SCID Mouse Xenograft Model Research Articles

Abstract C215: Sensitivity of PTEN-deficient prostate carcinoma xenografts to ionizing radiation through inhibition of treatment-induced CXCL8 signaling.

Abstract Background: CXCL8 signaling levels are elevated in localized high grade prostate cancer (CaP). We have shown exposure of CaP cells to chemical DNA-damaging agents potentiates CXCL8 signaling, which underpins an autocrine mode of therapeutic resistance. Data from in vitro and in vivo CaP models has shown loss of the tumor suppressor PTEN also increases expression of the pro-inflammatory chemokine CXCL8. Experiments were designed to determine whether exposure to ionising radiation (IR) selectively induced CXC-chemokine signaling in PTEN-deficient CaP cells, and to determine how CXCL8 signaling may modulate their radiosensitivity. Methods: shRNA-directed strategies were used to establish cell-based models of CaP with differential expression of PTEN. These clonal (DU145) and heterogeneous (22Rv1) populations were subjected to IR under normal or hypoxic culture conditions. PTEN was reconstituted in PC3 cells and was driven by tetracycline-regulated expression of the gene. Changes in chemokine expression were analyzed by RT-PCR, immunoblotting and ELISA. Autocrine CXCL8 signaling was attenuated using siRNA to down-regulate gene expression of its receptors, CXCR1 and CXCR2. The effects of modulating chemokine signaling on cell viability following exposure to clinically-relevant doses of IR were assessed by colony formation assays, cell proliferation assays, and analysis of apoptosis and cellular senescence. The effect of CXCL8 signaling inhibition in vivo was determined using a PC3 xenograft model in SCID mice. The cell penetrating pepducin, x1/2pal-i3, was used to attenuate both CXCR1 and CXCR2 and tumor growth was assessed via caliper measurements. Results: Exposure to IR (3 Gy) increased gene expression of CXCL8, CXCR1 and CXCR2 - with PTEN-deficiency enhancing this response. Loss of PTEN expression differentially modulated the radiosensitivty of CaP cell lines: PTEN-depletion conferred relative radioresistance in DU145 cells but made 22RV1 cells more radiosensitive. However, the inhibition of CXCL8 signaling in all PTEN-depleted models resulted in consistently increased radiosensitivity (SER 1.2, 1.13, and 1.39 in DU145, 22RV1, and PC3 CaP cells, respectively; p<0.05). Reconstitution of PTEN expression in PC3 cells abrogated the capacity of CXCL8 signaling blockade to confer radiosensitivity. The combination of IR and CXCL8 signaling-inhibition conferred increased apoptosis in DU145 cells (p53 mutant) but induced premature senescence in 22RV1 cells (p53 WT). In vivo, treatment of PC3 xenografts with x1/2pal-i3 combined with IR (3 Gy) resulted in a significant delay in tumor growth compared to control (median quadrupling time: 42 day x1/2pal-i3 & IR vs. 32 day control & IR; p<0.05). Subsequent analysis of gene expression in these tumors confirmed that CXCL8 signaling inhibition attenuated IR-induced expression of anti-apoptotic proteins Bcl-2 and C-FLIP. Conclusions: Our results demonstrate the role of treatment-induced CXCL8 signaling as a mechanism of resistance to IR in PTEN-depleted CaP cells. Inhibition of IR-induced CXCL8 signaling provides a novel therapeutic opportunity to enhance the radiosensitivity of PTEN-depleted CaP cells. However, mechanistic results indicate that other genetic factors including p53 status dictate the resultant induction of treatment-induced apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C215. Citation Format: Chris WD Armstrong, Jonathan A. Coulter, Steven Walker, Richard D. Kennedy, Kevin M. Prise, Joe M. O'Sullivan, David JJ Waugh. Sensitivity of PTEN-deficient prostate carcinoma xenografts to ionizing radiation through inhibition of treatment-induced CXCL8 signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C215.

Abstract B198: Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC).

Abstract Selinexor (KPT-330) is a novel small molecule inhibitor of nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IκB, resulting in selective death of cancer cells. Preclinical studies have shown that oral Selinexor is well tolerated, even with prolonged (4-8 month) administration. Human Phase 1 clinical studies with Selinexor in hematological and solid cancer patients are ongoing (Clinicaltrials.gov NCT01607892 and NCT01607905). In this study we evaluated the mechanisms involved in Selinexor-induced growth inhibition of NSCLC in vitro and in vivo. Methods: A panel of NSCLC cells with various genetic backgrounds were treated with Selinexor and tested in proliferation (MTT), clonogenic survival, and flow cytometric assays. The cellular localization of TSPs were evaluated by immunofluorescence (IF). Western blotting was used to study levels of cell cycle regulating proteins and quantify markers of cell survival. Functional inhibition of NF-κB was measured using a transcription factor assay kit. A SCID mouse xenograft model of NSCLC, adenocarcinoma A549 (wt p53, NF-κB IC50 of 1.19 uM), was used to evaluate Selinexor effects in vivo and tumors were evaluated by histology and immunohistochemistry (IHC). Results: Selinexor induced cell cycle arrest, inhibited tumor cell growth and clonogenic formation and induced apoptosis in lung cancer cell lines regardless of genetic background (IC50 25-700 nM). Treated normal human dermal fibroblasts entered into cell cycle arrest but resisted apoptosis induction (IC50 4.0 uM). IF of the TSPs p53, p21, IκB, E2F4, and survivin demonstrated strong nuclear localization after 4 to 24 hour treatment with Selinexor followed by apoptosis as determined by cleavage of PARP, caspases 3 and 8 by immunoblot analysis. Functional evaluation of NF-kB in A549 and H1299 revealed transcriptional repression with IC50 values similar to MTT assays. Finally, A549 xenografts treated with Selinexor (10 - 20 mg/kg QoDx3) showed marked tumor suppression when compared to vehicle and cisplatin-treated mice (growth inhibition was 78% at 20 mg/kg, p<0.0001). Histological and IHC analysis of treated tumors indicated increased nuclear accumulation of p53, p21, FOXO1, survivin, NF-κB and IκB, reduced cell proliferation, and tumor cell death. Conclusions: In NSCLC, Selinexor forces nuclear retention of TSPs, inhibits NF-κB transcriptional activity, inhibits tumor growth and induces cell death regardless of p53 status. Selinexor thus has therapeutic potential for the treatment of NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B198. Citation Format: William Senapedis, Marsha Crochiere, Tami Rashal, Trinayan Kashyap, Boris Klebanov, Jean-Richard Saint-Martin, Ori Kalid, Sharon Shechter, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Eran Shacham, Sharon Tamir, Dilara McCauley, Erkan Baloglu, Michael Kauffman, Sharon Shacham, Yosef Landesman. Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B198.

Immunohistochemical Vascular Factor Expression in Canine Inflammatory Mammary Carcinoma

Human inflammatory breast carcinoma (IBC) and canine inflammatory mammary carcinoma (IMC) are considered the most malignant types of breast cancer. IMC has similar characteristics to IBC; hence, IMC has been suggested as a model to study the human disease. To compare the angiogenic and angioinvasive features of IMC with non-IMC, 3 canine mammary tumor xenograft models in female SCID mice were developed: IMC, comedocarcinoma, and osteosarcoma. Histopathological and immunohistochemical characterization of both primary canine tumors and xenografts using cellular markers pancytokeratin, cytokeratin 14, vimentin, and α-smooth muscle actin and vascular factors (VEGF-A, VEGF-D, VEGFR-3, and COX-2) was performed. Tumor cell proliferation index was measured by the Ki-67 marker. The xenograft models reproduced histological features found in the primary canine tumor and preserved the original immunophenotype. IMC xenografts showed a high invasive character with tumor emboli in the dermis, edema, and occasional observations of ulceration. In addition, compared with osteosarcoma and comedocarcinoma, the IMC model showed the highest vascular factor expression associated with a high proliferation index. Likewise, IMC xenografts showed higher COX-2 expression associated with VEGF-D and VEGFR-3, as well as a higher presence of dermal lymphatic tumor emboli, suggesting COX-2 participation in IMC lymphangiogenesis. These results provide additional evidence to consider vascular factors, their receptors, and COX-2 as therapeutic targets for IBC.

Anti-CD30 antibody conjugated liposomal doxorubicin with significantly improved therapeutic efficacy against anaplastic large cell lymphoma

The use of nano-carriers has been shown to improve the delivery and efficacy of chemotherapeutic agents in cancer patients. Recent studies suggest that decoration of the surface of nano-carriers with various targeting moieties may further improve the overall therapeutic efficacy. In this study, we compared the therapeutic efficacy of Doxil® (commercial doxorubicin-loaded liposomes) and that of Doxil® conjugated with anti-CD30 antibodies (CD30-targeted Doxil®) in treating anaplastic large cell lymphoma (ALCL), a type of T-cell lymphoma characterized by a high CD30 expression. Compared to Doxil®, the CD30-targeted Doxil® showed a significantly higher binding affinity to ALCL cells (5.3% versus 27%, p = 0.005) and a lower inhibitory concentration at 50% (IC50) in-vitro (32.6 μg/mL versus 12.6 μg/mL, p = 0.006). In a SCID mouse xenograft model, CD30-targeted Doxil® inhibited tumor growth more significantly than the unconjugated formulation; specifically, tumors in mice treated with CD30-targeted Doxil® were significantly smaller than those in mice treated with Doxil® (average, 117 mm3 versus 270 mm3, p = 0.001) at 18 days after the tumors were inoculated. Our findings have provided the proof-of-principle of using CD30-targeted nano-carriers to treat cancers that are characterized by a high level of CD30 expression, such as ALCL.

Establishment and characterization of a canine xenograft model of inflammatory mammary carcinoma

Canine inflammatory mammary cancer (IMC) and human inflammatory breast cancer (IBC) are the most aggressive form of mammary/breast cancer. Both species naturally develop it, sharing epidemiological, clinical and histological characteristics. Thus, IMC has been suggested as a model to study the human disease. We have developed the first IMC xenograft model in SCID mice. Xenografts reproduced the histological features from the primary tumor, were highly aggressive and showed dermal tumor emboli, distinctive hallmarks of IMC/IBC. This model was hormone receptors positive and HER2 negative. Our findings showed that estrogens and androgens are locally produced in tissues. Factors related to tumor vascularization showed positive expression and xenografts with the highest expression of all analyzed vascular factors had the highest rate of tumor proliferation. The role of steroid hormones and the angio/lymphangiogenic properties found in this model, provide additional knowledge for future interventions in the diagnosis, treatment and prevention of the disease.

E- and P-Selectins Are Essential for Repopulation of Chronic Myelogenous and Chronic Eosinophilic Leukemias in a Scid Mouse Xenograft Model

In chronic myelogenous (CML) and chronic eosinophilic leukemia (CEL), neoplastic cells spread via the circulation into various extramedullary organs. As E- and P-selectin constitute the starting point for the leucocyte adhesion/invasion cascade, and CEL and CML cells share many properties with normal granulocytes, we investigated the role of these selectins in CEL and CML cell expansion and organ invasion in a xenotransplantation model using scid mice. Using two human leukemic cell lines (EOL-1 and K562), we were able to show that E- and P-selectins mediate leukemia cell tethering and adherence in a laminar flow assay. While E-selectin binding depended on sialylated carbohydrate moieties, P-selectin binding was completely (K562) or partially (EOL-1) independent of these carbohydrates indicating the involvement of non-canonical selectin ligands. In a xenograft model in scid mice, both cell lines invaded the bone marrow and other organs, formed chloromas, and ultimately produced an overt leukemia. In contrast, in E- and P-selectin knockout scid mice, the cells failed to show engraftment in 8 out of 10 animals and even if they did engraft, they produced only little organ invasion and chloroma formation. Together, these data suggest that E- and P-selectins play an important role in leukemic dissemination in CML and CEL.

Sterol regulatory element-binding protein 1 is required for ovarian tumor growth

Re-programming of lipogenic signaling is one of the most significant alterations of tumor cell pathology. Consistent with a large demand for lipids, tumor cells express high levels of lipogenic enzymes, most of which are transcriptional targets of sterol regulatory element-binding protein 1 (SREBP1). However, the expression levels and the function of SREBP1 in ovarian cancer are largely unknown. Our study aimed to assess the oncogenic potential of SREBP1 in ovarian cancer. In this study, we showed that the SREBP1 protein expression was significantly higher in human ovarian cancer compared to benign and borderline ovarian tumors by immunohistochemical staining. Knockdown of SREBP1 by small hairpin RNA (shRNA) in ovarian cancer cells retarded cell growth, migration and invasion and enhanced cell apoptosis without significant effects on cell cycle distribution. In a xenograft SCID mouse model, SREBP1 silencing inhibited tumor growth in vivo and reduced the expression of SREBP1 downstream lipogenic genes at both the protein and mRNA levels. Taken together, the results from this study demonstrate a crucial role of SREBP1 in ovarian cancer growth, which establish SREBP1 as a novel therapeutic target for antitumor therapy.

HM1.24/CD317-directed immunotoxin to eliminate malignant plasma cells in vitro and in vivo.

8604 Background: Targeted immunotherapy, based on antibodies against tumor-associated antigens, is a promising approach for the treatment of multiple myeloma (MM). Recently, antibody-based strategies delivering a toxic payload have documented impressive clinical activity in hematological malignancies. In particular, surface molecules overexpressed on malignant plasma cells and efficiently internalized represent promising targets for developing myeloma-directed immunoconstructs. Here, the identification of CD317 (HM1.24) as a potent target structure and the characterization of a novel CD317-directed single-chain immunotoxin, HM1.24-ETA', is described. Methods: Using a novel screening tool, a panel of antibodies against MM-associated antigens was evaluated for their ability to mediate antigen-dependent delivery of a truncated version of Pseudomonas exotoxin A (ETA’) to MM cells. HM1.24-ETA' was generated by genetic fusion of a CD317-specific single-chain Fv antibody and ETA'. The anti-myeloma activity of the E. coli-expressed immunotoxin was evaluated in vitro and in a xenograft mouse model. Results: By screening a panel of antibodies including CD38, CS1, IL-6R, CD138 and CD317, CD317 was identified as a suitable receptor to deliver ETA’ to MM cells. The subsequently designed recombinant HM1.24-ETA' immunotoxin efficiently inhibited growth of MM cell lines with halfmaximal growth inhibition at concentrations of less than 1 nM. Antigen-specific MM cell killing occurred via induction of apoptosis. The proliferation of IL-6 dependent INA-6 cells was completely inhibited by HM1.24-ETA' even in the presence of bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA' strongly triggered apoptosis (up to 80%) in freshly isolated tumor cells from 7 out of 7 MM patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA' immunotoxin (p < 0.04). Conclusions: The HM1.24-ETA' immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of MM using immunoconjugates with toxic payloads.

Downregulation of miR-497 promotes tumor growth and angiogenesis by targeting HDGF in non-small cell lung cancer

MicroRNAs (miRNAs) play important roles in the development of various cancers. MiRNA-497 functions as a tumor-suppressor that is downregulated in several malignancies; however, its role in non-small cell lung cancer (NSCLC) has not been examined in detail. Here, we showed that miR-497 is downregulated in NSCLC tumors and cell lines and its ectopic expression significantly inhibits cell proliferation and colony formation. Integrated analysis identified HDGF as a downstream target of miR-497, and the downregulation of HDGF by miR-497 overexpression confirmed their association. Rescue experiments showed that the inhibitory effect of miR-497 on cell proliferation and colony formation is predominantly mediated by the modulation of HDGF levels. Furthermore, tumor samples from NSCLC patients showed an inverse relationship between miR-497 and HDGF levels, and ectopic expression of miR-497 significantly inhibited tumor growth and angiogenesis in a SCID mouse xenograft model. Our results suggest that miR-497 may serve as a biomarker in NSCLC, and the modulation of its activity may represent a novel therapeutic strategy for the treatment of NSCLC patients.

Abstract 1024: Therapeutic efficacy of RNA interference targeting focal adhesion kinase against orthotropic xenograft of human malignant pleural mesothelioma in SCID mice.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive neop lasm of the pleura. The prognosis for MPM remains disappointing, although there have been some improvements in prognosis from newer chemotherapies and multimodality treatments. Focal adhesion kinase (FAK) is an important regulator of cellular signaling, migration, apoptosis, and cell cycle progression. We tested the hypothesis that FAK plays an important role in the pathogenesis of MPM and examined the effect of inhibiting FAK expression of MPM using small interfering RNA (siRNA) in vitro and in vivo. FAK expression was evaluated by Western and Northern blots. Knockdown of FAK resulted in detachment and proliferation inhibition of MPM cells in vitro. We assessed the therapeutic applicability of FAK siRNA in orthotopic xenograft model in SCID mice. FAK siRNA treatment inhibited the growth of MPM tumors and production of pleural effusion. These findings suggest that FAK may be a potential therapeutic target for human MPM and exogenous delivery of siRNA targeting FAK may be a useful strategy for MPM therapy. Citation Format: Jun Huang, Dai-Shun Liu, Soji Kakiuchi, Hisatsugu Goto, Masaki Hanibuchi, Yasuhiko Nishioka. Therapeutic efficacy of RNA interference targeting focal adhesion kinase against orthotropic xenograft of human malignant pleural mesothelioma in SCID mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2013-1024

Preventing the autophagic survival response by inhibition of calpain enhances the cytotoxic activity of bortezomib in vitro and in vivo.

Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo. Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion), and growth inhibition (MTT). The effects of calpain on bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects. Inhibition of the Ca(2+)-dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of bortezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in an SCID mouse xenograft model of myeloma.

CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

Pancreatic stellate cells (PSCs) play a crucial role in the aggressive behavior of pancreatic cancer. Although heterogeneity of PSCs has been identified, the functional differences remain unclear. We characterized CD271+ PSCs in human pancreatic cancer. Immunohistochemistry for CD271 was performed for 31 normal pancreatic tissues and 105 pancreatic ductal adenocarcinomas (PDACs). We performed flow cytometry and quantitative RT-PCR, and assessed CD271 expression in PSCs isolated from pancreatic tissues and the changes in CD271 expression in PSCs cocultured with cancer cells. We also investigated the pattern of CD271 expression in a SCID mouse xenograft model. In the immunohistochemical analyses, the CD271-high staining rates in pancreatic stroma in normal pancreatic tissues and PDACs were 2/31 (6.5%) and 29/105 (27.6%), respectively (p = 0.0069). In PDACs, CD271+ stromal cells were frequently observed on the edge rather than the center of the tumors. Stromal CD271 high expression was associated with a good prognosis (p = 0.0040). Flow cytometric analyses demonstrated CD271-positive rates in PSCs were 0–2.1%. Quantitative RT-PCR analyses revealed that CD271 mRNA expression was increased in PSCs after coculture with pancreatic cancer cells. However, the level of CD271 mRNA expression subsequently decreased after the transient increase. Furthermore, CD271 mRNA expression was decreased in PSCs migrating toward pancreatic cancer cells through Matrigel. In the xenograft model, CD271+ PSCs were present at tumor margins/periphery and were absent in the tumor core. In conclusion, CD271 was expressed in PSCs around pancreatic tumors, but not in the center of the tumors, and expression decreased after long coculture with pancreatic cancer cells or after movement toward pancreatic cancer cells. These findings suggest that CD271+ PSCs appear at the early stage of pancreatic carcinogenesis and that CD271 expression is significantly correlated with a better prognosis in patients with PDAC.

Small Molecule MIRA-1 Induces p53-Independent Apoptosis in Multiple Myeloma Cells Through Activation of the p38 MAPK Signaling Pathway.

Abstract 2937Mutation of p53, a tumor suppressor protein, is relatively rare (∼10% in newly diagnosed patients) in multiple myeloma (MM). However, p53 mutations/deletions are important risk factors for predicting the resistant to chemotherapy and no treatment is currently available for this subgroup of patients. MIRA-1, a novel class of small molecules with the ability to restore wild type conformation and function to mutant p53, induces apoptosis in different types of solid tumors harboring mutant p53. However, its effect on MM cells is not known. In this study we examined the ability of MIRA-1 to induce cytotoxic and apoptotic response in MM cells and inhibit tumor growth in MM mouse xenograft model. In addition, we explored the molecular mechanisms of MIRA-1-induced apoptosis in MM cells.Treatment of MM cells with MIRA-1 resulted in a time- and dose-dependent decrease in survival and increase in apoptosis of MM cells harboring either wild type (MM.1S, H929) or mutant (U266, 8226, and LP1) p53 suggesting that MIRA-induced apoptosis in MM cells is independent of p53 status. The IC50 of MIRA-1 observed in these cells was ranged between 10 and 15 μM. In addition, MIRA-1 elicited a dose-dependent inhibition of myeloma cell growth in seven primary MM samples with an average IC50of 10 μM. Two of the seven patient samples harbors p53 mutations/deletions. In contrast, MIRA-1 did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from three healthy donors at the concentrations (10–20 μM) that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Apoptosis induced by MIRA-1 in MM cells harbouring either wild type or mutant p53 was associated with time- and dose-dependent activation of caspas-8, caspase-3 and PARP with subsequent up-regulation of a pro-apoptotic protein, Noxa and down-regulation of an anti-apoptotic protein, Mcl-1. Interestingly, MIRA-1 did not significantly modulate the level of p53 expression, although immunoprecipitation studies confirmed the restoration of wild type conformation of mutant p53 in LP1 and 8226 cells. Importantly, genetic knockdown of p53 using siRNA against wild type or mutant p53 had only a little effect on apoptosis induction by MIRA-1 in MM.1S or LP1 cells, respectively, confirming that apoptosis induction by MIRA-1 in MM cells is independent of p53. Furthermore, the combination of MIRA-1 with current anti-myeloma agents, dexamethasone or doxorubicin displayed synergistic cytotoxic response in MM.1S or LP1 cells (CI<1; p<0.05).To delineate the molecular mechanisms of apoptosis in MM cells induced by MIRA-1, we performed RT2 profiler PCR array analysis for the differential expression of 84 genes related to mitogen activated protein kinase (MAPK) signaling pathway. A significant number of genes of the MAPK family including MAP3K: MAP3K2 (MEKK2), MAP3K4 (MEKK4), PAK1; MAP2K: MAP2K5 (MEK5); and MAPK: MAPK11 (p38bMAPK) as well as transcription factors such as c-Jun, c-FOS, EGR1, and MKNK1, whose expression is induced by MAPK signaling, were up-regulated by more than 2-fold in MIRA-1-treated 8226 cells. On the other hand, expression of the scaffolding/anchoring genes, MAPK8IP2 (JIP-1) was down-regulated by ∼2-fold. Up-regulations of c-Jun, c-Fos, and EGR1 at their protein levels were further confirmed by Western blot analysis of MM.1S and 8226 cells treated with MIRA-1. Importantly, Western blot analysis revealed that treatment of MIRA-1 resulted in a time- and dose-dependent increase of phosphorylated p38 MAPK level in both MM.1S and 8226 cells. Taken together, our data indicates that activation of the MAPK signaling pathway is, at least in part, associated with MIRA-1-induced apoptosis of MM cells.Finally, we evaluated anti-tumorigenic potential of MIRA-1 in MM xenograft SCID mouse models. 8266 cells were inoculated into SCID mice and the mice received i.p. injections of either 100 μL PBS (control) or 10 mg/kg MIRA-1 once daily for 18 days after tumor formation was evident. Administration of MIRA-1 resulted in significant inhibition of tumor growth (p<0.05) and increase in survival (p=0.007) of the mice with no apparent toxicity.Our study for the first time demonstrates potent in vitro and in vivo anti-myeloma activity of MIRA-1 and thus providing a framework for clinical evaluation of MIRA-1 either alone or in combination with current anti-myeloma agents. Disclosures:Reece:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Millennium Pharmaceuticals: Research Funding.

The CD317-Targeted Immunotoxin HM1.24-ETA′ Efficiently Eliminates Malignant Plasma Cells in Vitro and in Vivo

AbstractAbstract 1838Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma still needs improvement. Recently antibody-based targeted therapies with a toxic payload have documented impressing activity. HM1.24 (CD317), a surface molecule overexpressed on malignant plasma cells, is efficiently internalized and may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the generation and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described.HM1.24-ETA′ was generated by genetic fusion of a CD317-specific single-chain Fv antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and the protein was purified to homogeneity by affinity chromatography. HM1.24-ETA′ efficiently inhibited growth of myeloma cell lines (INA-6, RPMI8226, U266) analyzed in MTT assays. Half maximal growth inhibition was observed at low nanomolar concentrations. Target cell killing occurred via induction of apoptosis as indicated by annexin V / propidium iodide staining and analysis of PARP cleavage. The delivery of HM1.24-ETA′ to target cells is antigen-specific, because excess of unconjugated parental antibody completely blocked the cytotoxic effect. The proliferation of IL-6 dependent INA-6 was efficiently reduced by HM1.24-ETA′ even in co-culture experiments with bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA′ strongly triggered apoptosis (up to 80% annexin V-positive cells) of freshly isolated tumor cells from 5 of 5 myeloma patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA′ immunotoxin (p < 0.04).Thus, HM1.24-ETA′ immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of multiple myeloma using immunoconjugates with toxic payloads. Disclosures:No relevant conflicts of interest to declare.

Alisertib Added to Rituximab and Vincristine Is Synthetic Lethal and Curative of Double Hit Diffuse Large B-Cell Lymphoma in Mice

Abstract 159 About one-third of diffuse large B-cell lymphoma (DLBCL) patients develop relapsed/refractory disease, a major cause of morbidity and mortality. Of DLBCL, 5% are double-hit lymphomas (DHLs) with translocations of MYC and BCL2 characterized by poor outcomes with R-CHOP. Auroras (A and B) are a family of mitotic oncogenic serine/threonine kinases required for high fidelity regulation of the mitotic phase of the cell cycle. Aberrant over-expression of Auroras leads to genetic instability, polyploidy and resistance to microtubule targeted agents. Previously we showed Aurora inhibitor (MLN8237, alisertib) [M] plus vincristine [V] plus rituximab [R] is synthetic lethal and a potential curative therapy in a mantle cell lymphoma model (Granta-519) with several cell cycle genetic defects [t (11;14)], ATM (haploinsufficient), p16/p14 (Del)] but p53 (WT). We evaluated ABC-DLBCL cell lines TMD-8 and U-2932 where the BCR/NF-kB pathway is constitutively active. In TMD-8, CD79B is mutated and in U-2932, Bcl2 [3q27] and Bcl6 [18q21] are amplified, p53 (mutated) and c-Myc over-expressed [DHL]. Triple therapy with MVR abolished proliferation, disrupted cell cycle processes and induced apoptosis compared to single agent and doublet treatments. A Mouse xenograft model of U-2932 showed modest activity for M (30 mg kg, PO, QD, 3 weeks), V (0.375 mg/kg, IV, Q1Wx3) and MV with tumor growth inhibition (TGI) of ∼10% (p=0.01) respectively. However, R (10 mg/kg, IV, Q1Wx3) alone induced ∼ 50% TGI (p=0.01) due to effective targeting of Bcl2. Doublets MR and VR led to tumor regression (TR), but relapse soon after stopping therapy. In contrast, MVR demonstrated TR with no relapses &gt;40 days after stopping therapy. All treatments were well tolerated (no weight loss) and Kaplan-Meier analysis showed that mice treated with MR, VR and MVR had a statistically significant improvement in overall survival compared to control, M, V and MV (p&lt;0.0001). Moreover, with MVR, OS was 100% (Figure 1). Gene expression profiling, RT-PCR, IHC and Western blotting of harvested tumors at the end of treatment (3 weeks) has been interrogated to ascertain the mechanistic role of Aurora inhibition when added to R [R Vs MR] and VR [VR Vs MVR]. Thus, addition of R to MV represents a novel therapeutic strategy that is currently under clinical trial evaluation in aggressive B-NHL including double hit lymphomas sponsored by Millennium Pharmaceuticals (C14011). Figure 1: Kaplan-Meier Survival by Treatment: MLN8237 (M), Rituximab (R), Vincristine (V), MR, MV, VR, MVR in a U-2932 ABC-DLBCL SCID Mouse Xenograft Model Figure 1:. Kaplan-Meier Survival by Treatment: MLN8237 (M), Rituximab (R), Vincristine (V), MR, MV, VR, MVR in a U-2932 ABC-DLBCL SCID Mouse Xenograft Model Disclosures: No relevant conflicts of interest to declare.

Target-Directed Development of a Proposed Biosimilar Rituximab (GP2013): Comparability of Antibody-Dependent Cellular Cytotoxicity Activity and Pre-Clinical Pharmacokinetics and Pharmacodynamics with Originator Rituximab

AbstractAbstract 4867 Background:Biosimilars are biologics approved by highly-regulated markets to be similar to existing agents that aim to offer more affordable treatment, thereby increasing patient access. Development of a biosimilar involves extensive characterization of the originator product over several years and a target-directed iterative development process to ensure a product that is highly comparable to the originator with similar clinical efficacy, safety and quality. Using antibody-dependent cellular cytotoxicity (ADCC), a main mode of action of rituximab, we illustrate how functional/structural relationship can be engineered into a biosimilar to ensure comparability at the in vitro level. Here we also present pre-clinical data confirming in vivo comparability for the proposed biosimilar rituximab GP2013, in terms of pharmacokinetics (PK), pharmacodynamics (PD) and efficacy. Methods:By employing a highly sensitive glycan quantitation method, relevant post-translational glycosylation patterns were assessed for their impact on in vitro ADCC relative potency data using the Raji and NK3.3 cell lines as target- and effector cells, respectively. Subsequently, bioactivity of GP2013 and originator rituximab were evaluated in a dose-response manner across a wide concentration range against SU-DHL-4 (diffuse large B-cell lymphoma) and Daudi (Burkitt's lymphoma) cell lines using freshly purified human NK cells. In vivo anti-tumor activity was assessed in two xenograft SCID mouse models of non-Hodgkin's lymphoma (SU-DHL-4 and Jeko-1 cell lines). Comparative PK and PD were assessed in single (5 mg/kg, n=14) and multiple (20 or 100 mg/kg, n=8) dose studies in cynomolgus monkeys, the pharmacologically most relevant species. Results:GP2013 and originator rituximab showed similar ADCC potency against both SU-DHL-4 and Daudi cells, with ADCC being reflective of engineered glycosylation patterns and structure-function relationships. In both xenograft mouse models, GP2013 and originator rituximab inhibited tumor growth to a similar extent, including at the more sensitive sub-optimal dose levels that are most likely to identify any potential differences. In primates, PK analysis confirmed bioequivalence between GP2013 and originator rituximab with nearly identical AUC values and 90% CIs entirely within the standard acceptance range of 0.8–1.25. Bioequivalence of PD response (B-cell depletion) was also shown, with 95% CIs of areas under the effect-time curves (AUEC) ratios for relative change from baseline in B-cell populations within the 0.8–1.25 acceptance range. The use of different doses indicated that comparable exposure and PD response can be expected for GP2013 and originator rituximab using indication-specific dosing regimens. Conclusions:This pre-clinical comparability exercise confirms that GP2013 and originator rituximab are pharmacologically similar with regard to ADCC potency, anti-tumor activity, PK exposure (AUC) and B-cell depletion. As such, it is hypothesized that GP2013 will show similar efficacy and safety as the originator product in ongoing clinical trials across different clinical indications. Disclosures:da Silva:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Kronthaler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Meyer:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Papandrikopoulou:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Stangler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Visser:Sandoz Biopharmaceuticals/HEXAL AG: Employment.

447PD - Preclinical and Clinical Evaluation of the Combination of Sorafenib and Everolimus in Patients with Advanced Solid Tumors

ABSTRACT Background Using a patient-derived primary pancreatic cancer (PanCa) xenograft SCID mouse model, we found synergistic anti-tumor effects of sorafenib (S) 20 mg/kg + everolimus (E) 1 mg/kg, and greater efficacy than either drug alone. No significant mouse toxicity was observed (weight loss, fur quality, GI toxicity). Here, we conducted a phase I and pharmacokinetic (PK) study of S + E in patients with advanced solid tumors, intending to expand to a phase II PanCa trial. Methods: Patients with advanced solid tumors were treated at dose levels (DL): [DL1] S 400 mg bid + E 5 mg daily; [DL2] S 400 mg bid + E 10 mg daily every 28-day cycle using a 3 + 3 dose escalation design. Patients started treatment with either S or E alone during a 1-week run-in, and the other agent added from Cycle 1 Day 1 onwards. Plasma samples were collected and analyzed for S and E, and PK was compared to preclinical studies. Results Ten of 22 patients were evaluable for dose-limiting toxicity (DLT). Two of 6 and 3 of 4 patients developed DLT at DL1 and 2 respectively, including grade (G) 3 diarrhea, rash, hypophosphatemia, hand-foot syndrome and G4 fistula. Median cycles received were 1.5. Clinical E exposure (AUC profile) was significantly lower (1/100th) than in preclinical combination studies whereas S exposure was comparable. Best response was stable disease for 6 cycles (168 days) in a uterine carcinosarcoma patient at DL1 (prior therapy PFS 114 days). Intention-to-treat analysis of 12 advanced gemcitabine-refractory (GR) PanCa patients showed median survival of 2.7 mo (95%CI 0, 6.8 mo). No PK interaction between S and E was observed. Conclusions E at doses 5 mg and 10 mg daily combined with S 400 mg bid were not tolerable in patients. S toxicity appeared accentuated by E co- administration with no drug-drug PK interaction observed. Efficacy was observed in uterine carcinosarcoma but no significant efficacy in GR-PanCa. Although our preclinical model indicated efficacy of S + E in PanCa, it failed to predict the clinical toxicity profile. Disclosure All authors have declared no conflicts of interest.

Tumor Xenograft Response to Redox-Active Therapies Assessed by Magnetic Resonance Imaging Using a Thiol-Bearing DOTA Complex of Gadolinium

Gd-LC6-SH is a thiol-bearing DOTA complex of gadolinium designed to bind plasma albumin at the conserved Cys34 site. The binding of Gd-LC6-SH shows sensitivity to the presence of competing thiols. We hypothesized that Gd-LC6-SH could provide magnetic resonance imaging (MRI) enhancement that is sensitive to tumor redox state and that the prolonged retention of albumin-bound Gd-LC6-SH in vivo can be exploited to identify a saturating dose above which the shortening of MRI longitudinal relaxation time (T1) of tissue is insensitive to the injected gadolinium dose. In the Mia-PaCa-2 pancreatic tumor xenograft model in SCID mice, both the small-molecule Gd-DTPA-BMA and the macromolecule Galbumin MRI contrast agents produced dose-dependent decreases in tumor T1. By contrast, the decreases in tumor T1 provided by Gd-LC6-SH at 0.05 and 0.1 mmol/kg were not significantly different at longer times after injection. SCID mice bearing Mia-PaCa-2 or NCI-N87 tumor xenografts were treated with either the glutathione synthesis inhibitor buthionine sulfoximine or the thiol-oxidizing anticancer drug Imexon, respectively. In both models, there was a significantly greater increase in tumor R1 (=1/T1) 60 minutes after injection of Gd-LC6-SH in drug-treated animals relative to saline-treated controls. In addition, Mercury Orange staining for nonprotein sulfhydryls was significantly decreased by drug treatment relative to controls in both tumor models. In summary, these studies show that thiol-bearing complexes of gadolinium such as Gd-LC6-SH can serve as redox-sensitive MRI contrast agents for detecting differences in tumor redox status and can be used to evaluate the effects of redox-active drugs.

Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis

BackgroundMicroRNAs (miRs) are small non-coding RNAs that play an important role in cancer development where they can act as oncogenes or as tumor-suppressors. miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC).Methods and ResultsmiR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell-proliferation, migration and clonogenic survival was examined by MTT, Xcelligence system, scratch assay and colony formation assay. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. miR-34a overexpression also markedly downregulated E2F3 and survivin levels. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions.ConclusionsTaken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC.

Discovery of an Orally Efficacious Inhibitor of Anaplastic Lymphoma Kinase

Anaplastic lymphoma kinase (ALK) is a promising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and culminating in the recent FDA approval of the ALK inhibitor crizotinib. Through a series of targeted modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase selectivity, and overall pharmaceutical properties. Optimization of this scaffold has led to the identification of a potent and efficacious inhibitor of ALK, 25b. A striking feature of 25b over previously described ALK inhibitors is its >600-fold selectivity over insulin receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment in advanced preclinical models.