Serum neurofilament light-chain levels and long-term treatment outcomes in relapsing-remitting multiple sclerosis patients: A post hoc analysis of the randomized CombiRx trial.

Background: CombiRx was a randomized, double-blind, placebo-controlled phase 3 trial in treatment-naive relapsing-remitting multiple sclerosis (RRMS) patients randomized to intramuscular interferon beta-1a (IM IFN beta-1a), glatiramer acetate (GA), or both therapies. Objective: This analysis investigated changes in serum neurofilament light-chain (sNfL) levels in response to treatment and assessed baseline sNfL as a predictor of relapse. Methods: RRMS patients treated with IM IFN beta-1a 30 µg weekly + placebo (n = 159), GA 20 mg/mL daily + placebo (n = 172), or IM IFN beta-1a + GA (n = 344) were included. A linear mixed model compared sNfL values over time. Cox regression models analyzed baseline sNfL and gadolinium-enhancing (Gd+) lesions as predictors of relapse. Results: In all treatment arms, the proportion of patients with sNfL ≥16 pg/mL decreased significantly from baseline to 6 months and was maintained at 36 months. A significantly higher percentage of patients with both baseline sNfL ≥16 pg/mL and ≥1 Gd+ lesion experienced relapses within 90 days compared to patients with sNfL <16 pg/mL and/or no Gd+ lesions. Conclusion: sNfL levels were reduced within 6 months and remained low at 36 months. Results suggest that the combination of lesion activity and sNfL was a stronger predictor of relapse than either factor alone.

Pharmacokinetics/pharmacodynamics by race: Analysis of a peginterferon ß-1a phase 1 study.

BACKGROUND: Black/African American participants are underrepresented in clinical trials for multiple sclerosis but can experience a greater burden of disease than other racial groups in the United States. A phase 1, open-label, crossover study that demonstrated bioequivalence of subcutaneous and intramuscular injection of peginterferon ß-1a in healthy volunteers enrolled similar proportions of Black and White participants, enabling a post hoc subgroup analysis comparing these groups. METHODS: Peginterferon ß-1a (125 µg) was administered by subcutaneous or intramuscular injection, followed by a washout period before a second injection using the alternative method. The primary pharmacokinetic and pharmacodynamic endpoints were maximum observed concentration (Cmax) and area under the concentration-time curve from hour 0 to infinity (AUCinf) of study drug and serum concentration of neop-terin, respectively. Safety and tolerability were included as secondary endpoints. FINDINGS: This analysis included 70 (51.5%) Black and 59 (43.3%) White participants. Peginterferon ß-1a Cmax was 29.8% higher in Black than in White participants following subcutaneous administration but was similar following intramuscular administration. Mean AUCinf was 31.0% and 11.8% greater in Black than in White participants with subcutaneous and intramuscular administration, respectively. Pharmacodynamics and safety signals were similar between groups, although Black participants reported numerically fewer adverse events. CONCLUSIONS: No clinically meaningful differences were identified between Black and White participants related to peginterferon ß-1a administration, supporting the approved dose of 125 µg/mL peginterferon ß-1a. Future clinical studies should include sufficiently diverse populations to ensure accurate assessments of treatment response. FUNDING: Funding for medical writing support was provided by Biogen (Cambridge, MA, USA).

Safety and clinical effectiveness of peginterferon beta-1a for relapsing multiple sclerosis in the real-world setting: Interim results from the Plegridy Observational Program.

BACKGROUND: The Plegridy Observational Program (POP) is an ongoing, 5-year, phase 4 real-world study of the safety and effectiveness of subcutaneous peginterferon beta-1a in patients with relapsing multiple sclerosis (RMS). METHODS: This interim analysis from POP assessed the safety and effectiveness of peginterferon beta-1a, including subgroup analyses of patients aged < 50 and ≥ 50 years, newly diagnosed and non-newly diagnosed patients, and new and experienced peginterferon beta-1a users. RESULTS: A total of 1208 patients enrolled in POP. Mean (standard deviation) peginterferon treatment duration in the overall population was 757.0 (529.5) days. The overall incidence of treatment-emergent adverse events (AEs) was 65.5%, and the incidence was higher in new than experienced peginterferon beta-1a users (78.1 vs 52.4%). The overall incidence of treatment-emergent serious AEs was 7.6%, and the incidence was lower in younger than older patients (5.8 vs 11.1%). No new or unexpected safety signals were reported. Overall treatment discontinuation due to AEs occurred in 20.7% of patients, with a higher proportion of new than experienced peginterferon beta-1a users (27.0 vs 14.2%) discontinuing treatment due to AEs. Flu-like symptoms and injection site reactions were significant predictors of time to treatment discontinuation. The adjusted annualized relapse rate (ARR) was 0.12 (95% confidence interval 0.11-0.13) in the overall population and was similar across all subgroups. In the overall population at 4 years, 79.1% of patients were relapse free, the estimated cumulative proportion of patients with confirmed disability worsening was 1.8%, and > 67% of patients achieved clinical no evidence of disease activity (NEDA). CONCLUSIONS: Safety data of patients enrolled in POP are consistent with the established clinical safety profile of peginterferon beta-1a. In addition, the low ARR and high proportion of patients achieving clinical NEDA at 4 years across all subgroups indicates the effectiveness of peginterferon beta-1a in treating RMS in real-world clinical settings.

Early treatment responses to peginterferon beta-1a are associated with longer-term clinical outcomes in patients with relapsing-remitting multiple sclerosis: Subgroup analyses of ADVANCE and ATTAIN.

BACKGROUND: Early intervention with well-tolerated disease-modifying therapies (DMTs) for relapsing-remitting multiple sclerosis (RRMS) is recommended in order to delay disease progression, reduce neurologic damage, preserve brain volume, and optimize long-term patient outcomes. Lack of conversion of new/newly enlarging T2 (NET2) or gadolinium-enhancing (Gd+) lesions to chronic hypointensities (black hole conversion) and achievement of no evidence of disease activity (NEDA) early in the course of treatment are considered potential indicators of treatment effect and predictors of longer-term clinical outcomes. METHODS: Patients with RRMS who were treated with peginterferon beta-1a in the 2-year ADVANCE phase 3 clinical trial (NCT0090639) and its 2-year open-label extension study, ATTAIN (NCT01332019), were grouped as newly diagnosed (diagnosed ≤1 year prior to enrollment and DMT naive) or non-newly diagnosed. For analyses of the impact of early treatment and disease activity control, the newly diagnosed and non-newly diagnosed subgroups were further divided based on whether they initiated peginterferon beta-1a every 2 weeks (Q2W) starting in study year 1 (continuously treated) or peginterferon beta-1a Q2W or every 4 weeks in study year 2 (delayed treatment). Patient subgroups were evaluated for conversion of NET2 or Gd+ lesions to persistent black holes (PBHs), brain atrophy (percentage change in whole brain volume [WBV]), achievement of NEDA composite outcomes, and the association of these disease activity measures with longer-term clinical outcomes (annualized relapse rate [ARR] and confirmed disability worsening [CDW]). RESULTS: At 2 years, significantly fewer PBHs developed from NET2 lesions or Gd+ lesions in newly diagnosed and non-newly diagnosed patients continuously treated with peginterferon beta-1a than in the corresponding delayed-treatment groups (all p<0.0001). Percentage decrease in WBV from 6 months (rebaselined) to 2 years was significantly lower for newly diagnosed and non-newly diagnosed patients who received continuous peginterferon beta-1a treatment than for patients who received delayed treatment (both p ≤ 0.0442). In study year 1, a higher proportion of newly diagnosed and non-newly diagnosed patients treated with peginterferon beta-1a than those treated with placebo achieved NEDA (newly diagnosed: 28.3% vs 13.5% [p = 0.0010]; non-newly diagnosed: 40.8% vs 15.8% [p<0.0001]). NEDA rates remained stable over study years 2-4 for the newly diagnosed (range: 50.0%-53.9%) and non-newly diagnosed (range: 54.4%-57.0%) subgroups. Patients without PBH conversion had significantly lower ARR at 2 years (newly diagnosed: p = 0.0109; non-newly diagnosed: p = 0.0044) and a lower proportion of patients with 12-week CDW at 2 years (newly diagnosed: p = 0.2787; non-newly diagnosed: p = 0.0045) than the corresponding patient subgroups with PBH conversion. Patients who achieved NEDA in ADVANCE (study years 1-2) had a significantly lower ARR in ATTAIN (study years 3-4) than patients who did not achieve NEDA (newly diagnosed, p = 0.0003; non-newly diagnosed, p = 0.0001). Over 4 years, safety outcomes did not differ for the newly diagnosed and non-newly diagnosed patient subgroups. CONCLUSIONS: These results indicate that newly diagnosed and non-newly diagnosed patients treated continuously with peginterferon beta-1a Q2W experienced better disease control over time than those who received delayed treatment. Patients with NEDA or evidence of less radiological disease activity in the first 2 years of treatment had better longer-term clinical outcomes than those with evidence of greater disease activity.

Immunoglobulin G immune response to SARS-CoV-2 vaccination in people living with multiple sclerosis within Multiple Sclerosis Partners Advancing Technology and Health Solutions.

BACKGROUND: The impact of multiple sclerosis (MS) disease-modifying therapies (DMTs) on SARS-CoV-2 vaccination response is uncertain. METHODS: Post-SARS-CoV-2 vaccination blood samples across multiple DMTs were tested for SARS-CoV-2 immunoglobulin G (IgG) response. RESULTS: Three hundred twenty-two people with MS were included; 91.9% received an mRNA vaccine. Post-vaccination reactive IgG rates (IgG index > 1) were 40% for anti-CD20 (32/80 patients); 41% for sphingosine 1-phosphate receptor modulators (S1PRM, 16/39); and 100% for all other classes, including the no DMT group. CONCLUSION: Anti-CD20 therapies and S1PRMs reduce IgG response to SARS-CoV-2 vaccination; IgG response is preserved with other DMTs.

Global Peginterferon Beta-1a Tolerability Management Best Practices: A Nurse-Focused Delphi Approach.

INTRODUCTION: Injection site reactions (ISRs) and flu-like symptoms (FLS) are common in patients with relapsing forms of multiple sclerosis (MS) treated with peginterferon beta-1a. The purpose of this Delphi analysis was to explore peginterferon beta-1a discontinuation rates across MS treatment centers, to obtain consensus on effective mitigation and management strategies for ISRs and FLS, and to identify areas where additional training and education for nurses and patients could improve treatment outcomes. METHODS: In this modified Delphi process, an international steering committee of eight MS-certified nurses developed two rounds of surveys, which were completed by 262 and 188 MS nurses, respectively, representing nine countries. RESULTS: On average, nurses reported that 25% and 30% of patients treated with peginterferon beta-1a experienced ISRs and FLS, respectively. Discontinuation due to severe ISRs or FLS was most common in the first 6 months of treatment, yet follow-up visits typically took place 6 months after peginterferon beta-1a initiation. Preferred management strategies for ISRs included nonsteroidal anti-inflammatory drugs and rotation of the injection site, whereas preferred management strategies for FLS included acetaminophen/paracetamol and hydration/nutrition. Most nurses (77%) agreed that additional education and training on ISR and FLS management would bolster their confidence in treating patients with these symptoms. CONCLUSION: Delphi respondents reached consensus on ISR and FLS management strategies, which can help to inform treatment decisions. The results of this global Delphi analysis indicate that management of ISRs and FLS could be improved with more frequent follow-up visits and individualized training and education.

COVID-19 in Patients with Multiple Sclerosis: Associations with Disease-Modifying Therapies.

BACKGROUND: Disease-modifying therapies (DMTs) for multiple sclerosis (MS) target immunity and have the potential to increase the risk of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and alter its clinical course. We assessed these risks in patients with MS (PwMS). OBJECTIVE: The objective of this study was to describe the overall risk of coronavirus disease 2019 (COVID-19) infection, severe disease course, and potential population-level predictors of COVID-19 infection in PwMS, and to provide a context using a cohort of patients with systemic lupus erythematosus (SLE). In addition, the association of different MS DMTs with the incidence and clinical course of COVID-19 was evaluated. Safety data from the Biogen Global Safety Database are also presented on reported cases of COVID-19 in patients treated with Biogen MS therapies. METHODS: The IBM® Explorys electronic health record database of > 72,000,000 patients from US healthcare networks identified patients with MS or SLE, with and without polymerase chain reaction-confirmed COVID-19. COVID-19 cumulative incidence, hospitalization, and deaths among DMT classes were compared using logistic regression (adjusted for age, sex, body mass index, comorbidities, and race/ethnicity). As a secondary data source to assess safety data, COVID-19 reports for Biogen MS therapies were extracted and described from Biogen's Global Safety Database. RESULTS: 30,478 PwMS with an open DMT prescription were identified within Explorys; 344 were COVID-19 positive. The most significant risk factors for acquiring COVID-19 were comorbidity score ≥ 1, body mass index ≥ 30, and Black/African ancestry. Similar risk factors were also identified for patients with SLE. Patients with MS were less likely to develop COVID-19 when treated with interferons (0.61%) and glatiramer acetate (0.51%), vs all other MS DMTs (both p < 0.001); anti-CD20 therapy was associated with the highest risk (3.45%; p < 0.0001). In the Biogen Global Safety Database, we identified 1217 patients who were COVID-19 positive treated with intramuscular interferon beta-1a, peginterferon beta-1a, natalizumab, dimethyl fumarate, diroximel fumarate, or fampridine. CONCLUSIONS: Comorbidities, obesity, and Black/African ancestry, but not age, were associated with a higher risk of SARS-CoV-2 infection in PwMS. Interferons and glatiramer acetate were associated with a reduced COVID-19 risk, whereas anti-CD20 therapies were associated with an increased risk, within the treated MS cohort. COVID-19 safety reports for patients receiving Biogen MS therapies were consistent with the Explorys database and MS literature, illustrating the replicability and power of this approach.

Bioequivalence of intramuscular and subcutaneous peginterferon beta-1a: results of a phase I, open-label crossover study in healthy volunteers.

BACKGROUND: Peginterferon beta-1a administered every 2 weeks via subcutaneous (SC) injection is approved to treat adult patients with relapsing-remitting multiple sclerosis (RRMS) and relapsing forms of multiple sclerosis (RMS). However, associated injection site reactions (ISRs) can lead to treatment discontinuation. Prior studies with interferon beta-1a reported a lower frequency of ISRs with intramuscular (IM) administration than with SC administration. IM administration of peginterferon beta-1a may therefore represent a useful alternative treatment option. METHODS: A phase I, open-label, two-period crossover study randomized healthy volunteers to receive a single dose of peginterferon beta-1a 125 mcg administered IM followed by a single 125 mcg dose administered SC after a 28-day washout or vice versa. Blood samples were collected up to 504 h post dose to determine pharmacokinetic (PK) and pharmacodynamic (PD) profiles. The primary endpoint was assessment of bioequivalence based on maximum serum concentration (Cmax) and area under the curve from time zero extrapolated to infinity (AUCinf). Other PK parameters, as well as PD (serum neopterin) and safety profiles, were also evaluated. RESULTS: The study enrolled 136 participants. Bioequivalence of IM and SC peginterferon beta-1a was established for both Cmax ([least squares (LS)] mean IM/SC ratio: 1.083 [90% confidence interval (CI), 0.975-1.203]) and AUCinf (LS mean IM/SC ratio: 1.089 [90% CI, 1.020-1.162]). Other PK and PD parameters were similar between administration routes, although moderate to high inter-subject variability was observed for IM and SC. Safety profiles were generally balanced between IM and SC administration. ISRs occurred at a lower frequency with IM [14.4% (95% CI, 8.89-21.56%)] than with SC [32.1% (95% CI, 24.29-40.70%)] administration (p = 0.0005). CONCLUSIONS: These results demonstrate bioequivalence between peginterferon beta-1a IM and SC and support the consideration of IM injection of peginterferon beta-1a as a viable treatment option in patients with RRMS and RMS.

Matching comparisons of therapeutic efficacy suggest better clinical outcomes for patients treated with peginterferon beta-1a than with glatiramer acetate.

BACKGROUND: Peginterferon beta-1a and glatiramer acetate (GA) are approved first-line therapies for the treatment of relapsing forms of multiple sclerosis, but their therapeutic efficacy has not been compared directly. METHODS: Clinical outcomes at 2 years, including no evidence of disease activity (NEDA), for patients receiving peginterferon beta-1a 125 mcg every 2 weeks (Q2W) or GA 20 mg/ml once daily (QD) were compared by propensity score matching analysis using individual patient data from ADVANCE and CONFIRM phase III clinical trials. In addition, clinical outcomes at 1-3 years for patients receiving peginterferon beta-1a Q2W or GA 40 mg/ml three times a week (TIW) were evaluated using a matching-adjusted comparison analysis of individual patient data from ADVANCE and the ADVANCE extension study, ATTAIN, and aggregate patient data from the phase III GALA and the GALA extension studies. RESULTS: Propensity-score-matched peginterferon beta-1a patients (n = 336) had a significantly lower annualized relapse rate [ARR (0.204 versus 0.282); rate ratio = 0.724; p = 0.045], a significantly lower probability of 12-week confirmed disability worsening (10.0% versus 14.6%; hazard ratio = 0.625; p = 0.048), and a significantly higher rate of NEDA (20.3% versus 11.5%; p = 0.047) compared with GA 20 mg/ml QD patients after 2 years of treatment. Matching-adjusted peginterferon beta-1a patients (effective n = 276) demonstrated a similar ARR at 1 year (0.278 versus 0.318; p = 0.375) and significantly lower ARR at 2 years (0.0901 versus 0.203; p = 0.032) and 3 years (0.109 versus 0.209; p = 0.047) compared with GA 40 mg/ml TIW patients (n = 834). CONCLUSION: Results from separate matching comparisons of phase III clinical trials and extension studies suggest that peginterferon beta-1a 125 mcg Q2W may provide better clinical outcomes than GA (20 mg/ml QD or 40 mg/ml TIW).

Matching-adjusted comparisons demonstrate better clinical outcomes in patients with relapsing multiple sclerosis treated with peginterferon beta-1a than with teriflunomide.

BACKGROUND: Peginterferon beta-1a and teriflunomide are both first-line disease-modifying therapies (DMTs) approved for the treatment of relapsing multiple sclerosis (RMS); however, no head-to-head trials have directly compared their clinical efficacy. We performed a matching-adjusted comparison of individual patient data from the peginterferon beta-1a pivotal phase 3 study, ADVANCE, and its extension study, ATTAIN, with pooled aggregated data from the teriflunomide pivotal phase 3 studies, TEMSO and TOWER. METHODS: A total of 512 patients randomized to subcutaneous (SC) peginterferon beta-1a 125 mcg every 2 weeks in ADVANCE and 731 patients randomized to teriflunomide 14 mg daily (359 from TEMSO and 372 from TOWER) were matched on key baseline characteristics. After matching, weighted annualized relapse rate (ARR) and 24-week confirmed disability worsening (CDW) were calculated and compared for peginterferon beta-1a- and teriflunomide-treated patients. A subset analysis comparing weighted ARR in patients who were newly diagnosed with RMS (diagnosis ≤1 year before study enrollment and disease-modifying therapy naïve) was also performed. RESULTS: After matching, the peginterferon beta-1a and teriflunomide treatment groups were identically matched across baseline characteristics. The proportion of patients in the overall study populations with 24-week CDW at 108 weeks was significantly lower in the peginterferon beta-1a group than the teriflunomide group both before matching (8.5% vs 12.6%; P = 0.0249) and after matching (8.4% vs 12.6%; P = 0.0323). ARR at 108 weeks was numerically lower with peginterferon beta-1a than with teriflunomide both before matching (0.278 vs 0.354; P = 0.1326) and after matching (0.257 vs 0.354; P = 0.0510). Newly diagnosed patients treated with peginterferon beta-1a had numerically lower ARR than patients treated with teriflunomide both at 108 weeks (before matching: 0.225 vs 0.270; P = 0.587; after matching: 0.201 vs 0.270; P = 0.384) and at 5 years (before matching: 0.150 vs 0.196; after matching: 0.142 vs 0.196). CONCLUSIONS: In this matching-adjusted comparison of patients with RMS from three phase 3 trials, a significantly lower proportion of patients treated with SC peginterferon beta-1a 125 mcg every 2 weeks than with oral teriflunomide 14 mg once daily had 24-week CDW at 108 weeks. In addition, in both the overall population and newly diagnosed patient subgroups, ARR at 108 weeks was numerically lower with peginterferon beta-1a than with teriflunomide. The numerically lower ARR in newly diagnosed patients treated with peginterferon beta-1a compared with those treated with teriflunomide was sustained through up to 5 years of treatment.

Long-term outcomes of peginterferon beta-1a in multiple sclerosis: results from the ADVANCE extension study, ATTAIN.

BACKGROUND: ADVANCE was a phase III trial of the efficacy and safety of subcutaneous peginterferon beta-1a 125 µg every 2 or 4 weeks in patients with relapsing-remitting multiple sclerosis (RRMS). ATTAIN was a 2-year extension study of ADVANCE. The aim was to evaluate the long-term safety, tolerability, and efficacy of peginterferon beta-1a 125 µg every 2 or 4 weeks in ATTAIN. METHODS: ADVANCE dosing schedules were maintained in ATTAIN, except that every-4-weeks dosing patients were switched to every-2-weeks dosing after conversion of the study to an open-label protocol. ATTAIN was considered complete when the last patient completed the 96-week extension study. Primary endpoints included adverse event (AE) and serious AE (SAE) incidence. Secondary endpoints included relapse, magnetic resonance imaging, and disability outcomes. RESULTS: Of the 1512 patients randomized in ADVANCE, 1076 (71%) continued treatment in ATTAIN; of these, 842 (78%) completed the open-label extension study. During ATTAIN, 478 patients (87%) in the every-2-weeks group and 471 patients (89%) in the every-4-weeks group experienced an AE; SAEs were reported in 90 patients (16%) in the every-2-weeks group and 113 patients (21%) in the every-4-weeks group. The most frequent AEs reported were injection site reactions and flu-like symptoms, both of which numerically decreased over time. Peginterferon beta-1a every 2 weeks versus every 4 weeks significantly reduced the adjusted annualized relapse rate over 6 years (0.188 versus 0.263, p = 0.0052) and the risk of relapse over 5 years (36% versus 49%, p = 0.0018). Fewer new T1, new/newly enlarging T2, and gadolinium-enhancing magnetic resonance imaging lesions were observed with every-2-weeks dosing than every-4-weeks dosing over 4 years. CONCLUSIONS: Results from the ADVANCE extension study, ATTAIN, confirm the favorable long-term safety and tolerability profile of peginterferon beta-1a in patients with RRMS and provide additional evidence for the clinical and radiological benefits associated with this therapy.

Peginterferon ß-1a every 2 weeks increased achievement of no evidence of disease activity over 4 years in the ADVANCE and ATTAIN studies in patients with relapsing-remitting multiple sclerosis.

BACKGROUND: No evidence of disease activity (NEDA) is a composite measurement, incorporating clinical and magnetic resonance imaging (MRI) elements of disease activity to sensitively evaluate the therapeutic efficacy of treatments for relapsing-remitting multiple sclerosis (RRMS). OBJECTIVE: To assess the NEDA status of patients treated with peginterferon ß-1a in the ADVANCE and ATTAIN studies and explore its predictive value on longer-term clinical outcomes. METHODS: ATTAIN was a 2-year extension of the pivotal 2-year ADVANCE study of peginterferon ß-1a for RRMS. Achievement of clinical NEDA, MRI NEDA, or overall NEDA was calculated cumulatively and by year over 4 years. Clinical outcomes during ATTAIN were analyzed based on NEDA status at the end of ADVANCE. RESULTS: Significantly more patients treated with peginterferon ß-1a every 2 weeks than every 4 weeks achieved clinical NEDA (60.6% versus 50.6%, p = 0.0063) and MRI NEDA (28.3% versus 15.8%, p = 0.0005) through year 4 and overall NEDA through year 3 (20.9% versus 13.9%, p = 0.0160). Over 4 years, 15.8% of patients in the every 2 weeks group and 10.7% of patients in the every 4 weeks group maintained overall NEDA (p = 0.0584). Achievement of clinical NEDA, MRI NEDA, or overall NEDA in ADVANCE was predictive of annualized relapse rate in ATTAIN; achievement of clinical NEDA in ADVANCE was also predictive of NEDA achievement and confirmed disability worsening in ATTAIN. CONCLUSIONS: Peginterferon ß-1a every 2 weeks is associated with higher levels of NEDA compared with placebo in year 1 or peginterferon ß-1a every 4 weeks in years 2-4. Overall NEDA within the first 2 years of treatment may be prognostic of long-term clinical outcomes.Clinicaltrials.gov: NCT01332019.

Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.

Mrc1 phosphorylation in response to DNA replication stress is required for Mec1 accumulation at the stalled fork.

DNA replication stress activates a response pathway that stabilizes stalled forks and promotes the completion of replication. The budding yeast Mec1 sensor kinase, Mrc1 mediator, and Rad53 effector kinase are central to this signal transduction cascade in S phase. We report that Mec1-dependent, Rad53-independent phosphorylation of Mrc1 is required to establish a positive feedback loop that stabilizes Mec1 and the replisome at stalled forks. A structure-function analysis of Mrc1 also uncovered a central region required for proper mediator function and association with replisome components. Together these results reveal new insight into how Mrc1 facilitates checkpoint signal amplification at stalled replication forks.

RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion.

Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G(2)-arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

Essential role of limiting telomeres in the pathogenesis of Werner syndrome.

Mutational inactivation of the gene WRN causes Werner syndrome, an autosomal recessive disease characterized by premature aging, elevated genomic instability and increased cancer incidence. The capacity of enforced telomerase expression to rescue premature senescence of cultured cells from individuals with Werner syndrome and the lack of a disease phenotype in Wrn-deficient mice with long telomeres implicate telomere attrition in the pathogenesis of Werner syndrome. Here, we show that the varied and complex cellular phenotypes of Werner syndrome are precipitated by exhaustion of telomere reserves in mice. In late-generation mice null with respect to both Wrn and Terc (encoding the telomerase RNA component), telomere dysfunction elicits a classical Werner-like premature aging syndrome typified by premature death, hair graying, alopecia, osteoporosis, type II diabetes and cataracts. This mouse model also showed accelerated replicative senescence and accumulation of DNA-damage foci in cultured cells, as well as increased chromosomal instability and cancer, particularly nonepithelial malignancies typical of Werner syndrome. These genetic data indicate that the delayed manifestation of the complex pleiotropic of Wrn deficiency relates to telomere shortening.

Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression.

Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc(-/-) Ink4a/Arf(-/-) mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc(-/-) Ink4a/Arf(-/-) cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc(-/-) Ink4a/Arf(-/-) cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly, despite a marked increase in telomere reserve, cells derived from the ALT+ subcutaneous tumors were unable to generate lung metastases, indicating in vivo functional differences in these principal mechanisms of telomere maintenance. Together, these results are consistent with the model that although telomere dysfunction provokes chromosomal aberrations that initiate carcinogenesis, telomerase-mediated telomere maintenance enables such initiated cells to efficiently achieve a fully malignant endpoint, including metastasis.