Erick Riquelme

EZH2 Protein Expression Associates with the Early Pathogenesis, Tumor Progression, and Prognosis of Non–Small Cell Lung Carcinoma

Purpose: Enhancer of zeste homolog 2 (EZH2) promotes carcinogenesis by epigenetically silencing tumor suppressor genes. We studied EZH2 expression by immunohistochemistry in a large series of non–small cell lung carcinomas (NSCLC) in association with tumor characteristics and patient outcomes. Experimental Design: EZH2 immunohistochemistry expression was analyzed in 265 normal and premalignant bronchial epithelia, 541 primary NSCLCs [221 squamous cell carcinomas (SCC) and 320 adenocarcinomas] and 36 NSCLCs with paired brain metastases. An independent set of 91 adenocarcinomas was also examined. EZH2 expression was statistically correlated with clinico-pathological information, and EGFR/KRAS mutation status. Results: EZH2 expression was significantly (P < 0.0001) higher in SCCs compared with adenocarcinomas and in brain metastasis relative to matched primary tumors (P = 0.0013). EZH2 expression was significantly (P < 0.0001) elevated in bronchial preneoplastic lesions with increasing severity. In adenocarcinomas, higher EZH2 expression significantly correlated with younger age, cigarette smoking, and higher TNM stage (P = 0.02 to P < 0.0001). Higher EZH2 expression in adenocarcinoma was associated with worse recurrence-free survival (RFS; P = 0.025; HR = 1.54) and overall survival (OS; P = 0.0002; HR = 1.96). Furthermore, lung adenocarcinomas with low EZH2 levels and high expression of the lineage-specific transcription factor, TTF-1, exhibited significantly improved RFS (P = 0.009; HR = 0.51) and OS (P = 0.0011; HR = 0.45), which was confirmed in the independent set of 91 adenocarcinomas. Conclusion: In lung, EZH2 expression is involved in early pathogenesis of SCC and correlates with a more aggressive tumor behavior of adenocarcinoma. When EZH2 and TTF-1 expressions are considered together, they serve as a prognostic marker in patients with surgically resected lung adenocarcinomas. Clin Cancer Res; 19(23); 6556–65. ©2013 AACR.

Increased VEGFR-2 Gene Copy Is Associated With Chemoresistance and Shorter Survival in Patients with Non-small Cell Lung Carcinoma Who Receive Adjuvant Chemotherapy

VEGF receptor-2 (VEGFR-2 or kinase insert domain receptor; KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected non-small-cell lung carcinoma (NSCLC; n = 248). KDR copy number gains (CNG), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR = 5.16; P = 0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations, we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear hypoxia inducible factor-1α (HIF-1α) in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11, and 21 by PCR-based sequencing; however, two variant single nucleotide polymorphism genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.

Frequent coamplification and cooperation between C-MYC and PVT1 oncogenes promote malignant pleural mesothelioma.

Introduction: Malignant pleural mesothelioma (MPM) is a deadly disease with poor prognosis and few treatment options. We characterized and elucidated the roles of C-MYC and PVT1 involved in the pathogenesis of MPM. Methods: We used small interfering RNA (siRNA)-mediated knockdown in MPM cell lines to determine the effect of C-MYC and PVT1 abrogation on MPM cells undergoing apoptosis, proliferation, and cisplatin sensitivity. We also characterized the expression of microRNAs spanning the PVT1 region in MPM cell lines. Copy number analysis was measured by quantitative polymerase chain reaction and fluorescence in situ hybridization. Results: Copy number analysis revealed copy number gains (CNGs) in chromosomal region 8q24 in six of 12 MPM cell lines. MicroRNA analysis showed high miR-1204 expression in MSTO-211H cell lines with four copies or more of PVT1. Knockdown by siRNA showed increased PARP-C levels in MSTO-211H transfected with siPVT1 but not in cells transfected with siC-MYC. C-MYC and PVT1 knockdown reduced cell proliferation and increased sensitivity to cisplatin. Analysis of the expression of apoptosis-related genes in the MSTO-211H cell line suggested that C-MYC maintains a balance between proapoptotic and antiapoptotic gene expression, whereas PVT1 and, to a lesser extent, miR-1204 up-regulate proapoptotic genes and down-regulate antiapoptotic genes. Fluorescence in situ hybridization analysis of MPM tumor specimens showed a high frequency of both CNGs (11 of 75) and trisomy (three copies; 11 of 75) for the C-MYC locus. Conclusion: Our results suggest that C-MYC and PVT1 CNG promotes a malignant phenotype of MPM, with C-MYC CNG stimulating cell proliferation and PVT1 both stimulating proliferation and inhibiting apoptosis.

Modulation of EZH2 Expression by MEK-ERK or PI3K-AKT Signaling in Lung Cancer Is Dictated by Different KRAS Oncogene Mutations

EZH2 overexpression promotes cancer by increasing histone methylation to silence tumor suppressor genes, but how EZH2 levels become elevated in cancer is not understood. In this study, we investigated the mechanisms by which EZH2 expression is regulated in non-small cell lung carcinoma cells by oncogenic KRAS. In cells harboring KRAS(G12C) and KRAS(G12D) mutations, EZH2 expression was modulated by MEK-ERK and PI3K/AKT signaling, respectively. Accordingly, MEK-ERK depletion decreased EZH2 expression in cells harboring the KRAS(G12C) mutation, whereas PI3K/AKT depletion decreased EZH2 expression, EZH2 phosphorylation, and STAT3 activity in KRAS(G12D)-mutant cell lines. Combined inhibition of EZH2 and MEK-ERK or PI3K/AKT increased the sensitivity of cells with specific KRAS mutations to MEK-ERK and PI3K/AKT-targeted therapies. Our work defines EZH2 as a downstream effector of KRAS signaling and offers a rationale for combining EZH2 inhibitory strategies with MEK-ERK- or PI3K/AKT-targeted therapies to treat lung cancer patients, as stratified into distinct treatment groups based on specific KRAS mutations.

Immune Profiling of Premalignant Lesions in Patients With Lynch Syndrome

Importance Colorectal carcinomas in patients with Lynch syndrome (LS) arise in a background of mismatch repair (MMR) deficiency, display a unique immune profile with upregulation of immune checkpoints, and response to immunotherapy. However, there is still a gap in understanding the pathogenesis of MMR-deficient colorectal premalignant lesions, which is essential for the development of novel preventive strategies for LS. Objective To characterize the immune profile of premalignant lesions from a cohort of patients with LS. Design, Setting, and Participants Whole-genome transcriptomic analysis using next-generation sequencing was performed in colorectal polyps and carcinomas of patients with LS. As comparator and model of MMR-proficient colorectal carcinogenesis, we used samples from patients with familial adenomatous polyposis (FAP). In addition, a total of 47 colorectal carcinomas (6 hypermutants and 41 nonhypermutants) were obtained from The Cancer Genome Atlas (TCGA) for comparisons. Samples were obtained from the University of Texas MD Anderson Cancer Center and “Regina Elena” National Cancer Institute, Rome, Italy. All diagnoses were confirmed by genetic testing. Polyps were collected at the time of endoscopic surveillance and tumors were collected at the time of surgical resection. The data were analyzed from October 2016 to November 2017. Main Outcomes and Measures Assessment of the immune profile, mutational signature, mutational and neoantigen rate, and pathway enrichment analysis of neoantigens in LS premalignant lesions and their comparison with FAP premalignant lesions, LS carcinoma, and sporadic colorectal cancers from TCGA. Results The analysis was performed in a total of 28 polyps (26 tubular adenomas and 2 hyperplastic polyps) and 3 early-stage LS colorectal tumors from 24 patients (15 [62%] female; mean [SD] age, 48.12 [15.38] years) diagnosed with FAP (n = 10) and LS (n = 14). Overall, LS polyps presented with low mutational and neoantigen rates but displayed a striking immune activation profile characterized by CD4 T cells, proinflammatory (tumor necrosis factor, interleukin 12) and checkpoint molecules (LAG3 [lymphocyte activation gene 3] and PD-L1 [programmed cell death 1 ligand 1]). This immune profile was independent of mutational rate, neoantigen formation, and MMR status. In addition, we identified a small subset of LS polyps with high mutational and neoantigen rates that were comparable to hypermutant tumors and displayed additional checkpoint (CTLA4 [cytotoxic T-lymphocyte–associated protein 4]) and neoantigens involved in DNA damage response (ATM and BRCA1 signaling). Conclusions and Relevance These findings challenge the canonical model, based on the observations made in carcinomas, that emphasizes a dependency of immune activation on the acquisition of high levels of mutations and neoantigens, thus opening the door to the implementation of immune checkpoint inhibitors and vaccines for cancer prevention in LS.

Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells

BACKGROUND & AIMS Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to pancreatic interepithelial neoplasia (PanIN) initiation and progression. We investigated the effects that IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. METHODS We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KCiMist;G) and Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KCiMist mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of DCLK1 and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or short hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using The Cancer Genome Atlas database. RESULTS PanIN cells from KCiMist;G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17-neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern and had significantly decreased expression of DCLK1 and POU2F3, which regulate tuft cell development. KCiMist mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and mitogen-activated protein kinase signaling and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells) compared with cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 after incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C was higher in DCLK1-positive PanIN cells from mice compared with DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 were also associated with patient survival time. CONCLUSIONS In studies of mouse and human pancreatic tumors and precursors, we found that immune cell-derived IL17 regulated development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.

Immunotherapy for Pancreatic Cancer: More Than Just a Gut Feeling

Development of pancreatic cancer in spontaneous murine models is associated with enrichment of specific strains of gut and intratumoral bacteria that induce a tolerogenic immunosuppressive microenvironment favoring cancer progression and resistance to immunotherapies. Ablation of the microbiome with antibiotics reshapes the tumor microenvironment, inducing T-cell activation, improving immune surveillance, and increasing sensitivity to immunotherapy in established tumors. Cancer Discov; 8(4); 386-8. ©2018 AACR.See related article by Pushalkar et al., p. 403.

Abstract 1857: Early detection of pancreatic intraepithelial neoplasms (PanINs) in transgenic mouse model by hyperpolarized 13C metabolic magnetic resonance imaging

Pancreatic cancer, one of the most lethal solid tumors, is an aggressive disease that develops relatively symptom-free. Pre-invasive pancreatic intraepithelial neoplasias (PanINs) have been identified as precursor lesions to pancreatic cancer. While there is growing evidence supporting PanIN9s genetic links to pancreatic cancer, there is no non-invasive method to detect them. There is an unmet need of novel strategies for detection of pancreatic cancer at the earliest stages, preferably at the stage of PanINs. Hyperpolarized magnetic resonance imaging (HP-MRI) has shown potential for early detection of cancers and monitoring therapeutic efficacy. Here we have employed in-vivo 13C pyruvate metabolic imaging and ex-vivo nuclear magnetic resonance (1H-NMR) metabolomics to identify and understand metabolic changes, to enable detection of early PanINs and its progression to advanced PanINs and pancreatic cancer. Genetically engineered mouse (GEM) models with progression of PanIN lesions and control animals, with no pancreatic lesions, were employed in this study. Hyperpolarization of pyruvate and in-vivo 13C MRS were performed using Hypersense (Oxford Instruments) and 7T MRI scanner with a dual tuned 1H/13C volume coil respectively. Tissue alanine and lactate concentrations were determined using a Bruker 500 MHz NMR spectrometer coupled with cryo-probe. Histology and immunohistochemistry were performed on excised tissue samples. P48Cre mice were used as controls and P48Cre;LSLKrasG12D mice were used for detection of early and advanced PanINs, which usually develop in these mice between 12 and 20 months. The imaging experiments were performed at the different stages of the disease. Progression of disease from tissue containing predominantly low-grade PanINs to tissue with high-grade PanINs showed a decreasing alanine/lactate concentration ratio. Real time in-vivo 13C MRS was used to measure non-invasively changes of alanine and lactate metabolites with disease progression and in control mice, following injection of hyperpolarized pyruvate. The alanine-to-lactate (Ala/Lac) signal intensity ratio was found decreased as the disease progressed from low-grade PanINs to high-grade PanINs. These results demonstrate that there are significant alterations of alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities which favor the transformation of aggressive pancreatic cancer from PanINs lesion. Our results suggest that real-time conversion kinetic rate constants kPA(pyruvate-to-alanine) and kPL(pyruvate-to-lactate) can be used as metabolic imaging biomarkers for assessing the early stage of pancreatic diseases. Findings from this highly promising HP-MRI technique could be rapidly translated to the clinic for early detection of pancreatic cancer in patients at high risk for developing the disease. Citation Format: Prasanta Dutta, Yu Zhang, Michelle Zoltan, Marilina Mascaro, Erick Riquelme, Jaehyuk Lee, Anirban Maitra, Florencia McAllister, Pratip Bhattacharya. Early detection of pancreatic intraepithelial neoplasms (PanINs) in transgenic mouse model by hyperpolarized 13C metabolic magnetic resonance imaging [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 1857. doi:10.1158/1538-7445.AM2017-1857

Abstract 2332: Oncogenic mutant KRAS modulates EZH2 expression through MEK-ERK signaling by remodeling gene expression in NSCLC

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Abstract: Background. EZH2 overexpression occurs in lung cancer and is associated with a poor outcome. The mechanisms driving EZH2 expression in lung cancer are not fully understood. In this study, we investigated whether pharmacological disruption of signaling MEK-ERK pathway would affect EZH2 expression in a panel of NSCLC cell lines with and without KRAS mutation. Moreover, we analyzed the transcriptome expression following knockdown of EZH2 expression in NSCLC cell lines with different types of KRAS mutations. Methods. NSCLC cell lines were treated with different doses of MEK inhibitor AZD6244 (0, 0.5 and 1μM) and the expressions of EZH2, MEK and MAPK were determined by Western-blots. Cell lines were transfected with gene-specific EZH2 siRNA and control siRNA. Gene expression profiling was performed using the Illumina Human HT-12 v 4.0 platform. Functional pathway analysis was conducted using the software Ingenuity Pathways Analysis. Result. We found that pharmacological disruption of signaling MEK-ERK pathways with AZD6244 decreases the expression of MAPK p44/42 and phospho-MAPK p44/42 in all NSCLC cell lines analyzed wild-type and of KRAS mutant status. However, after AZD6244 treatment, EZH2 expression in NSCLC cell lines varied according to the KRAS mutation status: strong reduction only in NSCLC cell lines harboring GtoC mutation, a partial reduction in cells with GtoS and GtoR mutation, and no change in cell lines with GtoD or GtoV mutation and with wild-type KRAS. The gene expression analysis revealed that 3,235 genes were similarly regulated for mutant KRAS GtoC, GtoD and GtoV in NSCLC following EZH2 knockdown compared with KRAS wild-type (P < 0.05). Functional pathways analysis showed that EZH2 knockdown modulated differentially pathways commonly activated in cancer as growth factor signaling, molecular mechanism of cancer and cellular growth, proliferation and development, between cell lines that harbor mutant KRAS GtoC, GtoD or GtoV compared with KRAS wild-type. Conclusion. Our findings suggest that oncogenic mutant KRAS GtoC, GtoS and GtoR modulate EZH2 expression through MEK-ERK signaling contributing to changes of the expression of genes frequently altered in cancer (Grant support: 5 R01 CA155196 and P50CA70907) Citation Format: Erick M. Riquelme, Li Shen, Jing Wang, Carmen Behrens, John D. Minna, Ignacio I. Wistuba. Oncogenic mutant KRAS modulates EZH2 expression through MEK-ERK signaling by remodeling gene expression in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2332. doi:10.1158/1538-7445.AM2014-2332

Abstract 374: KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors.

Targeted therapies designed to inhibit the vascular endothelial growth factor (VEGF) pathway have been extensively evaluated in the treatment of malignancies including Non-Small Cell Lung Cancer (NSCLC). VEGF pathway inhibitors such as bevacizumab, or the multitargeted receptor tyrosine kinase inhibitors (TKIs) vandetanib and sorafenib, have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, seen only in subsets of patients. Thus, predictive markers for identifying which patients are likely to benefit are critically needed. Although expression of VEGF receptor-2 (VEGFR-2, also known as KDR) was initially thought to primarily occur in endothelial cells, VEGFR-2 has been detected on malignant cells, including lung cancer cells, and in NSCLC, overexpression of VEGFR-2 on tumor cells is associated with a poor clinical outcome. Amplification of KDR has been detected in lung cancer specimens at a relatively high frequency (9% and 32%). The consequences of KDR copy number gains (CNGs) are not yet understood. Recently, we have shown that NSCLC cell lines with KDR copy number gains (CNGs) were associated with in vitro resistance to platinum chemotherapy, and KDR CNG predicted worse overall survival in patients who received platinum adjuvant therapy but not in untreated patients. We investigated the hypothesis that NSCLC tumor cells with KDR CNG display increased sensitivity to VEGFR TKIs compared to tumor cells without KDR CNG. In tumor cell lines with KDR CNG, treatment with exogenous VEGF ligand enhanced cell motility and this was inhibited by VEGFR blockade with TKIs. Multiple receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxic conditions compared to NSCLC cell lines without KDR CNG. Here, we show that in NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α- regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG as determined by SNP array had a partial response to VEGFR inhibition with sorafenib. Citation Format: Monique B. Nilsson, Tina Cascone, Jayanthi Gudikote, Emily Roarty, Lixia Diao, Andrew Koo, Sumankalai Ramachandran, Erick Riquelme, Hai Tran, Ignacio Wistuba, David P. Carbone, John Heymach. KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors. [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 374. doi:10.1158/1538-7445.AM2013-374