bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-003: Androgen-regulated proteome reveals a therapeutically relevant androgen receptor coactivator target in prostate cancer
Authors: Asim Mohammad , Massie Charlie , Warren Anne , Luko Katarina , Chohan Brinder , Menon Suraj , Baridi Ajoeb , Orafidiya Folake , Zhao Wanfeng , Escriu Carlos , Mohammed Hisham , D’Santos Clive , Yang Xiaoping , Taylor Chris , Qureshi Arham , Watt Kate , Zecchini Vincent , Selth Luke , Dehm Scott , Mills Ian , Carroll Jason , Tilley Wayne , McEwan Iain , Baniahmad Aria , Neal David
Published: July 2016

Resistance to androgen deprivation therapy (ADT) and the development of castration resistant prostate cancer (CRPC) remains a major challenge in the treatment of advanced prostate cancer (PCa). Elucidation of resistance mechanisms leading to CRPC is essential to develop novel therapeutic interventions. Using a systems biology approach, we interrogated the androgen-regulated proteome and identified a mitotic Kinase as a novel androgen regulated kinase in PCa. The kinase was shown to be androgen-regulated in PCa and its nuclear localisation significantly increased in CRPC and was associated with biochemical relapse. Interestingly, the Kinase interacted directly with the amino terminus transactivation and carboxy terminus ligand binding domain of androgen receptor (AR), resulting in stabilisation of AR protein and its activity. The AR-driven global transcriptional program was regulated by this coactivator kinase including pathways involved in tumour invasion and metastasis. The inhibition of the kinase function decreased AR levels and activity, and slowed growth of PCa cell lines and human PCa explants. Thus the mitotic kinase is a novel AR-Stablised kinase with potential for clinical applications as both a candidate biomarker of PCa progression and a therapeutic target in advanced disease.

Citation Format: Mohammad Asim, Charlie Massie, Anne Warren, Katarina Luko, Brinder Chohan, Suraj Menon, Ajoeb Baridi, Folake Orafidiya, Wanfeng Zhao, Carlos Escriu, Hisham Mohammed, Clive D’Santos, Xiaoping Yang, Chris Taylor, Arham Qureshi, Kate Watt, Vincent Zecchini, Luke Selth, Scott Dehm, Ian Mills, Jason Carroll, Wayne Tilley, Iain McEwan, Aria Baniahmad, David Neal. Androgen-regulated proteome reveals a therapeutically relevant androgen receptor coactivator target in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-003.
Abstract LB-006: Macrophage-dependent activation of Notch1 signaling regulates breast tumor cell intravasation
Authors: Pignatelli Jeanine , Bravo-Cordero Jose Javier , Roh-Johnson Minna , Gandhi Saumil , Wang Yarong , Singer Robert , Hodgson Louis , Oktay Maja , Condeelis John
Published: July 2016

The process of intravasation, a key component to the metastatic cascade, remains poorly understood. How the multi-cell type tumor microenvironment facilitates tumor cell intravasation is largely unknown. Intravital imaging of rodent mammary tumors has shown that direct contact between a Mena expressing tumor cell, a perivascular macrophage and an endothelial cell forms a microanatomical structure named TMEM (Tumor Microenvironment of Metastasis) which is the site where intravasation occurs in mammary tumors. Clinical studies have shown that the number of TMEM is correlated with increased risk of developing distant metastasis in breast cancer patients. While TMEM is an excellent prognostic marker for predicting metastasis, the mechanisms of TMEM assembly and function are not well understood. Recently, we showed that heterotypic cell contact between tumor cells and macrophages induces the formation of invadopodia in tumor cells, invasive structures necessary for matrix degradation and required for tumor cell intravasation. In further work we found that Notch1, a known receptor involved in heterotypic cell contact signaling, is involved. In the absence of Notch1 signaling, macrophage-induced invadopodium formation and tumor cell intravasation are abolished. This heterotypic tumor cell - macrophage interaction regulates the expression profile of Mena in the tumor cell. Upon touching, the transcription of Mena shows a rapid kinetic response, with detectable changes in single transcriptome activity within 1 hour. Inhibition of Notch1 in vivo results in decreased intravasation of mammary tumor cells. Our findings indicate that Notch1 signaling regulates heterotypic cell contact mediated invadopodium formation, transendothelial migration and intravasation and reveals a novel Notch1/Mena pathway as a molecular target to prevent TMEM function and therefore metastasis.

Citation Format: Jeanine Pignatelli, Jose Javier Bravo-Cordero, Minna Roh-Johnson, Saumil Gandhi, Yarong Wang, Robert Singer, Louis Hodgson, Maja Oktay, John Condeelis. Macrophage-dependent activation of Notch1 signaling regulates breast tumor cell intravasation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-006.
Abstract LB-016: Nutlin and oxaliplatin induce p53-dependent addiction to FLIP
Authors: McIntyre Alexander , Falcone Fiammetta , Johnston Patrick G. , Longley Daniel B. , McDade Simon S.
Published: July 2016

Background: The tumour suppressive functions of the p53 transcription factor are inactivated through mutations or suppressed through non-mutational mechanisms in almost all cancer cells. This work focusses on identifying mechanisms through which, tumours retaining wild-type p53 evade apoptosis in response to chemotherapies and p53 agonists such as Nutlin-3A.

Methods: A panel of matched p53 wild-type and deficient colorectal cancer cell line models were studied, using Nutlin-3A and oxaliplatin as direct and indirect p53 activating agents. A number of molecular (Western blots, RT-PCR), phenotypic (cell death) and genomic analyses were used to investigate the importance of p53 and its downstream transcriptional programs.

Results: We demonstrate the importance of p53 in priming cancer cell sensitivity to apoptotic cell death following treatment with Nutlin-3A or oxaliplatin through activation of pro-apoptotic p53 targets, such as TRAIL-R2, Fas/CD95, PUMA and NOXA. Surprisingly, our analysis further reveals that this priming is accompanied by a parallel early p53-dependent pro-survival response that imposes a critical dependence on the caspase-8 inhibitor FLIP. Preventing FLIP up-regulation with siRNA resulted in synergistic increases in apoptosis in response to Nutlin-3A and oxaliplatin in p53 wild-type, but not p53 null cells. p53-mediated FLIP up-regulation was blocked by pharmacological inhibition of HDACs1-3 using Entinostat, which caused hyper-acetylation of the p53 C-terminus and altered transactivation of a number of other p53 apoptotic target genes. Notably, the synergy between Nutlin-3A or oxaliplatin and Entinostat was abrogated in cells over-expressing FLIP trans-genes or in cells in which caspase-8 was depleted using RNAi.

Conclusion: These results uncover a p53-regulated apoptosis priming event, in which a number of key pro-apoptotic genes are upregulated in response to direct p53 agonism (Nutlin-3A) or indirect activation downstream of DNA damage (oxaliplatin). However, immediate commitment to apoptosis is prevented by the concomitant p53-mediated up-regulation of FLIP. This may be a mechanism that has evolved to allow DNA damage repair time to take place, while priming the cell to undergo apoptosis if the damage is irreparable. From a cancer therapeutics point-of-view, this work identifies FLIP upregulation in p53 wild-type cancers as a key target for overcoming resistance to direct and indirect p53 activating therapies.

Citation Format: Alexander McIntyre, Fiammetta Falcone, Patrick G. Johnston, Daniel B. Longley, Simon S. McDade. Nutlin and oxaliplatin induce p53-dependent addiction to FLIP. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-016.
Abstract LB-027: Single-cell analysis of functional heterogeneity dynamics in premalignant progression revealed by combined interrogation of functional and transcriptional phenotypes
Authors: Kelbauskas Laimonas , Ashili Shashanka , Zeng Jia , Mohammadreza Aida , Lee Kristen , Derkach Dmitry , Ueberroth Benjamin , Gao Weimin , Paulson Thomas , Wang Hong , Tian Yanqing , Smith Dean , Reid Brian , Meldrum Deirdre
Published: July 2016

Intercellular heterogeneity is pivotal in carcinogenesis and tumor development and plays a central role in advanced metastatic disease, therapeutic resistance, and recurrence at the cellular, tissue and whole organism levels. Rapid advances in single-cell analysis technologies have enabled unprecedented insights into cellular machinery. Yet, the ultimate goal of directly correlating the cellular function with the transcriptional profile of a single cell for comprehensive interrogation of the dynamics of cellular variability remains elusive. We present a study on alterations in intercellular diversity in terms of functional and transcriptional phenotypes in the context of selective pressure based on integrated, multiplexed single-cell measurements of cellular respiration and gene transcription levels in the same single cells. Utilizing a Barrett's esophagus premalignant progression model, we investigated alterations in the intercellular diversity resulting from adaptation to the episodes of acute hypoxia that generated stringent selective pressure on a panel of human esophageal epithelial cell lines representing different stages (metaplasia and dysplasia) in the progression. We correlated alterations in cellular respiration with the expression levels of genes involved in the glycolysis and hypoxia response pathways in the same individual cells. Using dimensionality reduction approaches combined with multiparameter analysis we found evidence that population-level functional heterogeneity can be recapitulated and maintained by a small (∼10%) sub-population of cells that survive selective pressure. By combining functional and molecular profiling of the same individual cells we identified unique errant cellular functional phenotypes of individual cells that would remain hidden otherwise, but may harbor clues to phenotypic cancer progression dynamics. To our knowledge, this study is the first successful demonstration of an integrated, multiparameter approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells at different stages of pre-malignant progression.

Citation Format: Laimonas Kelbauskas, Shashanka Ashili, Jia Zeng, Aida Mohammadreza, Kristen Lee, Dmitry Derkach, Benjamin Ueberroth, Weimin Gao, Thomas Paulson, Hong Wang, Yanqing Tian, Dean Smith, Brian Reid, Deirdre Meldrum. Single-cell analysis of functional heterogeneity dynamics in premalignant progression revealed by combined interrogation of functional and transcriptional phenotypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-027.
Abstract LB-046: Mechanism and consequences of HDAC6 regulation by RanBPM
Authors: Salemi Louisa , Wang Xu , Hess David , Schild-Poulter Caroline
Published: July 2016

Ran-binding protein M (RanBPM) has been shown to interact with numerous proteins, implicating it in a variety of cellular processes including cell adhesion, migration, microtubule dynamics, and gene transcription. Work in our laboratory previously identified RanBPM as an activator of apoptosis induced by DNA damage. Our studies have also suggested a tumour suppressor role for RanBPM, as its downregulation not only disrupted apoptotic activation, but lead to loss of growth factor dependence and increased rates of cell migration. Here we show that RanBPM downregulation promotes tumour formation in orthotopic and metastasis mouse models, further suggesting a tumour-suppressive function for RanBPM. Recently, we characterized an interaction between RanBPM and Histone Deacetylase 6 (HDAC6). HDAC6 is a cytoplasmic deacetylase with an important role in cell migration. Its substrates include α-tubulin, cortactin and Hsp90. Increased HDAC6 expression and/or activity has been demonstrated to promote cell migration and tissue invasiveness. HDAC6 has also been shown to be required for oncogenic transformation and tumour formation. Upregulated HDAC6 has been observed in a number of different cancers and recently, specific HDAC6 inhibitors have been found to inhibit cell growth and prevent tumour formation in mouse models. We previously identified that the interaction between RanBPM and HDAC6 mediates the inhibition of HDAC6. Here we demonstrate that the LisH domain of RanBPM specifically, is responsible for the association with HDAC6. RanBPM also interacts with α-tubulin at microtubules and this localization is dependent on HDAC6, whereas HDAC6 localization at microtubules is independent of RanBPM. RanBPM has been identified to be part of a large protein complex, termed the CTLH complex. Components of the complex are conserved from the homologous yeast complex, the Gid complex, which functions as an E3 ubiquitin ligase complex. Here we evaluate HDAC6 regulation by the CTLH complex and asses co-localization of the CTLH complex with microtubules, specifically α-tubulin. Overall, our results suggest that the tumour suppressor functions of RanBPM could stem, at least in part from an inhibition of the oncogenic activities of HDAC6.

Citation Format: Louisa Salemi, Xu Wang, David Hess, Caroline Schild-Poulter. Mechanism and consequences of HDAC6 regulation by RanBPM. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-046.
Abstract LB-051: Cancer cell binding, internalization and intracellular localization of a novel therapeutic peptide SOR-C13 targeting the TRPV6 oncochannel
Authors: Davey Michelle , Dugourd Dominique , Lutes Tyler , Rice Christopher , Gormley Sean , St. Pierre Stephanie , Stewart John M.
Published: July 2016

SOR-C13, a 13-mer peptide derived from soricidin, the paralytic protein component of saliva of the Northern Short-tailed shrew, has recently completed a phase I clinical trial for the treatment of epithelial-derived cancers. SOR-C13 specifically targets and inhibits the TRPV6 calcium channel - a recognized oncochannel over-expressed in a number of epithelial cancers (e.g. breast, ovarian, prostate). SOR-C13 is the first TRPV6-targeting drug to enter clinical development. Xenograft and fluorescent imaging studies provide in vivo evidence of SOR-C13 efficacy in a range of epithelial tumors as well as SOR-C13's preferential accumulation at tumour sites. To demonstrate the specificity of the peptide for TRPV6 and determine its fate, modified SOR-C13 peptide was labeled with fluorescein to enable monitoring of cellular binding and internalization in high versus low TRPV6 cancer cell lines. Various organelle-specific fluorescent trackers for nuclei, actins, lysosomes, and endoplasmic reticula were paired with labeled SOR-C13 peptides and anti-TRPV6 antibody in fluorescence imaging studies to monitor intracellular trafficking. Rapid peptide binding and internalization was observed on incubation of fluorescently labeled SOR-C13 with a breast cancer cell line (T-47D) expressing high levels of the ion channel. Significantly less SOR-C13 binding was observed in an ovarian cancer cell line (SKOV-3) with low TRPV6 levels. Fluorescently labeled SOR-C13 scramble peptide was included as a control and did not bind to high TRPV6 cells. Co-localization studies did not demonstrate a clear association of the fluorescently labeled SOR-C13 with lysosomes, the ER or actin. This study demonstrates that SOR-C13 not only specifically binds to TRPV6-expressing cancer cells according to their level of TRPV6 expression but is also internalized. The correlation of TRPV6 expression in tumors and clinical efficacy will be evaluated during the next SOR-C13 clinical trials. Additionally, these results set the stage for further development of SOR-C13-based peptide drug conjugates for TRPV6 targeted anti-cancer therapies.

Citation Format: Michelle Davey, Dominique Dugourd, Tyler Lutes, Christopher Rice, Sean Gormley, Stephanie St. Pierre, John M. Stewart. Cancer cell binding, internalization and intracellular localization of a novel therapeutic peptide SOR-C13 targeting the TRPV6 oncochannel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-051.
Abstract LB-054: Small molecule inhibitors of the hexosamine biosynthetic pathway and the cancer O-GlcNAcome
Authors: Saeui Christopher T. , Yarema Kevin
Published: July 2016

In recent years, the long-known Warburg effect–wherein cancer cells utilize abnormally high levels of glucose by forgoing oxidative phosphorylation–has been rediscovered but its impact on flux through the hexosamine biosynthetic pathway (HBP) remains relatively unexplored. In this work, we reasoned that UDP-GlcNAc, the end product of the HBP, could promote cancer by increasing the activity of O-GlcNAc transferase (OGT) and subsequently perturb global patterns of protein O-GlcNAcylation. To combat increased O-GlcNAcylation, which is increasingly being linked to cancer cell survival, metastasis, and tumor aggressiveness, most efforts to date have focused on direct inhibition of OGT. In contrast, we take an alternate approach by developing first-in-class small molecules that target UDP-N-acetylglucosamine pyrophosphorylase 1/2 (UAP1/2), the final enzyme of the HBP; this strategy has the added benefit of also potentially reducing the production of highly-branched N-glycans associated with cancer and other diseases. In our first biological tests, presented here, we show that our UAP1/2 inhibitors decrease intracellular UDP-GlcNAc levels as intended and modulate the cancer O-GlcNAcome in a way consistent with reduced viability of difficult-to-treat pancreatic and brain cancer cell lines observed in treated cells. Finally, our strategy of targeting UAP1/2 and the cancer O-GlcNAcome decreased the expression of oncoproteins such as c-Myc, YAP, and TAZ which are all linked to highly aggressive and difficult-to-treat types of cancer. In summary, we have developed first-in-class of small molecules that successfully target UAP1/2, inhibit flux through the HBP, reduce global patterns of O-GlcNAcylation, and knock-down several endpoints linked to cancer progression.

Citation Format: Christopher T. Saeui, Kevin Yarema. Small molecule inhibitors of the hexosamine biosynthetic pathway and the cancer O-GlcNAcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-054.
Abstract LB-060: A novel target for genetic silencing of AXL mediates taxane resistance, invasion, and migration in uterine cancer
Authors: Palisoul Marguerite , Nguyen Mai , Pan Hua , Lohrey Anne , Wickline Samuel , Powell Matthew , Mutch David , Fuh Katherine
Published: July 2016

Objectives: We investigated the role of AXL expression in chemotherapy response in uterine papillary serous cancer, and sought to reverse chemoresistance through inactivation of AXL. Methods: In vitro cell viability (XTT) assays were performed to confirm response to chemotherapy in AN3CA (taxane sensitive) and ARK1 (taxane resistant) uterine cell lines. AXL knockdown by siRNA was performed with either a traditional transfection reagent (DharmaFECT) or with a novel serum-stable, cell-penetrating, endosomolytic amphipathic peptide delivery system (p5RHH). Following knockdown of AXL using siRNA and p5RHH, chemotherapy response was assessed by XTT assay and invasion and migration assays were performed. Cell viability assays were analyzed using multiple t-tests. P < 0.05 was considered statistically significant. Results: Expression of AXL was found to be associated with a worse prognosis in uterine cancer. Chemoresistance was validated with XTT assays for AN3CA and ARK1. Western blotting confirmed over-expression of AXL in the chemoresistant cell line, and lack of AXL expression in its chemosensitive counterparts. Reversal of paclitaxel resistance was achieved in ARK1 cells genetically by silencing AXL with shRNA. The novel delivery system, p5RHH, combined to siAXL, was shown to inactivate AXL in ARK1 cells at the RNA and protein level to a similar extent as traditional transfection technique (8.4-fold vs 8.7-fold decrease for RNA, respectively; 67% vs 83% knockdown at 48h and 84% vs 97% knockdown at 96 hours for protein, respectively). Reversal of paclitaxel resistance was achieved in ARK1 cells therapeutically and genetically using the p5RHH-siAXL complex. With AXL silencing using the p5RHH-siAXL complex, tumor cell invasion was inhibited by greater than 40%, and tumor cell migration was inhibited by greater than 60%. Conclusions: AXL expression is associated with chemoresistance in a uterine papillary serous cancer cells. Genetic inactivation of AXL in a taxane-resistant uterine cancer cells reverses chemoresistance. A novel siRNA nano-particle delivery system was successfully utilized to mimic genetic inactivation resulting in improved chemotherapy response and decreased invasion and migration.

Citation Format: Marguerite Palisoul, Mai Nguyen, Hua Pan, Anne Lohrey, Samuel Wickline, Matthew Powell, David Mutch, Katherine Fuh. A novel target for genetic silencing of AXL mediates taxane resistance, invasion, and migration in uterine cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-060.
Abstract LB-071: Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism
Authors: Lito Piro , Solomon Martha , Hansen Rasmus , Li Lian-Sheng , Rosen Neal
Published: July 2016

Oncogenic mutations impair the GTPase activity of RAS, causing it to accumulate in the activated, GTP-bound conformation. Here we describe the mechanism of action of a novel compound that binds to KRASG12C to selectively inhibit proliferation and induce apoptosis in cancer cells with this mutation. The inhibitor interacted preferentially with the GDP-bound form of the protein, yet it reduced the level of GTP-bound KRASG12C. This suggested that GTPase activity is required for inhibition, which turned out to be the case. The inhibitor traps GDP-bound KRASG12C and prevents nucleotide exchange to the GTP-bound form. Genetic experiments revealed that mutants completely lacking GTPase activity, and those promoting exchange, reduced the potency of the drug. In contrast, mutants with diminished exchange were more susceptible to inhibition. In agreement with these findings, targeting receptor signaling potentiated KRASG12C inhibition, whereas its activation had the opposite effect. This mechanism reveals that KRASG12C undergoes nucleotide cycle in cancer cells and provides a basis for the development of effective combination therapies to treat KRASG12C-driven cancers.

Citation Format: Piro Lito, Martha Solomon, Rasmus Hansen, Lian-Sheng Li, Neal Rosen. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-071.
Abstract LB-077: T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy
Authors: Raber Patrick , Sierra Rosa A. , Zhang Shuzhong , Thevenot Paul , Rodriguez Paulo C.
Published: July 2016

The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ T cells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of strategies to increase ACT responses in cancer.

Citation Format: Patrick Raber, Rosa A. Sierra, Shuzhong Zhang, Paul Thevenot, Paulo C. Rodriguez. T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-077.
Abstract LB-090: Sequence-based discovery of fully human anti-CD3 and anti-PDL1 single domain antibodies using novel transgenic rats
Authors: Trinklein Nathan , Force Aldred Shelley , Schellenberger Ute , Harris Kat , Clarke Starlynn , Dang Kevin , Pham Duy , Bruggemann Marianne , vanSchooten. Wim
Published: July 2016

We have created a new platform and method for discovering fully human single domain antibodies (UniDabs) by combining novel transgenic rats (UniRats), antibody repertoire sequencing, and high-throughput gene assembly, expression and screening. UniRats express a comprehensive human VH gene repertoire in a heavy chain only antibody format, and they mount robust immune responses to a variety of antigens. UniRats do not produce endogenous Ig as their heavy, kappa and lambda loci have been silenced, so the only Ig expression comes from the human heavy chain transgenes. Human VHs identified using high-throughput functional screening of heavy chain antibodies may also be converted to a single domain UniDab format. UniDabs are powerful and flexible modular binding domains that may be combined to form multivalent structures, nanoparticle conjugates, or antibody-drug conjugates. In our preclinical CD3 and PDL1 programs, we have identified hundreds of antigen-specific UniDabs. This large collection of UniDabs covers a diverse set of epitopes, a wide range of affinities, and is a valuable resource for creating next-generation multi-specific cancer therapeutics.

Citation Format: Nathan Trinklein, Shelley Force Aldred, Ute Schellenberger, Kat Harris, Starlynn Clarke, Kevin Dang, Duy Pham, Marianne Bruggemann, Wim vanSchooten. Sequence-based discovery of fully human anti-CD3 and anti-PDL1 single domain antibodies using novel transgenic rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-090.
Abstract LB-091: Inhibition of Siglec-15 prevents bone loss in a mouse model of multiple myeloma
Authors: Tremblay Gilles B. , Moraitis Anna , Vanderkerken Karin , Filion Mario
Published: July 2016

Multiple myeloma (MM) is a B-cell malignancy associated with infiltration and growth of plasma cells in the bone marrow resulting in end-organ damage including devastating osteolytic bone disease. We examined the ability of 25B2, an antibody targeting Siglec-15, to prevent bone loss in the 5T2MM syngeneic model of MM. C57BlKaLwRij male mice (n = 10/group) were injected with 5T2MM cells and 24h later treated for 12 weeks with the PBS, 25B2 (10 mg/kg, q3d), bortezomib (0.7 mg/kg, q3d) or a combination of 25B2 and bortezomib. 25B2 treatment caused a 3.7-, 5.4- and 1.2-fold increase in bone volume (%BV/TV) compared to PBS-treated mice in the tibia, femur and lumbar vertebra (LV), respectively. Increases in bone volume were also seen with bortezomib with 3.1-, 4.1- and 1.4-fold increases in the tibia, femur and LV, respectively. Combination of the two agents further increased%BV/TV by 6.9-, 10.5- and 1.5-fold in the tibia, femur and LV, respectively, when compared to the PBS treated mice. All of the changes were statistically significant with the exception of those in the LV. Unlike bortezomib treatment, which inhibits the growth of 5T2MM cells in this model, there was no direct effect of 25B2 on tumor burden in 5T2MM mice when the percentage of plasmacytosis and the serum paraprotein levels were measured. However, 25B2 caused a significant decrease in the number of bone lesions by 49.5%. The number of lesions in the bortezomib group decreased by 71.7%, but there was no significant difference between the 25B2 and bortezomib groups. The number of lesions decreased by 89.4% in the combination group. Consistent with the mechanism of action of Siglec-15 antibodies, osteoclasts were rendered inactive, however cross-talk with bone-forming osteoblasts was likely maintained. This unique mechanism of action would be particularly appropriate for MM, where osteolytic disease is not only the result of increased osteoclast activity but also decreased osteoblast-mediated bone formation. The 5T2MM model is one of the most representative of the human pathology and therefore, the effect observed in response to the Siglec-15 antibody holds the promise that such a treatment in MM patients could provide benefit and further delay skeletal related events.

Citation Format: Gilles B. Tremblay, Anna Moraitis, Karin Vanderkerken, Mario Filion. Inhibition of Siglec-15 prevents bone loss in a mouse model of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-091.
Abstract LB-134: Sequential validation of blood-based protein biomarker candidates for early-stage pancreatic cancer
Authors: Capello Michela , Bantis Leonidas E. , Scelo Ghislaine , Zhao Yang , Dhillon Dilsher S. , Wang Hong , Abbruzzese James L. , Maitra Anirban , Tempero Margaret A. , Brand Randall , Firpo Matthew A. , Mulvihill Sean , Katz Matthew H. , Brennan Paul , Feng Ziding , Hanash Samir M. , Taguchi Ayumu
Published: July 2016

Purpose: CA19-9, which is currently in clinical use as a pancreatic ductal adenocarcinoma (PDAC) biomarker, has limited performance in detecting early stage disease. We and others have identified protein biomarker candidates that have the potential to complement CA19.9. We have carried out sequential validations starting with 32 protein biomarker candidates to develop a marker panel to improve detection of early stage disease compared with CA19.9 alone.

Patients and Methods: Multiple independent sample sets consisting of PDAC cases, benign pancreatic disease and healthy controls were utilized for immunoassays of candidate markers which after sequential validations yielded a panel of three markers that was further tested in a blinded setting.

Results: The 32 biomarker candidates were initially tested in a sample set consisting of 75 PDAC cases, 29 healthy controls, and 19 chronic pancreatitis cases which narrowed the list to seven biomarker candidates. A second independent set of 73 early-stage PDAC cases, 60 healthy controls, and 74 benign pancreatic disease cases (60 chronic pancreatitis cases and 14 benign pancreatic cyst cases) yielded a panel of three markers, including CA19-9, with an AUC (95%CI) of 0.949 (0.917-0.981) and sensitivity of 0.849 at 95% specificity. Additional blinded testing of the model was done using an independent set of plasma samples from 39 resectable PDAC patients and 82 matched healthy controls, yielding an AUC of 0.887. The performance of the biomarker panel was significantly improved compared to CA19-9 alone (P < 0.001) in both plasma sample sets.

Conclusion: We validated a protein-based three marker panel with significantly improved performance compared to CA19-9 with the potential to contribute to strategies for detecting early-stage PDAC.

Citation Format: Michela Capello, Leonidas E. Bantis, Ghislaine Scelo, Yang Zhao, Dilsher S. Dhillon, Hong Wang, James L. Abbruzzese, Anirban Maitra, Margaret A. Tempero, Randall Brand, Matthew A. Firpo, Sean Mulvihill, Matthew H. Katz, Paul Brennan, Ziding Feng, Samir M. Hanash, Ayumu Taguchi. Sequential validation of blood-based protein biomarker candidates for early-stage pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-134.
Abstract LB-156: Delineating the pathological roles and molecular functions of histone methyltransferase G9a in hepatocellular carcinoma
Authors: Wei Lei , Tsang Felice Ho-Ching , Au Sandy Leung-Kuen , Lee Joyce , Wong Carmen , Ng Irene Oi-Lin , Wong Chun-Ming
Published: July 2016

Liver cancer, primarily hepatocellular carcinoma (HCC), is the third leading cause of cancer mortality worldwide. In HCC, epigenetic changes including aberrant DNA methylations and histone modifications occur far more frequently than genetic alternations. However, the underlying mechanism and specific gene targets are still unknown. Using RNA-Sequencing, we identified Euchromatic histone-lysine N-methyltransferase 2 (EHMT2/G9a) as one the most significantly deregulated epigenetic regulatory genes in human HCC. G9a is a SET domain containing histone methyltransferase specific for histone H3 lysine 9 methylation. However, little is known about the functional roles of G9a in human HCC. Herein, we showed that G9a expression was frequently up-regulated in primary HCC. Up-regulation of G9a was significantly associated with multiple clinicopathological features related to HCC metastasis. We demonstrated that the frequent G9a up-regulation in HCC was attributed to the G9a gene copy number gain. In addition, we identified miR-1 as a novel post-transcriptional regulator of G9a. Loss of miR-1 significantly contributed to G9a up-regulation in human HCC. Functionally, stable knockdown (by lenti-viral based shRNA) and genetic knock-out of G9a (by CRISPR/Cas9 gene editing system) significantly suppressed HCC cell proliferation, migration and colony formation. We also demonstrated that G9a specific inhibitors, UNC0638 and BIX01294, could effectively inhibit HCC cell growth. By utilizing nude mice model, we showed that depletion of G9a drastically inhibited in vivo tumorigenicity of HCC cells. Mechanistically, we found that up-regulation of G9a epigenetically silence the expression of tumor suppressive gene RARRES3 to promote HCC tumorigenicity and metastasis. In summary, our finding suggested that up-regulation of G9a contributed to the liver carcinogenesis and could be a therapeutic target for HCC treatment.

Citation Format: Lei Wei, Felice Ho-Ching Tsang, Sandy Leung-Kuen Au, Joyce Lee, Carmen Wong, Irene Oi-Lin Ng, Chun-Ming Wong. Delineating the pathological roles and molecular functions of histone methyltransferase G9a in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-156.
Abstract LB-164: DNA ligase IV modulates the cellular response to DNA replication stress
Authors: Hooks Grace , Anchondo Yasmin , Sharma Neelam , Nickoloff Jac , Ashley Amanda
Published: July 2016

Correctly repairing DNA damage is crucial to the survival and genomic integrity of cells. Double strand breaks (DSBs) are particularly problematic DNA damage; failure to properly repair DSBs can precede genomic instability leading to cancer. Because of the high proliferative index of cancer cells, replication toxins are successfully used to treat cancer. The two major DNA DSB repair pathways are homologous recombination and nonhomologous end-joining (NHEJ). DNA ligase IV is a central component of NHEJ, performing the final ligation step to reseal the broken DNA ends, in concert with XRCC4. We determined cells lacking the early acting NHEJ protein, and DNA damage response signaling kinase, DNA-PK, restarted DNA replication forks quicker than wild-type following replication stress. The role of other NHEJ proteins in replication fork restart is unclear. Our goal was to investigate whether ligase IV, a protein that appears to function exclusively in NHEJ, is involved in the replication stress response. We tested the hypothesis that ligase IV delays replication restart following exposure to replication toxins. Ligase IV knockdown was confirmed by qPCR and western blotting. siRNA knockdown of ligase IV alone reduced cell viability and it reduced EdU incorporation after replication stress. The opposite effects of DNA-PK and ligase IV on the speed of replication fork restart suggests that NHEJ, per se, does not directly affect replication fork restart. We previously demonstrated that DNA-PK regulates replication fork restart, at least in part, by phosphorylating RPA32, which in turn regulates ATR activation and downstream checkpoint functions. Ligase IV deficiency may influence replication fork restart indirectly, for example, by causing spontaneous DSBs to persist longer, limiting availability of repair factors needed for fork restart (saturation effect). A not mutually exclusive possibility is that fork restart is hindered by persistent DSBs due to checkpoint activation. Understanding how NHEJ proteins impact the replication stress response may provide opportunities for developing more effective cancer treatments.

Citation Format: Grace Hooks, Yasmin Anchondo, Neelam Sharma, Jac Nickoloff, Amanda Ashley. DNA ligase IV modulates the cellular response to DNA replication stress. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-164.
Abstract LB-175: ATM regulates radiation-induced translational control of gene expression
Authors: Lehman Stacey L. , Wahba Amy , Camphausen Kevin , Tofilon Philip J.
Published: July 2016

The cellular radioresponse is composed of processes regulated by constitutively expressed proteins and processes regulated by new gene expression. While processes regulated by constitutively expressed proteins, such as those participating in DNA repair and cell cycle arrest, have been extensively studied, the radiation-induced changes in gene expression are not well defined. Previously, we have demonstrated that translation plays an important role in regulating gene expression in response to radiation. In glioblastoma cells, treatment with radiation affects more genes at the translational level than at the transcriptional level. Specific transcripts were found to be recruited to or away from polysomes without global changes in the polysome profile of the cells. Here, we utilized microarray analysis of polysome-associated transcripts in the glioblastoma stem-like cell line NSC11 to demonstrate that the DNA damage response kinase ATM controls the translation of a subset of genes as part of the radioresponse. Cells were incubated with the ATM inhibitor KU-60019 at a dose of 500nM for one hour, exposed to 2 Gy, and harvested for polysome extraction one hour after irradiation. ATM inhibition blocked translational changes of approximately one third of genes that were affected by radiation. Gene enrichment analysis revealed that genes translationally regulated by ATM were significantly involved in a number of cellular processes, indicating that ATM-mediated translational control may have functional consequences on the cellular radioresponse. By GSEA, cells treated with radiation alone were highly enriched for terms involving mitochondrial structure and function, cellular respiration, and oxidative stress as compared to irradiated cells treated with the ATM inhibitor. Other significantly enriched categories of functional interest included the spliceosome, RNA catabolic processes, the small nuclear ribonucleoprotein complex, and phospholipid and phosphoinositide metabolism. Analysis of UTR regulatory elements revealed that a significant percentage of the genes translationally upregulated by ATM in irradiated cells were enriched for HuR binding sites, which suggests a potential mechanism for polysome recruitment of these transcripts. Overall, these findings uncover a novel network of post-transcriptional gene regulation orchestrated by ATM and provide a framework to identify potential new molecules and pathways that govern tumor cell radiosensitivity.

Citation Format: Stacey L. Lehman, Amy Wahba, Kevin Camphausen, Philip J. Tofilon. ATM regulates radiation-induced translational control of gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-175.
Abstract LB-194: AhR Arg554Lys impacts tumor ER expression and endocrine treatment response in breast cancer
Authors: Simonsson Maria , Markkula Andrea , Rose Carsten , Ingvar Christian , Jernström Helena C.
Published: July 2016

Introduction: Markers for treatment resistance in breast cancer are needed. The Aryl hydrocarbon receptor (AhR) is involved in the regulation of estrogen metabolism. Previous studies have observed a crosstalk between AhR and the estrogen receptor (ER), indicating that the AhR may be of importance in the response of endocrine treatment.

Materials and methods: A functional polymorphism in the AhR Arg554Lys (G>A) was analyzed in a cohort of 634 breast cancer patients included at their preoperative visits in Lund, Sweden between 2002 and 2008. AhR genotypes were studied in relation to ER status and risk for breast cancer events.

Results: The frequencies of the AhR GG/ GA/ AA genotypes were 82.0%, 16.7%, and 1.3%, respectively. There was a trend towards increasing frequency of ER+ tumors with increasing number of A-alleles (P-trend = 0.03). Since few patients had the A/A genotype, patients with the G/A and A/A genotypes were combined in the survival analyses of the 576 patients with invasive tumors, no preoperative treatment, and no events detected on the postoperative metastases screen.

Overall, AhR was not associated with event-free survival (LogRank P = 0.22). Among patients ever treated with chemotherapy or radiotherapy, AhR was not associated with event-free survival (LogRank P = 0.24 and P = 0.18, respectively). However, among tamoxifen-treated patients with ER+ tumors, AhR G/G carriers had a significantly lower risk for breast cancer events (LogRank P = 0.005), adjusted HR 0.53 (95% CI 0.29-0.94). This association was confined to patients who had received both tamoxifen and aromatase inhibitor (AI) switch treatment (LogRank P = 0.0002), adjusted HR 0.23 (0.09-055), while no association was seen in patients treated with tamoxifen only (LogRank P = 0.56).

AI-treated patients with the G/G genotype had an even lower risk for breast cancer events than tamoxifen-treated patients (LogRank P = 0.005), adjusted HR 0.39 (0.19-0.81). Again this was only seen in patients who had received both AI and tamoxifen, but not in patients who had received AI but no tamoxifen treatment. Adjustments were made for age at inclusion, axillary lymph node involvement, invasive tumor size, and histological grade. Further adjustment for smoking status did not materially change the results.

Conclusion: The functional AhR Arg554Lys polymorphism may impact tumor ER expression and response to endocrine switch treatment with both AI and tamoxifen in breast cancer patients.

Citation Format: Maria Simonsson, Andrea Markkula, Carsten Rose, Christian Ingvar, Helena C. Jernström. AhR Arg554Lys impacts tumor ER expression and endocrine treatment response in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-194.
Abstract LB-221: Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity, unique sequence homology and in vitro and in vivo antitumor efficacy
Authors: Eaverone David , Behrens Jeff , Prendergast Jillian
Published: July 2016

Siamab Therapeutics is developing monoclonal antibodies and antibody drug conjugates (ADCs) targeting tumor-associated carbohydrate antigens such as Sialyl-Tn (STn). These antibodies are selected using a proprietary glycan microarray that enriches for candidates whose binding is protein independent, highly specific and demonstrates exceptional target affinity. STn is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy.

Siamab has developed over 20 murine mAbs that bind STn with high affinity and specificity. When formatted as ADCs, anti-STn mAb-ADCs target and selectively kill STn expressing cells and also demonstrate efficacy in multiple xenograft studies. Multiple murine mAbs perform well in IHC studies with multiple solid tumor samples and tissue microarrays and demonstrate high tumor specificity. Remarkable sequence homology across all murine mAbs was observed in both heavy and light chains, and hot spots for hypermutation were identified. Humanization was successfully completed using a selection of mAbs and multiple humanized mAbs maintain their target specific binding, affinity and cytotoxicity in vitro. Our data demonstrates that high-affinity, STn-specific mAbs show promise both as therapies for solid tumors and as a potential tool for the development of biomarkers and companion diagnostics enabling selection of patients most likely to respond to therapy.

Citation Format: David Eaverone, Jeff Behrens, Jillian Prendergast. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates (ADCs) demonstrate tumor specificity, unique sequence homology and in vitro and in vivo antitumor efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-221.