Exploiting replication gaps for cancer therapy.

Defects in DNA double-strand break repair are thought to render BRCA1 or BRCA2 (BRCA) mutant tumors selectively sensitive to PARP inhibitors (PARPis). Challenging this framework, BRCA and PARP1 share functions in DNA synthesis on the lagging strand. Thus, BRCA deficiency or "BRCAness" could reflect an inherent lagging strand problem that is vulnerable to drugs such as PARPi that also target the lagging strand, a combination that generates a toxic accumulation of replication gaps.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency.

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

The effectiveness of poly (ADP-ribose) polymerase inhibitors (PARPi) in creating single-stranded DNA gaps and inducing sensitivity requires the FANCJ DNA helicase. Yet, how FANCJ relates to PARP1 inhibition or trapping, which contribute to PARPi toxicity, remains unclear. Here, we find PARPi effectiveness hinges on S-phase PARP1 activity, which is reduced in FANCJ deficient cells as G-quadruplexes sequester PARP1 and MSH2. Additionally, loss of the FANCJ-MLH1 interaction diminishes PARP1 activity; however, depleting MSH2 reinstates PARPi sensitivity and gaps. Indicating sequestered and trapped PARP1 are distinct, FANCJ loss increases PARPi resistance in cells susceptible to PARP1 trapping. However, with BRCA1 deficiency, the loss of FANCJ mirrors PARP1 loss or inhibition, with the detrimental commonality being loss of S-phase PARP1 activity. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA1 deficient cells and emphasize the importance of understanding drug mechanisms for enhancing therapeutic response.

FANCJ promotes PARP1 activity during DNA replication that is essential in BRCA1 deficient cells.

Single-stranded DNA gaps are postulated to be fundamental to the mechanism of anti-cancer drugs. Gaining insights into their induction could therefore be pivotal for advancing therapeutic strategies. For poly (ADP-ribose) polymerase inhibitors (PARPi) to be effective, the presence of FANCJ helicase is required. However, the relationship between FANCJ dependent gaps and PARP1 catalytic inhibition or trapping-both linked to PARPi toxicity in BRCA deficient cells-is yet to be elucidated. Here, we find that the efficacy of PARPi is contingent on S-phase PARP1 activity, which is compromised in FANCJ deficient cells because PARP1, along with MSH2, is "sequestered" by G-quadruplexes. PARP1's replication activity is also diminished in cells missing a FANCJ-MLH1 interaction, but in such cells, depleting MSH2 can release sequestered PARP1, restoring PARPi-induced gaps and sensitivity. Our observations indicate that sequestered and trapped PARP1 are different chromatin-bound forms, with FANCJ loss increasing PARPi resistance in cells susceptible to canonical PARP1 trapping. However, in BRCA1 null cells, the loss of FANCJ mirrors the effects of PARP1 loss or inhibition, with the common detrimental factor being the loss of PARP1 activity during DNA replication, not trapping. These insights underline the crucial role of PARP1 activity during DNA replication in BRCA deficient cells and emphasize the importance of understanding drug mechanisms for enhancing precision medicine.

Exploration of three different nutritional scores in predicting postoperative complications after pancreaticoduodenectomy.

INTRODUCTION: Objectives: we used the Controlling Nutritional Status score (CONUT), Geriatric Nutritional Risk Index (GNRI), and Prognostic Nutritional Index (PNI) to explore three different nutritional scores in predicting postoperative complications after pancreaticoduodenectomy (PD). Methods: data were retrospectively reviewed from 113 patients who underwent PD to treat pancreatic cancer and periampullary neoplasms at the Second Affiliated Hospital of Soochow University between 2015 and 2020. Nutritional status was assessed by the CONUT, GNRI, and PNI scores, and patients were categorized as either at risk or not at risk for malnutrition by each score. Postoperative complications were defined according to the Clavien-Dindo classification. Data were analyzed using Fisher's exact probability method and multivariate logistic regression analysis. The relationships between the three nutritional scoring systems and postoperative complications were examined. Results: CONUT, GNRI and PNI scores were closely related to the occurrence of postoperative complications. CONUT (OR = 0.92, 95 % CI, 0.75-1.12, p = 0.043), GNRI (OR = 0.98, 95 % CI, 0.93-1.02, p = 0.036), PNI (OR = 0.96, 95 % CI, 0.89-1.03, p = 0.024), and operation periods (OR = 1.01, 95 % CI, 0.99-1.02, p = 0.034) were independent risk factors for complications in patients after PD. The predictive value of the three nutritional screening methods for overall complications in patients with PD had a sensitivity of 31.8 %, 56.06 % and 74.24 %, a specificity of 85.10 %, 68.08 % and 76.81 %, a Youden index of 0.17, 0.24 and 0.71, and a kappa value of 0.460, 0.389 and 0.472, respectively. The predictive value of the three nutritional screening methods in predicting the severity of complications in patients with PD had a sensitivity of 82.11 %, 58.95 % and 65.26 %, a specificity of 38.89 %, 55.56 % and 66.67 %, a Youden index of 0.21, 0.15 and 0.36, and a kappa value of 0.664, 0.416 and 0.645, respectively. Among the three nutrition scoring systems, PNI score had better diagnostic efficiency (0.660 area under the AUC curve), higher specificity (66.67 %), and was consistent with postoperative complications (KCONUT = 0.664, KGNRI = 0.416, KPNI = 0.645) when compared to the GNRI and CONUT scores. Conclusions: CONUT, GNRI and PNI scores, especially PNI score, have good predictive values for the occurrence and severity of postoperative complications in PD patients, and should be used as preoperative nutritional risk screening tools for PD patients.

INTRODUCCIÓN: Objetivos: se utilizaron las escalas Controlling Nutritional Status (CONUT), Geriatric Nutritional Risk Index (GNRI) y Prognostic Nutritional Index (PNI) para explorar tres puntuaciones nutricionales diferentes en la predicción de las complicaciones posoperatorias después de la pancreaticoduodenectomía (PD). Métodos: en este estudio se revisaron retrospectivamente los datos de 113 pacientes después de una PD entre 2015 y 2020. El estado nutricional se evaluó mediante las puntuaciones CONUT, GNRI y PNI. Se examinaron las relaciones entre tres sistemas de puntuación nutricional y las complicaciones posoperatorias. Resultados: las puntuaciones CONUT, GNRI y PNI estuvieron estrechamente relacionadas con la aparición de complicaciones posoperatorias. CONUT (OR = 0,92, IC del 95 %: 0,75-1,12, p = 0,043), GNRI (OR = 0,98, IC del 95 %: 0,93-1,02, p = 0,036), PNI (OR = 0,96, IC del 95 %: 0,89-1,03, p = 0,024) y períodos de operación (OR = 1,01, IC del 95 %: 0,99-1,02, p = 0,034) fueron factores de riesgo independientes de aparición de complicaciones en los pacientes después de la DP. El valor predictivo de los tres métodos de cribado nutricional para las complicaciones globales de los pacientes con DP tuvo una sensibilidad del 31,8 %, 56,06 % y 74,24 %, una especificidad del 85,10 %, 68,08 % y 76,81 %, un índice de Youden de 0,17, 0,24 y 0,71, y un valor kappa de 0,460, 0,389 y 0,472, respectivamente. El valor predictivo de los tres métodos de cribado nutricional para predecir la gravedad de las complicaciones en pacientes con DP tuvo una sensibilidad del 82,11 %, 58,95 % y 65,26 %, una especificidad del 38,89 %, 55,56 % y 66,67 %, un índice de Youden de 0,21, 0,15 y 0,36, y un valor kappa de 0,664, 0,416 y 0,645, respectivamente. Entre los tres sistemas de puntuación nutricional, la puntuación PNI obtuvo una mejor eficiencia diagnóstica (área bajo la curva AUC: 0,660) y una mayor especificidad (66,67 %), y fue más consistente con respecto a las complicaciones posoperatorias (KCONUT = 0,664, KGNRI = 0,416, KPNI = 0,645), comparada con las puntuaciones GNRI y CONUT. Conclusiones: las puntuaciones CONUT, GNRI y PNI, especialmente la puntuación PNI, tienen buenos valores predictivos para la aparición y gravedad de las complicaciones posoperatorias en pacientes con DP, y deben utilizarse como herramientas de cribado de riesgo nutricional preoperatorio en dichos pacientes con DP.

The prognostic significance of LTBP2 for malignant tumors: Evidence based on 11 observational studies.

BACKGROUND AIMS: At present, increasing reports have shown that latent transforming growth factor-ß-binding protein 2 (LTBP2) was associated with the prognosis of many types of cancer. We performed rounded analysis to comprehensively analyze and evaluate the prognostic significance of LTBP2 for patients with malignant tumors. METHODS: We identified relevant studies by searching database including PubMed, Embase, Cochrane Library, and Web of Science. The odds ratio with its 95% confidence interval (CI) was used to assess the correlation between LTBP2 and clinicopathologic features or overall survival of patients with cancer. Hazard ratio with its 95% CI was used to explore the prognostic risk factors. The analysis was performed and assessed using Review Manager 5.2. RESULTS: A total of 11 studies including 2322 participants were included in this systematic review. Pooled results showed that malignant tissues experienced higher incidence of high LTBP2 expression when compared with adjacent or normal tissues. Patients with high LTBP2 expression experienced significantly lower 1-year, 2-year, 3-year, and 4-year overall survival rate, with the pooled odds ratios being 0.26 (95% CI 0.13-0.53; P = .0002), 0.27 (95% CI 0.14-0.50; P < .0001), 0.26 (95% CI 0.13-0.53; P = .0002), and 0.21 (95% CI 0.06-0.73; P = .01) respectively. Univariate analysis showed high LTBP2 expression, tumor node metastasis stage, T stage, and N stage were prognostic factors of patients with tumors. Multivariate analysis indicated high LTBP2 expression was an independent prognostic factor. CONCLUSIONS: The present analysis suggested that LTBP2 may have significant association with survival of patients with cancer. High LTBP2 expression was an independent prognostic factor and indicated poor survival.

Comparison between fluoropyrimidine-hepatic arterial infusion and systemic chemotherapy for unresectable liver metastases: A protocol for systematic review and meta-analysis based on 16 observational studies.

BACKGROUND: The benefit of loco-regional treatments such as hepatic arterial infusion (HAI) in terms of survival and response rate is unclear. The aim of this work is to quantitatively summarize the results of both randomized controlled trials (RCTs) and non-randomized studies of interventions (NRSIs) comparing fluoropyrimidine-HAI (F-HAI) to systemic chemotherapy (SCT) for the treatment of colorectal liver metastases (CRLMs). METHODS: We searched the Cochrane Library, PubMed, EMBASE, and Web of Science up to July 1, 2021. The outcome measures were tumor response rate and overall survival (OS). Both RCTs and NRSIs comparing HAI to SCT for patients with unresectable CRLMs were included. The outcome measures were tumor response rate and OS. Two reviewers assessed trial quality and extracted data independently. All statistical analyses were performed using standard statistical procedures provided in Review Manager 5.2. RESULTS: A total of 16 studies including 11 RCTs and 5 NRSIs were identified for the present meta-analysis. Nine RCTs compared F-HAI to SCT for patients with unresectable CRLMs and the pooled result indicated that patients who received F-HAI experienced more than twofold response rate than SCT, with a pooled risk ratio of 2.10 (95%CI 1.59-2.79; P < .00001). In addition, the pooled result based on RCTs showed that F-HAI had a significant benefit regarding OS, with a pooled HR of 0.83 (95% CI 0.70-0.99; P = .04). Similarly, the benefit of F-HAI in terms of OS was also observed in the results of NRSIs. CONCLUSIONS: Our results indicated that the F-HAI regimen had a greater tumor response rate and survival advantage than SCT for patients with unresectable CRLMs. Future propensity score-matched analyses with a large sample size should be conducted to support the evidence of our results based on RCTs and NRSIs.

Replication Gaps Underlie BRCA Deficiency and Therapy Response.

Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.See related commentary by Canman, p. 1214.

Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability.

The replication stress response, which serves as an anticancer barrier, is activated not only by DNA damage and replication obstacles but also oncogenes, thus obscuring how cancer evolves. Here, we identify that oncogene expression, similar to other replication stress-inducing agents, induces single-stranded DNA (ssDNA) gaps that reduce cell fitness. DNA fiber analysis and electron microscopy reveal that activation of translesion synthesis (TLS) polymerases restricts replication fork slowing, reversal, and fork degradation without inducing replication gaps despite the continuation of replication during stress. Consistent with gap suppression (GS) being fundamental to cancer, we demonstrate that a small-molecule inhibitor targeting the TLS factor REV1 not only disrupts DNA replication and cancer cell fitness but also synergizes with gap-inducing therapies such as inhibitors of ATR or Wee1. Our work illuminates that GS during replication is critical for cancer cell fitness and therefore a targetable vulnerability.

Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress.

The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.