USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

A Virtual Curriculum to Increase Exposure to Oncologic Subspecialties for Undergraduate Medical Students.

Medical student exposure to oncology is imperative given the prevalence of cancer, growing need for survivorship care, and ever-evolving therapies. Our institution offers a Cancer Care Elective for undergraduate medical students focused on clinical shadowing, but the COVID-19 pandemic necessitated completely redesigning a virtual alternative. In this study, we utilize a post-elective survey to 1) assess whether the novel virtual elective effectively promoted student learning and 2) identify which components were most impactful. We created an entirely virtual, semester-long course with structured mentorship, subspecialty panels, physician-led didactics, and patient exposure. Students attended multidisciplinary tumor boards and presented on oncologic topics. A post-elective survey assessed the course's impact on students' knowledge and the perceived value of each elective component. Of the 29 enrolled students, 12 responded to our survey (41%). Most students reported that the elective highly enhanced their understanding of medical (67%), surgical (75%), and pediatric (66%) oncology. The highest rated didactic involved patients discussing their cancer journeys, with 80% of students reporting that this session enhanced their understanding of patient-physician collaboration. Students reported that physician mentorship helped them better understand oncology (90%) and promoted interest in pursuing an oncologic career (100%). This study demonstrates that our virtual Cancer Care Elective was effective at increasing student understanding of oncology in practice. The results also suggest that patient exposure and physician mentorship are particularly educational and encouraging.

Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.

Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.

Human CHD1 is required for early DNA-damage signaling and is uniquely regulated by its N terminus.

CHD1 is a conserved chromatin remodeling enzyme required for development and linked to prostate cancer in adults, yet its role in human cells is poorly understood. Here, we show that targeted disruption of the CHD1 gene in human cells leads to a defect in early double-strand break (DSB) repair via homologous recombination (HR), resulting in hypersensitivity to ionizing radiation as well as PARP and PTEN inhibition. CHD1 knockout cells show reduced H2AX phosphorylation (γH2AX) and foci formation as well as impairments in CtIP recruitment to the damaged sites. Chromatin immunoprecipitation following a single DSB shows that the reduced levels of γH2AX accumulation at DSBs in CHD1-KO cells are due to both a global reduction in H2AX incorporation and poor retention of H2AX at the DSBs. We also identified a unique N-terminal region of CHD1 that inhibits the DNA binding, ATPase, and chromatin assembly and remodeling activities of CHD1. CHD1 lacking the N terminus was more active in rescuing the defects in γH2AX formation and CtIP recruitment in CHD1-KO cells than full-length CHD1, suggesting the N terminus is a negative regulator in cells. Our data point to a role for CHD1 in the DSB repair process and identify a novel regulatory region of the protein.

Human CHD2 is a chromatin assembly ATPase regulated by its chromo- and DNA-binding domains.

Chromodomain helicase DNA-binding protein 2 (CHD2) is an ATPase and a member of the SNF2-like family of helicase-related enzymes. Although deletions of CHD2 have been linked to developmental defects in mice and epileptic disorders in humans, little is known about its biochemical and cellular activities. In this study, we investigate the ATP-dependent activity of CHD2 and show that CHD2 catalyzes the assembly of chromatin into periodic arrays. We also show that the N-terminal region of CHD2, which contains tandem chromodomains, serves an auto-inhibitory role in both the DNA-binding and ATPase activities of CHD2. While loss of the N-terminal region leads to enhanced chromatin-stimulated ATPase activity, the N-terminal region is required for ATP-dependent chromatin remodeling by CHD2. In contrast, the C-terminal region, which contains a putative DNA-binding domain, selectively senses double-stranded DNA of at least 40 base pairs in length and enhances the ATPase and chromatin remodeling activities of CHD2. Our study shows that the accessory domains of CHD2 play central roles in both regulating the ATPase domain and conferring selectivity to chromatin substrates.

Adverse Childhood Experiences (ACEs) in Unhoused Children Increase Odds of Psychiatric Illness, Physical Illness, and Psychiatric Admission.

Unhoused children and adolescents have high rates of adverse childhood experiences (ACEs). The objective of this study was to characterize a large cohort of unhoused children and investigate rates of psychiatric diagnoses, medical diagnoses, and utilization of emergency department (ED) resources depending on the presence of additional documented ACEs. A retrospective cohort of all unhoused children who presented to the ED of a large Midwestern health system from January 2014 to July 2019 were included. Unhoused status was determined by address field or ICD-10 code for homelessness (Z59.0). Demographics and ED visits were extracted from the electronic health record. Past medical history, ACEs, chief complaint (CC), length of stay (LOS), imaging, and labs were extracted by chart review. T-tests, chi square tests, and Fisher's exact tests were completed for each sub-analysis. Unhoused children with at least one additional ACE had higher odds of the following psychiatric disorders: depression (OR = 5.2, 95% CI = 3.4- 7.9), anxiety (OR = 3.4, 95% CI = 32.1-5.5), behavioral disorder (OR = 7.2, 95% CI = 35.1- 10.4), psychoses (OR = 6.0, 1.9-18.4), bipolar disorder (OR = 19.8, 95% CI = 34.6-84.9), suicidal ideation (OR = 8.0, 95% CI = 34.8-13.4), post-traumatic stress disorder (OR = 10.1, 95% CI = 35.4-18.6), and attention deficit hyperactive disorder (OR = 4.1, 3.0-5.7). Patients with additional documented ACEs were also more likely to have a prior psychiatric admission (p < 0.001). Unhoused children and adolescents with exposure to additional documented ACEs are more likely to have some serious psychiatric and medical diagnoses compared to other unhoused children.

Consequences of anti-vascular endothelial growth factor treatment lapse in patients with retinal vein occlusion.

BACKGROUND: Anti-vascular endothelial growth factor (VEGF) treatments are the first-line treatment for Retinal Vein Occlusion (RVO). Although effectiveness and safety of these treatments is well documented, knowledge regarding the effect of lapses in anti-VEGF treatment among RVO patients is lacking. The purpose of this study is to analyse the anatomic and visual outcomes from a lapse in anti-VEGF treatment in patients with RVO. METHODS: This retrospective case-control study evaluated 136 patients diagnosed with RVO and treated with anti-VEGF between January 2012 and June 2020 at Cole Eye Institute, Cleveland Clinic. Patients were divided into two cohorts: RVO patients with no lapse in anti-VEGF treatment (control group) and RVO patients with a lapse ≥3 months (lapse group). Central subfield thickness (CST) and best corrected visual acuity (BCVA) were collected pre-lapse, the first appointment post-lapse, and at 3-, 6-, and 12-month follow-up appointments. RESULTS: Lapse patients (n = 68) and control patients (n = 68) had similar pre-lapse CST (p = 0.466) and BCVA (p = 0.303). Lapse patients experienced a significant increase in CST after discontinuing anti-VEGF therapy (lapse: 400.6 ± 192.1 µm, control: 333.0 ± 111.1 µm, p = 0.024). This persisted 12 months post-lapse after re-initiation of anti-VEGF agents (lapse: 381.6 ± 161.1 µm, control: 307.5 ± 95.4 µm, p = 0.030). Lapse patients also experienced a decrease in BCVA after lapse (lapse: 54.3 ± 25.1 ETDRS, control: 64.4 ± 17.8 ETDRS, p < 0.001) that recovered after 6 months of anti-VEGF treatment. CONCLUSIONS: RVO patients with any lapse of anti-VEGF treatment are at risk for poorer anatomic and visual outcomes. Though BCVA normalizes upon treatment resumption, patients experience a statistically significant increase in CST that does not recover.

Predictors of response to a lapse in anti-VEGF treatment in patients with macular edema secondary to retinal vein occlusion.

BACKGROUND: Macular edema (ME) in the setting of retinal vein occlusions (RVO) is a common cause of vision loss worldwide. Anti-vascular endothelial growth factor (anti-VEGF) injections are the gold standard for ME secondary to RVO. Despite their efficacy, anti-VEGF injections carry significant burdens for patients, resulting in high rates of loss to follow-up and treatment lapses. METHODS: A sub-analysis examining the effects of a treatment lapse in RVO patients was conducted. Sixty patients were included and separated into vision-loss and stable-vision groups based on change in vision after a lapse. A logistic regression with age, body mass index (BMI), history of dyslipidemia, and time since diagnosis of RVO as predictors was used to predict whether patients would experience vision loss after a lapse. RESULTS: The average lapse was 5.6 months and similar in the vision-loss and stable-vision groups. At baseline, the vision-loss group was older and had a lower BMI (p < 0.05). Age and history of dyslipidemia increased the odds of vision loss by factors of 1.23 (range, 1.10-1.45) and 8.40 (range, 1.62-66.2), respectively. BMI and time since RVO diagnosis decreased the odds of vision loss by factors of 0.83 (range, 0.69-0.95) and 0.95 (range, 0.90-0.99), respectively. The final model had a specificity of 87.5% and a sensitivity of 70.0%. CONCLUSIONS: Patients' responses to treatment lapses for ME secondary to RVO can be predicted with reasonable accuracy using readily available clinical data, particularly age, BMI, time since diagnosis, and history of dyslipidemia. Providers should consider these factors when counselling patients and determining follow-up schedules.

Sarcopenia in Patients With Rectal Adenocarcinoma: An Opportunity for Preoperative Rehabilitation.

BACKGROUND: Sarcopenia, defined as low skeletal muscle mass, affects up to 60% of rectal adenocarcinoma patients receiving neoadjuvant chemoradiation (NACRT), with negative impact on patient outcomes. Identifying modifiable risk factors may decrease morbidity and mortality. METHODS: A retrospective review of rectal cancer patients from a single academic center from 2006 to 2020 was performed. Sixty-nine patients with pre- and post-NACRT CT imaging were included. Skeletal muscle index (SMI) was calculated as total L3 skeletal muscle divided by height squared. Sarcopenia thresholds were 52.4 cm2/m2 for men and 38.5 cm2/m2 for women. Student T-test, chi-square test, multivariable regression, and multivariable Cox hazard analysis were performed. RESULTS: 62.3% of patients lost SMI from pre- to post-NACRT imaging, with a mean change of -7.8% (±19.9%). Eleven (15.9%) patients were sarcopenic at presentation, increasing to 20 (29.0%) following NACRT. Mean SMI decreased from 49.0 cm2/m2 (95% CI: 42.0 cm2/m2-56.0 cm2/m2) to 38.2 cm2/m2 (95% CI: 33.6 cm2/m2-42.9 cm2/m2) (P = .003). Pre-NACRT sarcopenia correlated with post-NACRT sarcopenia (OR 20.6, P = .002). Percent decrease in SMI was associated with a 5% increased mortality risk. CONCLUSION: The presence of sarcopenia at diagnosis and its association with post-NACRT sarcopenia suggests an opportunity for a high-impact intervention.

Outcomes in patients with retinal vein occlusion with good baseline visual acuity.

BACKGROUND: Intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) are first-line therapy for macular oedema in retinal vein occlusion (RVO). Appropriate management for RVO with good visual acuity at diagnosis has not been evaluated. The purpose of this study is to analyse the visual and anatomic outcomes from anti-VEGF treatment among RVO patients with good vision at baseline. METHODS: This retrospective cohort study evaluated patients diagnosed with macular oedema secondary to RVO from January 2012 to February 2021 at a tertiary ophthalmic centre. Patients had a Snellen acuity of 20/32 or better at diagnosis. Three cohorts were compared: patients with no anti-VEGF treatment, delayed anti-VEGF treatment (initial injection >30 days post-diagnosis) and immediate anti-VEGF treatment (initial injection ≤30 days post-diagnosis). Central subfield thickness (CST) and best visual acuity (BVA) were collected at diagnosis and 6-, 12- and 24-month follow-up appointments. RESULTS: Among 131 eyes, mean BVA values among treatment groups did not differ at 6-, 12- or 24-month follow up visits (P = 0.521, 0.426, 0.356, respectively). The percentage of eyes with at least a 5-letter BVA decrease at 24 months was 24.1%, 65.0% and 30.8% in the no treatment, delayed and immediate treatment groups respectively (P = 0.010). There was no significant difference in the percentage of eyes with at least a 10% decrease in CST at 24 months among groups (P = 0.095). CONCLUSIONS: Close observation with initiation of treatment in patients with good visual acuity with macular oedema secondary to RVO as indicated has similar outcomes in the setting of routine clinical practice.

Limited Effect of Indolamine 2,3-Dioxygenase Expression and Enzymatic Activity on Lupus-Like Disease in B6.Nba2 Mice.

B6.Nba2 mice spontaneously develop a lupus-like disease characterized by elevated levels of serum anti-nuclear autoantibody (ANA) immune complexes and constitutive type I interferon (IFNα) production. During disease progression, both plasmacytoid dendritic cells (pDCs) and antibody secreting plasma cells accumulate in spleens of B6.Nba2 mice. Indoleamine 2,3-dioxygenase (IDO) has been suggested to play a role in several autoimmune diseases including in the MRL/lpr model of mouse lupus-like disease; however, it remains unknown if IDO is involved in disease development and/or progression in other spontaneous models. We show here that IDO1 protein and total IDO enzymatic activity are significantly elevated in lupus-prone B6.Nba2 mice relative to B6 controls. IDO1 expression was restricted to PCs and SignR1+ macrophages in both strains, while significantly increased in B6.Nba2-derived SiglecH+ (SigH+) pDCs. Despite this unique expression pattern, neither pharmacologic inhibition of total IDO nor IDO1 gene ablation altered serum autoantibody levels, splenic immune cell activation pattern, or renal inflammation in B6.Nba2 mice. Interestingly, IDO pharmacologic inhibition, but not IDO1 deficiency, resulted in diminished complement factor C'3 fixation to kidney glomeruli, suggesting a possible therapeutic benefit of IDO inhibition in SLE patients with renal involvement.

Cyclic changes in metabolic state during the life of a yeast cell.

Budding yeast undergo robust oscillations in oxygen consumption during continuous growth in a nutrient-limited environment. Using liquid chromatography-mass spectrometry and comprehensive 2D gas chromatography-mass spectrometry-based metabolite profiling methods, we have determined that the intracellular concentrations of many metabolites change periodically as a function of these metabolic cycles. These results reveal the logic of cellular metabolism during different phases of the life of a yeast cell. They may further indicate that oscillation in the abundance of key metabolites might help control the temporal regulation of cellular processes and the establishment of a cycle. Such oscillations in metabolic state might occur during the course of other biological cycles.