bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 243: Expression of T-cell checkpoint ligands on circulating tumor cells is modulated following treatment of patients with an anti-tumor DNA vaccine
Authors: Rekoske Brian T. , Olson Brian M. , McNeel Douglas G.
Published: August 2015

Background: We have previously demonstrated in a murine tumor model that treatment with a DNA vaccine could lead to increased PD-1 expression on antigen-specific CD8+ T cells, led to upregulated levels of PD-L1 on tumors, and that the anti-tumor efficacy of the vaccine was enhanced when combined with PD-1/L1 blockade. We therefore examined whether prostate cancer patients previously treated with a DNA vaccine similarly upregulated these immunosuppresive ligands on their tumor cells, or the corresponding receptors on tumor-specific CD8+ T cells, and if augmented Th1-type immune responses were detectable following in vitro stimulation with checkpoint blockade.

Methods: Circulating tumor cells (CTCs, CD45-/EpCAM+) from 16 prostate cancer patients with castrate-resistant disease, and antigen-specific (tetramer+) CD8+ T cells from those patients that were HLA-A2+ (6/16), were analyzed for their expression of various checkpoint ligands (PD-L1, galectin-9, HVEM) or receptors (PD-1, TIM-3, LAG-3, BTLA, CD160) using flow cytometry before and after treatment with a DNA vaccine encoding prostatic acid phosphatase (PAP). Immune responses to the PAP target antigen were measured in vitro for cytokine secretion via ELISA, or by a trans-vivo DTH-footpad assay, in the presence or absence of PD-1/L1 blockade.

Results: Patients that developed either long-term immune responses or changes in PSA doubling time following vaccination had significantly higher levels of PD-L1 and HVEM, and lower levels of galectin-9, on their post-treatment CTCs than patients who did not develop responses. No significant changes in PD-1 or other regulatory receptors were detectable on tetramer+ CD8+ T cells. However, at least 25% of patients following immunization had augmented PAP-specific cytokine secretion in vitro when combined with PD-L1 blockade. PD-1 regulated PAP-specific responses were also observed in patients using the trans vivo DTH assay.

Conclusions: These results demonstrate that the development of an anti-tumor immune response via DNA vaccination can lead to changes in checkpoint ligand expression on tumor cells and checkpoint-regulated immune responses. This suggests that combining DNA vaccines with blockade of one or more of these checkpoint pathways could produce enhanced anti-tumor responses in patients with prostate cancer.

Citation Format: Brian T. Rekoske, Brian M. Olson, Douglas G. McNeel. Expression of T-cell checkpoint ligands on circulating tumor cells is modulated following treatment of patients with an anti-tumor DNA vaccine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2015-243
Abstract 2434: The anticancer activity of Japonicone A is mediated by inhibiting NFAT1-MDM2 pathway
Authors: Voruganti Sukesh , Qin Jiang-Jiang , Wang Wei , Zhang Ruiwen
Published: August 2015

NFAT1 plays a crucial role in cancer development and progression and has been suggested as a novel target for cancer therapy. We recently report NFAT1 promotes the expression of the MDM2 oncogene, independent of p53. The present study was designed to elucidate the mechanisms of action for a newly identified NFAT1 inhibitor, Japonicone A (JapA), and demonstrate the value of targeting NFAT1-MDM2 signaling in cancer therapy. JapA was identified as a novel NFAT1 inhibitor from our initial screening of a natural product library. Its effects on NFAT1 expression and stability were analyzed in several human breast cancer cell lines with different genetic background, including MCF-7 (estrogen receptor (ER) positive and p53 wild-type), MCF-7 p53−/− (ER positive and p53 knockout), MDA-MB-231 (triple negative and p53 mutant), and MDA-MB-468 (triple negative and p53 mutant) cell lines. The effects of JapA on NFAT1 expression were also determined in MCF-7 and MDA-MB-231 xenograft tumors in vivo. To further demonstrate the specificity of JapA in targeting NFAT1-MDM2 pathway and the importance of NFAT1-MDM2 inhibition in JapA's anticancer activity, MCF7 NFAT1 overexpression (OE) and knockdown (KD) cell lines, as well as pharmacological activators and inhibitors of NFAT1 signaling were used in this study. Our results indicated that JapA showed a significant, specific inhibition of NFAT1 in breast cancer cells in vitro and in vivo. It induced NFAT1 protein ubiquitination and proteasomal degradation, and inhibited NFAT1 nuclear translocation. JapA also disrupted the binding of NFAT1 to the MDM2 P2 promoter, leading to the inhibition of MDM2 transcription. Furthermore, the inhibition of NFAT1 and the NFAT1-MDM2 signaling are critical for JapA's anticancer activity, as shown in NFAT1 OE and KD cell lines. In conclusion, JapA represents a new class of NFAT1 inhibitor. It exerts its anticancer activity through inhibiting NFAT1 and the NFAT1-MDM2 pathway, providing evidence for the value of NFAT1 as a novel target in developing effective targeted therapy for cancers. (Supported by NIH R01 CA112029, R01 CA121211, and R01 CA186662)

Citation Format: Sukesh Voruganti, Jiang-Jiang Qin, Wei Wang, Ruiwen Zhang. The anticancer activity of Japonicone A is mediated by inhibiting NFAT1-MDM2 pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2434. doi:10.1158/1538-7445.AM2015-2434
Abstract 2441: Engineering and mapping difficult proteins using comprehensive mutagenesis
Authors: Paes Cheryl , Goodman Jason , Wescott Melanie , Thaker Yana , Thomas Anu , Couto Joseph , Rucker Joseph , Doranz Benjamin J.
Published: August 2015

Alanine (Ala) scanning is a widely used mutagenesis approach for systematic evaluation of protein function, which for large targets can be laborious and time-consuming. We have developed a platform technology, Shotgun Mutagenesis, for rapid and comprehensive alanine scanning mutagenesis for protein mapping and engineering applications. Using this technology, every residue in a target protein is individually mutated, expressed in human cells, and assayed for desired functions. Critical mutations are rapidly identified using high-throughput 384-well assays including flow cytometry. Here we used Shotgun Mutagenesis to create comprehensive mutation libraries for different classes of targets including the GPCR CXCR4, bitter taste receptor TAS2R16 and the therapeutic antibody Palivizumab (Synagis). For the cancer target CXCR4, we identified critical residues for antibody binding (epitope mapping) and ligand-dependent activation (functional domain mapping). We also discovered TAS2R16 residues that can be altered for increased expression (protein optimization) and Palivizumab residues which tolerate germline substitutions permitting engineering of a maximally humanized antibody variant (antibody humanization). This platform allows comprehensive protein mapping and engineering of challenging targets previously considered prohibitively time-consuming or expensive, enabling discovery pathways for many untapped therapeutic targets.

Citation Format: Cheryl Paes, Jason Goodman, Melanie Wescott, Yana Thaker, Anu Thomas, Joseph Couto, Joseph Rucker, Benjamin J. Doranz. Engineering and mapping difficult proteins using comprehensive mutagenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2015-2441
Abstract 2453: The role of p21 in the toxicity of mitomycin C and decarbamoylmitomycin C
Authors: Champeil Elise , Cheng Shu-Yuan , Huang Bik Tzu , Seo Jiwon
Published: August 2015

The mitomycins are a family of antitumor antibiotics made by Streptomyces. One member of this family, mitomycin C (MC), is currently used to treat certain cancers. Its mode of action has been extensively examined. In comparison, 10-decarbamoyl mitomycin C (DMC) has not been the object of such intense investigation. It has recently been found to form similar or identical adducts with DNA as mitomycin C (MC). In particular, DMC generates a unique stereoisomeric interstrand crosslink (beta isomer, β-ICL). Although the DNA-adducts of both drugs share common structural features, the biochemical responses to the two drugs are different. In particular, contrary to MC, the DNA-adducts generated by DMC treatment (β-ICL) rapidly activate a p53-independent signal transduction pathway. Thus, the study MC-DMC provides an ideal model for identifying structural features determining the cell signaling outcome in the presence or the absence of a functioning p53 pathway. In particular, we are interested in correlating MC and DMC-adducts structures with the role of p21 in the toxicity of the α-ICL and β-ICL. In this report, we present the following preliminary results 1) Synthesis of the beta DNA monoadduct of DMC via post-oligomerization methods 2) Role of p21 in the upstream p53-independent signaling pathway in response to MC/DMC and the α-ICL.

Citation Format: Elise Champeil, Shu-Yuan Cheng, Bik Tzu Huang, Jiwon Seo. The role of p21 in the toxicity of mitomycin C and decarbamoylmitomycin C. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2453. doi:10.1158/1538-7445.AM2015-2453
Abstract 2454: Targeting the translational enhancer complex MCT-1:DenR to disrupt NHL survival
Authors: Stowe Sean D. , Cavalier Michael C. , Godoy-Ruiz Raquel , Varney Kristen J. , Wilder Paul T. , Gartenhaus Ronald B. , Weber David J.
Published: August 2015

Despite the vast number of therapeutic advances that have been made, Non-Hodgkin Lymphoma (NHL) remains a leading cause of cancer-related deaths in the United States. In order to increase the NHL cure rate, new targeted options are needed. One such option that has been the focus of much recent interest is the post-transcriptional regulation in anti-apoptotic phenotypes of NHL, particularly the translational enhancer complex of MCT-1 and DENR. Involved in translation initiation and ribosomal recycling for transcripts regulated by 5′-uORF’s, the MCT-1:DENR complex has been shown to increase cell proliferation and the translation of survival-related transcripts through the combination of two critical RNA-binding domains. The goal of this project has been to biophysically characterize the formation and activity of this complex and to develop a method for evaluating the effects of disrupting its formation in vivo through the combination of NMR, X-ray crystallography, cell-free and cell-based assays. Ultimately, the information gained from this study will be used to evaluate a library of perturbagens that interfere with dimer formation and the development of MCT-1 driven anti-apoptotic phenotypes.

Citation Format: Sean D. Stowe, Michael C. Cavalier, Raquel Godoy-Ruiz, Kristen J. Varney, Paul T. Wilder, Ronald B. Gartenhaus, David J. Weber. Targeting the translational enhancer complex MCT-1:DenR to disrupt NHL survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2015-2454
Abstract 2468: Antibody targeting soluble NKG2D ligand sMIC induces regression of primary tumors and eliminates metastasis in multiple pre-clinical cancer models
Authors: Wu Jennifer D. , Basher Fahmin , Rubinstein Mark
Published: August 2015

Expression of the MHC I-chain related molecules A and B (MICA/B) on epithelial cell surface in response to transformation or DNA damage can signal the immune system of the abnormality and thus initiate active immune surveillance by Natural Killer (NK) cells and cytotoxicity T cells. We and others have shown that malignant tumor cells can shed MICA/B to down regulate NKG2D expression and negatively impact NK and CD8 T cell function in cancer patients. Increased tumor-specific shedding of cell surface NKG2D ligand, MHC I chain related molecule (MIC), is associated with advanced stage and metastasis in many types of epithelial cancer. High serum levels of soluble MIC (sMIC) insults the immune system not only by down-regulating NKG2D expression on natural killer (NK) cells and effector T cells but also perturbing NK cell peripheral maintenance. Whether sMIC is an effective cancer therapeutic target has not been addressed due to the lack of specific sMIC-blocking reagent and clinically relevant pre-clinical animal models. Using a “humanized” clinically relevant spontaneous prostate carcinoma TRAMP/MIC bi-transgenic mouse model and multiple engineered syngeneic transplantable tumor models, we unprecedentedly show that therapy with a sMIC-specific monoclonal antibody induced rapid regression of primary tumors and metastasis without systemic toxicity. The therapy revamped a myriad of anti-tumor immune responses including NK cell homeostatic renewal and function, CD4 T cells to Th1 and Th17 responses. Using the well-defined melanoma model, we further show that anti-sMIC therapy augments Pmel-1 antigen-specific CD8 T cell responses. Notably, depletion of NK cells mitigated the therapeutic effect of anti-sMIC antibody and the effector function of both CD4 and CD8 T cells. We conclude that sMIC is an effective therapeutic target to potentiate innate and adoptive immune responses against MIC+ malignancies.

Citation Format: Jennifer D. Wu, Fahmin Basher, Mark Rubinstein. Antibody targeting soluble NKG2D ligand sMIC induces regression of primary tumors and eliminates metastasis in multiple pre-clinical cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2468. doi:10.1158/1538-7445.AM2015-2468
Abstract 2487: The antitumor efficacy of anti-BST2 antibody is significantly increased in combination with CpG oligodeoxynucleotide in endometrial cancer
Authors: Takahashi Yusuke , Serada Satoshi , Hiramatsu Kosuke , Kobiyama Kouji , Fujimoto Minoru , Ishii Ken , Naka Tetsuji
Published: August 2015

Background:

CpG oligodeoxynucleotides (ODN) has been shown to exhibit a potent immunostimulatory activity. Therefore, CpG ODN has been considered to be a promising material for enhancing the therapeutic effect of anticancer monoclonal antibody with antibody-dependent cell-mediated cytotoxicity (ADCC) activity. We have previously identified bone marrow stromal antigen 2 (BST2) as a therapeutic target of endometrial cancer cells and also demonstrated that an anti-BST2 antibody showed a potent anti-tumor effect by ADCC and complement-dependent cytotoxicity (CDC) activities against BST2 positive cells. The aim of this study was to evaluate the synergistic anti-tumor effect by combination of anti-BST2 antibody and CpG ODN, and to clarify the mechanisms of its anti-tumor effect, using xenograft model of BST2 positive endometrial cancer cells.

Material and Method:

We implanted 7-8 week-old female SCID mice with 5×106 HEC-88 nu endometrial cancer cells subcutaneously. Mice with tumor were treated by intra-peritoneal injection of an anti-BST2 mAb alone (12.5 and 200 μg/mouse, twice a week for four weeks), intra-tumoral injection of CpG ODN alone (10 μg/mouse, twice a week for four weeks) or combination therapy (12.5 μg/mouse of anti-BST2 mAb and 10 μg/mouse of CpG ODN). Anti-tumor effect was evaluated by monitoring the changes of tumor volume. Finally, population of NK cells and macrophages infiltrated into the tumor were analyzed by the FACS analysis, how CpG ODN induces immunopotentiative action.

Results:

Intraperitoneal treatment by anti-BST2 antibody showed a significant anti-tumor effect, dose dependently. The combination therapy of anti-BST2 antibody and CpG ODN was more effective than the single-agent therapy (p = 0.0002). In addition, the combination therapy also resulted high population of NK cells and macrophages in tumor significantly (p = 0.00018).

Conclusion:

We showed that CpG ODN increases the efficacy of anti-tumor effect of anti-BST2 mAb induced ADCC activity via potentiating NK cells and macrophages mediated cancer cell injury. Combination of GpG ODN would be a promising approach for enhancing the activity of ADCC based monoclonal antibodies, which targeting anti-tumor antigen.

Citation Format: Yusuke Takahashi, Satoshi Serada, Kosuke Hiramatsu, Kouji Kobiyama, Minoru Fujimoto, Ken Ishii, Tetsuji Naka. The antitumor efficacy of anti-BST2 antibody is significantly increased in combination with CpG oligodeoxynucleotide in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2487. doi:10.1158/1538-7445.AM2015-2487
Abstract 2505: Therapeutic efficacy of the ZVex™ and GLAAS™ platforms in a B16-F10/hCAIX melanoma mouse model
Authors: Campbell David J. , Reeves Rebecca S. , Flynn Patrick A. , Robbins Scott H. , Berglund Peter , ter Meulen Jan H.
Published: August 2015

ZVex™ and GLAAS™ are two dendritic-cell (DC) targeting platform technologies designed to enhance immune responses through the in vivo induction of antigen specific CD8 and CD4 T-cells, respectively. ZVex™ is a lentiviral vector pseudotyped with a modified Sindbis virus envelope engineered to deliver tumor antigen-encoding nucleic acids to dendritic cells in vivo. GLAAS™(Glucopyranosyl Lipid A Adjuvant System) activates DC by binding to the TLR-4 receptor and inducing strong Th1 type CD4 responses against co-delivered recombinant proteins. Currently both platform technologies are being investigated in phase I clinical trials in cancer patients.

Human carbonic anhydrase 9 (hCAIX) is a tumor-associated transmembrane antigen that is over-expressed on various cancer cell types. We mapped hCAIX specific, multi-functional CD8 and CD4 T-cell epitopes within the extracellular and transmembrane regions of the protein for the mouse haplotype H-2b by intracellular cytokine staining. Mice lethally challenged s.c. on the flank with a B16-F10 tumor cell line expressing the hCAIX protein (designated BC.12) fully controlled large tumors (>100 mm2) when therapeutically immunized (subcutaneously at the base of tail) with ZVex™ encoding hCAIX or the recombinant hCAIX protein with GLAAS™ (either s.c. or i.m.). In both models, tumor control was dose-dependent. Additionally, the presence of a strong transmembrane H-2b restricted CD8 T-cell epitope was required for tumor control and regression. hCAIX-specific CD8 T-cell responses were detectable as far out as day 67 post challenge in mice displaying full regression of tumor. These results demonstrate proof of concept for ZVex™ and GLAAS™ platform technologies in an aggressive murine melanoma model.

Citation Format: David J. Campbell, Rebecca S. Reeves, Patrick A. Flynn, Scott H. Robbins, Peter Berglund, Jan H. ter Meulen. Therapeutic efficacy of the ZVex™ and GLAAS™ platforms in a B16-F10/hCAIX melanoma mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2015-2505
Abstract 2506: Preclinical characterization of LV305, a lentiviral vector targeting tumors expressing NY-ESO-1
Authors: Albershardt Tina C. , Campbell David J. , Parsons Andrea J. , ter Meulen Jan H. , Berglund Peter
Published: August 2015

The cancer-testis antigen, NY-ESO-1, is an attractive tumor antigen target for cancer immunotherapy, because it is expressed in numerous cancer types (e.g., melanoma, breast cancer, sarcoma, lung cancer) but not in normal adult tissues except the testis or placenta. Although recent studies using various NY-ESO-1 formulations show promising results, an optimal immunotherapeutic strategy to generate T cell responses for controlling tumor growth is still lacking. Lentiviral vectors (LVs) are proven tools for delivering nucleic acid payloads into cells due to their ability to transduce not only dividing cells but also quiescent cells, such as dendritic cells (DCs). Because DCs are essential for the initiation of T cell responses, we have engineered LV305 (a product candidate from ZVex™, our LV-based discovery platform) to selectively deliver the antigen-encoding NY-ESO-1 gene to human DCs in vivo. To develop tools to evaluate LV305 in a preclinical setting, we 1) generated a cell line, CIN.23, which is a CT26 murine colon cancer cell line that stably expresses human NY-ESO-1 protein; and 2) mapped and confirmed NY-ESO-1 CD8 and CD4 T cell epitopes recognized by H2d. Mice immunized with LV305 developed strong, dose-dependent, poly-functional, and cytotoxic NY-ESO-1-specific CD8 T cells within 14 days post-immunization and could be boosted with LV305 at least three times to recall peak-level CD8 T cell responses. Immunization with LV305 protected mice against CIN.23 tumor challenge, which was abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, together with CD4 T cells or NK cells, from LV305-immunized donor mice to tumor-bearing mice conferred significant protection against metastatic tumor growth. This protection was not observed when CD8 T cells alone were adoptively transferred. Taken together, these findings suggest that CD8 T cells are required but not sufficient for mediating LV305-induced anti-tumor efficacy. We have demonstrated the anti-tumor therapeutic potential of LV305-mediated in vivo DC immunization in murine models and are currently investigating LV305 in a phase I clinical trial in cancer patients with tumors expressing NY-ESO-1.

Citation Format: Tina C. Albershardt, David J. Campbell, Andrea J. Parsons, Jan H. ter Meulen, Peter Berglund. Preclinical characterization of LV305, a lentiviral vector targeting tumors expressing NY-ESO-1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2506. doi:10.1158/1538-7445.AM2015-2506
Abstract 252: Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of immune checkpoint blockades in K1735 and B16 mouse melanoma models
Authors: Freimark Bruce , Gong Jian , Ye Dan , Brekken Rolf , Yin Shen , Hutchins Jeff , Nguyen Van , Hughes Chris , Huang Xianming
Published: August 2015

Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed abundantly on tumor endothelial cells, tumor-secreted microvesicles, and tumor cells in response to chemo- and radiotherapy. Extensive studies have shown that PS is a global immune checkpoint and major contributor to tumor immunosuppression, which promotes the expansion of immunosuppressive cells, such MDSC and M2 macrophages, inhibits DCs maturation, while stimulates them to secrete immunosuppressive mediators. We have shown that PS targeting can override PS-mediated immunosuppression, reactivate innate tumor immunity, and evoke adaptive antitumor immunity. In the present study, we assessed the antitumor effect of the combination of PS blockade and anti-CTLA-4 or anti-PD-1 antibodies in B16 and K1735 melanoma models. Both combinations showed significantly superior tumor growth inhibition over single treatment, with many mice achieving complete tumor regression. Flow cytometry analysis showed that the combination treatment had significantly greater total and functional tumor-infiltrating CD8+ T, more IL-2- and IFNγ-producing splenic T cells, and lower number of splenic MDSCs than did single treatment. In addition, the ratio of M2 to M1 in the tumor was significantly lower in the combination treatment than that in single treatment. Finally, no toxicity was observed in any of the treatment groups following multiple treatment doses. These data suggest that combination of PS blockade with immune checkpoint blockade promotes strong, localized, enhanced therapeutic efficacy without the side-effects of systemic immune activation and represents a promising combinatorial strategy for cancer immunotherapy. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in multiple late-stage clinical trials. These data support our Phase I IST evaluation of our PS-targeting antibody bavituximab in combination with ipilimumab in advanced melanoma patients.

Citation Format: Bruce Freimark, Jian Gong, Dan Ye, Rolf Brekken, Shen Yin, Jeff Hutchins, Van Nguyen, Chris Hughes, Xianming Huang. Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of immune checkpoint blockades in K1735 and B16 mouse melanoma models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2015-252
Abstract 2520: Synergistic combination of lurbinectedin and PARP inhibitors in breast cancer tumor cell lines
Authors: Santamaría Gema , Avila Sonia , Moneo Victoria , Cuevas Carmen , García-Fernández Luis F. , Galmarini Carlos M.
Published: August 2015

Lurbinectedin (PM1183) is a new tetrahydroisoquinoline derivative currently evaluated in phase II trials in BRCA1-mutated breast cancer and NSCLC patients. Two pivotal trials (in platinum-resistant ovarian cancer and in SCLC) will be launched in 2015. PM1183 shows antitumor activity against a wide variety of tumor cells with mean IC50 values in the low nanomolar range. In living cells, PM1183 inhibits trans-activated transcription and blocks NER-dependent DNA repair inducing the formation of dsDNA breaks and the collapse of replication forks that need to be repaired by homologous recombination (HR). As expected, PM1183 is more active against homologous recombination (HR)-deficient cell lines. In this work, we evaluated the in vitro antitumor effect of PM1183 when combined with the PARP inhibitors ABT-88 (Veliparib, PARP-1 and -2), AZD-2281 (Olaparib, PARP-1), BSI-201 (Iniparib, PARP-1) and BMN-673 (PARP-1 and -2) (Selleck Chemicals, Houston, TX, USA) in a panel of human breast carcinoma cells with different BRCA1 status, including MCF-7 (BRCA1 +/+), MDA-MB-231 (BRCA1 +/-), HCC-1937 (BRCA1 -/-) and MDA-MB-436 (BRCA1 -/-). The combination index (CI) of each different drug-drug combination was calculated by applying the Chou and Talalay method. Different degrees of synergism (CI < 1) were recorded when PM1183 was combined with AZD-2281 (Olaparib) and BMN-673, depending on the tumor cell line and the conditions tested. The highest degree of synergism with Olaparib was observed in MDA-MB-436 cells followed by MDA-MB-231, MCF7 and HCC-1937 cells. The highest degree of synergism with BMN-673 was observed in MCF-7 cells followed by MDA-MB-231 and HCC-1937 cells. In MDA-MB-231 cells, both combinations led to a synergistic induction of γ-H2AX expression and PARP-1 cleavage, as analyzed by western blot. Collectively, our results indicate that the combination of PM1183 and PARP inhibitors (Olaparib and BMN673) induces an artificial synthetic lethality that can be advantageously used to kill several breast cancer cells, independently from their BRCA1 status.

Citation Format: Gema Santamaría, Sonia Avila, Victoria Moneo, Carmen Cuevas, Luis F. García-Fernández, Carlos M. Galmarini. Synergistic combination of lurbinectedin and PARP inhibitors in breast cancer tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2520. doi:10.1158/1538-7445.AM2015-2520
Abstract 2522: Molecular and cellular mechanisms underlying the therapeutic efficacy of the combination of JAK inhibitor, ruxolitinib, PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical model of myeloproliferative neoplasia
Authors: Pinzon-Ortiz Maria , Longmire Tyler , Rong Xianhui , Caponigro Giordano , Vanasse Gary , Lee Benjamin H. , Cao Z. Alexander
Published: August 2015

The JAK-STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. The genetic aberration of JAK2V617F and the associated activation of STAT in myeloproliferative neoplasia (MPN) is one example of the involvement of this pathway in human cancer. We have previously shown the combination benefits of combining ruxolitinib (Jak1,2 inhibitor), with LGH447 (PIM inhibitor) and LEE011 (CDK4/6 inhibitor) in a Ba/F3-JAK2V617F-driven MPN model. Here, we set to explore the molecular and cellular mechanisms underlying this combination benefit in the JAK2V617F UKE-1 model of MPN.

The triple combination of ruxolitinib, LGH447 and LEE011 resulted in sustained tumor regression in the UKE-1 model. Ruxolitinib, LGH447 and LEE011 inhibited pSTAT5, pBAD and pRb respectively. The monotherapies and the combination of ruxolitinib + LGH447 did not affect G1-S cycle. In contrast, the combination of ruxolitinib+LEE011, LGH447+LEE011 and the triple combination resulted in significant inhibition of G1-S progression. Consequently, after 72 hours of treatment, the percentage of cells in S phase decreased from 67% under DMSO to 15.8% under ruxolitinib+LEE011, and to 1.6% under the triple combination. Furthermore, monotherapies of LGH447 or LEE011 did not affect cell viability. Ruxolitinib monotherapy and ruxolitinib+LGH447 combination reduced viable cell% by 47% and 86%, respectively. The triple combination reduced it by 91%.

Our data suggest that the triple combination efficacy is the result of both cell killing and cell cycle blockade. The inhibition of JAK-PIM pathway significantly reduced cell viability. And the inclusion of CDK4/6 inhibition by LEE011 lead to more potent cell killing and cell growth arrest. Taken together, the triple combination achieves sustained tumor regression in preclinical models of MPN.

Citation Format: Maria Pinzon-Ortiz, Tyler Longmire, Xianhui Rong, Giordano Caponigro, Gary Vanasse, Benjamin H. Lee, Z. Alexander Cao. Molecular and cellular mechanisms underlying the therapeutic efficacy of the combination of JAK inhibitor, ruxolitinib, PIM inhibitor, LGH447, and CDK4/6 inhibitor, LEE011, in a preclinical model of myeloproliferative neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2522. doi:10.1158/1538-7445.AM2015-2522
Abstract 253: Synergistic antitumor activity of PD-1 signaling blockade and CD27 costimulation correlates with enhanced ratio of effector to regulatory T cells at the tumor site
Authors: Thomas Lawrence J. , He Li-Zhen , Wasiuk Anna , Gergel Lauren E. , Boyer James M. , Round Sarah M. , Marsh Henry C. , Keler Tibor
Published: August 2015

Varlilumab (CDX-1127) is a fully human agonist antibody to CD27, a TNF receptor superfamily member expressed on the majority of T cells and subsets of NK cells and B cells. As such, varlilumab is an immunotherapy designed to harness the body's natural immune response by enhancing the activation of T cells that can specifically recognize and kill cancer cells. In an ongoing Phase 1 clinical trial, varlilumab has been very well tolerated and has demonstrated clear biologic activity and promising signs of clinical activity in advanced, treatment-refractory patient populations. This clinical data provides the rationale for combination studies with other immunomodulatory therapies, including inhibitors of PD-1 signaling. In this study we investigated the activity of varlilumab in combination with anti-PD-L1 using a disseminated murine B cell tumor, BCL1 in human CD27 transgenic mice. In this model of i.v. inoculated BCL1 cells, we have previously shown that varlilumab can delay tumor growth, but rarely results in cures. Despite expression of PD-L1, the BCL1 tumors were completely resistant to anti-PD-L1 monotherapy. However, the combination of varlilumab and anti-PD-L1 resulted in a significant improvement in survival and approximately 50% long-term survivors. To investigate the mechanism resulting in the synergistic activity, we collected spleens (the major site of tumor growth) from tumor bearing animals treated with individual or combined regimens and processed them for flow cytometry analysis. We found that animals treated with the combination of varlilumab and anti-PD-L1 had a higher ratio of infiltrating immune cells to tumor cells relative to monotherapy or saline controls. In particular, the ratio of effector (CD8+) T cells to Treg and the number of IFNγ+ CD4 and CD8 T cells were highly increased with the combination treatment. These data demonstrate that CD27 costimulation combined with checkpoint inhibition with anti-PD-L1 can result in improved immune profile in the tumor and synergistic survival benefit. This study, along with the good safety profile of varlilumab reported in the Phase 1 study, support the design of future combination studies in patients with cancer.

Citation Format: Lawrence J. Thomas, Li-Zhen He, Anna Wasiuk, Lauren E. Gergel, James M. Boyer, Sarah M. Round, Henry C. Marsh, Tibor Keler. Synergistic antitumor activity of PD-1 signaling blockade and CD27 costimulation correlates with enhanced ratio of effector to regulatory T cells at the tumor site. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 253. doi:10.1158/1538-7445.AM2015-253
Abstract 2535: Synergistic anti-tumor activity of the Notch gamma secretase inhibitor BMS-906024 and paclitaxel in the treatment of lung adenocarcinoma
Authors: Morgan Katherine M. , Lee Francis , Michaud Erin , Bertino Joseph R. , Fischer Bruce S. , Pine Sharon R.
Published: August 2015

Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancer cases, primarily through loss of the endogenous Notch inhibitor, Numb, or via gain-of-function mutations in the Notch1 receptor. Notch activity is associated with poor overall survival among non-small cell lung cancer patients whose tumors are wildtype for TP53. We set out to evaluate the combination of Notch-targeted therapy with front-line chemotherapy as an effective treatment for non-small cell lung cancer. Our study focused on lung adenocarcinoma, the most common histological subtype in lung cancer. To target Notch, we utilized the gamma secretase inhibitor (GSI) BMS-906024 which inhibits Notch activation. BMS-906024 is currently in Phase 1 clinical trials for patients with T-cell acute lymphoblastic leukemia and metastatic solid tumors, including lung cancer. Human cell lines representing the major genetic subtypes of lung cancer, most of which were derived from adenocarcinomas, underwent MTS drug synergy assays consisting of treatment with BMS-906024, cisplatin or paclitaxel, or the combination of GSI and chemotherapy. The dosing and timing for BMS-906024 administration were optimized by examination of maximal Notch1 inhibition. Analysis of the drug effects with CalcuSyn yielded Combination Index (CI) values, in which a CI of 0.5 or less was considered as strong synergism for the drug combination. We found that there were significantly lower CI values for the GSI BMS-906024 combined with paclitaxel than with cisplatin (average CI = 0.54 vs 0.85, respectively; P = 0.001). We then grouped the cell lines by major genetic subtype (wildtype versus mutant or null for EGFR, Kras or TP53). The synergy between BMS-906024 and paclitaxel was significantly greater in Kras-wildtype than Kras-mutant cells (average CI = 0.39 vs 0.68, respectively; P = 0.009), while there was no correlation with EGFR or TP53 status. These results are a step toward identification of potential biomarkers that could be used to predict patient response to Notch-targeted therapy, which could have a positive impact on the care of lung adenocarcinoma patients and be informative for treatment decisions.

Citation Format: Katherine M. Morgan, Francis Lee, Erin Michaud, Joseph R. Bertino, Bruce S. Fischer, Sharon R. Pine. Synergistic anti-tumor activity of the Notch gamma secretase inhibitor BMS-906024 and paclitaxel in the treatment of lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2535. doi:10.1158/1538-7445.AM2015-2535
Abstract 2536: Assessing combinations of FDA approved chemotherapy in malignant peripheral nerve sheath tumor (MPNST) cell lines
Authors: Kahen Elliott , Yu Diana , Cubitt Christopher , Sullivan Dan , Reed Damon R.
Published: August 2015

Background: Malignant Peripheral Neural Sheath Tumors (MPNST) are a type of sarcoma that arise spontaneously from a peripheral nerve or plexiform neurofibroma. Approximately 1 in 10 people with a constitutional mutation in the NF1 gene, a negative regulator of RAS function, develop MPNST with a peak incidence in the adolescent and young adult years. Available chemotherapy treatment for MPNST has low response rates, and the prognosis for unresectable or metastatic tumors is poor. A clinically relevant system to rapidly evaluate the efficacy of many agents and combinations of agents on MPNSTs which can be translated into clinical trials is needed.

Methods: Here, we seek to identify promising single drug and combination therapies using FDA approved and other agents of interest via high-throughput screening platforms, based on established MPNST cell line models (SNF02.2, SNF10.1, SNF94.3, and SNF96.2). Taking into consideration past lessons learned from in vitro experiments, we designed stringent screening conditions that assess the candidate compounds and combinations at clinically-relevant concentrations and exposure times that mimic the in vivo pharmacokinetics in an effort to maximize the translational potential of these results. Fifty-four agents with roughly ¼ inhibitors of the RAS downstream pathways, another ¼ kinase inhibitors, and the remainder a representation of many classes of agents with diverse mechanisms of action were screened across 4 cell lines with biological replicates. The majority of the agents are FDA approved. Others are in active clinical trials, demonstrate a promising mechanism of action, or have published preclinical data in MPNST models. CellTiter Glo assays were used to assess cell viability at the maximal achievable serum concentration from phase I studies and at two lower concentrations. Promising agents were then studied in two-drug combinations to evaluate for synergy in addition to efficacy of the combination. The combinations were selected with careful attention to known drug-drug interactions, metabolism, schedule and non-overlapping toxicities.

Results: Our screening results indicate that a number of FDA approved chemotherapy agents, evaluated at clinically relevant concentrations and exposure times, significantly reduced cell viability across all cell lines tested. These findings suggest promising opportunities for developing combination clinical trials.

Citation Format: Elliott Kahen, Diana Yu, Christopher Cubitt, Dan Sullivan, Damon R. Reed. Assessing combinations of FDA approved chemotherapy in malignant peripheral nerve sheath tumor (MPNST) cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2536. doi:10.1158/1538-7445.AM2015-2536
Abstract 2560: The cisplatin and Chal-24 combination synergistically kills cancer cells through IAP and c-FLIP degradation, Ripoptosome formation and autophagy-mediated apoptosis
Authors: Shi Shaoqing , Wang Qiong , Xu Jennings , Padilla Mabel T. , Nyunoya Toru , Xing Chengguo , Zhang Lin , Lin Yong
Published: August 2015

Because chemoresistance is the major hurdle of anticancer therapy, sensitizing cancer cells to chemotherapy is a main challenge for improving survival in cancer patients. Combined therapy by using drugs with distinct functioning mechanisms may increase anticancer efficacy. We have recently identified the novel chalcone derivative chalcone-24 (Chal-24) as a potential therapeutic that kills cancer cells through activation of an autophagy-mediated necroptosis pathway. In this study, we investigated if Chal-24 can be combined with the frontline genotoxic anticancer drug cisplatin for cancer therapy. The combination of Chal-24 and cisplatin synergistically induced apoptotic cytotoxicity in lung cancer cell lines, which was dependent on Chal-24-induced autophagy. While cisplatin slightly potentiated the JNK/Bcl2/Beklin1 pathway for autophagy activation, it strongly triggered Chal-24-induced proteasomal degradation of the cellular inhibitor of apoptosis proteins (c-IAPs) and formation of the Ripoptosome complex that contains RIP1, FADD and caspase 8. Furthermore, cisplatin induced dramatic degradation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (cFLIPL) which suppresses Ripoptosome-mediated apoptosis activation. These results establish a dual mechanism for potentiation of anticancer activity with combination of Chal-24 and cisplatin: to enhance apoptosis signaling through Ripoptosome formation and to release the apoptosis brake through c-FLIP degradation. Altogether, our work suggests that combination of Chal-24 and cisplatin could be employed for improving chemotherapy efficacy. (Supported by R01ES017328 (NIEHS/NIH) and DE-FG02-09ER64783 (DOE).

Citation Format: Shaoqing Shi, Qiong Wang, Jennings Xu, Mabel T. Padilla, Toru Nyunoya, Chengguo Xing, Lin Zhang, Yong Lin. The cisplatin and Chal-24 combination synergistically kills cancer cells through IAP and c-FLIP degradation, Ripoptosome formation and autophagy-mediated apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2560. doi:10.1158/1538-7445.AM2015-2560
Abstract 2573: Investigation of factors involved in the hypersensitivity to KP1339-treatment
Authors: Alte Beatrix , Pirker Christine , Mohr Thomas , Kryeziu Kushtrim , Keppler Bernhard K. , Heffeter Petra , Berger Walter
Published: August 2015

The ruthenium-based compound KP1339 (trans-(tetrachlorobis(1H-indazole)ruthenate(III)), which is currently in Phase I/II trial, is already known for its promising anticancer activity. In course of a recent screening using diverse cancer cell lines of different origin (n = 25), a small subgroup with exceptional sensitivity to KP1339-treatment was discovered. The aim of the here presented study was to identify factors that underlie this sensitivity or which are connected to resistance to KP1339. Due to the fact that KP1339 comprises a metal center, interpretations of it's mode of action tend towards either generation of ROS or DNA binding. To avoid a biased approach, whole genome expression arrays were performed with mRNA isolated from hypersensitive and normal responsive cells that were either untreated or treated with KP1339 for 3 h and 6 h. Subsequent bioinformatic analysis of the gained data indicated that while hypersensitive cell lines activated pathways indicative for response to chemical stimuli (“metal ion”, “organic compound”), in normal responsive ones preferentially genes involved in pathways controlling cell cycle, DNA repair and metabolism were found. Subsequent investigations confirmed that upon KP1339-treatment hypersensitive cell lines showed pronounced apoptosis induction (indicated by caspase-mediated PARP cleavage and appearance of apoptotic nuclei visualized by DAPI staining). In contrast, normal responsive cells rather reacted with prolonged duration of the G2 phase to KP1339 treatment (video evaluation). This was confirmed by FACS analysis of ethanol-fixed and propidium iodide-stained cells indicating a profound increase in the percentage of cells with double DNA content upon KP1339 treatment. Moreover, in accordance to previous studies, KP1339 induced strong phosphorylation of ERK, p38 and JNK especially in normal responsive cell lines. This is of interest as p38 and JNK activation are known to be pro-survival factors within the extrinsic pathway of apoptosis. The relevance of this particular pathway is supported by preliminary results of activation of caspase 8 upon KP1339 treatment in hypersensitive cells. This knowledge can help to better understand the mechanisms underlying the promising anticancer activity of KP1339.

Citation Format: Beatrix Alte, Christine Pirker, Thomas Mohr, Kushtrim Kryeziu, Bernhard K. Keppler, Petra Heffeter, Walter Berger. Investigation of factors involved in the hypersensitivity to KP1339-treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2573. doi:10.1158/1538-7445.AM2015-2573