Ziyan Liu

SMARCA4/BRG1 Is a Novel Prognostic Biomarker Predictive of Cisplatin-Based Chemotherapy Outcomes in Resected Non–Small Cell Lung Cancer

Purpose: Identification of predictive biomarkers is critically needed to improve selection of patients who derive the most benefit from platinum-based chemotherapy. We hypothesized that decreased expression of SMARCA4/BRG1, a known regulator of transcription and DNA repair, is a novel predictive biomarker of increased sensitivity to adjuvant platinum-based therapies in non–small cell lung cancer (NSCLC). Experimental Design: The prognostic value was tested using a gene-expression microarray from the Directors Challenge Lung Study (n = 440). The predictive significance of SMARCA4 was determined using a gene-expression microarray (n = 133) from control and treatment arms of the JBR.10 trial of adjuvant cisplatin/vinorelbine. Kaplan–Meier method and log-rank tests were used to estimate and test the differences of probabilities in overall survival (OS) and disease-specific survival (DSS) between expression groups and treatment arms. Multivariate Cox regression models were used while adjusting for other clinical covariates. Results: In the Directors Challenge Study, reduced expression of SMARCA4 was associated with poor OS compared with high and intermediate expression (P < 0.001 and P = 0.009, respectively). In multivariate analysis, compared with low, high SMARCA4 expression predicted a decrease in risk of death [HR, 0.6; 95% confidence interval (CI), 0.4–0.8; P = 0.002]. In the JBR.10 trial, improved 5-year DSS was noted only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine [HR, 0.1; 95% CI, 0.0–0.5, P = 0.002 (low); HR, 1.0; 95% CI, 0.5–2.3, P = 0.92 (high)]. An interaction test was highly significant (P = 0.01). Conclusions: Low expression of SMARCA4/BRG1 is significantly associated with worse prognosis; however, it is a novel significant predictive biomarker for increased sensitivity to platinum-based chemotherapy in NSCLC. Clin Cancer Res; 22(10); 2396–404. ©2015 AACR.

Association of MGMT Promoter Methylation Status With Survival Outcomes in Patients With High-Risk Glioma Treated With Radiotherapy and Temozolomide: An Analysis From the NRG Oncology/RTOG 0424 Trial

Importance The initial report of NRG Oncology/Radiation Therapy Oncology Group (RTOG) 0424 demonstrated a 3-year overall survival benefit with the addition of temozolomide to radiotherapy compared with a historical control. However, an important end point of the trial—evaluation of the association between O6-methylgaunine-DNA-methyltransferase (MGMT) promoter methylation and survival outcomes—was not previously reported. Objective To examine the proportion of patients in NRG Oncology/RTOG 0424 with MGMT promoter methylation and its association with survival outcomes. Design, Setting, and Participants Specimens collected were analyzed after trial completion to determine MGMT promoter methylation and IDH1/2 status and the association between MGMT status and survival outcomes. A model derived from logistic regression (MGMT-STP27) was used to calculate MGMT promoter methylation status. Univariate and multivariable analyses were performed using the Cox proportional hazards regression model to determine the association of MGMT status with survival outcomes. Patient pretreatment characteristics were included as covariates in multivariable analyses. Main Outcomes and Measures Progression-free survival (PFS) and overall survival (OS). Results Of all 129 eligible patients in NRG Oncology/RTOG 0424, 75 (58.1%) had MGMT status available (median age, 48 years; age range, 20-76 years; 42 [56.0%] male): 57 (76.0%) methylated and 18 (24.0%) unmethylated. A total of 13 unmethylated patients (72.2%) had astrocytoma as opposed to oligoastrocytoma or oligodendroglioma, whereas 23 methylated patients (40.4%) had astrocytoma. On univariate analyses, an unmethylated MGMT promoter was significantly associated with worse OS (hazard ratio [HR], 3.52; 95% CI, 1.64-7.56; P < .001) and PFS (HR, 3.06; 95% CI, 1.55-6.04; P < .001). The statistical significances were maintained in multimarker multivariable analyses, including IDH1/2 status for both OS (HR, 2.70; 95% CI, 1.02-7.14; P = .045) and PFS (HR, 2.74; 95% CI, 1.19-6.33; P = .02). Conclusions and Relevance In this study, MGMT promoter methylation was an independent prognostic biomarker of high-risk, low-grade glioma treated with temozolomide and radiotherapy. This is the first study, to our knowledge, to validate the prognostic importance of MGMT promoter methylation in patients with grade II glioma treated with combined radiotherapy and temozolomide and highlights its potential prognostic value beyond IDH1/2 mutation status. Trial Registration ClinicalTrials.gov Identifier: NCT00114140

Abstract 5726: A novel tumor-promoting role for miR-4516 in glioblastoma

Background: Glioblastomas are the most aggressive high-grade brain tumors, which are often life-threatening due to their location and rapid growth. Although survival rates differ depending on a variety of genetic and environmental factors, the average survival rate for a glioblastoma (GBM) patient is less than 15 months. Although the mechanisms of tumorigenesis are still being elucidated, miRNAs are promising candidates to explore as novel and prognostic biomarkers in GBM. Here we demonstrate a novel role for miR-4516 in promoting growth and migration of GBM and establish the molecular mechanisms mediating these functions. Methods: Formalin-fixed, paraffin-embedded tissue blocks (n=268) were collected for all patients and total RNA was isolated. miRNAs were analyzed simultaneously using the nCounter human miRNA v2 assay (NanoString Technologies; Seattle,WA). Functional characterization studies were conducted in vitro and in vivo. The effect of miR-4516 on GBM cell growth and motility were evaluated by cell proliferation assay, migration and invasion assay, and Annexin-V assay. Realtime PCR, immunoblotting, and 3’ untranslated region luciferase assays were used to analyze miR-4516 targets and signaling pathways. Intracranial injection will be performed to investigate the role of miR-4516 in tumor growth in vivo. Results: Univariate analysis showed that miR-4516 expression in GBM patients was inversely correlated with overall survival (FDR=0.002, p=1.02E-05). Knockdown of miR-4516 blocked tumor growth and induced cell apoptosis. Tumor cell growth, migration and invasion were induced in both transient miR-4516 overexpressed GBM cells (LN229, LN18, and U87) and stable miR-4516 overexpressed GBM cells (U87-EGFRvIII). These miR-4516 tumor-promoting effects were mediated in part via direct targeting PTPN14 and CDKN1A. Investigation of the other miR-4516 targets and in vivo functional study are in process. Conclusion: Taken together, these results suggest that miR-4516 acts as a prognostic biomarker for GBM patients. Funding Information: 1R01CA169368 (PI: Houghton; Co-I:Chakravarti); 1R01CA11522358 Multiple-PI R01: Chakravarti (PI); Xia (PI); 1R01CA1145128 Baroukhim (PI); Chakravarti (Co-PI) 7/2015-6/2020; R01CA108633 (PI:Chakravarti); 1RC2CA148190 (Scientific PI: Chakravarti) Citation Format: Tiantian Cui, Ashley Gray, Ziyan Liu, Marjolein Geurts, Pierre Robe, Joseph McElroy, Erica Hlavin Bell, Arnab Chakravarti. A novel tumor-promoting role for miR-4516 in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5726. doi:10.1158/1538-7445.AM2017-5726

Abstract A47: Chromatin remodeling factor SMARCA4 as a predictive biomarker of cisplatin therapy in non-small cell lung cancer

Adjuvant cisplatin-based chemotherapy is recommended for patients with completely resected non-small cell lung cancer (NSCLC). However, due to limited efficacy of this therapy, studies are required to identify biomarkers to select patients who would derive the most benefit of the therapy. SMARCA4 (also known as BRG1) and SMARCA2 (also known as BRM) are two important mutually exclusive catalytic subunits with ATPase activity of the mammalian chromatin remodeling complex SWItch/Sucrose NonFermentable (SWI/SNF). These subunits are required for mammalian development and are altered in a variety of malignancies including lung, prostate, pancreatic, breast, and colon. Several studies have reported that both SMARCA4 and SMARCA2 are required for the full activation of DNA damage response by ATM-mediated activation of γ-H2AX. As SMARCA4 is frequently deleted in many primary tumors including NSCLC, we hypothesized that the loss of SMARCA4 may enhance the sensitivity of tumor cells to chemotherapeutic agents causing DNA damage and may serve as predictive biomarkers of sensitivity for these agents. To that end, in the present study we evaluated the association between SMARCA4 and/or SMARCA2 alterations and the outcome of DNA-damaging chemotherapeutic cisplatin in NSCLC. We used a gene expression profiling microarray (n=133) from both control and treatment arms of the North American Intergroup phase III trial of adjuvant cisplatin plus vinorelbine (JBR.10). Kaplan-Meier method and log-rank tests were used to estimate and test the differences of probabilities in overall survival and disease-specific survival between expression groups and treatment arms. Multivariate Cox regression models were used while adjusting for other baseline clinical covariates. Improved five-year disease-specific survival was detected only in patients with low SMARCA4 expression when treated with adjuvant cisplatin/vinorelbine compared with the observational arm (HR=0.1, 95% CI: 0.0.0·5, P=0.001 (low); HR 1.1, 95% CI: 0·5-2.4, P=0·76 [high]). The interaction test was statistically significant (P=0.007). In contrast, no significant survival benefits were noted in patients with high SMARCA4 or high SMARCA2 expression. Taken together, these results suggest that decreased expression of chromatin remodeling factors SMARCA4 may serve as putative predictive biomarkers of platinum-based therapy (cisplatin) in NSCLC and requires further validation. Citation Format: Arup R. Chakraborty, Erica Bell, Mo Xiaokui, Ziyan Liu, Konstantin Shilo, Simon Kirste, Petra Stegmaier, Maureen McNulty, Niki Karachaliou, Rafael Rosell, Gerold Bepler, David P. Carbone, Arnab Chakravarti. Chromatin remodeling factor SMARCA4 as a predictive biomarker of cisplatin therapy in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A47.

Abstract 2984: Understanding the relationship between a common mutation within the SWI/SNF chromatin remodeling complex in lung cancer and treatment response.

SWI/SNF is a chromatin remodeling complex that plays important roles in transcriptional regulation, DNA replication, and DNA repair. BRG1, one of its catalytic ATPase subunits, is a tumor suppressor that is mutated in approximately 20-30% of non-small cell lung cancers. To determine the clinical significance of BRG1 expression, non-small cell lung cancer data from The Cancer Genome Atlas was analyzed and showed that patients with stage II, high-BRG1 tumors have a higher overall survival than patients with stage II, low-BRG1 disease probably due to differences in genomic instability. BRG1’s involvement in multiple DNA repair pathways likely results in a decreased ability of BRG1-deficient tumor cells to repair damage, which underlies our hypothesis that DNA damaging therapies may be the best treatment for patients harboring deficient BRG1. In order to study the effects of DNA damage on BRG1-positive and BRG1-negative cells, we utilized ionizing radiation, ABT-888 (a PARP inhibitor), mitomycin C, and cisplatin to test BRG1’s role in double-strand break repair, base excision repair, nucleotide excision repair, and interstrand cross-link repair. Cell lines expressing a functional BRG1 (HeLa and H520) were approximately 2-4-fold less sensitive to radiation than cells without BRG1 (C33A and A427). Likewise, HeLa cells were about 4-fold less sensitive to cisplatin, a DNA crosslinking agent, than were C33A cells. We will present data from these assays in a panel of BRG1-positive and BRG1-negative non-small cell lung cancer cell lines, and in BRG1 knockdown and knock-in models. To ascertain the effect of BRG1 loss on the expression of genes involved in related pathways, BRG1-positive and BRG1-negative HeLa cells were analyzed with RT-PCR arrays. We found statistically significant dysregulation of genes involved in several pathways, including chromatin remodeling (SMARCA2, SMARCB1, and INO80) and DNA repair (XRCC4, CDK7, GTF2H1, and GTF2H2). We are also using RT-PCR arrays to study the panel of BRG1-positive and BRG1-negative cell lines. Elucidating novel roles for BRG1 in DNA repair pathways and determining its molecular targets will help identify the best treatment strategy for patients lacking functional BRG1, and may have the potential to increase the life expectancy of lung cancer patients. Citation Format: Maureen McNulty, Simon Kirste, Jinwei Hu, Udit Singhal, Ziyan Liu, Petra Stegmaier, Daniel Dressler, Arnab Chakravarti, Erica Bell. Understanding the relationship between a common mutation within the SWI/SNF chromatin remodeling complex in lung cancer and treatment response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2013-2984