Thomas J. Hayman

The mTORC1/mTORC2 inhibitor AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells

BACKGROUND The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs). METHODS mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation. RESULTS Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments. CONCLUSIONS These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.

Translation Initiation Factor eIF4E Is a Target for Tumor Cell Radiosensitization

A core component in the cellular response to radiation occurs at the level of translational control of gene expression. Because a critical element in translation control is the availability of the initiation factor eIF4E, which selectively enhances the cap-dependent translation of mRNAs, we investigated a regulatory role for eIF4E in cellular radiosensitivity. eIF4E silencing enhanced the radiosensitivity of tumor cell lines but not normal cells. Similarly, pharmacologic inhibition of eIF4E with ribavirin also enhanced tumor cell radiosensitivity. eIF4E attenuation did not affect cell-cycle phase distribution or radiation-induced apoptosis, but it delayed the dispersion of radiation-induced γH2AX foci and increased the frequency of radiation-induced mitotic catastrophe. Radiation did not affect 4E-BP1 phosphorylation or cap-complex formation but it increased eIF4E binding to more than 1,000 unique transcripts including many implicated in DNA replication, recombination, and repair. Taken together, our findings suggest that eIF4E represents a logical therapeutic target to increase tumor cell radiosensitivity.

The ATP-Competitive mTOR Inhibitor INK128 Enhances In Vitro and In Vivo Radiosensitivity of Pancreatic Carcinoma Cells

Purpose: Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines. Experimental Design: Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay. Results: INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation. Conclusion: These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair. Clin Cancer Res; 20(1); 110–9. ©2013 AACR.

Body Mass Index and Survival in Esophageal Adenocarcinoma Treated with Chemoradiotherapy Followed by Esophagectomy

BackgroundBody mass index (BMI) has been linked with inferior outcomes in gastrointestinal malignancies. The purpose of this study is to evaluate the effect of BMI on survival in patients with esophageal adenocarcinoma.MethodsMedical records were analyzed for patients who underwent esophagectomy after neoadjuvant chemoradiotherapy (nCRT) for adenocarcinoma from 2000 to the present. Patients were grouped into BMI ≤25, >25–30, >30–35, and BMI >35. Overall survival (OS) and disease-free survival (DFS) were analyzed using the Kaplan–Meier method. Multivariate analysis (MVA) was performed using Cox proportional hazard regression model.ResultsWe identified 303 patients for the analysis. The only difference in patient characteristics between groups was gender. We found no difference in OS and DFS associated with BMI (p = 0.3297 for OS; p = 0.5950 for DFS). There were no differences in postoperative complications or mortality between BMI groups. MVA revealed that higher stage and less than a complete response to nCRT were prognostic for worse OS and DFS, while age, gender, type of surgery, year of diagnosis, and BMI were not prognostic.ConclusionsBMI was neither associated with surgical complications nor survival in patients with esophageal adenocarcinoma treated with nCRT. BMI should not be considered a contraindication to surgical resection after nCRT.

Outcomes of resected pancreatic cancer in patients age ≥70.

OBJECTIVE To determine outcomes of patients ≥70 years with resected pancreatic cancer. METHODS A study was conducted to identify pancreatic cancer patients ≥70 years who underwent surgery for pancreatic carcinoma from 2000 to 2012. Patients were excluded if they had neoadjuvant therapy. The primary endpoint was overall survival (OS). RESULTS We identified 112 patients with a median follow-up of surviving patients of 36 months. The median patient age was 77 years. The median and 5 year OS was 20.5 months and 19%, respectively. Univariate analysis (UVA) showed a significant correlation for increased mortality with N1 (P=0.03) as well as post-op CA19-9 >90 (P<0.001), with a trend towards decreased mortality with adjuvant chemoradiation (P=0.08). Multivariate analysis (MVA) showed a statistically significant increased mortality associated with N1 (P=0.008), post-op CA19-9 >90 (P=0.002), while adjuvant chemoradiation (P=0.04) was associated with decreased mortality. CONCLUSIONS These data show that in patients ≥70, nodal status, post-op CA19-9, and adjuvant chemoradiation, were associated with OS. The data suggests that outcomes of patients ≥70 years who undergo upfront surgical resection are not inferior to younger patients.

Abstract 69: In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128.

Radiation-induced translational control of gene expression has been shown to be a component of the cellular radioresponse. Furthermore, recent data suggest strategies targeting eIF4E may be effective for tumor cell radiosensitization. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of eIF4E and mRNA translation. The newly developed ATP-competitive mTOR inhibitor, INK128, inhibits both mTORC1 and mTORC2 activity, and is in currently undergoing evaluation in Phase 1 clinical trials. In the study described here we evaluated the effects of INK128 treatment on tumor cell radiosensitivity in vitro using a clonogenic survival assay and in vivo according to tumor growth delay. For tumor cell lines initiated from different histologic origins in vitro exposure to INK128 resulted in the complete inhibition of both mTORC1 and 2. Treatment with the same concentration of INK128 immediately after irradiation increased tumor cell radiosensitivity with DEFs greater than 1.3. To determine whether the INK128 induced radiosensitization could be extended to an in vivo model we used subcutaneous xenografts initiated from U251 or PSN1 tumor cells. INK128 treatment (oral gavage) inhibited mTOR activity in tumors established from both cell lines as defined by loss of 4E-BP1 phosphorylation. Furthermore, m 7 -GTP batch chromatography showed that INK128 treatment decreased cap-binding activity in xenografts. To evaluate the effects of INK128 on in vivo tumor radiosensitivity a tumor growth delay assay was performed using mice bearing U251 tumor xenografts. When tumors reached approximately 210mm 3 mice were randomized into 4 groups: vehicle, radiation, INK128, and INK128 plus radiation. INK128 was delivered once a day (3 mg/kg, oral gavage) for four days with the tumor locally irradiated (2 Gy) 2h after each of the four drug treatments. Absolute growth delays were calculated as the time in days for tumors in treated mice to grow from 210 to 1000mm 3 minus the time in days for tumors to reach the same size in vehicle treated mice. The absolute growth delays for INK128 alone and radiation alone groups were 1.7 and 12.7 days respectively. The growth delay in mice treated with the combination of INK128 and radiation was 22.2 days, which is greater than the sum of the growth delays caused by INK128 and radiation alone. The DEF, obtained by dividing the normalized tumor growth delay in mice treated with the radiation/PP242 combination (20.5) by the absolute growth delay in mice treated with radiation only (12.7), was 1.6. These results suggest that mTOR inhibition by the clinically relevant ATP-competitive mTOR inhibitor, INK128, enhances tumor cell radiosensitivity both in vitro and in vivo. Citation Format: Thomas J. Hayman, Tamalee Kramp, Kevin Camphausen, Philip J. Tofilon. In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 69. doi:10.1158/1538-7445.AM2013-69

Abstract 2510: eIF4E is a potential tumor specific target for radiosensitization

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The translational control of gene expression has been implicated as a component of the cellular response to ionizing radiation (IR). However, the mechanisms mediating this process and its specific role in radiosensitivity have not been defined. eIF4E, an mRNA 5’ cap-binding protein, is a critical protein involved in regulating gene translation in response to a variety of environmental signals. In the study described here, we have determined the effects of IR on eIF4E activity. Moreover, using 3 tumor cell lines (MDA-MB-231, DU145 and A549) and 2 normal cell lines (MRC9 and HMEC), the potential role of eIF4E as a molecular determinant of radiosensitivity was investigated. eIF4E activity is regulated by eIF4E binding protein 1 (4E-BP1); 4E-BP1 phosphorylation releases eIF4E to enhance eIF4F complex formation and cap-dependent translation. Exposure of MDA-MB-231 or MRC9 cells to IR (2 Gy, 1h) resulted in an increase in 4E-BP1 phosphorylation. Consistent with this effect, m7-GTP batch chromatography showed that IR induced an increase in cap-bound eIF4G and a decrease in cap-bound 4E-BP1 proteins. Thus, these results indicate that IR, via 4E-BP1 phosphorylation, enhances eIF4E activity and cap-dependent translation. Whereas phosphorylation of eIF4E has also been shown to influence its translational activity, IR had no effect on eIF4E phosphorylation in any of the cell lines evaluated. To determine the significance of eIF4E as a regulator of radiosensitivity, tumor and normal cell lines were treated with siRNA to eIF4E and radiation survival curves were generated based on the colony formation assay. Knockdown of eIF4E in each of the 3 tumor cell lines resulted in an increase in radiosensitivity with DEFs (dose enhancement factors at 0.1 surviving fraction) ranging from 1.24 to 1.44. In contrast, eIF4E knockdown had no effect on the radiosensitivity of normal fibroblasts (MRC9) or normal mammary epithelial cells (HMEC). To investigate the mechanism through which eIF4E influences tumor cell radiosensitivity, we initially focused on MDA-MB-231 cells. Knockdown of eIF4E had no significant effect on MDA-MB-231 cell cycle distribution indicating that a loss of S-phase cells does not account for the observed radiosensitization. In addition, eIF4E knockdown had no effect on apoptotic cell death after IR (6 Gy, 24 and 48h). However, knockdown of eIF4E significantly enhanced the level of mitotic catastrophe induced by IR (2 Gy). These initial results suggest that eIF4E may influence the ability of tumor cells to respond to genotoxic injury. Whereas the specific mechanisms remain to be determined, these data suggest that eIF4E selectively regulates the radiosensitivity of tumor cells over normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2510. doi:10.1158/1538-7445.AM2011-2510