bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A30: The prevalence of RON isoforms in experimental and clinical tumor samples.
Authors: Amatulli Michael
Published: August 2014

The role of the RON receptor or MST1R in tumor development has been demonstrated in multiple tumor models. As a result of alternative pre-mRNA splicing, protein truncation and alternative transcription, several isoforms of RON (WT, delta-55, delta-110, delta-155, delta-160, delta-165, and delta-170) are known to exist. In contrast to WT RON, which localizes to the cell membrane, some RON variants (delta-155, delta-165) are constitutively active in the cytoplasm. Despite the previously documented role of RON in tumor development, the prevalence of individual RON isoforms in different tumor tissues remains largely unknown. Due to a high level of conservation between the RON isoforms which results largely from in frame exon 5, 6 and/or 11 deletions, protein-based variant characterization is unfeasible. We therefore decided to focus on an mRNA-based detection technique using a PCR-based sizing assay. In total 63 samples, including 28 tumor cell lines, 16 patient-derived tumor models and 19 colorectal tumors were analyzed. Total RNA was isolated and converted to cDNA followed by standard PCR amplification and Bio-Analyzer quantification and analysis for specific isoforms including RON delta-155, delta-160, delta-165, and delta-170/WT. In 35/48 samples tested (73%), the most prevalent isoform was RON delta-165 followed by WT RON. FACS analysis of lung adenocarcinoma A549 cells, which displayed only RON delta-165 mRNA expression, confirmed intracellular localization of RON protein.

Analysis of RNA sequencing data in TCGA data sets which include over 5,000 patient tumor samples also demonstrated high prevalence of RON delta-165 expression across different tumor indications. More than 98% of samples, both tumor and normal, showed the RON delta-165 variant. Prevalence, defined as a delta-165: WT ratio > 1, was seen in 28.6% and 24.3% of cases, respectively. Analysis of the lung and colon subsets revealed differentially higher RON delta-165 prevalence in lung (26%) and colon (25%) tumors, compared to respective normal tissue (7% and 0% respectively). Taken together, this data indicates that RON delta-165 is expressed in multiple tumor types and alludes to an altered RON isoform expression pattern in lung and colon cancer. Given a unique biology of this variant (constitutive activation and intracellular localization), isoform-specific targeted therapies should be developed for the inhibition of this pathway.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A30.

Citation Format: Michael Amatulli. The prevalence of RON isoforms in experimental and clinical tumor samples. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A30.
Abstract A83: Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy.
Authors: Gilabert-Oriol Roger , Thakur Mayank , von Mallinckrodt Benedicta , Hug Thomas , Wiesner Burkhard , Eichhorst Jenny , Melzig Matthias F. , Fuchs Hendrik , Weng Alexander
Published: August 2014

Antitumor therapy with monoclonal antibodies has shown great therapeutic benefits in clinics and a number of therapeutic antibodies have been approved for use in patients. Nevertheless, their efficacy is limited due to their insufficient inherent cell killing activity. In order to augment their antitumor efficacy, monoclonal antibodies may be modified with natural toxins to create a drug conjugate (immunotoxin) that comprises the dual functionality of monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) and the cell-killing activity of the toxin. In our investigations, two immunotoxins were prepared by chemically coupling the monoclonal antibodies trastuzumab (Herceptin®) and cetuximab (Erbitux®) to the plant derived toxin saporin via a cleavable disulfide bond. Impedance-based real-time viability assays and confocal live cell imaging revealed that the toxin is efficiently delivered to the targeted tumor cells. In addition, immunotoxin cytotoxicity was tremendously enhanced in combination with certain plant glycosides, which specifically mediate the endo/lysosmal release of the toxin moiety into the cytosol without damaging the biological membranes. The combinatorial approach was successful in achieving a complete cell death of human breast ductal carcinoma BT-474 cells, at a concentration of 1 nM of trastuzumab-saporin or 0.001 nM cetuximab-saporin in the presence of the glycosides. Furthermore, immunotoxins retained their ability to interact with the innate immune system and trigger antibody-dependent cell-mediated cytotoxicity. The combinatorial approach is conceived as a novel and promising way to increase the efficacy of monoclonal antibodies in targeted tumor therapies.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A83.

Citation Format: Roger Gilabert-Oriol, Mayank Thakur, Benedicta von Mallinckrodt, Thomas Hug, Burkhard Wiesner, Jenny Eichhorst, Matthias F. Melzig, Hendrik Fuchs, Alexander Weng. Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A83.
Abstract B245: Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody.
Authors: Ayrolles-Torro Adeline , Vezzio-Vie Nadia , Denis Vincent , Boissiere-Michot Florence , Garambois Véronique , Busson Muriel , Ait Arsa Imade , Mollevi Caroline , Pugniere Martine , Bibeau Frédéric , Martineau Pierre , Gongora Céline , Del Rio Maguy
Published: August 2014

In metastatic colorectal cancer (CRC) patients, chemotherapy in combination with targeted therapies such as Avastin, Erbitux, and Vectibix (monoclonal antibodies) has improved response rates with prolongation of progression free survival and overall survival. Despite these major therapeutic progresses, at least 30 to 50% of patients remain non-responders.

Our project aimed to identify and validate new molecular targets of CRC. Comparison of gene expression profiles between normal mucosa and primary tumor of CRC patients followed by immunohistochemistry analysis identified claudin-1 (CLDN1) as potential therapeutic target for antibody-targeted therapy. CLDN1 is a major tight junction transmembrane protein, already found overexpressed in CRC in several studies and associated with tumor progression.

We have generated monoclonal antibodies (mAb) directed against extracellular domains of CLDN1 and demonstrated their specificity against human CLDN1 in vitro and in vivo. Then, using mice xenografted with the CRC cell line SW620 we tested the therapeutic effect of the 6F6C3mAb. The mice were treated by i.p. injections with vehicle or 6F6C3mAb at two different doses. The results showed that 6F6C3mAb treated-groups had a significant (p= 0,018) reduced growth compared to the control group and this effect was dose-dependent. The in vitro effects of the binding of 6F6C3mAb to CLDN1 were also assessed by clonogenic assays. Treatment by 6F6C3mAb induced a significant (p< 0.05) reduction of the number of colony-forming cells for CLDN1high colorectal cancer cells (SW620, Caco2) but also for CLDN1high ovarian and pancreatic cancer cells (PANC1, BXPC3, SKOV3, IGROV1). Thus, 6F6C3mAb binding had a profound effect on cancer cell survival.

In conclusion, CLDN1 seems to be a promising target for antibody-based therapy. The 6F6C3mAb is able to inhibit tumor growth and tumor survival by binding the extracellular domains of CLDN1. It can be used in the treatment of cancer diseases such as colorectal cancer as well in adjuvant as in metastatic setting.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B245.

Citation Format: Adeline Ayrolles-Torro, Nadia Vezzio-Vie, Vincent Denis, Florence Boissiere-Michot, Véronique Garambois, Muriel Busson, Imade Ait Arsa, Caroline Mollevi, Martine Pugniere, Frédéric Bibeau, Pierre Martineau, Céline Gongora, Maguy Del Rio. Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B245.
Abstract B246: Overcoming cancer resistance to death receptor targeted therapies.
Authors: Zhang Baolin , Chen Junjie , Di Xu , Zhang Yaqin
Published: August 2014

Multiple clinical trials are underway to evaluate the antitumor activity of a new class of protein products that target the cell surface death receptors (DRs) 4 and 5. These include the recombinant human TNF-related apoptosis inducing ligand (TRAIL) and agonistic antibodies specific to DR4 or DR5. The early-phase clinical data is encouraging in suggesting a tolerated safety profile for these products. However, there is a major concern about the occurrence of drug resistance that has been seen in a large number of human cancer cell lines and primary tumors. There are unmet needs in the development of DR-targeted therapies, in terms of both the ability to predict a tumor response and the selection of combinational drugs for overcoming the resistance towards a better clinical efficacy.

Our laboratory investigates cell death and cell survival mechanisms involved in cancer drug action and resistance. Over the past eight years, we have made significant findings on the molecular determinants of TRAIL resistance. We have shown that deficiency of DR4 and/or DR5 on surface membrane is a critical mechanism of cancer cell resistance to the targeted therapies. In some cancer cells, the two receptors are found to be not properly expressed on surface membrane and, instead, are mislocalized in intracellular compartments. We provide evidence for novel mechanisms of DR surface deficiency, which involve deregulation of endocytosis and autophagy pathways in the TRAIL-resistant cells. These data could help identify biomarkers for predication of a tumor response and novel molecular targets for combination drugs to overcome or bypass the resistance mechanisms. This presentation will give an update of the relevant clinical trails and our research findings on DR-targeted therapies.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B246.

Citation Format: Baolin Zhang, Junjie Chen, Xu Di, Yaqin Zhang. Overcoming cancer resistance to death receptor targeted therapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B246.
Abstract B248: Antitumor activity mediated by N-myristoylated cationic peptides (myrCP).
Authors: Krotova Karina , Aslanidi George
Published: August 2014

Although new chemotherapeutic agents are improving the survival rate of cancer patients, all of them have serious toxic side effects. Thus, it is still necessary to search for new agents with minimal side effects or to enhance the effectiveness of known cytotoxic agents at a reduced dose. The modification of peptides by adding myristol group is commonly used to facilitate intracellular delivery. It has been shown recently that the combination of myristoylation and the cationic charge of peptide confer new biological activity to the peptide. We examined the possibility of using such peptides for the treatment of cancer. In present studies, we evaluated the effect of two N-myristoylated cationic peptides (myrCP) on several human cancer cell lines: H1299 (lung), MCF7 (breast), and PC3 (prostate). The first peptide mimics the endogenous Protein kinase C (PKC) pseudosubstrate region and it is considered as a selective inhibitor of PKC (myr-SIYRRGARRWRKL-OH). This peptide has a dual effect on cancer cells as it prevents proliferation by inhibiting PKC and also serves as a cytotoxic agent. The second peptide that is a scrambled version of the first peptide (myr-RLYRKRIWRSAGR-OH) was used as a control to ensure that cytotoxic effect is not only dependent on PKC inhibition. We found that both myrCP had superior cytotoxic effect on cancer cells. Moreover, they dramatically enhanced the activity of commonly used chemotherapeutic agents such as mitoxantrone and cisplatin which were active in combination with peptides at significantly lower concentration. Since the lipophilic nature of myristoylation facilitates penetration of these peptides across cell membranes, we analyzed what lipids can be involved in such interaction. Membrane lipid arrays showed that myrCP preferably binds to PtdIns (3, 4, 5) P3, phosphatidic acid and at less extent to PI (4, 5) P2. Our results suggested that lipid membrane composition of cells could play an essential role in efficacy of myrCP. In summary, the potential outcome of the current studies will be the development of a useful therapeutic tool for efficient killing of tumor cells. Considering that cytotoxic effects of myrCP are non-specific to the type of cancer, a similar strategy can be applied for many different malignancies.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B248.

Citation Format: Karina Krotova, George Aslanidi. Antitumor activity mediated by N-myristoylated cationic peptides (myrCP). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B248.
Abstract B259: Novel indolizino[6,7-b]indoles suppress the growth of human non-small cell lung cancer cells in xenografted and orthotopic mouse models via induction of DNA crosslinks and inhibition of topoisomerase I and II.
Authors: Chen Chi-Wei , Chen Yi-Fan , Chao Shu-Hsin , Tala Satishkumar , Su Tsann-Long , Lee Te-Chang
Published: August 2014

Non-small cell lung cancer (NSCLC) remains a global health problem due to the limited therapeutic responses and drug resistance. We have previously demonstrated that indolizino[6,7-b]indoles are potent antitumor agents with DNA cross-linking and topoisomerase I and II inhibition activities (JMC, 56: 1544-1563, 2013). The present study explores its therapeutic activity against NSCLC. Our results showed that BO-1922 and BO-1978, the selected derivatives of indolizino[6,7-b]indoles, effectively repressed the cell growth of several NSCLC lines, including as H460 (KRAS mutation), H1299 (TP53 deficient), A549 (KRAS mutation), CL141T (TP53 mutation), PC9 (EGFR mutation), PC9/gef B4 (EGFR mutation and acquired resistant to TKI), CL100 (EGFR mutation), and CL97 (EGFR mutation and acquired resistant to TKI). Using mouse xenograft models, we found that BO-1922 and BO-1978 could significantly suppress the tumor growth of H460, PC9, and PC9/gef B4 in mice. Furthermore, we demonstrated that these 2 compounds could remarkably inhibit the growth of CL1-5/GFP-Luciferase cells in orthotopic lung cancer model. In addition, we performed preclinical toxicity studies. Accordingly, the LD50 of BO-1922 and BO-1978 to ICR mice are 77.4 and 93.7 mg/kg, respectively. Based on blood cell counts, blood biochemistry, histopathological examination of major organs, BO-1922 and BO-1978 did not cause obvious toxicity at the doses of 20 and 40 mg/kg on day 2 and 14 after injections. Taken together, our present study revealed that BO-1922 and BO-1978 are highly prospective leads for treatment of NSCLC.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B259.

Citation Format: Chi-Wei Chen, Yi-Fan Chen, Shu-Hsin Chao, Satishkumar Tala, Tsann-Long Su, Te-Chang Lee. Novel indolizino[6,7-b]indoles suppress the growth of human non-small cell lung cancer cells in xenografted and orthotopic mouse models via induction of DNA crosslinks and inhibition of topoisomerase I and II. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B259.
Abstract B299: The role of Necl-5 in hepatocarcinogenesis.
Authors: Chen Xue , Yano Yoshihiko , Hayashi Yoshitake
Published: August 2014

Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy of adults and the third leading cause of cancer mortality in the world. Hepatocarcinogenesis is a multistep process, usually caused by disturbing cell-cell adhesion and associated with a change of surface molecules. Recently, it is noticed that Necl-5 (Nectin-like 5) could regulate cell adhesion and migration, moreover, overexpressed in many cancers, such as colon cancer and lung cancer. However, the expression of Necl-5 in HCC is still unknown. Hence, we examined the chemically induced hepatocarcinogenesis using Necl-5 knock out mice and the expression of Necl-5 in human HCC. It was significantly showed that HCC was easily generated in Necl-5 knock out mice compared with wild type mice. In human cancerous tissues, the expression of Necl-5 was significantly higher in well differentiated HCC than moderately and poorly differentiated HCC. The expression of CD226, the ligand of Necl-5, was significantly lower in cancerous tissues compared with non-cancerous tissue. It was suggested that there was a negative regulation between Necl-5 and CD226. It was known that NK cells were activated by CD226 and played a role in early stage of tumorigenesis and participated in immune surveillance. Lacked of the Necl-5 gene indirectly impair immune surveillance to induce mice was easier generated cancer. Those findings suggested that Necl-5 might be a potential target of anti-cancer drugs in the future.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B299.

Citation Format: Xue Chen, Yoshihiko Yano, Yoshitake Hayashi. The role of Necl-5 in hepatocarcinogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B299.
Abstract B4: Evaluation of VEGFxxxb isoforms as a predictive biomarker for anti-VEGF cancer therapy.
Authors: Zhang Wen , Abrahams Cristina , Huang Ying , Li Baosheng , Parveen Asma , Ni Min , Wei Yi , Ding Yueming , Zhang Eric , Lin Hsin Chieh , Thurston Gavin , Zhang Qing
Published: August 2014

Biomarkers are critical tools for measuring efficacy of treatments or predicting which patients are mostly likely to respond to a particular therapy. Recently, the VEGF isoform, VEGF165b, was proposed to be a predictive marker for bevacizumab in metastatic colorectal cancer patients. VEGF165b is an anti-angiogenic splice variant that differs from its pro-angiogenic counterpart, VEGF165, by only six amino acids at the C-terminal end of the protein. Members of the VEGFxxxb isoform family are produced by alternative splicing of the 3’ untranslated region (UTR) to generate exon 8b, instead of the conventional exon 8 which produces exon 8a. The study of VEGF165b in colorectal cancer (Bates et al, Clinical Cancer Research, 2012) reported that patients with low VEGF165b:VEGFtotal ratio had improved PFS following bevacizumab + FOLFOX4 treatment, while individuals with higher VEGF165b:VEGFtotal ratios did not benefit. On the other hand, another recent study (Harris et al, PLoS One, 2012) reported that VEGFxxxb isoforms may be a RT-PCR artifact. In this study, we sought to test the role of VEGF165b in the response to anti-VEGF therapy using preclinical human tumor models, and to evaluate the prevalence of VEGFxxxb isoform in human cancer cells and normal tissues using RNA-Seq, a next generation sequencing (NGS) platform that is independent of RT-PCR. Our results show that xenograft tumors engineered to overexpress VEGF165b had similar growth kinetics, tumor vascular density and were equally sensitive to aflibercept or bevacizumab treatment in humanized VEGF SCID mice compared to their parental counterparts. These results indicate that VEGF165b does not have anti-angiogenic properties, and VEGF165b levels did not correlate with the efficacy of anti-VEGF therapy. Furthermore, although we were able to detect VEGF165b splice variants by NGS profiling in cells engineered to overexpress VEGF165b, no NGS evidence supporting the VEGFxxxb isoforms was found in a panel of 24 human cancer cell lines and 12 different types of human normal tissues. To our knowledge, this is the first report to survey VEGFxxxb isoforms using NGS RNA-seq technology. Overall, these studies suggest that closer inspection of VEGFxxxb isoforms may be required before employing VEGF165b as a predictive biomarker for anti-VEGF therapy.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B4.

Citation Format: Wen Zhang, Cristina Abrahams, Ying Huang, Baosheng Li, Asma Parveen, Min Ni, Yi Wei, Yueming Ding, Eric Zhang, Hsin Chieh Lin, Gavin Thurston, Qing Zhang. Evaluation of VEGFxxxb isoforms as a predictive biomarker for anti-VEGF cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B4.
Abstract B50: Regulation of non-small cell lung cancer stem cells by STAT3 and WNT pathways.
Authors: Shao Chunli , DeSevo Chris , Girard Luc , Yenerall Paul , Behrens Carmen , Wistuba Ignacio , Minna John D.
Published: August 2014

Background: Accumulating evidence demonstrates that the malignant phenotype is driven and maintained by cancer stem cells (CSCs), which can self-renew, differentiate, and contribute to drug resistance and tumor metastasis. Our group has identified CSCs in both non-small cell lung cancer cell (NSCLC) lines and patient tumor samples by their enhanced ALDH activity and demonstrated that ALDH+ lung cancer cells are highly tumorigenic and clonogenic. We also found that ALDH1A3 is the major isozyme responsible for the ALDH activity and functionally important for lung CSCs.

Aims and Methods: The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. We performed a siRNA screen of 40 stem cell pathway genes in a panel of NSCLC lines to identify potential targets related to stem cell self-renewal pathways and other regulators. qRT-PCR, western blot and flow cytometry based Aldefluor assay were used to validate the promising hits. To further explore their biological function in lung CSCs we manipulated their expression genetically or pharmacologically followed by colony formation and tumor formation assays.

Results: siRNA screen revealed that knocking down ALDH1A3, Signal Transducers and Activators of Transcription 3 (STAT3), or certain components of WNT and Notch pathway caused a reduction of cell viability in many NSCLC lines. Western blot analysis confirmed that ALDH+ cells contained more activated STAT3 compared to ALDH− lung cancer cells. Inhibition of STAT3 activation by Stattic (a specific STAT3 inhibitor), GSK126 (an EZH2 inhibitor), or siRNA attenuated ALDH1A3 expression, ALDH+ lung cancer cells and tumor cell clonogenicity. In addition, FZD2, DVL3 and LBH were more commonly upregulated in ALDH+ cells across the penal of NSCLC lines. shRNA mediated knockdown of DVL3 in NSCLCs resulted in a significant reduction in ALDH activity, clonogenicity, and tumorigenicity.

Conclusions: Our data suggest that ALDH1A3 expression, STAT3 pathway activation (via EZH2), and an active WNT pathway are all essential for maintenance of lung CSCs. Blocking ALDH1A3, WNT or STAT3 pathways appear to be rationale therapeutic strategies for eliminating NSCLC stem cells.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B50.

Citation Format: Chunli Shao, Chris DeSevo, Luc Girard, Paul Yenerall, Carmen Behrens, Ignacio Wistuba, John D. Minna. Regulation of non-small cell lung cancer stem cells by STAT3 and WNT pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B50.
Abstract B56: Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency.
Authors: Gagou Mary E. , Ganesh Anil , Meuth Mark
Published: August 2014

Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly replication and genome stability in lower and higher eukaryotes. While mouse pif1-/- knockouts show no phenotype and PIF1 is dispensable in non-cancerous human cells, Pif1 knockdowns sensitize many tumor cell lines to replication inhibitors, slow S-phase progression, and trigger apoptosis. Here we investigated the effects of PIF1 depletion on a non-tumor cell line, MRC5-SV2, and HRAS-transformed derivatives. We show that PIF1 depletion reduces the growth and viability of RAS-transformed MRC5-SV2 cells relative to parental and increases sensitivity to the therapeutic DNA replication inhibitor gemcitabine. Since previous work suggested that PIF1 depletion affected S-phase entry and transition in cultured tumor cells, we used single fiber analysis of pulse-labeled DNA to investigate the potential role of PIF1 in these events. Here we show that PIF1 depletion triggers replication stress characterized by slower fork rates and increased fork arrest during normal cycling conditions in non-tumor MRC5-SV2 cells. These effects were enhanced in the RAS-transformed derivative. Impaired fork movement upon PIF1-depletion did not augment DNA double-stranded break formation or trigger DNA damage responses. However PIF1 depletion affected resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and S-phase entry. Taken together, our data establish a novel functional role for human PIF1 in DNA replication that becomes critical for cell growth during oncogenic stress.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B56.

Citation Format: Mary E. Gagou, Anil Ganesh, Mark Meuth. Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B56.
Abstract B65: Antitumor activity of imatinib in GIST is attenuated by FGFR signaling.
Authors: Li Fang , Growney Joseph , Battalagine Linda , Qiu Shumei , Manley Paul , Schlegel Robert , Monahan John
Published: August 2014

Activating mutations in either KIT or PDGFR are present in approximately 90% of gastrointestinal stromal tumors (GISTs). Treatment with the KIT and PDGFR inhibitor imatinib can control advanced disease in about 80% of GIST patients. However, the beneficial effect is not durable with more than 50% of patients progressing after 2 years of imatinib first line therapy. Gene expression data has revealed that FGF2 and FGFR1 are highly expressed in all primary GIST samples examined and at significantly higher levels than the other forty tumor types tested, suggesting that FGFR signaling may limit the antitumor response of imatinib. The effect of inhibiting FGFR signaling in GIST cell lines was tested by combining imatinib with BGJ398, a potent and selective small-molecule inhibitor of FGFR1, FGFR2, and FGFR3. This combination showed increased growth inhibition in imatinib-sensitive GIST cell lines. This effect was observed in the presence or absence of added FGF2. The degree of synergy generated by imatinib and BGJ398 combination correlated with the level of added FGF2. In addition, inhibition of mitogen-activated protein kinase (MAPK) was transient in GIST cells treated with imatinib. The re-activation of extracellular signal-regulated kinase (ERK) in GIST cells was accompanied by increased FGFR signaling, and can be suppressed by BGJ398 treatment. These results suggest that imatinib treatment induces feedback activation of FGFR signaling that can attenuate the antitumor effects of imatinib. This provides a rationale for combining imatinib and FGFR inhibitors, such as BGJ398, in the first line therapy of GIST.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B65.

Citation Format: Fang Li, Joseph Growney, Linda Battalagine, Shumei Qiu, Paul Manley, Robert Schlegel, John Monahan. Antitumor activity of imatinib in GIST is attenuated by FGFR signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B65.
Abstract B66: A novel cre-versible approach for identifying synthetic lethal interactions in the DNA damage response in vivo.
Authors: Morandell Sandra , Reinhardt H.-Christian , Cannell Ian G. , Yaffe Michael B.
Published: August 2014

A fundamental limitation in the ability to devise new therapeutic strategies for manipulating the DNA damage response to treat cancer is the identification of synthetic lethal interactions that function in tumors in vivo. We recently identified the stress activated p38MAPK/MK2 pathway as a critical component of the DNA damage response network in tumor cells in vitro. When treated with genotoxic chemotherapy, MK2 is essential for the survival of tumor cell lines that lack functional p53, but is dispensable in p53-proficient cells. A critical function of MK2 in p53-deficient cells is the post-transcriptional regulation of gene expression, namely the stabilization of the mRNA of cell cycle regulators such as Gadd45α. Intriguingly, MK2 also plays an essential role in the biosynthesis of inflammatory cytokines, including IL-1, IL-6 and TNFα that are key signaling components in the tumor microenvironment. These MK2-regulated cytokines are likely to play important roles in promoting tumor development, as many cancers arise in setting of chronic inflammation, as well as in the creation of chemo-resistant niches where therapeutic resistance develops. The fact that p53 is commonly lost in tumors suggests that the therapeutic targeting of MK2 may be a useful strategy to enhance destruction of tumors by genotoxic chemotherapy.

To explore the role of the p38MAPK/MK2 pathway in the integration of the DNA damage response and cytokine signaling during tumor development and chemotherapeutic response in vivo, we have generated a novel mouse model in which Cre-mediated recombination can be used to create MK2-proficient and deficient tumors within a single animal. This strategy allows direct comparison of MK2 synthetic lethality with genetic mutations that promote tumor development and with the tumor response to DNA damaging chemotherapy in a novel internally controlled setting in vivo. Furthermore, this approach potentially allows tumor autonomous signaling events after DNA damage to be distinguished from signaling events within the stromal microenvironment. Using this approach in a murine autochthonous model of non-small-cell lung cancer (NSCLC) we demonstrate that synthetic lethality between p53 and MK2 can successfully be exploited for enhanced chemo-sensitization of tumors to DNA damaging chemotherapy in vivo.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B66.

Citation Format: Sandra Morandell, H.-Christian Reinhardt, Ian G. Cannell, Michael B. Yaffe. A novel cre-versible approach for identifying synthetic lethal interactions in the DNA damage response in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B66.
Abstract B8: Targeting CXCR4/CXCL12 axis by AMD3100 inhibits ECTC-associated metastasis in hepatocellular carcinoma.
Authors: Yan Jing , Liang Jing , Xu Jing , Zheng Limin
Published: August 2014

Background: Hepatocellular carcinoma (HCC) is characterized by high incidence of blood-borne metastasis. We have recently found that endothelium-coated tumor cell cluster (ECTC), a distinct vascular morphology, is significantly correlated with micrometastatic emboli and might represent a novel metastatic pathway of HCC. The aim of this study is to uncover the mechanisms of ECTC formation and the therapeutic potential of ECTC inhibition.

Methods: ECTC was evaluated by immunohistochemical staining (IHC) of CD34. CXCR4 and CXCL12 expression in tumor vessels were examined by immunofluorescence staining. Human umbilical venous endothelial cells (HUVEC) were treated with CXCL12 and cell branches were assessed. HUVEC migration towards CXCL12 was tested by transwell assay. Orthotopic HCC was induced by intrahepatic injection of Hepa1-6 in male C57/BL6 mice. To block CXCR4/CXCL12 interaction, AMD3100 was injected i.p. every other day one week after the implantation. Tumor volumes and intrahepatic metastases were evaluated at the 3rd week after the injection. Endothelial cell branching and ECTC formation were measured by IHC.

Results: ECTC-associated endothelial cells specifically expressed CXCR4 while endothelial cells from non-ECTC samples did not. Furthermore, CXCL12 (the unique CXCR4 ligand) was more abundant in ECTC vessels in comparison to non-ECTC vessels, suggesting a possible involvement of CXCR4/CXCL12 axis in ECTC formation. In vitro experiments revealed that CXCL12 promoted the branching and chemotactic migration of HUVEC. Mouse orthotopic HCC model showed that the intrahepatic metastasis incidence was inhibited by the specific CXCR4 antagonist AMD3100 (25% vs. 100%), accompanied with smaller tumor volumes. Moreover, endothelial cell branching (34% reduction, P<0.05) and ECTC formation (62% reduction, P<0.01) were significantly reduced by AMD3100 administration.

Conclusion: CXCR4/CXCL12 interaction may promote ECTC formation by facilitating endothelial cell branching and directional migration. Disrupting CXCR4-CXCL12 interaction with AMD3100 reduced tumor metastasis by decreasing ECTC formation. Therefore, remodeling tumor vascular morphology by targeting CXCR4/CXCL12 axis may serve as a novel therapy for HCC treatment.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B8.

Citation Format: Jing Yan, Jing Liang, Jing Xu, Limin Zheng. Targeting CXCR4/CXCL12 axis by AMD3100 inhibits ECTC-associated metastasis in hepatocellular carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B8.
Abstract B84: Can propensity score analyses be alternatives to randomized trials.
Authors: Ishizuka Naoki , Yamanaka Takeharu
Published: August 2014

Introduction: The use of propensity Score (PS) in oncology is increasing. Previous reports have, however, shown that, in the case of a normal or binary response, the inclusion of variables which affect treatment choice but do not correlate with the response into the statistical model for calculating PS needs a caution because the statistical power to detect the association between treatment and response gets decreased. In this study, we evaluated the PS method for survival response in terms of power loss under various scenarios.

Method: We conducted simulation studies to compare statistical power between the Cox models with and without PS. We assumed 4 strata, each having a unique PS (a probability of choosing test drug) as in Table 1. Patients belong to one of the 4 strata and received either test drug or control drug according to his/her PS. Every stratum has an exponential survival distribution with a mean of 1.25 years for the test drug and 1 year for the control drug. Of the 3 scenarios we considered, Scenario 1 is closest to the condition of "clinical equipoise," while Scenario 3 reflects the situation that variables unrelated to survival strongly affect treatment choice.

Result: See Table 2. Statistical power was decreased in any scenario and Scenario 3 was most serious.

Conclusion: The indiscriminate inclusion of variables into PS will lead to a power loss. It would be inevitable in an evaluation for anti-cancer drugs where it is not easy to exclude variables which are unrelated to survival because the choice of drug depends on risk as well as benefit for each patient.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B84.

Citation Format: Naoki Ishizuka, Takeharu Yamanaka. Can propensity score analyses be alternatives to randomized trials. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B84.
Abstract B93: Preclinical evaluation of BMN 673 in combination with temozolomide (TMZ) in various tumor types including small cell lung cancer (SCLC) cells.
Authors: Feng Ying , Post Leonard , Wang Bing , Shen Yuqiao
Published: August 2014

BMN 673 is a potent, specific PARP1/2 inhibitor that shows promising clinical activity as a monotherapy agent against cancers that harbor BRCA1 or BRCA2 mutations (de Bono et al. 2013). A large body of evidence also indicates PARP inhibitors may be combined with cytotoxic chemotherapy agents (such as TMZ, topoisomerase inhibitors, platinum drugs, etc.) for greater antitumor activity.

In this study, BMN 673 was evaluated in combination with TMZ against solid tumor cell lines representing breast cancer, small cell lung cancer (SCLC), and other cancer types. The breast cancer cell lines include those that carry BRCA1/2 mutations (MX-1, MDA-MB-436) and cell lines that are wild-type for BRCA1/2 (MDA-MB-231, MDA-MB-468). None of the SCLC lines have been reported to have BRCA1/2 mutations. Cells were treated for 5-7 days with single agent TMZ or BMN 673, or in combination. Cell viability was assessed by CellTiter-Glo. In all the cell lines we have tested, regardless of their BRCA1/2 mutation status, BMN 673 markedly sensitized tumor cell cells to TMZ cytotoxicity. Compared to cells that are relatively resistant to BMN 673, cells that are sensitive to single-agent BMN 673 are more likely to show complete growth inhibition when treated with BMN 673/TMZ combination. Similarly, a fixed concentration of TMZ (10 μM) reduced IC50 of BMN 673 in the tested cell lines by as much as 1,000 fold. This dose of TMZ by itself had no effect in cultured cells under the assay condition. These observations support a treatment scheme in which small doses of TMZ may be combined with full dose BMN 673 for potent antitumor activity. Encouraged by the in vitro assay results, we are currently evaluating BMN 673 in combination with TMZ in vivo using various subcutaneous xenograft mouse models. Data will be presented at the 2013 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B93.

Citation Format: Ying Feng, Leonard Post, Bing Wang, Yuqiao Shen. Preclinical evaluation of BMN 673 in combination with temozolomide (TMZ) in various tumor types including small cell lung cancer (SCLC) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B93.
Abstract B94: Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor.
Authors: Finlay M. Raymond V. , Anderton Mark , Ashton Susan , Ballard Peter G. , Bradbury Rob H. , Butterworth Sam , Chuaqui Claudio E. , Colclough Nicola , Cross Darren A. E. , McFarland Heather L. , Mellor Martine J. , Ward Richard A. , Waring Mike J.
Published: August 2014

Small molecule inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations in the kinase domain such as T790M is emerging as a growing clinical issue. This presentation will describe the discovery of AZD9291, an orally bioavailable, irreversible EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 <0.025 μM) and activating mutations (PC9, cell phosphorylation IC50<0.025 μM) that also spares inhibition of the wild type form of the receptor (A431, cell phosphorylation IC50>0.5 μM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be discussed for the first time including the medicinal chemistry program that led to the identification of AZD9291, details of significant in vivo oral activity in pre-clinical xenograft models (including tumor regression in the L858R/T790M double mutant setting at a dose of 5 mpk) and the first disclosure of the candidate drug structure. The pre-clinical findings from this work strongly supported selection of AZD9291 as a clinical candidate, and first dose in man was achieved with AZD9291 in March 2013.

L858R/T790M Double Mutant (resistance) cell IC50 (μM)EGFRm+ Single Mutant (activating) cell IC50 (μM)EGFR Wild Type cell IC50 (μM)Aqueous solubility, (salt) (pH=6.8, μg/mL)L858R/T790M Double Mutant efficacy (% TGI at 5mpk PO QD for 14 days)AZD9291<0.025<0.025>0.5>490119

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B94.

Citation Format: M. Raymond V. Finlay, Mark Anderton, Susan Ashton, Peter G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A. E. Cross, Heather L. McFarland, Martine J. Mellor, Richard A. Ward, Mike J. Waring. Discovery of and first disclosure of the clinical candidate AZD9291, a potent and selective third-generation EGFR inhibitor of both activating and T790M resistant mutations that spares the wild type form of the receptor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B94.
Abstract B99: Discovery of 2,4-diarylaminopyrimidine SOMCL-12-81 as a potent inhibitor targeting both wild and mutant ALK kinases.
Authors: Song Zilan , Ai Jing , Liu Zhiqing , Peng Xia , Chen Yi , Ding Jian , Geng Meiyu , Zhang Ao
Published: August 2014

Anaplastic lymphoma kinase (ALK) is an orphan receptor tyrosine kinase (RTK), structurally belonging to the insulin receptor family. The identification of ALK fusion genes NPM-ALK and EML4-ALK in ALCL and NSCLC respectively, and clinical outcomes resulting from aberrant ALK signalling in different human cancers has confirmed the emergence of ALK as a novel and promising ‘personalized’ anticancer drug target. The development of small molecule ALK inhibitors has been accelerated and validated by the recent approval of crizotinib. Unfortunately, similar to other RTK inhibitors, resistance to crizotinib has been reported within a year of starting therapy. Different from the acquired resistance to EGFR inhibitors, the resistance to crizotinib was much more complex and driven by more than twenty secondary mutations, including the gate-keeper L1196M, and the C1156Y near the α-C-helix. In the course of our drug discovery program toward the discovery of novel ALK inhibitors, we took advantage of the 2,4-diarylaminopyrimidine (DAAP) scaffold that has long been recognized as a classical kinase inhibitor motif (TAE684) by introducing an amino acid side chain to the solvent interaction region to bring in additional H-bondings. Therefore, a series of new DAAP analogues (DAAPalogues) were developed and one compound SOMCL-12-81 was identified with high potency against both wild type ALK and L1196M mutations with IC50 values of 2.7 nM and 15.3 nM, respectively. In cellular assay, SOMCL-12-81 displayed an IC50 value of 3.0 nM against the proliferation of EML4-ALK dependent Karpass-299 cells. It was relatively selective (>10-fold) against a panel of in-house 20 RTKs. Dose-dependent tumor growth inhibition was observed in the BF3 rat xenograft model carrying the EML4-ALK fusion gene with daily administration of SOMCL-12-81. The high potency of SOMCL-12-81 both in vitro and in vivo allows it for further investigation.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B99.

Citation Format: Zilan Song, Jing Ai, Zhiqing Liu, Xia Peng, Yi Chen, Jian Ding, Meiyu Geng, Ao Zhang. Discovery of 2,4-diarylaminopyrimidine SOMCL-12-81 as a potent inhibitor targeting both wild and mutant ALK kinases. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B99.
Abstract C102: Identify novel pathways conferring PARP inhibitor resistance in BRCA-associated breast and ovarian cancer.
Authors: He Lei , Chen Li , Ramakrishnan Saranya , Torres-Lockhart Kristine , Ellisen Leif
Published: August 2014

Inhibitors of poly(ADP-ribose) polymerase (PARP) enzymes hold great promise in treating BRCA1/2 associated breast and ovarian cancers, which have profound homologous recombination (HR) defect. However, not all BRCA1/2 patients respond to PARP inhibitor treatment, and the majority of the responders eventually relapsed. Little is known about the resistance mechanisms that are clinical relevant.

We recently developed a highly versatile insertional mutagenesis system, using transposons to insert a CMV promoter randomly into the genome and thereby activate nearby genes. We constructed genome-wide insertional libraries using both human and mouse BRCA1-deficient cancer cell lines. When these mutated cells were treated with the PARP inhibitor, resistant colonies formed at extremely low frequency. Resistant colonies were re-grown and maintained in drug-free medium for extended period of time and robust resistance was retained. Insertion sites for these colonies were then determined, many of which are near genes whose alteration in expression can up-regulate activities of MEK/ERK pathway or PI3K/AKT pathway. Next we hope to investigate the resistance mechanism. We hypothesize that resistance is mediated via decreased DNA damage, improved DNA repair, or increase in general survival pathway after PARP inhibitor treatment. We have access to tumor specimens from a recently-completed clinical trial using ABT-888 in combination with temozolomide in a BRCA1/2 carrier-enriched population. The expression level and mutational status of these genes will be surveyed and correlated with clinical response. Finally based on the insight gained from aforementioned mechanism study, we hope to find inhibitors that specifically target the resistance mechanism; by combining this inhibitor with PARP inhibitor, we could develop effective therapy to treat the subset of patients resistant to PARP inhibitor.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C102.

Citation Format: Lei He, Li Chen, Saranya Ramakrishnan, Kristine Torres-Lockhart, Leif Ellisen. Identify novel pathways conferring PARP inhibitor resistance in BRCA-associated breast and ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C102.