bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3949: Rapid 5-marker multiplex phenotyping of breast cancer subtypes & tumor-infiltrating leukocytes “in situ” in FFPE sections
Authors: Levin Matt , Kron Stephen J. , Schwartz David , Snyder Helen
Published: July 2016

Breast cancer can be divided into four subgroups HER2+, Luminal A, Luminal B and triple negative using multiple clinically validated monoclonal antibodies. Each subtype presents a different prognosis and treatment option. It is also increasingly clear that immune subsets within the tumor microenvironment may also have a bearing on prognosis, with certain subsets being associated with increased patient survival. Determining the tumor and immunologic phenotype of initial tumor biopsies both in the composition of cells and their relative spatial distribution is becoming key in selecting optimal treatment. The aim of our research has been to develop a multiplex immunofluorescence technology that can rapidly and simultaneously detect 5+ markers on a single section, allowing multiparametric quantitative analysis of cell phenotype and tissue composition as well as providing important spatial information of cells relative to each other. Our multiplex immunofluorescence (mxIF), modified-hapten based technology allows the simultaneous detection of multiple biomarkers using a standard two-step procedure. The technology is antibody species independent and produces signals 3-4X stronger than direct fluor-labeled secondary antibodies. It can detect multiple co-localized markers and while it has been engineered to detect four biomarkers and DAPI, it has the potential for significantly higher multiplexing. In addition it is modular and can be readily modified for any multiplex panel. The data generated from a staining experiment can be readily exported into any image analysis program. In comparison to existing technologies from GE and PerkinElmer, our technology allows multiple targets to be interrogated rapidly and simultaneously without the need for multiple rounds of stripping and imaging. The breast cancer phenotyping panel (BC-1) provides rapid phenotyping information allowing identification of Luminal A, Luminal B, HER2+ or triple negative cancer. Our immuno-oncology panel (IO-1) identifies CD4 and CD8 T cells, CD20+ B cells and CD68+ macrophages. Application of each panel to one of two sequential sections and then image overlay allows both tumor and immunophenotyping. The modular and non-species specific nature of this technology means that panels can be modified and expanded to characterize multiple different tumors and immune cell subpopulations in a rapid, sensitive and quantitative manner.

Citation Format: Matt Levin, Stephen J. Kron, David Schwartz, Helen Snyder. Rapid 5-marker multiplex phenotyping of breast cancer subtypes & tumor-infiltrating leukocytes “in situ” in FFPE sections. [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 3949.
Abstract 4094: Exosomes secreted from the noncancerous microenvironment promote proliferation, migration, and invasion in breast cancer cells
Authors: Yearby Letitia A. , Cunningham Terri , Matthews Jacenta , Lemieux. KiTani Parker
Published: July 2016

Introduction: Triple negative breast cancer (TNBC) is one of the most aggressive forms of five major types of breast cancer. Because TNBC mortality disproportionately affects premenopausal African American women, a health disparity exists within this group. Our research has provided insight on how the noncancerous microenvironment plays a role in TNBC proliferation, migration, and invasion. Exosomes, extracellular vesicles (30-300 nm) produced ubiquitously by mammalian cells, are secreted by noncancerous breast epithelial cells, and have been noted to enhance proliferation, migration and invasion in TNBC. Therefore, the purpose of this study is to determine if exosome-free conditioned media (EFCM), which contains noncancerous MCF10A exosomes, enhances proliferation, migration and invasion in TNBC.

Methods: MDA-MB-231 and MCF10A breast epithelial cells were used as cell line models. Proliferation was measured by the alamar blue proliferation assay. The transwell migration assay was used to assess migration, and the Matrigel invasion assay was used to measure migration. ExoELISA assays were also used to detect exosomes in exosome-free conditioned media (EFCM) from MCF10A cells. MCF10A cells were serum-starved for 24 hours, using DMEM/F-12 with 0.5% standard or exosome-depleted FBS (edFBS), to produce exosomal conditioned media (CM) and EFCM. This media was harvested and sterile filtered before use. MDA-MB-231 cells were treated with EFCM in each of the bioassays, and RPMI-1640 was used as a control.

Results: MCF10A exosomes introduced via EFCM enhanced proliferation, migration and invasion of MDA-MB-231 cells.

Conclusions: Taken together, our findings demonstrate that secreted factors from noncancerous MCF10A cells that have been stressed as a direct result of serum starvation are exosomes. These exosomes recruit TNBC cells to increase proliferation, migration, and invasion. The role of these exosomes in the noncancerous microenvironment is poorly understood, but these data suggest that exosomes from the noncancerous microenvironment have potential to be used in targeted therapies against triple negative breast disease.

Citation Format: Letitia A. Yearby, Terri Cunningham, Jacenta Matthews, KiTani Parker Lemieux. Exosomes secreted from the noncancerous microenvironment promote proliferation, migration, and invasion in breast cancer cells. [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 4094.
Abstract 4123: Thymoquinone inhibits bone metastasis in a breast cancer mouse model by modulating CXCR4/CXCL12 signaling axis
Authors: Shanmugam Muthu K. , Hsu Annie , Hui Kam Man , Tan Benny K.H. , Sethi Gautam
Published: July 2016

Several lines of evidence(s) indicate that CXCR4 overexpression has been correlated with distant site metastasis and poor overall survival rate in patient with breast cancer. The tumor metastasis promoting molecule CXCR4 is considered as a potential therapeutic target for inhibiting breast cancer metastasis. Thus, novel agents that can down-regulate CXCR4 expression have potential against breast cancer metastasis. In the present report we investigated the effect of thymoquinone (TQ), derived from the seeds of medicinal plant Nigella sativa, on the expression and regulation of CXCR4 in breast cancer cells. In addition, we evaluated the effect of TQ in a metastasis mouse model established by intracardiac injection of luciferase tagged MDA-MB-231 breast cancer cells that metastasize to the bones. We observed that TQ could inhibit the expression of CXCR4 in MCF-7 and MDA-MB-231 cells in a dose and time dependent manner. TQ (2 mg/kg or 4 mg/kg) treatment for four weeks significantly inhibited tumor growth and significantly reduced metastasis to multiple vital organs, including lungs, brain and bone. Immuno-histochemical analysis of the lung and brain tissue showed significant reduction in CXCR4, Ki67, MMP9, VEGFR2 and COX2 expression compared to control tissues. TQ treatment also reduced the overall bone tumor burden. Overall, our results show that TQ exerts its antitumor and anti-metastatic effects by downregulation of CXCR4 expression both in vitro and in vivo thus may have possible potential for the treatment of breast cancer.

Acknowledgement: This work was supported by NUHS Basic Seed Research grant to Prof. Benny Tan.

Citation Format: Muthu K. Shanmugam, Annie Hsu, Kam Man Hui, Benny K.H. Tan, Gautam Sethi. Thymoquinone inhibits bone metastasis in a breast cancer mouse model by modulating CXCR4/CXCL12 signaling axis. [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 4123.
Abstract 4187: Loss of SLFN11 or gain of TWIST1 promote chemotherapy resistance in small cell lung cancer
Authors: Gardner Eric E. , Lok Benjamin H. , Schneeberger Valentina E. , de Stanchina Elisa , Poirier John T. , Rudin Charles M.
Published: July 2016

The majority of patients with small cell lung cancer (SCLC) who are treated with first-line platinum/etoposide chemotherapy develop therapeutic resistance, but little is mechanistically understood how resistance develops in patients. This is partly due to limited access to historic paired tissue samples, few investigational re-biopsy programs and the aggressive nature of this disease.

In this study we used a collection of 10 treatment-naïve, patient-derived xenograft (PDX) models of SCLC to determine how resistance to chemotherapy can be acquired in vivo, by cycling weekly cisplatin and etoposide, followed by serial passaging of tumor cells capable of surviving and proliferating through treatment. Applying whole transcriptome (RNAseq), exome and high-depth targeted sequencing approaches to paired parental and resistant populations revealed minimal recurrent mutations; however, two consistent gene expression changes were observed, implicating either a decrease in SLFN11 or an increase in TWIST1 transcript levels may promote resistance. We validated both mechanisms in vitro using conditional gain and loss of function experiments in SCLC cell lines of human and mouse origin. Where suppression of SLFN11 was associated with resistance to select classes of DNA damaging agents, gaining TWIST1 produced a broad resistance phenotype, consistent with an epithelial-to-mesenchymal transition.

Increased sensitivity to chemotherapy through overexpression of SLFN11 was observed only in cell lines with low levels of TWIST1. Importantly, TWIST1 knockdown or knockout did not dramatically change the sensitivity to chemotherapy in multiple TWIST1-high cell lines tested, suggesting that directly targeting TWIST1 may be of limited utility in SCLC. We extended these results by modeling acquired resistance to cisplatin and etoposide in a triple knockout (p53/Rb/p130) mouse allograft model of SCLC, finding that increased expression of TWIST1 was associated with chemotherapy resistance, but did not confer a greater metastatic potential in a tumor type with an already high predilection for metastasis. Understanding the relative expression of SLFN11 and TWIST1 at baseline and upon clinical relapse may be informative in determining therapeutic options for SCLC patients that no longer respond to platinum and etoposide.

Citation Format: Eric E. Gardner, Benjamin H. Lok, Valentina E. Schneeberger, Elisa de Stanchina, John T. Poirier, Charles M. Rudin. Loss of SLFN11 or gain of TWIST1 promote chemotherapy resistance in small cell lung 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 4187.
Abstract 4192: The association between gastric Mucin 5AC (MUC5AC) expressions, transforming growth factor-β (TGF-β) signal, and histopathological phenotypes of lung cancer in xenograft models
Authors: Sato Ryo , Semba Takashi , Kohrogi Hirotsugu , Saya Hideyuki , Arima Yoshimi
Published: July 2016

According to the 2015 World Health Organization (WHO) classification of lung tumors, invasive lung adenocarcinomas are pathologically classified as lepidic, acinar, papillary, micropapillary, or solid types. Adding the cribriform pattern as an additional subtype to the original WHO classification has recently been proposed, because patients with cribriform predominant tumors have a poorer prognosis than that for patients with acinar or papillary predominant tumors. Understanding the molecular mechanisms contributing to the pathological subtypes may provide a basis for the development of new therapeutic strategies and an improved prognosis for lung cancer patients.

We found that the human lung adenocarcinoma A549 cells developed distinct histological types of tumors; acinar types and mucinous cribriform types, when cells were inoculated into immunodeficient mice. Acinar type tumors showed a gland-like pattern containing stromal cells, and cribriform type tumors contained abundant acidic mucins stained with Alcian blue. We also found that the cribriform type tumors, but not acinar type tumors, expressed Mucin 5AC (MUC5AC), which is one of the common mucin core proteins. In contrast, acinar type tumors upregulated the phosphorylation of Smad3, which is the downstream of the transforming growth factor-β (TGF-β) signal, indicating that the TGF-β/Smad pathway is activated in acinar type tumors. We went on to culture A549 cells in several conditions and found that MUC5AC is upregulated in serum-free floating culture conditions. To determine the relationship between TGF-β and MUC5AC expressions, TGF-β1 was added to the culture medium, and we found that TGF-β1 completely inhibited MUC5AC induction.

These data imply that cancer cells and/or cancer associated fibroblasts (CAFs) release TGF-β1 and inhibit MUC5AC expressions, leading to acinar type tumor formations.

Citation Format: Ryo Sato, Takashi Semba, Hirotsugu Kohrogi, Hideyuki Saya, Yoshimi Arima. The association between gastric Mucin 5AC (MUC5AC) expressions, transforming growth factor-β (TGF-β) signal, and histopathological phenotypes of lung cancer in xenograft models. [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 4192.
Abstract 4422: Extracellularly secreted APE1/Ref-1 triggers apoptosis in triple-negative breast cancer cells via RAGE binding, which is mediated through acetylation
Authors: Lee Yu Ran , Joo Hee Kyoung , Kim Ki Mo , Jeon Byeong Hwa , Choi Sunga
Published: July 2016

The present study evaluated the mechanism of apoptosis caused by posttranslational modification, hyperacetylation in triple-negative breast cancer (TNBC) cells. We previously showed that trichostatin A (TSA) induced secretion of acetylated apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1). This is the first report showing that Ac-APE1/Ref-1 initiates apoptosis in TNBC cells by binding to the receptor for advanced glycation end products (RAGE). The functional significance of secreted Ac-APE1/Ref-1 was studied by induction of intracellular hyperacetylation through co-treatment with acetylsalicylic acid and TSA in MDA-MB-231 cells. In response to hyperacetylation, secretion of Ac-APE1/Ref-1 in vesicles was observed, resulting in significantly decreased cell viability and induction of apoptosis with increased expression of RAGE. The hyperacetylation-induced apoptosis was similar in two other TNBC cell lines: BT-459 and MDA-MB-468. Therefore, hyperacetylation may be a therapeutic target for treatment of TNBCs. This study introduces a novel paradigm whereby posttranslational modification induces apoptotic cell death in breast cancer cells resistant to standard chemotherapeutic agents through secretion of auto- or paracrine molecules such as Ac-APE1/Ref-1.

Citation Format: Yu Ran Lee, Hee Kyoung Joo, Ki Mo Kim, Byeong Hwa Jeon, Sunga Choi. Extracellularly secreted APE1/Ref-1 triggers apoptosis in triple-negative breast cancer cells via RAGE binding, which is mediated through acetylation. [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 4422.
Abstract 4647: BET protein inhibition blocks growth of triple-negative breast cancer by inducing mitotic and cytokinetic dysfunction
Authors: Brancato Jennifer M. , Gayle Sylvia , Weber-Bonk Kristen , Summers Matthew , Bebek Gurkan , Keri Ruth
Published: July 2016

Bromodomain and Extra Terminal (BET) proteins are epigenetic “readers” that recognize acetylated histones and mark areas of the genome for transcription. BRD4, a BET family member protein, has been implicated in a number of types of cancer. It has been recently found to associate with super-enhancers, and elevated levels of BRD4 have been linked to increased expression of MYC as well as other oncogenes. BET protein inhibitors are currently being tested for their potential use in the treatment of HIV, heart failure, and cancer, all of which are diseases of aberrant transcription. However, little is known regarding their efficacy in triple-negative breast cancer (TNBC). We found JQ1, a prototypical BET inhibitor, impedes growth of seven TNBC cell lines in a dose-dependent manner within 72 hours of treatment. JQ1 also suppresses growth of three different TNBC xenografts, including a patient-derived model of basal-like breast cancer. Growth arrest, in vitro, is followed by either apoptosis or senescence. However, prior to the induction of these two terminal responses, prolonged treatment results in polyploidy in many of the cell lines, suggesting BETi disrupts mitosis/cytokinesis. Live-cell imaging revealed JQ1 significantly increased the duration of mitosis, and microarray analyses showed a significant JQ1-mediated downregulation of genes critical for cell cycle progression, mitosis, and cytokinesis. In all TNBC cell lines tested, Aurora kinases, proteins critical for proper progression through mitosis and cytokinesis, are suppressed in response to JQ1. Treatment with AZD1152, an Aurora kinase B inhibitor, elicits the same cellular responses as BET inhibition, indicating that the suppression of Aurora kinases plays a key role in the response of TNBC cells to BET inhibitors. These findings reveal that BET inhibitors block the growth of highly aggressive TNBC cells by inducing mitotic dysfunction and that these drugs are promising potential therapeutics for the treatment of TNBC.

Citation Format: Jennifer M. Brancato, Sylvia Gayle, Kristen Weber-Bonk, Matthew Summers, Gurkan Bebek, Ruth Keri. BET protein inhibition blocks growth of triple-negative breast cancer by inducing mitotic and cytokinetic dysfunction. [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 4647.
Abstract 4694: ABBV-075, a novel BET family inhibitor, disrupts critical transcription programs that drive prostate cancer growth to induce potent anti-tumor activity in vitro and in vivo
Authors: Faivre Emily J. , Wilcox Denise M. , Hessler Paul , Uziel Tamar , Tapang Paul , Magoc Terry , Albert Daniel H. , Fang Guowei , Rosenberg Saul , McDaniel Keith , McDaniel Keith , Kati Warren , Shen Yu
Published: July 2016

The novel bromodomain inhibitor, ABBV-075, is being tested in a Phase I study for the treatment of solid tumors. Here, we show that potent inhibition of the BET (bromodomain and extra-terminal) family with ABBV-075 is a highly efficacious therapy in pre-clinical models of prostate cancer. The single-digit to low nanomolar anti-proliferative IC50s and potent in vivo tumor growth inhibition of ABBV-075 is mediated in part via inhibition of androgen receptor (AR)-dependent transcription. Prostate tumor incidence and CRPC clinical progression are driven by aberrant activation of the AR transcription program. Gene expression profiling and qPCR results indicate that ABBV-075 inhibited DHT-stimulated transcription of AR target genes without significant effect on AR protein expression. Further, ABBV-075 disrupted DHT-stimulated recruitment of the BET family member BRD4 to gene regulatory regions co-occupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition led to the disassembly of AR occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and additionally downregulated enhancer RNA (eRNA) transcription. ABBV-075 displayed potent anti-proliferative activity in multiple models of resistance to the second generation anti-androgen Enzalutamide, including the F876L, L702H AR ligand binding domain mutations and the AR-V7 splicing variant. In addition to blocking the transcription activation downstream of AR, ABBV-075 is also a potent inhibitor of MYC and the TMPRSS2-ETS fusion proteins. Thus, we propose that ABBV-075 may provide a promising therapeutic option for CRPC patients who have developed resistance to second-generation anti-androgens.

Citation Format: Emily J. Faivre, Denise M. Wilcox, Paul Hessler, Tamar Uziel, Paul Tapang, Terry Magoc, Daniel H. Albert, Guowei Fang, Saul Rosenberg, Keith McDaniel, Keith McDaniel, Warren Kati, Yu Shen. ABBV-075, a novel BET family inhibitor, disrupts critical transcription programs that drive prostate cancer growth to induce potent anti-tumor activity in vitro and in vivo. [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 4694.
Abstract 4746: Pharmacological characterization of BAY-876, a novel highly selective inhibitor of glucose transporter (GLUT)-1 in vitro and in vivo
Authors: Kopitz Charlotte , Toschi Luisella , Algire Carolyn , Héroult Mélanie , Frisk Anna-Lena , Meyer Kirstin , Schmitz Arndt , Lagkadinou Eleni , Petrul Heike , Heisler Iring , Neuhaus Roland , Buchmann Bernd , Himmel Herbert , Bauser Marcus , Haegebarth Andrea , Ziegelbauer Karl
Published: July 2016

One hallmark of cancer is the accelerated metabolism, high energy requirements, and increased glucose uptake by the tumor cells, the latter being the first and rate-limiting step for glucose metabolism. Glucose transport into the tumor cell is mediated by facilitative high-affinity glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, expression of GLUT1 in normal organs is nearly exclusively restricted to the blood brain barrier, while other GLUTs are also expressed in a wide variety of vital organs such as liver and heart. Interestingly, GLUT1 expression is highly regulated by hypoxia-inducible factor (HIF)-1α, a key driver of tumor progression. In line with this finding, GLUT1 over-expression was found to be associated with tumor progression and poor overall survival in various tumor indications. Consequently, GLUT1 represents a potential target for cancer treatment. Therefore, we have developed a highly-selective GLUT1 inhibitor, namely BAY-876, with selectivity over GLUT2, 3, and 4 of 4700-, 800-, and 135-fold, respectively. We here show for the first time the pharmacological characterization of BAY-876, comprising inhibition of glucose-uptake, anti-proliferative activity in vitro, and anti-tumor efficacy in vivo in models of different tumor indications in monotherapy as well as first results on the combinability of BAY-876. Furthermore, at the therapeutic dose, BAY-876 treatment did not show any relevant finding on the behavior of treated mice in the Irwin test, assuming no or only minor effects on brain function. In conclusion, BAY-876 is the first GLUT1-selective inhibitor which reduces glucose uptake and growth of tumor cells with sufficient tolerability at the efficacious dose in preclinical models.

Citation Format: Charlotte Kopitz, Luisella Toschi, Carolyn Algire, Mélanie Héroult, Anna-Lena Frisk, Kirstin Meyer, Arndt Schmitz, Eleni Lagkadinou, Heike Petrul, Iring Heisler, Roland Neuhaus, Bernd Buchmann, Herbert Himmel, Marcus Bauser, Andrea Haegebarth, Karl Ziegelbauer. Pharmacological characterization of BAY-876, a novel highly selective inhibitor of glucose transporter (GLUT)-1 in vitro and in vivo. [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 4746.
Abstract 4798: Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL
Authors: Balasubramanian Wesley Roy , Gummadi Venkateshwar Rao , Nellore Kavitha , Mukherjee Subhendu , Marappan Sivapriya , Basavaraju Aravind , Ainan Bharathi Raja , Daginakatte Girish , Gopinath Sreevalsam , Giri Sanjeev , Antony Thomas , Chelur Shekar , Samajdar Susanta , Pandit Chetan , Ramachandra Murali
Published: July 2016

Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to the tyrosine-like kinase (TLK) family. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. TLR/IL-1R stimulation leads to recruitment of MYD88, an adaptor molecule, to the activated receptor complex, which then complexes with IRAK4 and activates IRAK1. TRAF6 is then activated by IRAK1 leading to NFkB activation. Recent studies have reported the occurrence of gain of function oncogenic mutation (L265P) in MYD88 in ∼30% of activated B cell diffuse large B-cell lymphoma(ABC DLBCL) and ∼90% of Waldenstrom's macroglobulinemia (WM) leading to constitutive activation of IRAK4 and NFkB pathway. Among the DLBCL subtypes (GCB, ABC DLBCL and PMBL), ABC DLBCL is the most refractory. Inhibition of constitutive IRAK4 signalling can be used as a therapeutic strategy to treat ABC DLBCL

Small molecule inhibitors of IRAK4 were synthesized based on hits originating from Aurigene's compound library. Structure guided drug design approach was used to further improve the potency. Lead compounds demonstrated moderate to very high selectivity towardsIRAK4 (S35 score of 0.03) when screened against a large panel of 329 kinases. Aurigene's lead compounds have excellent PK profile and good oral bioavailability in mice, leading to good in-vivo activity in TLR4 induced cytokine release model. Selected lead compounds were tested in a OCI-Ly3 xenograft model, which has a MYD88(L265P) mutation leading to constitutive activation of IRAK4 signaling. An advanced lead compound has demonstrated excellent efficacy in OCI-Ly3 model, with tumor stasis at low doses and tumor regression at higher doses. The compound is well tolerated and has a good therapeutic window as determined in a 14 day rodent toxicity study. In summary, a selective IRAK4 inhibitor has been identified with excellent efficacy and good safety profile.

Citation Format: Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Kavitha Nellore, Subhendu Mukherjee, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL. [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 4798.
Abstract 4800: Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia
Authors: Versele Matthias , Haefner Burkhard , Wroblowski Berthold , Stansfield Ian , Mevellec Laurence , Gilissen Ron , Neumann Lars , Augustin Martin , Jacobs Kris , Cools Jan , Barluenga S , Röthlingshöfer M , Karthikeyan G , Attar Ricardo , Meerpoel Lieven , Winssinger Nicolas
Published: July 2016

Inhibition of Flt3 kinase activity is a promising strategy for the treatment of Flt3 mutant acute myeloid leukemia (AML). However, clinical studies with Flt3 kinase inhibitors have shown that mutations of the gate keeper or an activation loop residue in the Flt3 catalytic domain limits the duration of response. Such mutations generally reduce kinase domain binding affinity and residence time of the kinase inhibitor, and consequently its efficacy. We therefore investigated whether covalent, irreversible binding to Flt3 could overcome some of the limitations of classic non-covalent Flt3 inhibitors. Here, we report for the first time on the high-affinity Cys828-covalent binding mode of a resorcylic acid lactone to the isolated Flt3 kinase domain using X-ray crystallography and kinetic binding assays. In a cellular context (Ba/F3-Flt3-ITD), mutation of Cys828 to Ala reduces potency (IC50) from low nM to microM, demonstrating all relevant Flt3 inhibition in cells critically depends on Cys828. Consistently, the molecule is a low nM inhibitor of both Flt3-ITD and Flt3D835Y in vitro and in BaF3 cells, translating to potent nM inhibition of Flt3-ITD driven AML cell line proliferation, with only microM antiproliferative activity in non-Flt3 driven AML and unrelated leukemia cell lines. Finally, in spite of its fast clearance when dosed to MV4-11 (Flt3-ITD AML) xenograft bearing mice, robust anti-tumor activity was observed using a once-daily treatment schedule. These data demonstrate the feasibility of covalent Flt3 inhibition, and suggest it represents an attractive novel therapeutic approach for the treatment of Flt3-driven AML.

Citation Format: Matthias Versele, Burkhard Haefner, Berthold Wroblowski, Ian Stansfield, Laurence Mevellec, Ron Gilissen, Lars Neumann, Martin Augustin, Kris Jacobs, Jan Cools, S Barluenga, M Röthlingshöfer, G Karthikeyan, Ricardo Attar, Lieven Meerpoel, Nicolas Winssinger. Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia. [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 4800.
Abstract 4855: Discovery of NVP-HDM201 - First disclosure of a Next-Generation Mdm2 inhibitor with superior characteristics
Authors: Holzer Philipp , Chène Patrick , Ferretti Stéphane , Furet Pascal , Gabriel Tobias , Gruenenfelder Bjoern , Guagnano Vito , Hofmann Francesco , Kallen Joerg , Mah Robert , Masuya Keiichi , Ramos Rita , Ruetz Stephan , Rynn Caroline , Stachyra-Valat Thérèse , Stutz Stefan , Vaupel Andrea , Jeay. Sébastien
Published: July 2016

Activation of p53 by blocking the p53-Mdm2 interaction using non-peptidic small-molecule inhibitors has been pursued for many years as a promising cancer therapeutic strategy.

We disclose the identity of NVP-HDM201, a novel, highly optimized and selective inhibitor of the p53-Mdm2 interaction. NVP-HDM201 binds to human Mdm2 protein with a sub-nanomolar Ki value, activates p53 and induces robust p53-dependent cell cycle arrest and apoptosis in human p53 wild-type tumor cells. The activity and selectivity of NVP-HDM201 have been tested and confirmed across a panel of cancer cell lines and the molecule displays desirable pharmacokinetic and pharmacodynamic profiles in animals together with excellent oral bioavailability. Application of NVP-HDM201 using various dosing schedules triggers rapid and sustained activation of p53-dependent pharmacodynamic biomarkers resulting in tumor regression in multiple xenografted models of p53 wild-type human cancers.

We report here how a promising lead series was discovered and how innovative medicinal chemistry efforts led to further optimization of the potency and physico-chemical properties, culminating in the discovery of NVP-HDM201. The superior characteristics of the compound allowed the fast progression of the compound into the clinic where NVP-HDM201 is currently in Phase 1 clinical trials both as a single agent and as a combination partner in patients pre-selected for p53 wild-type tumors.

Citation Format: Philipp Holzer, Patrick Chène, Stéphane Ferretti, Pascal Furet, Tobias Gabriel, Bjoern Gruenenfelder, Vito Guagnano, Francesco Hofmann, Joerg Kallen, Robert Mah, Keiichi Masuya, Rita Ramos, Stephan Ruetz, Caroline Rynn, Thérèse Stachyra-Valat, Stefan Stutz, Andrea Vaupel, Sébastien Jeay. Discovery of NVP-HDM201 - First disclosure of a Next-Generation Mdm2 inhibitor with superior characteristics. [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 4855.
Abstract 4908: Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires Batf3-dependent dendritic cells
Authors: Sánchez-Paulete Alfonso R. , Cueto Francisco J. , Martínez-López María , Labiano Sara , Morales-Kastresana Aizea , Rodríguez-Ruiz Maria E. , Jure-Kunkel Maria , Azpilikueta Arantza , Quetglas José I. , Sancho David , Melero Ignacio
Published: July 2016

Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory monoclonal antibodies (mAbs) targeting PD-1 or CD137. Using Batf3-/- mice, which are defective for cross-presentation of cell-associated antigens, we show that Batf3-dependent dendritic cells (DCs) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3-/- mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFlt3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-OVA-derived melanomas, whereas this function was lost in Batf3-/- mice. These experiments show that cross-priming of tumor antigens by Flt3L- and Batf3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects.

Citation Format: Alfonso R. Sánchez-Paulete, Francisco J. Cueto, María Martínez-López, Sara Labiano, Aizea Morales-Kastresana, Maria E. Rodríguez-Ruiz, Maria Jure-Kunkel, Arantza Azpilikueta, José I. Quetglas, David Sancho, Ignacio Melero. Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires Batf3-dependent dendritic cells. [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 4908.
Abstract 4914: Auristatin-based antibody drug conjugates activate multiple ER stress response pathways resulting in immunogenic cell death and amplified T-cell responses
Authors: Cao Anthony , Heiser Ryan , Law Che-Leung , Gardai Shyra J.
Published: July 2016

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate (ADC) directed against CD30. This ADC consists of monoclonal antibodies conjugated to the auristatin monomethyl auristatin E (MMAE) a microtubule-disrupting agent. ADC antitumor activity is thought to be primarily the result of intracellular payload release leading to mitotic arrest and apoptotic cell death. We have recently demonstrated that brentuximab vedotin induces surface expression of calreticulin and HSP70 as well as activation of the transcription factor ATF6 which are classical hallmarks of immunogenic cell death (ICD) associated with unfolded protein/ER stress response. In this study, we provide evidence that additional ER stress response pathways are activated.

Treatment of CD30+ Hodgkin lymphoma (HL) cells or xenografts with brentuximab vedotin resulted in the phosphorylation of the ER-associated, unfolded-protein response transmembrane protein inositol-requiring transmembrane kinase/endonuclease 1 (IRE1). Activation of IRE1 was confirmed by the increase in phosphorylation of the downstream effector Jun N-terminal kinase (JNK). Kinetics of tubulin disruption, ER stress induction, evidence of ICD, mitotic arrest, and apoptosis was assessed. Onset of ICD occurred concurrently with induction of ER stress response pathways including induction of pIRE1 and pJNK. Interestingly, induction of ER stress response and ICD preceded mitotic arrest and caspase activation. These results indicate that brentuximab vedotin-mediated disruption of the microtubule network contributes to ER stress, which maybe an apoptotic mechanism mediated by auristatin-ADCs in addition to mitotic arrest at G2/M phase of the cell cycle. Induction of ICD may also be a class effect of the auristatin-based ADCs, as the small molecule payload MMAE, was able to activate IRE1 and JNK and induced surface expression of calrecutilin on target cells.

Previously, brentuximab vedotin-killed HL cells were shown to induce innate immune cell activation in vitro and in vivo. Addition of ADC-killed tumor-like cells to autologous PBMCs results in the expansion of INFã-secreting cytotoxic T-cells. Via induction of ER stress, tumor cells killed by auristatin-based ADCs may initiate an antitumor immune response and provides a rationale for exploring therapeutic strategies that combine ADCs with other immune stimulatory regimens.

Citation Format: Anthony Cao, Ryan Heiser, Che-Leung Law, Shyra J. Gardai. Auristatin-based antibody drug conjugates activate multiple ER stress response pathways resulting in immunogenic cell death and amplified T-cell responses. [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 4914.
Abstract 4957: Keratin 13 drives the dissemination of bone and brain metastases of human prostate and breast cancer cells
Authors: Yin Lijuan , Li Qinlong , Huang Jen-Ming , Lewis Michael , Garraway Isla P. , Li Quanlin , Bu Hong , Chung Leland W. K , Zhau Haiyen E.
Published: July 2016

Introduction and Objective: Bone and brain metastases are prevalent in human prostate and breast cancer patients. Keratin 13 (KRT13), a member of the intermediate filament proteins and an epithelial stem cell differentiation marker, induces cancer cell migration and progression toward increased malignant state. The objectives of this study are: 1) to determine KRT13 expression in human prostate and breast cancer and its clinical correlation; 2) to determine the functional cell signaling role of KRT13 in prostate and breast cancer cells using gene transfer technology.

Methods and Results: We evaluated KRT13 protein expression in primary prostate cancer tissues from 44 cases with well-documented overall survival by quantitative quantum dot immuno-labelling. We found that KRT13 expression in primary prostate cancer correlated with patient overall survival and bone metastasis (p<0.05). We next evaluated KRT13 protein expression in 36 prostate and 58 breast cancer tissue specimens by immunohistochesmistry (IHC) and found that KRT13 is overexpressed in breast cancer bone (5/8) and brain (7/10) metastases and prostate cancer bone (6/12) metastasis specimens (p<0.05). KRT13 expression in primary prostate and breast cancer specimens is associated with decreased patient overall survival based on our data as well as publicly available Oncomine gene expression datasets. To elucidate the mechanisms by which KRT13 promotes cancer invasion, migration and metastasis, we genetically overexpressed KRT13 in human prostate CWR22 Rv1 and LNCaP, and human breast MCF7 and MCF10A cells, with respectively high or low malignant potential. KRT13-overexpressing prostate and breast cancer cells increase differential expression of genes associated with epithelial to mesenchymal transition (EMT; E-Cadherin and Vimentin), stemness (Nanog), neuromimicry (Chromagranin A and Synaptophysin), osteomimicry (RANKL) related genes as analyzed by qRT-PCR, western blot, and IHC. KRT13 also promotes increased cancer cell proliferation, migration and invasion in vitro, and bone and brain metastases in vivo.

Conclusion: These results suggest that KRT13 drives prostate and breast cancer bone and brain metastases via osteomimicry and neuromimicry associated genes resulting in downstream cell signaling network convergence between EMT, stemness, cell growth, and survival pathways.

Funded in part by NCI-P01-CA098912

Citation Format: Lijuan Yin, Qinlong Li, Jen-Ming Huang, Michael Lewis, Isla P. Garraway, Quanlin Li, Hong Bu, Leland W. K Chung, Haiyen E. Zhau. Keratin 13 drives the dissemination of bone and brain metastases of human prostate and breast cancer cells. [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 4957.
Abstract 5057: Mena at the nexus of chemotaxis and haptotaxis during tumor progression
Authors: Oudin Madeleine J. , Miller Miles A. , Kosciuk Tatsiana , Klazen Joelle , Lussiez Alisha , Lauffenburger Douglas , Bear James E. , Gertler Frank B.
Published: July 2016

Directed-cell migration is key step in the metastatic cascade, and multiple cues can promote local invasion. The most well studied mode of cell motility for metastasizing tumor cells is chemotaxis, the directional movement of cells attracted to a source of soluble cues. In contrast, haptotaxis, migration of cells on gradients of substrate-bound factors, remains poorly understood. Mena, an actin regulatory protein, plays an important role in cell motility and is upregulated in various cancers, driving migration in response to chemotactic and haptotactic cues. We have recently shown expression of the pro-metastatic isoform of Mena, MenaINV, increases sensitivity to growth factors (EGF, HGF and IGF) via dysregulation of the phosphatase PTP1B, as well as high concentrations of fibronectin (FN) via its interaction with the integrin α5β1. We hypothesized that MenaINV may also promote crosstalk between these two pathways to drive metastasis. First, we show that MenaINV-driven haptotaxis on FN gradients and FN-driven invasion requires crosstalk between α5β1 and EGFR. Indeed, we show evidence that MenaINV expression is associated high phosphorylation of EGFR in high FN environments in vitro and in human breast cancer databases. Second, we find that MenaINV promotes synergy between EGF and FN in vitro and in vivo, leading to hyper-invasive phenotypes that drive more invasion than which each cue alone. Finally, we show that MenaINV-driven haptotaxis, ECM reorganization and 3D invasion requires RCP-dependent recycling of α5β1 and EGFR. Together, our data demonstrate that MenaINV is a shared component of the machinery that mediates both haptotactic responses to FN and chemotactic responses to EGF, two pathways that promote tumor progression.

Citation Format: Madeleine J. Oudin, Miles A. Miller, Tatsiana Kosciuk, Joelle Klazen, Alisha Lussiez, Douglas Lauffenburger, James E. Bear, Frank B. Gertler. Mena at the nexus of chemotaxis and haptotaxis during tumor progression. [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 5057.
Abstract 5102: Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment
Authors: Brasil Fabio H. , Ferrer Valeria , Navone Nora M. , Farach-Carson Mary C. , Carson Daniel D.
Published: July 2016

We previously showed that the heparan sulfate proteoglycan, perlecan (PLN)/HSPG2, is deposited into the matrix of reactive stroma surrounding tumors, where it concentrates heparin binding growth factors (HBGFs) and cytokines at the tumor-stromal border both in primary prostatic tumors and bone metastatic lesions. Heparan sulfate (HS) modifying enzymes produced in tumor-associated stroma play key roles in the delivery of these perlecan-sequestered cancer, stromal and tumor macrophage-produced HBGFs and cytokines, serving to enhance tumorigenesis and recruit other bystander cells to sites of metastasis. Studies presented here focused on the production of secreted HS-modifying enzyme heparanase and the two extracellular sulfatases, SULF1 and SULF2, in the prostatic tumor microenvironment. Our studies showed that SULF1 and heparanase mRNA levels are elevated in prostate cancer (PCa) cells of the LNCaP lineage in the presence of stroma, indicating that both stroma and PCa cells can be sources of these enzymes. We are presently examining a number of PCa patient derived xenografts to determine the prevalence of SULF-1 and heparanase elevation in castrate resistant disease. To study function, we developed a modified 3D hyaluronate gel system that contains HS-bearing recombinant perlecan domain I (PlnDI) to retain and deliver HBGFs and cytokines to various PCa cells cultured with bone marrow stromal cells, osteoblasts, or tumor macrophages. We are using this system to study the expression and individual functions of heparanase and the two SULFs in promoting HBGF-dependent PCa progression in a physiologically relevant system. Overexpression and gene deletion techniques allow us to determine the role that heparanase or SULFs play in each relevant cell type present in the tumor microenvironment, and thus determine the need for co-targeting of both tumor and reactive stroma. (Supported by NIH P01 and CAPES).

Citation Format: Fabio H. Brasil, Valeria Ferrer, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment. [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 5102.