SCID Mouse Xenograft Model Research Articles

Disruption of Aneuploidy and Senescence Induced by Aurora Inhibition Promotes Intrinsic Apoptosis in Double Hit or Double Expressor Diffuse Large B-cell Lymphomas.

Double hit (DH) or double expressor (DE) diffuse large B-cell lymphomas (DLBCL) are aggressive non-Hodgkin's lymphomas (NHL) with translocations and/or overexpressions of MYC and BCL-2, which are difficult to treat. Aurora kinase (AK) inhibition with alisertib in DH/DE-DLBCL induces cell death in ∼30%, while ∼70% are aneuploid and senescent cells (AASC), a mitotic escape mechanism contributing to drug resistance. These AASCs elaborated a high metabolic rate by increased AKT/mTOR and ERK/MAPK activity via BTK signaling through the chronic active B-cell receptor (BCR) pathway. Combinations of alisertib + ibrutinib or alisertib + ibrutinib + rituximab significantly reduced AASCs with enhanced intrinsic cell death. Inhibition of AK + BTK reduced phosphorylation of AKT/mTOR and ERK-1/2, upregulated phospho-H2A-X and Chk-2 (DNA damage), reduced Bcl-6, and decreased Bcl-2 and Bcl-xL and induced apoptosis by PARP cleavage. In a DE-DLBCL SCID mouse xenograft model, ibrutinib alone was inactive, while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ∼25%. However, TGI for ibrutinib + rituximab was ∼50% to 60%. In contrast, triple therapy showed a TGI rate of >90%. Kaplan-Meier survival analysis showed that 67% of mice were alive at day 89 with triple therapy versus 20% with ibrutinib + rituximab. All treatments were well tolerated with no changes in body weights. A novel triple therapy consisting of alisertib + ibrutinib + rituximab inhibits AASCs induced by AK inhibition in DH/DE-DLBCL leading to a significant antiproliferative signal, enhanced intrinsic apoptosis and may be of therapeutic potential in these lymphomas. Mol Cancer Ther; 16(10); 2083-93. ©2017 AACR.

Invitro and invivo anti-primary effusion lymphoma activities of fucoidan extracted from Cladosiphon okamuranus Tokida.

Primary effusion lymphoma (PEL) caused by Kaposi's sarcoma-associated herpesvirus (KSHV) is characterized by lymphomatous effusion in body cavities and poor prognosis. There is still no effective treatment for PEL. Fucoidan, a major sulfated polysaccharide isolated from brown seaweeds, has an attractive array of bioactivities such as cancer inhibition. However, the effects of fucoidan on PEL cells remain unclear. We investigated the anti-PEL effects of fucoidan obtained from Cladosiphon okamuranus Tokida cultivated in Okinawa. Fucoidan dose-dependently inhibited the proliferation of KSHV-infected PEL cell lines, and provoked G1 cell cycle arrest, which was accompanied by CDK4 and CDK6 downregulation. Fucoidan also induced apoptosis of PEL cells through caspase-3, -8 and -9 activation; this occurred partly through the downregulation of anti-apoptotic Bcl-xL, Mcl-1 and XIAP proteins. Fucoidan also suppressed nuclear factor-κB, activator protein-1 (AP-1), and T-lymphokine-activated killer cell-originated protein kinase (TOPK) signaling pathways through inhibition of phosphorylation of IκBα and TOPK, and the expression of AP-1 family proteins, JunB and JunD. Oral administration of fucoidan effectively inhibited the development of PEL cells and ascites in a xenograft SCID mouse model, with minimal serious adverse effects. Notably, native fucoidan exhibited a more efficient anti-PEL effect than nanoparticle fucoidan. These preclinical findings highlight the anti-PEL actions of fucoidan, suggesting it could be potentially useful for the prevention and treatment of PEL.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

Abstract 4057: Inhibition of polyploidy and senescence induces intrinsic apoptosis in double hit or double expresser DLBCL

Abstract Introduction: Double Hit (DH) or Double Expresser (DE) DLBCL are aggressive Non-Hodgkin’s Lymphomas (NHLs) with translocation or over-expression of MYC and BCL-2 which are incurable with current therapies. MYC is a critical driver of oncogenic phenotypes and regulates expression of numerous genes including aurora kinases (AKs), which are validated drug targets. AKs are over-expressed in DLBCL and AK inhibition in two Phase-II trials of relapsed and refractory B/T-NHL resulted in 20%-30% overall response rate. Bruton’s tyrosine kinase (BTK) plays a central role in B-cell receptor (BCR) signaling and inhibitors of BTK have shown efficacy in B-NHL clinical trials. Targeting CD20 (rituximab) in DLBCL inhibits BCL-2 and induces apoptosis via caspase-3 activation. We hypothesized that DH/DE-DLBCL are amenable to a novel therapeutic strategy of AK, BTK and CD20 inhibition. Methods: Four DH/DE DLBCL cell lines were evaluated for viability by MTS; polyploidy, senescence and apoptosis by flow cytometry; DNA damage by TUNEL assay. Phospho-BCL-2, -AK and -BTK analysis by western. Safety and efficacy of drug combinations in a mouse xenograft model. Analysis of patient samples and mouse tumors by IHC. Results: DH/DE-DLBCL cell lines and patient samples displayed over-expression of MYC, BCL-2, BTK and AK-A proteins. Cell viability assays with alisertib alone showed ~30% cell death, while majority of remaining ~70% cells were polyploid and senescent at day-4 (a mechanism of treatment failure). These polyploid and senescent cells (PASC) showed increased levels of phospho-BTK indicating increased BCR signaling (a rationale for combination therapy). We show that alisertib + ibrutinib + rituximab significantly reduced PASCs leading to intrinsic cell death compared to single/double combinations. Dual inhibition of AK + BTK reduced phospho-ERK-1/2, -Bcl-6 and -MYC with increased DNA breaks indicated by up regulation of phospho-H2A-X, -Chk-2, and pro-apoptotic Noxa and Puma proteins. Triple therapy reduced Bcl-2, Bcl-xL and increased Noxa and Puma compromising the mitochondrial outer membrane potential, release of Cyt-C, activation of caspase-3 and induction apoptosis by PARP cleavage. A DLBCL SCID mouse xenograft model showed ibrutinib alone is inactive while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ~25%. TGI for ibrutinib + rituximab was ~50-60%. However, triple therapy showed a TGI of >90% and was well tolerated. Survival analysis: 67% of mice alive at day-89 with triple therapy versus 20% with ibrutinib + rituximab. Other treatments showed no survival past day-52. Conclusions: A novel triple therapy consisting of alisertib + ibrutinib + rituximab inhibits PASCs induced by AK inhibition in DH/DE-DLBCL leading to a significant anti-proliferative signal and intrinsic apoptosis. Our work provides a rationale for exploring these combinations in the clinic. Citation Format: Shariful Islam, Wenqing Qi, Carla Morales, Laurence Cooke, Catherine M. Spier, Daruka Mahadevan. Inhibition of polyploidy and senescence induces intrinsic apoptosis in double hit or double expresser DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2017-4057

Abstract B89: The chimeric antibody NZ-12 or CAR T cells targeting human podoplanin possesses antitumor activity against orthotopic xenograft models

Abstract Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, which is expressed in lymphatic endothelium. PDPN is also highly expressed in many cancers such as malignant pleural mesothelioma (MPM), lung cancer, and malignant brain tumors including glioblastoma (GBM). We previously developed a rat anti-PDPN monoclonal antibody NZ-1 (rat IgG2a, lambda), and a rat-human chimeric anti-PDPN antibody NZ-8 (human IgG1, kappa), which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against PDPN-positive cancer cells. Firstly, we demonstrated the antitumor effect of not only NZ-1/NZ-8 but also NZ-12 (human IgG1, lambda), a novel rat-human chimeric anti-PDPN antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a+) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human NK (CD56+) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Moreover, NZ-12 induced potent ADCC mediated by human MNC, compared with NZ-1/NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56+) cells in MPM orthotopic xenograft mice. Secondly, we constructed a lentiviral vector expressing chimeric antigen receptor (CAR) comprising NZ-1-based single chain variable fragment (scFv) with CD28, 4-1BB, and CD3zeta intracellular domains. CAR-transduced peripheral blood monocytes (PBMCs) were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. Taken together, PDPN-targeting antibody therapy or PDPN-targeting CAR T cell therapy might provide an efficacious therapeutic strategy for the treatment of MPM or GBM. Citation Format: Yukinari Kato, Satoshi Shiina, Atsushi Natsume, Shinji Abe, Yasuhiko Nishioka, Satoshi Ogasawara, Mika K. Kaneko. The chimeric antibody NZ-12 or CAR T cells targeting human podoplanin possesses antitumor activity against orthotopic xenograft models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B89.

Treating B Cell Lymphomas with Anti CD81 Antibodies

The tetraspanin CD81 associates with CD19 on B cells; this molecular complex functions as co-receptor to lower the threshold of BCR-initiated B cell activation.Recently we have shown the importance of CD81 in tumor growth and metastasis of solid tumors (Vences-Catalan et al., 2015). However, the role of CD81 in lymphoid malignancies has not been explored. Previous studies demonstrated anti-proliferative effects of anti-CD81 antibodies on human B cell lymphomas using in vitro assays (Oren et al., 1990). Here we tested the therapeutic effect of an anti-human CD81 antibody in vivo against Raji and SUP-B8 B cell lymphomas using a xenograft model in SCID mice. Our studies demonstrated that our anti-human CD81 antibody (mouse IgG1) had therapeutic effect comparable to Rituximab (human IgG1) (Figure 1A). Yet, the two antibodies differ in their ability to mediate antibody-dependent cell cytotoxicity (ADCC), Rituximab is known to be highly effective, whereas the mouse IgG1 anti-CD81 antibody is not expected to mediate ADCC.To enhance the anti-CD81-mediated ADCC, we class switched the hybridoma to mouse IgG2a; we also engineered a chimeric antibody containing human IgG1ADCC-HIGH Fc constant region. Indeed, mouse IgG2a and the chimeric human IgG1 anti-CD81 mAb showed a remarkable increase in NK cell-mediated ADCC as well as complement-dependent cytotoxicity (CDC) when compared to Rituximab in vitro (data not shown) and in vivo (Figure 1B). These results suggest that CD81 can be a potential therapeutic target on B cell lymphomas by virtue of both its direct cytotoxic effect and as a mediator of ADCC and CDC. The humanized IgG1 version is being developed as a therapeutic candidate. [Display omitted] DisclosuresLevy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

A Novel Fusion of ALT-803 (Interleukin (IL)-15 Superagonist) with an Antibody Demonstrates Antigen-specific Antitumor Responses

IL-15 and its receptor α (IL-15Rα) are co-expressed on antigen-presenting cells, allowing transpresentation of IL-15 to immune cells bearing IL-2RβγC and stimulation of effector immune responses. We reported previously that the high-affinity interactions between an IL-15 superagonist (IL-15N72D) and the extracellular IL-15Rα sushi domain (IL-15RαSu) could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes. The IL-15N72D·IL-15RαSuFc complex, also known as ALT-803, is a multimeric complex constructed by fusing IL-15N72D·IL-15RαSu to the Fc domain of IgG1. ALT-803 is an IL-15 superagonist complex that has been developed as a potent antitumor immunotherapeutic agent and is in clinical trials. Here we describe the creation of a novel fusion molecule, 2B8T2M, using the ALT-803 scaffold fused to four single chains of the tumor-targeting monoclonal antibody rituximab. This molecule displays trispecific binding activity through its recognition of the CD20 molecule on tumor cells, stimulation via IL-2RβγC displayed on immune effector cells, and binding to Fcγ receptors on natural killer cells and macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies.

Abstract B061: CD81: A new target for therapy of B cell lymphoma

Abstract CD81 belongs to an evolutionary conserved family of proteins named tetraspanins and it was originally described as a B cell target. Tetraspanins associate in multi-molecular complexes in tetraspanin-enriched microdomains (TEMS). Indeed, in B cells CD81 associates with CD19 and CD21, and together function as co-receptor to lower the cell activation threshold initiated by the BCR. Recently, we have demonstrated the importance of CD81 in tumor growth and metastasis of solid tumors1. However, the role of CD81 in B cell malignancies has not been explored. Previously, we have shown that anti-CD81 antibodies had anti-proliferative effects on human B cell lymphoma cell lines in vitro2. Here we tested the therapeutic effect of a mouse anti-human CD81 antibody in vivo against Raji and SUP-B8 B cell lymphomas using a xenograft model in SCID mice. Our studies demonstrated that our anti-CD81 antibody had therapeutic effect comparable to rituximab. Moreover, we found ADCC as one of the mechanism of action. Furthermore, to enhance the anti-CD81-mediated ADCC we engineered chimeric antibodies containing human IgG1ADCC-HIGH and human IgG4 ADCC-LOW Fc constant regions. Indeed, the chimeric human IgG1 anti-CD81 mAb showed a remarkable increase in NK cell-mediated ADCC compared to Rituximab in vitro. These results suggest that CD81 can be a potential therapeutic target on B cell lymphomas by virtue of both its direct cytotoxic effect and as a mediator of ADCC. The Human IgG1 version is being developed as a therapeutic candidate. 1. Vences-Catalan, F., et al. Cancer Res 75, 4517-4526 (2015). 2. Oren, R., Takahashi, S., Doss, C., Levy, R. & Levy, S. Mol Cell Biol 10, 4007-4015 (1990). Citation Format: Felipe Vences-Catalán, Chiung-Chi Kuo, Ranjani Rajapaksa, Ronald Levy, Shoshana Levy. CD81: A new target for therapy of B cell lymphoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B061.

Discovery of ursolic acid prodrug (NX-201): Pharmacokinetics and in vivo antitumor effects in PANC-1 pancreatic cancer

The aim of our study was to develop ursolic acid (UA) prodrugs in order to overcome UA’s weakness, which has an extremely low bioavailability. UA-medoxomil (NX-201), one of our UA prodrugs, showed an improved bioavailability about 200times better than UA in rodent model. According to in vivo test performed with PANC-1 xenograft SCID mouse model, tumor growth rate decreased dose-dependently and 100mg/kg dose of NX-201 had an anticancer effect comparable to gemcitabine. Most of all the combination of NX-201 (50mg/kg, po, daily) and gemcitabine (40mg/kg, iv, 2timesperweek) even reduced tumor size after three weeks.

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer.

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) – a novel sphingolipid-like marine compound – was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti‐human podoplanin mAb, NZ‐1, and a rat–human chimeric anti‐human podoplanin antibody, NZ‐8, derived from NZ‐1, which induced antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity against podoplanin‐positive MPM cell lines. In this study, we showed the antitumor effect of NZ‐1, NZ‐8, and NZ‐12, a novel rat–human chimeric anti‐human podoplanin antibody derived from NZ‐1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ‐1 and rat NK (CD161a+) cells inhibited the growth of tumors and the production of pleural effusion in NCI‐H290/PDPN or NCI‐H226 orthotopic xenograft mouse models. NZ‐8 and human natural killer (NK) (CD56+) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ‐12 induced potent ADCC mediated by human MNC, compared with either NZ‐1 or NZ‐8. Antitumor effects were observed following treatment with NZ‐12 and human NK (CD56+) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ‐12 mediated by human NK (CD56+) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin‐targeting immunotherapy using both NZ‐12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM.

Bazedoxifene enhances the anti-tumor effects of cisplatin and radiation treatment by blocking IL-6 signaling in head and neck cancer.

Recent studies have shown that IL-6 signaling plays an important role in the aggressive and metastatic phenotype of head and neck squamous cell carcinoma (HNSCC). Therefore, we hypothesized that targeting of IL-6 signaling in HNSCC could enhance the therapeutic efficacy of standard chemoradiation treatment. We used both in vitro and in vivo models to test the efficacy of Bazedoxifene (BZA), a drug that was originally developed as a newer-generation selective estrogen receptor modulator (SERM) for the treatment of postmenopausal osteoporosis. Recently, BZA was also shown to exhibit potent anti-cancer effects that were both estrogen receptor (ER)-dependent and ER-independent. Our results suggest that BZA inhibits IL-6 signaling by disrupting IL-6R/gp130 protein-protein interactions. BZA treatment of CAL27-IL-6 (IL-6 overexpressing cells) or UM-SCC-74A (naturally expressing high levels of IL-6) significantly inhibited cell proliferation, migration and colony formation ability in a dose-dependent manner. In addition, BZA significantly decreased IL-6-mediated tumorsphere formation by markedly reducing nanog expression. BZA treatment also markedly reduced chemo and radioresistance in head and neck cancer cells by downregulating ERCC-1, XRCC-1 and survivin expression. In a SCID mouse xenograft model, BZA significantly enhanced the anti-tumor effects of cisplatin and radiation treatment with no added systemic toxicity. Furthermore, combination treatments significantly decreased tumor metastasis, pSTAT3 expression and nanog expression, in vivo. Taken together, our results suggest that targeting IL-6 signaling with bazedoxifene could be an effective treatment strategy for the treatment of HNSCC patients.

Low-Dose Paclitaxel Reduces S100A4 Nuclear Import to Inhibit Invasion and Hematogenous Metastasis of Cholangiocarcinoma.

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubule-stabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma. Cancer Res; 76(16); 4775-84. ©2016 AACR.

Abstract 286: Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma

Abstract Objective Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastasic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of pan-HDACi alone and in combination with gemcitabine in LMS. Methods Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included a pan-HDAC inhibitor (HDACi) and nucleoside analog, gemcitabine (Gem). MTS and clonogenic assays were used to evaluate the effect of HDACi on LMS cell growth. Cell cycle FACS and annexin V PI/FACS analysis were used to determine the effects of HDACi on cell cycle and apoptosis, respectively. Compusyn software was used to determine in vitro synergy studies for the combination of HDACi + Gem. A LMS xenograft model in SCID mice was used to test the impact of HDACi alone, Gem alone and the combination of HDACi + Gemcitabine. Results Pan-HDAC inhibition abrogated LMS cell growth and clonogenic potential. HDACi induced G2/M cell cycle growth arrest and enhanced apoptosis in LMS cell lines. The combination of HDACi + Gem exhibited a synergistic effect in LMS cells in vitro. Similarly, HDACi combined with Gem resulted in superior anti-LMS effects in vivo. Conclusion LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of pan-HDAC inhibition combined with gemcitabine for the treatment of LMS. Citation Format: Gonzalo Lopez, Edwin Choy, Shreyaskumar Patel, Hemant Bid, Danielle Braggio, Dina Lev, Raphael Pollock. Preclinical study: HDAC inhibition combined with gemcitabine for the treatment of leiomyosarcoma. [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 286.

Abstract 4993: Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma

Abstract Background: Gangliosides such as GD2, GD3 and GM2 are promising targets for antibody-mediated cancer therapy since they are expressed at high levels on the surface of several cancers, including neuroblastomas, sarcomas and melanomas. Anti-GD2 monoclonal antibodies have shown promising clinical outcomes in neuroblastoma and a chimeric anti-GD2 antibody (Unituxin™) was recently approved by FDA. However, murine-derived antibodies discovered so far show adverse effects that limit their clinical utility. Fully human antibodies derived from immunized patients might be able to overcome these limitations, since epitope specificity has evolved in the human tissue environment. Here we describe the characterization and functional evaluation of two potential development candidates. Methods: PBMCs from patients immunized with GD2 lactone-keyhole limpet hemocyanin (GD2L-KLH) conjugate vaccine were utilized to isolate fully human monoclonal antibodies (humAbs). HumAbs were selected for 1) specificity against GD2 initially by ELISA and cell surface binding by flow cytometry 2) by glycan array and affinity assays and 3) by effector functions in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) assays. The therapeutic potential of lead antibodies was further evaluated in xenograft models in SCID mice using GD2 positive CHLA255 neuroblastoma as well as with TC71 and SaoS2 sarcoma cell lines. Results: Antibodies that bind specific to purified GD2 by ELISA were expressed as recombinant antibodies in CHO cells. Many recovered antibodies showed cross-reactivity with several gangliosides in ELISA assays. Two antibodies with high specificity for GD2, 1B7 and 31F9 were selected for further studies. Binding to native antigen expressed on the cell surface of different cell lines was confirmed by FACS analysis. 31F9 was monospecific for GD2 (affinity by Surface Plasmon Resonance was ∼4 nM) while 1B7 showed dual specificity for GD2 and GM2 with affinities of ∼1 nM for GD2 and ∼ 370 nM for GM2, respectively. These two antibodies induced CDC ranged between 35% and 80%, and ADCC with human PBMCs between 40% and 65% on CHLA255, TC71 and SaoS2 cell lines. Survival following IV or SC challenge with SaoS2 and TC71 cells (respectively) was at least doubled following six IP treatments with 200 mcg of either 1B7 or 31F9 twice weekly. Inhibition of CHLA255 tumor growth was less dramatic. Conclusions: Vaccinated patients produce antigen-specific IgG antibodies that were further analyzed on the molecular level. Antibodies with single and dual specificity (GD2 and GM2) were recovered and shown to be very active in functional assays. Based on these favorable in vitro and in vivo studies, 1B7 and 31F9 humAbs merit further development efforts to evaluate potential utility for treatment of GD2 positive cancers. Citation Format: Govind Ragupathi, Xiaohong Wu, Ritsuko Sawada, Philip O. Livingston, Wolfgang W. Scholz. Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma. [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 4993.

In vitro and in vivo effects of a recombinant anti-PSMA immunotoxin in combination with docetaxel against prostate cancer.

Docetaxel (DOC) is used for the first-line treatment of castration resistant prostate cancer (CPRC). However, the therapeutic effects are limited, only about one half of patients respond to the therapy and severe side effects possibly lead to discontinuation of treatment. Therefore, actual research is focused on the development of new DOC-based combination treatments.In this study we investigated the antitumor effects of a recombinant immunotoxin targeting the prostate specific membrane antigen (PSMA) in combination with DOC in vitro and in vivo. The immunotoxin consists of an anti-PSMA single chain antibody fragment (scFv) as binding and a truncated form of Pseudomonas aeruginosa Exotoxin A (PE40) as toxin domain. The immunotoxin induced apoptosis and specifically reduced the viability of androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cells. A synergistic cytotoxic activity was observed in combination with DOC with IC50 values in the low picomolar or even femtomolar range. Moreover, combination treatment resulted in an enhanced antitumor activity in a C4-2 SCID mouse xenograft model. This highlights the immunotoxin as a promising therapeutic agent for a future DOC-based combination therapy of CPRC.

Oxidative stress induces the acquisition of cancer stem-like phenotype in breast cancer detectable by using a Sox2 regulatory region-2 (SRR2) reporter.

We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expression of 3 signature genes of RR cells (CD133, GPR49 and MUC15). Lastly, the H2O2-induced RU/RR conversion was detectable in a SCID mouse xenograft model and primary tumor cells. To conclude, the H2O2-induced RU/RR conversion has provided a novel model to study the acquisition of cancer stemness and plasticity.

SL-801, a Novel, Reversible Inhibitor of Exportin-1 (XPO1) / Chromosome Region Maintenance-1 (CRM1) with Broad and Potent Anti-Cancer Activity

XPO1/CRM1, the principal nuclear export protein in eukaryotic cells, is required for the nuclear-cytoplasmic transport of both proteins and RNAs. Overexpression of XPO1 is reported in many cancers, causing dysregulated protein localization, aberrant cell proliferation, and resistance to apoptosis, and is associated with aggressive characteristics and poor patient outcome. Recent work has revealed XPO1 to be a clinically relevant target, and nuclear export inhibitors have emerged as a new class of anti-cancer agents with clinical activity in multiple hematologic and solid malignancies. SL-801 is a novel small molecule that binds covalently to Cys528 of XPO1, blocking the ability of XPO1 to interact with substrate cargos (e.g., p53, FOXO, p21, p27, and others). In contrast to the prototypical XPO1 inhibitor leptomycin B, which binds irreversibly to XPO1 and caused significant toxicities in Phase 1 trials, SL-801 binding to XPO1 is reversible, a characteristic that may be exploited to maximize its therapeutic index. Exposure to SL-801 results in potent inhibition of XPO1-dependent nuclear export, cell cycle arrest, and induction of apoptosis in a time- and dose-dependent manner. Here, the anti-tumor activity of SL-801 was investigated against a panel of 240 cell lines representing a broad range of solid and hematologic malignancies and confirmed in several SCID xenograft models.The OncoPanelTM high content screening platform was used to evaluate the cytotoxicity of SL-801 against 205 solid tumor and 35 liquid tumor cell lines. SL-801 demonstrated potent activity, with 50% growth inhibitory (GI50) values ≤ 10 nM in 51/240 (21.3%) cell lines and GI50 values ≤ 100 nM in 230/240 (95.8%) cell lines. SL-801 sensitivity was independent of cell proliferation rate or XPO1 expression levels. While SL-801 was broadly cytotoxic, cell lines of hematopoietic origin exhibited greater sensitivity. GI50s in hematologic cancers ranged from 3-93 nM in leukemias, 1-103 nM in lymphomas, and 3-11 nM in multiple myelomas. SL-801 also inhibited solid tumor growth, with GI50s ≤ 10 nM in several breast, brain, cervical, ovarian, gastric, kidney, liver, lung, melanoma, prostate, and sarcoma lines. In addition, a 5-fold increase in active caspase-3 staining was observed at SL-801 concentrations ≤ 100 nM in 117/240 (48.8%) cell lines, consistent with induction of apoptosis. To understand tumor sensitivity to SL-801, results of the cell line cytotoxicity screen were analyzed against publicly available genomic datasets. This analysis revealed that SL-801 was cytotoxic towards cell lines regardless of mutation status of key oncogenes (e.g., KRAS) and tumor suppressor genes (e.g., TP53).The in vitro cytotoxicity of SL-801 against tumor cell lines was further validated in several xenograft models in SCID mice. In the RPMI-8226 multiple myeloma xenograft model, tumor growth was significantly inhibited at oral SL-801 doses of 31.25 mg/kg administered daily for five days for two weeks. In the ARH-77 human multiple myeloma xenograft model, overall survival was significantly prolonged by daily oral administration of 125 mg/kg SL-801 for ten days. This dose and regimen were well tolerated, and 90% of SL-801-treated mice survived > 150 days, whereas median survival was 39.5 days in the vehicle-treated group (p < 0.001). Significant tumor growth inhibition was also observed in the NCI-H226 non-small cell lung cancer and 22RV1 prostate cancer xenograft models.These data demonstrate that SL-801 is a promising clinical candidate that inhibits a novel, clinically validated target and support its clinical development in a broad range of oncologic indications. The reversible binding of SL-801 to XPO1 may offer the potential to develop dosing schedules to enable recovery in normal tissues, thus broadening the therapeutic index of this class of agents. IND-enabling work is underway to support entry into clinical trials, and a Phase I trial design will be discussed. DisclosuresChen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. McDonald:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Schwartz:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Schneider:Stemline Therapeutics, Inc.: Employment. Sakakibara:CanBas Co., Ltd.: Employment, Equity Ownership. Saito:CanBas Co., Ltd.: Employment, Equity Ownership. Sato:CanBas Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Kawabe:CanBas Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership.

STEM-20MAPPING DYNAMICS OF CELL DIVISION AND GENE EXPRESSION AT THE SINGLE CELL LEVEL TO UNDERSTAND GLIOMA CELL STATES

GBM tumors are recognized as being heterogeneous in nature, consisting of many different cell types and cell states. This presents a major hurdle during the treatment of tumors as radiation and chemotherapeutic agents preferentially target proliferative cells. Notably, quiescent and slowly-dividing cancer stem cells are thought to provide a reserve of cells that drive cancer relapse following treatment, making them more malignant than their rapidly dividing counter-parts. To determine whether quiescent and slow dividing stem cells are a common feature within patient GBMs we profiled the dynamics of cell proliferation. GBM tumors surgically resected from patients were used to generate Patient Derived Xenograft (PDX) models in NOD SCID mice. During the two months that it takes for xenografts to establish, mice were feed with regimes of thymidine analogues (BrdU, CldU or IdU) in their drinking water, effectively tagging dividing cells, to profile the history of each glioma cell. Adapting the regimes that mice were feed thymidine analogues facilitated the characterization of cancer cell proliferation. We observe diverse proliferation dynamics within each PDX mouse, representing rapidly dividing, slowly dividing and quiescent cells. To then establish a single cell gene expression map of proliferating versus non-proliferating glioma cell states we have generated a custom Glioma Specific Epigenetic Expression (GSEE) qPCR array. The GSEE qPCR array has been utilized for the analysis of gene expression at the single cell level in patient derived glioma cells. We have subsequently generated a core network of epigenetic modifier genes that reliably represent glioma cell state. This has enabled the mapping of glioma cell states with respect to dynamics of cell division, revealing novel therapeutic targets.

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells.

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6-12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.