bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 32: Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3)
Authors: Lea Michael A. , Guzman Yolanda , desBordes. Charles
Published: July 2016

The tendency for enhanced glycolysis in cancer cells presents a target for chemotherapy. In previous studies we observed that proliferation of colon and bladder cancer cells can be inhibited by treatment with either phenformin or an inhibitor of PFKFB3 namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work we have examined the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15). The LDHA inhibitors that we chose to study in increasing order of IC50s were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. A synergistic anti-cancer effect of phenformin and oxamate has been reported. In the present work with colon and bladder cancer cells, additive but not synergistic growth inhibitory effects were seen with the LDHA inhibitors of which NHI-2 was effective at the lowest concentrations. Growth inhibition with PQP and PFK15 was compared in colon (Caco-2 and HT29) and bladder cancer cells (5637, HT1376, RT4, SW780, T24, TCCSUP and UM-UC-3). Apart from RT4 cells where the effects were similar, the effects were somewhat greater with PFK15 than with PQP and for both compounds the actions were seen at lower concentrations than in previous studies with 3PO. Actions on medium acidification and glucose uptake are more readily observed in the most rapidly growing cell lines. Effects of phenformin on medium pH and glucose concentration were decreased by the PFKFB3 and LDHA inhibitors that were examined. In accord with our previous studies on inhibitors of glycolysis, the increased medium acidification and glucose uptake caused by phenformin could be blocked by combined treatment with PFKFB3 or LDHA inhibitors. At the same time additive growth inhibitory effects were observed. The results supported the concept that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation while mitigating the lactic acidosis caused by phenformin as a single agent.

Citation Format: Michael A. Lea, Yolanda Guzman, Charles desBordes. Inhibition of cancer cell growth by combined treatment with lactate dehydrogenase (LDHA) inhibitors and either phenformin or inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). [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 32.
Abstract 3202: Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity
Authors: Schirmer David , Klar Richard , Schmidt Oxana , Wohlleber Dirk , Uckert Wolfgang , Thiel Uwe , Bohne Felix , Busch Dirk H. , Krackhardt Angela M. , Burdach Stefan , Richter Günther H.
Published: July 2016

Background: Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology targetable tumor associated antigens (TAA) are identified and exploited for engineered T cell therapy. Here, the specific recognition and lytic potential of transgenic, allo-restricted CD8+ T cells directed against the ES-associated antigen STEAP1 was examined.

Methods: Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells, allo-restricted HLA-A*02:01- CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, TCR α- and β-chains were identified. They were cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01- primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2-/-γc-/- mouse model.

Results: Initially generated and transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific IFNγ release. They lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01- ES lines. In vivo tumor growth was inhibited more effective with transgenic STEAP1130-specific T cells than with unspecific T cells.

Conclusion: Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1 expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1 expressing tumors.

Citation Format: David Schirmer, Richard Klar, Oxana Schmidt, Dirk Wohlleber, Wolfgang Uckert, Uwe Thiel, Felix Bohne, Dirk H. Busch, Angela M. Krackhardt, Stefan Burdach, Günther H. Richter. Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity. [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 3202.
Abstract 3206: Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies
Authors: Takahashi Miho , Watanabe Satoshi , Sato Ko , Tanaka Tomohiro , Saida Yu , Baba Junko , Ohtsubo Aya , Sato Miyuki , Kondo Rie , Okajima Masaaki , Tanaka Junta , Kagamu Hiroshi , Yoshizawa Hirohisa , Kikuchi Toshiaki
Published: July 2016

Cytotoxic lymphodepletion therapies, such as chemotherapy and radiotherapy, have been established to augment antitumor immunity. Naïve T cells elicit effector-like phenotypes and functions during recovery from lymphopenia. We and others have repeatedly demonstrated that transfer of naïve T cells into lymphopenic-tumor bearing mice delayed tumor growth. This enhancement of naïve T cells is required an activation through T-cell receptor (TCR).

Programmed death receptor-1 (PD-1)/programmed death receptor ligand-1 (PD-L1) blockade therapy has been demonstrated to augment antitumor immunity. Previous studies have shown that PD-1/PD-L1 blockade therapy stimulates or restores the function of antitumor T cells in the effector phase. Because engagement of programmed death receptor-1 (PD-1) by ligand suppresses TCR signaling and inhibits T cell activation and function, there is a possibility that PD-1 regulates activation of T cells during recovery from lymphopenia after cytotoxic therapies.

In the current study, we transferred naïve T cells into tumor-bearing mice following whole-body irradiation for lymphodepletion. These mice were further treated with anti-PD-1 antibodies. PD-1/PD-L1 blockade therapy after lymphodepletion significantly suppressed tumor progression. Next, we tested several kinds of cytotoxic agents to induce lymphopenia in mice. We found that the kind of cytotoxic agents affected the augmentation of antitumor efficacies of PD-1/PD-L1 blockade. Analyses of tumor-draining lymph-nodes (TDLNs) revealed that the number of tumor-specific effector T cells was significantly increased in mice treated with anti-PD-1 antibodies. By contrast, the number of effector T cells in spleens was not increased by PD-1/PD-L1 blockade therapy. These results indicate that PD-1 regulates a priming of antitumor effector T cells in TDLNs after cytotoxic lymphodepletion therapies. PD-1/PD-L1 blockade therapy is able to enhance antitumor T cell responses not only in the effector phase but also in the priming phase.

Citation Format: Miho Takahashi, Satoshi Watanabe, Ko Sato, Tomohiro Tanaka, Yu Saida, Junko Baba, Aya Ohtsubo, Miyuki Sato, Rie Kondo, Masaaki Okajima, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Toshiaki Kikuchi. Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies. [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 3206.
Abstract 3210: An in vitro approach to study T cell exhaustion
Authors: Hastings William , Dranoff Glenn , Cao Alexander
Published: July 2016

Immune checkpoint inhibition is now a validated modality in the treatment of multiple cancers. In vitro immune cell assays are a key tool in the study of pharmacological agents for immune modulation. Assays often involve isolating lymphocytes from human peripheral blood followed by a T cell stimulus such as anti-CD3, staph enterotoxin B, or allogeneic APCs. While this approach is useful to probe the events of immune stimulation, it likely reflects a primary or secondary immune activation, rather than a state of exhaustion. As intra-tumoral T cells are thought to be exhausted by chronic/repeated exposure to self-antigen, we sought to define experimental conditions that would reflect this state in vitro. We reasoned that repeated stimulation of T cells using anti-CD3/anti-CD28 coated microbeads would induce an exhausted phenotype. We found that, using PBMC from normal healthy donors, frequent and repeated stimuli resulted in a loss of T cells’ ability to produce IL-2, TNFa, and IFNg as measured by intracellular cytokine staining. Interestingly, only one restimulation was necessary to confer this phenotype, as repeated stimuli over a longer time course did not result in further suppression. We also found that repeat stimulation conferred a surface marker phenotype suggestive of exhausted cells. Finally, we used cell death inhibitors z-7AAD-cmk and EGTA and found no effect on cytokine loss, supporting the hypothesis that cells are exhausted and not deleted by activation induced cell death. Thus we have defined experimental conditions that possibly reflect an exhausted T cell phenotype in vitro. This may be useful for the pharmacological evaluation of different therapeutic agents for their immune modulatory activities in cancer.

Citation Format: William Hastings, Glenn Dranoff, Alexander Cao. An in vitro approach to study T cell exhaustion. [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 3210.
Abstract 3222: Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy
Authors: Augier Severine , Perrot Ivan , Dejou Cecile , Joly Rachel , Delahaye Stephane , Rispaud Blanc Helene , Denis Caroline , Gauthier Laurent , Bensussan Armand , Eliaou Jean-francois , Morel Yannis , Bonnefoy Nathalie , Bastid Jeremy , Paturel Carine
Published: July 2016

CD39 (ENTPD1) is a cell membrane ectonucleotidase that hydrolyzes extracellular immunoactivating ATP and ADP into AMP, which can be further hydrolyzed by ectonucleotidase CD73 into immunosuppressive adenosine. Within the tumor microenvironment, adenosine accumulation causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. The role of CD39 expression on both Tregs and on tumor cells in promoting immunosuppression has been demonstrated in several reports. Blockade of CD39 may promote anti-tumor immunity by directly accumulating immunostimulating ATP and indirectly by reducing adenosine accumulation. Here, we describe the discovery and preclinical development of an anti-huCD39 blocking antibody for cancer immunotherapy. Parental anti-huCD39 mouse monoclonal antibody was humanized. The humanized mAb specifically binds huCD39 protein, but not CD39-like proteins. Nanomolar affinities for human CD39 were measured in SPR studies on recombinant CD39 protein and in flow cytometry titration studies on CD39 expressing transfectants and tumor cell lines. The humanized mAb blocked human CD39 ATPase activity in vitro in the nanomolar range, as demonstrated using transfected cells, CD39-expressing tumor cell lines, as well as human PBMC and ex-vivo isolated fresh tumor samples. The humanized mAb cross-reacted on cynomolgus CD39 and blocked ATPase activity on cynomolgus PBMC with similar efficacy as on human PBMC. Finally, treatment with blocking anti-CD39 mAb inhibited tumor growth in vivo in mouse tumor models. Taken together, these data support the clinical development of anti-CD39 neutralizing mAb for cancer immunotherapy.

Citation Format: Severine Augier, Ivan Perrot, Cecile Dejou, Rachel Joly, Stephane Delahaye, Helene Rispaud Blanc, Caroline Denis, Laurent Gauthier, Armand Bensussan, Jean-francois Eliaou, Yannis Morel, Nathalie Bonnefoy, Jeremy Bastid, Carine Paturel. Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer 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 3222.
Abstract 3238: Imbalance of circulating monocyte subsets in patients with squamous cell carcinoma of the head and neck
Authors: Sakakura Koichi , Takahashi Hideyuki , Motegi Sei-ichiro , Chikamatsu Kazuaki
Published: July 2016

Introduction: Human circulating monocyte is classified as three populations according to CD14 and CD16 expression: CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes and CD14dimCD16+ nonclassical monocytes. Recent studies have unveiled the specific surface molecules and functions of each subset; however their dynamics in patients with cancer still remains unclear. In this study, we evaluated the proportions and the expressions of immunological/angiogenic molecules of the monocyte subsets in peripheral circulation in patients with squamous cell carcinoma of the head and neck (SCCHN).

Materials and Methods: Peripheral blood mononuclear cells were isolated from 9 patients with SCCHN and age-matched 4 healthy donors. The 3 monocyte subsets were identified according to CD14/CD16 expressions using multi-color flow cytometry. Mean fluorescence intensities of various HLA molecules and angiogenic markers were analyzed.

Results: Percentage of CD14+CD16+ intermediate monocyte among total monocyte tended to decrease in SCCHN patients than that in normal donors (p = 0.079). Higher surface expression of immunosuppressive HLA-G molecule on circulating classical (p = 0.045) and intermediate (p = 0.052) monocyte was observed in patients with SCCHN. An immunosuppressive receptor ILT4 showed tendency to increase on nonclassical monocyte (p = 0.061). Moreover, intracytoplasmic level of milk fat globule-EGF factor 8 (MFG-E8) in association with phagocytosis and angiogenesis tended to increase in intermediate (p = 0.064) and nonclassical (p = 0.052) monocytes of SCCHN patients.

Discussion and Conclusion: CD16+ intermediate and nonclassical monocytes have been reported as mature, proinflammatory and angiogenic population in periphery. In the present study, we showed possible alternations of these subsets in patients with SCCHN. Furthermore, increasing surface expressions of immunosuppressive molecules HLA-G and ILT4 on circulating monocytes implied immunological deterioration in cancer patients. However the sample size is still small, our preliminary data strongly suggests proportional and functional changes of peripheral monocyte subsets in SCCHN patients. Further analyses in more patients are currently underway.

Citation Format: Koichi Sakakura, Hideyuki Takahashi, Sei-ichiro Motegi, Kazuaki Chikamatsu. Imbalance of circulating monocyte subsets in patients with squamous cell carcinoma of the head and neck. [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 3238.
Abstract 3239: Cancer-associated fibroblasts induce immunosuppressive macrophages in head and neck squamous cell carcinoma
Authors: Takahashi Hideyuki , Sakakura Koichi , Chikamatsu Kazuaki
Published: July 2016

Background

Cancer-associated fibroblasts (CAFs) are one of main elements in the tumor microenvironment (TME). Among the innate and adaptive immune cells in the TME, tumor-associated macrophages (TAMs) are particularly abundant and play a protumoral role. However, an interaction between CAFs and TAMs in head and neck squamous cell carcinoma (HNSCC) still remains unclear. In the present study, we investigated whether CAFs skewed macrophage function toward the immune suppressive phenotype.

Materials and Methods

In vitro assay: CAFs were prepared from surgical tissue of HNSCC patients, and culture supernatants of CAFs were collected. Using this supernatants and peripheral blood mononuclear cells collected from healthy donors, immunological interactions between CAFs and TAMs was investigated.

In vivo assay: The immunohistochemistry (IHC) of 73 patients with HNSCC was performed. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs, respectively. The relationship between immunohistochemical parameters and clinical parameters was evaluated.

Results

The expression levels of CD68, CD163, CD14, CD80, CD86, and HLA-G were up-regulated in CD14+ cells co-cultured with the supernatants of CAFs (CAF-educated cells) compared with control cells. Moreover, the gene expressions of ARG1, IL6, IL10 and TGFB1 were also increased in CAF-educated cells. The CAF-educated cells suppressed T-cell proliferation more intensively than control cells, and neutralization of TGF-β and IL-10 led to significant restoration of T-cell proliferation. In IHC, the intensity of CAFs correlated with the number of CD68-positive macrophages, especially with CD163-positive macrophages. It also correlated with several clinical parameters such as lymphatic invasion, vascular invasion, nodal status, tumor stages and recurrence rate. Moreover, the high intensity of CAFs correlated with poor PFS and OS. The high intensity of CAFs and M2 macrophage infiltration were independent prognostic factors of PFS and OS in HNSCC.

Conclusion

The present study suggests that CAFs can induce an immunosuppressive phenotype of macrophages and contribute to establishing the immunosuppressive networks in TME, resulting poor prognosis in HNSCC patients.

Citation Format: Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. Cancer-associated fibroblasts induce immunosuppressive macrophages in head and neck squamous cell 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 3239.
Abstract 3241: M1 macrophages promote ovarian cancer metastasis through NF-κB/TWIST axis signaling pathway
Authors: Jo Un-Tek , Song. YongSang
Published: July 2016

The importance of tumor microenvironment in cancer progression has been recognized recently. However, factors associated with the tumor microenvironment are diverse and the molecular mechanisms are needed to be further defined for therapeutic intervention. Here we focused on the interaction between obesity related inflammatory cell, macrophage type 1 (MФ-1), and ovarian cancer cells in relation to metastasis. Two types of ovarian cancer cell lines; PA-1 and SKOV3, and monocyte-derived macrophages were used in this study. In the treatment with MФ-1 conditioned media, only SKOV3 cells were affected in proliferation activity. However migration and invasion were promoted by treatment with MФ-1 conditioned media in both ovarian cancer cells. In parallel, MФ-1 conditioned media treatment increased TWIST, EMT related gene. Interestingly, TWIST expression was correlated with protein expression levels of nuclear NF-κB (p65 and p50). Upon MФ-1 conditioned media treatment, nuclear translocation level and transcriptional activity of the NF-κB increased. Also, TPCK, NF-κB inhibitor suppressed invasion activity by MФ-1 conditioned media, in parallel with down-regulation of TWIST expression. Taken together, MФ-1 promoted EMT related metastatic potential in ovarian cancer cells through NF-κB signaling pathway. These results provide a new insight into the obesity related tumor microenvironment in ovarian cancer cell progression.

Citation Format: Un-Tek Jo, YongSang Song. M1 macrophages promote ovarian cancer metastasis through NF-κB/TWIST axis signaling pathway. [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 3241.
Abstract 3245: IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas
Authors: Kim Peter S. , Kwilas Anna R. , Xu Wenxin , Alter Sarah , Jeng Emily K. , Wong Hing C. , Schlom Jeffrey , Hodge James W.
Published: July 2016

Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded NK and CD8+ T cells. In examining natural killer (NK) cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1-) phenotype. In 4T1 breast tumor-bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors.

Citation Format: Peter S. Kim, Anna R. Kwilas, Wenxin Xu, Sarah Alter, Emily K. Jeng, Hing C. Wong, Jeffrey Schlom, James W. Hodge. IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas. [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 3245.
Abstract 3250: Caspase-1 from MDSC promote MyD88 dependent carcinogenesis that is T-cell independent
Authors: Zeng Qi , Qualliotine Jesse R. , Blosser Lee , Pardoll Drew , Kim Young J.
Published: July 2016

Myeloid-derived-suppression cells (MDSC) are functionally defined to suppress T-cells in their primary means of immune evasion mechanism in cancer. However, we have discovered that MDSC can have direct, T-cell independent pro-carcinogenic effect on tumor cells. Sorted monocytic CD14+/CD11b+/HLA-DRlow MDSCs from head and neck squamous cell carcinoma (HNSCC) patients were found to increase the proliferation index of HNSCC cells. This induction of tumor proliferation was not dependent on MDSC-tumor cell contact. Exome analysis showed that these human MDSC from cancer patients expressed high levels of inflammasome complex, and supernatant from the MDSCs were found to secrete IL-1b and IL-18. When we probed the functional significance of these inflammasome complex, we found that MDSC's promotion of tumor proliferative index of the tumor was found to be Caspase-1 dependent. To test this in vivo, T-cell depleted caspase-1 null mice showed significant decrease in tumor growth rate. To confirm the importance of myeloid inflammasome signaling in carcinogenesis, we suppressed MyD88 gene in tumor cell line Cal27 and found that the ability of MDSC to promoting tumor proliferation is diminished. Taken together, our findings demonstrate that tumor infiltrating MDSCs may play a prominent role in chronic inflammation associated carcinogenesis.

Citation Format: Qi Zeng, Jesse R. Qualliotine, Lee Blosser, Drew Pardoll, Young J. Kim. Caspase-1 from MDSC promote MyD88 dependent carcinogenesis that is T-cell independent. [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 3250.
Abstract 3259: LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis
Authors: Chintharlapalli Sudhakar R. , Croy Johnny E. , Leung Donmienne , Gerald Damien , Lu Jirong , Iversen Philip W. , Lee Linda N. , Huber Lysiane , Tetreault Jonathan , Almonte-Baldonado Rowena , Xu Jianghuai , Ramamurthy Bharathi , Pereira Jennifer A. , Chow Chi-Kin , Hanauske Axel-Rainer , Wacheck Volker , Benjamin Laura , Liu Ling
Published: July 2016

Angiopoeitin-2 (Ang2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. Combination of an anti-Ang2 antibody and aflibercept, a VEGF trap, has been shown to inhibit tumor growth and decrease tumor vascularity in mouse xenograft tumor models (Daly et al., Cancer Res (2013) 73(1):108). Multiple investigational anti-Ang2 antibody therapies are currently in clinical trials. LY3127804 is a humanized and engineered IgG4 isotype antibody that selectively binds to Ang2 with high affinity and neutralizes Ang2 induced phospho-Tie2. LY3127804 inhibits sprouting angiogenesis and increases pericyte coverage in a mouse developmental retinal angiogenesis model and in mice bearing PC3 xenograft tumors. Combination of LY3127804 and DC101, a potent anti-VEGFR2 antibody, exhibits enhanced efficacy when compared to monotherapy in multiple patient derived xenograft models including NSCLC and ovarian cancers. Anti-Ang2 antibody monotherapy alone resulted in marginal reduction of tumor growth and improved overall survival, while DC101monotherapy had greater reduction in tumor volume with no survival benefit in MDA-MB-231 breast orthotopic model. Combination of anti-Ang2 antibody with anti-VEGFR2 antibody shows reduction in tumor volume and improved overall survival. This robust pre-clinical evidence supports testing the combination of anti-Ang2 and anti-VEGFR2 antibodies in the clinic. LY3127804 is currently in Phase 1 clinical trials (NCT02597036)

Citation Format: Sudhakar R. Chintharlapalli, Johnny E. Croy, Donmienne Leung, Damien Gerald, Jirong Lu, Philip W. Iversen, Linda N. Lee, Lysiane Huber, Jonathan Tetreault, Rowena Almonte-Baldonado, Jianghuai Xu, Bharathi Ramamurthy, Jennifer A. Pereira, Chi-Kin Chow, Axel-Rainer Hanauske, Volker Wacheck, Laura Benjamin, Ling Liu. LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis. [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 3259.
Abstract 3292: Manipulating osteoblast differentiation in order to inhibit protection of AML cells within the bone marrow
Authors: Sterner Rosalie , Kremer Kimberly , Dudakovic Amel , Kaufmann Scott , Westendorf Jennifer , van Wijnen Andre , Hedin Karen
Published: July 2016

While standard chemotherapeutics can initially induce remission in patients with acute myelogenous leukemia (AML), patients often relapse in part due to the protective bone marrow microenvironment, which promotes the survival of AML cells. Using an in vitro cell culture model of the bone marrow, our lab recently showed that differentiating osteoblasts potently protect AML cells from apoptosis. In addition, we found that differentiating osteoblasts treated with histone deacetylase inhibitors (HDACi) fail to protect AML cells in this model. Since HDACi decreased the expression of genes required for osteoblast differentiation, we hypothesize that blocking specific stage(s) of osteoblast differentiation may make AML cells within the bone marrow more susceptible to chemotherapy. We are currently disrupting specific osteoblast differentiation stages via molecular or therapeutic drug manipulation and exploring the impact on AML cell survival both in cell culture models of the bone marrow microenvironment and in a mouse model of AML. The results of our studies will characterize novel bone marrow molecular and cellular interactions that promote the survival of AML cells. Targeting these interactions in combination therapies may more completely eradicate the AML cell reservoir and better prevent relapse.

Citation Format: Rosalie Sterner, Kimberly Kremer, Amel Dudakovic, Scott Kaufmann, Jennifer Westendorf, Andre van Wijnen, Karen Hedin. Manipulating osteoblast differentiation in order to inhibit protection of AML cells within the bone marrow. [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 3292.
Abstract 3299: Bacterial DNA integrations in the genomes of gastric tumor and adjacent samples
Authors: Robinson Kelly , Kumar Nikhil , Torres Javier , Dunning Hotopp Julie C.
Published: July 2016

There are 10 times more bacterial cells in the human body than human cells, and various bacteria are known to influence carcinogenesis. While viral DNA integrations in the human genome have been shown to promote carcinogenesis, bacterial DNA integrations (BDI) into the human genome are rarely investigated. Our previous analysis of publicly available sequencing data from the Cancer Genome Atlas showed BDIs from Pseudomonas spp. rRNA into the 5’-UTR of four proto-oncogenes as well as in Ig (the immunoglobulin gene) of gastric cancer samples. However, we were left with many unanswered questions when we were unable to obtain the materials required to validate our findings. Therefore, we sought to sequence a different cohort of gastric cancer patients in order to identify BDIs. Whole exome sequencing was completed on seven tumor samples and six adjacent gastric samples, as well as one intestinal metaplasia sample and one non-atrophic gastritis sample. Four of those samples were also investigated with transcriptomics and whole genome sequencing. Using our previously published BWA-based pipeline, we identified putative BDIs from Helicobacter pylori rRNA into numerous genes in one tumor sample as well as in its adjacent matched sample, including BDIs in the Ig locus. Helicobacter pylori is considered a carcinogen by the World Health Organization due to its ability to promote carcinogenesis in gastric tissue, causing us to speculate that these integrations could be carcinogenic. Similarly to our analysis of the Cancer Genome Atlas data, we also identified a few samples with BDIs from Pseudomonas spp. and about half of the samples possessed BDIs from other bacteria. We identified at least one BDI in each sample regardless of the sequencing data type. Validation of these results is ongoing and will be presented. Given these results, more consideration should be given to BDIs into the human genome when bacterial associations with diseases are suspected.

Citation Format: Kelly Robinson, Nikhil Kumar, Javier Torres, Julie C. Dunning Hotopp. Bacterial DNA integrations in the genomes of gastric tumor and adjacent samples. [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 3299.
Abstract 3308: Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice
Authors: Bouamar Hakim , Zhang Fuchuang , Gu Xiang , Dong Qiaoxiang , Sun Lu-Zhe
Published: July 2016

Breast cancer is a commonly diagnosed cancer during pregnancy. However, how endogenous and exogenous endocrine factors may contribute to the development of pregnancy-associated mammary tumorigenesis is not clear. There is growing evidence that mammary stem cells (MaSCs) may initiate neoplastic transformation when dysregulated in mouse models. We investigated the effect of the environmental endocrine disruptor bisphenol A (BPA) on mouse mammary gland morphology, epithelial cell composition, pre-neoplastic lesions, and the regenerative function of MaSCs. Pregnant FVB mice with GFP transgene on Day E8.5 were implanted with osmotic pumps that constantly release BPA at 0, 25 or 250 ng/kg/day for 28 days and the mice were euthanized one month after weaning. In agreement with the literature, we observed an abnormality of the morphology of the mammary gland after BPA treatment characterized by higher duct density and abnormal secondary and tertiary branching. Quantification of percent hyperplastic mammary ducts in H&E-stained tissue slides revealed a significant increase of ducts with hyperplastic lesions after BPA treatment, particularly with the low dose. To investigate the effects of BPA treatment on MaSCs, we used enzyme digestion to isolate the CD24hi/CD49f+ luminal epithelial cells (also termed as colony forming cell or CFC) and the CD24+/CD49fhi basal epithelial cells (also termed as mammary repopulating unit or MRU) from mammary gland tissues by FACS and found no significant difference in percent of luminal or basal cell population after BPA treatment. Because the basal cells are enriched with MaSCs that can form mammospheres in suspension culture and subsequently form solid 3D organoids when cultured in Matrigel, we transplanted the solid 3D organoids into cleared mammary fat pads of syngeneic FVB mice and immune-compromised nude mice to examine how BPA treatment might alter MaSC function. Significantly, similar to the results from the primary mammary glands, the regenerated mammary glands by MaSCs from mice treated with the low dose of BPA showed increased duct density, secondary and tertiary branching, and a significantly greater number of hyperplastic lesions. Taken together, our study demonstrated that BPA exposure at very low dose could induce pre-neoplastic lesions in the mammary gland of pregnant mice, apparently by directly targeting MaSCs, and implicates BPA as an exogenous endocrine factor that may promote pregnancy-associated mammary tumorigenesis.

Citation Format: Hakim Bouamar, Fuchuang Zhang, Xiang Gu, Qiaoxiang Dong, Lu-Zhe Sun. Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice. [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 3308.
Abstract 3318: Exploring the utility of natural and synthetic resveratrol derivatives for bone regrowth following loss due to breast cancer therapies
Authors: Robert Evan , Rhodes Lyndsay V.
Published: July 2016

Many of the most widely used therapies for the treatment of breast cancer have been associated with enhanced rate of bone loss, including chemotherapies and hormone therapies, due to alteration of normal hormone signaling. Bone loss is due to a shift in the balance between osteoblast and osteoclast cells that are derived from a common precursor, mesenchymal stem cells (MSC), found in the bone marrow. Estrogen is an important mediator of both hormone-responsive breast cancers and normal bone development and regeneration, favoring MSC differentiation down the osteoblast lineage. Stilbenes, defense compounds produced by plants including red grapes, peanuts, and blueberries, have been popularized in recent years based on observed health benefits. Resveratrol, the most widely studied stilbene, has been credited with many health benefits including cardiovascular health, anti-cancer and antioxidant effects, and inhibition of obesity and diabetes. Many stilbenes, including resveratrol can be classified as phytoestrogens due to their ability to bind and alter the activity of the estrogen receptor. A series of 28 stilbene compounds, including resveratrol and pterostilbene, were obtained by the U.S. Department of Agriculture and screened for effects on MSC differentiation. MSC were treated with stilbene compounds (both E and Z isoforms) and cultured in either osteogenic or adipogenic differentiation media for 7-21 days. Following differentiation, cells were stained with (1) Alizarin Red S to identify calcium deposits or (2) Oil Red O to identify lipid droplets. Several compounds were found to stimulate osteogenesis while inhibiting adipogenesis and were selected for further study and development. Realtime PCR for known genes associated with osteogenesis (ALPL, RUNX2, OCN, OPN) and adipogenesis (ADIPOQ, GLUT4, LEP, PPARG) were used to confirm differentiation and gain insight into the mechanism of altered differentiation. Unlike estrogen, stilbenes do not appear to negatively impact breast and reproductive organs. Stilbenes thus represent a novel option for inducing a preferential shift of MSCs toward the osteogenic lineage while potentially avoiding the negative health effects of estrogen. Targeting the differentiation of MSC to favor bone formation represents a viable target for the development of novel therapeutics for bone loss following cancer treatment. Further, identifying compounds that stimulate osteogenesis while repressing adipogenesis could benefit other metabolic disorders like obesity associated with increased breast cancer risk.

Citation Format: Evan Robert, Lyndsay V. Rhodes. Exploring the utility of natural and synthetic resveratrol derivatives for bone regrowth following loss due to breast cancer therapies. [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 3318.
Abstract 3321: PUMA expression targets αvβ3/Slug+ “stem-like” tumor cells to prevent breast cancer progression independent of subtype
Authors: Sun Qi , Lesperance Jacqueline , Desgrosellier Jay S.
Published: July 2016

Breast cancer cells bearing similarities to normal mammary stem cells represent some of the most aggressive tumors cells. Here, we show that αvβ3/Slug+ “stem-like” tumor cells are highly vulnerable to cell death induced by PUMA (p53-upregulated modulator of apoptosis). In human breast cancers, we were surprised to find that αvβ3/Slug+ cells represent a unique subset of tumor cells associated with progression that are present in all major histological subtypes. Analysis of “stem-like” cell lines showed that αvβ3 directs an EMT-independent role for Slug in suppressing PUMA expression and promoting tumor cell survival and stemness. In fact, αvβ3 is both necessary and sufficient for PUMA suppression and PUMA knockdown specifically rescued defective colony formation and tumor initiation caused by decreased β3 expression. Inducing PUMA expression with specific therapies selectively ablated αvβ3/Slug+ cells, preventing the formation of new colonies or tumors, with no effect on established disease. These new findings define an unexpected role for αvβ3/Slug-mediated PUMA suppression in driving stemness and suggest that inducing PUMA with specific therapies can selectively target “stem-like” breast cancer cells to prevent aggressive disease.

Citation Format: Qi Sun, Jacqueline Lesperance, Jay S. Desgrosellier. PUMA expression targets αvβ3/Slug+ “stem-like” tumor cells to prevent breast cancer progression independent of subtype. [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 3321.
Abstract 3325: Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance
Authors: Pathania Rajneesh , Kolhe Ravindra B. , Ramachandran Sabarish , Mariappan Gurusamy , Thakur Priyanka , Prasad Puttur D. , Ganapathy Vadivel , Thangaraju Muthusamy
Published: July 2016

In recent years, impressive technical advancements have been made in the isolation and validation of the mammary stem cells (MaSCs) and cancer stem cells (CSCs). However, the signaling pathways that regulate stem cell self-renewal are largely unknown. Further, CSCs are believed to contribute to resistance to chemotherapy and radiation therapy. However, an effective therapeutic strategy to overcome this resistance is yet to be identified. We have recently discovered that the DNA methyltransferases, especially DNMT1, play critical role in MaSCs and CSCs self-renewal and targeted deletion of this gene impaired mammary tumor formation by inhibiting CSCs formation. However, the molecular mechanism(s) by which DNMTs control CSCs and the therapeutic relevance of DNMTs inhibitors in regulation of CSCs and overcome drug resistance are also largely unknown. In this study, using MMTV-Neu-Tg mouse mammary tumor model, we found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which leads to activation of un-activated Neu-Tg into transformed tumor forming phenotype. Combination of 5-Azacytidine, a DNMT inhibitor, and butyrate, a HDAC inhibitor, markedly reduces CSCs and consequently increases the overall survival of the animal. RNA-seq analysis of the CSCs treated with 5-AzaC+butyrate provides evidence that combined inhibition of DNMTs and HDACs reduces CSCs pool in the mammary gland by blocking growth-promoting signaling molecules like RAD51AP1 and SPC25. RAD51AP1 and SPC25, which are known to play a key role in DNA damage repair and kinetochore assembly, are significantly overexpressed in breast tumor tissues and associated with decreased overall patients’ survival. Further, these two genes are overexpressed in Tamoxifen and Taxol resistant human breast cancer cell lines. Functional inactivation of these genes in breast caner cells facilitates chemotherapy-induced apoptosis and reduces tumor growth. Overall, our studies provide strong evidence that breast CSCs (both basal stem cells and luminal progenitor cells) are susceptible for genetic and epigenetic modifications and associated with resistance to chemo- and radiotherapy. Thus, combination of DNMT and HDAC inhibitors can serve as an effective therapeutic strategy to block mammary tumor growth and to overcome drug resistance by inhibiting CSCs.

Citation Format: Rajneesh Pathania, Ravindra B. Kolhe, Sabarish Ramachandran, Gurusamy Mariappan, Priyanka Thakur, Puttur D. Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance. [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 3325.
Abstract 3331: RP6530, a dual PI3K δ/γ inhibitor attenuates cancer stem cell proliferation in serous adenocarcinoma of ovary
Authors: S Sneha , R Nivetha , Viswanadha Srikant , Vakkalanka Swaroop , TS Ganesan
Published: July 2016

The overall survival of patients with advanced serous adenocarcinoma of the ovary (EOC) has not significantly improved for the past 3 decades despite advances in treatment. In part, this is due to resistance of malignant cells and it is currently hypothesized that this could be due to persistence of cancer stem cells (CSC). Elimination of these cells may improve the survival of patients. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor that demonstrated high potency against PI3Kδ (IC50 = 25 nM) and γ (IC50 = 33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. A Phase-1 dose-escalation study to evaluate the safety and efficacy of RP6530 in patients with hematological malignancies in currently underway at several sites in Europe. The objective of this study was to test the efficacy of RP6530 against the CSC of EOC. A spheroid assay enriched for CSC was employed for this study. Initially, we have analyzed 3 cell lines (PEO4, OVCAR-5 and CAOV-2) with and without addition of 5 μM RP6530. In addition, MTT assay for cell viability was performed for the molecule to evaluate for non-selective cytotoxicity. To confirm that the target was CSC, we performed analysis of Side population (SP) based on H33342 dye exclusion. In the screening assay (triplicate and repeated thrice), RP6530 significantly inhibited spheroid formation in PEO4 and OVCAR-5 (p<0.05) and CAOV-2 (p<0.0005). In the cell viability assay, RP6530 had no cytotoxic effect in any of the cell lines. To evaluate the effect of the compound on CSC, we performed analysis of SP cells with Hoechst 33342 at 355 nm by flow cytometry. RP6530 either caused a reduction in the fraction of SP cells (PEO4 and CAOV-2) or was neutral (OVCAR-5) indicating that the compound preferentially targets CSC in ovarian cancer cell lines. Further in vitro and in vivo experiments are ongoing to conclusively prove this effect.

Citation Format: Sneha S, Nivetha R, Srikant Viswanadha, Swaroop Vakkalanka, Ganesan TS. RP6530, a dual PI3K δ/γ inhibitor attenuates cancer stem cell proliferation in serous adenocarcinoma of ovary. [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 3331.