Description: Purpose: Our goal was to identify circulating micro RNA (miRNA) levels that could distinguish patients with low-stage pancreatic cancer from healthy and disease controls. Experimental Design: We measured 735 miRNAs in pancreatic cancer case and control sera by QRTPCR using TaqMan MicroRNA Arrays. After array analysis, we selected 18 miRNA candidates for validation in an independent set of cases and control samples. Results: Of the significantly elevated circulating miRNAs in patients with pancreatic cancer compared with controls, miR-1290 had the best diagnostic performance: receiver operating characteristic (ROC) analysis on miR-1290 serum level yielded curve areas (AUC) of 0.96 [95% confidence interval (CI), 0.91–1.00], 0.81 (0.71–0.91), and 0.80 (0.67–0.93), for subjects with pancreatic cancer ( n = 41) relative to healthy controls ( n = 19), subjects with chronic pancreatitis ( n = 35), and pancreatic neuroendocrine tumors ( n = 18), respectively. Serum miR-1290 levels were also significantly higher than healthy controls among patients with intraductal papillary mucinous neoplasm (IPMN; n = 20; AUC = 0.76, 0.61–0.91). Serum miR-1290 levels distinguished patients with low-stage pancreatic cancer from controls better than CA19-9 levels, and like CA19-9, higher miR-1290 levels predicted poorer outcome among patients undergoing pancreaticoduodenectomy. Greater numbers of miR-1290 transcripts were detected by FISH in primary pancreatic cancer and IPMN than normal pancreatic duct cells. miR-1290 influenced in vitro pancreatic cancer cell proliferation and invasive ability. Several other circulating miRNAs distinguished sera of patients with pancreatic cancer from those of healthy controls with AUCs 〉0.7, including miR-24, miR-134, miR-146a, miR-378, miR-484, miR-628-3p, and miR-1825. Conclusions: The detection of elevated circulating miR-1290 has the potential to improve the early detection of pancreatic cancer. Clin Cancer Res; 19(13); 3600–10. ©2013 AACR .

Description: Platinum and PARP inhibitor (PARPi) sensitivity commonly coexist in epithelial ovarian cancer (EOC) due to the high prevalence of alterations in the homologous recombination (HR) DNA repair pathway that confer sensitivity to both drugs. In this report, we describe a unique subset of EOC with alterations in another DNA repair pathway, the nucleotide excision repair (NER) pathway, which may exhibit a discordance in sensitivities to these drugs. Specifically, 8% of high-grade serous EOC from The Cancer Genome Atlas dataset exhibited NER alterations, including nonsynonymous or splice site mutations and homozygous deletions of NER genes. Tumors with NER alterations were associated with improved overall survival (OS) and progression-free survival (PFS), compared with patients without NER alterations or BRCA1/2 mutations. Furthermore, patients with tumors with NER alterations had similar OS and PFS as BRCA1/2-mutated patients, suggesting that NER pathway inactivation in EOC conferred enhanced platinum sensitivity, similar to BRCA1/2-mutated tumors. Moreover, two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in the two most platinum-sensitive tumors, were functionally associated with platinum sensitivity in vitro. Importantly, neither NER alteration affected HR or conferred sensitivity to PARPi or other double-strand break–inducing agents. Overall, our findings reveal a new mechanism of platinum sensitivity in EOC that, unlike defective HR, may lead to a discordance in sensitivity to platinum and PARPi, with potential implications for previously reported and ongoing PARPi trials in this disease. Cancer Res; 75(4); 628–34. ©2015 AACR.

Description: Purpose: MET amplification, responsible for 20% of acquired resistance to EGFR tyrosine kinase inhibitor (TKI) in patients with advanced non–small cell lung cancer (NSCLC), presents an attractive target. Numerous studies have conferred susceptibility of MET mutations and focal amplification to targeted MET-TKIs. However, the mechanism underlying MET-TKIs–induced resistance remains elusive. Experimental Design: We conducted a cohort of 12 patients with advanced NSCLC who developed resistance to a combinatorial therapy consisting of gefitinib and a type I MET-TKI. We performed capture-based targeted ultra-deep sequencing on serial tumor biopsies and plasmas ctDNA samples to detect and quantify genetic alterations. Results: We identified 2 newly acquired MET mutations, Y1248H and D1246N, in 2 patients and further confirmed their resistance against type I MET-TKIs in silco, in vitro , and in vivo . Interestingly, NIH3T3 cells harboring either mutation exhibited responses to type II MET-TKIs, suggesting sequential use of MET-TKIs may offer a more durable response. In addition, we also discovered that EGFR amplification may act as an alternative MET-TKI resistance mechanism. Conclusions: Our study provides insight into the diversity of mechanisms underlying MET-TKI–induced resistance and highlights the potential of sequential use of MET-TKIs. Clin Cancer Res; 23(16); 4929–37. ©2017 AACR .

Description: Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti–PD1-resistant lung tumors. Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti–PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes. Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4 + and CD8 + T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103 + dendritic cells. Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti–PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735–43. ©2018 AACR .

Description: Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients. Experimental Design: To investigate TG02, an agent with known penetration of the blood–brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays. Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue. Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124–37. ©2017 AACR .

Description: Cancer stem cells (CSC) play critical roles in cancer initiation, progression, and therapeutic refractoriness. Although many studies have focused on the genes and pathways involved in stemness, characterization of the factors in the tumor microenvironment that regulate CSCs is lacking. In this study, we investigated the effects of stromal fibroblasts on breast cancer stem cells. We found that compared with normal fibroblasts, primary cancer-associated fibroblasts (CAF) and fibroblasts activated by cocultured breast cancer cells produce higher levels of chemokine (C-C motif) ligand 2 (CCL2), which stimulates the stem cell–specific, sphere-forming phenotype in breast cancer cells and CSC self-renewal. Increased CCL2 expression in activated fibroblasts required STAT3 activation by diverse breast cancer–secreted cytokines, and in turn, induced NOTCH1 expression and the CSC features in breast cancer cells, constituting a cancer-stroma-cancer signaling circuit. In a xenograft model of paired fibroblasts and breast cancer tumor cells, loss of CCL2 significantly inhibited tumorigenesis and NOTCH1 expression. In addition, upregulation of both NOTCH1 and CCL2 was associated with poor differentiation in primary breast cancers, further supporting the observation that NOTCH1 is regulated by CCL2. Our findings therefore suggest that CCL2 represents a potential therapeutic target that can block the cancer–host communication that prompts CSC-mediated disease progression. Cancer Res; 72(11); 2768–79. ©2012 AACR.

Description: In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression. Cancer Res; 74(13); 3617–29. ©2014 AACR.

Description: Glioblastoma multiforme is the most common type of primary malignant brain tumor and may arise from a cell with neural stem-like properties. Deregulation of the retinoblastoma, phosphoinositide-3 kinase (PI3K), and p53 pathways are molecular hallmarks of this disease. Recent work has shown that p53−/−Pten−/− mice form gliomas in a c-Myc–dependent manner. To explore the role of the INK4A/ARF locus and Pten deletions in gliomagenesis, we generated Pten−/−Ink4a/Arf−/− mouse neural stem cells (mNSC) and such cells were highly proliferative, self-renewing, relatively refractory to differentiation, and induced both low- and high-grade glioma formation in vivo. In contrast to p53−/− Pten−/− mNSCs, however, Pten−/−Ink4a/Arf−/− mNSCs do not express appreciable levels of c-Myc in vitro, although glioma stem cells derived from thesecells did. Sequencing of Pten−/−Ink4a/Arf−/− mNSC–derived tumors revealed spontaneous mutations in Tp53 in vivo with subsequent downregulation of Fbxw7. Expression of p53 mutants in Pten−/−Ink4a/Arf−/− mNSC or knockdown of Fbxw7 resulted in reexpression of c-Myc with enhanced Pten−/−Ink4a/Arf−/− mNSC tumorigenecity. We propose that p53 mutations contribute to gliomagenesis by both allowing the overexpression of c-Myc through downregulation of Fbxw7 and by protecting against c-Myc–induced apoptosis. Cancer Res; 72(22); 6065–75. ©2012 AACR.