Subunit Of Ribonucleotide Reductase Research Articles

Inhibition of Ribonucleotide Reductase Subunit M2 Enhances the Radiosensitivity of Metastatic Pancreatic Neuroendocrine Tumor

Ribonucleotide Reductase (RNR) is a rate-limiting enzyme in the production of deoxyribonucleoside triphosphates (dNTPs), which are essential substrates for DNA repair after radiation damage. We explored the radiosensitization property of RNR and investigated a selective RRM2 inhibitor, 3-AP, as a radiosensitizer in the treatment of metastatic pNETs. We investigated the role of RNR subunit, RRM2, in pancreatic neuroendocrine (pNET) cells and responses to radiation in vitro. We also evaluated the selective RRM2 subunit inhibitor, 3-AP, as a radiosensitizer to treat pNET metastases in vivo. Knockdown of RNR subunits demonstrated that RRM1 and RRM2 subunits, but not p53R3, play significant roles in cell proliferation. RRM2 inhibition activated DDR pathways through phosphorylation of ATM and DNA-PK protein kinases but not ATR. RRM2 inhibition also induced Chk1 and Chk2 phosphorylation, resulting in G1/S phase cell cycle arrest. RRM2 inhibition sensitized pNET cells to radiotherapy and induced apoptosis in vitro. In vivo, we utilized pNET subcutaneous and lung metastasis models to examine the rationale for RNR-targeted therapy and 3-AP as a radiosensitizer in treating pNETs. Combination treatment significantly increased apoptosis of BON (human pNET) xenografts and significantly reduced the burden of lung metastases. Together, our results demonstrate that selective RRM2 inhibition induced radiosensitivity of metastatic pNETs both in vitro and in vivo. Therefore, treatment with the selective RRM2 inhibitor, 3-AP, is a promising radiosensitizer in the therapeutic armamentarium for metastatic pNETs.

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

Reduced ribonucleotide reductase RRM2 subunit expression increases DNA damage and mitochondria dysfunction in woody breast chickens.

The aim of this study was to investigate the roles of ribonucleotide reductase subunit M2 (RRM2; subunit of ribonucleotide reductase) in severe woody breast (WB) and normal breast muscles. 40 8-week-old male Ross-708 broiler chickens. Quantitative PCR was performed to determine gene expression, and commercial ELISA/assay kits were used to obtain several enzymatic activities. Results showed that RRM2 activity (P = .0002) and RRM2 (P = .05) and hydroxymethylbilane synthase expression (impaired oxygen transport and metabolism, P = .002) were reduced in WB, while caveolin-3 (defected membrane integrity, P = .09), endoglin (increased fibrosis, P = .06), and secreted protein acidic rich in cysteine (metabolic dysregulation, P = .09) expression tended to increase in WB. WB tended to have increased levels of homocysteine (P = .06), aspartate aminotransferase mitochondria (P = .02), pyruvate kinase (P = .04), DNA damage (P = .06), creatine kinase (P = .05), and triglyceride (P = .002) but decreased ATPase activity (P = .01), all indicating mitochondria dysfunction and tissue damage. In this study, differences in various enzyme activities and increased DNA damage suggest that RRM2-mediated mitochondrial abnormalities may play a role in WB myopathy.

N6-methyladenosine facilitates mitochondrial fusion of colorectal cancer cells via induction of GSH synthesis and stabilization of OPA1 mRNA.

Mitochondria undergo fission and fusion that are critical for cell survival and cancer development, while the regulatory factors for mitochondrial dynamics remain elusive. Herein we found that RNA m6A accelerated mitochondria fusion of colorectal cancer (CRC) cells. Metabolomics analysis and function studies indicated that m6A triggered the generation of glutathione (GSH) via the upregulation of RRM2B-a p53-inducible ribonucleotide reductase subunit with anti-reactive oxygen species potential. This in turn resulted in the mitochondria fusion of CRC cells. Mechanistically, m6A methylation of A1240 at 3'UTR of RRM2B increased its mRNA stability via binding with IGF2BP2. Similarly, m6A methylation of A2212 at the coding sequence (CDS) of OPA1-an essential GTPase protein for mitochondrial inner membrane fusion-also increased mRNA stability and triggered mitochondria fusion. Targeting m6A through the methyltransferase inhibitor STM2457 or the dm6ACRISPR system significantly suppressed mitochondria fusion. In vivo and clinical data confirmed the positive roles of the m6A/mitochondrial dynamics in tumor growth and CRC progression. Collectively, m6A promoted mitochondria fusion via induction of GSH synthesis and OPA1 expression, which facilitated cancer cell growth and CRC development.

Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs.

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known. Using NMR spectroscopy, in vivo assays, X-ray crystallography, calorimetry, and Monte Carlo simulations, an additional PCNA binding motif in Spd1, a PIP-box, is revealed. The two tandemly positioned, low affinity sites exchange rapidly on PCNA exploiting the same binding sites. Increasing or decreasing the binding affinity between Spd1 and PCNA through mutations of either motif compromised the ability of Spd1 to cause checkpoint activation in yeast. These results pinpoint a role for PCNA in Spd1-mediated checkpoint activation and suggest that its tandemly positioned short linear motifs create a neatly balanced competition-based system, involving PCNA, Spd1 and the small ribonucleotide reductase subunit, Suc22R2. Similar mechanisms may be relevant in other PCNA binding ligands where divergent binding motifs so far have gone under the PIP-box radar.

Abstract B024: The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Abstract The replication stress (RS) response protein ataxia telangiectasia and Rad3-related protein (ATR) and its main downstream factor, checkpoint kinase 1 (CHK1), play important roles in cell survival under RS. The inhibitors targeting CHK1 (CHK1i’s) have been shown to be a powerful strategy for treating solid tumors and hematological malignancies in preclinical studies. However, in most clinical trials, including those for treating non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer cases, CHKi’s have failed to achieve their primary endpoints and have shown cumulative tissue toxicities in normal tissues. These findings significantly limit the clinical benefit of these agents. Thus, identifying novel combinational strategies that can enhance the sensitivity of tumor cells to CHKi’s, while limiting their toxicities, might be the key to improve the safety and efficacy of these compounds. To explore novel combinational strategies that can overcome these limitations, we performed an unbiased high-throughput screen in a NSCLC cell line and identified thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a novel determinant of CHK1i sensitivity. We established 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 RS. In addition, we also demonstrate that CHK1-mediated E2F1-RRM2 induction was triggered during Trx1 or TrxR1 depletion-induced RS. CHK1 inhibition abrogates the Trx1 or TrxR1 depletion induced RRM2 expression and leads to the more profound dNTP pool scarcity. Further, the TrxR1 inhibitor auronafin, an anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. In summary, our study reveals an underappreciated mechanism responsible for the Trx or TrxR1 inhibition-induced RS. We propose that increased RRM1 oxidation following Trx system inhibition and the abrogation of RRM2 pathway by CHKi contributes to the synergistic interaction by the severe loss of RNR function. Our findings identify a new pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity. Citation Format: Chandra Bhushan Prasad, Adrian Oo, Zhaojun Qiu, Na Li, Deepika Singh, Xiwen Xin, Young-Jae Cho, Zaibo Li, Xiaoli Zhang, Chunhong Yan, Qingfei Zheng, Jimin Shao, Qi-En Wang, Baek Kim, Junran Zhang. The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B024.

A comprehensive analysis of ribonucleotide reductase subunit M2 for carcinogenesis in pan-cancer.

Although there is evidence that ribonucleotide reductase subunit M2 (RRM2) is associated with numerous cancers, pan-cancer analysis has seldom been conducted. This study aimed to explore the potential carcinogenesis of RRM2 in pan-cancer using datasets from The Cancer Genome Atlas (TCGA). Data from the UCSC Xena database were analyzed to investigate the differential expression of RRM2 across multiple cancer types. Clinical data such as age, race, sex, tumor stage, and status were acquired to analyze the influence of RRM2 on the clinical characteristics of the patients. The role of RRM2 in the onset and progression of multiple cancers has been examined in terms of genetic changes at the molecular level, including tumor mutational burden (TMB), microsatellite instability (MSI), biological pathway changes, and the immune microenvironment. RRM2 was highly expressed in most cancers, and there was an obvious correlation between RRM2 expression and patient prognosis. RRM2 expression is associated with the infiltration of diverse immune and endothelial cells, immune checkpoints, tumor mutational burden (TMB), and microsatellite instability (MSI). Moreover, the cell cycle is involved in the functional mechanisms of RRM2. Our pan-cancer study provides a comprehensive understanding of the carcinogenesis of RRM2 in various tumors.

Targeting of RRM2 suppresses DNA damage response and activates apoptosis in atypical teratoid rhabdoid tumor

BackgroundAtypical teratoid rhabdoid tumors (ATRT) is a rare but aggressive malignancy in the central nervous system, predominantly occurring in early childhood. Despite aggressive treatment, the prognosis of ATRT patients remains poor. RRM2, a subunit of ribonucleotide reductase, has been reported as a biomarker for aggressiveness and poor prognostic conditions in several cancers. However, little is known about the role of RRM2 in ATRT. Uncovering the role of RRM2 in ATRT will further promote the development of feasible strategies and effective drugs to treat ATRT.MethodsExpression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used shRNA knockdown RRM2 in three different ATRT cells to elucidate the oncogenic role of RRM2. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro and in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT.ResultsRRM2 was found to be significantly overexpressed in multiple independent ATRT clinical cohorts through comprehensive bioinformatics and clinical data analysis in this study. The expression level of RRM2 was strongly correlated with poor survival rates in patients. In addition, we employed shRNAs to silence RRM2, which led to significantly decrease in ATRT colony formation, cell proliferation, and migration. In vitro experiments showed that treatment with COH29 resulted in similar but more pronounced inhibitory effect. Therefore, ATRT orthotopic mouse model was utilized to validate this finding, and COH29 treatment showed significant tumor growth suppression and prolong overall survival. Moreover, we provide evidence that COH29 treatment led to genomic instability, suppressed homologous recombinant DNA damage repair, and subsequently induced ATRT cell death through apoptosis in ATRT cells.ConclusionsCollectively, our study uncovers the oncogenic functions of RRM2 in ATRT cell lines, and highlights the therapeutic potential of targeting RRM2 in ATRT. The promising effect of COH29 on ATRT suggests its potential suitability for clinical trials as a novel therapeutic approach for ATRT.

Theoretical Insights into the Generation Mechanism of the Tyr122 Radical Catalyzed by Intermediate X in Class Ia Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides in all organisms. There is an ∼35 Å long-range electron-hole transfer pathway during the catalytic process of class Ia RNR, which can be described as Tyr122β ↔ [Trp48β]? ↔ Tyr356β ↔ Tyr731α ↔ Tyr730α ↔ Cys439α. The formation of the Y122• radical initiates this long-range radical transfer process. However, the generation mechanism of Y122• is not yet clear due to confusion over the intermediate X structures. Based on the two reported X structures, we examined the possible mechanisms of Y122• generation by density functional theory (DFT) calculations. Our examinations revealed that the generation of the Y122• radical from the two different core structures of X was via a similar two-step reaction, with the first step of proton transfer for the formation of the proton receptor of Y122 and the second step of a proton-coupled long-range electron transfer reaction with the proton transfer from the Y122 hydroxyl group to the terminal hydroxide ligand of Fe1III and simultaneously electron transfer from the side chain of Y122 to Fe2IV. These findings provide an insight into the formation mechanism of Y122• catalyzed by the double-iron center of the β subunit of class Ia RNR.

The key cellular senescence related molecule RRM2 regulates prostate cancer progression and resistance to docetaxel treatment

BackgroundProstate cancer is a leading cause of cancer-related deaths among men worldwide. Docetaxel chemotherapy has proven effective in improving overall survival in patients with castration-resistant prostate cancer (CRPC), but drug resistance remains a considerable clinical challenge.MethodsWe explored the role of Ribonucleotide reductase subunit M2 (RRM2), a gene associated with senescence, in the sensitivity of prostate cancer to docetaxel. We evaluated the RRM2 expression, docetaxel resistance, and ANXA1 expression in prostate cancer cell lines and tumour xenografts models. In addition, We assessed the impact of RRM2 knockdown, ANXA1 over-expression, and PI3K/AKT pathway inhibition on the sensitivity of prostate cancer cells to docetaxel. Furthermore, we assessed the sensitivity of prostate cancer cells to the combination treatment of COH29 and docetaxel.ResultsOur results demonstrated a positive association between RRM2 expression and docetaxel resistance in prostate cancer cell lines and tumor xenograft models. Knockdown of RRM2 increased the sensitivity of prostate cancer cells to docetaxel, suggesting its role in mediating resistance. Furthermore, we observed that RRM2 stabilizes the expression of ANXA1, which in turn activates the PI3K/AKT pathway and contributes to docetaxel resistance. Importantly, we found that the combination treatment of COH29 and docetaxel resulted in a synergistic effect, further augmenting the sensitivity of prostate cancer cells to docetaxel.ConclusionOur findings suggest that RRM2 regulates docetaxel resistance in prostate cancer by stabilizing ANXA1-mediated activation of the PI3K/AKT pathway. Targeting RRM2 or ANXA1 may offer a promising therapeutic strategy to overcome docetaxel resistance in prostate cancer.Graphical

Impact of N221S missense mutation in human ribonucleotide reductase small subunit b on mitochondrial DNA depletion syndrome

The impact of N221S mutation in hRRM2B gene, which encodes the small subunit of human ribonucleotide reductase (RNR), on RNR activity and the pathogenesis of mitochondrial DNA depletion syndrome (MDDS) was investigated. Our results demonstrate that N221 mutations significantly reduce RNR activity, suggesting its role in the development of MDDS. We proposed an allosteric regulation pathway involving a chain of three phenylalanine residues on the αE helix of RNR small subunit β. This pathway connects the C-terminal loop of β2, transfers the activation signal from the large catalytic subunit α to β active site, and controls access of oxygen for radical generation. N221 is near this pathway and likely plays a role in regulating RNR activity. Mutagenesis studies on residues involved in the phenylalanine chain and the regulation pathway were conducted to confirm our proposed mechanism. We also performed molecular dynamic simulation and protein contact network analysis to support our findings. This study sheds new light on RNR small subunit regulation and provides insight on the pathogenesis of MDDS.

Atypical genotoxicity of carcinogenic nickel(II): Linkage to dNTP biosynthesis, DNA-incorporated rNMPs, and impaired repair of TOP1-DNA crosslinks

Cancer is a genetic disease requiring multiple mutations for its development. However, many carcinogens are DNA-unreactive and nonmutagenic and consequently described as nongenotoxic. One of such carcinogens is nickel, a global environmental pollutant abundantly emitted by burning of coal. We investigated activation of DNA damage responses by Ni and identified this metal as a replication stressor. Genotoxic stress markers indicated the accumulation of ssDNA and stalled replication forks, and Ni-treated cells were dependent on ATR for suppression of DNA damage and long-term survival. Replication stress by Ni resulted from destabilization of RRM1 and RRM2 subunits of ribonucleotide reductase and the resulting deficiency in dNTPs. Ni also increased DNA incorporation of rNMPs (detected by a specific fluorescent assay) and strongly enhanced their genotoxicity as a result of repressed repair of TOP1-DNA protein crosslinks (TOP1-DPC). The DPC-trap assay found severely impaired SUMOylation and K48-polyubiquitination of DNA-crosslinked TOP1 due to downregulation of specific enzymes. Our findings identified Ni as the human carcinogen inducing genome instability via DNA-embedded ribonucleotides and accumulation of TOP1-DPC which are carcinogenic abnormalities with poor detectability by the standard mutagenicity tests. The discovered mechanisms for Ni could also play a role in genotoxicity of other protein-reactive carcinogens.

Prognostic significance and immune cell infiltration analysis of differentially expressed genes in malignant pleural mesothelioma

Objective: To explore and analyze differential expressed genes in malignant pleural mesothelioma (MPM) by bioinformatics method, and to study their prognostic value in MPM and their potential role in immunotherapy. Methods: In January 2022, the dataset GSE51024 was downloaded from the GEO database, and MPM (55 cases) and normal tissue (41 cases) samples were obtained. Using R software and HMDD and miRNet database, MPM-related differential genes were screened and co-expressed genes were identified. Co-expressed genes were enriched and functionally annotated, and protein-protein interaction (PPI) networks were constructed and key genes were identified using the STRING database and Cytoscape software. TRRUST and GEPIA databases were used to predict transcription factors of key genes and to analyze prognosis and survival. The correlation between key genes and the degree of infiltration of immune cells was analyzed using TIMER. Results: A total of 435 co-expressed genes were obtained, which were mainly concentrated in the extracellular matrix tissue and the signaling pathways of cell adhesion molecules. Combined with PPI and TRRUST database, seven key MPM prognostic genes were identified. Among them, cyclin 20 (CDC20) , cell cycle checkpoint kinase 1 (CHEK1) , enhancer of Zeste homolog 2 (EZH2) , ribonucleotide reductase subunit M2 (RRM2) , topoisomerase 2A (TOP2A) , ubiquitin like plant homeodomain and ring finger domain 1 (UHRF1) were significantly up-regulated in MPM, while cyclin A1 (CCNA1) was significantly down-regulated. The expressions of CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 genes were significantly associated with MPM overall survival (P<0.05) . The expressions of CDC20, CHEK1, EZH2, RRM2 and TOP2A genes were positively correlated with B cells and dendritic cells (P<0.05) , and negatively correlated with neutrophils (P<0.05) . Conclusion: CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 may be potential prognostic markers in MPM patients, and their expressions may be related to MPM tumor immunity.

APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels.

Precision genome editing has become a reality with the discovery of base editors. Cytosine base editor (CBE) technologies are improving rapidly but are mostly optimized for TC dinucleotide targets. Here, we report the development and implementation of APOBEC Reporter Systems for Evaluating diNucleotide Editing Levels (ARSENEL) in living cells. The ARSENEL panel is comprised of four constructs that quantitatively report editing of each of the four dinucleotide motifs (AC/CC/GC/TC) through real-time accumulation of eGFP fluorescence. Editing rates of APOBEC3Bctd and AIDΔC CBEs reflect established mechanistic preferences with intrinsic biases to TC and GC, respectively. Twelve different (new and established) base editors are tested here using this system with a full-length APOBEC3B CBE showing the greatest on-target TC specificity and an APOBEC3A construct showing the highest editing efficiency. In addition, ARSENEL enables real-time assessment of natural and synthetic APOBEC inhibitors with the most potent to-date being the large subunit of the Epstein-Barr virus ribonucleotide reductase. These reporters have the potential to play important roles in research and development as precision genome engineering technologies progress toward achieving maximal specificity and efficiency.

PAK4 inhibition significantly potentiates Gemcitabine activity in PDAC cells via inhibition of Wnt/β-catenin, p-ERK/MAPK and p-AKT/PI3K pathways

Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most difficult to treat cancers. Gemcitabine is still the standard of care treatment for PDAC but with modest survival benefit and well reported resistance. Here we explored potential of inhibiting p21 activated kinase 4 (PAK4), a downstream protein of KRAS oncogenic pathway, in combination with Gemcitabine in PDAC cells. PAK4 inhibition by KPT-9274 led to significant potentiation of Gemcitabine activity in PDAC cells, with an increase in apoptosis, DNA damage and cell cycle arrest. At molecular level, PAK4 inhibition dose dependently inhibited Gemcitabine-induced β-catenin, c-JUN and Ribonucleotide Reductase subunit 2 (RRM2) levels. PAK4 inhibition further inhibited levels of phosphorylated ERK (p-ERK); Gemcitabine-induced phosphorylated AKT (p-AKT), phosphorylated and total c-Myc. These results suggest possible role of β-catenin, p-ERK and p-AKT, key effector proteins of Wnt/β-catenin, MAPK and PI3K pathways respectively, in sensitisation of Gemcitabine activity with PAK4 inhibition. Our data unravel probable molecular mechanisms behind combination of PAK4 inhibition with Gemcitabine to counter PDAC, which may be unequivocally proved further with knock down of PAK4. Our findings provide a strong rationale to exploit the combination therapy of Gemcitabine and PAK4 inhibitor for PDAC at pre-clinical and clinical levels.

Targeting CK2-mediated phosphorylation of p53R2 sensitizes BRCA-proficient cancer cells to PARP inhibitors.

Poly[ADP-ribose] polymerase (PARP) inhibitors, which selectively kills homologous recombination (HR) repair-deficient cancer cells, are widely employed to treat cancer patients harboring BRCA1/2 mutations. However, they display limited efficacy in tumors with wild-type (WT) BRCA1/2. Thus, it is crucial to identify new druggable HR repair regulators and improve the therapeutic efficacy of PARP inhibitors via combination therapies in BRCA1/2-WT tumors. Here, we show that the depletion of ribonucleotide reductase (RNR) subunit p53R2 impairs HR repair and sensitizes BRCA1/2-WT cancer cells to PARP inhibition. We further demonstrate that the loss of p53R2 leads to a decrease of HR repair factor CtIP, as a result of dNTPs shortage-induced ubiquitination of CtIP. Moreover, we identify that casein kinase II (CK2) phosphorylates p53R2 at its ser20, which subsequently activates RNR for dNTPs production. Therefore, pharmacologic inhibition of the CK2-mediated phosphorylation of p53R2 compromises its HR repair capacity in BRCA1/2-WT cancer cells, which renders these cells susceptible to PARP inhibition in vitro and in vivo. Therefore, our study reveals a novel strategy to inhibit HR repair activity and convert BRCA1/2-proficient cancers to be susceptible to PARP inhibitors via synthetic lethal combination.

Analysis of the Arabidopsis venosa4‐0 mutant supports the role of VENOSA4 in dNTP metabolism

Human Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) functions as a dNTPase to maintain dNTP pool balance. In eukaryotes, the limiting step in de novo dNTP biosynthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR). In Arabidopsis, the RNR1 subunit of RNR is encoded by CRINKLED LEAVES 8 (CLS8), and RNR2 by three paralogous genes, including TSO MEANING 'UGLY' IN CHINESE 2 (TSO2). In plants, DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1 (DOV1) catalyzes the first step of the de novo biosynthesis of purines. Here, to explore the role of VENOSA4 (VEN4), the most likely Arabidopsis ortholog of human SAMHD1, we studied the ven4‐0 point mutation, whose leaf phenotype was stronger than those of its insertional alleles. Structural predictions suggested that the E249L substitution in the mutated VEN4-0 protein rigidifies its 3D structure. The morphological phenotypes of the ven4, cls8, and dov1 single mutants were similar, and those of the ven4 tso2 and ven4 dov1 double mutants were synergistic. The ven4‐0 mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in the de novo purine biosynthesis. Our results reveal high functional conservation between VEN4 and SAMHD1 in dNTP metabolism.

Inhibition of RRM2 radiosensitizes glioblastoma and uncovers synthetic lethality in combination with targeting CHK1

Glioblastoma (GBM) is an aggressive malignant primary brain tumor. Radioresistance largely contributes to poor clinical outcomes in GBM patients. We targeted ribonucleotide reductase subunit 2 (RRM2) with triapine to radiosensitize GBM. We found RRM2 is associated with increasing tumor grade, is overexpressed in GBM over lower grade gliomas and normal tissue, and is associated with worse survival. We found silencing or inhibition of RRM2 by siRNA or triapine sensitized GBM cells to ionizing radiation (IR) and delayed resolution of IR-induced γ-H2AX nuclear foci. In vivo, triapine and IR reduced tumor growth and increased mouse survival. Intriguingly, triapine led to RRM2 upregulation and CHK1 activation, suggesting a CHK1-dependent RRM2 upregulation following RRM2 inhibition. Consistently, silencing or inhibition of CHK1 with rabusertib abolished the triapine-induced RRM2 upregulation. Accordingly, combining rabusertib and triapine resulted in synthetic lethality in GBM cells. Collectively, our results suggest RRM2 is a promising therapeutic target for GBM, and targeting RRM2 with triapine sensitizes GBM cells to radiation and independently induces synthetic lethality of GBM cells with CHK1 inhibition. Our findings suggest combining triapine with radiation or rabusertib may improve therapeutic outcomes in GBM.

Effect of siRNA-mediated silencing of p53R2 gene on sensitivity of T-ALL cellsto Daunorubicin

Introductionp53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. MethodsTransfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. ResultsWe found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. ConclusionThe results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL.

Ribonucleotide reductase subunit M1 blocks glucose catabolism via phosphorylation of pyruvate kinase M2 in human esophageal squamous cell cancer

Ribonucleotide reductase subunit M1 blocks glucose catabolism via phosphorylation of pyruvate kinase M2 in human esophageal squamous cell cancer