Katie Beam

In vitro and In vivo Radiosensitization of Glioblastoma Cells by the Poly (ADP-Ribose) Polymerase Inhibitor E7016

Purpose: Poly (ADP-ribose) polymerase (PARP) inhibitors are undergoing clinical evaluation for cancer therapy. Because PARP inhibition has been shown to enhance tumor cell sensitivity to radiation, we investigated the in vitro and in vivo effects of the novel PARP inhibitor E7016. Experimental Design: The effect of E7016 on the in vitro radiosensitivity of tumor cell lines was evaluated using clonogenic survival. DNA damage and repair were measured using γH2AX foci and neutral comet assay. Mitotic catastrophe was determined by immunostaining. Tumor growth delay was evaluated in mice for the effect of E7016 on in vivo (U251) tumor radiosensitivity. Results: Cell lines exposed to E7016 preirradiation yielded an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 from 1.4 to 1.7. To assess DNA double-strand breaks repair, γH2AX measured at 24 hours postirradiation had significantly more foci per cell in the E7016/irradiation group versus irradiation alone. Neutral comet assay further suggested unrepaired double-strand breaks with significantly greater DNA damage at 6 hours postirradiation in the combination group versus irradiation alone. Mitotic catastrophe staining revealed a significantly greater number of cells staining positive at 24 hours postirradiation in the combination group. In vivo, mice treated with E7016/irradiation/temozolomide had an additional growth delay of six days compared with the combination of temozolomide and irradiation. Conclusions: These results indicate that E7016 can enhance tumor cell radiosensitivity in vitro and in vivo through the inhibition of DNA repair. Moreover, enhanced growth delay with the addition of E7016 to temozolomide and radiotherapy in a glioma mouse model suggests a potential role for this drug in the treatment of glioblastoma multiforme.

In vitro and In vivo Radiosensitization Induced by the DNA Methylating Agent Temozolomide

Purpose: Temozolomide, a DNA methylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomide has been shown to increase survival rates of patients with malignant gliomas when given combined with radiation, and there is conflicting preclinical data concerning the radiosensitizing effects of temozolomide, we further investigated the possible temozolomide-induced enhancement of radiosensitivity. Experimental Design: The effects of temozolomide on the in vitro radiosensitivity of U251 (a human glioma) and MDA-MB231BR (a brain-seeking variant of a human breast tumor) cell lines was evaluated using clonogenic assay. DNA damage and repair were evaluated using phosphorylated histone H2AX (γH2AX), and mitotic catastrophe was measured using nuclear fragmentation. Growth delay was used to evaluate the effects of temozolomide on in vivo (U251) tumor radiosensitivity. Results: Exposure of each cell line to temozolomide for 1 h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. As a measure of DNA double strand breaks, γH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of γH2AX foci per cell was significantly greater at 24 h after the combined modality compared with the individual treatments. Mitotic catastrophe, measured at 72 h, was also significantly increased in cells receiving the temozolomide + irradiation combination compared with the single treatments. In vivo studies revealed that temozolomide administration to mice bearing U251 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay with a dose enhancement factor of 2.8. Conclusions: These results indicate that temozolomide can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.

Postradiation Sensitization of the Histone Deacetylase Inhibitor Valproic Acid

Purpose: Preclinical studies evaluating histone deacetylase (HDAC) inhibitor-induced radiosensitization have largely focused on the preirradiation setting based on the assumption that enhanced radiosensitivity was mediated by changes in gene expression. Our previous investigations identified maximal radiosensitization when cells were exposed to HDAC inhibitors in both the preradiation and postradiation setting. We now expand on these studies to determine whether postirradiation exposure alone affects radiosensitivity. Experimental Design: The effects of the HDAC inhibitor valproic acid (VA) on postirradiation sensitivity in human glioma cell lines were evaluated using a clonogenic assay, exposing cells to VA up to 24 h after irradiation. DNA damage repair was evaluated using γH2AX and 53BP1 foci and cell cycle phase distribution was analyzed by flow cytometry. Western blot of acetylated γH2AX was done following histone extraction on AUT gels. Results: VA enhanced radiosensitivity when delivered up to 24 h after irradiation. Cells accumulated in G2-M following irradiation, although they returned to baseline at 24 h, mitigating the role of cell cycle redistribution in postirradiation sensitization by VA. At 12 h after irradiation, significant γH2AX and 53BP1 foci dispersal was shown in the control, although cells exposed to VA after irradiation maintained foci expression. VA alone had no effect on the acetylation or phosphorylation of H2AX, although it did acetylate radiation-induced γH2AX. Conclusions: These results indicate that VA enhances radiosensitivity at times up to 24 h after irradiation, which has direct clinical application.

Vorinostat enhances the radiosensitivity of a breast cancer brain metastatic cell line grown in vitro and as intracranial xenografts

Vorinostat (suberoylanilide hydroxamic acid), a histone deacetylase inhibitor, is currently undergoing clinical evaluation as therapy for cancer. We investigated the effects of vorinostat on tumor cell radiosensitivity in a breast cancer brain metastasis model using MDA-MB-231-BR cells. In vitro radiosensitivity was evaluated using clonogenic assay. Cell cycle distribution and apoptosis was measured using flow cytometry. DNA damage and repair was evaluated using γH2AX. Mitotic catastrophe was measured by immunostaining. Growth delay and intracranial xenograft models were used to evaluate the in vivo tumor radiosensitivity. Cells exposed to vorinostat for 16 hours before and maintained in the medium after irradiation had an increase in radiosensitivity with a dose enhancement factor of 1.57. γH2AX, as an indicator of double-strand breaks, had significantly more foci per cell in the vorinostat plus irradiation group. Mitotic catastrophe, measured at 72 hours, was significantly increased in cells receiving vorinostat plus irradiation. Irradiation of s.c. MDA-MB-231-BR tumors in mice treated with vorinostat resulted in an increase in radiation-induced tumor growth delay. Most importantly, animals with intracranial tumor implants lived the longest after combination treatment. These results indicate that vorinostat enhances tumor cell radiosensitivity in vitro and in vivo. There was a greater than additive improvement in survival in our intracranial model. Combining vorinostat with radiation may be a potential treatment option for patients with breast cancer who develop brain metastases. [Mol Cancer Ther 2009;8(6):1589–95]

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

Development of multiplex serological assays to detect oncoviral infections

Serological markers of infection (antibodies or antigens) of viruses that cause cancer are most often detected using ELISA-based methodologies. In many cases, multiple markers of infection must be assessed to determine a final sero-status. Volume requirements and costs of reagents for single analyte ELISAs are high and studies which include multiple viruses can require milliliters of plasma, often not available from archived cohorts. Thus, we sought to develop a Luminex® bead-based customizable panel initially including Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Epstein-Barr Virus (EBV) and often the concomitant infection, Human Immunodeficiency Virus (HIV) to reduce sample volume requirements, overall cost and increase flexibility. Peptides, antigens and antibodies were sourced from multiple manufacturers and tested for suitability in this platform. Where necessary, suitable reagents were designed and produced in-house. The HCV assay multiplexes four HCV peptides designed to detect antibodies raised to HCV Core (2), HCV NS4, HCV NS5 gene regions. The HBV assay multiplexes a HBV early antigen peptide, a recombinant HBV core protein and either a recombinant HBV surface antigen or an antibody specific for HBV surface antigen to assess HBV infection. The EBV assay multiplexes peptides specific to viral capsid antigen, EBNA-1 and early antigen (Cyto-Barr, Zuidhorn, The Netherlands). HIV-1 assay development is ongoing and the list of antigens to be included in the assay has not been finalized. These assays can be run singly or with any combination (multi-plex) of the above listed targets. Each target has been independently validated using samples of known molecular and serological status to determine specificity (false positive versus false negative) and re-evaluated under multiplex conditions to confirm assay performance. In addition, where possible, samples were assayed on commercial testing platforms as well as our multiplex assay to assess concordance (94-99%). Dependent on the panel selected and the expected antibody titers in a particular population, plasma or serum volumes in the range of 10 µL to 125 µL per subject would be required to determine the HBV, HCV, EBV and/or HIV serostatus of a subject. This assay platform is inherently flexible and the benefits include amenability to expansion to include other oncogenic viruses as well as screening large epidemiological cohorts or smaller subsets of samples in an economical and high throughput manner.

Enhancement of Tumor Cell Radiosensitivity by a Novel Functional Class of Thalidomide Analogs

Materials/Methods: Clonogenic survival assays were used to evaluate cell survival after exposure to CPS45 followed by radiation. DNA damage and repair were evaluated using phosphorylated histone H2AX (gH2AX) and the neutral comet assay. Mechanism of cell death was determined by immunostaining for mitotic catastrophe and apoptosis by flow cytometry. Cell cycle changes were evaluated by staining of phospho-H3 and flow cytometry. In vivo activity was measured using a tumor growth delay assay. Results: Exposure of each cell line to CPS45 for 16 h before irradiation (IR) resulted in an increase in radiosensitivity with dose enhancement factors of 1.8 and 1.5 for U251 and MiaPaCa cells, respectively (PF = 0.43, 0.31 respectively in CPS45 treated cells). CPS45 had no effect on IR-induced apoptosis or on the activation of the G2 cell cycle checkpoint. However, CPS45 did modify the time course ofgH2AX expression in IR cells. There was significant difference in IR-inducedgH2AX foci count at both 6 h and at 24 h after IR in the CPS45 + IR group than either individual treatment. In the neutral comet assay, there was a significant increase in the percentage of DNA damage remaining in cells exposed to CPS45 before IR compared to IR alone. Mitotic catastrophe, measured at 48 and 72 hour, was also significantly increased in cells receiving the CPS45 + IR combination compared with the individual treatments. Tumor growth delay demonstrated a greater than additive effect of the combination of CPS45 and IR than either treatment alone. Conclusions: These results indicate that CPS45 can enhance tumor cell radiosensitivity and suggest that this effect involves an inhibition of double strand breaks repair leading to an increase in mitotic catastrophe.