bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3540: Novel targeted combinational therapies for melanoma
Authors: Suriano Robert , Tuli Neha Y. , Geliebter Jan , Tiwari Raj K. , Wallack Marc
Published: August 2015

The BRAFV600E mutation is frequently observed in over 60% of melanomas. Development of the small molecule inhibitor PLX4032, which specifically targets the mutated BRAF protein, offers a novel therapeutic approach by effectively blocking the activation of key signaling molecules of the mitogen-activated protein kinase (MAPK) pathway, such as Mek and Erk, effectively leading to arrest in cell proliferation. Provided that MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesized that the inhibition of BRAFV600E can modulate the expression of MAAs. Since MAAs are essential in the development of a melanoma vaccine, due to their increased immunogenicity, altering their expression can be a useful immunotherapeutic approach to targeting melanoma. Using patient derived melanoma cell lines, which express various MAAs; our laboratory has developed a vaccinia virus based immunotherapeutic vaccine. These melanoma cell lines have been characterized by their status of BRAFV600E mutation and found to be either homozygous or heterozygous for the mutation. Inhibition of two key MAPK pathway molecules, namely Mek and Erk, denotes that the melanoma cell lines are responsive to PLX4032. Phenotypically, the cells’ invasive and migratory properties were also modulated upon treatment with PLX4032. Moreover, we observed that the expression of various MAAs was enhanced upon treatment with PLX4032. An amplification of gp100, MART-1 and tyrosinase expression on the RNA level as well as on the protein level was observed. Lastly, expression of MMAs was conserved in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. Based on our observations in that PLX4032 modulates MMA expression, further studies are warranted to determine the clinical effectiveness of combinational immunologic therapy using BRAFV600E inhibitors with our vaccinia virus melanoma cell line derived preparation.

Citation Format: Robert Suriano, Neha Y. Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Novel targeted combinational therapies for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3540. doi:10.1158/1538-7445.AM2015-3540
Abstract 3548: Oncolytic adenovirus Ad5dlE1b replication efficiency is dependent on p53 mutation type in cancer cells
Authors: Abdalla Moemen , Abbas Basma M. , Haj-Ahmad Yousef
Published: August 2015

Adenoviruses are the most popular viral DNA vectors in the development of gene therapies and oncolytic therapy. Oncolytic adenoviruses are genetically modified to provide a means of tumor-selectivity by conditionally replicating in tumor cells and substantially killing them via their viral lytic cycle. The p53 molecule is a tumor suppressor protein that regulates the genomic integrity of the cell by controlling apoptosis and cell cycle events. Upon viral infection, normal cell (p53 intact cell) responds through the activation of apoptosis by the p53 dependent pathway. The adenovirus E1B 55kDa protein inactivates p53 and prevents apoptosis leading to progressive viral lytic cycle. Although demonstrating a moderate successes rate in some cancers, Ad deleted E1B 55kDa has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1B deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this relationship has not been investigated deeply on the molecular level. However, this relationship has not been investigated deeply on the molecular level. Therefore, the objective of this study is to understand the correlation between p53 status, levels, and functional activity, and oncolytic Ad5dl E1B 55kDa replication efficiency at the molecular level. We evaluated the effect of p53's functional status on the replication efficiency of oncolytic Ad5dl E1B 55kDa. Multiple mutant p53 were engineered and viral growth was evaluated with each mutant form in H1299 cells. P53-R175H mutant successfully rescued viral growth and its transcriptome analysis showed partial loss of wild type activity as well as gain of new function by allowing the virus to grow and to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific cellular genes in the p53 pathway, cell cycle and apoptosis, to promote its production leading to efficient oncolytic effect. These results confirmed that onclytic Ad5dl E1B 55kDa sensitivity is mutation type-specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined and also more screening for the effect of other mutations should be done for successful rescuing and efficient antitumor effect.

Citation Format: Moemen Abdalla, Basma M. Abbas, Yousef Haj-Ahmad. Oncolytic adenovirus Ad5dlE1b replication efficiency is dependent on p53 mutation type in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2015-3548
Abstract 3551: A microRNA-1280/JAG2 network comprises a novel biological target in high-risk medulloblastoma
Authors: Wang Fengfei , Remke Marc , Bhat Kruttika , Wong Eric , Zhou Shuang , Ramaswamy Vijay , Dubuc Adrian , Fonkem Ekokobe , Salem Saeed , Zhang Hongbing , Hsieh Tze-chen , O'Rourke Stephen , Wu Lizi , Li David , Hawkins Cynthia , Kohane Isaac , Wu Joseph , Wu Min , Taylor Michael , Wu Erxi
Published: August 2015

Over-expression of PDGF receptors (PDGFRs) has been previously implicated in high-risk medulloblastoma (MB) pathogenesis. However, the exact biological functions of PDGFRα and PDGFRβ signaling in MB biology remain poorly understood. Here, we report the subgroup specific expression of PDGFRα and PDGFRβ and their associated biological pathways in MB tumors. c-MYC, a downstream target of PDGFRβ but not PDGFRα, is involved in PDGFRβ signaling associated with cell proliferation, cell death, and invasion. Concurrent inhibition of PDGFRβ and c-MYC blocks MB cell proliferation and migration synergistically. Integrated analysis of miRNA and miRNA targets regulated by both PDGFRβ and c-MYC reveals that increased expression of JAG2, a target of miR-1280, is associated with high metastatic dissemination at diagnosis and a poor outcome in MB patients. Our study may resolve the controversy on the role of PDGFRs in MB and unveils JAG2 as a key downstream effector of a PDGFRβ-driven signaling cascade and a potential therapeutic target.

Citation Format: Fengfei Wang, Marc Remke, Kruttika Bhat, Eric Wong, Shuang Zhou, Vijay Ramaswamy, Adrian Dubuc, Ekokobe Fonkem, Saeed Salem, Hongbing Zhang, Tze-chen Hsieh, Stephen O'Rourke, Lizi Wu, David Li, Cynthia Hawkins, Isaac Kohane, Joseph Wu, Min Wu, Michael Taylor, Erxi Wu. A microRNA-1280/JAG2 network comprises a novel biological target in high-risk medulloblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3551. doi:10.1158/1538-7445.AM2015-3551
Abstract 3553: xCT is a new cancer stem cell immunotherapeutic target for breast cancer
Authors: Macagno Marco , Lanzardo Stefania , Conti Laura , Ruiu Roberto , Calogero Raffaele A. , Cavallo Federica
Published: August 2015

The several unsuccessful treatments in metastatic cancers might miss cancer stem cells (CSC), a sub-population of cells with a critical role in cancer. By contrast, a vaccine-elicited immune response against CSC might be particularly effective. The identification of oncoantigens (OA) expressed by CSC may provide new targets for effective anticancer vaccines.

To this purpose, the transcription profile of the ErbB2+ TUBO mammary cancer cell line was compared with that of TUBO cells grown under specific conditions to generate CSC-enriched mammospheres.

Integrating the data obtained with meta-analyses of seven independent human breast tumor data sets we identified new CSC mammary OA that were validated both in vitro and in vivo.

Among the identified OA, we focused on xCT, a channel that supports glutathione synthesis regulating the intracellular redox balance. We observed that xCT expression increases in TUBO and 4T1 derived mammospheres; its silencing, obtained with specific siRNA, or incubating mammospheres with its inhibitor sulfasalazine (2), significantly reduces TUBO cells ability to generate mammospheres. In vivo xCT inhibition impaired CSC ability to generate lung metastases derived both from an intravenous injection of TUBO-derived mammospheres and from subcutaneous 4T1 tumors; furthermore, this effect was improved when combined with the cytotoxic drug doxorubicin. Moreover, the anti xCT vaccination of mice bearing mammosphere-derived tumors both from TUBO and 4T1 cells reduced the speed of tumor growth.

In conclusion, this study provides a genomic characterization of mammary CSC and identifies fresh target for new and potentially effective anticancer vaccines, thus providing a new tool for the design of combined therapeutic approaches that efficaciously target both CSC and more differentiated cells in breast cancers, leading to both cancer treatment and prevention of metastases and recurrence.

BIBLIOGRAPHY

1. Nagano O et al., Oncogene 2013.

2. Chen RS et al., Oncogene 2009.

Citation Format: Marco Macagno, Stefania Lanzardo, Laura Conti, Roberto Ruiu, Raffaele A. Calogero, Federica Cavallo. xCT is a new cancer stem cell immunotherapeutic target for breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3553. doi:10.1158/1538-7445.AM2015-3553
Abstract 3555: Akt kinase-interacting protein1 as a potential therapeutics target in CREB1 signaling in malignant pleural mesothelioma
Authors: Yamada Tadaaki , Amann Joseph M. , Shilo Konstantin , Fujita Naoya , Yano Seiji , Carbone David P.
Published: August 2015

Background:

Diffuse malignant mesothelioma (DMM) is an aggressive tumor arising from the mesothelial cells of serosal membranes. As conventional treatment regimens are commonly unsuccessful in changing its outcome, novel effective therapeutic strategies based on the molecular mechanisms of disease are needed to improve the prognosis of DMM. Akt kinase-interacting protein1 (Aki1), known as a scaffold protein of PI3K/PDK1/Akt that determines receptor signal selectivity for EGFR, has been suggested as a therapeutic target in lung cancer. The aim of this study was to evaluate Aki1 as a potential therapeutic target for treatment of DMM.

Methods:

DMM cell lines (211H, H290, H2052, H2373, H2452, Y-meso8A, and Y-meso14) and immortalized human mesothelial cells (Met-5A) were employed for evaluation of effects of treatment with Aki1 or CREB1 siRNAs on cell viability by MTT assay, cell cycle by FACS analysis, cell signaling by WB, and CREB transcriptional activity. The efficacy of Aki1 siRNA against growth of 211H cells was investigated in an orthotropic implantation model using SCID mice. We further examined Aki1 and p-CREB1 expression in DMM tumors from 35 patients utilizing tissue microarray and resection specimens.

Results:

Cell based assay showed that silencing of Aki1 inhibited cell viability and caused cell arrest of mesothelioma cell lines but not immortalized mesothelial cells (Met-5A). Importantly, we identified that the efficacy of Aki1 is regulated by CREB1 signaling which is involved in cell viability, cell cycle, and transcriptional activity. Application of Aki1 siRNA significantly inhibited growth of 211H cell line within pleural cavity compared with that of control siRNA in an orthotropic implantation model (p = 0.006). Aki1 and phosphorylated CREB1 were frequently expressed in DMM patients (65/68 and 30/35 cases, respectively). Furthermore, the expression of Aki1 correlated with phosphorylation of CREB1 (Spearman rank correlations = 0.521; p = 0.002).

Conclusions:

Our data suggest that Aki1/CREB axis may regulate the survival signaling, and may therefore be a potent target for DMM patients.

Citation Format: Tadaaki Yamada, Joseph M. Amann, Konstantin Shilo, Naoya Fujita, Seiji Yano, David P. Carbone. Akt kinase-interacting protein1 as a potential therapeutics target in CREB1 signaling in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3555. doi:10.1158/1538-7445.AM2015-3555
Abstract 3558: uPAR mediated endothelial targeting by oncolytic measles virus
Authors: Toro Bejarano Marcela M. , Jing Yuqi , Kovacs Krisztina , Merchan Jaime
Published: August 2015

Background: The urokinase receptor (uPAR) is over-expressed in tumor cells as well as in tumor endothelium and plays a critical role in tumor neovascularization. We have recently rescued an oncolytic measles virus fully retargeted against human uPAR (MV-h-uPA). The aim of this study was to further characterize the endothelial targeting capabilities of MV-h-uPA over MV-GFP.

Methods: In vitro cytotoxicity assays were performed in unstimulated and stimulated HUVEC, and the effects of both viruses were analyzed. The effects of MV-GFP and MV-h-uPA on actively forming vs. established capillaries was investigated using the matrigel tube formation assay. Endothelial to tumor transfer of MV-uPA or MV-GFP was investigated in breast, colon cnd kidney cancer cell lines expressing RFP by overlaying infected HUVEC on tumor cell monolayers. The mechanisms/kinetics of viral transfer from HUVEC to tumor cells was characterized. Microvessel density of tumors treated with MV-h-uPA was quantitated by immunohistochemistry using an antibody against CD31.

Results: MV-h-uPA and MV-GFP induced cytotoxicity at 24, 48 and 72 hours in stimulated HUVECs. MV-h-uPA replicated more efficiently in growth factor stimulated, compared to resting HUVECs. Interestingly, MV-h-uPA, but not MV-GFP successfully infected actively forming (angiogenic) HUVEC capillaries, as assessed by mean fluorescence quantification of GFP expression of infected capillaries. On the other hand, the non-targeted MV-GFP predominantly infected already established HUVEC capillaries (infected 24 hours after tubes were formed), an effect not observed with MV-h-uPA. Importantly, MV-h-uPA did not induce vascular disrupting effects in HUVEC capillaries. MV-h-uPA was successfully transferred from infected endothelial cells to tumor cells. This effect was mediated by heterofusion, as chemical inhibition of virus induced fusion at different time points during co-culture completely blocked EC to TC transfer. Heterofusion occurred as early as 4 hours after endothelial cell overlay into tumor cell cultures. EC to TC viral transfer resulted in significant tumor cell cytotoxicity.

Conclusion: The above results demonstrate the improved ability of uPAR retargeted oncolytic measles virus to infect, replicate and successfully infect angiogenic endothelial cells, as well as successful EC to TC transfer in vitro, resulting in tumor cell cytotoxicity. Vascular targeting by oncolytic MVs may result in improved tumor cell delivery in vivo, and enhanced antitumor effects.

Citation Format: Marcela M. Toro Bejarano, Yuqi Jing, Krisztina Kovacs, Jaime Merchan. uPAR mediated endothelial targeting by oncolytic measles virus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2015-3558
Abstract 3562: Protein palmitoylation in breast cancer: DHHC5 palmitoyltransferase as a potential therapeutic target
Authors: Tian Hui , Qi Xiaoxia , Huffman Kenneth , Minna John , Hofmann Sandra L.
Published: August 2015

Protein S-palmitoylation is a widespread and dynamic post-translational modification that regulates protein-membrane interactions, protein-protein interactions, and protein stability. S-palmitoylation is catalyzed by members of a family of 23 palmitoyl acyl transferases, termed the DHHC family due to the presence of a common catalytic motif. The role of protein palmitoylation in cancer is largely unexplored.

In our previous study, we have shown that DHHC5 palmitoyltransferase supports the growth of non-small cell lung cancer. To further investigate the role of DHHC5 in cancer, we expanded our study to include breast cancer cell lines. Stable knockdowns of DHHC5 were undertaken in 8 breast cancer cell lines (including 4 “triple-negative” breast cancer lines) as well as in two normal human mammary epithelial cell lines (HMECs) using lentiviral shRNAs. The protein levels of DHHC5 were analyzed by immunoblotting, which all showed efficient knockdown. Cell proliferation and anchorage-dependent/ independent colony formation assays indicated that DHHC5 knockdown led to cell growth arrest and decreased cell clonogenicity in most of the tested breast cancer cell lines in vitro, but not in the HMECs. BT-549, SkBr3 and T-47D cell lines were particularly dependent on DHHC5 expression, and rescue experiments involving ectopic plasmid-driven DHHC5 and its catalytic mutant DHHS expression are in progress. In vivo tumor formation assays will be carried out by injecting MCF-7 and T-47D control cells and DHHC5 stable knockdown cells as well as rescued cells subcutaneously into NOD/SCID mice. Tumor xenograft formation will be measured.

As DHHC5 function is needed for optimal breast cancer cell growth, it will be important to identify physiologically relevant substrates in order to develop inhibitors of DHHC5 function. Future plans include microarray and protein kinase array analysis to identify possible signaling pathways in which DHHC5 may be involved, as well as palmitoyl-protein profiling of control and knockdown DHHC5 cell lines by mass spectrometry.

Citation Format: Hui Tian, Xiaoxia Qi, Kenneth Huffman, John Minna, Sandra L. Hofmann. Protein palmitoylation in breast cancer: DHHC5 palmitoyltransferase as a potential therapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2015-3562
Abstract 3588: Emergence of multiple EGFR extracellular mutations during cetuximab treatment in colorectal cancer
Authors: Lazzari Luca , Arena Sabrina , Bellosillo Beatriz , Siravegna Giulia , Martínez Alejandro , Cañadas Israel , Ferruz Noelia , Russo Mariangela , Misale Sandra , González Iria , Iglesias Mar , Gavilan Elena , Corti Giorgio , Hobor Sebastijan , Crisafulli Giovanni , Salido Marta , Sánchez Juan , Dalmases Alba , Bellmunt Joaquim , De Fabritiis Gianni , Rovira Ana , Di Nicolantonio Federica , Albanell Joan , Bardelli Alberto , Montagut Clara
Published: August 2015

Colorectal cancer (CRC) patients who respond to the anti-EGFR antibody cetuximab often develop resistance within several months of initiating therapy. To design new lines of treatment the molecular landscape of resistant tumors must be ascertained. We investigated the role of mutations in the EGFR signalling axis on the acquisition of resistance to cetuximab in patients and cellular models.

Mutational profiling was performed on both biopsies collected from 37 CRC patients who became refractory to cetuximab and on a collection of cetuximab-resistant derivatives obtained from CRC cells sensitive to EGFR blockade.

The genetic profile of tumor specimens obtained after cetuximab treatment revealed the emergence of a complex pattern of mutations in EGFR, KRAS, NRAS, BRAF and PIK3CA genes, including two novel EGFR ectodomain mutations. Mutational profiling of cetuximab resistant cells recapitulated the molecular landscape observed in clinical samples and revealed three additional EGFR ectodomain alleles.

Structural modelling showed that these mutations are located in the cetuximab-binding region, except for one mutant. Functionally, EGFR ectodomain mutations prevent binding to cetuximab but a subset is permissive for interaction with panitumumab.

In conclusion, we reported that colorectal tumors evade EGFR blockade by constitutive activation of downstream signalling effectors and through mutations affecting receptor-antibody binding. Both mechanisms of resistance may occur concomitantly. Our data have implications for designing additional lines of therapy for CRC patients who relapse upon treatment with anti-EGFR antibodies.

Citation Format: Luca Lazzari, Sabrina Arena, Beatriz Bellosillo, Giulia Siravegna, Alejandro Martínez, Israel Cañadas, Noelia Ferruz, Mariangela Russo, Sandra Misale, Iria González, Mar Iglesias, Elena Gavilan, Giorgio Corti, Sebastijan Hobor, Giovanni Crisafulli, Marta Salido, Juan Sánchez, Alba Dalmases, Joaquim Bellmunt, Gianni De Fabritiis, Ana Rovira, Federica Di Nicolantonio, Joan Albanell, Alberto Bardelli, Clara Montagut. Emergence of multiple EGFR extracellular mutations during cetuximab treatment in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3588. doi:10.1158/1538-7445.AM2015-3588
Abstract 359: STAT3-axis activated snail expression and promoted drug resistance in pediatric brain tumor
Authors: Chi-Chang Tsai
Published: August 2015

Atypical teratoid/rhabdoid tumor (ATRT) is a malignant pediatric brain tumor with great recurrence after complete surgery and chemotherapy. Here, we demonstrate that cisplatin treatment select not only for resistance but also for a more oncogenic phenotype characterized by high self-renewal and invasive capabilities. These phenomena are likely due to STAT3 upregulation which occurred simultaneously with higher expression of Snail, an activator of epithelial-mesenchymal transition (EMT), in ATRT-CisR cells. STAT3 knockdown effectively suppressed Snail expression and blocked motility and invasion in ATRT-CisR cells, while overexpressing Snail reversed these effects. Chromatin immunoprecipitation assay indicated that STAT3 directly bound to Snail promoter. Moreover, STAT3 knockdown effectively suppressed cancer stem-like properties, synergistically enhanced the chemotherapeutic effect, and significantly improved survival rate in ATRT-CisR-transplanted immunocompromised mice. Finally, immunohistochemistrical analysis showed that STAT3 and Snail were coexpressed at high levels in recurrent ATRT tissues. Thus, the STAT3/Snail pathway plays an important role in oncogenic resistance, rendering cells not only drug-resistant but also increasingly invasion and recurrence. Therefore, the STAT3/Snail could be a target for ATRT treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Tsai Chi-Chang. STAT3-axis activated snail expression and promoted drug resistance in pediatric brain tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 359. doi:10.1158/1538-7445.AM2015-359
Abstract 3590: Resistance mechanisms to ALK inhibitors
Authors: Katayama Ryohei , Yanagitani Noriko , Koike Sumie , Sakashita Takuya , Kitazono Satoru , Nishio Makoto , Okuno Yasushi , Engelman Jeffrey A. , Shaw Alice T. , Fujita Naoya
Published: August 2015

Purpose: ALK-rearranged non-small cell lung cancer (NSCLC) was first reported in 2007. Approximately 3-5% of NSCLCs harbor an ALK gene rearrangement. The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with advanced ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising antitumor activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to one next-generation ALK-TKI - alectinib - and potential strategies to overcome this resistance.

Experimental Procedure: We established a cell line model of alectinib resistance, and analyzed resistant tumor specimens from patients who had relapsed on alectinib. Cell lines were also established under an IRB-approved protocol when there was sufficient fresh tumor tissue. We established Ba/F3 cells expressing EML4-ALK and performed ENU mutagenesis to compare potential crizotinib or alectinib-resistance mutations. In addition, we developed Ba/F3 models harboring ALK resistance mutations and evaluated the potency of multiple next-generation ALK-TKIs including 3rd generation ALK inhibitor in these models and in vivo. To elucidate structure-activity-relationships of ALK resistance mutations, we performed computational thermodynamic simulation with MP-CAFEE.

Results: We identified multiple resistance mutations, including ALK I1171N, I1171S, and V1180L, from the ENU mutagenesis screen and the cell line model. In addition we found secondary mutations at the I1171 residue from the Japanese patients who developed resistance to alectinib or crizotinib. Both ALK mutations (V1180L and I1171 mutations) conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Based on thermodynamics simulation, each resistance mutation is predicted to lead to distinct structural alterations that decrease the binding affinity of ALK-TKIs for ALK.

Conclusions: We have identified multiple alectinib-resistance mutations from the cell line model, patient derived cell lines, and tumor tissues, and ENU mutagenesis. ALK secondary mutations arising after alectinib exposure are sensitive to other next generation ALK-TKIs. These findings suggest a potential role for sequential therapy with multiple next-generation ALK-TKIs in patients with advanced, ALK-rearranged cancers.

Citation Format: Ryohei Katayama, Noriko Yanagitani, Sumie Koike, Takuya Sakashita, Satoru Kitazono, Makoto Nishio, Yasushi Okuno, Jeffrey A. Engelman, Alice T. Shaw, Naoya Fujita. Resistance mechanisms to ALK inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3590. doi:10.1158/1538-7445.AM2015-3590
Abstract 3604: Targeting the PIM kinases in combination with BTK inhibition is synergistic in preclinical models of B-cell malignancies
Authors: Bearss Jeremiah J. , Bahr Brigham L. , Soh Katie K. , Peterson Peter W. , Whatcott Clifford J. , Siddiqui-Jain Adam , Bearss David J. , Warner Steven L.
Published: August 2015

BTK inhibitors (e.g. ibrutinib) have significantly impacted the treatment of B-cell malignancies in a positive way. Single agent response rates with ibrutinib are 65% or higher in B-cell lymphomas and chronic lymphocytic leukaemia with the majority of patients enjoying a prolonged duration of response. Continued clinical development is needed, however, as most patients achieve only a partial response from their treatment and ultimately patients become refractory to ibrutinib leading to relapse and disease progression. Targeted combinations with ibrutinib could potentially increase the number of patients undergoing complete remission and combat emergent resistant mechanisms. The PIM family (1, 2, and 3) are serine/threonine kinases that have proven to be oncogenic in-part due to their ability to suppress c-Myc induced apoptosis. The PIM kinases have emerged as important regulators of drug resistance in multiple cancer types. Tolero Pharmaceutical's second generation PIM Kinase inhibitor, TP-3654 has exhibited favorable activity in preclinical models of prostate cancer, AML, and lymphoma. Due to the signaling crosstalk between BTK and PIM through the STAT transcription factors, we hypothesized that synergies may arise through the simultaneous targeting of both kinases. Here, we report a significant increase in drug activity when a BTK inhibitor (ibrutinib) was combined with TP-3654 in various lymphoma cell lines. In Granta-519 cells, the IC50 of ibrutinib decreased 3.5-fold, from 0.7 μM to 0.2 μM, when cultured in combination with a subtoxic concentration of TP-3654 (300 nM). Similarly, the IC50 of TP-3654 decreased 6-fold, from 2.4 μM to 0.4 μM, when cells were cultured in combination with a subtoxic concentration of ibrutinib (100 nM). BTK is known to attenuate the activity of the transcription factor STAT3, a major regulator of PIM kinase levels in cells. Due to this, mechanistic studies focused on analyzing the STAT3 pathway are ongoing to determine the downstream effects of using ibrutinib and TP-3654 in combination. Several lymphoma xenograft studies are also ongoing to further explore this combination in vivo. These results provide a strong rationale that inhibitors of PIM and BTK could be used in combination for the treatment of B-cell malignancies and other B-cell mediated diseases.

Citation Format: Jeremiah J. Bearss, Brigham L. Bahr, Katie K. Soh, Peter W. Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. Targeting the PIM kinases in combination with BTK inhibition is synergistic in preclinical models of B-cell malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3604. doi:10.1158/1538-7445.AM2015-3604
Abstract 361: Opposite regulation of stromelysin expression in prostate tumor and its microenvironment
Authors: Sung Shian-Ying , Hsieh Chia-Ling
Published: August 2015

Crosstalk between cancer and milieu cells plays a central role during cancer progression, which includes determine the faith of increasing metalloproteinase secretion by cancer epithelial cells. Here, we showed prostate tumor and stromal fibroblasts expressed opposite levels of Stromelysin-1 and -2 in both 3D co-culture and paired patient samples. Macroarray and quantitative RT-PCR studies demonstrated significantly decreased of Stromelysin-1 expression in co-cultured HS27AC4-2 and 4 paired tumor associated fibroblasts that were collected in previous report. We noticed the Stromelysin-1 expression level was decreased in stromal region of prostate cancer. By contrast, Stromelysin-1 level was increased in malignant cancer epithelial cells in both cells and tissue staining. Canonical pathway study revealed of differentially gene expression between HS27AC4-2 and HS27ARWV including the majority of pathway enrichment in TGF-β and hydrogen peroxide as central mediators. Induction of Stromelysin-1 and -2 by TGF-β and/or hydrogen peroxide were preliminary validated in the conditioned medium of prostate cancer cells, PC3 and DU145, and paired patient stromal fibroblasts that confirmed the opposite regulation of Stromelysin-1 and -2 expression by hydrogen peroxide but not TGF-β. Our results demonstrate the differential regulation of Stromelysin-1 and -2 expression in prostate cancer cells and stromal fibroblasts through ROS, such as hydrogen peroxide, as mediator and shed the light on the potentially useful as indication for the differential regulation between cancer and stromal cells.

Citation Format: Shian-Ying Sung, Chia-Ling Hsieh. Opposite regulation of stromelysin expression in prostate tumor and its microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2015-361
Abstract 3613: P300 inhibition enhances cytotoxic effect of Gemcitabine through E2F1 activation in pancreatic cancer
Authors: Ono Hiroaki , Basson Marc D. , Ito Hiromichi
Published: August 2015

Background:

Transcriptional cofactor, P300 has been reported to regulate cell cycle on G1/S transition. Previously, we shown that prolonged S-phase is associated with increased apoptosis induced by Gemcitabine (GEM) in pancreatic cancer cells. Thus, we hypothesized that targeting P300 enhances the cytotoxic effect of GEM on pancreatic cancer.

Methods:

We studied 2 human pancreatic cancer cell lines, Panc1 and MIAPaCa2. P300 was gene-silenced using siRNA, and P300 histone acetyltransferase (HAT) activity was inhibited by specific inhibitor C646. Cell viability was measured by WST-8 assay and GEM-induced apoptosis was measured using Caspase3 and PARP by Western blotting, Tunel assay by immunofluorescence staining, and Annexin V-PI staining by flow cytometry. CDK4/6 inhibitor Palbociclib was used to block S-phase entry.

Results:

Robust P300 expression was observed in pancreatic cancer cell lines and human pancreatic adenocarcinoma tissues. When P300 expression was depleted by specific P300 siRNA, cell viability after GEM treatment was decreased significantly compared with the control pancreatic cancer cells (43% decrease in MIAPaCa2 and 39% decrease in Panc1, respectively, p<0.05). Increased GEM-induced apoptosis was confirmed by increased cleaved forms of Caspase3 and PARP, increased tunel positive cells, and increased Annexin positive cells in P300-depleted cells compared to the control cells. When S phase entry was blocked by Palbociclib, the effect of gene silencing of P300 on GEM-induced apoptosis was abolished. As Rb-E2F1 complex is known as the key regulator of the S phase specific gene transcriptions, we evaluated the effect of P300 gene silencing on Rb and E2F1. While P300 gene-silencing did not affect Rb phosphorylation after GEM treatment, it increased E2F1 expression significantly (56% increase in MIAPaCa2 and 119% increase in Panc1, respectively, p<0.05). When pancreatic cancer cells were treated with combination of GEM and C646, P300 specific HAT inhibitor, the cell viability was reduced significantly compared with when treated with GEM alone (48% decrease for Panc1, and 58% for MIAPaCa2, respectively, p<0.05).

Conclusion:

Targeting P300 by siRNA or HAT inhibitor enhances the efficacy of GEM on pancreatic cancer cells. Our observation suggests the presence of Rb-independent E2F1 regulatory mechanism in pancreatic cancer cells. This pathway may be an important therapeutic target to potentiate GEM chemotherapy in the treatment of pancreatic cancer.

Citation Format: Hiroaki Ono, Marc D. Basson, Hiromichi Ito. P300 inhibition enhances cytotoxic effect of Gemcitabine through E2F1 activation in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2015-3613
Abstract 3616: Development of a Sox2 targeting therapy for the treatment of lung squamous cell carcinoma
Authors: Ishida Naomasa , Fukazawa Takuya , Takaoka Munenori , Yamatsuji Tomoki , Morita Ichiro , Haisa Minoru , Takaoka Nagio , Naomoto Yoshio
Published: August 2015

Despite the recent development of several effective molecular targeted agents, lung cancer is the most common cause of cancer related deaths worldwide. Recently, molecular targeted therapies for pulmonary adenocarcinoma with mutant EGFR or ALK fusions have reduced non-tumor toxicity and have extended patient survival time compared to conventional chemotherapies. However, the development of molecular targeting drugs for NSCLC has made apparent the fact that histology is an important factor and molecularly targeted therapies have been more effective in pulmonary adenocarcinoma than in lung squamous cell carcinoma. Therefore, specific therapies that target lung squamous cell carcinoma are desperately needed.

Sox2 is a master pluripotency controller that was recently identified as a novel major oncogene, recurrently amplified and activated in lung squamous cell carcinoma. Immunohistochemistry shows that Sox2 expression is seen in approximately 55% of human pulmonary adenocarcinoma sections. On the other hand Sox2 expression is detected in more than 90% of lung squamous cell carcinoma sections. Inhibition of Sox2 by siRNA suppresses cell viability and colony formation of Sox2 expressing EBC2 and LK2 lung squamous cell carcinoma cells. Moreover, Sox2 siRNA inhibits lung squamous cell carcinoma growth in vivo in a xenograft mouse model derived from EBC2 cells. Flowcytometric analysis demonstrated that Annexin V positive and PI negative cell populations were not changed 48 hours after Sox2 silencing in EBC2 lung squamous cell carcinoma cells. Microarray analysis demonstrated that the expression of several cell cycle related genes were changed after Sox2 silencing in Sox2 expressing lung squamous cell carcinoma cells. Cell cycle analysis showed that Sox2 silencing significantly increased the G1 population in the cells. These results indicate that Sox2 silencing might produce an antitumor effect via G1 cell cycle arrest.

Citation Format: Naomasa Ishida, Takuya Fukazawa, Munenori Takaoka, Tomoki Yamatsuji, Ichiro Morita, Minoru Haisa, Nagio Takaoka, Yoshio Naomoto. Development of a Sox2 targeting therapy for the treatment of lung squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3616. doi:10.1158/1538-7445.AM2015-3616
Abstract 3618: SGKs survival signal via inhibition of pro-apoptotic Mixed Lineage Kinase 3 (MAP3K11) in cancer cells
Authors: Sondarva Gautam V. , Rangasamy Velusamy , Viswakarma Navin , Das Subhasis , Nair Rakesh Sathish , Rana Basabi , Rana Ajay
Published: August 2015

Survival of cancer cells requires activation of pro-survival and inhibition of pro-apoptotic pathways in concert. It is utmost important to understand the underlying molecular pathways that regulate cancer cell survival and death, so that specific inhibitor could be identified and used to target cancer cell death pathways. Dexamethasone (DEX) is routinely administered to cancer patients, in order to reduce allergic responses. It is reported that DEX use in certain cancer patients could lead to early cancer relapse and detrimental. Earlier, we reported that insulin regulated PI3K-AKT pathway down regulated pro-apoptotic Mixed Lineage Kinase 3 (MLK3) and this down regulation of kinase activity, favored cancer cell survival. Since SGK family members are also regulated by PI3K via Glucocorticoid Receptor (GR) in response to DEX, and SGK is reported to have similar function like AKT, we hypothesized that SGKs might also regulate pro-apoptotic function of MLK3, similar to AKT but probably via a non-redundant pathway.

Here we report that MLK3 is a direct target of SGK in cancer cells. Endogenous SGK physically associated with and phosphorylated MLK3 in HepG2 cells and this interaction was regulated by synthetic glucocorticoid, dexamethasone. JNK and MLK3 kinase activities were significantly inhibited either by over expression of SGK3 or by dexamethasone. The SGK3 and dexamethasone-mediated attenuation of MLK3, MKK7 and JNK kinase activities were blocked by PI3K inhibitor, LY294002, suggesting a link between PI3K-SGK and MLK3-JNK signaling pathways. SGK phosphorylated MLK3 both in vivo and in vitro. SGK1 and SGK2/3 phosphorylated MLK3 on Ser674 and Thr738, respectively. Furthermore, SGK3 inhibited MLK3-mediated cell death and JNK kinase activity in a manner, dependent on Thr738 phosphorylation of MLK3. Taken together, these data define the mechanisms of glucocorticoid-mediated inhibition of MLK3-JNK axis and cell death pathways. Furthermore, our data also identify MLK3 as an indirect target of glucocorticoids that might play a significant role in the pro-survival and anti-inflammatory actions of glucocorticoid in cancer cell. Therefore, it is conceivable that targetting MLK3 with its agonist, ceramide (by nano-ceramide particles) could possibly overcome DEX-induced cancer relapse.

This work is supported by VA Grant #: BX000312 and NIH, R01 CA 176846 to AR; and VA Grant #: BX000571to BR.

Citation Format: Gautam V. Sondarva, Velusamy Rangasamy, Navin Viswakarma, Subhasis Das, Rakesh Sathish Nair, Basabi Rana, Ajay Rana. SGKs survival signal via inhibition of pro-apoptotic Mixed Lineage Kinase 3 (MAP3K11) in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3618. doi:10.1158/1538-7445.AM2015-3618
Abstract 3619: Arsenic trioxide downregulates NPM-ALK and inhibits the proliferation of ALK-positive anaplastic large cell lymphoma
Authors: Piao Wenying , Chau David HW , Yue Kevin LM , Kwong Yok Lam , Tse Eric WC
Published: August 2015

INTRODUCTION

Anaplastic Large Cell Lymphoma (ALCL) is a mature T-cell lymphoma. Depending on the presence or absence of chromosomal translocations involving the anaplastic lymphoma kinase ALK gene and hence the expression of ALK, ALCL is further subdivided into ALK positive (ALK+) or ALK negative (ALK-) types. NPM-ALK chimeric fusion oncogenic protein is found in around 80% of ALK+ ALCL, as a result of the chromosomal translocation t(2;5)(p23;q35). The current standard of treatment for ALK+ ALCL is combination chemotherapy. Crizotinib, an ALK tyrosine kinase inhibitor, is the only agent available to target NPM-ALK in ALCL. The identification of other targeting agents would potentially improve the treatment outcome of ALK+ ALCL especially for relapsed and/or refractory cases. In this study, we investigated the effect of arsenic trioxide (As2O3) on NPM-ALK in ALCL.

Methods

We treated ALK+ ALCL cell lines with various clinically achievable concentrations of As2O3 (0, 0.5, 1 and 2μM) for 24 hours and examined its effects on NPM-ALK mRNA expression by semi-quantitative RT-PCR and protein expression by Western immunoblotting. The downstream signaling pathway of NPM-ALK, phosphorylation of STAT3 on Tyr705, was also examined. Next, we analyzed apoptotic cell death and growth arrest induced by As2O3 in ALK+ and ALK- ALCL cell lines using Annexin V-7AAD staining and cell cycle analysis. The effect of As2O3 on cell viability was also measured with WST-1 assay.

RESULTS

Our data showed that As2O3 downregulated NPM-ALK at the protein level without affecting the transcription. It also inhibited the phosphorylation of STAT3 in all ALK+ ALCL cell lines. We also found that blocking protein degradation through proteasome with MG132 abrogated the effect of As2O3 on NPM-ALK. Furthermore, As2O3 induced apoptotic cell death, cell cycle arrest at G2/M phase and growth inhibition in ALK+ but not in ALK- ALCL cell lines, with a GI50 of less than 1μM, which was a level achievable with clinical use of As2O3.

CONCLUSIONS.

Our data demonstrated that As2O3 downregulated NPM-ALK by facilitating protein degradation through the proteasomal pathway, thereby inhibited cell proliferation and induced cell death in ALK+ ALCL. As2O3 may therefore represent a potential therapeutic agent for the treatment of ALK+ ALCL.

Citation Format: Wenying Piao, David HW Chau, Kevin LM Yue, Yok Lam Kwong, Eric WC Tse. Arsenic trioxide downregulates NPM-ALK and inhibits the proliferation of ALK-positive anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3619. doi:10.1158/1538-7445.AM2015-3619
Abstract 3620: An oral procaspase activating drug, PAC-1, shows preclinical promise for glioblastoma therapy
Authors: Joshi Avadhut D. , Botham Rachel C. , Roth Howard S. , Fan Timothy M. , Tarasow Theodore M. , Hergenrother Paul J. , Riggins Gregory J.
Published: August 2015

PAC-1 is an oral administered procaspase-3 activating drug developed to overcome the malignant cell's barrier to apoptosis. The promising preclinical safety and efficacy of PAC-1 has led to its consideration for a first in human phase 1 clinical trial for recurrent and advanced malignancies. We have found that because PAC-1 is able to reach intracranial brain tumors in effective concentrations, that it is particularly useful for glioblastoma (GBM), an aggressive and invasive malignancy responsible for the most deaths due to brain cancer. In glioblastoma patients a worse prognosis was correlated with high expression of procaspase-3 mRNA, indicating that procaspase-3 activation may be involved in the pathology of the tumor progression. In preclinical testing of PAC-1, it showed significant survival benefit and low systemic toxicity, in orthotopic animal models of glioblastoma. An aqueous suspension of PAC-1 delivered orally to rats with intracranial 9L glioblastoma improved survival compared to untreated controls. Additionally, when PAC-1 was administered as an oral capsule to rats with 9L glioblastoma, median survival increased over three fold. Oral PAC-1 improved survival significantly also in a human xenograft model (p = 0.04) compared to controls. PAC-1 combined effectively with standard of care, and in some animals, either alone or in combination resulted in long term survival. PAC-1 has the ability to penetrate sufficiently within intracranial tumors and its ability to overcome resistance to apoptosis and activate widespread cell death specifically in the malignant cells makes it a promising small molecule for further preclinical and clinical development.

Citation Format: Avadhut D. Joshi, Rachel C. Botham, Howard S. Roth, Timothy M. Fan, Theodore M. Tarasow, Paul J. Hergenrother, Gregory J. Riggins. An oral procaspase activating drug, PAC-1, shows preclinical promise for glioblastoma therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3620. doi:10.1158/1538-7445.AM2015-3620