bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4419: Pilot toxicology study of the B-cell lymphoma drug candidate SH7139 demonstrates lack of toxicity in beagles.
Authors: Balhorn Rod , Hok Saphon , Balhorn Monique
Published: August 2014

A group of small molecule drug candidates for non-Hodgkin's lymphoma, called Selective High Affinity Ligands (SHALs), have been developed to target the cell surface antigen HLA-DR10 that is known to be over expressed on many B-cell derived malignancies. These compounds are created by chemically linking together two or three small molecule “recognition elements” that have been identified to bind to neighboring unique sites on the surface of the target protein. One of these compounds, SH7139, has been shown previously to be effective in treating human B cell lymphoma in a mouse xenograft model at extremely low doses (5μg/kg). Earlier experiments conducted in mice have also shown the compound to exhibit no signs of toxicity at 2000 times (10mg/kg) this dose. In an effort to obtain preliminary toxicology and safety data on SH7139 and assess the reversibility or persistence of any potential toxic effects prior to its being evaluated in a pet dog B-cell lymphoma trial for treating canine B-cell lymphomas, a pilot 28-day toxicology study was performed in three naïve Beagles. SH7139 was administered intravenously in three doses injected on days 0, 7 and 14. The dose tested, 50μg/kg, was ten times the dose required to cure 69% of mice carrying Raji xenografts. During the in-life period, animals were observed daily for mortality, moribundity, and food consumption, detailed clinical examinations were conducted on the days of dose, 24 hrs post-dose and weekly thereafter, and individual body weights and temperatures were recorded on the days of dosing and selected days after dosing. Clinical pathology samples (urinalysis, hematology, coagulation, clinical chemistry) were collected prior to dose administration on the days of dose and during the recovery phase (Days 21 and 28). On Day 29, all animals were euthanized and a complete necropsy was conducted with tissue collection and preservation. The key tissues were embedded in paraffin, H&E stained and examined microscopically by a board-certified pathologist. The results of this study show the treatment with SH7139 is well tolerated by healthy, naïve dogs. No treatment-related effects on body weight, body temperature, food consumption or clinical pathology parameters were observed. In addition, the lack of significant changes in hematology, clinical chemistry, urinalysis or coagulation parameters also indicate SH7139 exhibited no apparent systemic toxicity or side effects.

Citation Format: Rod Balhorn, Saphon Hok, Monique Balhorn. Pilot toxicology study of the B-cell lymphoma drug candidate SH7139 demonstrates lack of toxicity in beagles. [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 4419. doi:10.1158/1538-7445.AM2013-4419
Abstract 443: PKC epsilon is activated by ionizing radiation in a reactive nitrogen species dependent manner in tumor endothelial cells.
Authors: Alam Asim , Rabender Chris , Mikkelsen Ross
Published: August 2014

PKCε is activated by ionizing radiation in a reactive nitrogen species dependent manner in tumor endothelial cells.

Ionizing radiation (IR) has been shown to increase angiogenic markers and migration in endothelial cells involving reactive oxygen/nitrogen species and eNOS, but the exact mechanism is poorly understood. This activation of endothelial cells can increase angiogenesis post radiation and confer cytoprotection to tumor cells. Thus targeting this IR induced activation of endothelial cells should increase therapeutic efficacy of radiation therapy. Protein Kinase C Epsilon is a molecule that has been shown to be important in cancer cell motility, proliferation, hormone sensitivity and angiogenesis. PKCε activates downstream pathways involved in tumorigenicity such as Akt and Stat3. PKCε has also been shown to be nitrated in response to NO donors and inflammatory conditions in cardiac myocytes. During inflammation, hypoxia and irradiation, levels of reactive nitrogen species are elevated due to uncoupling of Nitric Oxide Synthase (NOS). NOS can be recoupled with tetrahydrobiopterin precursor (sepiapterin) which decreases RNS/ROS in cells. We have shown in 2H11 cells (mouse tumor endothelial cells) that PKCε levels are increased due to exposure to clinically relevant doses of ionizing radiation or levels of NO found in chronic inflammatory conditions. Also in these cells PKCε is nitrated in response to reactive nitrogen species which leads to its activation and activation of Stat3 and Akt. Using sepiapterin, we have shown decreased levels of PKCε, Stat3 and Akt. We have also blocked ionizing radiation induced migration in vitro in 2H11 cells using sepiapterin and a PKCε inhibitor. We hope to show that PKCε is nitrated at specific residue(s) after ionizing radiation and activates tumor endothelial cells to promote the survival of the tumor. We believe sepiapterin with ionizing radiation therapy will result in increase cytotoxicity in a spontaneous mouse model of lung cancer, by decreasing endothelial activation, migration, proliferation and angiogenesis.

Citation Format: Asim Alam, Chris Rabender, Ross Mikkelsen. PKC epsilon is activated by ionizing radiation in a reactive nitrogen species dependent manner in tumor endothelial cells. [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 443. doi:10.1158/1538-7445.AM2013-443

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 4430: Protective effects of rebamipide liquid on radiation-induced glositis in rats.
Authors: Nakashima Takako , Uematsu Naoya , Shibamori Masafumi , Sakurai Kazushi , Sato Masayuki , Matsuda Takakuni , Sako Nobutomo
Published: August 2014

[Background & Aims] Rebamipide is widely used for mucosal protection, healing of gastric ulcers and treatment of gastritis. Recently, it was reported that Rebamipide gargle inhibited oral mucositis induced by chemoradiotherapy in head and neck cancer patients. In this study, we examined protective effects of Rebamipide Liquid on radiation-induced glossitis rat model and analyzed the expression level of pro-inflammatory cytokines and chemokines in the injury area of tongue.

[Materials & Methods] Rebamipide Liquid, comprising with Rebamipide crystals less than 500 nm particles, was prepared by a neutralizing crystallization technique. This formulation is a stable homogenous aqueous suspension provided with appropriate viscosity by adding viscosity enhanced agents to improve the retention in the oral cavity.

The glossitis was induced by X-ray with 15Gy irradiation only around the snout (Day 0) in rats. Rebamipide Liquid was administered intraorally at doses of 5, 10 or 20 mg/kg (1, 2 or 4%, respectively), 6 times a day for 14 days from Day -7 to Day 6. These tongue tissue specimens were obtained at Day 7 and their images were recorded by a digital camera for efficacy evaluations. Gene expression analysis was performed with quantitative real-time PCR (ABI) and protein level was evaluated with Bio-Plex system(BioRad) or ELISA in a different study which was conducted at the two doses of 0 and 20 mg/kg (0 and 4%).

[Result & Conclusions] The ulcer-like areas (10.8 ± 1.2, 7.9 ± 1.1 and 7.0 ± 0.7%) at doses of 5 - 20 mg/kg (1 - 4%) of Rebamipide Liquid administration, were statistically smaller than the vehicle control at 14.7±1.6%. Another study revealed that the expression of pro-inflammatory cytokines (TNF-α, IL-6 and Il-1β) and chemokines (MCP-1 and Gro-α) were dramatically elevated in the irradiated tongue at Day7, as compared to normal. But these elevated expressions were significantly suppressed by the 4% Rebamipide treatment. These immuoassays elucidated that the production of these cytokines and chemokines were significantly suppressed by the Rebamipide administration. These results demonstrated that the Rebamipide Liquid has a potent pharmacological action for the radiation-induced oral mucositis mediated by the suppression of pro-inflammatory cytokines (TNF-α, IL-6 and Il-1β) and chemokines (MCP-1 and Gro-α).

Citation Format: Takako Nakashima, Naoya Uematsu, Masafumi Shibamori, Kazushi Sakurai, Masayuki Sato, Takakuni Matsuda, Nobutomo Sako. Protective effects of rebamipide liquid on radiation-induced glositis in rats. [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 4430. doi:10.1158/1538-7445.AM2013-4430
Abstract 4432: MMP9 reinforces radiation-induced delayed invasion and metastasis of neuroblastoma through second-signaling positive feedback with NFκB via both ERK and IKK activation.
Authors: Major Ryan , Aravindan Sheeja , Natarajan Mohan , Herman Terence S. , Aravindan Natarajan
Published: August 2014

Tumor recurrences continue to remain high in neuroblastoma (NB), and a substantial fraction of those develop metastatic disease with only 10% survival in systemic recurrences. Efficient and improved therapeutic strategy can be developed through targeted drug delivery approach and by intervening molecular pathways operating in response to tumor recurrence. Herein, we investigated the onset and maintenance of metastatic signal transduction in neuroblastoma (SH-SY-5Y, MC-IXC, SK-PN-DW and IMR-32) cells that survive after radiotherapy. Gelatin zymography revealed that exposure to radiation induces significant and consistent increase in MMP2 and MMP9 activity. Further, muting radiation induced NFκB with dominant negative mutant IkB and/or forced expression of NFκB (p50/p65 sub-units) in these cells affirmed the influence of radiation-induced NFκB in MMP9 transactivation (QPCR), translation (immunoblotting) and activity (sequence specific substrate based high sensitive fluorogenic assay). Interestingly, blocking radiation-induced MMP9 with GM-6001 profoundly inhibited p65 transactivation and nuclear localization. More importantly, blocking radiation-induced MMP9 greatly introverted radiation-induced phosphorylation of IKK in these cells. Furthermore, radiation increased both transactivation and activity of ERK-1 (active ERK/phosphor-transferase immunoprecipitation) in neuroblastoma cells. This pathway was further defined (MMP9 blocking and activation, PMA studies) to show that radiation-induced functional MMP9 is necessary for activation of ERK- and NFκB-dependent onset of metastasis (tumor invasion and metastasis transcriptome profiling) in surviving NB cells. Selective MMP9-dependent ERK regulation was confirmed with futile inhibition of ERK activity in aprotinin treated cells. Together, these data strongly suggest that radiation induced NFκB-dependent MMP9 reinforces radiation-induced delayed invasion and metastasis. Further, these results imply that the MMP9 dependent reinforcement may involve dual signaling feedback activation of NFκB though ERK and IKK phosphorylation.

Citation Format: Ryan Major, Sheeja Aravindan, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. MMP9 reinforces radiation-induced delayed invasion and metastasis of neuroblastoma through second-signaling positive feedback with NFκB via both ERK and IKK activation. [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 4432. doi:10.1158/1538-7445.AM2013-4432
Abstract 4436: The down-regulation of ferritin heavy chain as an adjuvant therapy in human glioma.
Authors: Pang Min , Madhankumar A.B. , Webb Becky , Liu Xiaoli , Connor James R.
Published: August 2014

Increased iron requirements and heightened iron metabolism is among the key characteristics of highly proliferative cancer cells. As the major iron storage protein, ferritin expression is elevated in cancer cells and we have previously shown that ferritin down-regulation in human glioblastoma cells increases their sensitivity to chemotherapeutic agent both in vitro and in vivo. In this study, we report that the sensitization of H-ferritin down-regulation in glioma cells against radiation and suggest the potential of H-ferritin down-regulation as an adjuvant therapy in human glioma.

Down-regulation of H-ferritin was performed in human glioma cell line U251 through a nanotechnology-based transfection platform and effective knockdown efficiency was achieved. A transient decrease in transferrin receptor level was also observed, suggesting a release of intracellular iron with loss of ferritin. This iron release in turn produced intracellular oxidative stress, demonstrated by protein oxidation damage, as well as a decrease in the stability of hypoxia-inducible factors (HIFs), which is an indicator of radioresistance. In U251 cells, exposure to radiation resulted in protein oxidation which could be exacerbated by H-ferritin down-regulation.

Previously we have demonstrated a DNA protection role for H-ferritin. Here we report that the down-regulation of H-ferritin is associated with increased DNA vulnerability when cells were exposed to ionizing radiation. Moreover, the activation of DNA repair mechanisms was impaired when H-ferritin was absent. The in vitro data suggest a synergistic effect of radiation and H-ferritin down-regulation.

Additionally, we expand our research into CD133-positive glioma stem cells (GSCs), which are notoriously resistant to anti-cancer treatment. Down-regulation of H-ferritin in these cells inhibited cell proliferation in vitro. With an intracranial glioma model established by GSC implantation, we demonstrated that the survival was significantly prolonged by H-ferritin siRNA transfection through intravenous injection, in tumor bearing mice treated with first-line drug Temodar. This study supports the potential of H-ferritin siRNA as an adjuvant therapy in glioma treatment.

Citation Format: Min Pang, A.B. Madhankumar, Becky Webb, Xiaoli Liu, James R. Connor. The down-regulation of ferritin heavy chain as an adjuvant therapy in human glioma. [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 4436. doi:10.1158/1538-7445.AM2013-4436
Abstract 444: Mitochondrial metabolism and phosphorylation of Stat3 in radioresistant cancer stem cells isolated from glioma U87 cells.
Authors: Fan Ming , Shi Yan , Li Jian Jian
Published: August 2014

Abstract

Radiation therapy continues to play a central role in the management of brain tumors. The radioresistance of high-grade gliomas remains one of the reasons for the failure of treatment, which would otherwise require tumoricidal doses beyond the normal tissue tolerance and beyond safe clinical practice. Thus, determining the cellular, molecular and biochemical mechanisms underlying radiation resistance becomes critical for successful treatment of these tumors. For this purpose, glioma cell line U87 were treated with fractionated doses of radiation and the resistant clones isolated from the surviving tumor cells were characterized. The radioresistant U87 cells showed enhanced aggressiveness and clonogenic survival with an increased expression of HER2 and CD133, glioma cancer stem cell surface markers, as well as elevated mitochondrial membrane potential but lower oxygen consumption and ATP generation. In addition, compared with the wild type U87 cells, the phosphorylation of mitochondrial Stat3 at Serine 727 was decreased in the radioresistant U87 cells, indicating a role for mitochondrial Stat3 in the radioresistance mechanisms. Additionally, mutation of Serine 727 of Stat3 into Alanine resulted in increased radiosensitivity. These results demonstrate that the radioresistant glioma cells surviving routine radiotherapy are enriched with cancer stem cells expressing CD133 and HER2. The radioresistant phenotype is linked with a lowered mitochondrial metabolism and increased anti-apoptotic function due to inhibition of mitochondrial Stat3 phosphorylation. Thus, improving the mitochondrial metabolism by enhancing mitochondrial Stat3 phosphorylation may serve as an effective approach to re-sensitize the radioresistant glioma tumors.

Citation Format: Ming Fan, Yan Shi, Jian Jian Li. Mitochondrial metabolism and phosphorylation of Stat3 in radioresistant cancer stem cells isolated from glioma U87 cells. [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 444. doi:10.1158/1538-7445.AM2013-444
Abstract 4443: Small cell transformation of lung adenocarcinoma in acquired resistance to EGFR-TKI.
Authors: So Kwang Sup , Choi Yun Jung , Kim Sun Ye , Choi Chang-Min , Lee Jae Cheol , Kim Woo Sung , Rho Jin Kyung
Published: August 2014

Small cell transformation during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer has been suggested as one of possible resistant mechanisms. We evaluated whether such phenomenon can be found in the cell line model. In addition, its effect on response to conventional chemotherapeutic drugs for small cell lung cancer treatment was investigated. We established resistant cells to various kinds of EGFR-TKIs such as gefitinib, erlotinib, CL387,785 and ZD6474 with A549, PC-9 and HCC827 lung adenocarcinoma cell lines. Among them, two resistant cells, A549/GR (resistant to gefitinib) and PC-9/ZDR (resistant to ZD6474) showed increased expression of CD56 while increased synaptophysin was found in A549/GR in Western blotting, suggesting that small cell transformation occurred in A549/GR. A549/GR cells were more sensitive to etoposide and cisplatin, most commonly using drugs for treatment of small cell lung cancer, compared to parental cells. Treatment with cAMP and IBMX induced synaptophysin and chromogranin A expression in A459 cells, which also made them more sensitive to etoposide and cisplatin. Further, we found a patient showing increased expression of CD56 and synaptophysin after development of resistance to erlotinib. In conclusion, small cell transformation of lung adenocarcinoma can occur during EGFR-TKI treatment leading to resistance.

Citation Format: Kwang Sup So, Yun Jung Choi, Sun Ye Kim, Chang-Min Choi, Jae Cheol Lee, Woo Sung Kim, Jin Kyung Rho. Small cell transformation of lung adenocarcinoma in acquired resistance to EGFR-TKI. [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 4443. doi:10.1158/1538-7445.AM2013-4443
Abstract 4446: Activation of HER Family signaling as a mechanism of acquired resistance to ALK Inhibitors in EML4-ALK-positive Non-Small Cell Lung Cancer.
Authors: Tanizaki Junko , Okamoto Isamu , Okabe Takafumi , Sakai Kazuko , Tanaka Kaoru , Hayashi Hidetoshi , Kaneda Hiroyasu , Takezawa Ken , Nishio Kazuto , Nakagawa Kazuhiko
Published: August 2014

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown.

Experimental Design: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristic.

Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of the epidermal growth factor receptor (EGFR), HER2, and HER3 was up-regulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion.

Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK.

Citation Format: Junko Tanizaki, Isamu Okamoto, Takafumi Okabe, Kazuko Sakai, Kaoru Tanaka, Hidetoshi Hayashi, Hiroyasu Kaneda, Ken Takezawa, Kazuto Nishio, Kazuhiko Nakagawa. Activation of HER Family signaling as a mechanism of acquired resistance to ALK Inhibitors in EML4-ALK-positive Non-Small Cell Lung Cancer. [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 4446. doi:10.1158/1538-7445.AM2013-4446
Abstract 4449: Resistance to insulin-like growth factor 1 receptor antibody associated with downregulated IGFBP2.
Authors: Yu Yunkai , Kang Zhigang , Helman Lee , Meltzer Paul , Cao Liang
Published: August 2014

Agents targeting the insulin-like growth factor 1 receptor are in various stages of clinical development. Despite some initial response with single agent trials, the responses are limited and durations are short. It is important to understand the mechanics of innate and adaptive responses of the tumors that lead to resistance. An in vivo mouse model was used for the development of IGF1R antibody-resistant rhabdomyosarcoma tumor. All tumors, despite remarkable initial response, all eventually grow in the presence of the antibody. The isolated cell lines show cross-resistance to IGF1R antibodies, and are presented with increased AKT activity in the presence of an IGF1R antibody. Expression analysis indicates that IGFBP2 is the most affected gene in the IGF signaling process that is significantly down-regulated in two independently generated resistant lines. In addition, the recombinant IGFBP2 is capable of inhibiting of IGF1R phosphorylation and AKT signaling. Further, the inhibitory activity is amplified in the presence of an IGF1R antibody, against both sensitive and resistant RMS cells. Finally, the addition of antibodies against IGF1/2 or IGF2 enhances the activity of an IGF1R antibody against RMS cells. The findings suggest that IGFBP2 down-regulation may represent a novel mechanism to escape the blockage of IGF1R signaling by a therapeutic antibody.

Citation Format: Yunkai Yu, Zhigang Kang, Lee Helman, Paul Meltzer, Liang Cao. Resistance to insulin-like growth factor 1 receptor antibody associated with downregulated IGFBP2. [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 4449. doi:10.1158/1538-7445.AM2013-4449
Abstract 4450: Identification of IGF1R as a responsible factor and a therapeutic target for overcoming acquired and intrinsic resistance to PI3K inhibitors.
Authors: Dan Shingo , Isoyama Sho , Nishimura Yumiko , Nakamura Naoki , Kajiwara Gensei , Tamaki Naomi , Okamura Mutsumi , Yamori Takao
Published: August 2014

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers. Therefore PI3K is thought to be a promising target for cancer therapy, and many compounds targeting PI3K have been developed and tested in clinical trials. Acquired resistance is a major obstacle for conventional cancer chemotherapy, and also for some of the targeted therapies approved to date. Long-term administration of protein tyrosine kinase inhibitors (TKIs), such as gefitinib and imatinib, gives rise to resistant cancer cells carrying a drug-resistant gatekeeper mutation in the kinase domain of the respective target genes, EGFR and BCR-ABL. As for the PI3K inhibitors (PI3Kis), little is known about their acquired resistance. To address this issue, we established four human cancer cell lines that exhibited resistance to ZSTK474, a PI3Ki we developed previously, after drug exposure for a period of more than 1-year in vitro. None of the PI3Ki-reistant cells, however, contained any mutation in the kinase domain of the PIK3CA gene. Instead, we found that insulin-like growth factor 1 receptor (IGF1R) was overexpressed in all four resistant cells. Furthermore, PI3Ki-naïve cancer cells expressing high levels of IGF1R also exhibited resistance to PI3Kis. Interestingly, IGF1R-TKIs strongly enhanced the efficacy of PI3Ki in these cells. Thus, we propose that IGF1R could serve as a biomarker to predict the inefficacy of PI3K inhibitors, and also could be a target for overcoming acquired and intrinsic resistance in IGF1R-overexpressing cancers.

Citation Format: Shingo Dan, Sho Isoyama, Yumiko Nishimura, Naoki Nakamura, Gensei Kajiwara, Naomi Tamaki, Mutsumi Okamura, Takao Yamori. Identification of IGF1R as a responsible factor and a therapeutic target for overcoming acquired and intrinsic resistance to PI3K inhibitors. [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 4450. doi:10.1158/1538-7445.AM2013-4450
Abstract 4451: Long-term resistance to PI3K/mTOR inhibition requires increased expression of c-myc and YAP.
Authors: Muranen Taru E. , Coloff Jonathan , Selfors Laura , Bui Amy , Mills Gordon B. , Brugge Joan S.
Published: August 2014

We have shown that extracellular matrix (ECM) contact contributes to PI3K/mTOR inhibitor resistance, where drug treatment leads to cytostasis but not cell death in ECM-attached cells. These cells do undergo proliferation arrest and this may reflect the situation in vivo, where PI3K/mTOR inhibitor treatment initially leads to growth arrest of the tumor cells. However this is often followed by re-initiation of proliferation, leading to relapse. In this study we have investigated the underlying molecular mechanisms that mediate long-term resistance to PI3K/mTOR inhibitors by selecting for cancer cells that proliferate after chronic inhibitor exposure in 3D cell cultures. To assess the mechanism of resistance, we performed reverse phase protein and RNA-seq arrays to identify signaling pathways that were altered in proliferating drug resistant cells. The protein arrays revealed up-regulated levels of c-myc and the Hippo pathway effector, YAP, in the resistant cells, and introduction of exogenous c-myc and YAP into drug sensitive cells rendered them resistant. While expression of c-myc alone induced partial resistance, YAP was also required, as knock-down of endogenous YAP resulted in loss of resistance. These data suggest that c-myc alone is not sufficient for generating resistance to PI3K/mTOR inhibitors, but an additional driver is required. Since the Hippo pathway is another major growth-promoting pathway (in addition to mTOR), it is conceivable that YAP compensates for the loss of mTOR signaling, contributing to resistance. Our current efforts are aimed at elucidating the mechanism by which YAP and c-myc mediate drug resistance to PI3K/mTOR inhibitors. Preliminary data suggests that c-myc-dependent glutamine metabolism contributes to drug resistance, as inhibition of glutaminase prevents resistance.

Citation Format: Taru E. Muranen, Jonathan Coloff, Laura Selfors, Amy Bui, Gordon B. Mills, Joan S. Brugge. Long-term resistance to PI3K/mTOR inhibition requires increased expression of c-myc and YAP. [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 4451. doi:10.1158/1538-7445.AM2013-4451
Abstract 4452: p90RSK mediates resistance to PI3K-pathway inhibitors in breast cancer.
Authors: Serra Violeta , Eichhorn Pieter JA , García-García Celina , Ibrahim Yasir , Prudkin Ludmila , Rodríguez Olga , Marlow Sara , Prat Aleix , Hahn William C. , Baselga José
Published: August 2014

The PI3K signaling pathway regulates diverse cellular processes including proliferation, survival and metabolism and is aberrantly activated in human cancer. As such, numerous compounds targeting the PI3K pathway are currently being clinically evaluated for the treatment of cancer and they have shown some early indications of efficacy in breast cancer. However, resistance against these agents, both de novo and acquired, may ultimately limit the efficacy of these compounds. Here, we have taken a systematic functional approach to uncover potential mechanisms of resistance to PI3K inhibitors and have identified several genes whose expression promotes survival under conditions of PI3K/mTOR blockade, including the ribosomal S6 kinases RPS6KA2 (RSK3) and RPS6KA6 (RSK4). We demonstrate that overexpression of RSK3 or RSK4 supports proliferation upon PI3K inhibition both in vitro and in vivo, in part through the attenuation of an apoptotic response and upregulation of protein translation. The addition of MEK- or RSK-specific inhibitors can extinguish these resistance phenotypes, both in cells engineered to overexpress RSK and in breast cancer cells that have elevated levels of RSK activity. These observations provide a strong rationale for the combined use of RSK and PI3K pathway inhibitors to elicit favorable responses in breast cancer patients with activated RSK.

Citation Format: Violeta Serra, Pieter JA Eichhorn, Celina García-García, Yasir Ibrahim, Ludmila Prudkin, Olga Rodríguez, Sara Marlow, Aleix Prat, William C. Hahn, José Baselga. p90RSK mediates resistance to PI3K-pathway inhibitors in breast cancer. [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 4452. doi:10.1158/1538-7445.AM2013-4452
Abstract 4463: Activation of FGFR signaling as a mechanism of acquired resistance to erlotinib in EGFR-mutant lung cancer associated with an EMT.
Authors: Ye Xiaofen , Yue Peng , Patel Rajesh , Modrusan Zora , Settleman Jeff , Ashkenazi Avi , Yauch Robert L.
Published: August 2014

Treatment of NSCLC patients harboring activating EGFR mutations with EGFR TKIs results in anti-tumor responses to therapy, however patients ultimately progress on therapy due to the acquisition of drug resistance. Known mechanisms of resistance include secondary mutations in the EGFR gene (EGFRT790M) or MET gene amplification, however the underlying mechanism of resistance remains to be elucidated in ∼40-45% of cases. Previous studies provide evidence for an epithelial-to-mesenchymal transition (EMT) in a subset of cases that is not associated with the known mechanisms of resistance. In this study, we identify 2 models of acquired resistance to erlotinib that are associated with an EMT phenotype and a dependence on autocrine FGF-FGFR signaling for resistance. Evidence for FGFR pathway activation in the erlotinib-resistant cell population included an increase in FGF2, FGFR1 and phospho-FRS2. Inhibition of FGFR signaling using a small molecule kinase inhibitor or FGFR1-Fc neutralization of FGFR1 ligands re-sensitized resistant cells to erlotinib. Re-sensitization was accompanied by an inhibition of downstream signaling. In contrast, inhibition of AXL, a kinase that has recently been identified as an EMT-associated driver of erlotinib resistance in some pre-clinical models, failed to re-sensitize erlotinib-resistant cells. Our findings are consistent with autocrine FGFR pathway activation, as FGF ligand neutralization in the absence of an exogenous source of ligand can re-sensitize cells to erlotinib. Finally, FGFR pathway inhibition suppressed the development of resistance to erlotinib in sensitive parental cells. These data indicate that FGFR activation could potentially serve as a mechanism of acquired resistance to erlotinib in cases associated with an EMT and suggest that combined inhibition of EGFR and FGFR signaling could be beneficial in treating such cases.

Citation Format: Xiaofen Ye, Peng Yue, Rajesh Patel, Zora Modrusan, Jeff Settleman, Avi Ashkenazi, Robert L. Yauch. Activation of FGFR signaling as a mechanism of acquired resistance to erlotinib in EGFR-mutant lung cancer associated with an EMT. [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 4463. doi:10.1158/1538-7445.AM2013-4463
Abstract 4464: Elucidation of crizotinib resistance in NCI-H3122 and strategies to circumvent bypass resistance.
Authors: Lee Nathan V. , Cao Joan , Scheffelin Timothy , Engstrom Lars , Huang Stephen , Lira Maruja , Garza Scott , Yuan Jing , Enyart Blake , Christensen James , Kan Julie
Published: August 2014

The Anaplastic Lymphoma Kinase (ALK) fusion proteins such as EML4-ALK act as oncogenic drivers in approximately 5-7% of non-small cell lung carcinomas (NSCLC). The rationale for targeting this kinase as a means of treatment has been validated by the clinical efficacy and subsequent approval of the ALK inhibitor, crizotinib (Xalkori ®). The overall response rate of 61% suggests potential intrinsic resistance to crizotinib. In addition, like many tyrosine kinase inhibitors, acquired resistance inevitably leads to treatment refractory cancers. To better understand resistance mechanisms (both intrinsic and acquired), and circumvent resistance to crizotinib, we developed in vitro resistance models through prolonged crizotinib exposure. We isolated resistance clones and characterization identified different resistance mechanisms, including pathway bypass as well as an ALK kinase mutation (G1269A), similar to those that have been reported from refractory patient samples. The majority of the resistance clones escaped crizotinib suppression via pathway bypass. In several resistance clones, simultaneous activation of EGFR and IGF1R receptors limited the efficacy of double combination strategies and required triple combinations. In addition, phospho-proteomic analysis revealed simultaneous activation of different receptor tyrosine kinases, suggesting that combination treatment with inhibitors of downstream mediators such as PI3K and MEK may be effective strategies.

Our resistance models demonstrate the heterogeneity of cancers and reveal the multiplicity of escape mechanisms. Our results provide support for the evaluation of combination therapy with downstream mediator inhibitors as a way to restore as well as enhance sensitivity to crizotinib.

Citation Format: Nathan V. Lee, Joan Cao, Timothy Scheffelin, Lars Engstrom, Stephen Huang, Maruja Lira, Scott Garza, Jing Yuan, Blake Enyart, James Christensen, Julie Kan. Elucidation of crizotinib resistance in NCI-H3122 and strategies to circumvent bypass resistance. [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 4464. doi:10.1158/1538-7445.AM2013-4464
Abstract 4472: PI3K pathway dependencies in endometrioid endometrial cancer.
Authors: Weigelt Britta , Warne Patricia H. , Lambros Maryou B. , Reis-Filho Jorge S. , Downward Julian
Published: August 2014

Background: Endometrioid endometrial cancers (EECs) frequently harbor coexisting mutations in PI3K pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to determine the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cells rely on p110β signaling for survival. Methods: Twenty-four human EEC cell lines were characterized for their mutation profile using the Sequenom OncoCarta Panel v1.0 and Sanger sequencing for PTEN, PIK3CA, and PIK3R1 transcripts, and their protein and phospho-protein expression levels of PI3K and MAPK signaling pathway components. The cell line panel was treated with pan-class I PI3K, p110α and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, MEK and RAF inhibitors, and the correlations between protein expression, gene mutations, and sensitivity to targeted therapeutics determined. RNA interference was employed to assess effects of KRAS silencing on EEC cells. Results: The prevalence of PI3K pathway mutations in the EEC cell line panel studied mirrored that reported for primary EECs. PI3K pathway activation as determined by levels of AKTSer473 phosphorylation was significantly associated with PTEN but not PIK3CA or KRAS mutation status. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor Temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition. Only 2/6 KRAS-mutant EEC cell lines showed response to MEK inhibition, and KRAS siRNA silencing did not induce apoptosis in KRAS-mutant EEC cells. EEC cell lines harboring PTEN mutations were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only combination with the p110α selective inhibitor A66 resulted in a decrease in cell viability. Conclusions: Our findings provide evidence to suggest that selective pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant cancers, respectively, and that these PI3K inhibitors may even be effective in a subset of EECs concurrently harboring KRAS mutations. Our results further demonstrate that p110β inhibition alone is not sufficient to sensitize PTEN-mutant EEC cells, suggesting that combination with other targeted agents may be required to increase efficacy.

Citation Format: Britta Weigelt, Patricia H. Warne, Maryou B. Lambros, Jorge S. Reis-Filho, Julian Downward. PI3K pathway dependencies in endometrioid endometrial cancer. [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 4472. doi:10.1158/1538-7445.AM2013-4472
Abstract 4474: Suppression of gefitinib-resistant human lung cancer cells by indolizino[6,7-b]indole derivatives.
Authors: Chen Chi-Wei , Tala Satishkumar , Su Tsann-Long , Lee Te-Chang
Published: August 2014

According to the most prevalent, poor prognosis, and lowest 5 year survival rate, patients with non-small cell lung cancer (NSCLC) are one of the most common issues in global health problem. Although tyrosine receptor inhibitors (TKI), cetuximab, erlotinib, and gefitinib, which target epidermal growth factor receptor (EGFR), are only effective in certain subpopulations, TKI resistance is developed after a median of 10 to 14 months via acquisition of second mutation as well as several other mechanisms. Developing therapeutic agent against TKI resistance is of critical and essential. In this study, we demonstrated that the derivatives of 1,2-bis-hydroxy- indolizino[6,7-b]indiole diols, a novel class of DNA crosslinking agent, effectively kill NSCLC cell lines including PC9 (exon 19 deletion), PC9/gefb4 (exon 19 deletion and resistant to gefitinib), CL100 (exon 19 deletion), and CL97 (exon 19 deletion and T790M). Our results also showed that BO-1922, one of derivatives, was almost equally effective in suppression the growth of various H1299 cell lines which ectopically expressed wild type or a variety of mutant EGFR (such as including L858R, exon 19 deletion, L858R/T790M, and exon 19 deletion/T790M, respectively). The selected derivatives, BO-1922 and BO-1978, were shown to induce DNA double-strand crosslink by modified comet assay and modulate the expression of DNA repair proteins. By aid of flow cytometric assay, we found that exposure of PC9 and PC9/gefb4 cells to BO-1922 resulted in accumulation of cells at the G2/M phase, while BO-1978 causes cell cycle delay at the G1 and S phase. Furthermore, BO-1922 and BO-1978 given at 25 mg/kg significantly suppressed the growth of PC9/gefb4 xenograft tumors. Our present results revealed that BO-1922 and BO-1978 may have potential against NSCLC with EGFR mutations and gefitinib resistance.

Citation Format: Chi-Wei Chen, Satishkumar Tala, Tsann-Long Su, Te-Chang Lee. Suppression of gefitinib-resistant human lung cancer cells by indolizino[6,7-b]indole derivatives. [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 4474. doi:10.1158/1538-7445.AM2013-4474
Abstract 4480: New approaches to changing an enzyme into a cytotoxic agent: A novel way to stimulate DNA cleavage by topoisomerase II.
Authors: Rogojina Anna , Nitiss Karin C. , Nitiss John L.
Published: August 2014

Topoisomerase poisons are important anti-cancer drugs, and include agents such as etoposide and doxorubicin that target eukaryotic topoisomerase II. Most topoisomerase II poisons act as interstitial inhibitors. This mode of inhibition depends on the coordination of the enzyme and DNA substrate to form a drug binding site. While recent structural studies have localized topoisomerase II poisons to the drug/DNA interface, our understanding of the mechanism of enzyme inhibition remains incomplete. We have combined genetic, biochemical, and structural approaches to identify regions of topoisomerase II that are important for drug action and identified sites near the active site tyrosine that contribute to drug sensitivity. Interestingly, we also identified amino acids near the C-terminal dimerization domain that also control DNA cleavage. Mutations in the C-terminal dimerization domain exhibit several unique characteristics. Importantly, we identified two mutants in the dimerization domain of yeast Top2 that could not be viably expressed in yeast mutants defective in double strand break repair. Expression of these mutants in repair proficient strains led to elevated rates of homologous recombination, suggesting that these mutant proteins are able to generate DNA damage even in the absence of topoisomerase II targeting agents. We purified the mutant proteins and demonstrated that they possess elevated levels of drug independent DNA cleavage, compared to wild type proteins. Elevated cleavage was only seen in the presence of ATP, indicating that the mutant proteins are likely defective in DNA religation at a specific step in the catalytic cycle, and that the C-terminal dimerization and DNA religation occurs by a coordinated process. Finally, we have also shown that homologous mutations in human topoisomerase II alpha generate effects similar to those described for the yeast protein. Taken together, these results suggest that it may be possible to identify potent allosteric inhibitors of topoisomerase II directed against the C-terminal catalytic domain of the protein. These hypothetical inhibitors may have unique therapeutic potential, especially because they represent an approach for isoform specific topoisomerase poisons.

Citation Format: Anna Rogojina, Karin C. Nitiss, John L. Nitiss. New approaches to changing an enzyme into a cytotoxic agent: A novel way to stimulate DNA cleavage by topoisomerase II. [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 4480. doi:10.1158/1538-7445.AM2013-4480
Abstract 4490: Triapine sensitizes chemoresistant ovarian cancer cells to platinum therapy.
Authors: Ratner Elena S. , Whicker Margaret , Lin Z. Ping , Sartorelli Alan C.
Published: August 2014

Objectives:

Epithelial ovarian cancer (EOC) is the second most common female pelvic reproductive organ cancer in the United States, and carries the highest mortality in this category in the Western world. The progression free survival and overall survival depend greatly on tumor sensitivity to a platinum chemotherapy. Once platinum resistance is encountered, response rates of only 6-30% are achieved. Triapine, a novel small-molecule drug developed in our laboratory, potently inhibits the activity of ribonucleotide reductase involved in the key step of DNA synthesis and replication. Given that triapine caused profound disruption of dNTP levels and S phase progression, we examined the effects of triapine on the repair of replication-associated DSBs and on the sensitivity of platinum resistant EOC to DNA damaging and platinum therapy.

Methods:

Cell sensitivity to varying ratios of treating drug combinations (triapine/6-thio-guanine alone, triapine/6-thioguanine with cisplatin, carboplatin, doxorubicin, or paclitaxel) was carried out by clonogenic survival assays using multiple EOC cells lines (A2780, CP70, CAOV-3, IGROV-1, BG-1, PEO1, PEO4, SKOV-3). Colonies were stained and counted to determine % survival. DNA replication was determined by EdU incorporation into DNA using flow cytometry and confocal microscopy.

Results:

Treatment with triapine leads to synergistic sensitization of BRCA wild-type EOC cells to platinum drugs. The degree of sensitization by triapine is more pronounced toward the lower concentrations of platinum drugs, at which DNA replication is still ongoing. Platinum drugs induce DNA double strand breaks (DSBs) and Rad51 foci in BRCA wild type EOC cells. Triapine attenuates formation of Rad51 foci, a marker of DSB repair, in these cells. For EOC cells that are highly platinum resistant, combination of triapine and 6-thioguanine is required to achieve significant sensitization to platinum drugs.

Conclusions:

Platinum drugs induce replication-associated DSBs and Rad51-dependent repair in BRCA wild-type EOC cells. Triapine inhibits Rad51-dependent repair of DSBs and therefore sensitizes BRCA wild-type EOC cells to platinum drugs. The mechanism by which 6-thioguanine enhances the effects of triapine is currently being investigated. Our findings indicate for the first time that triapine and 6-thioguanine can be used in combination with platinum agents to target BRCA wild-type and platinum-resistant EOC.

Citation Format: Elena S. Ratner, Margaret Whicker, Z. Ping Lin, Alan C. Sartorelli. Triapine sensitizes chemoresistant ovarian cancer cells to platinum therapy. [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 4490. doi:10.1158/1538-7445.AM2013-4490