Nicole Fer

Small Cell Lung Cancer Screen of Oncology Drugs, Investigational Agents, and Gene and microRNA Expression

BACKGROUND Small cell lung carcinoma (SCLC) is an aggressive, recalcitrant cancer, often metastatic at diagnosis and unresponsive to chemotherapy upon recurrence, thus it is challenging to treat. METHODS Sixty-three human SCLC lines and three NSCLC lines were screened for response to 103 US Food and Drug Administration-approved oncology agents and 423 investigational agents. The investigational agents library was a diverse set of small molecules that included multiple compounds targeting the same molecular entity. The compounds were screened in triplicate at nine concentrations with a 96-hour exposure time using an ATP Lite endpoint. Gene expression was assessed by exon array, and microRNA expression was derived by direct digital detection. Activity across the SCLC lines was associated with molecular characteristics using pair-wise Pearson correlations. RESULTS Results are presented for inhibitors of targets: BCL2, PARP1, mTOR, IGF1R, KSP/Eg5, PLK-1, AURK, and FGFR1. A relational map identified compounds with similar patterns of response. Unsupervised microRNA clustering resulted in three distinct SCLC subgroups. Associating drug response with micro-RNA expression indicated that lines most sensitive to etoposide and topotecan expressed high miR-200c-3p and low miR-140-5p and miR-9-5p. The BCL-2/BCL-XL inhibitors produced similar response patterns. Sensitivity to ABT-737 correlated with higher ASCL1 and BCL2. Several classes of compounds targeting nuclear proteins regulating mitosis produced a response pattern distinct from the etoposide response pattern. CONCLUSIONS Agents targeting nuclear kinases appear to be effective in SCLC lines. Confirmation of SCLC line findings in xenografts is needed. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sclccelllines.cancer.gov.

Evaluation of the selectivity and sensitivity of isoform- and mutation-specific RAS antibodies

Validation studies reveal the reliability of isoform- and mutation-specific anti-RAS antibodies. A resource on RAS antibodies Researchers rely largely on antibodies to measure the abundance, activity, and localization of a protein, information that provides critical insight into both normal and pathological cellular functions. However, antibodies are not always reliable or universally valid for the methods in which they are used; in particular, the reliability of commercial antibodies against RAS is highly variable. Waters et al. rigorously assessed 22 commercially available RAS antibodies for their utility to detect the distinct RAS isoforms in various cell types and for their use in specific analytical methods. Their findings show how reliably one can interpret the data acquired from each reagent. There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform–specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from “Rasless” cells expressing the oncoprotein BRAFV600E. Using our validated antibodies, we identified RAS isoform–specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.

The HIF-1α-c-Myc pathway and tumorigenesis: Evading the apoptotic gate-keeper

Comment on: Hayashi M, et al. Cell Cycle 2011; 10:2364–72

The HIF-1α-c-Myc pathway and tumorigenesis

Comment on: Hayashi M, et al. Cell Cycle 2011; 10:2364–72

Abstract 1131: Uncovering oncogenic KRAS allele specific phenotypes using an isogenic MEF cell line system

The RAS Initiative at the Frederick National Laboratory has generated an isogenic mouse embryonic fibroblast (MEFs) cell line panel containing single transgene alleles using the RAS-less MEF system developed by Matthias Drosten and Mariano Barbacid (CNIO). These MEFs are H, N and K-RAS null, growth is dependent on exogenous RAS or MAPK pathway activation. The panel includes: KRAS 4A WT, KRAS 4B WT, KRAS 4B G12C, G12D, G12V, G13D, Q61L, Q61R, HRAS WT, NRAS WT or BRAF V600E. The workflow to generate the lines requires cre-lox removal of endogenous KRAS and the cells arrest in the G1 phase of the cycle. Proliferation is resumed through the delivery of the transgene to the cells using lentiviral transduction. Clonal cell lines are derived from the initial pools and are thoroughly characterized including confirmation of endogenous KRAS gene removal, identification of the transgene insertion site(s), calculation of proliferation rates and doubling times, analysis of signaling pathways, response to tool compounds and exome sequenced to exclude lines with mutations in onco-relevant genes. This panel is a unique resource that can be used to ask allele and isoform specific questions (gene expression profiles, GTP-loading, etc.) and screen for RAS inhibitors as well as determine novel compound specificity. This panel is available to the academic RAS research community. Citation Format: Nicole Fer, Brian Smith, Leslie Garvey, William Burgan, Katie Powell, Kanika Sharma, Andrew Waters, Xiaolin Wu, Dan Soppet, Robert Stephens, Dwight Nissley, Matthew Holderfield, Rachel K. Bagni. Uncovering oncogenic KRAS allele specific phenotypes using an isogenic MEF cell line system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1131.

Abstract 2147: Correlating RAS oncogenic allele dependence with drug sensitivity

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA KRAS is a small G-protein that is a highly important member of the EGFR pathway. KRAS is frequently mutated in human cancers and predicts a shorter survival for patients with lung and colorectal cancers. Although any mutation at codon 12 or 13 is potentially oncogenic, Cysteine-12, Aspartate-12, Valine-12, and Aspartate 13 mutations predominate in human cancers, and the various oncogenic alleles correlate with distinct clinical outcomes. There is even some indication that Glycine 12 mutations may benefit from EGFR targeted therapy, whereas codon 12 mutations are contraindicated. We hypothesize that these clinical observations may be explained by subtle differences in effector pathway activation. It has been reported that G12C and G12V mutations preferentially activate the Ral-GDS pathway, while cell lines with G12D mutations elevate the PI3′ kinase and MAPK pathways. However, it is difficult to correlate clinical response to differences in biochemical signaling amidst a myriad of potentially confounding genetic alterations present across a panel of cell lines or patient tumor samples. In order to normalize the contributions of cell line heterogeneity, we tested pairs of isogenic cell lines to isolate KRAS allele specific effects, and to identify compounds that selectively inhibit RAS dependent growth in an allele specific manner. To do this, we used an engineered mouse embryonic fibroblast cell line - H-Ras−/−, N-Ras−/− and K-Raslox/lox, which expresses a transgene cre-recombinase - estrogen receptor fusion. Treatment with 4OHT results in excision of the endogenous K-Ras alleles, loss of KRAS expression, suppression of the MAPK pathway and G1 arrest. Reconstitution with any RAS isoform (K- H- or N-Ras), including any of the KRAS oncogenic alleles using a lentiviral vector system, allows the cells to re-enter the cell cycle and proliferate. The doubling time and basal pERK and pAKT levels are comparable across all isogenic uniclonal cell lines regardless of the RAS allele introduced. This system takes advantage of its dependency on a single RAS mutant to help screen for effector pathway dependence or synthetic lethal interactions unilaterally relevant to one mutant KRAS allele. Majority of compounds tested were unbiased towards the cell lines, but a few classes of drug showed selectivity. Cells expressing wild-type RAS were more sensitive to RTK inhibitors compared with cells expressing a mutant KRAS allele. Farnesyl Transferase Inhibitors (FTIs) such as Tipifarnib (R115777/Zarnestra) and Lonafarnib (SCH66336/Sarasar) were more potent against the HRAS WT dependent cell lines as compared to the KRAS dependent lines, validating the escape mechanism by which K-Ras and N-Ras get geranylgeranylated in the presence of FTIs. This strategy provides us with tools to delineate the subtle allele specific sensitivity differences which may translate to meaningful biology and may be used for tailoring mutant specific drug regimens in the clinic. Citation Format: Kanika Sharma, Katie Beam, Nicole Fer, Matthew Holderfield. Correlating RAS oncogenic allele dependence with drug sensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2147. doi:10.1158/1538-7445.AM2015-2147

Abstract 4690: Next-generation screen for integrative subtyping and target discovery for KRAS-mutant cancer

Mutations in the small GTPase, KRAS, are found in ∼140,000 new cases of cancer every year in the United States. This heterogeneous class of cancers manifests primarily as adenocarcinomas of the lung, colon and pancreas. These cancers display a wide spectrum of KRAS-dependency and differentially activate downstream effector signaling. The tumors further diverge in their array of co-occurring secondary mutations, expression signatures and KRAS mutant allele. Ultimately, the sole trait these cancers share in common is an obstinate resistance to chemo- and targeted-therapies, making identification of effective treatments an urgent need. To identify treatments for such a heterogeneous class of cancers, we developed a strategy to stratify KRAS-mutant cell lines into subtypes by integrating next-generation RNAi screening and “Omics” database mining. Each subtype is characterized by unique biomarkers and distinct patterns of effector dependency, both of which represent potential targets for personalized therapeutic strategies. Our RNAi screen systematically evaluates sensitivity to siRNA-mediated knockdown of 40 KRAS effector nodes in a panel of 135 lung, colorectal and pancreatic cancer cell lines. Data is analyzed on the single cell level, through the simultaneous measurement of 5 functional parameters. This single-cell, multi-dimensional approach allows for a comprehensive assessment of cellular homeostasis, with unprecedented depth and dynamic range that allows robust classification of cell lines by similarity. We identify subtypes of KRAS-mutant cell lines that rely on particular effector pathways such as the RAL, RSK, MTOR and autophagy pathways, which are not engaged by all KRAS-mutant cell lines, and thus may represent targets for personalized treatment. We further identify widely shared dependencies such as on the RAF, glycolytic and cell cycle pathways. Through integrative data mining of exome, transcriptome and drug/siRNA sensitivity databases for each KRAS-mutant subtype, we can identify unique biomarkers that will serve to stratify patients in the clinic and recommend personalized treatment strategies. Citation Format: Tina L. Yuan, Rachel Bagni, Ming Yi, Arnaud Amzallag, Shervin Afghani, Katie Beam, William Burgan, Nicole Fer, Leslie Garvey, Brian Smith, Andrew Waters, Robert Stephens, Cyril Benes, Frank McCormick. Next-generation screen for integrative subtyping and target discovery for KRAS-mutant cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4690. doi:10.1158/1538-7445.AM2015-4690

Exploiting “Hif Addiction” For Cancer Therapy

Intratumor hypoxia is a common and unifying characteristic of solid tumors that are associated with altered cellular metabolism, an invasive and metastatic phenotype, as well as resistance to radiation and chemotherapy. The discovery of hypoxia-inducible factor-1 (HIF-1), a transcription factor critically involved in cellular responses to hypoxia and tumor progression, has provided a molecular target of the hypoxic tumor microenvironment that has been exploited for the development of novel cancer therapeutics. However, HIF-1 expression in human cancers is focal and heterogeneous, strongly arguing against the clinical efficacy of single agent HIF-1 inhibitors and opening the door to combinational therapies. A therapy-induced hypoxic tumor microenvironment, and the subsequent activation of HIF-1-dependent pathways, may create an “HIF addiction” that will represent the perfect setting to fully exploit HIF-1 inhibitors. Interestingly, therapies targeting tumor vasculature have been shown to increase intratumor hypoxia in some instances and hypoxia has been suggested to mediate several mechanisms of resistance to antiangiogenic therapies, providing a rationale for combination therapies with HIF-1 inhibitors. In this chapter, we discuss the potential of exploiting a therapy-induced “HIF addiction” by using known HIF inhibitors to improve clinical efficacy keeping in mind that patient selection is essential.

Abstract 3741: Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors

Recurrent isocitrate dehydrogenase (IDH) mutations have recently been identified in several cancer types. These point mutations specifically affect IDH1 and IDH2 active site arginine residues and confer the ability to reduce α-ketoglutarate to R-2-hydroxyglutarate (2HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Accumulating evidence indicates that mIDH enzymes are attractive targets for the development of novel cancer therapeutics. To validate potential mIDH inhibitors in a cellular assay, we acquired several cell lines that harbor mIDH1 R132H and R132C: HCT116 mIDH1 (colon cancer, mIDH1 R132H +/-, Horizon Discovery), U87 MG mIDH1 (glioma, m IDH1 R132H +/+, Japan), HT1080 (fibrosarcoma, mIDH1 R132C +/-) and JJ012 (chondrosarcoma, mIDH1 R132C +/-, Rush University Medical Center). Cell lines were sequenced to confirm the presence of mIDH1 and 2HG production was measured in cellular supernatants by GC-MS. We observed that cells harboring mIDH1 R132C produce higher amounts of 2HG compared to lines harboring mIDH1 R132H, in agreement with recent reports. In addition, we show that accumulation of 2HG is time dependent and that its half-life in cellular supernatant is over 48 hours. Moreover, we demonstrate that 2HG is a stable analyte in media (through five sample free-thaw cycles) and that our samples are within linear range, providing a good intra-assay and inter-assay variability (CV Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Nicole D. Fer, Erik Harris, Stephen Fox, Ming Zhou, Catherine Simpson, Jing Liu, Ilia Korboukh, Emily A. Hull-Ryde, William P. Janzen, Stephen V. Frye, Anne Monks, Beverly Teicher, Annamaria Rapisarda. Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors. [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 3741. doi:10.1158/1538-7445.AM2014-3741

Abstract 890: Interleukin 11: A novel therapeutic target in IL-11Rα expressing sarcomas

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Interleukin-11 (IL-11) is a member of the IL-6 cytokine family, which mediate signaling via a common signal-transducing gp130 component and a cytokine-specific subunit (IL-11Rβ). Upon binding to the IL-11Rα, a signaling cascade activates several pathways, including JAK kinases, STAT transcription factors (STAT3 and STAT1), MAPK, src-family kinases and PI3K. IL-11 has been implicated in experimental models of chronic inflammation and associated tumorigenesis. Oncomine analysis of publicly available microarray data from patient biopsies showed that IL-11Rα mRNA is expressed in several sarcomas and not in the matched normal tissue counterparts. Surgical resection and cytotoxic chemotherapy are the standard of care for most locally advanced and metastatic sarcomas, the identification of new markers and the development of targeted therapies are needed to increase long-term survival of these patients. Previous studies demonstrated IL-11Rα is expressed in human primary and metastatic osteosarcomas but not in normal bone, suggesting that IL-11Rα is a potential target for sarcoma therapy. We analyzed a panel of 45 human sarcoma cell lines for the expression of IL-11Rα and IL-11. Interestingly, IL-11Rα protein is expressed at high levels in the majority of cell lines examined. In addition, we found that nearly all osteosarcoma, rhabdomyosarcoma, fibrosarcoma and Ewing sarcoma cell lines express high constitutive levels of IL-11 mRNA, indicating that IL-11 autocrine signaling may play a role in these tumor types. Two lentiviral vectors, used for the delivery of shRNA targeting IL-11 in HT-1080, produced an 80-90% decrease in IL-11 mRNA expression when compared to the luciferase control shRNA; however, IL-11 mRNA levels remained significantly high in HT1080 knock-down cells when compared to the constitutive levels of non sarcoma cancer types. Decreasing levels of IL-11 in HT-1080 resulted in a 50% inhibition of cell growth, demonstrating that IL-11 has a role in cell proliferation. Further experiments aimed to assess the expression of IL-11 and IL-11Rβ in xenografts and to explore the role of IL-11 in sarcoma pathogenesis, propagation and response to therapy are ongoing. These data suggest that neutralization of IL-11 or IL-11Rα may be a potential target for the development of cancer therapeutics for sarcomas. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 890. doi:1538-7445.AM2012-890