bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5755: In vitro reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells
Authors: Nakamura Kohei , Nakayama Kentaro , Kiyono Tohru , Ishikawa Noriyoshi , Minamoto Toshiko , Ishibashi Tomoka , Sanuki Kaori , Yamashita Hitomi , Ono Ruriko , Iida Kouji , Sasamori Hiroki , Sultana Razia , Hossain Mohammad Mahmud , Ishikawa Masako , Kyo Satoru
Published: July 2017

Purpose: Recent studies suggest that fallopian tube secretory epithelial cells (FTSECs) are potential cells-of-origin for high-grade serous ovarian carcinoma (HGSOC). Several genetic alterations are involved in HGSOC carcinogenesis, but the minimal requirement for tumor initiation remains unclear. Therefore, we sought to identify oncogenic mutations indispensable for HGSOC carcinogenesis in a stepwise model using immortalized FTSECs.

Experimental Design: We established an in vitro stepwise carcinogenesis model using immortalized FTSECs overexpressing cyclin D1, CDK4R24C, and hTERT, and mimicked select genetic abnormalities identified as gene alterations essential for carcinogenesis, including p53, c-Myc, or RAS/PI3K pathway mutations.

Results: Our analyses revealed two distinct patterns of gene alterations essential for HGSOC carcinogenesis: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53 expression alone or in combination with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), or c-Myc in immortalized cells failed to induce a tumorigenic phenotype; however, overexpression of either CA-AKT or c-Myc in combination with dominant-negative p53 and KRASV12 was sufficient to confer tumorigenic potential. Importantly, all transformed FTSECs formed tumors in xenograft mice, which were grossly, histologically, and immunohistochemically similar to human HGSOC. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination observed in their human counterparts. C-Myc is associated with cell proliferation in vitro; therefore, this genetic abnormality may promote HGSOC progression.

Conclusions: Collectively, our data showed that aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling is the minimal requirement for FTSEC carcinogenesis. Moreover, the model generated with this evidence will likely facilitate analysis of early events in HGSOC carcinogenesis.

Citation Format: Kohei Nakamura, Kentaro Nakayama, Tohru Kiyono, Noriyoshi Ishikawa, Toshiko Minamoto, Tomoka Ishibashi, Kaori Sanuki, Hitomi Yamashita, Ruriko Ono, Kouji Iida, Hiroki Sasamori, Razia Sultana, Mohammad Mahmud Hossain, Masako Ishikawa, Satoru Kyo. In vitro reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5755. doi:10.1158/1538-7445.AM2017-5755
Abstract 5758: Elevated level of estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells
Authors: Chang Yu-Wei , Singh Kamaleshwar
Published: July 2017

Effectiveness of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen sensitive and the levels of ovarian estrogen are age-dependent variable among the breast cancer patients. Whether this variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is unclear. Therefore, the objective of this study was to determine the effects of natural estrogen 17 beta-estradiol on the efficacy of chemotherapy in breast cancer cells. Estrogen responsive MCF-7 breast cancer cells were long-term exposed to 100 pg/ml estrogen for three months. Using these estrogen exposed MCF-7 cells, the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased efficacy of doxorubicin and cisplatin in estrogen-exposed MCF-7 cells as compared to control MCF-7 cells. Gene expression analysis of marker genes for cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-Aza-2-deoxycytidine and Trichostatin A before doxorubicin and cisplatin treatments. The enhanced efficacy of doxorubicin and cisplatin in estrogen-treated cells was significantly decreased by 5-aza-2 deoxycytidine pre-treatment, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In conclusion, results of this study demonstrate that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the type and dose of chemotherapy in breast cancer patients depending on the serum estrogen levels that varies in between pre- and post-menopausal age of the patients.

Citation Format: Yu-Wei Chang, Kamaleshwar Singh. Elevated level of estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5758. doi:10.1158/1538-7445.AM2017-5758
Abstract 5762: PR55-alpha associated PP2A promotes pancreatic tumorigenesis
Authors: Yan Ying , Hein Ashley L. , Seshacharyulu Parthasarathy , Rachagani Satyanarayana , Sheinin Yuri M. , Ouellette Michel M. , Ponnusamy Moorthy P. , Batra Surinder K.
Published: July 2017

PP2A holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Loss of function analysis using PP2A catalytic inhibitors or inhibition via tumor viral proteins suggest PP2A as a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α, a PP2A regulatory subunit, in pancreatic cancer cells compared to normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared to adjacent normal pancreatic tissues (P<0.0001) and correlated with poor survival of pancreatic cancer patients (P<0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cells resulted in diminished phosphorylation of both AKT and ERK1/2 and decreased protein levels of β-catenin. Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice showed a marked reduction in tumorigenicity (P<0.001) and distant metastases. These results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK and Wnt.

Citation Format: Ying Yan, Ashley L. Hein, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Yuri M. Sheinin, Michel M. Ouellette, Moorthy P. Ponnusamy, Surinder K. Batra. PR55-alpha associated PP2A promotes pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5762. doi:10.1158/1538-7445.AM2017-5762
Abstract 5767: Ex vivo three-dimensional tumor growth assay: 3DX-TGA
Authors: Nair Praveen , Nair Dileep , Hinata Kaede , Mirsaidi Cyrus , Wu Junjie , Hu Yong , Hall Brett M.
Published: July 2017

With a 7% likelihood of regulatory approval, oncology drug registration failure rates lead all therapeutic areas. Further complicating the drug development process, preclinical oncology models poorly reflect tumor tissue biology and produce a high level of false positive data. Yet, monocellular two-dimensional (2D) tissue culture remains the preferred platform for most laboratory preclinical studies. The popularity of 2D tissue culture was driven predominantly by fast and dependable proliferation of human tumor cells rather than alignment to the pathophysiology of human cancer. As a consequence of this artificial selection bias, many registered oncology drugs today are highly toxic with a propensity to attack any dividing cell (i.e., non-targeted). Fortunately, recent advances in genomics, metabolomics and immunology have aided in development of new targeted agents, many of which have achieved regulatory approval.

Nevertheless, rational drug combinations and identification of clinically meaningful drug targets persist as major challenges in oncology, and each can be meaningfully addressed using more diverse and biologically relevant preclinical models. Ex vivo tumor tissue transplant models, such as patient-derived xenografts (PDX) offer greater model diversity and more faithfully reflect patient tumor genetics. However, cost and scalability barriers tend to limit widespread and practical model utility. In addition, there remains a strong selection bias for autocrine human tumors that can quickly adapt to a cross-species mouse host.

As a practical alternative to PDX models, we established, serially propagated and molecularly characterized 300 ex vivo 3D (3DX) models spanning 15 tumor indications. Given patient tumor seeding success rates of over 95%, the 3DX-TGA model enabled diverse pharmacologic evaluation for the vast majority of cancer patient models attempted. After achieving sufficient tumor biomass, typically within 7 to 8 weeks, thirty-one drugs were screened for proliferation and viability endpoints over a three-log dose range. Resistance and sensitivity profiles mirrored population-based response rates for indication-aligned FDA-registered drugs. Taken together, the 3DX-TGA model represents a powerful preclinical ex vivo model that: (1.) more faithfully recapitulates human tumor biology, (2.) can be scaled at a fraction of time and cost compared to PDX models, and (3.) provide a superior screening platform for novel drugs and drug-drug combinations.

Citation Format: Praveen Nair, Dileep Nair, Kaede Hinata, Cyrus Mirsaidi, Junjie Wu, Yong Hu, Brett M. Hall. Ex vivo three-dimensional tumor growth assay: 3DX-TGA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5767. doi:10.1158/1538-7445.AM2017-5767
Abstract 5769: Modeling the tumor invasion front using 3D fluidic tumoroid culture of cancer cells
Authors: Yong Koh Meng Aw , Oliver Christopher , Altemus Megan , Wu Zhi Fen , Merajver Sofia
Published: July 2017

To realize the promise of precision medicine, it is important to integrate phenotypic assessment of cell populations to genomic data. The analysis of invading leader cells at the tumor invasion front is of interest as they may be guided by a targetable molecular phenotype. However, there is a lack of suitable platforms on which to analyze the tumor invasion front. In this study, we have designed and constructed a fluidic device for long-term (several days to weeks) 3-dimensional tumoroid culture of diverse cancer cells. Using this device, we can recapitulate the tumor invasion front and at the same time quantify the invasive potential of different breast cancer cell line models. Analyses of the tumor invasion front indicated a region of higher proliferation and suggest that the leader cells possess a different molecular phenotype from the tumoroid mass. Interestingly, significant heterogeneity among invading cells was still observed, suggesting that there could be: 1) the presence of multiple subpopulations of invasive cells, each with a different clonal genetic signature; and/or 2) reversible phenotypic switching occurring among invading cells due to phenotypic plasticity. These results obtained using this innovative device highlight and present a promising solution to the challenges developing adequate therapeutics accounting for tumor phenotypic heterogeneity. There is potential for the device for use in personalized medicine at diagnosis, allowing for both the quantification of disease progression risk as well as the molecular characterization of the invasive subpopulations from patient samples and their response to tailored therapies.

Citation Format: Koh Meng Aw Yong, Christopher Oliver, Megan Altemus, Zhi Fen Wu, Sofia Merajver. Modeling the tumor invasion front using 3D fluidic tumoroid culture of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5769. doi:10.1158/1538-7445.AM2017-5769
Abstract 5774: Effect of extrinsic and intrinsic solid stress on tumor spheroid growth
Authors: Guillaume Ludivine , Desmaison Annaick , Ducommun Bernard , Lobjois Valérie
Published: July 2017

Growth-induced intrinsic solid stress and adaptive response to extrinsic mechanical cues have been recognized as major hurdles to overcome in cancer treatment and any approach that could significantly modulate these parameters would have an impact in improving therapies. An attracting in vitro model to explore this issue is the Multicellular Tumor Spheroid, a 3D culture model reproducing the non-vascularized microtumor environment, proliferation gradient and cellular interactions. To explore and assess spheroid growth-induced solid stress, we couple biological approach to the application of mechanics and modeling. We adapted a method, which consist in partial sectioning of the microtumor and measuring tissue relaxation parameters. Using this strategy, we showed that cell proliferation induced the accumulation of intrinsic mechanical constraint. To investigate the consequence of the accumulation of solid stress at the cell scale, we analyzed 3D cellular organization within spheroids. Using 3D light sheet microscopy (LSM) visualization on optically cleared spheroids combined with 3D reconstruction and automatic algorithms, we were able to determine cell density, nuclear orientation and nuclear shape in whole 3D spheroids. We found that stress accumulation is associated with an increase in cell density and a regionalization of nuclei organization. We also explored the impact of extrinsic stress in 3D culture condition under constraint. We found that mechanical constraint and cytoskeleton control nuclei orientation and using live 3D LSM microscopy we show that mechanical constraint induces an actin cytoskeleton-dependent prometaphase delay. We anticipate that this work will provide key information and parameters to improve our knowledge of the crosstalk between mechanical parameters and tumor growth.

Citation Format: Ludivine Guillaume, Annaick Desmaison, Bernard Ducommun, Valérie Lobjois. Effect of extrinsic and intrinsic solid stress on tumor spheroid growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5774. doi:10.1158/1538-7445.AM2017-5774
Abstract 5790: Assessing the effects of TIMP2 knockout on lung cancer cell lines cultured in 3D
Authors: Peeney David
Published: July 2017

Tissue inhibitor of matrix metalloproteinases (TIMPs) are a small family of endogenous proteins that classically function to inhibit metalloproteinase activity. Since the original description of this protein family in the 80s and 90s, various MMP-independent biological functions of TIMPs have been described. This built the impression that MMP/TIMP ratios may play an important role in tissue homeostasis, an idea which is supported by the observation that altered MMP/TIMP expression ratios are often associated with a number of human conditions such as cancer, cardiovascular and CNS disease. TIMP2 is the most abundantly expressed protein in this family and has previously been shown to interact with several membrane proteins including MT1-MMP, insulin-like growth factor-1 receptor (IGF-I-R) and alpha3 beta1 integrin (α3β1) to mediate downstream intracellular signaling. In addition, TIMP2 has been shown to inhibit growth factor stimulated proliferation, angiogenesis and tumor cell invasion and metastasis, highlighting the potential for TIMP2-based cancer bio-therapies that can be used in conjunction with conventional treatments. Recent studies in our lab highlight that syngeneic lung tumors (LL2 cells; Lewis lung carcinoma) developed in C57BL mice harboring a loss-of-function mutation in TIMP2 are significantly larger than tumors grown in their WT counterparts. To gain a deeper understanding of the role of TIMP2 in tumor initiation and progression we have used CRISPR-Cas9 to develop stable TIMP2 knockout (T2KO) human lung cancer cell lines. Although indistinguishable in 2D culture, T2KO tumor cells display a morphologically distinct phenotype when grown in spheroids. Preliminary data show that, when grown in spheroids, T2KO cells exhibit enhanced EGFR activation in comparison to WT cells. By assessing the functional characteristics and gene expression of T2KO cells grown in 3D culture conditions we hope to gain further insight into the biological functions of TIMP2 and to provide a mechanistic link between the loss of TIMP2 activity and enhanced tumor formation that is observed in our mouse model.

Citation Format: David Peeney. Assessing the effects of TIMP2 knockout on lung cancer cell lines cultured in 3D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5790. doi:10.1158/1538-7445.AM2017-5790
Abstract 5795: Loss of CDCP1 in patient prostate cancer metastasis leads to uncoupling of beta-1 integrin from its cytoplasmic signaling through FAK
Authors: Pollan Sara G. , Huang Fangjin , Lang Joshua M. , Sperger Jamie M. , Shah Kavita , Knudsen Beatrice S.
Published: July 2017

The Cub-Domain Containing Protein - 1, CDCP1, is a transmembrane glycoprotein, which is able to sequester Src and PKCδ into unique microdomains within the plasma membrane. CDCP1 can either promote or suppress tumor metastasis, dependent on the cancer type and experimental system. How the loss of CDCP1 leads to tumor metastasis is not well understood. We demonstrated for the first time in patients with prostate cancer (PCA), a significant reduction of CDCP1 expression in circulating cancer cells (CTC) and tumor metastasis relative to primary tumors and concordant analytical results. To investigate how the loss of CDCP1 facilitates PCA metastasis, we determined the consequences of CDCP1 loss in non-adherent cancer cells, which provide an experimental model system of CTCs. In this system, CDCP1 silenced cells exhibit 3-fold higher proliferation, 4-fold greater anchorage independent growth with colonies exceeding 5 microns in diameter, and a 2-fold reduction in migration ability. In 10% human plasma, these cells up-regulate p-FAK, p-SRC, p-AKT and p-MAPK expression, and at the same time loose expression of activated β1-integrin. The loss of inside-out activation of β1-integrin occurs through prevention of TALIN phosphorylation. Upon loss of CDCP1, Talin is no longer phosphorylated by CDK5 and this causes the disassembly of the β1 integrin - Talin complex. In addition, β1-integrin dissociates from CDK5 and from the CDK5-regulatory subunit, p35, but remains bound to p-FAK. We determined the pathway by which the loss of CDCP1 arrests CDK5 kinase activity. Upon loss of CDCP1, SRC phosphorylates p35. This generates a binding site for the C2 domain of PKCδ and phosphorylation of CDK5-T77 by PKCδ. The subsequent dissociation of the regulatory subunit abolishes the activity of CDK5. We show that both SRC inhibition and silencing of PKCδ reestablish the inside-out activation of β1-integrin. Altogether we discovered a new mechanism of regulation of CDK5 in prostate cancer cells, which leads to the uncoupling of β1-integrin and FAK. The potential biological and clinical consequences of this mechanism are (1) a switch from cell-matrix to cell-cell adhesion, (2) increased sensitivity of CTCs to FAK inhibitors and (3) improved adaptation and survival of cancer cells in the circulation and at the metastatic sites.

Citation Format: Sara G. Pollan, Fangjin Huang, Joshua M. Lang, Jamie M. Sperger, Kavita Shah, Beatrice S. Knudsen. Loss of CDCP1 in patient prostate cancer metastasis leads to uncoupling of beta-1 integrin from its cytoplasmic signaling through FAK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5795. doi:10.1158/1538-7445.AM2017-5795
Abstract 5799: Targeting CX3CR1 impairs the reseeding of cancer cells recirculating from metastatic tumors
Authors: Qian Chen , Worrede-Mahdi Asurayya , Kaur Ramanpreet , Shen Fei , Salvino Joseph , Meucci Olimpia , Fatatis Alessandro
Published: July 2017

Cancer cells re-enter systemic blood from established metastatic tumors. Recent evidence indicates that these recirculating cancer cells further seed and colonize skeleton and soft tissues to expand metastatic dissemination, thus precipitating the clinical progression to terminal disease. We have previously shown that the chemokine receptor CX3CR1 is implicated in the metastatic seeding of breast cancer cells and that novel small-molecule antagonists for this receptor effectively contains the number and size of secondary tumors in animal models. Using the same models, we now report that targeting CX3CR1 also restrains the re-seeding of skeleton and soft-tissue by circulating cancer cells (CTCs) departing from existing metastatic lesions. Notably, we also found that CTCs unable to re-seed are forced to remain in the blood circulation longer, eventually succumbing to apoptotic death. It has been proposed that when either spontaneously or forcibly (re)circulating in the blood, cancer cells from solid tumors are more vulnerable to the effects of both cytotoxic and targeted therapeutics. Given the role of CX3CR1 antagonists in prolonging the time spent by CTCs in the blood, we sought to assess whether these compounds could synergize with docetaxel by extending bioavailability and its cytotoxic effects. Thus, we tested this paradigm on CTCs departing from skeletal and soft-tissue lesions generated by breast and prostate cancer cells in mice. Based on the results obtained from these experiments, it can be concluded that implementing the development of CX3CR1 antagonists and promoting their clinical use will provide novel and effective tools to contain the progression of metastatic disease in cancer patients.

Citation Format: Chen Qian, Asurayya Worrede-Mahdi, Ramanpreet Kaur, Fei Shen, Joseph Salvino, Olimpia Meucci, Alessandro Fatatis. Targeting CX3CR1 impairs the reseeding of cancer cells recirculating from metastatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5799. doi:10.1158/1538-7445.AM2017-5799
Abstract 58: Local intratumoral treatment with anti-mesothelin Immunotoxins overcomes resistance to anti-CTLA-4 therapy
Authors: Leshem Yasmin , Liu Xiu-fen , Bera Tapan , Terabe Masaki , Berzofsky Jay A. , Bossenmaier Birgit , Niederfellner Gerhard , O'Brien James , Reiter Yoram , Pastan Ira
Published: July 2017

Background: SS1P and RG7787 are immunotoxins composed of anti-mesothelin antibody fused to a fragment from pseudomonas exotoxin A. We previously observed delayed tumor responses in patients treated with SS1P leading us to hypothesis that immunotoxins can provoke anti-tumor immunity. We hypothesize that intra-tumoral injection of SS1P or RG7787 will produce immunologic death and convert living tumors to a source of antigens and boost the therapeutic effect of immune check point blockade.

Method: A BALB/c breast cancer cell line was transfected with human mesothelin (66C14-M) and grown in BALB/c mice transgenic for human mesothelin. SS1P or RG7787 was injected directly into established tumors (80-100 mm3) and anti-CTLA-4 administered IP.

Results: We observed total tumor regressions in 23 out of 38 mice (60%) treated with anti-CTLA-4 and either RG7787 or SS1P. No cure was achieved when the drugs were given as a monotherapy supporting the case for synergic anti-tumor activity. Pathologic evaluation of regressing tumors showed a massive inflammation of admixed eosinophils and mononuclear cells located in a collar surrounding the tumors. Tumor regression was associated with increased number of CD8+ cells and was blocked when CD8+ cells were depleted by an antibody. In addition, cured mice were resistant to re-implantation of 66C14 cells not expressing mesothelin indicating that a long-term anti-tumor immunity was formed. To determine if pathogen associated molecular patterns contributed to the response, we combined anti-CTLA-4 with an inactive RG7787 and observed a major reduction in anti-tumor activity (P<0.01) indicating that the effect is not mediated by recognition of a foreign bacterial protein.

Conclusions: We suggest that injecting immunotoxins into tumors is a useful approach to boost the activity of anti-CTLA-4. Based on the synergistic anti-tumor effect demonstrated in this study we believe that there is a strong rational to explore such combination therapies in patients.

Citation Format: Yasmin Leshem, Xiu-fen Liu, Tapan Bera, Masaki Terabe, Jay A. Berzofsky, Birgit Bossenmaier, Gerhard Niederfellner, James O'Brien, Yoram Reiter, Ira Pastan. Local intratumoral treatment with anti-mesothelin Immunotoxins overcomes resistance to anti-CTLA-4 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 58. doi:10.1158/1538-7445.AM2017-58
Abstract 5818: Disrupting the Aurora kinase A interactome in pediatric cancer
Authors: Mukherjee Sucheta , Tu Carolyn , Gustafson Clay
Published: July 2017

Neuroblastoma is the most common extra-cranial solid tumor of childhood. Amplification of the MYCN proto-oncogene occurs in 50% of the high-risk neuroblastoma and indicates an aggressive and lethal form of the disease. As a transcription factor with no apparent small-molecule surfaces, direct inhibition of MYCN has proven challenging. Proteolytic cleavage of MYCN is regulated in part by an activity-independent scaffold function of Aurora Kinase A (AURKA). The dynamic expression and activation of AURKA allows for precise cell cycle regulation of several substrates identified by protein-protein interaction studies that are also viable anti-cancer targets, such as the aforementioned MYCN oncoprotein. Utilizing co-immunopreciptation, flow cytometry, and other pertinent cell biology techniques, we aim to delineate a novel mechanism of targeting the AURKA interactome by conformation disruption of AURKA. We have recently described a neoteric class of confirmation disrupting “amphosteric” inhibitors of AURKA (CD-AURKAi) that orthosterically inhibit the ATP-binding pocket to dramatically disrupt the active conformation of AURKA to dissociate and degrade MYCN, a critical oncogenic driver of the pediatric cancer neuroblastoma. In addition to MYCN, our preliminary results show CD532 will also dissociate other oncogenic proteins that interact with AURKA. We hypothesize that conformation disruption of AURKA and subsequent blockade of an array of protein-protein interactions will delineate the roles of AURKA, MYC, and MYCN in the cell cycle and expand the clinical applications for CD-AURKAi in cancer to include non-MYCN driven diseases.

Citation Format: Sucheta Mukherjee, Carolyn Tu, Clay Gustafson. Disrupting the Aurora kinase A interactome in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5818. doi:10.1158/1538-7445.AM2017-5818
Abstract 5821: Loss of Pin1 suppresses Hedgehog-driven medulloblastoma tumorigenesis
Authors: Rual Jean-Francois M.
Published: July 2017

Medulloblastoma is the most common malignant brain tumor in children. Therapeutic approaches to medulloblastoma (combination of surgery, radiotherapy and chemotherapy) have led to significant improvements, but are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed. Genetic mutations leading to the activation of the Hedgehog pathway drive tumorigenesis in ~30% of medulloblastoma. In a yeast two-hybrid proteomic screen, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hedgehog signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the post-phosphorylation fate of its targets. The GLI1/PIN1 interaction was validated by reciprocal pulldowns using epitope-tagged proteins in HEK293T cells as well as by co-immunoprecipiations of the endogenous proteins in a medulloblastoma cell line. Our results support a molecular model in which PIN1 promotes GLI1 protein abundance, thus contributing to the positive regulation of Hedgehog signals. Most importantly, in vivo functional analyses of Pin1 in the GFAP-tTA;TRE-SmoA1 mouse model of Hedgehog-driven medulloblastoma demonstrate that the loss of Pin1 impairs tumor development and dramatically increases survival. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in Hedgehog-driven medulloblastoma tumorigenesis.

Citation Format: Jean-Francois M. Rual. Loss of Pin1 suppresses Hedgehog-driven medulloblastoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5821. doi:10.1158/1538-7445.AM2017-5821
Abstract 5823: Modulation of the Hippo pathway protein YAP as a mechanism of resistance to MEK inhibition in MAPK hyperactivated neuroblastoma
Authors: Coggins Grace E. , Hart Lori S. , Elias Jimmy , Maris John M.
Published: July 2017

Background: Current therapies for high-risk neuroblastoma are highly toxic and ineffective, as a majority of patients will face therapy-resistant relapses. Our recent whole genome sequencing analysis revealed that a majority of relapsed tumors had mutations causing RAS-MAPK pathway activation. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP in relapsed neuroblastoma as well as resistance to MEK inhibition in other cancer models. Based on these findings, we hypothesized that YAP activity may lead to acquired resistance to MEK inhibition, and that dual inhibition of these two pathways may have synergistic therapeutic efficacy.

Methods: We selected the cell line NLF from a panel of NB cell lines due to its high YAP expression and NF1 loss. NLF cells were treated with MEK inhibitor trametinib (20 nM) over a 72-hour period and whole cell lysates and nuclear lysates were collected for immunoblot. Effects of YAP knockdown via siRNA transfection with and without trametinib co-treatment on survival were assessed by CellTiter-Glo and RT-CES. Cell signaling analyses of tumor lysates from mouse SKNAS (NRAS Q61K) xenografts treated with vehicle, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg for YAP, ERK, and apoptosis pathways were performed via immunoblot.

Results: Immunoblots of 72-hour time course treatments of trametinib in NLF showed decreased pYAP, the cytosolic inactive form of the YAP protein, indicating increased YAP capable of translocating to the nucleus, which was confirmed by analyses of nuclear extracts. siRNA-mediated knockdown of YAP alone in NLF cells had a modest effect on survival, while combinatorial inhibition of MEK and YAP signaling caused a significant reduction in cellular proliferation and survival. Immunoblotting of trametinib-treated SKNAS xenografts indicated decreased pYAP and pERK in response to increasing trametinib dose, while total YAP levels remained constant.

Conclusion: Our results demonstrate that prolonged trametinib exposure causes a marked nuclear enrichment of unphosphorylated YAP and co-inhibition of MEK and YAP in neuroblastoma models effectively reduces proliferation and survival. We are currently investigating the effects of MEK inhibition on the expression of YAP target genes and on cellular proliferation upon modulation of YAP protein expression.

Note: This abstract was not presented at the meeting.

Citation Format: Grace E. Coggins, Lori S. Hart, Jimmy Elias, John M. Maris. Modulation of the Hippo pathway protein YAP as a mechanism of resistance to MEK inhibition in MAPK hyperactivated neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5823. doi:10.1158/1538-7445.AM2017-5823
Abstract 5828: Targeting the polyamine-hypusine nexus for the treatment of neuroblastoma
Authors: Bachmann Andre S. , El-Khawaja Raid , Schultz Chad
Published: July 2017

The highly conserved and unique post-translational hypusine modification is essential for the activation of a single cellular protein called eukaryotic initiation factor 5A (eIF5A). Hypusination of eIF5A represents an essential mechanism in the control of cell proliferation and has been linked to cancer. The hypusine modification of eIF5A requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) and, importantly, uniquely depends on the polyamine spermidine as a selective substrate. Polyamines including spermidine are frequently dysregulated in neoplastic disease and are critical for cell proliferation. Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. We pioneered the repurposing of the irreversible ODC inhibitor alpha-difluoromethylornithine (DFMO) for neuroblastoma (NB), a devastating pediatric cancer of the sympathetic nervous system. Our preclinical work was confirmed by other groups and spurred two consortia (NMTRC and NANT) to test DFMO in independent phase I/II NB clinical trials.

The oncogene MYCN is commonly amplified in high-risk NB patients and predicts poor prognosis. MYCN directly activates ODC (and eIF5A) and drives NB tumor progression, presumably through the activation of polyamines, but the precise molecular mechanisms are not fully understood. Since spermidine is the only molecule that can activate eI5FA through hypusine modification, we hypothesized that the polyamine-hypusine nexus provides a rational target site to identify new drug combinations that trigger synergistic anti-tumor effects.

In this study we found that DFMO combined with DHPS inhibitor GC7 induced synergistic drug reactions in NB at concentrations that are not lethal on their own. While each drug alone at higher concentrations induced p27/Rb-mediated G1/S cell cycle arrest, the combination of both drugs resulted in the induction of programmed cell death (apoptosis). The intracellular free polyamine levels (putrescine, spermidine, and spermine) were quantified by C-18 RP-HPLC and responded directly to drug treatments, suggesting specific drug targeting effects.

This dual-pronged drug combination approach that specifically targets the blockade of the unique polyamine/spermidine-dependent hypusine-eIF5A signaling circuit known to be involved in cell proliferation might lead to more effective DFMO treatment options by reducing high DFMO doses currently needed while achieving better therapeutic outcomes in NB.

Citation Format: Andre S. Bachmann, Raid El-Khawaja, Chad Schultz. Targeting the polyamine-hypusine nexus for the treatment of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5828. doi:10.1158/1538-7445.AM2017-5828
Abstract 5833: Targeting the transcription co-activator TAZ inhibits MYC-driven medulloblastoma
Authors: Xue Yingchao , Ying Mingyao
Published: July 2017

Medulloblastoma (MB), the most common malignant pediatric brain tumor, results in significant neurological, intellectual and physical disability or death. Four MB subtypes (WNT, SHH, Group 3 and 4) have recently been identified. Group 3 MB, the most aggressive subtype, is more frequently associated with gene amplification and/or protein overexpression of the MYC (c-Myc) oncogene, hereby referred to as MYC-driven MBs. It is still unclear how MB cells activate and maintain high MYC expression, as only a small portion of MYC-high MBs is caused by MYC genomic amplification. MYC-targeted MB therapies are also lacking. Targeting MYC upstream activators is a promising strategy for inhibiting MYC-driven MBs, when compared to direct MYC inhibition that is still challenging. Here, we focus on the transcription co-activator TAZ, a novel MYC upstream activator and a potential therapeutic target for inhibiting MYC-driven MBs. TAZ (transcriptional co-activator with PDZ-binding motif) is an oncogenic driver in multiple human cancers. TAZ and its paralog YAP form complexes with other transcription factors, such as TEAD, to activate downstream gene targets. TAZ and YAP are transcriptional effectors of various signaling pathways, such as the Hippo pathway. We found for the first time that TAZ is an essential MYC activator in MB cells. TAZ but not its paralog YAP is highly expressed in MYC-amplified MB cells and their xenografts. TAZ silencing downregulates MYC and inhibits MB cell proliferation. Enforced TAZ expression induces MYC expression in MB cells and promotes MB cell growth. We further showed that TAZ forms complex with transcription activator TEAD to bind to and transactivate MYC enhancers. For pharmacological TAZ targeting, we found that the FDA-approved drug Verteporfin (VP) inhibits TAZ-TEAD interaction, suppresses MYC expression and inhibits MB cell tumor propagation in vivo. We have also developed a nanoparticle formulation by using DSPE-mPEG2000 micelles to achieve VP delivery cross the blood-brain barrier in mice bearing brain tumor xenografts. In summary, our results provide novel insights into MYC upstream activators in MB, and also identify TAZ as a potential therapeutic target for inhibiting MYC-driven MBs. This knowledge may also be widely applicable to other TAZ/MYC-expressing human cancers, such as glioblastoma. Moreover, our studies support the effectiveness of VP treatment in pre-clinical MB models. These results and also the nanocarrier system for systemic VP delivery provide a solid foundation for VP clinical trials in patients with MB and likely other MYC-driven cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Yingchao Xue, Mingyao Ying. Targeting the transcription co-activator TAZ inhibits MYC-driven medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5833. doi:10.1158/1538-7445.AM2017-5833
Abstract 5847: SIRT1 protects against radiation-induced lung injury via de-acetylating the Ku70 and P65
Authors: Xue Jianxin , Zhou Lin , Na Feifei , Deng Lei , Lan Jie , Lu Bo , Dicker Adam , Lu You
Published: July 2017

Radiation-induced lung injury (RILI) is a common and serious complication in the radiation therapy of thoracic cancer, which significantly restricts the radiation doses of tumor. The prevention and treatment of RILI have long been clinical challenges. SIRT1 is an NAD+ dependent protein deacetylase and the exact role of SIRT1 in RILI remains unclear. The aim of this study is to investigate the prevention and repairing efficacy of RILI by adenovirus-mediated SIRT1 gene overexpression in a mice model. Our studies confirmed that the protein expression and activity of SIRT1 in lung tissues were decreased in RILI mice models in a time-dependent manner, but the mRNA expression level was not obviously changed. The activity of SIRT1 was elevated by orally given resveratrol, an agonist of SIRT1. The extent of RILI could be evidently alleviated by orally given resveratrol, as assayed by histopathology, malondialdehyde (MDA) and plasma cytokines (IL-1β, TNF-α and active TGF-β1). Overexpressing SIRT1 could obviously mitigate the extent of RILI (H&E staining and CT scan), the concentration of MDA and ROS in lung, IL-1β and active TGF-β1 in plasma; as well as improve the mice survival. Moreover, SIRT1 could inhibit the radiation-induced DNA damage in vivo and in vitro, and decreased the expression of acetylated- Ku70 in the lung. In vitro Co-IP experiment showed that the expression of SIRT1 in irradiated cells was reduced and the acetylated- Ku70 was increased; over-expressing SIRT1 promoted the de-acetylation of Ku70; interference of Ku70 could reverse the effect of SIRT1 in DNA repair. Furthermore, overexpressing SIRT1 could inhibit the acetylation level of NF-κB subunit RELA (P65) in the lung; in vitro report gene experiment showed that SIRT1 suppressed the NF-κB transcriptional activity. In conclusion, our results suggest that SIRT1 could evidently mitigate the RILI, repair the irradiation-induced DNA damage and inflammation via de-acetylating the Ku70 and P65. Our study may provide new avenue for the prevention and treatment of RILI in clinic.

Note: This abstract was not presented at the meeting.

Citation Format: Jianxin Xue, Lin Zhou, Feifei Na, Lei Deng, Jie Lan, Bo Lu, Adam Dicker, You Lu. SIRT1 protects against radiation-induced lung injury via de-acetylating the Ku70 and P65 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5847. doi:10.1158/1538-7445.AM2017-5847
Abstract 5849: Exosomes promote survival and migration in squamous head and neck cancer cells after ionizing radiation: Evidence for a bystander effect
Authors: Mutschelknaus Lisa , Azimzadeh Omid , Heider Theresa , Winkler Klaudia , Vetter Marcus , Tapio Soile , Merl-Pham Juliane , Kell Rosemarie , Huber Stephan , Edalat Lena , Radulovic Vanja , Anastasov Natasa , Peters Carsten , Atkinson Michael John , Moertl Simone
Published: July 2017

Exosomes are important intercellular communicators in the acute radiation stress-response. Here we show that exosomes from head and neck tumor cells confer protective signals to their neighboring cells. This promotes the tumorigenic and radioresistant phenotype of head and neck cancer cells.

Isolation of exosomes was performed by serial ultracentrifugation of conditioned medium collected from irradiated or non-irradiated head and neck cancer cell lines BHY and FaDu. Quantification by NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response, exosomes isolated from non-irradiated and irradiated donor cells were transferred on non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes, when transferred to irradiated recipient cells compared to the transfer to non-irradiated cells.

Functional analyses indicate increased survival of irradiated recipient cells after exosome transfer. These findings mesh with increased DNA double strand break repair that we found after the transfer of exosomes isolated from irradiated cells. Moreover we observed an increased migration and enhanced chemotaxis induced motility after the transfer of exosomes isolated from irradiated cells. To investigate whether differential protein loading into exosomes is responsible for the functional differences we performed proteome analysis of the exosomes. We have identified 679 proteins, of which 39 were up- and 36 were downregulated by irradiation of the donor cells. In a good agreement with our functional assays, the proteome profiling showed that exosomal proteins were mainly involved in wound healing.

Exosomes are functional components of the response of tumor cells to therapeutic radiation exposure. In a clinical context these results suggests that radiotherapy modified exosomes may influence the cancer progression, metastasis and therapeutic success.

Citation Format: Lisa Mutschelknaus, Omid Azimzadeh, Theresa Heider, Klaudia Winkler, Marcus Vetter, Soile Tapio, Juliane Merl-Pham, Rosemarie Kell, Stephan Huber, Lena Edalat, Vanja Radulovic, Natasa Anastasov, Carsten Peters, Michael John Atkinson, Simone Moertl. Exosomes promote survival and migration in squamous head and neck cancer cells after ionizing radiation: Evidence for a bystander effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5849. doi:10.1158/1538-7445.AM2017-5849
Abstract 585: Chromosome 9p24.1 deletions as a determinant of tumor immune surveillance and immune checkpoint blockade therapy in non-small cell lung cancer
Authors: Shen Tao , Wu Jie
Published: July 2017

Anti-PD-1/PD-L1 immune checkpoint blockade therapy requires the presence of PD-L1/PD-L2 and MHC class I antigens on the tumor cell surface. We previously found JAK1 truncating mutations in endometrial cancer cells that impairs JAK1/JAK2-STAT1-IRF1-mediated signaling pathway, which regulates MHC class I antigen presentation. To determine the potential role and genetic defects in the IFN-γ-IRF1 pathway in non-small cell lung cancer (NSCLC), we analyzed the Cancer Genome Atlas (TCGA) data. Expression of IRF1 correlated with cytolytic activity markers GZMA and PRF1. Loss-of-function (LOF) genetic alterations in the IFN-γ-IRF1 pathway genes were found in 64 cases (6.3%) among 1016 patients. These genetic defects occurred prevalently in the JAK2 gene (33 cases) and often through deletions (29 cases) of chromosome 9p24.1. JAK2 gene deletions were frequently associated with deletions of CD274 and PDCD1LG2 genes that encode PD-L1 and PD-L2, respectively, whereas CD274 and PDCD1LG2 deletions were always accompanied by JAK2 deletions. Chromosome 9p is frequently deleted in NSCLC. This has been attributed to CDKN2A/CDKN2B and PTPRD tumor suppressor genes located at the chromosome 9p21.3-9p24.1. Interestingly, JAK2, CD274, and PDCD1LG2 genes are co-localized at chromosome 9p24.1 near the CDKN2A/CDKN2B and PTPRD genes. JAK2, CD274, and PDCD1LG2 deletion often but not always coincided with CDKN2A/CDKN2B and PTPRD deletions. IFN-γ induced IRF1 expression and cell surface HLA-ABC levels in A549 cells that contain wildtype JAK2 but not in H1573 cells that had a truncating JAK2. Deletion of JAK2 or inhibition of JAK2 kinase activity in A549 cells resulted in loss of IFN-γ-regulated IRF1 and cell surface HLA-ABC, whereas expression of exogenous JAK2 in H1573 cells restored IFN-γ responses. These findings reveal a previously unrecognized significance of chromosome 9p deletion in NSCLC and point to chromosome 9p24.1 as a determinant of immune checkpoint therapy.

Citation Format: Tao Shen, Jie Wu. Chromosome 9p24.1 deletions as a determinant of tumor immune surveillance and immune checkpoint blockade therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 585. doi:10.1158/1538-7445.AM2017-585