Beige Mice Research Articles

Abstract 2460: Dissecting the in vitro and in vivo mechanism of action of obinutuzumab (GA101) in preclinical models using an immune effector-dead version of obinutuzumab

Abstract Introduction: Obinutuzumab (GA101) is a glycoengineered type II CD20 antibody mediating superior direct cell death induction, ADCC and ADCP compared to rituximab while CDC is strongly reduced. Obinutuzumab is approved for 1st line treatment of CLL in combination with chlorambucil in the US and Europe. The contribution of these mechanisms of action (MOA) to the efficacy of obinutuzumab in patients is unclear. In order to further dissect the MOA GA101 and rituximab and variants with abolished FcγR and C1q binding were assessed in vitro and in vivo. Methods: Glycoengineered GA101, non-glycoengineered wild type (wt) GA101, effector-dead GA101 and rituximab with P329G LALA mutations in the Fc were generated and characterized i) for binding to FcγRs and CD20 positive cells by FACS, ii) for cell death induction by AnnexinV/PI staining, iii) in ADCC, ADCP and CDC assays, and iv) for depletion of CD19 positive B cells in whole blood from healthy volunteers. Anti-tumoral efficacy was assessed using established 300 mm2 s.c. SU-DHL4 xenograft tumors in Scid beige mice (30 mg/kg, q7d x 4, ip). Results: Variants of GA101 and rituximab retain CD20 binding and cell death inducing properties of the respective parental antibody. The introduction of P329G LALA in the Fc part abolishes binding to Fcγ receptors and C1q, so that they do not mediate ADCC, ADCP or CDC any longer, while direct cell death induction is not affected as compared to GA101 and rituximab, respectively. In whole blood assays GA101 showed the best B cell depletion in terms of potency and absolute B cell depletion followed by wtGA101 and rituximab. Notably, GA101 P329G LALA retained significant B cell depletion, while rituximab P329G LALA did not mediate B cell depletion. Thus, in whole blood B cell depletion, ADCC/ADCP plays an important role, but direct cell death induction is equally important for the MOA of GA101, whereas it does not contribute to B cell depletion of rituximab. In the SU-DHL4 xenograft model GA101 (TGI > 100%, non-parametric TCR (npTCR) 0 with 95% CI 0-0) and wtGA101 (TGI > 100%, npTCR 0, 95% CI 0-0.006) showed equivalent efficacy with complete tumor remission in 9/10 mice for GA101 and 8/10 mice for wtGA101. GA101 P329G LALA showed anti-tumoral efficacy (TGI 62%, npTCR 0.43, 95% CI 0.30-0.60) comparable to rituximab (TGI 71%, npTCR 0.37, 95% CI 0.29-0.50), while rituximab P329G LALA showed only residual anti-tumoral activity (TGI 26%, npTCR 0.73, 95% CI 0.44-0.92). Summary: These data indicate that in whole blood B cell depletion and xenograft models, direct cell death and ADCC/ADCP through the glycoengineered Fc part are required for anti-tumoral efficacy of obinutuzumab. Most notably, the effector dead version of obinutuzumab that completely lacks ADCC, ADCP and CDC activity, but has retained direct cell death induction, is as efficacious as rituximab in vivo supporting the role of direct effects for the efficacy of obinutuzumab. Citation Format: Sylvia Herter, Frank Herting, Gunter Muth, Erwin van Puijenbroek, Claudia Ferrara, Sabine Lang, Marina Bacac, Ekkehard Moessner, Pablo Umana, Christian Klein. Dissecting the in vitro and in vivo mechanism of action of obinutuzumab (GA101) in preclinical models using an immune effector-dead version of obinutuzumab. [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 2460. doi:10.1158/1538-7445.AM2015-2460

Abstract 1235: Inducible overexpression of bHLH transcriptional regulator Id4 abrogates tumorigenicity of prostate cancer xenografts

Abstract Introduction: Inhibitor of DNA binding/differentiation 4 (Id4) has been shown to be highly expressed during prostate development. However, in many cancers including prostate, Id4 is epigenetically silenced due to promoter hyper-methylation. In this study we developed a doxycycline inducible system for Id4 overexpression and show that controlled increase of Id4 in an aggressive metastatic prostate cancer cell line DU145, which lacks Id4 expression results in attenuation of tumor forming ability of this cell line and confirming its role as a tumor suppressor. Methods: Tetracycline inducible system was used to stably overexpress Id4 in DU145 cells. Empty vector backbone was used as a control. The Id4 overexpressed (DU(+)iId4) and vector control (DU(+)iVC) cells were used to investigate migration (scratch wound assay) and anchorage independent growth (soft agar assay). Tumorigenicity of DU(+)iId4 and DU(+)iVC cells was investigated in Scid beige mice. The gene expression profile between DU(+)iId4 and DU(+)iVC cells was analyzed by RNAseq. The DU(+)iId4 and DU(+)iVC tumors from mice were used for immunohistochemistry to validate prostate cancer pathways and RNAseq data. Results: Id4 was cloned in pcDNA4/TO/myc-His version A and stably transfected and clonally selected for overexpression in DU145 cells stably expressing pcDNA6/TR. The overexpression of Id4 on induction by doxycycline (a tetracycline analogue) was confirmed by qRT-PCR and western blotting. A clone with maximum expression was selected for further studies. On inducing the DU145 cells with doxycycline, an increase in apoptosis was observed as compared to un-induced DU145 as well as cells expressing the empty control vector. Decrease in migration and anchorage independent colony forming ability was also observed upon induction of Id4 in scratch wound and soft agar assay, respectively. Subcutaneous injection of DU145 cells in Scid beige mice resulted in formation of tumors, however upon induction of Id4 expression by doxycycline from day 0 or 2 weeks after injection of the cells showed a significant decrease in the tumor forming ability of the DU145 cells as evident from the reduction in the tumor weight and volume. Immunohistochemistry on tumor tissues was found to be in support of the RNAseq data. Conclusion: This study reports for the first time that conditional overexpression of tumor suppressor Id4 in prostate cancer cell lines DU145 results in diminution of tumor formation. The data describes use of a system that allows for the temporal regulation of Id4 expression in vivo which could act as a preliminary step in mimicking controlled delivery of Id4 for therapeutic purpose. Acknowledgement: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12MD007590. Citation Format: Divya Patel, Derrick J. Morton, Maxwell Korang-Yeboah, Yamini Gorantla, Sushma Bhosle, Perri Nagappan, Nathen Bowen, Jaideep Chaudhary. Inducible overexpression of bHLH transcriptional regulator Id4 abrogates tumorigenicity of prostate cancer xenografts. [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 1235. doi:10.1158/1538-7445.AM2015-1235

Abstract 4086: HMG Box Domain Containing 3 (HMGXB3), a novel TGFβ-induced cancer gene that influences cancer stem cells and metastasis in triple negative breast cancer

Abstract The role of HMGXB3 (HMG Box Domain Containing 3), member of the non-canonical high mobility group (HMG) genes, in cancer remains largely unknown. A preliminary “mutual exclusivity” analysis of the Breast Invasive TCGA database showed a significant association between HMGXB3 and TGFβ (Transforming Growth Factor β). TGFβ plays a significant role in metastasis and increases stem-like properties of breast cancer cells. This work aimed at elucidating whether there is a relationship between HMGXB3 and TGFβ, and their role on breast cancer stem cells (BCSC) and metastasis in triple negative breast cancer (TNBC). Changes in HMGXB3 protein levels (Abgent) were assessed by Western blot in MCF10A cells treated with human recombinant (hr) TGFβ1 (R&D) with/without the TGFβR1 kinase inhibitor, LY2157299. Knockdown was performed in MCF10A and two TNBC cell lines, SUM159 and MDA-MB-231. Cells transfected with two different siRNAs against HMGXB3, siRNA#1 and siRNA#2, and one negative control (siRNA-SCR) were assayed for proliferation as well as primary and secondary mammosphere (MS) formation for 48h with hrTGFβ1 (10ng/ml). Cell migration was determined in transfected TNBC cell lines treated with 10ng/ml hrTGFβ1 for 72h. For in vivo experiments, MDA-MB-231, stably transfected with a luciferase/GFP reporter plasmid, were injected in the right mammary fat pad of female SCID Beige mice (n = 10). Tumor-bearing mice were treated with DOPC (dioleoyl phosphatidylcholine) liposomes loaded with 5μg of either siRNA-SCR or HMGXB3-siRNA#2 with/without docetaxel (20mg/kg) for 21 days. Tumors were digested and cells isolated. Single cells were assayed for aldefluor (ALDF+) by flow cytometry and MS formation. Lung metastases were determined in lungs by ex vivo imaging of luciferase positive cells. Our results show a concentration- and time-dependent induction of HMGXB3 protein levels under treatment with hrTGFβ1 in MCF10A cells. Such an enhancement was efficiently abrogated by the addition of LY2157299. Knockdown of HMGXB3 in presence of hrTGFβ1 significantly reduced cell proliferation, migration and MS formation in the TNBC cell lines tested herein. In vivo experiments demonstrated that silencing of HMGXB3 correlates with reduced tumor growth, ALDF+ BCSC subpopulation, and formation of secondary MS compared to siRNA-SCR. Finally, silencing HMGXB3 significantly abrogated progression of metastasis from primary tumor to lungs in contrast to siRNA-SCR with/without chemotherapy. In conclusion, we are the first to identify HMGXB3 as a TGFβ-induced cancer gene that impacts tumor cell proliferation, migration, BCSC self-renewal and lung metastases in TNBC. Our preliminary results suggest HMGXB3 as a novel target for prevention of metastasis. Future experiments will elucidate the role and mechanism of HMGXB3 in cancer. This work will give rise to novel therapeutic targets for the treatment of breast cancer. Citation Format: Sergio Granados-Principal, Yi Liu, Cristian Rodriguez-Aguayo, Anil K. Sood, Jenny C. Chang. HMG Box Domain Containing 3 (HMGXB3), a novel TGFβ-induced cancer gene that influences cancer stem cells and metastasis in triple negative breast 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 4086. doi:10.1158/1538-7445.AM2015-4086

The antitumor effects of oncolytic adenovirus H101 against lung cancer.

Lung cancer is the leading cause of cancer mortality in both men and women, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. The new treatment options with completely novel mechanism of therapeutic activity are needed for lung cancer to improve patient outcome. The present study was aimed at testing the efficacy of recombinant adenovirus H101 as an oncolytic agent for killing human lung cancer cell lines in vitro and in vivo. We assessed the coxsackievirus adenovirus receptor (CAR) expression on human lung cancer cell lines by RT-PCR and immunocytochemistry staining. Viral infectivity and viral replication in lung cancer cells was assayed by flow cytometry and real-time fluorescent quantitative PCR. After H101 treatment, cytotoxic effect, cell cycle progression and apoptosis were further examined by lactate dehydrogenase release assay and flow cytometry in vitro, respectively. In vivo, antitumor effects of H101 were assessed on SCID Beige mice xenografted with human lung cancer cells. Receptor characterization confirmed that human lung cancer cell lines expressed CAR receptor for adenovirus type 5. Lung cancer cells were sensitive to infection by the H101 virus. H101 infection and replication resulted in very potent cytotoxicity, G2/M phase arrest and cell lysis. In vivo, we also showed that H101 significantly inhibited tumor growth following intratumoral injection, with virus replication, cell degeneration and necrosis in the tumor tissue. These results have important implications for the treatment of human lung cancer.

Establishment of the human acute promyelocytic leukemia pathological model in the SCID beige mice by using NB4 cell line

This study was to establish a stable, effective and reproducible human acute promyelocytic leukemia model in severe combined immunodeficient (SCID) mice by using NB4 cell line, and to investigate the disease course character and biological behaviors. Three-five-week-old SCID beige mice were divided randomly into two groups: experimental and control group. SCID mice of experimental group were transplanted by tail vein (iv) injection of 5×10(6) NB4 cells. The WBC cell count and the positive rate of promyelocytes in peripheral blood were dynamically monitored by using smears. Morphological examination and histopathological assay were employed to confirm NB4 cell infiltration in organs (liver, spleen, lung, kidney and brain). The expression level of PML-RARα fusion protein was detected by Western blot. Within two weeks there was no significant difference in peripheral blood WBC count between two groups (P > 0.05), meanwhile, NB4 cells were not found. At the day 21 and 28 after inoculation, the peripheral blood white blood cell count of experimental group reached to (4.79 ± 1.13)×10(9)/L and (7.62 ± 2.24)×10(9)/L respectively, which were significantly higher than that in control group (P < 0.05); simultaneously, the positive rates of promyelocytes on smears were (2.14 ± 0.63)% and (6.6 ± 2.76)%, respectively. Morphological observation showed single or multiple tumor lumps at day 21 after inoculation; HE staining of tissue biopsies demonstrated a large number of promyelocyte in the liver, spleen, lung, kidney and brain tissue. Cell immunofluorescence results showed that the CD33 expression of bone marrow cells in mice of experimental group was strongly positive (P < 0.05). Western blot confirmed that the PML-RARα fusion protein was expressed variously in liver, kidney and brain tissue. The human acute promyelocytic leukemia SCID mouse model is succesfully established by tail vein injection of NB4 cells. This model can mimic the characters of involved bone marrow and diffuse growth of cells. This model is a useful tool to explore the pathogenic mechanism and experimental treatment of human leukemia.

Characterization of Mouse Models of Mycobacterium avium Complex Infection and Evaluation of Drug Combinations

The Mycobacterium avium complex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen for M. avium complex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment of M. avium lung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) of M. avium infection and to assess the anti-M. avium activity of single and combination drug regimens, in vitro, ex vivo, and in mice. In vitro, clarithromycin and moxifloxacin were most active against M. avium, and no antagonism was observed between these two drugs. Nude mice were more susceptible to M. avium infection than the other mouse strains tested, but the impact of treatment was most clearly seen in M. avium-infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective for M. avium infection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy.

Newer-Generation HSP90 Inhibitors Can Overcome Ibrutinib Resistance and Suppress Proliferation in Human Mantle Cell Lymphoma in Vitro and in Vivo

Mantle cell lymphoma (MCL) makes up approximately 6% of non-Hodgkin’s lymphoma cases and has an aggressive disease course with a particularly poor outcome. To date there is no standard treatment and despite good initial response to chemotherapy, most patients relapse with long-term survival achieved in less than 40%. In almost all cases, MCL is distinguished by overexpression of Cyclin D1 (CCND1) usually caused by a translocation between chromosomes 11 and 14. Both CCND1 and BTK are known clients of heat shock protein 90 (HSP90). In previous studies, we demonstrated potent cytotoxic activity of second- (NVP-AUY922, PU-H71) and third-generation (NVP-HSP990) HSP90 inhibitors against the MCL cell lines Granta519, JeKo1, MAVER1, Rec1, and Z-138 (IC50s for AUY922, 3-11nM; HSP990, 5-24nM; PU-H71, 40-287 nM). Immunobloting of MCL cell lines exposed to 50nM AUY922 demonstrated reduced expression of client proteins CCND1, CDK4 and AKT and induction of G0/G1 cell cycle arrest within 6-8h followed by apoptosis within 27h. We now demonstrate that combining Ibrutinib with AU922 results in synergistic or additive effects in all lines except for the highly Ibrutinib- sensitive JEKO-1. In fact, AUY922 overcame ibrutinib-resistance due to activation of non B-cell receptor (BCR)-driven alternative NFkB signaling in Granta519 and Z-138 cells. Due to the promising results of AUY922 we conducted two in vivo studies. Current mouse MCL models are generated by injecting MCL cell lines in matrigel subcutaneously. The MCL grows as a solid tumor and treatment is typically initiated when mean tumor volume is approximately 200 mm3. First, we xenografted luciferized MAVER1 (ibrutinib-sensitive) and Z-138 (ibrutinib-resistant) cells into SCID beige mice (10 million cells per mouse). Mice were randomized to receive either AUY922 (50 mg/kg by tail vein injection thrice weekly) or vehicle. Tumors analyzed from mice sacrificed after 5 days of treatment showed complete loss of cyclin D1 and Ki67 staining by immunohistochemistry in those receiving AUY922. Tumor growth was significantly slowed in AUY922 treated animals in both lines, which translated into a survival advantage (p<0.01) for both lines. To advance beyond xenografting of cell lines, we generated a MCL patient-derived xenograft (PDX) model. Peripheral blood from a patient with relapsed MCL and peripheral blood involvement was xenografted by tail-vein injection. Within 28 days, fatal MCL developed in the spleen and bone marrow as evidenced by flow cytometry, but was also seen in liver and intestines. Xenografted splenocytes were secondarily transplanted into NSG mice. Eighteen days after injection, mice were randomized to receive either AUY922 (50 mg/kg by tail vein injection thrice weekly) or vehicle. After the third dose, mice underwent [18F]-FLT PET imaging. Vehicle mice had PET-avid disease diffusely within lymph nodes, spleen, bone marrow and intestine. Mice treated with 5 days of AUY922 had nearly complete resolution of PET activity in lymph nodes, spleen and bone marrow, as well as approximately 50% reduction in the intestines. Mice were euthanized and immunobloting of the spleens showed pronounced downregulation of both CCND1 and BTK in AUY922-treated mice compared with vehicle. In conclusion, newer-generation HSP90 inhibitors such as AUY922 have significant single-agent activity across a genetically diverse spectrum of MCLs, can target cyclin D1, CDK4, and BTK, and may overcome Ibrutinib resistance. DisclosuresWeinstock:Novartis: Consultancy, Research Funding.

Daratumumab, a Novel Anti-CD38 Monoclonal Antibody Shows Anti-Tumor Activity in CLL and hampers Leukemia-Microenvironment Interactions

Daratumumab (DARA) is a anti-human CD38 antibody with Fc-mediated cell killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M. J Immunol 2011), and antibody-dependent cellular phagocytosis (ADCP) by macrophages (mΦ), both murine and human in multiple myeloma (MM) and Burkitt lymphoma cells. DARA is currently being evaluated in phase III clinical trials in patients with MM. We have previously reported that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Chronic Lymphoctic Leukemia (CLL), and significantly prolongs overall survival of animals in a systemic CLL mouse model. Here, we present additional data on in vivo mechanism of DARA and its effect on tumor-microenvironment interactions in CLL.We first evaluated whether ADCP contributes to DARA activity both in vitro and in vivo. For in vitro ADCP, mΦ were generated from monocytes of normal PBMCs and stimulated with GM-CSF (10ng/mL, 7 days). CLL cell lines and primary cells were labeled with calcein and incubated for 4h with mΦ at an effector:target ratio of 2:1 in the presence of a fixed mAb concentration of 1 μg/mL, followed by flow cytometric analysis. The amount of remaining CLL target cells (CD19+, CD11b-) was reduced by 3-16%. ADCP defined as percentage of mϕ which had phagocytosed, referred to as double positive mΦ (CD11b+, calcein+, CD19-), ranged from 3-10%. To analyze ADCP in vivo, SCID beige mice, devoid of NK cells but with active macrophages, were inoculated intraperitoneally with CLL cells (20×106) and simultaneously treated with a single dose of DARA or isotype control (20mg/kg, n=3-5 per group). Forty-eight hours later, CLL cells were recovered from the intraperitoneal cavity and counted in a flow cytometer (identified as human CD45+/CD19+/CD5+cells). In DARA-treated mice the number of CLL cells recovered was reduced by 42% (n=2, p<0.05) compared to the isotype control group. Remarkably, the decrease in cell number was already detectable 2h after DARA administration.CLL pathogenesis relies on supportive tumor-microenvironment interactions both in the bone marrow (BM) and in the lymph node (LN), and CD38 constitutes a molecular hub integrating proliferative and migratory signals for CLL (Malavasi, F. Blood 2011). We evaluated the effect of DARA on migration and adhesion. In in vitro migrations assays, we have demonstrated that DARA (10-30 μg/mL) inhibited CXCL12/SDF1α-mediated migration up to 70% (n=5). In addition, DARA reduced up to 55% (n=2) of downstream pERK activation, that peaked after 5min of CXCL12/SDF1α stimulation. We analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to BM and spleen in vivo, using NOD/SCID/gamma (NSG) null mice (lacking NK cells and effective macrophages). In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, prior to injection of fresh primary CLL cells (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for human CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p<0.05).In addition to migration, CD38 also plays a key role in cell adhesion through interaction with integrins (CD49d/CD29) and with extracellular matrix proteins. We analyzed the effect of DARA on the adhesion of CLL cells to the extracellular matrix vascular-cell adhesion molecule-1 (VCAM-1) mediated by CD49d/CD29. DARA reduced adhesion of CLL cells (n=4), to VCAM-1 by 46±13% (range 27-57) compared to isotype control. By RT-PCR we observed an up-regulation of MMP9 transcripts (average 2 fold, n=2), and DARA abrogated both constitutive MMP9 expression (90% reduction) and VCAM-derived (94% reduction) MMP9 expression.In summary, DARA shows a positive effect on ADCP-mediated anti-tumor activity on CLL cells both in vitro and in vivo. In addition DARA exhibits a strong effect on CLL cell migration and adhesion. Based on these data, we hypothesize that DARA may exert unique and substantial effects on CLL tumor cell growth and contributes to potent therapeutic efficacy in a clinical setting. DisclosuresDoshi:Janssen R&D: Employment. Parren:Genmab: Employment, Equity Ownership. Lammerts van Bueren:Genmab : Employment. Pérez-Galán:Genmab: Research Funding.

Combination of the Glycoengineered Type II CD20 Antibody Obinutuzumab (GA101) and the MDM2 Selective Antagonist RG7388 Results in Superior Anti-Tumor Activity

Abstract Introduction: Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody that strongly induces direct cell death. As a result of glycoengineering, obinutuzumab has increased affinity for FcgRIII on innate immune effector cells resulting in enhanced induction of ADCC and ADCP. Obinutuzumab has been approved for first line treatment of CLL patients in combination with chlorambucil in the US and Europe and is currently in pivotal clinical trials in indolent NHL and DLCBL. RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound are novel, orally bioavailable, selective MDM2 antagonists which reactivate p53 and thereby mediate cell cycle arrest and subsequent apoptotic cell death in solid and hematologic tumors. RG7388 is currently in clinical trials for the treatment of AML and prostate cancer. Based on the fact that the majority of B lymphoid malignancies including NHL and CLL bear wildtype p53, and the complementary mechanisms of action involving increased apoptosis (MDM2 antagonist) or direct cell death (obinutuzumab), the combination of both compounds has the potential for superior efficacy in treating B lymphoid malignancies. Experimental methods: The combination of obinutuzumab and RG7388 (or obinutuzumab and rituximab with RG7112, the frontrunner compound with identical mode of action) was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (Annexin V/PI positivity) on p53 wildtype Z138 Mantle cell lymphoma (MCL) and DoHH-2 Diffuse large B-Cell lymphoma (DLBCL) cells by FACS and the impact of MDM2 inhibition on ADCC induction and whole blood B cell depletion. In vivo efficacy of the combination of obinutuzumab or rituximab with RG7122 and RG7388 was evaluated in the s.c. Z138 MCL xenograft model in immunodeficient SCID beige mice. Results: RG7338 induced concentration-dependent cell death of Z-138 and DOHH-2 cell lines. At concentrations &gt; 10-100 nM RG7388 resulted in enhanced cell death induction of DOHH-2 and Z-138 cells in combination with obinutuzumab. Notably, RG7388 did not influence obinutuzumab mediated ADCC during 4 h up to concentrations of 1000 nM and did not affect obinutuzumab mediated NK cell activation (CD16 downregulation, CD107a upregulation). Similarly, addition of RG7388 did not interfere with obinutuzumab mediated B cell depletion in healthy human whole blood at concentrations up to 1000 nM. In the Z-138 xenograft model, the combination of suboptimal doses of 0.5 mg/kg obinutuzumab or 1 mg/kg rituximab with 150 mg/kg RG7112 (three times a week p.o. for 3 weeks) resulted in superior tumor growth inhibition by obinutuzumab as compared to rituximab including the induction of complete tumor remission. In a second Z-138 study, the combination of the suboptimal dose of 0.5 mg/kg obinutuzumab with 80 mg/kg RG7388 yielded similar anti-tumor activity. In summary, the combination of either obinutuzumab or rituximab with RG7112 or the combination of obinutuzumab with RG7388 showed superior in vivo efficacy with no clinical signs of toxicity. Conclusions: The combination of obinutuzumab with MDM2 antagonists results in enhanced cell death of p53 wildtype NHL tumor cells while not affecting obinutuzumab mediated ADCC of tumor cells or B cell depletion in whole blood from healthy donors. In vivo the combination of obinutuzumab with MDM2 inhibitors RG7112 and RG7388 results in robust combined anti-tumor efficacy in xenograft models. Taken together, these preclinical data strongly support the investigation of obinutuzumab and RG7388 combination therapy in clinical trials. Disclosures Herting: Roche: Employment, Patents &amp; Royalties. Herter:Roche: Employment. Friess:Roche: Employment, Patents &amp; Royalties. Lehmann:Roche: Employment. Bacac:Roche: Employment. Dangl:Roche: Employment, Patents &amp; Royalties. Klein:Roche: Employment, Equity Ownership, Patents &amp; Royalties.

Adoptive immunotherapy of epithelial ovarian cancer with Vγ9Vδ2 T cells, potentiated by liposomal alendronic acid.

Adoptive immunotherapy using γδ T cells harnesses their natural role in tumor immunosurveillance. The efficacy of this approach is enhanced by aminobisphosphonates such as zoledronic acid and alendronic acid, both of which promote the accumulation of stimulatory phosphoantigens in target cells. However, the inefficient and nonselective uptake of these agents by tumor cells compromises the effective clinical exploitation of this principle. To overcome this, we have encapsulated aminobisphosphonates within liposomes. Expanded Vγ9Vδ2 T cells from patients and healthy donors displayed similar phenotype and destroyed autologous and immortalized ovarian tumor cells, following earlier pulsing with either free or liposome-encapsulated aminobisphosphonates. However, liposomal zoledronic acid proved highly toxic to SCID Beige mice. By contrast, the maximum tolerated dose of liposomal alendronic acid was 150-fold higher, rendering it much more suited to in vivo use. When injected into the peritoneal cavity, free and liposomal alendronic acid were both highly effective as sensitizing agents, enabling infused γδ T cells to promote the regression of established ovarian tumors by over one order of magnitude. Importantly however, liposomal alendronic acid proved markedly superior compared with free drug following i.v. delivery, exploiting the "enhanced permeability and retention effect" to render advanced tumors susceptible to γδ T cell-mediated shrinkage. Although folate targeting of liposomes enhanced the sensitization of folate receptor-α(+) ovarian tumor cells in vitro, this did not confer further therapeutic advantage in vivo. These findings support the development of an immunotherapeutic approach for ovarian and other tumors in which adoptively infused γδ T cells are targeted using liposomal alendronic acid.

Abstract 4024: iNOS inhibition increases survival in triple negative breast cancer by targeting metastasis and epithelial-mesenchymal transition

Abstract Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in breast cancer patients by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. We also propose an effective clinically relevant-targeted therapeutic regimen by re-purposing the iNOS inhibitor L-NMMA which has been extensively investigated in thousands of patients with cardiogenic shock. Cell migration was determined in confluent MDA-MB-231 cells with a “wound healing assay” treated with different concentrations of the specific iNOS inhibitor 1400W (0, 0.0001, 0.001, 0.01, 0.1, 1, 2, 4 mM) in low serum conditions (1%) for 72h. EMT-inducing factors (Snail, Slug, Twist1, and Zeb1) were determined by Western blot in MDA-MB-231 cells. For in vivo studies, 3x106 MDA-MB-231 and SUM159 cells were injected in the right mammary fat pad of female SCID Beige mice (n=10/group). The clinically relevant dose regimen consisted on two cycles of docetaxel (20mg/kg, i.p, on day 0) twelve hours before being combined with L-NMMA (400mg/kg on day 1, and 200mg/kg for 4 additional days by oral gavage) and amlodipine on day 0 (10mg/kg, i.p, daily, for 6 days). Docetaxel alone, as well as saline (i.p) + sterile water (oral gavage) were used as controls. Our results show that the specific iNOS inhibitor 1400W was able to diminish cell migration in vitro for all concentrations tested. Importantly, impact of iNOS inhibition on cell migration was further correlated with a dose-dependent decrease of the EMT transcription factors- Snail, Slug, and Twist1. Zeb1 was mainly affected at millimolar concentrations. In vivo, the combination of L-NMMA and docetaxel was able to decrease tumor growth in a MDA-MB-231 and SUM159 orthotopic models. Additionally, the combination with amlodipine did significantly prevent the L-NMMA-dependent increase of blood pressure. More importantly, this dose regimen also improved survival compared to docetaxel alone. In conclusion, targeted therapy with iNOS inhibitor impacts cell migration and changes in epithelial-mesenchymal transition might influence metastatic events. Based on these findings, we have planned a phase Ib/II targeted therapeutic trial by re-purposing L-NMMA as an anti-cancer indication. Targeting TNBC with L-NMMA could significantly alter clinical practice for treatment of breast cancer in the near future. Citation Format: Sergio M. Granados, Yi Liu, Bhuvanesh Dave, Melissa D. Landis, Steven S. Gross, Jenny C. Chang. iNOS inhibition increases survival in triple negative breast cancer by targeting metastasis and epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2014-4024

A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells.

The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/beige mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.

Enhanced diacylglycerol production by phospholipase D activation is responsible for abnormal increase in concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mice.

We previously reported that enhanced ceramide production induces calpain-mediated proteolysis of protein kinase C (PKC) in leukocytes from Chediak-Higashi syndrome (CHS). In the present study, we demonstrated that phospholipase D (PLD) inhibitors ameliorated abnormal increases in concanavalin A (Con A) cap formation in polymorphonuclear leukocytes (PMNs) from beige mouse, an animal model of CHS. PLD activity in PMNs from beige mice enhanced at 30 to 60s after Con A stimulation. In Con A-stimulated beige PMNs, both neutral sphingomyelinase (N-SMase) and acidic sphingomyelinase (A-SMase) activities enhanced, and ceramide levels are also increased. We found that ceramide levels were reversed by the treatment of beige PMNs with propranolol which inhibits phosphatidic acid phosphohydrolase. In addition, we showed that diacylgycerol (DAG) analogs enhance both N-SMase and A-SMase activities in PMNs from normal mice. We subsequently examined the association of CHS1 with PLD, and showed that expression of a truncated mutant of CHS1 in 293T cells induced abnormally rapid activation of PLD after phorbol ester stimulation. Moreover, we showed that specific inhibitors of 14-3-3 proteins, which interact with CHS1/LYST and bind PKC, did not affect abnormal increases in Con A cap formation in beige PMNs. These results suggest that the enhanced DAG production via the PLD pathway is associated with abnormal increases in Con A cap formation in beige PMNs, and that CHS1 may be involved in the regulation of PLD activity.

In-vivo testing of PSMA-targeted T-cell immunotherapy for prostate cancer (TUM7P.920)

Abstract Introduction: Skeletal metastases are a significant cause of morbidity and mortality and overall greatly affect the quality of life of prostate cancer patients. Examining impact of bone metastasis in vivo remains a significant challenge, as animal models that closely mimic the natural history and malignant progression of clinical prostate cancer are not available. Objectives: To develop an animal model of human metastatic prostate cancer. Once a model has been developed and optimized, it was this to test efficacy of immunotherapy using T-cells that have been genetically targeted against prostate-specific membrane antigen (PSMA). Material and methods: PCR, western blot, flow cytometry and ELISA, we performed functional anaylsis of fucosyltransferase 3(FT3) in PC3LN3(PL)and PC3LN3-PSMA (PLP) tumour cell lines. In vivo bioluminescent imaging (BLI) was used to detect metastases. Results: In preliminary studies, we have observed that delivery of a FT3-encoding retroviral vector to PL and PLP enables them to express sialyl Lewis X and to acquire E-selectin binding activity. We also showed that FT3 promotes increased PLP motility and invasiveness in vitro. Bioluminescent animal model of metastasised prostate cancer is established to determine the effect of this upon their pattern of metastatic spread in SCID Beige mice. Conclusion: We have established an in-vivo model of PSMA-expressing prostate cancer. This will serve as a platform to test immunotherapy using P28z+ T-cells.

Redox-responsive targeted gelatin nanoparticles for delivery of combination wt-p53 expressing plasmid DNA and gemcitabine in the treatment of pancreatic cancer

BackgroundPancreatic adenocarcinoma is one of the most dreaded cancers with very low survival rate and poor prognosis to the existing frontline chemotherapeutic drugs. Gene therapy in combination with a cytotoxic agent could be a promising approach to circumvent the limitations of previously attempted therapeutic interventions.MethodWe have developed a redox-responsive thiolated gelatin based nanoparticle system that efficiently delivers its payload in the presence of glutathione-mediated reducing intra-cellular environment and could be successfully used for site-specific wt-p53 expressing plasmid DNA as well as gemcitabine delivery by targeting epidermal growth factor receptor (EGFR). Efficacy studies were performed in subcutaneous human adenocarcinoma bearing SCID beige mice along with molecular level p53 plasmid and apoptotic marker expression by PCR and western blot for all study groups.ResultsEfficacy studies demonstrate an improved in vivo targeting efficiency resulting in increased transfection efficiency and tumor growth suppression. In all the treatment groups, the targeted nanoparticles showed better anti-tumor activity than their non-targeted as well as non-encapsulated, naked therapeutic agent counterparts (50.1, 61.7 and 77.3% tumor regression by p53 plasmid alone, gemcitabine alone and in combination respectively). Molecular analysis revealed a higher mRNA expression of transfected p53 gene, its corresponding protein and that the tumor cell death in all treatment groups was due to the induction of apoptotic pathways.ConclusionsGene/drug combination treatment significantly improves the therapeutic performance of the delivery system compared to the gene or drug alone treated groups. Anti-tumor activity of the thiolated gelatin loaded wt-p53 plasmid or gemcitabine-based therapy was attributed to their ability to induce cell apoptosis, which was confirmed by a marked increase in mRNA level of proapoptotic transcription factors, as well as, protein apoptotic biomarker expression and significant decrease in the anti-apoptotic transcription factors.

Host specificity and in vivo infectivities of the mouse coccidian parasites Eimeria krijgsmanni.

In the present study, infection experiments of E. krijgsmanni using various hosts were conducted to elucidate the host specificity among some animals and the infectivity to mouse strains. According to the results, the infection was not found in most animals, except for rats, in which some oocyst shedding was detected, and there was no significant difference in infectivity among mouse strains. Additionally, oocyst shedding was hardly detectable in a secondary infection to immunocompetent mice, although it was found in immunodeficient mice. These results indicated that only immunocompetent mice could develop adaptive immunity against reinfection by stimuli of the primary infection. Furthermore, the infection experiments were performed with splenic macrophage (Mφ)-depleted mice with a reagent and Beige (Bg) mice known to be a strain of mice with low NK cell activity. No significant effect was found in primary or secondary infections in the Mφ-depleted mice, whereas the mortality rate was clearly increased in Bg mice inoculated with a large number of oocysts. Their oocyst shedding was similar to that of immunocompetent hosts. Taken together, these results suggested that Mφ has only a minor role in the immune response, but the NK cell has an important function in resistance to primary infection of E. krijgsmanni.

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type-1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow-derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA-LPA1 signalling in this is yet unclear. Using a bleomycin-induced mouse lung-fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived-mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin-injured lung. The migrated BMSC contributed significantly to α-smooth muscle actin (α-SMA)-positive myofibroblasts. By transplantation of GFP-labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative-RT-PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA-induced BMSC differentiation into myofibroblast and the secretion of ECM viaLPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.

Nanoparticle Design For Bone-Specific Chemotherapy and Microenvironmental Targeting In Multiple Myeloma

IntroductionThe bone marrow (BM) niche is known to exert a protective effect on lymphoid tumors, such as multiple myeloma (MM), where mesenchymal stem cell interactions with clonal plasma cells increase tumor proliferation and survival. However, certain cells within the BM milieu, such as mature osteoblasts and osteocytes, have demonstrated the potential to inhibit tumor growth; utilizing these cells presents a promising new anti-cancer approach. Hence, designing better methods of bone-specific delivery for both direct cancer cell treatment and indirect treatment through the modulation of bone cells may result in a potent, two-pronged anti-cancer strategy. Our work aimed to develop a novel system to target both MM and bone cells to induce greater osteogenesis and hamper tumor growth. MethodsPEG–PLGA nanoparticles (NPs) coupled to alendronate (“bone-targeted”) or alone (“non-targeted”) were formulated and loaded with bortezomib (“BTZ-NPs”) or left empty (“BTZ-free”). NPs were characterized for their physiochemical properties, including size (using dynamic light scattering; surface charges (Zeta potential); and bone affinity (using hydroxyapatite binding). NPs were engineered with different formulation methods and those with the optimal physiochemical characteristics and drug encapsulation efficiency were used for further studies. BTZ release kinetics were analyzed using HPLC. Anti-MM effects were assessed in vitro using MTT, bioluminescence (BLI) and Annexin V/PI apoptosis flow cytometry analysis on MM1S cells. In vivo, efficacy was measured by mouse weight, BLI and survival after i.v. cancer cell injections in mice. Cellular uptake was assessed in vitro by flow cytometry and in vivo biodistribution was assessed using fluorescent whole body and fixed section imaging. Bone specificity was assessed in vitro by co-culture of bone-targeted and non-targeted NPs with bone chips or hydroxyapatite using fluorescence and TEM imaging.In an in vivo model of myeloma treatment, female Nod/SCID beige mice were injected i.v. with 4 × 106 Luc+/GFP+ MM1S cells and, at day 21, treated with a) BTZ, b) BTZ-bone-targeted NPs, c) BTZ-non-targeted NPs or d) BTZ-free bone-targeted NPs. Using an in vivo model of pre-treatment for cancer prevention, mice were pre-treated with i.p. injections of BTZ-bone-targeted NPs and appropriate controls thrice weekly for 3 weeks. They were then injected i.v. with Luc+/GFP+ 5TGM1 or MM1S cells and monitored for BLI and survival. Static and dynamic bone histomorphometry and μCT were used to assess effects of pre-treatment on bone formation and osteolysis prevention. ResultsOur biodegradable, NPs had uniform size distribution within the range of 100 to 200 nm based on the type of formulation, with a zeta potential of ±5mV. Bone- targeted NPs showed high affinity towards bone mineral in vitro and better skeletal accumulation in vivo compared to non-targeted NPs. NPs were easily up-taken by cells in vitro, and BTZ release kinetics showed a burst followed by a sustained-release pattern over 60 hrs. BTZ-NPs induced apoptosis in MM cells in vitro. Importantly, BTZ-bone-targeted-NP pre-treated mice showed significantly less tumor burden (BLI) and longer survival than free drug or drug-free bone-targeted NPs, thus demonstrating a tumor-inhibiting effect unique to the BTZ-bone-targeted-NPs. Pre-treatment with BTZ increased bone formation in tibias and femurs, as measured by μCT of bone volume/total volume, and trabecular thickness and number, suggesting that increased bone volume may inhibit MM. In a second mouse model, both BTZ-bone-targeted NPs and BTZ-free NPs were equally able to reduce tumor growth in vivo when given after tumor formation. ConclusionBone-targeted nanoparticles hold great potential for clinical applications in delivering chemotherapies to bone marrow niches, reducing off-target effects, increasing local drug concentrations, and lengthening the therapeutic window. BTZ-bone-targeted NPs are able to slow tumor growth and increase survival in mice when used as a pre-treatment. This may result, at least in part, from BTZ-induced increased bone formation. These findings indicate that BTZ-bone-targeted NPs exert a chemopreventive effect in MM in vivo, thus suggesting their potential use in the clinical setting. Disclosures:Basto:BIND Therapeutics: Patent licensed by BIND, Patent licensed by BIND Patents & Royalties. Farokhzad:BIND Therapeutics: Employment, Equity Ownership; Selecta Biosciences: Employment, Equity Ownership. Ghobrial:Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Sanofi: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Inducible Silencing Of eIF4E Using a Tet-On System Results In Myeloma Growth In Vivo That Correlates With eIF4E Expression

IntroductionOverexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and ResultsWe stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P<0.01) cell growth accompanied by a decrease of c-myc, cyclin D1, C/EBP beta and IRF4 all critical for myeloma cell growth. Cell cycle analysis revealed increased cells population in G0/G1 phase (62% vs 80%) in doxycycline-induced eIF4E shRNA cells with a significant reduction (P<0.001) of clonogenic tumor growth reflected by a decrease in colony numbers (27.6 ± 4.2 vs 5.3 ± 3.4) and size. To determine the role of high expression of eIF4E in MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the inducible U266-Tet-CT-shRNA and U266-Tet-eIF4E-shRNA cells. In contrast to vehicle or doxycycline-treated control shRNA tumors, doxycycline treated animals bearing U266-Tet-eIF4E-shRNA xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 21 days. The transient inhibition of tumor growth correlated with the transient doxycycline-induced eIF4E knockdown further confirming the critical role of eIF4E. Immunohistochemical staining of tumors confirmed the decreased of eIF4E expression in doxycycline-treated mice bearing U266-Tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non-doxycyclin treated mice. ConclusionHere we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures:Schecter:Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.