bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A58: KRAS mutations: Variable incidences in a Brazilian cohort of 8,234 metastatic colorectal cancer patients
Authors: Aran Veronica , Vieira Fernando M. , Victorino Ana Paula , Salem Jonas H. , Zalcberg-Renault Ilana , Bonamino Martin H. , Zalis Mariano , Ferreira Carlos Gil
Published: May 2015

Personalised medicine is an evolving field that seeks to target cancer therapies based on unique genetic characteristics of the tumour and/or the patient. One of the most significant advances towards personalised care in the field of oncology was the establishment of KRAS gene mutation as a validated biomarker predicting efficacy in epidermal growth factor receptor (EGFR) targeted therapies in the treatment of metastatic colorectal cancer (mCRC). KRAS mutations are frequently found in CRC indicating the importance of its genotyping in the study of the molecular mechanisms behind this disease. Although major advances have occurred over the past decade, there are still important gaps in our understanding of CRC carcinogenesis, particularly whether sex-linked factors play any role. Therefore, in this study the profile of KRAS mutations in the Brazilian population was analysed by conducting direct sequencing of KRAS codons 12 and 13 belonging to 8,234 metastatic CRC patient samples from different Brazilian regions. The median age was 59 years and the overall percentage of wild-type and mutated KRAS was 66.4% and 33.6%, respectively. Interestingly, a significant difference in the percentage of mutated KRAS patients was observed between male and female (32.5% versus 34.8%, p=0.03). However, there were no differences in the distribution of any specific KRAS mutation according to gender. This is one of the largest cohorts of KRAS genotyping in CRC patients indicating a higher incidence of KRAS mutations in females compared to males.

Citation Format: Veronica Aran, Fernando M. Vieira, Ana Paula Victorino, Jonas H. Salem, Ilana Zalcberg-Renault, Martin H. Bonamino, Mariano Zalis, Carlos Gil Ferreira. KRAS mutations: Variable incidences in a Brazilian cohort of 8,234 metastatic colorectal cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A58. doi: 10.1158/1557-3125.RASONC14-A58
Abstract B01: Improve MEK/ERK targeting in mutant KRAS lung cancer
Authors: Chen Zhao , Weisberg Ellen , Cheng Katherine , Kung Andrew L. , Bradner James , Griffin James , Wong Kwok-Kin
Published: May 2015

KRAS activating mutations are detected in 30% Non-Small Cell Lung Cancer (NSCLC) cases, and represent the subset of patients with the worst prognosis. Current efforts to develop treatments for this type of lung cancer are partially focused on inhibiting the Raf/MEK/ERK signaling cascade downstream of KRAS. Inhibition of the Raf/MEK/ERK pathway results in death of many mutant KRAS-driven lung cancer cell lines in vitro, and we have previously reported a murine active Kras lung cancer model that showed significant tumor regression upon treatment with the MEK inhibitor, selumetinib, when it was combined with other agents, such as PI3K/mTOR dual inhibitor BEZ235 or the cytotoxic agent docetaxel. The activity of the latter combination was validated in a recent phase II clinical trial in NSCLC patients harboring KRAS mutations. However, the responses were not durable in either human patients or mice, as resistance develop rapidly following chronic MEK inhibition. We have developed two approaches to reduce or delay the emergence of resistant clones. First, we have identified a number of novel treatment strategies that would directly improve the initial efficacy of MEK/ERK inhibition to dramatically enhance the initial response to MEK inhibition. Second, we have designed chemical screening strategies that identify compounds that act against active Kras lung cancer independently from the Raf/MEK/ERK pathway. We are using the first approach to delay the development of chronic resistance, while using the second approach to overcome resistance to MEK/ERK inhibition.

Citation Format: Zhao Chen, Ellen Weisberg, Katherine Cheng, Andrew L. Kung, James Bradner, James Griffin, Kwok-Kin Wong. Improve MEK/ERK targeting in mutant KRAS lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B01. doi: 10.1158/1557-3125.RASONC14-B01
Abstract B07: Ras-driven HCCs are addicted to FoxM1
Authors: Kopanja Dragana , Pandey Akshay , Kiefer Megan , Yu Dae-Yeul , Raychaudhuri Pradip
Published: May 2015

Hepatocellular carcinoma (HCC) is the fifth most fatal malignancy worldwide. In the US alone, according to American Cancer Society, about 17,550 new cases and over 15,000 deaths are reported annually. The fork-head box gene FoxM1 is highly significant in that regard, as it is over-expressed in HCC, and its over-expression coincides with highly aggressive, poorly differentiated HCC. Moreover, over-expression of FoxM1 correlates with poorer overall survival after surgery. Previous work demonstrated that FoxM1 is essential for chemical carcinogen (DEN)-induced development of HCC. Since the Ras-pathway is frequently mutated in human liver cancers, we investigated the effects of FoxM1 deletion on the progression of oncogenic Ras-induced HCC. In the mouse model we used, HRas-V12 is expressed specifically in the adult liver under control of the Alb-promoter. In this mouse model, majority (80 to 90%) of the male mice develop HCC by 8/9 months of age. We crossed that transgenic line with FoxM1 fl/fl MxCre strain. The triple transgenic strain of mice allowed us to delete FoxM1 at specific times after Ras driven tumorigenesis, by i.p. injection of double-stranded RNA (polyIpolyC). We deleted FoxM1 at 8 months of age, and observed significant decrease in the number of tumor nodules per liver. Deletion of FoxM1 inhibited proliferation of the tumor cells. Surprisingly, we did not observe increase in cell death in FoxM1 deleted tumors. Because FoxM1 is over-expressed in highly aggressive, poorly differentiated liver cancers, we investigated whether FoxM1 is needed for maintenance of hepatic cancer stem cells. Interestingly, we discovered almost complete depletion of hepatic cancers stem cells, CD90+CD45- cells, in FoxM1 deleted tumors. These studies have exciting implications in further considerations of FoxM1 as a target of Ras depended malignancies.

Citation Format: Dragana Kopanja, Akshay Pandey, Megan Kiefer, Dae-Yeul Yu, Pradip Raychaudhuri. Ras-driven HCCs are addicted to FoxM1. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B07. doi: 10.1158/1557-3125.RASONC14-B07
Abstract B09: Loss of PTEN cooperates with mutant KRAS initiating EMT and increased stemness in a mouse model of lung cancer
Authors: Malaney Prerna , Dave Vrushank
Published: May 2015

We have developed a preclinical model of lung cancer associated with hyper-activated PI3K/mTOR and KRAS. Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers; of which >50% have aberrant PI3K/AKT/mTOR signaling and ∼30% harbor oncogenic KRAS, conferring chemo/radio-resistance and poor prognosis. KRAS inhibitors are difficult to develop, and rapalogs targeting mTOR are toxic and induce compensatory activation of AKT, causing resistance to apoptosis. Hence, development of effective inhibitors of KRAS and PI3K/mTOR pathways is imperative. Further, clinical studies implicate that tumors with aberrant KRAS and PI3K/mTOR signaling are associated with epithelial-mesenchymal transition (EMT), metastasis, poor differentiation, and chemo/radio-resistance. However, molecular details of EMT mediated by KRAS and PI3K/mTOR pathway is lacking, limiting the development of drugs targeting EMT-mediators. Using a CCSP-promoter driven Cre/LoxP mediated deletion of PTEN in an oncogenic KRAS status (PTENΔΔ/KRasG12D), we have developed a preclinical mouse model of lung cancer with hyper-activation of PI3K/mTOR and KRAS pathways. Microarray based RNA profiling followed by bioinformatic analysis on PTEN-null lung epithelial cells revealed transcriptional activation of RAS/RAF/MAPK/ERK pathway associated candidate genes. On the other hand, Dox-induced PTENΔΔ/KRasG12D lung tumors showed induction of SNAIL1, SLUG, ZEB-2 and SOX2 genes. Taken together, our analysis reveals that PTEN loss predisposes lung tumors to hyper-activation of the KRAS pathway, which is further accentuated by oncogenic mutation in KRAS, initiating EMT and increased stemness.

Citation Format: Prerna Malaney, Vrushank Dave. Loss of PTEN cooperates with mutant KRAS initiating EMT and increased stemness in a mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B09. doi: 10.1158/1557-3125.RASONC14-B09
Abstract B16: Activated glucocorticoid signaling in pancreatitis contributes to acinar-to-ductal metaplasia and KrasG12D-driven tumorigenesis
Authors: Shen Chia-Ning , Su Chien-Chang , Hsieh Chi-Chih , Liao Wen-Ying , Mao Wan-Yu , Shen Chia-Rui , Hsiao Michael
Published: May 2015

Patients with chronic pancreatitis are at increased risk of developing pancreatic cancer. However, the key inflammatory action mediating pancreatic tumorigenesis has not been clearly determined. Here, we initially identified evaluated levels of activated glucocorticoid receptors (GR) were observed in patients of chronic pancreatitis and pancreatic ductal adenocarcinomas (PDAC). Utilizing mice that conditionally expressed KrasG12D in acinar cell compartment with or without loss of GR, we demonstrated that activation of GR was required for generation of metaplastic ductal lesion and development of pancreatic intraepithelial neoplasia (PanIN) in mice with experimental pancreatitis. Administration of dexamethason, a potent synthetic glucocoticoid, was sufficient to induce development of advanced PanINs and metastatic PDACs in KrasG12D mice that harbour single-allele deletion of p53. We further revealed that nongenomic activation of GR was a key action that led to evaluating c-Src signaling and triggering a pathological threshold of Ras activity necessary for neoplastic transformation. In contrast, treatment of dasatinib, a Src kinase inhibitor, could suppressed development of PanIN and PDAC induced by dexamethasone. Importantly, cre-lox based lineage-tracing mice was applied and identified that GR activation directly transformed GFP-tagged KrasG12D-expressing acinar cells to tumorigenic cells that posse features of cancer initiating cells. The findings explained why chronic inflammation increase pancreatic cancer risk and raised concerns on the use of glucocorticoids on patients of chronic pancreatitis

Citation Format: Chia-Ning Shen, Chien-Chang Su, Chi-Chih Hsieh, Wen-Ying Liao, Wan-Yu Mao, Chia-Rui Shen, Michael Hsiao. Activated glucocorticoid signaling in pancreatitis contributes to acinar-to-ductal metaplasia and KrasG12D-driven tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B16. doi: 10.1158/1557-3125.RASONC14-B16
Abstract B49: Role of RAS palmitoyl-acyltransferase DHHC9 in hematopoiesis and NRAS leukemogenesis
Authors: Liu Ping , Jiao Bo , Linder Maurine , Ren Ruibao
Published: May 2015

Hyperactivation of RAS is common in human cancer, including hematological malignancies. Most oncogenic RAS proteins have been difficult to target. Identification of alternative targets that block RAS signaling is critical to develop therapies for RAS-related cancer. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Protein palmitoylation regulates the membrane targeting, subcellular trafficking, and functions of proteins. We have previously examined the importance of PTMs in NRAS leukemogenesis and found for the first time that palmitoylation is essential for NRAS leukemogenesis. These studies suggest that targeting RAS palmitoylation may be an effective therapy for cancers involving RAS proteins that rely on palmitoylation for plasma membrane binding. In the previous studies, we blocked NRAS palmitoylation by mutating the palmitoylation site in NRAS. Therapeutic intervention of RAS palmitoylation requires targeting enzymes that mediate RAS palmitoylation. The reaction of protein S-palmitoylation is catalyzed by a family DHHC protein palmitoyl-acyltransferases (PATs). Thus far 24 mamalian PATs have been identified. It has been shown that DHHC9 (a 364-amino acid protein encoded by ZDHHC9, an X-linked gene), is the ortholog of yeast Ras2 PAT and constitute a mammalian PAT with specificity for H- and NRAS in vitro. Increased expression of DHHC9 has been found in various cancer. Here we investigate the role of DHHC9 in hematopoiesis and NRAS leukemogenesis in vivo. We found that ectopic expression of DHHC9 in mouse bone marrow cells hinders hematopoiesis, but is incapable of inducing hematological malignancies in mice. We also found that frequency of lineage-specific populations and hematopoietic stem cell phenotype were similar in mice with knockout alleles of ZDHHC9 as that of wild type mice, suggesting that DHHC9 is dispensable for normal hematopoiesis. Expression of oncogenic NRAS in bone marrow cells from ZDHHC9 knockout mice still induced leukemia but the mice survived longer that the wild type control mice. These results suggest that DHHC9 plays a role in the pathogenesis of NRAS-induced leukemia, but it is not the only PATs for RAS palmitoylation. We are identifying additional RAS PATs and developing cancer therapies targeting RAS palmitoylation.

Citation Format: Ping Liu, Bo Jiao, Maurine Linder, Ruibao Ren. Role of RAS palmitoyl-acyltransferase DHHC9 in hematopoiesis and NRAS leukemogenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B49. doi: 10.1158/1557-3125.RASONC14-B49
Abstract IA10: Signaling through individual Ras proteins in the absence of the Nf1-RASGAP
Authors: Ratner Nancy
Published: May 2015

Neurofibromatosis type 1 (NF1) patients are predisposed to develop neurofibromas, benign peripheral nerve tumors resulting from loss of function mutations in the NF1 gene. NF1 (neurofibromin), the protein product of the NF1 gene, is a GTPase activating protein (GAP) that inactivates the Ras proteins H-, N-, K-, M-, and R-Ras, and R-Ras2/TC21 (TC21). We hypothesized that relevant Ras proteins would be activated in vivo by deletion of NF1, so that removing single Ras proteins in Nf1 mutant cells would reveal specific functions of individual Ras proteins. We found that activation of TC21 accounts for migration defects in NF1 mutant Schwann cells (Huang et al, 2004), through AKT and not ERK. Removing TC21 in Nf1 mutant Schwann cell precursors identified an Nf1/TGFβ/TGFβRII/AKT autocrine survival loop (Patmore et al, 2012). Implying that delayed benign tumor formation resulted from effects on tumor initiation, benign tumor formation was delayed in mice lacking Nf1 and TC21. In Schwann cell precursors, activation of P-ERK, not P-AKT, was blocked by dnH-Ras. To define the role of ERK signaling in neurofibroma, we used a DhhCre;Nf1fl/fl mouse model of neurofibroma formation. Preclinical testing of specific signaling pathways with Phase I drug candidates in the neurofibroma mouse model was read out neurofibroma growth by 7T MRI imaging, in conjunction with pharmacokinetic and pharmacodynamic readouts (Jessen et al., 2013). MEK inhibition was highly effective although not curative in this model; we established minimum effective doses of the MEK inhibitor PD0325901 and effects of early MEK inhibition on peripheral nerve tumor growth. The data support the idea that NF1 GAP loss can be used to identify roles of individual Ras proteins and downstream signaling pathways in specific cell types, and that combinations of Ras proteins can collaborate to drive tumorigenesis. Supported by R01-NS28840, P50-NS057531 and the Children's Tumor Foundation/ Neurofibromatosis Therapeutic Acceleration Program.

Citation Format: Nancy Ratner. Signaling through individual Ras proteins in the absence of the Nf1-RASGAP. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA10. doi: 10.1158/1557-3125.RASONC14-IA10
Abstract PR01: Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer
Authors: Kapoor Avnish , Yao Wantong , Ying Haoqiang , Hua Sujun , Liewen Alison , Wang Qiuyun , Zhong Yi , Wu Chang-Jiun , Sadanandam Anguraj , Hu Baoli , Chang Qing , Chu Gerald , Al-Khalil Ramsey , Jiang Shan , Xia Hongai , Fletcher-Sananikone Eliot , Lim Carol , Horwitz Gillian , Viale Andrea , Pettazzoni Piergiorgio , Sanchez Nora , Wang Huamin , Protopopov Alexei , Zhang Jianhua , Heffernan Timothy , Johnson Randy , Chin Lynda , Wang Alan , Draetta Giulio , DePinho Ronald
Published: May 2015

Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible KrasG12D-driven mouse model of PDAC has established a critical role for sustained KrasG12D expression in tumor maintenance, providing a model to determine the potential for, and underlying mechanisms of, KrasG12D–independent PDAC recurrence. Here we show that majority tumors will undergo spontaneous regrowth following Kras oncogene extinction and, importantly, a subset of relapsed tumors remained devoid of KrasG12D expression and canonical MAPK signaling and acquired amplification and over-expression of Hippo effector, the Yap1 transcriptional co-activator. Functional studies established Yap1 and the Tead2 transcriptional factor in driving KrasG12D–independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to promote a cell cycle and DNA replication program and a quasimesenchymal phenotype with increased metastatic potential. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

This abstract is also presented as Poster B21.

Citation Format: Avnish Kapoor, Wantong Yao, Haoqiang Ying, Sujun Hua, Alison Liewen, Qiuyun Wang, Yi Zhong, Chang-Jiun Wu, Anguraj Sadanandam, Baoli Hu, Qing Chang, Gerald Chu, Ramsey Al-Khalil, Shan Jiang, Hongai Xia, Eliot Fletcher-Sananikone, Carol Lim, Gillian Horwitz, Andrea Viale, Piergiorgio Pettazzoni, Nora Sanchez, Huamin Wang, Alexei Protopopov, Jianhua Zhang, Timothy Heffernan, Randy Johnson, Lynda Chin, Alan Wang, Giulio Draetta, Ronald DePinho. Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR01. doi: 10.1158/1557-3125.RASONC14-PR01
Abstract PR03: Global quantitative proteomic and phosphoproteomic analysis of oncogenic Kras-driven mouse pancreatic cancer
Authors: Taguchi Ayumu , Kapoor Avnish , Momin Amin , Katayama Hiroyuki , Yao Wantong , Wang Hong , Ying Haoqiang , DePinho Ronald , Hanash Samir
Published: May 2015

Background: The most common genetic abnormality in pancreatic cancer is oncogenic mutation of KRAS, which is an initial key event in pancreatic carcinogenesis. Oncogenic KRAS activates downstream signaling pathways, such as PI3K and MAPK pathways, indicating the essential role of phosphorylation in pancreatic cancer. However, the effect of KRAS on the global phosphoproteome in pancreatic cancer has not been fully elucidated. We investigated proteomic and phosphoproteomic alterations induced by oncogenic Kras using mouse pancreatic cancer cell lines established from doxycycline-inducible KrasG12D pancreatic cancer mouse model.

Materials and Methods: Three mouse pancreatic cancer cell lines were established from triple transgenic inducible KrasG12D pancreatic cancer mouse model (tetO_LSL- KrasG12D; ROSA26A-LSL-rtTA-IRES-GFP; p48-Cre). To identify direct downstream pathways of Kras, we performed two different experiments for each cell line: (1) 20 hours Kras-off followed by 5 hours Kras-on (designated “Re-on”) vs. 25 hours Kras-off, and (2) Continuous Kras-on (designated “On”) vs. 24 hours Kras-off. Whole cell lysates were fractionated and subjected to analysis with using mass spectrometry.

Results and Conclusion: Overall 4,133 phosphorylation sites were identified. Phosphorylation levels of 2,670 sites were quantified in at least one sample, including 2,347 (87.9%) at serine, 293 (11.0%) threonine, and 30 (1.1%) tyrosine residues. Phosphorylation levels were more than 1.25 fold higher at 386 sites (139 proteins) and 558 sites (256 proteins) in “Re-on” and “On” samples, respectively. 190 sites were common between “Re-on” and “On”, 196 sites were “Re-on” unique, and 368 sites were “On” unique. mTOR and MAPK pathways were activated in both “Re-on” and “On”, while Rho kinase, Phospholipase C, and Rac signaling pathways were predominantly associated with “On”, suggestive of differential downstream pathway activation at different phases of Kras signaling.

This abstract is also presented as Poster A21.

Citation Format: Ayumu Taguchi, Avnish Kapoor, Amin Momin, Hiroyuki Katayama, Wantong Yao, Hong Wang, Haoqiang Ying, Ronald DePinho, Samir Hanash. Global quantitative proteomic and phosphoproteomic analysis of oncogenic Kras-driven mouse pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR03. doi: 10.1158/1557-3125.RASONC14-PR03
Abstract A20: Sphingosine 1-phosphate promotes tumor progression in a survivin-dependent manner
Authors: Samadi Nasser , Nezhad Maryam Tabasi , Ghanbari Parisa , Mohseni Mahsa
Published: May 2015

Introduction: sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that involves in tumor progressions, metastasis and chemo-resistance. We investigated the effects of S1P and its signaling mechanism in induction of tumor progression in chronic lymphoblastic leukemia SKW3 cells and non-small cell lung cancer H1299 cells.

Methods: We investigated the effects of S1P on proliferation with MTT assay, invasion by soft-agar colony forming assay and cell migration by applying trans-well migration assay. The expression of survivin gene was assessed by Real-time PCR.

Results: We showed that low concentrations of S1P induced proliferation and migration in both cell lines. S1P also stimulated invasiveness in H1299 cancer cells. We showed that the effects of S1P in tumor progression are S1P receptor-dependent. Survivin also plays a key role in S1P tumorogenic functions.

Conclusion: Our results suggest that identifying cancer patients with higher levels of S1P and survivin and inhibiting their activities can be considered as an important strategy to increase the efficacy of chemotherapeutic agents.

Note: This abstract was not presented at the conference.

Citation Format: Nasser Samadi, Maryam Tabasi Nezhad, Parisa Ghanbari, Mahsa Mohseni. Sphingosine 1-phosphate promotes tumor progression in a survivin-dependent manner. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A20.
Abstract A23: eIF4F is a key and targetable convergence nexus of multiple mechanisms of resistance to anti-BRAF and anti-MEK cancer therapies
Authors: Boussemart Lise , Malka-Mahieu Helene , Girault Isabelle , Allard Delphine , Hemmingson Oskar , Kamsu-Kom Nyam , Agoussi Sandrine , Eggermont Alexander , Desaubry Laurent , Robert Caroline , Vagner Stephan
Published: May 2015

The V600E mutation in BRAF is the most frequent oncogenic mutation in human cancers, and can be targeted by specific anti-BRAF agents. However, several mechanisms of resistance have been identified, leading to reactivation of the initially blocked MAP-kinase pathway (known as ERK-dependent mechanisms) or reactivation of alternative ones, like the PI3K-AKT-mTOR pathway, or inhibition of the apoptotic cascade (ERK-independent). These pathways converge to regulate the formation of the eIF4F translation initiation complex that binds to the 7-methylguanosine cap at the 5′ end of mRNAs, thereby modulating mRNA translation of specific mRNAs. We addressed the potential role of the eIF4F eukaryotic translation initiation complex in resistance or sensitivity to anti-BRAF and anti-MEK agents in several BRAF mutant cells, xenografts and tumors. Our data demonstrate that the formation of the eIF4F complex is associated with most resistance mechanisms to these targeted therapies, independently of their capacity to reactivate or not the MAP-kinase pathway. Formation of this complex can be explored in tumor samples using a new in situ technology. Inhibitors of the eIF4F complex synergize with anti-BRAF agents and can thus reverse resistance.

Citation Format: Lise Boussemart, Helene Malka-Mahieu, Isabelle Girault, Delphine Allard, Oskar Hemmingson, Nyam Kamsu-Kom, Sandrine Agoussi, Alexander Eggermont, Laurent Desaubry, Caroline Robert, Stephan Vagner. eIF4F is a key and targetable convergence nexus of multiple mechanisms of resistance to anti-BRAF and anti-MEK cancer therapies. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A23.
Abstract A38: Optimization of whole-genome amplification for analysis of single cells using next-generation sequencing
Authors: Amiss Terry J. , Tong Frances , Snowden Eileen , Kelly Richard , Blaesius Rainer , Herrmann Nick , Hahn Friedrich , Porter Warren , Ferguson Mitchell , Chang Chen , Clancy Daphne , Jurgensen Stewart
Published: May 2015

The accurate analysis of both genotypic variation in tumors and tumor evolution due to selective pressure is important in determining treatment options in the clinic. Next generation sequencing (NGS) of single cells from solid tumors and circulating tumor cells has the potential to dramatically increase the amount of genotypic detail obtained from these samples. For the analysis of single cells the challenge is to employ whole genome amplification (WGA) to obtain enough DNA for analysis while minimizing amplification bias and maintaining copy accuracy. We are developing tools for single cell sequencing to improve these parameters while also increasing sample throughput. Initially, three commercially available WGA methods were tested on 5-10 genome quantities of HCT-15 genomic DNA. Using the Ion Torrent AmpliSeq Cancer Hotspot Panel, the amplification accuracy, amplicon coverage and DNA yield were determined. When compared to controls significant differences were observed in the three WGA methods ability to accurately amplify DNA. The most accurate kit produced a specificity and sensitivity of 98.5% and 100% respectively. This protocol also produced a substantial DNA yield of 25 µgs and although, the average amplicon coverage indicated some bias, greater than 99% of the amplicons had at least 100-fold coverage. This WGA kit was then tested on single cells from a human breast cancer; an ER/PR/Her2 negative spindle cell metaplastic carcinoma. To obtain single cells the tumor was grown in a PDX mouse model and index sorted using a BD FACSAria II flow cytometer. The WGA produced an average DNA yield of 2.7± 0.8 µg, with 100-fold amplicon coverage of 92±7%. For the variants identified, the amplification bias differed by as much as 3,000 reads/variant. We are now taking steps to improve WGA of single cells and will report on these efforts in detail. To date when monitoring variant allele frequency and coverage depth in the triple negative tumor, KRAS and MET mutations were identified in single cells, as well as in the bulk sample. Interestingly, a TP53 mutation was identified only in single cells. This data supports the utility of WGA and single cell analysis to identify mutations, some having clinically relevant role for targeted therapy.

Citation Format: Terry J. Amiss, Frances Tong, Eileen Snowden, Richard Kelly, Rainer Blaesius, Nick Herrmann, Friedrich Hahn, Warren Porter, Mitchell Ferguson, Chen Chang, Daphne Clancy, Stewart Jurgensen. Optimization of whole-genome amplification for analysis of single cells using next-generation sequencing. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A38.
Abstract A42: Exogenous normal mammary epithelial mitochondria increase the drug sensitivity of human breast cancer MCF-7 cells
Authors: Jiang Xian-Peng , Philips Jeff , Elliott Robert L. , Head Jonathan F.
Published: May 2015

The mitochondrial dysfunction of cancer cells leads to the suppression of glucose oxidation, increased glycolysis, proliferated advantage, resistance to apoptosis and drug resistance. We hypothesize that the introduction of normal mitochondria into cancer cells will restore the mitochondrial function of cancer cells and increase drug sensitivity. We stained the mitochondria of the normal human mammary epithelial cell line MCF-12A with JC-1 dye, and found orange fluorescence (JC-1 aggregates in mitochondria due to high mitochondrial membrane potential) in the MCF-12A cells. We then isolated the JC-1 stained-mitochondria of the MCF-12A cells and co-cultured them with the cells of human breast cancer MCF-7 cell line. At 30 minutes after co-culture of MCF-7 cells with the isolated JC-1-stained mitochondria, the orange fluorescence was observed in the MCF-7 cells. The fluorescence continued to increase for at least 6 hours and was still detected at 24 hours. This demonstrated that exogenous normal mitochondria of MCF-12A can enter into MCF-7 breast cancer cells. In order to determine whether mitochondria of normal MCF-12A cells improve the sensitivity of breast cancer cells to chemotherapeutic drugs, we co-cultured MCF-7 cells for 24 hours with normal mitochondria of MCF-12A, aspirated off the media and treated the MCF-7 cells with the drugs doxorubicin, abraxane or carboplatin. We found that MCF-7 cells, treated with the normal mitochondria of MCF-12A, were more sensitive to doxorubicin, abraxane and carboplatin than non-treated MCF-7 cells. We also have RT-PCR results demonstrating that uptake of normal mitochondria by cancer cells down-regulates the expression of membrane glucose transporter GLUT3 in MCF-7 cells. In conclusion, we suggest that the introduction of normal cell mitochondria into cancer cells could restore cancer cell mitochondrial function and significantly impact cancer therapy.

Citation Format: Xian-Peng Jiang, Jeff Philips, Robert L. Elliott, Jonathan F. Head. Exogenous normal mammary epithelial mitochondria increase the drug sensitivity of human breast cancer MCF-7 cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A42.
Abstract A44: S100A4 drives the invasive potential of non-small cell lung cancer cells and is targeted by FDA approved drugs
Authors: Chen Min , Stewart Rachel , Knifley Teresa , Carpenter Brittany L.
Published: May 2015

The metastatic nature of advanced non-small cell lung cancer (NSCLC), often associated with therapeutic resistance, accounts for the majority of cancer death with the 5-year survival rate less than 10%. This dismal outcome will remain the same until we gain better understanding of the crucial drivers of the metastatic process and gain the power to effectively target them. S100A4 is a tumor metastasis associated protein and an epithelial to mesenchymal transition (EMT) marker. S100A4 contributes to several hallmarks of cancer such as anti-apoptosis, proliferation and therapeutic resistance, and has been implicated in the progression of different types of cancer, including breast, colon and pancreatic cancer. However, the impact of S100A4 signaling in lung cancer progression and its potential use as a target for therapy in lung cancer has not been properly explored. Here, by using established lung cancer cell lines, we demonstrate that S100A4 is upregulated in a subset of lung cancer cell lines both at the mRNA and protein levels. We further found that inhibition of S100A4 by shRNA in A549 and H460 lung cancer cell lines reduced cell proliferation and decreased 3D invasive growth, while exogenous overexpression of S100A4 in H1299 cells increased invasive potential. Interestingly, we showed that Niclosamide, an FDA-approved anti-tapeworm agent, suppressed S100A4 expression in A549 and H358 cells and led to decreased cell proliferation, invasion and invasive growth. Similarly, we found that a CDC25 inhibitor, NSC95397, disrupts the interaction of S100A4 and non-muscle myosin IIA and also decreased A549 cell invasion. Collectively, these data highlight the importance of S100A4 in lung cancer invasion and metastasis and provide the rationale for targeting S100A4 as a potential agent to combat lung cancer.

Citation Format: Min Chen, Rachel Stewart, Teresa Knifley, Brittany L. Carpenter. S100A4 drives the invasive potential of non-small cell lung cancer cells and is targeted by FDA approved drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A44.
Abstract B11: Stromal response to prostate cancer therapeutics
Authors: Gales Dominique Nicole , Fadlalla Khalda , Manne Upender , Yates Clayton , Samuel Temesgen
Published: May 2015

Docetaxel (D) is a clinically used front-line chemotherapeutic agent for castration resistant advanced prostate cancer (Pca). However, its long-term benefits are limited, with most patients eventually progressing because of inherent or acquired drug resistance. The treatment of advanced Pca is a significant challenge and there are no effective treatments that stably suppress the disease. The mechanisms behind the resistance to Docetaxel are not fully understood. It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at different stages of cancer development or during chemotherapy are largely unidentified. The central goal is to identify the cellular and biochemical responses of stromal cells in tumor microenvironment with which prostate cancer cells interact. Specifically, the interaction of prostate cancer cells with stromal cells of the prostate or the bone microenvironment in the presence of clinically used cancer therapeutics will be examined. Key regulators of these interactions will also be identified. Such regulators include cytokines, growth factors, proteins, and their receptors. By identifying these regulators and their contribution to tumor drug response, novel therapeutics targeted to the microenvironment can be developed. Therefore, we hypothesize that the response of prostate stromal cells exposed to chemotherapeutic agents could modify drug response or therapeutic outcome of prostate cancer. Cellular proliferation was analyzed by MTT assay. The IC50 of Docetaxel for WPMY-1 (normal stromal), LNCaP (androgen dependent), DU-145 (androgen independent), and HS27A (bone stromal) showed inhibitory effects between 2-10nM. Cell cycle profiles were assessed by flow cytometry. The analysis showed that Docetaxel (2-50nM) induced prominent G2-M arrest in prostate and stromal cells within 24 hours. Gene expression profiling of prostate cancer and stromal cells lines were evaluated. To obtain the expression profiles, we conducted a human cytokines and chemokines array, which consisted of 84 specific genes. Analysis of the prostate cancer and stromal cells treated with Docetaxel revealed differential expression patterns. These results suggest Docetaxel exhibits strong anti-proliferative effects and analysis of the gene array suggest regulation of genes associated with, cell growth, survival, proliferation, metastasis, angiogenesis, and apoptosis.

Note: This abstract was not presented at the conference.

Citation Format: Dominique Nicole Gales, Khalda Fadlalla, Upender Manne, Clayton Yates, Temesgen Samuel. Stromal response to prostate cancer therapeutics. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B11.
Abstract B22: Potential combinatorial strategies to overcome drug resistance in BRAF-mutant melanoma
Authors: Dompe Nicholas A. , Lin Eva , Peng Jing , Chan Grace , Chan Grace , Chan Grace , Tien Janet , Merchant Mark , Bourgon Richard , Settleman Jeffrey , Wilson Timothy , Neve Richard M.
Published: May 2015

RAF and MEK inhibitors are used to target malignant melanomas harbouring BRAF V600E mutations, but resistance to these therapies remains a significant challenge. Here we conducted a screen of 450 secreted peptides to identify possible cell-autonomous or microenvironment-driven mechanisms of resistance to RAF and MEK inhibitors in ten melanoma cell lines. We identified multiple receptor tyrosine kinase (RTK)-mediated resistance mechanisms and show that drug-resistance in vitro was driven by mechanisms predicted by the screen and that co-targeting FGFR and BRAF prevents tumour regrowth in vivo. We also found that cells can become resistant to rational combinations of targeted inhibitors in a serial manner, again predicted by the screen. In many cases resistance is mediated by reactivation of MAPK-signalling, however, there is also evidence for non-MAPK-dependent mechanisms of resistance once these are exhausted. These data reveal multiple, defined mechanisms of resistance as well as highlight potential challenges of overcoming therapeutic resistance in a highly adaptive tumour type.

Citation Format: Nicholas A. Dompe, Eva Lin, Jing Peng, Grace Chan, Grace Chan, Grace Chan, Janet Tien, Mark Merchant, Richard Bourgon, Jeffrey Settleman, Timothy Wilson, Richard M. Neve. Potential combinatorial strategies to overcome drug resistance in BRAF-mutant melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B22.
Abstract IA16: Acquired resistance to targeted therapeutics in hematologic malignancies
Authors: Shah Neil P.
Published: May 2015

Targeted treatment approaches of hematologic malignancies provided many of the first inroads into the successful treatment of human malignancy with the emerging class of targeted therapeutics. Initial responsiveness to targeted therapy provides compelling evidence for the importance of specific targets for the maintenance of disease. Understanding mechanisms of acquired resistance further informs how malignant cells can circumvent target inhibition in patients, and in some instances, these insights have facilitated the successful development of next generation targeted therapeutics, best exemplified by BCR-ABL tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia (CML). As detailed genomic analyses are becoming more readily available, opportunities for developing and incorporating targeted therapeutics are expected to increase. It is becoming clear, however, that tumor heterogeneity in advanced hematologic malignancies can be substantial, and resistant subclones can compromise duration of response to targeted therapy. Several examples of acquired resistance to targeted therapeutics in human hematopoietic malignancies will be presented, as well as the implications of these molecular mechanisms toward future successful management of these diseases.

Citation Format: Neil P. Shah. Acquired resistance to targeted therapeutics in hematologic malignancies. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA16.
Abstract IA22: Investigating breast cancer evolution and diversity with single-cell sequencing
Authors: Navin Nicholas E.
Published: May 2015

Sequencing breast tumor cohorts has identified many prevalent mutations, but provides limited insight into the genomic diversity within tumors. Here, we developed a whole-genome and exome single-cell sequencing approach called Nuc-Seq that can achieve 83-97% coverage breadth with low error rates. We applied this method to sequence single normal and tumor cells from an estrogen-receptor positive breast cancer and a triple-negative ductal carcinoma, and performed copy number profiling in parallel. Our data shows that aneuploid rearrangements occurred early in tumor evolution and remained highly stable as the tumor masses expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Many of the diverse mutations were shown to occur at low frequencies (0.03 -10%) in the tumor mass by targeted single-molecule sequencing. Using mathematical modeling we found that the triple-negative tumor cells had an increased mutation rate (13.3X) while the ER+ tumor cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

Citation Format: Nicholas E. Navin. Investigating breast cancer evolution and diversity with single-cell sequencing. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA22.
Abstract PR06: A co-clinical assessment of patterns of BRAF inhibitor resistance
Authors: Kwong Lawrence N. , Boland Genevieve , Frederick Dennie T. , Helms Timothy , Miller John P. , Jiang Shan , Cooper Zachary A. , Protopopov Alexei , Mills Gordon B. , Flaherty Keith , Wargo Jennifer A. , Chin Lynda
Published: May 2015

The acquired resistance of melanomas to BRAF inhibitors is now well-described, indicating many mechanisms including point mutations, amplifications, and alternative splicing. Here, we present a combined assessment of patient samples at both the RNA and protein levels. In parallel, we conducted a co-clinical mouse trial using a novel BRAF-driven model. We demonstrate that the response and resistance of the mouse melanomas closely parallels that of the human patient samples on both phenotypic and molecular levels, demonstrating multiple mechanisms of resistance that mirror the patient findings. On the basis of the combined data, we suggest that BRAF inhibitor-resistant melanomas can readily be categorized using economical and rapid protein array technology. This suggests that analyzing protein patterns offers a means of categorizing tumor resistance mechanisms even in the absence of identified driving genetic or epigenetic lesion(s). In summary, our results demonstrate the utility of a co-clinical assessment system and offer a cost-efficient means of categorizing patterns of BRAF inhibitor resistance in human patient samples within a clinically-meaningful timeframe.

Citation Format: Lawrence N. Kwong, Genevieve Boland, Dennie T. Frederick, Timothy Helms, John P. Miller, Shan Jiang, Zachary A. Cooper, Alexei Protopopov, Gordon B. Mills, Keith Flaherty, Jennifer A. Wargo, Lynda Chin. A co-clinical assessment of patterns of BRAF inhibitor resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR06.
A Rapid Embryonic Stem Cell–Based Mouse Model for B-cell Lymphomas Driven by Epstein–Barr Virus Protein LMP1
Authors: Ba Zhaoqing , Meng Fei-Long , Gostissa Monica , Huang Pei-Yi , Ke Qiang , Wang Zhe , Dao Mai N. , Fujiwara Yuko , Rajewsky Klaus , Zhang Baochun , Alt Frederick W.
Published: May 2015

The Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells (“CLT” mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation (“RDBC”) approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. Cancer Immunol Res; 3(6); 641–9. ©2015 AACR.