Immune and behavioral correlates of protection against symptomatic post-vaccination SARS-CoV-2 infection.

Introduction: We sought to determine pre-infection correlates of protection against SARS-CoV-2 post-vaccine inzfections (PVI) acquired during the first Omicron wave in the United States. Methods: Serum and saliva samples from 176 vaccinated adults were collected from October to December of 2021, immediately before the Omicron wave, and assessed for SARS-CoV-2 Spike-specific IgG and IgA binding antibodies (bAb). Sera were also assessed for bAb using commercial assays, and for neutralization activity against several SARS-CoV-2 variants. PVI duration and severity, as well as risk and precautionary behaviors, were assessed by questionnaires. Results: Serum anti-Spike IgG levels assessed by research assay, neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against PVIs after multivariable regression analysis. Commercial assays did not perform as well as research assay, likely due to their lower dynamic range. Discussion: In the 32 participants that developed PVI, anti-Spike IgG bAbs correlated with lower disease severity and shorter duration of illness.

Natural killer cells and BNT162b2 mRNA vaccine reactogenicity and durability.

Introduction: Natural killer (NK) cells can both amplify and regulate immune responses to vaccination. Studies in humans and animals have observed NK cell activation within days after mRNA vaccination. In this study, we sought to determine if baseline NK cell frequencies, phenotype, or function correlate with antibody responses or inflammatory side effects induced by the Pfizer-BioNTech COVID-19 vaccine (BNT162b2). Methods: We analyzed serum and peripheral blood mononuclear cells (PBMCs) from 188 participants in the Prospective Assessment of SARS-CoV-2 Seroconversion study, an observational study evaluating immune responses in healthcare workers. Baseline serum samples and PBMCs were collected from all participants prior to any SARS-CoV-2 infection or vaccination. Spike-specific IgG antibodies were quantified at one and six months post-vaccination by microsphere-based multiplex immunoassay. NK cell frequencies and phenotypes were assessed on pre-vaccination PBMCs from all participants by multi-color flow cytometry, and on a subset of participants at time points after the 1st and 2nd doses of BNT162b2. Inflammatory side effects were assessed by structured symptom questionnaires, and baseline NK cell functionality was quantified by an in vitro killing assay on participants that reported high or low post-vaccination symptom scores. Results: Key observations include: 1) circulating NK cells exhibit evidence of activation in the week following vaccination, 2) individuals with high symptom scores after 1st vaccination had higher pre-vaccination NK cytotoxicity indices, 3) high pre-vaccination NK cell numbers were associated with lower spike-specific IgG levels six months after two BNT162b2 doses, and 4) expression of the inhibitory marker NKG2A on immature NK cells was associated with higher antibody responses 1 and 6 months post-vaccination. Discussion: These results suggest that NK cell activation by BNT162b2 vaccination may contribute to vaccine-induced inflammatory symptoms and reduce durability of vaccine-induced antibody responses.

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti-lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.

Evaluating SARS-CoV-2 Saliva and Dried Blood Spot Surveillance Strategies in a Congregate Population.

The optimal approach to COVID-19 surveillance in congregate populations remains unclear. Our study at the US Naval Academy in Annapolis, Maryland, USA, assessed the concordance of antibody prevalence in longitudinally collected dried blood spots and saliva in a setting of frequent PCR-based testing. Our findings highlight the utility of salivary-based surveillance.

Antigenic cartography of well-characterized human sera shows SARS-CoV-2 neutralization differences based on infection and vaccination history.

The rapid emergence of SARS-CoV-2 variants challenges vaccination strategies. Here, we collected 201 serum samples from persons with a single infection or multiple vaccine exposures, or both. We measured their neutralization titers against 15 natural variants and 7 variants with engineered spike mutations and analyzed antigenic diversity. Antigenic maps of primary infection sera showed that Omicron sublineages BA.2, BA.4/BA.5, and BA.2.12.1 are distinct from BA.1 and more similar to Beta/Gamma/Mu variants. Three mRNA COVID-19 vaccinations increased neutralization of BA.1 more than BA.4/BA.5 or BA.2.12.1. BA.1 post-vaccination infection elicited higher neutralization titers to all variants than three vaccinations alone, although with less neutralization to BA.2.12.1 and BA.4/BA.5. Those with BA.1 infection after two or three vaccinations had similar neutralization titer magnitude and antigenic recognition. Accounting for antigenic differences among variants when interpreting neutralization titers can aid the understanding of complex patterns in humoral immunity that informs the selection of future COVID-19 vaccine strains.

SARS-CoV-2 BA.1 variant is neutralized by vaccine booster-elicited serum but evades most convalescent serum and therapeutic antibodies.

The rapid spread of the highly contagious Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with its high number of mutations in the spike gene has raised alarms about the effectiveness of current medical countermeasures. To address this concern, we measured the neutralization of the Omicron BA.1 variant pseudovirus by postvaccination serum samples after two and three immunizations with the Pfizer/BioNTech162b2 SARS-CoV-2 mRNA (Pfizer/BNT162b2) vaccine, convalescent serum samples from unvaccinated individuals infected by different variants, and clinical-stage therapeutic antibodies. We found that titers against the Omicron variant were low or undetectable after two immunizations and in many convalescent serum samples, regardless of the infecting variant. A booster vaccination increased titers more than 30-fold against Omicron to values comparable to those seen against the D614G variant after two immunizations. Neither age nor sex was associated with the differences in postvaccination antibody responses. We also evaluated 18 clinical-stage therapeutic antibody products and an antibody mimetic protein product obtained directly from the manufacturers. Five monoclonal antibodies, the antibody mimetic protein, three antibody cocktails, and two polyclonal antibody preparations retained measurable neutralization activity against Omicron with a varying degree of potency. Of these, only three retained potencies comparable to the D614G variant. Two therapeutic antibody cocktails in the tested panel that are authorized for emergency use in the United States did not neutralize Omicron. These findings underscore the potential benefit of mRNA vaccine boosters for protection against Omicron and the need for rapid development of antibody therapeutics that maintain potency against emerging variants.

Durability of Antibody Response and Frequency of SARS-CoV-2 Infection 6 Months after COVID-19 Vaccination in Healthcare Workers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies decay but persist 6 months postvaccination; lower levels of neutralizing titers persist against Delta than wild-type virus. Of 227 vaccinated healthcare workers tested, only 2 experienced outpatient symptomatic breakthrough infections, despite 59/227 exhibiting serologic evidence of SARS-CoV-2 infection, defined as presence of nucleocapsid protein antibodies.

Prospective Assessment of Symptoms to Evaluate Asymptomatic SARS-CoV-2 Infections in a Cohort of Health Care Workers.

BACKGROUND: The frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs). METHODS: A prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline. RESULTS: Two hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection. CONCLUSIONS: These results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.

Adverse Effects and Antibody Titers in Response to the BNT162b2 mRNA COVID-19 Vaccine in a Prospective Study of Healthcare Workers.

BACKGROUND: The relationship between postvaccination symptoms and strength of antibody responses is unclear. The goal of this study was to determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. METHODS: We conducted a single-center, observational cohort study consisting of generally healthy adult participants that were not severely immunocompromised, had no history of coronavirus disease 2019, and were seronegative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein before vaccination. Severity of vaccine-associated symptoms was obtained through participant-completed questionnaires. Testing for immunoglobulin G antibodies against SARS-CoV-2 spike protein and receptor-binding domain was conducted using microsphere-based multiplex immunoassays performed on serum samples collected at monthly visits. Neutralizing antibody titers were determined by microneutralization assays. RESULTS: Two hundred six participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers 1 month after vaccination. We also observed that (1) postvaccination symptoms were inversely correlated with age and weight and more common in women, (2) systemic symptoms were more frequent after the second vaccination, (3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and (4) older age was associated with lower titers. CONCLUSIONS: Lack of postvaccination symptoms after receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies 1 month after vaccination.

SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells.

SAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.

SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster.

The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants. ONE SENTENCE SUMMARY: Third dose of Pfizer/BioNTech COVID-19 vaccine significantly boosts neutralizing antibodies to the Omicron variant compared to a second dose, while neutralization of Omicron by convalescent sera, two-dose vaccine-elicited sera, or therapeutic antibodies is variable and often low.

Enhanced enzyme kinetics of reverse transcriptase variants cloned from animals infected with SIVmac239 lacking viral protein X.

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (-) SIVmac239-infected animals revealed that Vpx (-) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (-) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

Mechanistic cross-talk between DNA/RNA polymerase enzyme kinetics and nucleotide substrate availability in cells: Implications for polymerase inhibitor discovery.

Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.

SAMHD1 Functions and Human Diseases.

Deoxynucleoside triphosphate (dNTP) molecules are essential for the replication and maintenance of genomic information in both cells and a variety of viral pathogens. While the process of dNTP biosynthesis by cellular enzymes, such as ribonucleotide reductase (RNR) and thymidine kinase (TK), has been extensively investigated, a negative regulatory mechanism of dNTP pools was recently found to involve sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1, SAMHD1. When active, dNTP triphosphohydrolase activity of SAMHD1 degrades dNTPs into their 2'-deoxynucleoside (dN) and triphosphate subparts, steadily depleting intercellular dNTP pools. The differential expression levels and activation states of SAMHD1 in various cell types contributes to unique dNTP pools that either aid (i.e., dividing T cells) or restrict (i.e., nondividing macrophages) viral replication that consumes cellular dNTPs. Genetic mutations in SAMHD1 induce a rare inflammatory encephalopathy called Aicardi-Goutières syndrome (AGS), which phenotypically resembles viral infection. Recent publications have identified diverse roles for SAMHD1 in double-stranded break repair, genome stability, and the replication stress response through interferon signaling. Finally, a series of SAMHD1 mutations were also reported in various cancer cell types while why SAMHD1 is mutated in these cancer cells remains to investigated. Here, we reviewed a series of studies that have begun illuminating the highly diverse roles of SAMHD1 in virology, immunology, and cancer biology.

Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing cytarabine efficacy.

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

Efficient pre-catalytic conformational change of reverse transcriptases from SAMHD1 non-counteracting primate lentiviruses during dNTP incorporation.

Unlike HIV-1, HIV-2 and some SIV strains replicate at high dNTP concentrations even in macrophages due to their accessory proteins, Vpx or Vpr, that target SAMHD1 dNTPase for proteasomal degradation. We previously reported that HIV-1 reverse transcriptase (RT) efficiently synthesizes DNA even at low dNTP concentrations because HIV-1 RT displays faster pre-steady state kpol values than SAMHD1 counteracting lentiviral RTs. Here, since the kpol step consists of two sequential sub-steps post dNTP binding, conformational change and chemistry, we investigated which of the two sub-steps RTs from SAMHD1 non-counteracting viruses accelerate in order to complete reverse transcription in the limited dNTP pools found in macrophages. Our study demonstrates that RTs of SAMHD1 non-counteracting lentiviruses have a faster conformational change rate during dNTP incorporation, supporting that these lentiviruses may have evolved to harbor RTs that can efficiently execute the conformational change step in order to circumvent SAMHD1 restriction and dNTP depletion in macrophages.

A Novel Mechanism Driving Poor-Prognosis Prostate Cancer: Overexpression of the DNA Repair Gene, Ribonucleotide Reductase Small Subunit M2 (RRM2).

PURPOSE: Defects in genes in the DNA repair pathways significantly contribute to prostate cancer progression. We hypothesize that overexpression of DNA repair genes may also drive poorer outcomes in prostate cancer. The ribonucleotide reductase small subunit M2 (RRM2) is essential for DNA synthesis and DNA repair by producing dNTPs. It is frequently overexpressed in cancers, but very little is known about its function in prostate cancer. EXPERIMENTAL DESIGN: The oncogenic activity of RRM2 in prostate cancer cells was assessed by inhibiting or overexpressing RRM2. The molecular mechanisms of RRM2 function were determined. The clinical significance of RRM2 overexpression was evaluated in 11 prostate cancer clinical cohorts. The efficacy of an RRM2 inhibitor (COH29) was assessed in vitro and in vivo. Finally, the mechanism underlying the transcriptional activation of RRM2 in prostate cancer tissue and cells was determined. RESULTS: Knockdown of RRM2 inhibited its oncogenic function, whereas overexpression of RRM2 promoted epithelial mesenchymal transition in prostate cancer cells. The prognostic value of RRM2 RNA levels in prostate cancer was confirmed in 11 clinical cohorts. Integrating the transcriptomic and phosphoproteomic changes induced by RRM2 unraveled multiple oncogenic pathways downstream of RRM2. Targeting RRM2 with COH29 showed excellent efficacy. Thirteen putative RRM2-targeting transcription factors were bioinformatically identified, and FOXM1 was validated to transcriptionally activate RRM2 in prostate cancer. CONCLUSIONS: We propose that increased expression of RRM2 is a mechanism driving poor patient outcomes in prostate cancer and that its inhibition may be of significant therapeutic value.