapy of pancreatic cancer.

Circ_0008285 knockdown represses tumor development by miR-384/RRM2 axis in hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Circular RNA (circRNA) has attracted extensive attention in studies related to the malignant progression of cancer, including hepatocellular carcinoma (HCC). Therefore, its molecular mechanism in HCC needs to be further explored. MATERIALS AND METHODS: The expression levels of circ_0008285, microRNA (miR)-384 and ribonucleotide reductase subunit M2 (RRM2) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was analyzed using cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine assay, cell apoptosis was analyzed by flow cytometry, and cell migration and invasion were detected by transwell assay. Protein level was detected by western blot. The relationships between miR-384 and circ_0008285 or RRM2 were predicted by bioinformatics software and validated by dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0008285 expression is elevated to HCC tissues and cell lines. Silencing of circ_0008285 inhibited the proliferation, migration and invasion of HCC cells but accelerated cell apoptosis in vitro and impeded HCC tumorigenesis in vivo. Mechanistically, circ_0008285 directly interacted with miR-384, and miR-384 silencing attenuated the effects of circ_0008285 interference on cell proliferation, migration, invasion, and apoptosis. RRM2 was a direct target of miR-384, and RRM2 overexpression reversed the effects of miR-384 overexpression on cell proliferation, migration, invasion, and apoptosis. In addition, circ_0008285 regulated RRM2 expression by sponging miR-384. CONCLUSION: In this study, circ_0008285 could promote the malignant biological behaviors of HCC cells through miR-384/RRM2 axis and has the potential to become a therapeutic target for HCC, providing a new idea for targeted therapy of HCC.

E2F2 enhances the chemoresistance of pancreatic cancer to gemcitabine by regulating the cell cycle and upregulating the expression of RRM2.

Both pro-oncogenic and anti-oncogenic effects of E2F2 have been revealed in different malignancies. However, the precise role of E2F2 in pancreatic cancer, in particular in relation to therapeutic intervention with gemcitabine, remains unclear. In this study, the effect of E2F2 on the proliferation and cell cycle modulation of pancreatic cancer cells, and whether E2F2 plays a role in the treatment of pancreatic cancer cells by gemcitabine, were investigated. The expression of E2F2 in pancreatic cancer was assessed by various methods including bioinformatics prediction, Western blotting, and real-time PCR. The effect of E2F2 on the proliferation and cell cycling of pancreatic cancer cells was analyzed by tissue culture and flow cytometry. In addition, the effect of E2F2 on the intervention of pancreatic cancer by gemcitabine was investigated using both in vitro and in vivo approaches. The expression of E2F2 was found to be significantly increased in pancreatic cancer tissues and cell lines. The pathogenic capacity of E2F2 lied in the fact that this transcription factor promoted the transformation of pancreatic cancer cell cycle from G1-phase to S-phase, thus enhancing the proliferation of pancreatic cancer cells. Furthermore, the expression of E2F2 was increased in pancreatic cancer cells in the presence of gemcitabine, and the augmented expression of E2F2 upregulated the gemcitabine resistance-related gene RRM2 and its downstream signaling molecule deoxycytidine kinase (DCK). The resistance of pancreatic cancer cells to gemcitabine was confirmed using both in vitro and in vivo models. In this study, E2F2 has been demonstrated for the first time to play a pro-oncogenic role in pancreatic cancer by promoting the transition of the cell cycle from G1-phase to S-phase and, therefore, enhancing the proliferation of pancreatic cancer cells. E2F2 has also been demonstrated to enhance the chemotherapy resistance of pancreatic cancer cells to gemcitabine by upregulating the expression of RRM2 and DCK that is downstream of RRM2.

LncRNA PART1 Stimulates the Development of Ovarian Cancer by Up-regulating RACGAP1 and RRM2.

Ovarian cancer (OC) is a kind of gynecologic malignancy with a high mortality rate. Long non-coding RNAs (lncRNAs) have been reported to exert regulatory roles in multiple diseases. However, the role of lncRNA prostate androgen-regulated transcript 1 (PART1) has not been investigated in the development of OC. In this study, from RT-qPCR analysis, we discovered that PART1 demonstrated high expression in OC cells. Moreover, data from functional assays manifested that PART1 reduction hindered the proliferative, migratory, and invasive capabilities of OC cells. In vivo uncovered that PART1 knockdown impeded OC tumor growth. Furthermore, from the experimental results of RNA pull down, RIP, and luciferase reporter assays, we discovered that PART1 served as a sponge for microRNA-6884-5p (miR-6884-5p) to modulate the expression of Rac GTPase activating protein 1 (RACGAP1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Finally, rescue assays proved that overexpression of RACGAP1 or RRM2 abrogated the suppressive role of PART1 knockdown on OC cell malignant behaviors. RACGAP1 and RRM2 were also revealed to act as oncogenes in OC cells. In summary, our research verified the PART1/miR-6884-5p/RACGAP1/RRM2 axis in OC cells, which signified that PART1 might act as a novel biomarker in OC.

RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis.

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

Ribonucleotide reductase subunit M2 is a potential prognostic marker and therapeutic target for soft tissue sarcoma.

Soft tissue sarcomas (STSs) are highly aggressive malignant tumors that exhibit poor therapeutic outcomes. Hence, we aimed to track down a potential gene that can be used as a prognostic marker and therapeutic target for this malignancy. We integrated omics analysis of clinical data and in vitro studies and identified Ribonucleotide reductase subunit M2 (RRM2) as a potential oncogene associated with STS prognosis. We found RRM2 is highly expressed in STS cell lines and tissues. STS patients with increased RRM2 levels showed worse overall survival, disease-free survival, progression-free survival, and disease-specific survival. Further, overexpression of RRM2 in HT1080 cells induces proliferation, migration, invasion, and colony formation, whereas its silencing arrest the cell cycle at G0/G1 phase and induces apoptosis. Taken together, we established RRM2 to be positively associated with oncogenesis and prognosis of STS and therefore could be a promising prognostic marker and therapeutic target.

Bone marrow ribonucleotide reductase mRNA levels and methylation status as prognostic factors in patients with myelodysplastic syndrome treated with 5-Azacytidine.

Ribonucleotide Reductase (RNR) is a two-subunit (RRM1, RRM2) enzyme, responsible for the conversion of ribonucleotides to deoxyribonucleotides required for DNA replication. To evaluate RNR as a biomarker of response to 5-azacytidine, we measured RNR mRNA levels by a quantitative real-time PCR in bone marrow samples of 98 patients with myelodysplastic syndrome (MDS) treated with 5-azacytidine with parallel quantification of the gene promoter's methylation. Patients with low RRM1 levels had a high RRM1 methylation status (p = 0.005) and a better response to treatment with 5-azacytidine (p = 0.019). A next-generation sequencing for genes of interest in MDS was also carried out in a subset of 61 samples. Splicing factor mutations were correlated with lower RRM1 mRNA levels (p = 0.044). Our results suggest that the expression of RNR is correlated with clinical outcomes, thus its expression could be used as a prognostic factor for response to 5-azacytidine and a possible therapeutic target in MDS.

Ribonucleotide reductase subunit M2 promotes proliferation and epithelial-mesenchymal transition via the JAK2/STAT3 signaling pathway in retinoblastoma.

Retinoblastoma (RB) is an intraocular malignant tumor that often occurs in children. Along with the improvement of treatment strategies, the cure rate of RB has increased significantly. However, the treatment of advanced and recurrent RB remains as a critical challenge. Therefore, studying the molecular mechanisms underlying the progression of RB is essential for the development of novel and effective therapeutic strategies. Through the analysis of a previously published microarray study, we found that ribonucleotide reductase subunit M2 (RRM2) was highly expressed in RB tissues as compared to normal tissues. The purpose of this study is to clarify the role and mechanism of RRM2 in regulating the progression of RB. We first demonstrated that RRM2 expression level in RB tissues and cell lines was significantly higher when compared to that in normal retinal tissue and cell lines, and high RRM2 expression level was associated with a poorer overall survival of patients. In RB cells, RRM2 overexpression promoted cell proliferation, migration, invasion and epithelial-mesenchymal transformation (EMT), while RRM2 silencing suppressed these biological features. Silencing RRM2 reduced the activation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, and the presence of JAK2/STAT3 signaling pathway inhibitor INCB attenuated the effect of RRM2 overexpression. Collectively, our data indicate that RRM2 promotes the progression of RB by activating JAK2/STAT3 signaling pathway. Targeting RRM2/JAK2/STAT3 axis lays a theoretical foundation for the formulation of novel RB therapy.

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript.

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.