Natural Product Graveoline Modulates Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) Membrane Association: Insights from Advanced Spectroscopic Studies.

The KRAS gene plays a pivotal role in numerous cancers by encoding a GTPase that upon association with the plasma membrane activates the MAPK pathway, promoting cellular proliferation. In our study, we investigated small molecules that disrupt KRAS's membrane interaction, hypothesizing that such disruption could in turn inhibit mutant RAS signaling. Native mass spectrometry screening of KRAS-FMe identified compounds with a preference for interacting with the hypervariable region (HVR), and surface plasmon resonance (SPR) further refined our selection to graveoline as a compound exhibiting preferential HVR binding. Subsequent nuclear magnetic resonance (NMR) analysis showed that graveoline's interaction with KRAS depends on C-terminal O-methylation. Moreover, our findings revealed multiple interaction sites, suggesting weak engagement with the KRAS G domain. Using nanodiscs as a membrane mimetic, further characterization through NMR and Förster resonance energy transfer (FRET) studies demonstrated graveoline's ability to perturb KRAS membrane interaction in a biochemical setting. Our biophysical approach sheds light on the intricate molecular mechanisms underlying KRAS-ligand interactions, providing valuable insights into understanding the KRAS-associated pathophysiology. These findings contribute to the translational aspect of our study, offering potential avenues for further research targeting KRAS membrane association with the potential to lead to a new class of RAS therapeutics.

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior.

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.

Differential Effector Engagement by Oncogenic KRAS.

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.

A Gene Expression Signature Associated with Overall Survival in Patients with Hepatocellular Carcinoma Suggests a New Treatment Strategy.

Despite improvements in the management of liver cancer, the survival rate for patients with hepatocellular carcinoma (HCC) remains dismal. The survival benefit of systemic chemotherapy for the treatment of liver cancer is only marginal. Although the reasons for treatment failure are multifactorial, intrinsic resistance to chemotherapy plays a primary role. Here, we analyzed the expression of 377 multidrug resistance (MDR)-associated genes in two independent cohorts of patients with advanced HCC, with the aim of finding ways to improve survival in this poor-prognosis cancer. Taqman-based quantitative polymerase chain reaction revealed a 45-gene signature that predicts overall survival (OS) in patients with HCC. Using the Connectivity Map Tool, we were able to identify drugs that converted the gene expression profiles of HCC cell lines from ones matching patients with poor OS to profiles associated with good OS. We found three compounds that convert the gene expression profiles of three HCC cell lines to gene expression profiles associated with good OS. These compounds increase histone acetylation, which correlates with the synergistic sensitization of those MDR tumor cells to conventional chemotherapeutic agents, including cisplatin, sorafenib, and 5-fluorouracil. Our results indicate that it is possible to modulate gene expression profiles in HCC cell lines to those associated with better outcome. This approach also increases sensitization of HCC cells toward conventional chemotherapeutic agents. This work suggests new treatment strategies for a disease for which few therapeutic options exist.

Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.

Cell line models have been widely used to investigate glioblastoma multiforme (GBM) pathobiology and in the development of targeted therapies. However, GBM tumours are molecularly heterogeneous and how cell lines can best model that diversity is unknown. In this report, we investigated gene expression profiles of three preclinical growth models of glioma cell lines, in vitro and in vivo as subcutaneous and intracerebral xenografts to examine which cell line model most resembles the clinical samples. Whole genome DNA microarrays were used to profile gene expression in a collection of 25 high-grade glioblastomas, and comparisons were made to profiles of cell lines under three different growth models. Hierarchical clustering revealed three molecular subtypes of the glioblastoma patient samples. Supervised learning algorithm, trained on glioma subtypes predicted the intracerebral cell line model with one glioma subtype (r = 0.68; 95% bootstrap CI -0.41, 0.46). Survival analysis of enriched gene sets (P < 0.05) revealed 19 biological categories (146 genes) belonging to neuronal, signal transduction, apoptosis- and glutamate-mediated neurotransmitter activation signals that are associated with poor prognosis in this glioma subclass. We validated the expression profiles of these gene categories in an independent cohort of patients from 'The Cancer Genome Atlas' project (r = 0.62, 95% bootstrap CI: -0.42, 0.43). We then used these data to select and inhibit a novel target (glutamate receptor) and showed that LY341595, a glutamate receptor specific antagonist, could prolong survival in intracerebral tumour-implanted mice in combination with irradiation, providing an in vivo cell line system of preclinical studies.

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody, AMG102.

The hepatocyte growth factor (HGF)/Met signalling pathway is up-regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti-HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U-87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.

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 gammaH2AX. 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. gammaH2AX, 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.

In vitro and in vivo radiosensitization with AZD6244 (ARRY-142886), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 kinase.

PURPOSE: The mitogen-activated protein (MAP) kinase pathway is important for cell proliferation, survival, and differentiation, and is frequently up-regulated in cancers. The MAP kinase pathway is also activated after exposure to ionizing radiation. We investigated the effects of AZD6244 (ARRY-142886), an inhibitor of MAP kinase/extracellular signal-regulated kinase 1/2, on radiation response. EXPERIMENTAL DESIGN: The effects of AZD6244 on the in vitro radiosensitivity of human cancer cell lines (A549, MiaPaCa2, and DU145) were evaluated using clonogenic assays. DNA damage repair was evaluated using gammaH2AX, and mitotic catastrophe was measured using nuclear fragmentation. Cell cycle effects were measured with flow cytometry. Growth delay was used to evaluate the effects of AZD6244 on in vivo tumor radiosensitivity. RESULTS: Exposure of each cell line to AZD6244 before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1, ranging from 1.16 to 2.0. No effects of AZD6244 on radiation-induced apoptosis or persistence of gammaH2AX foci after irradiation were detected. Cells treated with AZD6244 had an increased mitotic index and decreased Chk1 phosphorylation at 1 and 2 hours after irradiation. Mitotic catastrophe was increased in cells receiving AZD6244 and irradiation compared with the single treatments. In vivo studies revealed that AZD6244 administration to mice bearing A549 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay (dose enhancement factor of 3.38). CONCLUSIONS: These results indicate that AZD6244 can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an increase in mitotic catastrophe.

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 gammaH2AX 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, gammaH2AX 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.

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 gammaH2AX and 53BP1 foci and cell cycle phase distribution was analyzed by flow cytometry. Western blot of acetylated gammaH2AX was done following histone extraction on AUT gels. RESULTS: VA enhanced radiosensitivity when delivered up to 24 h after irradiation. Cells accumulated in G(2)-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 gammaH2AX 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 gammaH2AX. CONCLUSIONS: These results indicate that VA enhances radiosensitivity at times up to 24 h after irradiation, which has direct clinical application.

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 (gammaH2AX), 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 G(2) cell cycle checkpoint. As a measure of DNA double strand breaks, gammaH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of gammaH2AX 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.

Inhibition of hsp90 compromises the DNA damage response to radiation.

Inhibitors of the molecular chaperone Hsp90 have been shown to enhance tumor cell radiosensitivity. To begin to address the mechanism responsible, we have determined the effect of the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) on the DNA damage response to radiation. Exposure of MiaPaCa tumor cells to 17DMAG, which results in radiosensitization, inhibited the repair of DNA double-strand breaks according to gammaH2AX foci dispersal and the neutral comet assay. This repair inhibition was associated with reduced DNA-PK catalytic subunit (DNA-PKcs) phosphorylation after irradiation and a disruption of DNA-PKcs/ErbB1 interaction. These data suggest that the previously established 17DMAG-mediated reduction in ErbB1 activity reduces its interaction with DNA-PKcs and thus accounts for the attenuation of radiation-induced DNA-PK activation. 17DMAG was also found to abrogate the activation of the G(2)- and S-phase cell cycle checkpoints. Associated with these events was a reduction in radiation-induced ataxia-telangiectasia mutated (ATM) activation and foci formation in 17DMAG-treated cells. Although no interaction between ATM and Hsp90 was detected, Hsp90 was found to interact with the MRE11/Rad50/NBS1 (MRN) complex. 17DMAG exposure reduced the ability of the MRN components to form nuclear foci after irradiation. Moreover, 17DMAG exposure reduced the interaction between NBS1 and ATM, although no degradation of the MRN complex was detected. These results suggest that the diminished radiation-induced activation of ATM in 17DMAG-treated cells was the result of a compromise in the function of the MRN complex. These data indicate that Hsp90 can contribute to the DNA damage response to radiation affecting both DNA repair and cell cycle checkpoint activation.

Inhibition of Akt by the alkylphospholipid perifosine does not enhance the radiosensitivity of human glioma cells.

Akt has been implicated as a molecular determinant of cellular radiosensitivity. Because it is often constitutively activated or overexpressed in malignant gliomas, it has been suggested as a target for brain tumor radiosensitization. To evaluate the role of Akt in glioma radioresponse, we have determined the effects of perifosine, a clinically relevant alkylphospholipid that inhibits Akt activation, on the radiosensitivity of three human glioma cell lines (U87, U251, and LN229). Each of the glioma cell lines expressed clearly detectable levels of phosphorylated Akt indicative of constitutive Akt activity. Exposure to a perifosine concentration that reduced survival by approximately 50% significantly reduced the level of phosphorylated Akt as well as Akt activity. Cell survival analysis using a clonogenic assay, however, revealed that this Akt-inhibiting perifosine treatment did not enhance the radiosensitivity of the glioma cell lines. This evaluation was then extended to an in vivo model using U251 xenografts. Perifosine delivered to mice bearing U251 xenografts substantially reduced tumor phosphorylated Akt levels and inhibited tumor growth rate. However, the combination of perifosine and radiation resulted in a less than additive increase in tumor growth delay. Thus, in vitro and in vivo data indicate that the perifosine-mediated decrease in Akt activity does not enhance the radiosensitivity of three genetically disparate glioma cell lines. These results suggest that, although Akt may influence the radiosensitivity of other tumor types, it does not seem to be a target for glioma cell radiosensitization.

In vitro and in vivo radiosensitization induced by the ribonucleotide reductase inhibitor Triapine (3-aminopyridine-2-carboxaldehyde-thiosemicarbazone).

PURPOSE: Because ribonucleotide reductase (RR) plays a role in DNA repair, it may serve as a molecular target for radiosensitization. Unlike previously investigated RR inhibitors, Triapine potently inhibits both RR holoenzymes. Therefore, the effects of Triapine on tumor cell radiosensitivity were investigated. EXPERIMENTAL DESIGN: The effects of Triapine on the in vitro radiosensitivity of three human tumor cell lines and one normal cell line were evaluated using a clonogenic assay. Growth delay was used to evaluate the effects of Triapine on in vivo tumor radiosensitivity. The levels of the RR subunits were determined using immunoblot analysis and DNA damage and repair were evaluated using gammaH2AX foci. RESULTS: Exposure of the tumor cell lines to Triapine before or immediately after irradiation resulted in an increase in radiosensitivity. In contrast, Triapine enhanced the radiosensitivity of the normal fibroblast cell line only when the exposure was before irradiation. There were no consistent differences between cell lines with respect to the expression of the RR subunits. Whereas Triapine had no effect on radiation-induced gammaH2AX foci at 1 hour, the number of gammaH2AX foci per cell was significantly greater in the Triapine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Triapine administration to mice bearing tumor xenografts immediately after irradiation resulted in a greater than additive increase in radiation-induced tumor growth delay. CONCLUSIONS: These results indicate that Triapine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.

ErbB3 expression predicts tumor cell radiosensitization induced by Hsp90 inhibition.

The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient.