Chemical Inhibition of RPA by HAMNO Alters Cell Cycle Dynamics by Impeding DNA Replication and G2-to-M Transition but Has Little Effect on the Radiation-Induced DNA Damage Response.

Replication protein A (RPA) is the major single-stranded DNA (ssDNA) binding protein that is essential for DNA replication and processing of DNA double-strand breaks (DSBs) by homology-directed repair pathways. Recently, small molecule inhibitors have been developed targeting the RPA70 subunit and preventing RPA interactions with ssDNA and various DNA repair proteins. The rationale of this development is the potential utility of such compounds as cancer therapeutics, owing to their ability to inhibit DNA replication that sustains tumor growth. Among these compounds, (1Z)-1-[(2-hydroxyanilino) methylidene] naphthalen-2-one (HAMNO) has been more extensively studied and its efficacy against tumor growth was shown to arise from the associated DNA replication stress. Here, we study the effects of HAMNO on cells exposed to ionizing radiation (IR), focusing on the effects on the DNA damage response and the processing of DSBs and explore its potential as a radiosensitizer. We show that HAMNO by itself slows down the progression of cells through the cell cycle by dramatically decreasing DNA synthesis. Notably, HAMNO also attenuates the progression of G2-phase cells into mitosis by a mechanism that remains to be elucidated. Furthermore, HAMNO increases the fraction of chromatin-bound RPA in S-phase but not in G2-phase cells and suppresses DSB repair by homologous recombination. Despite these marked effects on the cell cycle and the DNA damage response, radiosensitization could neither be detected in exponentially growing cultures, nor in cultures enriched in G2-phase cells. Our results complement existing data on RPA inhibitors, specifically HAMNO, and suggest that their antitumor activity by replication stress induction may not extend to radiosensitization. However, it may render cells more vulnerable to other forms of DNA damaging agents through synthetically lethal interactions, which requires further investigation.

Study on the allosteric activation mechanism of SHP2 via elastic network models and neural relational inference molecular dynamics simulation.

As a ubiquitous protein tyrosine phosphatase, SHP2 is involved in PD-1/PD-L1 mediated tumor immune escape and undergoes substantial conformational changes. Therefore, it is considered an ideal target for tumor intervention. However, the allosteric mechanisms of SHP2 binding PD-1 intracellular ITIM/ITSM phosphopeptides remain unclear, which greatly hinders the development of novel structure-based anticancer allosteric inhibitors. In this work, the open and closed structural models of SHP2 are first constructed based on this knowledge; next their motion modes are investigated via elastic network models such as the Gaussian network model (GNM), anisotropic network model (ANM) and adaptive anisotropic network model (aANM); and finally, a possible allosteric signaling pathway is proposed using a neural relational inference molecular dynamics (NRI-MD) simulation embedded with an artificial intelligence (AI) strategy. In GNM and ANM, the N-SH2, C-SH2 and PTP domains all exhibit distinct dynamics partitions, and the N-SH2/C-SH2 regions show a rigid rotation relative to PTP. According to a series of intermediate snapshots given by aANM, N-SH2 is first identified with pY223 specifically, inducing a D'E-loop to change from ß-sheets to random coils, and then, C-SH2 serves as a fulcrum to drive N-SH2 to rotate 110° completely away from the original active sites of PTP. Finally, a possible allosteric signaling-transfer path for SHP2, namely R220-R138-T108-R32, is proposed based on NRI-MD sampling. This work provides a possible allosteric mechanism of SHP2, which is helpful for the following design of novel allosteric inhibitors and is expected to be used in clinical synergies with PD-1 monoclonal antibody.

The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G2-Checkpoint Activation in Cells Exposed to Ionizing Radiation.

We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.

Preliminary results of randomized phase II study of etoposide plus lobaplatin or etoposide plus cisplatin with concurrent thoracic radiotherapy in the treatment of limited-stage small cell lung cancer.

The purpose is to compare the clinical efficacy and toxicity of etoposide plus lobaplatin (EL) or etoposide plus cisplatin (EP) with concurrent thoracic radiotherapy during the treatment of limited-stage small cell lung cancer (LS-SCLC). Forty-two patients with LS-SCLC were randomly divided into EL ( n = 19) or EP ( n = 23) regimens combined with thoracic intensity-modulated radiotherapy. The primary endpoint was 1-year progression-free survival (PFS) rate. The 1-, 2-, and 3-year PFS rates in the EL and EP cohorts were 50.8, 38.1, and 12.7%; and 56.5, 43.5, and 29.0%, respectively ( P = 0.527), whereas the 1-, 2-, and 3-year overall survival (OS) rates were 72.2, 52.5, and 43.8%; and 73.9, 48.4, and 48.4%, respectively ( P = 0.923). The hematological toxicities were similar in two cohorts. However, gastrointestinal reactions were more severe in the EP group. The incidence of nausea and vomiting in EL and EP cohorts were 31.6% vs. 73.9% ( P = 0.006) and 20.1% vs. 60.9% ( P = 0.009), respectively. The two cohorts did not show ≥grade 4 radiation esophagitis and ≥grade 3 radiation pneumonitis. The incidence of acute radiation esophagitis in EL group was lower ( P = 0.038), both groups showed a similar incidence of radiation pneumonitis ( P = 1.000). EL or EP chemotherapy with concurrent thoracic radiotherapy showed similar PFS and OS. The EL group showed milder gastrointestinal toxicity and radiation esophagitis. Radiation pneumonitis and hematological toxicity were similar in the two regimens, which can be tolerated by patients.

Recombinant human endostatin combined with radiotherapy promotes cardiomyocyte apoptosis in rats via TGFß1/Smads/CTGF signaling pathway.

PURPOSE: The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of transforming growth factor beta1 (TGF-ß1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling. METHOD: The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-ß1 siRNA (gene silencing) group, ES + siTGF-ß1 siRNA group, and 10 Gy radiation + ES + siTGF-ß1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC50) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-ß1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-ß1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis. RESULTS: ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-ß1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-ß1 gene (P < 0.05). CONCLUSION: The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-ß1/Smad3/CTGF signaling pathway.

Loss of TGFß signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types.

Among the pleotropic roles of transforming growth factor-ß (TGFß) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFß signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFß broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFß and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFß and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFß competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFß and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFß signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFß biology.

Pemetrexed plus cisplatin versus docetaxel plus cisplatin for stage IV lung adenocarcinoma based on propensity score matching.

The aim of this study was to compare the clinical efficacy of pemetrexed+cisplatin (PP) versus docetaxel+cisplatin (DP) for the treatment of stage IV lung adenocarcinoma. We retrospectively analyzed the clinical data of 147 patients with stage IV lung adenocarcinoma treated between January 2011 and December 2015, 100 of which were in the DP group whereas 47 were in the DP group. Main inclusion criteria were treatment-naive patients, first-line treatment with PP or DP with no molecular targeted therapy during treatment, 2-6 cycles of first-line chemotherapy with unknown status of epidermal growth factor receptor (EGFR) mutation, 18-75 years of age, and Karnofsky performance status score of at least 70. Prognostic factors for survival were identified by using univariate and multivariate analyses. Propensity score matching was performed to further adjust for confounding. A total of 47 pairs were successfully matched between the two groups. The median overall survival was 9.0 months in the DP group and 17.0 months in the PP group; the 1-year survival rate was 29.8 and 59.6%, respectively; the 2-year survival rate was 12.8 and 21.1%, respectively (χ=4.128, P=0.042); and median progression-free survival was 6.0 and 8.0 months, respectively (χ=4.839, P=0.028). Cox multivariate analysis showed that chemotherapy regimen and number of metastatic organs were independent factors for OS. The effect of the radiotherapy dose on the primary tumor on OS was close to statistically significant. The incidence of grade 3-4 neutropenia was more significantly reduced in the DP group than in the PP group after matching (61.7 vs. 27.7%, P=0.002), with no between-group difference for adverse effects on platelets or hemoglobin. For patients with stage IV lung adenocarcinoma and unknown EGFR mutation status, PP was more effective than DP in prolonging survival and had a less adverse effect on neutrophils.

Primary mediastinal sarcoma: surgical outcomes of 21 cases.

OBJECTIVES: Primary sarcomas of the mediastinum are relatively rare. This article reviews the surgical outcomes of 21 cases diagnosed with localized mediastinal sarcomas receiving multidisciplinary treatment modalities in Sichuan province, China, from January 1996 to January 2011. METHODS: Twenty-one cases of histologically diagnosed primary mediastinal sarcoma undergoing surgical treatment were reviewed retrospectively. Disease-free survival (DFS) and overall survival (OS) were statistically analysed. All the patients presented with localized tumours consisting of 5 females and 16 males with a median age of 41.0 years (range: 9.0-68.0 years). Among all cases, 17 (81.0%) had an Eastern Cooperative Oncology Group performance status score of ≤1 at diagnosis. Eight (38.1%) underwent macroscopically complete resection (R0-R1) and 13 (61.9%) had incomplete resection (R2). Ten (47.6%) received postoperative radiotherapy and 7 (33.3%) postoperative chemotherapy. RESULTS: The median DFS was 17 months (range: 0.4-79.8 months) and the median OS was 27.2 months (range: 0.4-79.8 months). Patients receiving complete resection showed significantly improved DFS (P = 0.031) and OS (P = 0.035) compared with those with incomplete resection. Neither postoperative radiotherapy nor chemotherapy significantly improved DFS (P = 0.770, P = 0.756) or OS (P = 0.905, P = 0.738). However, 7 patients (R2) and 2 (R0-R1 and grade 3) had improved local control with a local recurrence-free survival of 28.9 months (range: 7.6-73.2 months). CONCLUSIONS: Complete resection should be preferentially attempted compared with incomplete resection and postoperative radiotherapy might yield good local control.

Salvage treatment with erlotinib after gefitinib failure in advanced non-small-cell lung cancer patients with poor performance status: A matched-pair case-control study.

PURPOSE: Gefitinib plays an important role in non-small-cell lung cancer (NSCLC) treatment; however, progression of the disease occurs in most patients even after an initial response. The role of erlotinib after gefitinib failure has been investigated but continues to be debated, especially in heavily treated patients with poor performance status (PS). Therefore, a retrospective matched-pair case-control study was carried out to evaluate the role of erlotinib after gefitinib failure in advanced NSCLC patients. METHODS: A total of 58 patients were identified. The two groups were balanced with demographic and baseline clinical characteristics. All patients had PS ≥2 and most of them (89.7%) had received more than two systemic therapies before erlotinib or best supportive care (BSC). The epidermal growth factor receptor (EGFR) and KRAS genotypes were analyzed in 36 (62.1%) patients, 19 of them were in the erlotinib group. RESULTS: Median overall survival (OS) for all patients was 6 months. Median OS for patients who received erlotinib and BSC was 10 and 3 months, respectively (P= 0.001). Disease control rate (DCR) and objective response rate (ORR) were 51.7% and 10.3% in patients receiving erlotinib, respectively, while median time to progression (TTP) was 3 months. Among the 19 patients in the erlotinib group with biomarker results available, those with EGFR mutation achieved longer median TTP (P= 0.016) and better DCR (P= 0.177) than those with wild-type EGFR. CONCLUSIONS: A switch to erlotinib after gefitinib failure may represent a better therapeutic option for advanced NSCLC patients with poor PS, and an EGFR mutation seemed to be associated with better survival rate.