Enzyme In DNA Synthesis Research Articles

An insight into thymidylate synthase inhibitor as anticancer agents: an explicative review.

Cancer, a widespread challenge to global health, remains a puzzle of intricate molecular dynamics. This review article delves into the mystery of cancer, with a keen focus on understanding the contributory role of thymidylate synthase (TS) in cancer. TS, a vital enzyme in DNA synthesis and repair, emerges as a significant player in the narrative of cancer development. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is a major step in producing DNA. Numerous malignancies, including those of the breast, colon, lung, and ovary, have been linked to dysregulation of TS activity. Overexpression or mutations of TS lead to uncontrolled cell proliferation and tumorigenesis molecular interactions and signalling pathways involving TS come under scrutiny, revealing the nuanced connections that propel its involvement in cancer progression. Beyond overexpression and mutations, there emerges a subtle layer of regulation that involves microRNAs (miRNAs). These tiny particles attach to the TS messenger RNA, causing translational repression or its degradation, which in turn affects TS activity. Moving towards the therapeutic realm, thymidylate synthase inhibition acts as a promising anti-cancer strategy. Targeting TS with small-molecule inhibitors could provide a novel approach to treat various cancers. By reducing the number of available nucleotides, TS inhibition would slow down or halt cancer cell division, thus depriving the tumor of the building blocks required for its proliferation and growth. The aim is to assess the viability and effectiveness of targeting TS to halt or slow down cancer progression. There is growing evidence that, in comparison to traditional TS inhibitors, few novel antifolate TS inhibitors are effective against a wider variety of neoplasms, such as lung carcinomas. It has been discovered that TS inhibitors increase cancer tissues' sensitivity to chemotherapy and radiation, increasing their vulnerability to these treatments. This article aims to provide a comprehensive insight into TS, examining its cellular details, detailing the heterocyclic moieties and molecular foundations, and providing a promising future outlook.

An In-Silico Investigation on the Molecular Interactions between Ellagic Acid and PfDHFR-TS

Ellagic acid (EA) is an antiplasmodial polyphenol with reported in-vitro activity against Plasmodium falciparum. Studies have reported that EA acts in the late erythrocytic stages of P. falciparum (Pf) when DNA synthesis is taking place. Pf dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important enzyme for DNA synthesis as its inhibition can kill the parasite. As there is no reported study on the molecular interactions between EA and PfDHFR-TS, we aim to study the molecular interactions between EA and PfDHFR-TS (PDB ID: 3DGA) through molecular docking, molecular dynamics (MD) simulations, binding free energy (MM-GBSA) calculations, and density functional theory (DFT) studies. Site-specific and blind docking revealed that EA has a high binding affinity for the active site of PfDHFR-TS. EA formed hydrogen bonds with multiple active site residues. MD simulations for 100 ns revealed that the PfDHFR-TS-EA complex was stable. The average binding free energy of the PfDHFR-TS-EA complex throughout the 100 ns MD simulations was −39.84 kcal/mol. The energy difference (ΔE = 0.04089 eV) obtained from DFT studies indicates the electrical stability and reactivity of EA at the active site of PfDHFR-TS. We conclude that the antiplasmodial activity of EA might be attributed to its ability to potentially bind with PfDHFR-TS.

C11orf54 promotes DNA repair via blocking CMA-mediated degradation of HIF1A

C11orf54 is an ester hydrolase highly conserved across different species. C11orf54 has been identified as a biomarker protein of renal cancers, but its exact function remains poorly understood. Here we demonstrate that C11orf54 knockdown decreases cell proliferation and enhances cisplatin-induced DNA damage and apoptosis. On the one hand, loss of C11orf54 reduces Rad51 expression and nuclear accumulation, which results in suppression of homologous recombination repair. On the other hand, C11orf54 and HIF1A competitively interact with HSC70, knockdown of C11orf54 promotes HSC70 binding to HIF1A to target it for degradation via chaperone-mediated autophagy (CMA). C11orf54 knockdown-mediated HIF1A degradation reduces the transcription of ribonucleotide reductase regulatory subunit M2 (RRM2), which is a rate-limiting RNR enzyme for DNA synthesis and DNA repair by producing dNTPs. Supplement of dNTPs can partially rescue C11orf54 knockdown-mediated DNA damage and cell death. Furthermore, we find that Bafilomycin A1, an inhibitor of both macroautophagy and chaperone-mediated autophagy, shows similar rescue effects as dNTP treatment. In summary, we uncover a role of C11orf54 in regulating DNA damage and repair through CMA-mediated decreasing of HIF1A/RRM2 axis.

Comparing the prognostic and predictive utility of serum thymidine kinase 1 and CA 15-3 in patients with hormone receptor positive metastatic breast cancer starting first-line endocrine therapy in SWOG S0226.

1076 Background: Serum levels of thymidine kinase 1 activity (TKa), a fundamental enzyme in DNA synthesis and cellular proliferation, are prognostic of benefit from endocrine therapy (ET) in patients (pts) with hormone receptor positive (HR+) metastatic breast cancer (MBC) who participated in SWOG S0226, a prospective randomized trial comparing anastrozole vs. anastrozole and fulvestrant given in 4 week cycles. The assay for TKa (DiviTum TKa) was FDA approved in July 2022 for monitoring of postmenopausal HR+ MBC pts. The assay for circulating MUC1, CA 15-3, is routinely utilized in monitoring of pts with MBC. We compared the prognostic and predictive utilities of CA 15-3 and TKa in pts from S0226. Methods: TKa was measured in 1725 sera from 432 pts while CA 15-3 was measured in 1298 sera from 326 pts at baseline (BL), cycles 2, 3, 4 and 7. Prespecified cutoff levels of ≥ 250 DuA and ≥ 30 U/mL were considered high for TKa and CA 15-3 respectively. Progression-free survival (PFS) and overall survival (OS) were analyzed by Kaplan-Meier and Cox regression stratified by prior adjuvant tamoxifen use. To examine markers over time, a landmarked Cox regression analysis was done starting at initiation of Cycle 4. BL and recent (Cycle 3 if available, otherwise Cycle 2) markers were assessed for simultaneous prediction of subsequent PFS and OS. Results: BL TKa was elevated in 190/432 (44%) and CA 15-3 was elevated in 254/326 (78%). Agreement between assays was 53%. Pts with high versus low BL TKa had significantly worse PFS (median 11.6 vs. 17.2 months, HR = 1.68, 95% confidence interval (CI) 1.37-2.06) and OS (median 34 vs. 58 months, HR = 2.16, 95% CI 1.73-2.70) whereas pts with high versus normal BL CA 15-3 had no significant difference in PFS (median 13.6 vs. 16.1 months, HR = 1.23, 95% CI 0.93-1.62) but worse OS (median 45 vs. 66 months, HR = 1.82, 95% CI 1.30-2.53). A multivariable Cox model with both markers shows only TKa as being prognostic for PFS (TKa HR = 1.61, 95% CI 1.28-2.03; CA 15-3 HR = 1.21, 95% CI 0.91-1.59), but both prognostic for OS (TKa HR = 2.09, 95% CI 1.61-2.71; CA 15-3 HR = 1.70, 95% CI 1.22-2.38). In the landmarked multivariable analysis, positive TKa at BL was a strong predictor of PFS (HR = 1.65, 95% CI 1.28-2.14), but recent TKa was not (HR = 1.25, 95% CI 0.93-1.68). In contrast, positive CA15-3 at BL was not a predictor of PFS (HR = 0.73, 95% CI 0.46-1.17), but recent CA15-3 was (HR = 1.97, 95% CI 1.26-3.06). Conclusions: BL TKa is highly prognostic in pts with HR+ MBC initiating first-line systemic ET, with low BL TKa conferring superior prognosis. In contrast, CA 15-3 is only modestly prognostic at BL, but is prognostic after 3 cycles of treatment. These hypothesis-generating data suggest further study is needed to determine how these biomarkers should be employed in a complementary manner to monitor response to systemic therapy in HR+ MBC. Clinical trial information: NCT00075764 .

Abstract 5989: Targeting RRM2 & cell cycle for breast cancer treatment

Abstract Endocrine treatment is an effective first-line therapy for targeting ER positive, HER2 negative breast cancer. But success is limited by development of acquired resistance due to long-term therapy. Cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led the development of the first CDK4/6 inhibitor palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Owing to intrinsic and acquired drug resistance development, success is limited despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. Combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple negative breast cancer. Previously, we have shown that didox (DDX) can significantly halt malignant breast cancer cell division in combination with the anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. Recently, we confirmed that didox in combination with palbociclib significantly lowers ER positive and ER negative breast cancer and their palbociclib resistant counterparts’ growth compared to no treatment or palbociclib treatment. We also confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells IC50 of palbociclib drug is lower than their palbociclib resistant counterparts. Here, we are reporting that didox decreases cell cycle proteins alone or in combination with palbociclib in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment modulates cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data presents a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2 and cell cycle that merits further clinical investigation in animal and human models. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 & cell cycle for breast cancer treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5989.

Abstract P1-13-01: Targeting RRM2 for the treatment of palbociclib resistant breast cancer

Abstract Hormone receptor (HR) positive breast cancer (BC) is a prevalent disease accounting for almost 2 million new cases globally. Almost 70-80% of breast cancer patients are women with a positive score for the estrogen receptor (ER). HR positive BCs of all stages are selectively treated with endocrine therapy targeting ER activity. Intrinsic and acquired resistance are common phenomena, impacting patient outcomes negatively. Palbociclib is an FDA approved checkpoint inhibitor for the treatment of HR+/HER2- breast cancer in combination with aromatase inhibitors or selective estrogen receptor degraders. However, patients responding well to the therapy in the beginning, lose sensitivity to palbociclib with time. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. In our previous study, we have shown that RRM2 is upregulated in ER positive tamoxifen resistant BC. Previously, we have also reported that didox (DDX) which is a unique RRM2 enzyme inhibitor can significantly halt malignant breast cancer cell division in combination with an anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. In this study, we target palbociclib resistant BC with DDX to circumvent palbociclib resistance. For this purpose, we have developed palbociclib resistant ER positive MCF7 and ER negative MDA-MB-468 breast cancer cell lines. Here, we report morphological changes in parental as compared to palbociclib resistant cells after treatment with palbociclib alone, DDX alone or with palbociclib as compared to non-treated cells. Cell proliferation studies confirm the synergistic combinatorial effect of palbociclib and DDX compared to palbociclib or DDX alone. We also report that DDX alone or in combination with palbociclib downregulates cell cycle and NFkB proteins in palbociclib resistant ER positive MCF7 and ER negative MBA-MB-468 breast cancer cell lines. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 for the treatment of palbociclib resistant breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-01.

Biochemistry-Not Oncogenes-May Demystify and Defeat Cancer.

The presence of mutated genes strongly correlates with the incidence of cancer. Decades of research, however, has not yielded any specific causative gene or set of genes for the vast majority of cancers. The Cancer Genome Atlas program was supposed to provide clarity, but it only gave much more data without any accompanying insight into how the disease begins and progresses. It may be time to notice that epidemiological studies consistently show that the environment, not genes, has the principal role in causing cancer. Since carcinogenic chemicals in our food, drink, air, and water are the primary culprits, we need to look at the biochemistry of cancer, with a focus on enzymes that invariably facilitate transformations in a cell. In particular, attention should be paid to the rate-limiting enzyme in DNA synthesis, ribonucleotide reductase (RnR), whose activity is tightly linked to tumor growth. Besides circumstantial evidence that cancer is induced at this enzyme's vulnerable free-radical-containing active site by various carcinogens, its role in initiating retinoblastoma and human papillomavirus (HPV)-related cervical cancers has been well documented in recent years. Blocking the activity of malignant RnR is a certain way to arrest cancer.

Inhibition of Ribonucleotide Reductase Subunit 2 (RRM2) Induces Radiosensitization in Gastroenteropancreatic Neuroendocrine Tumors

<h3>Purpose/Objective(s)</h3> Novel radiotherapy, such as Peptide Radionuclide Receptor Therapy, is becoming a milestone treatment for advanced Gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Ribonucleotide Reductase (RnR) subunit M2 (RRM2) activates RnR, the rate-limiting enzyme of DNA synthesis and repair. The purpose of this study was to determine the therapeutic rationale of RnR-targeted therapy and to evaluate 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) as a radiosensitizer in treating GEP-NETs <i>in vitro</i> and <i>in vivo.</i> <h3>Materials/Methods</h3> (1) RRM2 inhibition was achieved by siRNA knockdown or a RRM2 specific inhibitor, 3-AP, in three GEP-NET cell lines (QGP-1, BON and NT-3) and patient-derived spheroids. Radiation (XRT) was delivered via an X-ray irradiator. Immunohistochemistry confirmed RRM2 expression in cell lines and spheroids, and western immunoblotting was used to examine phosphorylation of DNA repair pathways (DNA-PK, ATM, ATR), cell cycle arrest markers (Rb, Chk1, Chk2) and apoptotic protein (cleaved PARP). Proliferation assays and cell cycle analyses were used to evaluate cytostatic cellular response. Colorimetric survival and DNA fragmentation assays were used to investigate radiosensitization of RRM2 inhibition. (2) For <i>in vivo</i> studies<i>,</i> we utilized an athymic nude mouse GEP-NET xenograft model to assess tumor growth using a combination of 3-AP and XRT. <h3>Results</h3> (1) RRM2 receptor expression, predominantly in the cytoplasm, was noted in all GEP-NET cell lines and spheroids. Knockdown of RRM2 significantly inhibited cellular proliferation at 48 and 72h. Similarly, 3-AP inhibited cellular proliferation with an absolute IC-50 of 1240nM and 1297nM for QGP-1 and BON cells, respectively. Induction of ATM and DNA-PK phosphorylation, but not ATR, was detected with RRM2 inhibition. Importantly, siRNA RRM2 knockdown and 3-AP increased expression of cell cycle arrest markers (pRb, pChk1, and pChk2), and 3-AP (2500nM) arrested cells before entering the G2/M phase, significantly reducing the percentage of G2/M cells from 8.4% to 0.5% at 72h. Combination of RRM2 inhibition with XRT increased apoptosis as noted by increased expression of cleaved PARP. RRM2 inhibition by 3-AP potentiated XRT by reducing 14d cellular survival and inducing DNA fragmentation at 96h post-XRT. (2) We observed a significant decrease in tumor size in mice treated with XRT (2Gy) alone compared to control (146 mm² vs. 280 mm², respectively); combination treatment (2Gy XRT, followed by immediate 3-AP [10mg/kg] i.p. injection) demonstrated a further 48% decrease in tumor size compared to XRT alone. <h3>Conclusion</h3> Our studies demonstrate that inhibition of RRM2 potentiates XRT and results in a significant increase in apoptosis <i>in vitro</i> and a decrease in tumor size <i>in vivo</i>, suggesting enhanced cytotoxicity and radiosensitization. Selective RRM2 inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs.

RRM2 regulates osteogenesis of mouse embryo fibroblasts via the Wnt/β‑catenin signaling pathway.

Osteoporosis is a widespread bone metabolic disease characterized by reduced bone mass and bone microstructure deterioration. Ribonucleotide reductase M2 (RRM2) is a key enzyme in DNA synthesis and repair. The present study investigated the effect of RRM2 on osteogenesis of mouse embryo fibroblasts (MEFs) and its molecular mechanism. Bioinformatics analysis revealed that RRM2 expression was increased during osteogenesis of MEFs triggered by bone morphogenetic protein 9. Subsequently, MEFs were used as a mesenchymal stem cell model and osteogenic inducing medium was used to induce osteogenic differentiation. RRM2 protein expression was measured by western blotting during osteogenic differentiation induction of MEFs. RRM2 levels in MEFs were upregulated and downregulated by RRM2-overexpressing recombinant adenovirus and small interfering RNA-RRM2, respectively. Bone formation markers (RUNX family transcription factor 2, osterix, distal-less homeobox 5, collagen type I α1 chain, osteopontin and osteocalcin) were detected by reverse transcription-quantitative (RT-q) PCR and alkaline phosphatase (ALP) and Alizarin Red S staining were examined. The protein expression levels of β-catenin and the ratio of phosphorylated (p-)GSK-3β to GSK-3β were detected by western blotting and the RNA expression of downstream related target genes (β-catenin, axis inhibition protein 2 (AXIN2), transcription factor 7 like 2, lymphoid enhancer binding factor 1, c-MYC and Cyclin D1) in the Wnt/β-catenin signaling pathway was measured by RT-qPCR. RRM2 protein expression increased as the osteogenic differentiation induction period was extended. RRM2 overexpression increased osteogenic marker RNA expression, ALP activity, bone mineralization, the protein expression levels of β-catenin, the ratio of p-GSK-3β to GSK-3β and the RNA expression of downstream related target genes in the Wnt/β-catenin signaling pathway, whereas RRM2 knockdown had the opposite effect. The findings of the present study revealed that RRM2 overexpression enhanced osteogenic differentiation, while RRM2 knockdown reduced osteogenic differentiation. RRM2 may regulate osteogenic differentiation of MEFs via the canonical Wnt/β-catenin signaling pathway, providing a possible therapeutic target for osteoporosis.

SvSTL1 in the large subunit family of ribonucleotide reductases plays a major role in chloroplast development of Setaria viridis.

Ribonucleotide reductases (RNRs) are essential enzymes in DNA synthesis. However, little is known about the RNRs in plants. Here, we identified a svstl1 mutant from the self-created ethyl methanesulfonate (EMS) mutant library of Setaria viridis. The mutant leaves exhibited a bleaching phenotype at the heading stage. Paraffin section analysis showed the destruction of the C4 Kranz anatomy. Transmission electron microscopy results further demonstrated the severely disturbed development of some chloroplasts. MutMap analysis revealed that the SvSTL1 gene is the primary candidate, encoding a large subunit of RNRs. Complementation experiments confirmed that SvSTL1 is responsible for the phenotype of svstl1. There are two additional RNR large subunit homologs in S. viridis, SvSTL2 and SvSTL3. To further understand the functions of these three RNR large subunit genes, a series of mutants were generated via CRISPR/Cas9 technology. In striking contrast to the finding that all three SvSTLs interact with the RNR small subunit, the phenotype varied along with the copies of chloroplast genome among different svstl single mutants: the svstl1 mutant exhibited pronounced chloroplast development and significantly fewer copies of the chloroplast genome than the svstl2 or svstl3 single mutants. These results suggested that SvSTL1 plays a major role in the optimal function of RNRs and is essential for chloroplast development. Furthermore, through the analysis of double and triple mutants, the study provides new insights into the finely tuned coordination among SvSTLs to maintain normal chloroplast development in the emerging C4 model plant S. viridis.

One-Step Enzymatic Labeling Reveals a Critical Role of O-GlcNAcylation in Cell-Cycle Progression and DNA Damage Response.

O-linked N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous post-translational modification of proteins that is essential for cell function. Perturbation of O-GlcNAcylation leads to altered cell-cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one-step enzymatic strategy for capture and profiling O-GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O-GlcNAcylation. FEN1 O-GlcNAcylation is dynamically regulated during the cell cycle. O-GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O-GlcNAcylated proteins, and reveals an unknown mechanism of O-GlcNAcylation in regulating cell cycle progression and DNA damage response.

One‐Step Enzymatic Labeling Reveals a Critical Role of O‐GlcNAcylation in Cell‐Cycle Progression and DNA Damage Response

AbstractO‐linked N‐acetylglucosamine (O‐GlcNAcylation) is a ubiquitous post‐translational modification of proteins that is essential for cell function. Perturbation of O‐GlcNAcylation leads to altered cell‐cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one‐step enzymatic strategy for capture and profiling O‐GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O‐GlcNAcylation. FEN1 O‐GlcNAcylation is dynamically regulated during the cell cycle. O‐GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O‐GlcNAcylated proteins, and reveals an unknown mechanism of O‐GlcNAcylation in regulating cell cycle progression and DNA damage response.

Deferasirox Induces Apoptosis in Mantle Cell Lymphoma in a Mechanism Involving Cyclin-D1, p53 and the Ribonucleotide Reductase

Mantle cell lymphoma (MCL) is a difficult to treat B-cell malignancy characterized by a rapid progressive course and poor prognosis. Despite advances in conventional and novel agents such as bortezomib, lenalidomide and ibrutinib, many MCL patients are still subject to rapid relapses of the disease, responsible for the relatively short overall survival. MCL is characterized by the t(11,14) translocation, resulting in cyclin-D1 (CD1) overexpression and dysregulation of the cell cycle. Recently, it has been demonstrated that depletion of CD1 results in cell cycle arrest, apoptosis and cell death of MCL cells thus labeling CD1 as a therapeutic target of this aggressive malignancy. Accumulating findings indicate that not only overexpressed CD1 but also deregulated signaling pathways such as PI3K/AKT/mTOR and Wnt are accountable for the tumorigenesis of MCL and for the maintenance of its proliferative and survival capacity. We thus believe that MCL treatment should target multiple deregulated signaling pathways and key molecules. Iron chelators have emerged as a novel strategy to treat tumors due to their capacity to target a multitude of key molecules, signaling pathways and cellular processes. Yet, the application of common iron chelators is limited due to their low bioavailability and high toxicities. Deferasirox (DFX), a clinically approved iron chelator applied for the treatment of iron overload, in contrast to the majority of iron chelators, is highly bioavailable and exhibits little to no side effects. To date, DFX is reported as being an orally effective antitumor agent against solid tumors by various mechanisms. In our project, we examined the in vitro impact of DFX on three MCL cell lines; Jeko-1, REC-1 and Z138; aiming thereby to resolve its molecular mode of action. DFX inhibited the growth of the MCL cell lines with 50% inhibitory concentrations (IC50) of 8.07±1.08µM, 1.8±1.03µM and 1.52±1.54µM for Jeko-1, REC-1 and Z138 cell lines, respectively. Moreover, we demonstrated that DFX treatment trigged a rapid and intense (~2-fold) production of reactive oxygen species (ROS), induced severe DNA damage and arrested cells at the G1 phase within 6 hrs. Ultimately, a 72h treatment of 10µM DFX led to apoptosis in 60% to 86% of the MCL cells. Most importantly, we found DFX to substantially reduce CD1 protein levels in a dose- and time-dependent manner. The depletion of CD1 levels was accompanied with its phosphorylation at Thr-286 (pCD1), a phosphorylation known to sentence CD1 to proteasomal degradation. In agreement with previous studies that have shown a correlation between ROS induction, CD1-phosphorylation, CD1 depletion and DNA damage, we found CD1 levels to negatively correlate with increasing DNA damage levels following DFX treatment.Interestingly, the efficacy of DFX treatment was dependent on the p53-status of the respective cell line. The wild-type p53 expressing lines, REC-1 and Z138, were found to be ~5-fold more sensitive to DFX than the p53 negative Jeko-1 cell line. In an attempt to understand this intriguing finding, we found DFX treatment to post-translationally elevate and stabilize p53 levels, most likely by depleting the intracellular heme pool which serves as a negative p53-regulator. Furthermore, we showed that DFX plays an important role in the attenuation of the iron dependent ribonucleotide reductase enzyme (RR); the rate limiting enzyme in dNTP biosynthesis and thus the key enzyme in DNA synthesis, cell cycle regulation and proliferation. DFX inhibited the enzymatic activity of RR with an IC50 of ~25 µM; which is ~20-fold more effective than other established inhibitors such as deferoxamine.In conclusion and pending further analysis, we demonstrated a potent impact of DFX on MCL cells. The vigorous anti-tumoral activity of DFX is most likely mediated via its ability to induce oxidative stress, to inhibit the RR enzyme and to eliminate the main oncogene in MCL pathogenesis; CD1. The depleting of intracellular iron/heme pools and the modulation of p53 only intensify the effect of DFX. The ability to inhibit the RR activity qualifies DFX as a sensitizer to existing DNA damaging radio- and chemotherapy options of MCL. DFX possesses the unique capacity to affect a multitude of cellular mechanisms in an auto-potentiating manner that propose this clinically established molecule as a potential therapeutic option for mantle cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Glutamate Mediates Proton-Coupled Electron Transfer Between Tyrosines 730 and 731 in Escherichia coli Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis for all living organisms. It reduces ribonucleotides to the corresponding deoxyribonucleotides by a reversible radical transfer mechanism. The active form of E. coli Ia RNR is composed of two subunits, α and β, which form an active asymmetric α2β2 complex. The radical transfer pathway involves a series of proton-coupled electron transfer (PCET) reactions spanning α and β over ∼32 Å. Herein, quantum mechanical/molecular mechanical free energy simulations of PCET between tyrosine residues Y730 and Y731 are performed on the recently solved cryo-EM structure of the active α2β2 complex, which includes a pre-turnover α/β pair with an ordered PCET pathway and a post-turnover α'/β' pair. The free energy surfaces in both the pre- and post-turnover states are computed. According to the simulations, forward radical transfer from Y731 to Y730 is thermodynamically favored in the pre-turnover state, and backward radical transfer is favored in the post-turnover state, consistent with the reversible mechanism. E623, a glutamate residue that is near these tyrosines only in the pre-turnover state, is discovered to play a key role in facilitating forward radical transfer by thermodynamically stabilizing the radical on Y730 through hydrogen-bonding and electrostatic interactions and lowering the free energy barrier via a proton relay mechanism. Introduction of fluorinated Y731 exhibits expected thermodynamic trends without altering the basic mechanism. These simulations suggest that E623 influences the directionality of PCET between Y731 and Y730 and predict that mutation of E623 will impact catalysis.

Abstract PO-014: 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors

Abstract Background: Peptide receptor radionuclide therapy (PRRT) is a novel radiotherapy approved for gastroenteropancreatic neuroendocrine tumors (GEP-NETs); however, PRRT has a relatively low treatment response rate of 20-30%. We hypothesize that addition of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a ribonucleotide reductase (RnR) inhibitor that inhibits the rate-limiting enzyme of DNA synthesis and repair, can improve radiotherapy outcomes. The purpose of this study is to determine the therapeutic rationale of RnR-targeted therapy and evaluate 3-AP as a radiosensitizer in treating GEP-NET in vitro and in vivo. Methods: We utilized two human GEP-NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) Cells were treated with 3-AP for 96h and cell proliferation assays were performed using a sulforhodamine B (SRB) kit. Next, cells were treated with 3-AP for 24, 48, 72, 96h and cell cycle analyses were performed with Beckman Coulter cell cycle kit and quantified by flow cytometry. Expression of cell cycle arrest markers (i.e., pCdk-1, cyclin D1) was assessed by immunoblotting. (ii) To determine radiosensitivity in vitro, cells were treated with 3-AP for 16h, followed by X-ray radiotherapy (XRT), and incubation in drug-free medium. Cell survival was assessed by colony formation assay and quantified by SRB kit at 14d post-XRT. Apoptosis was measured at 72 and 96h post-XRT, and expression of apoptotic proteins (i.e., cleaved PARP and cleaved CASPASE 3) was examined by immunoblotting. (iii) To determine radiosensitivity in vivo, we randomized 20 nude mice bearing QGP-1 xenografts into four groups: control; 3-AP (10mg/Kg); XRT (2 Gy); and combination (XRT immediately followed by 3-AP); each mouse received 5 consecutive treatments. Results: (i) A dose of 2500nM 3-AP arrested cells before entering G2/M phase, significantly reducing percentage of G2/M cells from 8.4% to 0.5% at 72h. Concurrently, we observed increased expression of checkpoint proteins (pRb, pCdk1, pCdk2, pCdc2) and decreased expression of cell cycle inhibitory proteins (cyclin D1, p21, p27 Kip). (ii) Exposure of QGP-1 and BON cells to 3-AP prior to XRT resulted in a synergistic decrease (i.e., 85% and 90%, respectively) in clonogenicity. Furthermore, the combination of 3-AP and XRT increased apoptosis 5-fold and cleaved PARP expression in BON cells. (iii) A significant decrease in tumor size was noted in mice with XRT alone compared to control (146mm2 vs. 280 mm2, respectively); combination treatment (3-AP + XRT) demonstrated a further 48% decrease in tumor size compared to XRT alone. There was no significant decrease in tumor size with 3-AP treatment alone. Conclusion: 3-AP treatment prior to XRT therapy resulted in significant increase in apoptosis in vitro and decrease in tumor size in vivo, suggesting synergistic cytotoxicity and radiosensitivity. Selective RNR inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs. Citation Format: Zeta Chow, Jeremy Johnson, Aman Chauhan, Tadahide Isumi, Lowell Anthony, Courtney M. Townsend, B. Mark Evers, Piotr Rychahou. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-014.

Reticulocyte hemoglobin content as a best indicator of iron deficiency in female patients with diffuse non-scarring hair loss

Iron deficiency is a well-known cause of diffuse nonscarring hair loss in women. One of the rapidly diving cells in the body is the hair follicle matrix cells and iron is a cofactor for the ribonucleotide reductase which is the ratelimiting enzyme for DNA synthesis and, also is a regulator for multiple genes in the hair follicles. Iron deficiency without anemia is far more prevalent and about two-folds higher than iron deficiency anemia and iron deficiency is more prevalent in women than men. Iron deficiency with or without anemia is more common in menstruating women, black race, athletes, vegetarians and obese or overweight

Impact of sickle cell disease on presentation and progression of paediatric HIV: a retrospective cohort study.

HIV and sickle cell disease (SCD) are significant causes of morbidity and mortality in sub-Saharan Africa. Given their separate roles in immune dysregulation, our objective was to characterise the impact that SCD has on the presentation and progression of paediatric HIV. The study was a retrospective cohort study (study period 2004-2018). Cases of HIV+and SCD-afflicted patients (HIV+/SCD+) were obtained via electronic chart review from a paediatric HIV clinic in Kampala, Uganda and matched 1:3 with HIV+controls without SCD (HIV+/SCD-). Thirty-five HIV+/SCD+subjects and 95 HIV+/SCD- controls were analysed (39% female (51/130), age 3.6years (SD3.9)). At baseline, WHO clinical stage (64% total cohort Stage III/IV) and nutritional status (9.4% severe acute malnutrition) were similar for both groups, whereas HIV+/SCD+had higher though non-significant baseline CD4 count (1036 (SD713) vs 849 (SD638) cells/microlitre, P=0.20, two-tailed t-test). There were 19 deaths, 6 (17%) HIV+/SCD+and 13 (14%) HIV+/SCD-, with unadjusted/adjusted models showing no significant difference. Nutritional progression and clinical stage progression showed no significant differences between groups. Kaplan-Meier analysis showed a slower rate of treatment failures in the HIV+/SCD+cohort (P=0.11, log-rank survival test). Trajectory analysis showed that in the time period analysed, the HIV+/SCD+cohort showed a more rapid rise and higher total CD4 count (P=0.012, regression analysis). The study suggests that SCD does not adversely affect the progression of HIV in patients on ART. Further, HIV+/SCD+achieved higher CD4 counts and fewer HIV treatment failures, suggesting physiological effects due to SCD might mitigate HIV progression.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

Modulation of iron homeostasis with hepcidin ameliorates spontaneous murine lupus nephritis

Lupus nephritis is the end organ manifestation of systemic lupus erythematosus. Iron metabolism and its master regulator, hepcidin, are known to regulate cell proliferation and inflammation, but their direct role in the pathophysiology of lupus nephritis remains under-investigated. Exogenous hepcidin reduced the severity of lupus nephritis in MRL/lpr mice, a preclinical model of spontaneous systemic lupus erythematosus without worsening anemia of inflammation. Hepcidin treatment reduced renal iron accumulation, systemic and intrarenal cytokines, and renal immune cell infiltration, independent of glomerular immune complex deposits and circulating autoantibodies. Hepcidin increased renal H-ferritin (a ferroxidase), reduced expression of free iron dependent DNA synthesis enzymes, Ribonucleotide Reductase 1 and 2, and intra-renal macrophage proliferation. These findings were recapitulated invitro upon treatment of macrophages with hepcidin and murine colony stimulation factor-1. Furthermore, hepcidin-treated macrophages secreted less IL-1β and IL-6 upon stimulation with the TLR3 agonist polyinosine-polycytidylic acid. Of clinical relevance, hepcidin reduced progression and severity of nephritis in old mice with established systemic autoimmunity and overt proteinuria, highlighting its therapeutic potential. Thus, our findings provide a proof-of-concept that targeting cellular iron metabolism with hepcidin represents a promising therapeutic strategy in lupus nephritis.

Integrative Analysis Reveals Across-Cancer Expression Patterns and Clinical Relevance of Ribonucleotide Reductase in Human Cancers.

Mining cancer-omics databases deepens our understanding of cancer biology and can lead to potential breakthroughs in cancer treatment. Here, we propose an integrative analytical approach to reveal across-cancer expression patterns and identify potential clinical impacts for genes of interest from five representative public databases. Using ribonucleotide reductase (RR), a key enzyme in DNA synthesis and cancer-therapeutic targeting, as an example, we characterized the mRNA expression profiles and inter-component associations of three RR subunit genes and assess their differing pathological and prognostic significance across over 30-types of cancers and their related subtypes. Findings were validated by immunohistochemistry with clinical tissue samples (n = 211) collected from multiple cancer centers in China and with clinical follow-up. Underlying mechanisms were further explored and discussed using co-expression gene network analyses. This framework represents a simple, efficient, accurate, and comprehensive approach for cancer-omics resource analysis and underlines the necessity to separate the tumors by their histological or pathological subtypes during the clinical evaluation of molecular biomarkers.