bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3253: A novel small-molecule inhibitor of Stat3 induces antitumor cell effects in human glioma and breast cancer cells.
Authors: Yue Peibin , Haftchenary Sina , Gunning Patrick T. , Turkson James
Published: August 2014

Constitutive activation of Stat3, a member of signal transducer and activator of transcription (Stat) family of proteins, occurs with a high frequency in human tumors and contributes to carcinogenesis and tumor progression. Compelling evidence shows aberrant activation of Stat3 constitutes a point of convergence in oncogenic tyrosine kinase signaling, functioning as a master regulator of events crucial for tumorigenesis and malignant progression. We present a series of novel small-molecules that abrogate constitutive activation of Stat3 in human tumors, including glioma and breast cancer. The small-molecule, SH5-07, inhibits in vitro Stat3 DNA-binding activity, with IC50 value of 3.9 μM. Further, treatment with SH5-07 induces dose-dependent inhibition of constitutively-active Stat3 in human glioma U251MG and U373MG and breast cancer MDA-MB-231 cells. Moreover, treatment with low micromolar SH5-07 of U251MG, U373MG, and MDA-MB-231 cells blocks constitutive Stat3 phosphorylation, DNA-binding, and transcriptional activities by as early as 30 minutes. Furthermore, the inhibition of aberrantly-active Stat3 by SH5-07 blocks the anchorage-dependent and independent growth, survival, migration, and invasion in vitro of human glioma and breast cancer cells. Data together indicates SH5-07 is a promising small-molecule Stat3 inhibitor and a suitable drug candidate for clinical development.

Citation Format: Peibin Yue, Sina Haftchenary, Patrick T. Gunning, James Turkson. A novel small-molecule inhibitor of Stat3 induces antitumor cell effects in human glioma and breast cancer 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 3253. doi:10.1158/1538-7445.AM2013-3253
Abstract 3262: Targeting PI3K-coupled MEK/ERK pathway for therapy in pediatric B-cell acute lymphocytic leukemia.
Authors: Wang Xiang , Li Ben-shang , Ding Li-xia , Liang Chris , Ding Jian , Meng Ling-hua
Published: August 2014

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy diagnosed in children and B cell type is prevalent among pediatric ALL. At present, cytotoxic agents are the major components for chemotherapy against ALL, which are limited by their severe side effects. PI3Kδ was found to be over activated in most B cell type leukemia, and one of its inhibitor CAL-101 (GS-1101) is in active clinical trials for B cell type acute or chronic leukemia with promising outcomes. However, the mechanism of action of PI3Kδ inhibitor against B-ALL needs to be further studied to optimize the therapeutic success. Herein, for the first time we found that blockade of PI3K-coupled MEK/ERK pathway by a novel PI3Kδ selective inhibitor X-370 is essential for its efficacy against B cell acute lymphocytic leukemia. X-370, which was obtained through rational design and screening displayed potent activity against PI3Kδ (IC50=7 nM), while sparing class II/III PI3Ks as well as a panel of protein kinases representative of the human kinome. X-370 selectively blocked constitutively activated and stimuli-induced PI3Kδ signaling among four isoforms of class I PI3Ks. X-370 abrogated both Akt and Erk signaling in human B cell leukemia cells, which is accompanied with induction of cell cycle arrest and apoptosis. Mechanism studies revealed that X-370 obstructed PDK1 from binding to and/ or phosphorylating MEK in a PI3Kδ-dependent manner. Accordingly, forced expression of constitutively activated MEK attenuated the antiproliferative activity of X-370. Furthermore, X-370 preferentially inhibited the survival of primary B-ALL cells containing PI3Kδ-coupled Erk phosphorylation, which were obtained from pediatric B-ALL patients.

In summary, with a novel PI3Kδ selective inhibitor X-370, we found that inhibition of PI3Kδ led to abrogation of ERK signaling via an atypical PI3K/ PDK1/ MEK/ Erk signaling cascade. Inhibition of PI3Kδ-coupled Erk by a PI3Kδ inhibitor may predict its efficacy against B cell acute lymphocytic leukemia. These findings support the rationale for clinical testing of PI3Kδ inhibitor for the treatment of pediatric B-ALL and provide insight to optimize therapeutic strategy for such treatment.

Citation Format: Xiang Wang, Ben-shang Li, Li-xia Ding, Chris Liang, Jian Ding, Ling-hua Meng. Targeting PI3K-coupled MEK/ERK pathway for therapy in pediatric B-cell acute lymphocytic leukemia. [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 3262. doi:10.1158/1538-7445.AM2013-3262
Abstract 3294: Apoptotic effect of thymus extract plus Lactobacillus kefiri P-IF on human myeloid leukemia (HL60/AR) cancer cells.
Authors: Ghoneum Mamdooh H. , Henary Mark , Seto Yoshikazu
Published: August 2014

Apoptosis involves a cascade of biochemical events that is tightly regulated. Induction of apoptosis in tumor cells is a promising approach to curing cancer. Therefore, new and safe treatment methods that maximize cancer cells apoptosis are therefore highly sought after. In this study, we examined the effect of gross thymus extract (GTE) combined with freeze dried, heat-killed Lactobacillus kefiri P-IF in the induction of apoptosis in human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and evaluated the mechanism underlying its effect. L. kefiri P-IF is a specific strain of lactic acid bacteria that has a unique DNA sequence and PET scans show a 99.6% homology with regular kefiris. To identify any synergistic effect of these two products, tumor cells were cultured for three days with 1) GTE at a concentration of 1.25 mg/ml or 2) L.kefiri P-IF at a concentration of 1.25 mg/ml or 3) a combination of both products at the same concentrations. The apoptotic response was assessed by propidium iodide assay and expression of Bcl2, anti-apoptotic protein, was determined by flow cytometry. Results showed that GTE and L.kefiri P-IF induced apoptosis in HL60/AR cells; GTE caused 37% apoptosis while L.kefiri P-IF caused 17% apoptosis, as compared to 7% for control cancer cells without treatment. When cancer cells were cultured in the presence of both GTE and L.kefiri P-IF, 60% apoptosis of cancer cells was detected. This data was confirmed by giemsa stained cytospin preparation. The synergistic apoptotic effect was associated with decreased expression of Bcl2. MFC: 74, 68, 61 and 50 for control untreated cells, GTE, L.kefiri P-IF and GTE + kefiri P-IF respectively. Atomic Force Microscopy studies were performed and showed clear signs of apoptotic cancer cells including membrane blebbing. These results demonstrate that GTE + L.kefiri P-IF induces apoptosis on cancer cells that exceed the effect of either agent alone, and this agent may represent a new class of adjuvants for the treatment of leukemia. GTE (Thymax) was provided by the YS Nature Company, Tokyo, Japan; L.kefiri P-IF was provided by Paitos Co., Ltd. Yokohama, Kanagawa, Japan.

Citation Format: Mamdooh H. Ghoneum, Mark Henary, Yoshikazu Seto. Apoptotic effect of thymus extract plus Lactobacillus kefiri P-IF on human myeloid leukemia (HL60/AR) cancer 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 3294. doi:10.1158/1538-7445.AM2013-3294

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 330A: Differential expression of angiogenic genes in invasive high grade serous ovarian carcinomas.
Authors: Siamakpour- Reihani Sharareh , Jiang Chen , Turner Taylor , Owzar Kouros , Berchuck Andrew , Dewhirst Mark , Alvarez Secord Angeles
Published: August 2014

Our objective is to establish quantifiable and reproducible biomarkers that can aid in prognosis, and predicting treatment outcome in invasive high-grade serous ovarian cancer (HGSC). The goal of this project is to identify genomic determinants that affect the tumor angiogenesis as defined by microvascular density (MVD) count. Our central hypothesis is: differences in angiogenic gene expression profiles are associated with variations in MVD. This is a retrospective pilot study. Under an approved IRB protocol, between 1988 and 2001, sixty five snap frozen invasive serous ovarian cancer tissue samples were gathered at Duke University Medical Center. RNA from those samples were extracted and analyzed by the Affymetrix U133A microarray. Fifty-one of those samples belonged to patients with HGSC. MVD counts were determined for those samples using CD31 and CD105. CD31 and 105 are markers of proliferating endothelial cells and neoangiogenesis. Approximately 285 probe sets (associated with 148 genes) involved in angiogenesis and vasculogenesis were screened. Association between the gene expression levels and MVD counts based on a continuous variable and overall survival of the patients were evaluated. Our results indicate that: i) median CD105-MVD and CD31-MVD counts were 30 and 33, respectively; ii) there was a marginal association between CD105-MVD and overall survival (p-value =0.045) and CD31-MVD and overall survival (OS) (p-value =0.028); iii) CUL7, a member of CULLIN family of ubiquitin ligases, is associated with overall survival of the HGSC patients. Higher expression levels of CUL7 is related to worsening of OS (p-value= 8.17E−05); iv) a stronger association between CD105-MVD count and CUL7 expression (p-value= 0.001), compared to the association between CD31-MVD count and CUL7 expression (p-value= 0.024). In conclusion: CUL 7 has previously been implicated as an antiapoptotic oncogene and a P53 suppressor. Mutations in CUL7 have been linked to abnormal vasculogenesis. CUL2, another member of CULLIN family of ubiquitin ligases has also been implicated in the activity of Hypoxia-Inducible Factor (HIFα) and vasculogenesis. In this study we report that higher expression of CUL7 is significantly associated with worsening of the OS of patients with HGSC. These are considered preliminary results which need to be further verified and validated in an independent data set in future studies.

Citation Format: Sharareh Siamakpour- Reihani, Chen Jiang, Taylor Turner, Kouros Owzar, Andrew Berchuck, Mark Dewhirst, Angeles Alvarez Secord. Differential expression of angiogenic genes in invasive high grade serous ovarian carcinomas. [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 330A. doi:10.1158/1538-7445.AM2013-330A
Abstract 3342: Characterization and targeting of BRIT1 deficiency in liver cancer.
Authors: Liang Yulong , Yu Lihou , Zhang Dongxiao , Gao Hong , Lin Shiaw-Yih , Li Kaiyi
Published: August 2014

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and there are no safe and effective therapeutic agents yet to treat HCC after diagnosis. However, in HCC, genomic instability is the major remarkable hallmark, especially in the tumors at the late stage and metastatic tumors.

Thus, we wonder if targeting genomic instability can inhibit tumor growth in

HCC. BRIT1/MCPH1 has been identified to be one of the key DNA damage and repair proteins, which harbors three BRCT domains, conserved in BRCA1 and some other proteins involved in DNA damage response pathways. Our previous studies and other reports have illustrated

BRIT1’s essential roles in mitotic and meiotic homologous recombination DNA repair and maintaining genomic stability, which functions through interacting with BRCA2/Rad51 and/or chromatin structure remodeling protein SWI/SNF. Recently, we identified that BRIT1 is aberrantly expressed in ∼25% of HCC samples (n=28).

In these samples, the locus of BRIT2 gene showed notable loss of heterogeneity (nearly

50%). We also found a somatic mutation in BRIT1, which occurred in the splicing donor site of intro 10 in the BRIT1 gene, leading to a truncated protein.

Functional analysis showed that this mutation can cause failure of foci formation of BRIT1 under irradiation. Importantly, given that BRIT1-deficient cells exhibited defective DNA repair described above, our in vitro studies showed that the BRIT1-low expressing HCC cells HepG2 and Hep3B are more sensitive to olaparib, the inhibitor of poly(ADP-ribose) polymerase (PARPi), compared to the BRIT1-proficient cell SNU-449, which was determined by WST-1 and colony formation assays.

Furthermore, we also test the inhibitory effect of olaparib in the xenograft mouse model, and found that PARPi can significantly suppress tumor growth of Hep3B xenografts. Collectively, our results clearly demonstrate that

BRIT1 is mutated or aberrantly expressed in HCC, and targeting BRIT1 deficiency by PARP inhibitors may lead to novel and effective targeted therapies to treat BRIT1-deficient

HCC.

Citation Format: Yulong Liang, Lihou Yu, Dongxiao Zhang, Hong Gao, Shiaw-Yih Lin, Kaiyi Li. Characterization and targeting of BRIT1 deficiency in liver 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 3342. doi:10.1158/1538-7445.AM2013-3342

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3369: The importance of dose schedule with Hsp90 inhibitors: Results from a Phase II study in dogs with mast cell tumors.
Authors: London Cheryl A. , Bear Misty D. , Proia David A.
Published: August 2014

Purpose: STA-1474 is the prodrug of ganetespib, currently the most advanced Hsp90 inhibitor in human clinical trials. In a Phase I clinical trial, STA-1474 demonstrated biologic activity in dogs with spontaneous tumors. Data from this trial suggested that longer drug infusion times (8 hours versus 1 hour) were potentially associated with greater biologic activity. The purpose of the following study was to extend these observations and identify the optimum dosing schedule of STA-1474 in dogs with spontaneous mast cell tumors (MCT).

Experimental Design: This was a Phase 2 trial in which dogs with spontaneous MCT received STA-1474 (6 mg/kg, per week) under one of four different dosing schedules: once weekly with a 1 hour infusion (cohort A) or 8 hour infusion (cohort B), or twice weekly with a 1 hour infusion given 24 hour (cohort C) or 72 hour (cohort D) between doses. Toxicities, biomarker changes from pre- and two post-treatment biopsies, and tumor responses were assessed.

Results: Twenty-four chemotherapy and radiation naïve dogs with MCT were enrolled. Toxicities were mild and primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Measurable responses were observed from dogs in cohort A (SD=5, PD=1), cohort B (PR=2, SD=4), cohort C (PR=3, SD=3) and cohort D (PR=2, SD=1, PD=1). Biopsies are currently being evaluated by reverse phase protein array and results will be presented.

Conclusions: This study provides further evidence that prolonged exposure to STA-1474 improves biological activity. Dosing STA-1474 on two consecutive days yielded the greatest objective responses (50%) and a disease control rate of 100%. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 synthesis following its inhibition, it is likely that more frequent exposure to ganetespib will demonstrate comparable anti-cancer activity in human patients and is currently being evaluated in a Phase I study.

Citation Format: Cheryl A. London, Misty D. Bear, David A. Proia. The importance of dose schedule with Hsp90 inhibitors: Results from a Phase II study in dogs with mast cell tumors. [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 3369. doi:10.1158/1538-7445.AM2013-3369
Abstract 3379: Overexpression of CTGF is associated with chemoresistance in esophageal adenocarcinoma.
Authors: Wang Zhuwen , Lin Lin , Thomas Dafydd G. , Orringer Mark B. , Chang Andrew C. , Beer David G. , Lin Jules
Published: August 2014

Esophageal adenocarcinoma is the most rapidly increasing solid malignancy with an overall 5-year survival of only 19%. Neoadjuvant chemoradiation is standard practice in most large centers but results in a complete pathologic response in only 21%, although this subset has an improved 5-year survival. In this study, connective tissue growth factor (CTGF), which is associated with increased cell migration, tissue invasion, and angiogenesis, was evaluated for its role in chemoresistance in esophageal adenocarcinoma. CTGF was found to be overexpressed in 33.3% (5 of 15; mean 3.1-fold) of esophageal adenocarcinomas on oligonucleotide microarray relative to Barrett's metaplasia. Elevated expression of CTGF was also found in 5/5 advanced stage IV esophageal adenocarcinomas on Western blot. Overexpression of CTGF was observed in 70.6% of chemoresistant esophageal adenocarcinomas (n=17) as determined using qRT-PCR and was significantly higher compared to chemo-naïve esophageal adenocarcinomas (n=102; p<0.0005). Stable inhibition with an shRNA against CTGF in the esophageal adenocarcinoma cell line Flo, which expresses endogenous CTGF, led to a significantly increased response to the chemotherapeutic agents 5-FU (9.4-fold increase), cisplatin (5.5-fold), and etoposide (4.4-fold) at 24 hours (p<0.005). These findings suggest that CTGF may be important in mediating chemoresistance in esophageal adenocarcinoma and that inhibition of CTGF could be useful in modulating chemoresistance to current neoadjuvant regimens in esophageal adenocarcinomas overexpressing CTGF.

Citation Format: Zhuwen Wang, Lin Lin, Dafydd G. Thomas, Mark B. Orringer, Andrew C. Chang, David G. Beer, Jules Lin. Overexpression of CTGF is associated with chemoresistance in esophageal adenocarcinoma. [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 3379. doi:10.1158/1538-7445.AM2013-3379
Abstract 3401: Vemurafenib-induced RhoB expression leads to melanoma cell resistance.
Authors: Delmas Audrey , Cherier Julia , Medale-Giamarchi Claire , Pradines Anne , Favre Gilles
Published: August 2014

Most of melanoma harbour mutations leading to the constitutive activation of the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway (i.e: activating mutations of BRAF, NRAS, c-KIT). Addiction of melanoma to the MAPK pathways led to the development of inhibitors targeting either mutant BRAF, such as Vemurafenib (PLX4032), or MEK. Melanoma treatment recently met a breakthrough with the clinical evaluation of Vemurafenib that demonstrated an unprecedented 80% response rate among patients with BRAF V600E melanomas treated with Vemurafenib, but rapidly limited by drug resistance, which still requires to be elucidated.

The MAPK pathway regulates the expression of many genes which can mediate therapeutic response but also resistance, such as RhoGTPases. We analysed the mRNA expression of the RhoGTPases in two BRAF mutated melanoma cell lines, WM266-4 and A375, in response to inhibition of the MAPK pathway by PLX4032. We observed that the mRNA of RhoB was increased by PLX4032 treatment. This result was confirmed in many other BRAF mutated melanoma cell lines by inhibition of the MAPK pathway with BRAF inhibitors (PLX4032 or SB-590885) as well as MEK inhibitors (AZD6244 or AS703026). MEK inhibitors (AZD6244 or AS703026) upregulate RhoB in BRAF wild-type melanoma cells harbouring N-Ras, K-Ras or c-KIT mutation suggesting that inhibiting MAPK pathway is critical for RhoB expression modulation. It is noteworthy that this modulation is cell type dependent, while it was not found in BRAF mutated non-melanoma cells as colon carcinoma cells (Colo205 and RKO).

To investigate the role of RhoB induction in cellular responses to BRAF and MEK inhibitors, the RhoB induction was suppressed using RNA interference. The cells treated with siRNA targeting RhoB, WM266-4 and A375 cells, were more sensitive to PLX4032 and AZD6244. Moreover the blockade of RhoB induction led to a significant increase of PLX4032-induced apoptosis as shown by the increase of the subG1 population and PARP cleavage.

Our findings show that RhoB upregulation is involved in PLX4032 response and both inhibitions of MAPK pathway and RhoB expression trigger cell death and should prompt a search for agents. Indeed co-targeting MAPK and RhoB pathways, could lead to a synthetic lethality and prevent resistance to either BRAF or MEK inhibitors. Ongoing work focuses on confirming this role in tumour-xenografted mouse models.

Citation Format: Audrey Delmas, Julia Cherier, Claire Medale-Giamarchi, Anne Pradines, Gilles Favre. Vemurafenib-induced RhoB expression leads to melanoma cell 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 3401. doi:10.1158/1538-7445.AM2013-3401
Abstract 3404: Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma.
Authors: Girotti Maria R. , Pedersen Malin , Sanchez-Laorden Berta , Viros Amaya , Turajlic Samra , Niculescu-Duvaz Dan , Zambon Alfonso , Sinclair John , Hayes Andrew , Gore Martin , Springer Caroline , Larkin James , Jorgensen Claus , Marais Richard
Published: August 2014

We generated melanoma cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specificity tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed a tumor from a BRAF mutant patient with intrinsic resistance to vemurafenib. Vemurafenib treatment induced SFK activation in this tumor and we show that dasatinib blocked its growth and metastasis in immunocompromised mice. Our data shows that BRAF inhibitor-mediated activation of EFGR/SFK/STAT3 signaling can mediate resistance in BRAF mutant melanoma patients. We describe two treatments that appear to overcome this resistance and could deliver therapeutic efficacy in drug-resistant BRAF mutant melanoma patients.

Citation Format: Maria R. Girotti, Malin Pedersen, Berta Sanchez-Laorden, Amaya Viros, Samra Turajlic, Dan Niculescu-Duvaz, Alfonso Zambon, John Sinclair, Andrew Hayes, Martin Gore, Caroline Springer, James Larkin, Claus Jorgensen, Richard Marais. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. [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 3404. doi:10.1158/1538-7445.AM2013-3404
Abstract 3409: Inhibition of checkpoint kinase 2 enhances sensitivity of pancreatic adenocarcinoma cells to Gemcitabine.
Authors: Duong Hong-Quan , Kim Jung Soon , Lee Hee-Seok , Seong Yeon-Sun , Bae Insoo ,   Dept. Oncol and Dept. of Rad. Medicine, Lombardi Comprehensive Cancer Center, Georgetown University.
Published: August 2014

Checkpoint kinase 1/2 (Chk1/2) plays pivotal function as effectors of cell cycle checkpoint regulation following DNA damage. Small molecule inhibitors of Chk1/2 are under clinical evaluation in combination with chemotherapeutic agents. We examined whether NSC109555, a competitive inhibitor of Chk2 inhibitor could enhance the synergistic antitumor effect(s) of Gemcitabine(GEM) in pancreatic adenocarcinoma cell model. In the present study, the combination treatment by NSC109555 plus GEM demonstrated strong synergistic antitumor effect in four pancreatic cancer cells. In addition, the combination treatment of NSC109555 plus GEM significantly increased the generation of intracellular reactive oxygen species (ROS), followed by induction of apoptotic cell death. Furthermore, the inhibition of ROS generation by N-acetyl cysteine (NAC) significantly prevented the decrease in cell survival and increase in apoptotic cell death by combination treatment of NSC109555 plus GEM. Small interfering RNA (siRNA) mediated knockdown of Chk2 also enhanced GEM-inhibited cell survival and GEM-induced apoptotic cell death. Our findings suggest that NSC109555 would be beneficial therapeutic agents when combined with GEM and support the novel synthetical lethal approach for cancer therapy by these combinations for preclinical and clinical treatment of pancreatic cancer.

Citation Format: Hong-Quan Duong, Jung Soon Kim, Hee-Seok Lee, Yeon-Sun Seong, Insoo Bae, Dept. Oncol and Dept. of Rad. Medicine, Lombardi Comprehensive Cancer Center, Georgetown University. Inhibition of checkpoint kinase 2 enhances sensitivity of pancreatic adenocarcinoma cells to Gemcitabine. [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 3409. doi:10.1158/1538-7445.AM2013-3409
Abstract 3441: Dynamin inhibitors as anticancer agent in cervical carcinoma.
Authors: Lee Jeong-Won , Jeon Hye Kyung
Published: August 2014

Dynamin is a 96 kDa GTPase enzyme involved in membrane constriction and fission during endocytosis. Among the three human dynamin genes, dynamin I is expressed in neurons and neuro-endocrine cells, dynamin II is expressed in most cell types, and dynamin III is strongly expressed in the testis, brain, and lung tissue. Dynamin II plays an essential role in the completion of the final stage of mitosis, cytokinesis. MiTMAB (myristyl trimethyl ammonium bromides) and OcTMAB (octadecyltrimethyl ammonium bromide) are small-molecule inhibitors of dynamin that competitively interfere with the ability of dynamin to bind phospholipids and prevent receptor-mediated endocytosis. In this study, we investigated whether dynamin inhibitor could inhibit survival, proliferation, and invasion of cervical cancer cell lines. To evaluate the role of dynamin II in cell proliferation, we performed MTT assays with Hela and SiHa cells after incubation with various doses of MiTMAB or OcTMAB. To confirm the apoptosis after MiTMAB and OcTMAB treatment, active caspase-3 ELISA and FACS were performed. And to identify the role of dynamin II in cell invasion, we evaluated the secretion of MMP-9 after dynamin II inhibitor treatment by ELISA assay. We found that MiTMAB and OcTMAB had a growth-inhibiting effect at 2 or 3 day after treatment in both cells. Active caspase-3 expression and Annexin V intensity were increased by MiTMAB and OcTMAB. And the secretion of MMP-9 was increased after dynamin II inhibitor treatment. MiTMAB and OcTMAB are the first compounds reported to exclusively block cytokinesis without affecting progression through any other stage of the cell cycle. Cytokinesis failure induces cell death. These data revealed that dynamin II modulates cancer cell survival, proliferation, and invasion and suggest that dynamin II could be a key target for new strategies in cervical cancer treatment.

Citation Format: Jeong-Won Lee, Hye Kyung Jeon. Dynamin inhibitors as anticancer agent in cervical carcinoma. [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 3441. doi:10.1158/1538-7445.AM2013-3441
Abstract 3450: ABT-348 in combination with inhibition of CDK4/6 highlights a strategy to target RB mutant cells while sparing RB wild-type cells.
Authors: Guo Jun , Curtin Michael L. , Chen Zehan , Albert Daniel H. , Tapang Paul , Dai Yujia , Bui Mia-Ha , Kovar Peter , Michaelides Michael R. , Glaser Keith B. , Tse Chris , Shah O. Jameel
Published: August 2014

ABT-348 is a novel, potent and orally bioavailable inhibitor of the Aurora kinases as well as the VEGF and PDGF families of receptor tyrosine kinases. ABT-348 is broadly efficacious preclinically as a single agent against a wide range of tumor types and is currently in Phase I/II clinical trials. Several published studies have supported the potential utility of combining targeted anti-proliferative agents with cytotoxic chemotherapies. Herein, the efficacy of combining the neocytotoxic agent, ABT-348, with CDK4/6 inhibition in the treatment of solid tumor cells has been evaluated. Inhibition of CDK4/6 via siRNA elicits a potent cytostatic response in cells that harbor a wild-type, functional RB. We demonstrate that features of cellular senescence are induced in human tumor cells with siRNAs targeting both CDK4 and CDK6. Dual inhibition of CDK4 and CDK6 is required for antiproliferative activity in most cancer cell lines and this is not accompanied by induction of apoptosis. In RB wild-type cells, CDK4/6 inhibition antagonizes the activity of ABT-348 by inducing G0/G1 arrest. Conversely, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient cells to ABT-348, indicating that the effects of ABT-348 are dependent on cells progressing into mitosis. A preponderance of evidence from previous publications and our studies indicate that CDK4/6 inhibition attenuates the cellular response to cytotoxic chemotherapies in RB wild-type cells providing a potential to expand the therapeutic window for ABT-348. Thus combination with ABT-348 and a CDK4/6 inhibitor may provide an opportunity to selectively target RB mutant cells.

Citation Format: Jun Guo, Michael L. Curtin, Zehan Chen, Daniel H. Albert, Paul Tapang, Yujia Dai, Mia-Ha Bui, Peter Kovar, Michael R. Michaelides, Keith B. Glaser, Chris Tse, O. Jameel Shah. ABT-348 in combination with inhibition of CDK4/6 highlights a strategy to target RB mutant cells while sparing RB wild-type 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 3450. doi:10.1158/1538-7445.AM2013-3450
Abstract 3454: Bcl-xL anti-apoptotic network is dispensable for emergence and maintenance of CML but required for disease progression, and represents an alternative target for halting survival of blast crisis CML progenitors.
Authors: Harb Jason , Neviani Paolo , Ellis Justin E. , Ferenchak Gregory J. , Chyla Brenda I. , Hokland Peter , Roy Denis Claude , Caligiuri Michael A. , Marcucci Guido , Huettner Claudia S. , Perrotti Danilo
Published: August 2014

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug-resistance and disease progression. We generated an inducible mouse model in which simultaneous expression of p210 BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect the development of the chronic phase-like myeloproliferative disease; however, none of the deficient mice progressed to an advanced phenotype, suggesting the importance of the anti-apoptotic Bcl-xL signaling network for the progressing early progenitor cell survival. Indeed, pharmacologic inhibition of Bcl-xL with the Bcl-xL/Bcl-2 antagonist ABT-263, combined with activation of the Bcl-xL-regulator BAD, through the PP242-mediated inhibition of mTORC1/2, markedly augmented apoptosis of BCR-ABL1+ cell lines and primary CD34+ progenitors from CML-BC but not from healthy donors, regardless of the drug-resistance effect induced by bone marrow stromal cell-generated signals. Accordingly, shRNA-mediated downregulation of BAD and the BCR-ABL1-target hnRNP A1, which promotes translation of Bcl-xL but not Bcl-2, impaired the ability of PP242 to potentiate the effect of ABT-263, and mimicked the anti-leukemic activity of ABT-263, respectively, in primary leukemic CD34+ progenitors and/or CML-BC cell lines. Thus, suppression of the antiapoptotic potential of Bcl-xL together with activation of Bad represents a novel potential pharmacologic approach for treatment of those patients undergoing blastic transformation.

Citation Format: Jason Harb, Paolo Neviani, Justin E. Ellis, Gregory J. Ferenchak, Brenda I. Chyla, Peter Hokland, Denis Claude Roy, Michael A. Caligiuri, Guido Marcucci, Claudia S. Huettner, Danilo Perrotti. Bcl-xL anti-apoptotic network is dispensable for emergence and maintenance of CML but required for disease progression, and represents an alternative target for halting survival of blast crisis CML progenitors. [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 3454. doi:10.1158/1538-7445.AM2013-3454
Abstract 3475: Progranulin as a prognostic biomarker of advanced epithelial ovarian cancer.
Authors: Fitzgerald Sharon , Yu Minshu , Hays John , Di Santo Nicola , Havrilesky Laura , Kohn Elise
Published: August 2014

Late diagnosis and early recurrence make epithelial ovarian cancer (EOC) the leading cause of gynecologic cancer death in the US. Indicators of early relapse may allow triage of high-risk patients to earlier therapy. Progranulin (PGRN) is a growth and survival factor overexpressed in EOC. We hypothesize that elevated serum (s)PGRN concentrations after clinical complete response (CCR) to primary platinum and taxane-based combination chemotherapy prognosticates disease recurrence and decreased survival in advanced EOC patients. We previously demonstrated high plasma (p)PGRN concentration (cutoff 59ng/ml) at first CCR distinguished early from late recurrence and predicted long-term survival (ROC AUC=0.944; sensitivity 93%; specificity 100%). In multivariate analysis, high pPGRN concentration predicted early recurrence (HR=23.5; 95%CI: 2.49-221; P<0.006). As PGRN is cleaved into proinflammatory granulins by neutrophil elastase, we wished to investigate the reliability of sPGRN as a biomarker. We compared PRGN concentrations in ten blinded, paired serum and plasma samples, using commercial PRGN ELISA assays; paired two-tailed t-test demonstrated comparability (P=0.90, R2= 0.002). A cohort of 35 blinded serum samples from a different institution was analyzed using the ELISA. The optimal dichotomization concentration of sPGRN, 49.5ng/ml, was determined by ROC statistics at a specificity of 90%, and applied to outcome analyses using Cox proportional hazards and Kaplan-Meier modeling. This PGRN cutoff significantly differentiated progression-free survival (PFS; P=0.0013). Subjects with low sPGRN had 100% survival (OS) at 2 years (N=11; range 2-12). Further validation of PGRN's discriminatory value could lead to its clinical investigation as a prognostic biomarker to stratify those at high risk for recurrence to closer monitoring or maintenance therapy, as well as its examination as a predictive biomarker for response to initial therapy.

Citation Format: Sharon Fitzgerald, Minshu Yu, John Hays, Nicola Di Santo, Laura Havrilesky, Elise Kohn. Progranulin as a prognostic biomarker of advanced epithelial ovarian 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 3475. doi:10.1158/1538-7445.AM2013-3475
Abstract 3488: Surveying colorectal cancer genome for clinically actionable genomic amplifications with droplet digital PCR.
Authors: Rumma Rowza T. , Miotke Laura , Nadauld Lincoln , Natsoulis Georges , DiMaio Michael , Jalali Moe , Ji Hanlee
Published: August 2014

New cancer therapies are increasingly geared towards exploiting critical genetic and genomic features specific to the tumor. These mutations and genomic aberrations are the basis for precision cancer medicine. Thus, rapid molecular characterization of clinical cancer samples has become increasingly important for cancer targeted therapy development. Our study addresses this need by identifying ‘druggable’ gene amplifications in colorectal cancer (CRC). Along these lines, we developed an accurate and rapid droplet-digital PCR (ddPCR) assay to analyze cancer-specific copy number variations (CNV) in 13 genes with oncogenic function that are the targets of specific cancer therapies. These genes were selected based on the availability of approved and early-phase targeted therapeutics that inhibit their oncogenic function. Using TCGA data, we vetted our targets by identifying frequent CRC amplifications and a correlation with gene expression.

The ddPCR method involves emulsifying matched-normal cancer sample DNA that provides specific advantages for highly sensitive and specific detection of cancer CNVs. Post-amplification, emulsion droplets are streamed single-file into a capillary that leads past a two-color detector, where the positive droplets for the target and reference genes are “counted” for quantitation. This technology requires nanogram amounts of genomic DNA, thus facilitating the study of clinical cancer samples from small biopsies. It works well with genomic DNA extracted from formalin fixed paraffin embedded tumor samples. Furthermore, we demonstrated high sensitivity for detecting copy number amplifications even in samples containing a prominent fraction of normal tissue.

We are extending our analysis to the full 13 “druggable” gene targets and evaluating patterns of mutual exclusivity and co-occurrence among a cohort of over 200 CRC tumors with information on clinical outcome. For example, we have demonstrated FGFR1 amplifications in 5.2% of our CRC tumor population. For ERBB2 variations in the same cohort, we detected amplifications in 3.6% of our population. ERBB2 and FGFR1 amplification events displayed a mutually exclusive pattern of segregation (p value <0.0001 per Chi-squared test with Yates correction). This efficient and inexpensive assay offers a significant potential to extend personalized therapeutic options available to CRC patients.

Citation Format: Rowza T. Rumma, Laura Miotke, Lincoln Nadauld, Georges Natsoulis, Michael DiMaio, Moe Jalali, Hanlee Ji. Surveying colorectal cancer genome for clinically actionable genomic amplifications with droplet digital PCR. [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 3488. doi:10.1158/1538-7445.AM2013-3488
Abstract 349: Molecular pathogenesis of mucinous adenocarcinoma of the lung.
Authors: Maeda Yutaka , Whitsett Jeffrey
Published: August 2014

Human mucinous adenocarcinoma of the lung is a subtype of highly invasive pulmonary tumors and is associated with decreased or absent expression of the respiratory epithelium transcription factor NKX2-1 (NK2 homeobox 1, also known as TTF-1). Here, we show that haploinsufficiency of Nkx2-1 in combination with oncogenic Kras.G12D (hereafter; Kras.G12D;Nkx2-1+/-), but not with oncogenic EGFR.L858R, causes pulmonary tumors in transgenic mice that are phenotypically similar to human mucinous adenocarcinomas. Expression of both CK7 and CK20, biomarkers for human mucinous adenocarcinoma of the lung, were confirmed in lungs of the Kras.G12D;Nkx2-1+/- mice. Expression of mucins, such as MUC5AC, and mucin/metastasis-related protein AGR2 was observed in tumorigenic mucous cells in the Kras.G12D;Nkx2-1+/- mice as well as non-tumorigenic airway mucous cells induced by allergen; however, mucous cell master regulators, such as SPDEF and FOXA3, were seen only in the non-tumorigenic airway mucous cells but not in the tumorigenic mucous cells, indicating a distinct mucous gene regulation in the tumorigenic mucous cells. NKX2-1 cell-autonomously inhibited mRNA expression of MUC5AC and AGR2 in A549 human lung carcinoma cells that harbor a KRAS mutation. Chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) analysis identified a direct association of NKX2-1 with the promoter and intron of MUC5AC and AGR2 genes in A549 cells, indicating that NKX2-1 functions as a transcriptional repressor for the mucous gene expression. The ChIP-seq analysis further revealed that NKX2-1 associated with the AP-1 binding element (TGAnTCA) as well as the canonical NKX2-1 binding element (CTTG). NKX2-1 inhibited AP-1-mediated activity and tumor colony formation in vitro, suggesting that NKX2-1 suppresses mutant KRAS-driven lung tumorigenesis in part by inhibiting AP-1 activity. These results demonstrate that NKX2-1 suppresses mutant KRAS-driven mucinous pulmonary adenocarcinoma in part by directly inhibiting expression of genes induced in mucinous tumors.

Citation Format: Yutaka Maeda, Jeffrey Whitsett. Molecular pathogenesis of mucinous adenocarcinoma of the lung. [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 349. doi:10.1158/1538-7445.AM2013-349
Abstract 3493: A single-tube, multiplexed, transcript-based assay to detect ALK, ROS1 and RET fusions in lung cancer.
Authors: Lira Maruja , Choi Yoon-La , Deng Shibing , Huang Donghui , Ozeck Mark , Han Joungho , Jeong Ji Yun , Park Keunchil , Kim Jhingook , Ahn Myung-Ju , Mao Mao
Published: August 2014

Oncogenic fusions involving ALK, ROS1 and RET define a small subpopulation (∼7%) of non-small cell lung carcinomas (NSCLC). Tumors harboring ALK and ROS1 rearrangements are highly sensitive to crizotinib whereas RET inhibitors may be of potential benefit for the treatment for RET-fusion positive tumors1. Current methods for detection of ALK, ROS1, and RET rearrangements involve fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) or RT-PCR, each test offering its own advantages and disadvantages, and making it difficult for screening large numbers of patient samples for all three targetable fusions.

To explore screening modalities for the simultaneous detection of ALK, ROS1 and RET fusions from a single analyte, we designed a multiplexed, transcript-based assay to detect for presence or absence of fusions. Utilizing a combined 3’ over-expression and fusion-specific detection strategy, we developed a single-tube multiplex assay with a quantitative scoring modality that is highly sensitive, reproducible and capable of detecting low abundant ALK, ROS1 and RET fusion transcripts. We successfully validated the assay in 273 NSCLC specimens. For ALK, our results were highly concordant (97.4%) to prior results obtained by IHC (n=189). We confirmed ROS1 (n=7) and RET (n=11) fusion-positive tumors by RT-PCR plus sequencing. There is a significant enrichment for ROS1 and RET fusions in triple negative NSCLC (i.e. KRAS/EGFR/ALK wildtype) (Table 1). ALK, ROS1, RET, KRAS and EGFR mutations are mutually exclusive in this study. Our assay offers an easy to perform, high-throughput, and FFPE-compatible screening method for detection of ALK, ROS1 and RET fusions.

Table 1. Incidence of ALK, ROS1 and RET fusions cohorts clinical/molecular feature # cases ALK+ ROS1+ RET+ Set 1 ALK IHC+, Kras/EGFR wt 95 91 0 0 Set 2 ALK IHC−, Kras/EGFR wt 94 1 5 11 Set 3 never smokers 84 10 2 1

Citation Format: Maruja Lira, Yoon-La Choi, Shibing Deng, Donghui Huang, Mark Ozeck, Joungho Han, Ji Yun Jeong, Keunchil Park, Jhingook Kim, Myung-Ju Ahn, Mao Mao. A single-tube, multiplexed, transcript-based assay to detect ALK, ROS1 and RET fusions in 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 3493. doi:10.1158/1538-7445.AM2013-3493
Abstract 3497: Pathway profiling for personalized medicine: Comparison of two immunoassays.
Authors: Stresemann Antje , von Ahsen Oliver , Politz Oliver , Kim Phillip , Singh Sharat , Asadullah Khusru , Ziegelbauer Karl , Krahn Thomas
Published: August 2014

Cancer therapy is evolving to a detailed molecular analysis of the patient's tumor followed by treatment with selective drug(s) targeting the individual properties of the tumor based upon identification of prognostic and predictive biomarkers. This requires comprehensive, highly sensitive test systems. We therefore sought to develop a sensitive diagnostic test for functionally profiling a spectrum of signaling pathway proteins in tumor samples. Here, we evaluated and compared two assay platforms: CEER (Collaborative Enzyme Enhanced Reactive-Immunoassay; Prometheus Labaratories) and MSD (Meso Scale Discovery).

Breast, lung, and prostate cancers as well as glioblastoma and melanoma model cell lines (in all 10 cell lines) with various oncogenic pathway signatures were treated with different concentrations of PI3K inhibitor (BAY 806946) or an inhibitor targeting HER1 and HER2 (Lapatinib). Cells were lysed and the activation status as well as abundance of ten pathway proteins (HER1, HER2, cMET, PI3K, AKT, ERK, MEK, PRAS40, RPS6, and P70S6K) was measured by MSD at Bayer in Berlin and by CEER in a blinded fashion at Prometheus Laboratories. Target-specific inhibition (IC50) and downstream signal modulations were determined and compared. Overall we found a high concordance between the two assays. While target specific inhibitions were observed in relevant cell lines, varying mechanisms of treatment resistance due to redundant pathway activation, feedback loop or pathway cross talks were observed between the PI3K/AKT and RAS/ERK pathways.

This study shows that sensitive immunoassays are a suitable tool for the detection and monitoring of biomarkers and provides new insights into the mode of action of targeted agents. Comprehensive profiling of signaling pathways holds promise as an approach to personalize selection of anticancer therapy.

Citation Format: Antje Stresemann, Oliver von Ahsen, Oliver Politz, Phillip Kim, Sharat Singh, Khusru Asadullah, Karl Ziegelbauer, Thomas Krahn. Pathway profiling for personalized medicine: Comparison of two immunoassays. [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 3497. doi:10.1158/1538-7445.AM2013-3497