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RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular switch that enables the genome-protective function of RIF1 during DNA replication stress.

Purpose For patients with long bone metastases who undergo orthopedic stabilization surgery followed by radiotherapy (RT), it is unclear what extent of hardware coverage by the radiation field is needed for optimal tumor control. Methods and Materials Long bone metastases treated with surgical intervention followed by radiation between August 2011 to May 2019 from a single institution were reviewed. Local recurrence, defined as any in-bone recurrence, was identified by chart review. Accompanying demographic and treatment characteristics were recorded. Statistical analysis to evaluate factors associated with tumor recurrence included univariate analysis, multivariate analysis, and propensity score matching. Results Among 138 patients with 145 long bone metastases undergoing postoperative RT with a median follow-up of 29.5 months, 36 bone metastases experienced a local recurrence. Most patients (92%) were treated with conventional RT and the median delivered dose was 30 Gy (interquarile range, 20-30 Gy). On univariate analysis, whole hardware RT field coverage and higher dose (biologically effective dose 10 ≥39 Gy) were associated with reduced local recurrence (0.44 hazard ratio [HR]; 95% confidence interval [CI], 0.22%-0.86%; P = .017; 0.5 HR; 95% CI, 0.26%-0.96%; P = .038, respectively). Covariates of time from surgery to RT start, histology of primary tumor (categorized as resistant vs sensitive), intramedullary hardware placement, reaming procedure, and margin status did not reach statistical significance. To adjust for confounding effects, we also conducted a propensity score matched analysis which confirmed that whole hardware coverage was statistically associated with a decreased risk of recurrence on the matched dataset (0.24 HR; 95% CI, 0.07%-0.84%; P = .026). Conclusions In this analysis of mostly patients undergoing conventional radiation, coverage of the whole hardware was associated with reduced local recurrence for patients with long bone metastases, consistent with prior reports. Investigation of approaches to further reduce local recurrence, such as preoperative stereotactic radiation, may be warranted.

Purpose Local therapy for patients with nonspine bone metastases is evolving, with data supporting the use of single-fraction treatments, and more recently, showing possible benefit from stereotactic body radiation therapy (SBRT). However, the rate of local salvage therapy (LST) after each technique has not been characterized in real-world clinic settings where patients are selected at physician discretion. We examined rates of LST in patients with nonspine bone metastases. Methods and Materials We reviewed records of RT for nonspine bone metastases at our institution from January 1, 2016, to December 31, 2018. We defined LST as the first occurrence of RT or surgery for oncologic progression to a bone metastasis after initial RT. Cumulative incidence functions for retreatment were generated. We conducted multivariate analysis to identify variables associated with LST. Results A total of 1754 patients were analyzed, with median follow-up of 16.2 months (range, 0-36.8 months). Of all episodes of RT, 51.5% were multifraction external beam radiation therapy (EBRT), 7.0% were single-fraction EBRT, and 41.4% were SBRT. Altogether, 88 patients (5.0%) required LST, with an incidence at 6 months of 2.5%. Incidence of LST at 6 months was 2.1% for SBRT, 5.3% for single-fraction conventional regimens, and 2.4% for multifraction conventional regimens (P = .26). Patients of younger age, who had a higher Karnofsky performance status, and/or who had lesions in the pelvis had a higher risk of retreatment. Conclusions In this large institutional cohort, the rate of LST was low, with no difference between RT techniques. The findings indicated that SBRT for patients at high risk for treatment failure may reduce the rate of retreatment overall. When treatment modality was selected based on patient characteristics, rates of LST were lower than when treatment was randomly selected.

To study how severity and progression of coronavirus disease (COVID-19) affect cytokine profiles in pregnant women.69 third-trimester, pregnant women were tested for COVID-19 infection and SARS-CoV-2 specific IgM and IgG antibodies. Patients were stratified according to SARS-CoV-2 Reverse Transcriptase-PCR (RT-PCR) status and serology (IgM and IgG) status. Cytokines G-CSF, HGF, IL-18, IL-1Ra, IL-2Ra, IL-8, and IP-10 were measured via ELISA. Retrospective chart review for COVID-19 symptoms and patient vitals was conducted, and cytokine levels were compared between SARS-CoV-2 positive and negative cohorts, by seronegative and seropositive infection, by time course since onset of infection, and according to NIH defined clinical severity.IL-18, IL-1Ra, and IP-10 increased in the 44 RT-PCR positive pregnant women compared to the 25 RT-PCR negative pregnant controls. Elevated cytokine levels were found in early infections, defined by positive RT-PCR and seronegative status, and higher cytokine levels were also associated with more severe disease. By IgM seroconversion, IL-8 and IP-10 returned to levels seen in uninfected patients, while IL-18 levels remained significantly elevated.Cytokine profiles of third-trimester pregnant women vary with the time course of infection and are correlated with clinical severity.

Purpose/Objective(s) Local therapy for patients with non-spine bone metastases is evolving, with recent data suggesting that stereotactic body radiotherapy (SBRT) may improve pain response in the palliative setting. However, the rate of local salvage therapy (LST) after SBRT compared to conventional RT is unknown. We therefore characterized rates and risk factors for LST in patients with non-spine bone metastases. Materials/Methods We reviewed records of all courses of RT for non-spine bone metastases at our institution from 1/1/2016 to 12/31/2018. LST was defined as the first occurrence of either RT or surgery for progression to a bone metastasis following initial RT. Functions of the cumulative incidence of re-treatment were generated for each RT technique. We identified associations with various predictors using the Fine and Gray method for competing risk regression. Results Of the 1,754 patients included, 2,648 bone metastases were treated with RT and analyzed with a median follow up of 15 months (IQR = 6.4 – 25.0 months). 51% of episodes were multi-fraction EBRT, 10% were single fraction EBRT, and 39% were SBRT. Of the treated metastases, 120 (4.5%) episodes required LST (median time to LST = 6.2 months, range = 0.3-35.6 months), with a cumulative incidence at 6 and 12 months of 2.4% and 3.9%, respectively. Cumulative incidence of re-treatment at 6 months was 2.6% for SBRT, 3.5% for single-fraction conventional, and 2.1% for multi-fraction conventional regimens (P = 0.76). On multivariable analysis, younger age, higher Karnofsky performance status (KPS), and location of the metastasis in the pelvis had higher hazard of re-treatment (Table 1). There was no significant difference in hazard of LST among RT techniques (P = 0.44), nor was there a significant interaction between RT technique and radioresistant histology (P = 0.33). Conclusion In our large institutional cohort, the rate of LST was low, with no difference between RT techniques. Our results suggest that judicious selection of patients at high risk for eventual treatment failure (i.e., those with longer expected survival) for SBRT may reduce the rate of retreatment rate in the population. When patients are appropriately selected for single fraction conventional RT, such as those who are older or have poor KPS, rates of LST remain low. Further studies to validate our findings are warranted to help clinicians to select the appropriate treatment technique for patients with bone metastases.

Summary Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3′ Igh super-enhancer, 3′ regulatory region (3′RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3′RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3′RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3′RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3′RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3′ Igh super-enhancer., Graphical Abstract, Highlights • ZMYND8 is required for GLT of acceptor S regions and Class Switch Recombination • ZMYND8 supports efficient somatic hypermutation of the Igh variable regions • ZMYND8 binds B cell super-enhancers, including the 3′ Igh enhancer • ZMYND8 modulates the transcriptional status and activity of the 3′ Igh enhancer, Antibody diversity is essential for the establishment of an effective immune response. Delgado-Benito and Rosen et al. report that diversification of the immunoglobulin heavy chain (Igh) gene in mature B lymphocytes is regulated by the chromatin reader ZMYND8, which binds to and modulates the activity of the 3′ Igh locus super-enhancer.