Multiple Myeloma Xenograft Mouse Model Research Articles

Fucoidan inhibits angiogenesis induced by multiple myeloma cells.

Multiple myeloma (MM) remains an incurable hematological neoplasms. Our previous studies showed that Fucoidan possessed anti-myeloma effect by inducing apoptosis and inhibiting invasion of myeloma cells. In this study, we evaluated the effect of Fucoidan on angiogenesis induced by human myeloma cells and elucidated its possible mechanisms. Multiple myeloma cells were treated with Fucoidan at different concentrations, then the conditioned medium (CM) was collected. The levels of VEGF in the CM were tested by ELISA. The results showed that Fucoidan significantly decreased VEGF secretion by RPMI-8226 and U266 cells. The tube formation assay and migration test on human umbilical vein endothelial cells (HUVECs) were used to examine the effect of Fucoidan on angiogenesis induced by human myeloma cells. The results showed that Fucoidan decreased HUVECs formed tube structures and inhibited HUVECs migration, and suppressed the angiogenic ability of multiple myeloma RPMI-8226 and U266 cells in a dose-dependent manner. The study also showed that Fucoidan downregulated the expression of several kinds of proteins, which may be correlated with the reduction of angiogenesis induced by myeloma cells. Moreover, results were compared from normoxic and hypoxic conditions, they showed that Fucoidan had anti-angiogenic activity. Furthermore, in a multiple myeloma xenograft mouse model, it indicated that Fucoidan negatively affected tumor growth and angiogenesis in vivo. In conclusion, our results demonstrate that Fucoidan was able to interfere with angiogenesis of multiple myeloma cells both in vitro and invivo and may have a substantial potential in the treatment of MM.

Abstract 2912: The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment

Abstract Introduction: The activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. In multiple myeloma (MM), the role of AP-1 is largely unknown. Experimental procedures: MM cell lines and primary tumor cells were co-cultured with primary bone marrow stromal cells (BMSCs) or BMSC lines. AP-1 expression was measured by western blot analysis and real-time PCR. AP-1 activation was determined using TransAM AP-1 assay kit. To investigate the upstream regulators of JunB, cytokine array and specific inhibitors were used followed by 3H-thymidine incorporation, western blot and TransAM AP-1 assays. To delineate the specific functional role of JunB in MM pathogenesis, we used pLKO.1- JunB shRNA (shJunB) and pLKO.1- scrambled shRNA (SCR) vectors for constitutive knockdown, as well as pMSCV-JunB-ER-IRES-GFP and empty vectors for inducible overexpression, together with 3H-thymidine incorporation, alamarBlue, flow cytometry and western blot analysis, as well as gene expression profiling (GEP). To evaluate the functional role of JunB in vivo, a MM xenograft mouse model was used. Results: Co-cultures of MM cells with BMSCs rapidly and strongly induced sustained expression and activation of JunB, but not of other AP-1 family members. Induction of JunB was predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, IL-6 stimulation of MM.1S cells resulted in rapid and strong upregulation of JunB. Conversely, anti-IL-6 receptor antibody tocilizumab blocked BMSC-induced JunB expression and activation. Pharmacologic inhibition identified the requirement of the MEK/ERK and NF-κB pathways for BMSC-induced JunB expression. Functionally, significant inhibition of proliferation was observed in MM cells carrying pLKO.1- shJunB, but not pLKO.1-SCR. Importantly, knockdown of other AP-1 family members had minor effects on MM cell proliferation. Moreover, GEP performed on MM.1S- shJunB cells co-cultured with BMSCs as well as data analysis of a patient cohort using Gene Set Enrichment Analysis (GSEA) suggested a key role for JunB in the regulation of Mcl-1 and c-Myc expression. Furthermore, knockdown of JunB overcame resistance of MM cells to dexamethasone. Conversely, 4-OHT treatment of MM cell lines transduced with JunB-ER but not control vector induced significant JunB/AP-1 luciferase activity and protected MM cells against bortezomib-induced apoptosis and ER stress. Confirming our in vitro data, preliminary results show significant inhibition of tumor growth in a xenograft mouse model inoculated with inducible Tet-shJunB-GFP but not Tet-SCR-GFP MM.1S cells upon treatment with doxycycline. Conclusion: Taken together, our data demonstrate for the first time an important and surprising role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Muhammad Hasan Bashari, Mostafa Jarahian, Eugenio Morelli, Dirk Hose, Latifa Bakiri, Claudia Ball, Hanno Glimm, Martin Sattler, Hartmut Goldschmidt, Giovanni Tonon, Pierfrancesco Tassone, Erwin F. Wagner, Dirk Jäger, Klaus Podar. The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2912.

Role of Myeloma-Derived MIF in Myeloma Cell Adhesion to Bone Marrow and Chemotherapy Response.

Multiple myeloma (MM) remains an incurable cancer characterized by accumulation of malignant plasma cells in the bone marrow (BM). The mechanism underlying MM homing to BM is poorly elucidated. The clinical significance of migration inhibitory factor (MIF) expression was examined by analyzing six independent gene expression profile databases of primary MM cells using the Student's t test and Kaplan-Meier test. Enzyme-linked immunosorbent assay was used to examine MIF expression. In vivo bioluminescent imaging was used to determine MM cell localization and treatment efficacy in human MM xenograft mouse models, with three to four mice per group. MM cell attachment to BM stromal cells (BMSCs) was monitored by cell adhesion assay. MIF regulation of the expression of adhesion molecules was determined by chromatin immunoprecipitation (ChIP) assay. Statistical tests were two-sided. High levels of MIF were detected in MM BM (MIF level in BM plasma: healthy = 10.72 ± 5.788 ng/mL, n = 5; MM = 1811 ± 248.7 ng/mL, n = 10; P < .001) and associated with poor survival of patients (Kaplan-Meier test for MM OS: 87 MIF(high) patients, 86 MIF(low) patients, P = .02). Knocking down MIF impaired MM cell adhesion to BMSCs in vitro and led to formation of extramedullary tumors in SCID mice. MIF acted through surface receptor CXCR4 and adaptor COPS5 to regulate the expression of adhesion molecules ALCAM, ITGAV, and ITGB5 on MM cells. More importantly, MIF-deficient MM cells were sensitive to chemotherapy in vitro when cocultured with BMSCs and in vivo. MIF inhibitor 4-IPP sensitized MM cells to chemotherapy. MIF is an important player and a novel therapeutic target in MM. Inhibiting MIF activity will sensitize MM cells to chemotherapy.

Regulation of Sclerostin Expression in Multiple Myeloma by Dkk-1: A Potential Therapeutic Strategy for Myeloma Bone Disease.

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl-Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD-SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S-Luc-GFP cells) demonstrated significantly higher concentrations of mouse-derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated β-catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high-affinity grade scl-Ab reversed osteolytic bone disease in this animal model. Because scl-Ab did not demonstrate significant in vitro anti-MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk-1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl-Ab combined with proteasome inhibitors in MM. © 2016 American Society for Bone and Mineral Research.

Anti-tumor activity of the proteasome inhibitor BSc2118 against human multiple myeloma

Introduction of bortezomib, the first generation of proteasome inhibitor, has significantly improved the median overall survival of patients with multiple myeloma (MM). However, the dose-limiting adverse events and acquired drug resistance limit its long-term usage. Here, we report in vitro and in vivo anti-MM activity of the irreversible proteasome inhibitor BSc2118. BSc2118 inhibited the chymotrypsin-like (CT-L) proteasome activity, accompanied by accumulation of ubiquitinated proteins. BSc2118 suppressed tumor cell growth through induction of G2/M phase arrest and induced apoptosis via activation of the apoptotic signaling cascade, in association with up-regulation of p53 and p21. Importantly, BSc2118 was active in vitro against MM cells' acquired bortezomib resistance. Of note, BSc2118 also displayed a novel anti-angiogenesis activity both in vitro and in vivo. Lastly, BSc2118 exhibited a broader safety dose range and higher anti-tumor efficacy in vivo in a human MM xenograft mouse model, compared to bortezomib. Together, these findings indicate the in vitro and in vivo anti-MM activities of BSc2118 through induction of cell cycle arrest and apoptosis, as well as inhibition of tumor angiogenesis. They also suggest that BSc2118 might, at least in vitro, partially overcome acquired bortezomib resistance, likely associated with inhibition of autophagy.

Icaritin suppresses multiple myeloma, by inhibiting IL-6/JAK2/STAT3.

Icaritin is an active prenylflavonoid derived from Epimedium genus, a traditional Chinese medicine. Icaritin has a wide range of pharmacological and biological activities, including cardiovascular function improvement, hormone regulation and antitumor activity. Here, we investigated the effect of icaritin on multiple myeloma (MM) in vitro and in vivo. Icaritin inhibited cell growth of MM cell line and primary MM cells. In contrast, icaritin had low or no cytotoxic effect on normal hematopoiesis. We also demonstrated that in MM xenograft mouse models, icaritin suppressed tumor growth and decreased serum IL-6 and IgE levels, but did not show adverse reactions such as body weight loss. The anti-MM activity of icaritin was mainly mediated by inhibiting IL-6/JAK2/STAT3 signaling. We suggest that icaritin can be further tested in clinical trials in MM.

Farnesol inhibits tumor growth and enhances the anticancer effects of bortezomib in multiple myeloma xenograft mouse model through the modulation of STAT3 signaling pathway

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is frequently observed in multiple myeloma (MM) cancer and can upregulate the expression of several genes involved in proliferation, survival, metastasis, and angiogenesis. The effect of farnesol (FOH) on STAT3 activation, associated protein kinases, its regulated gene products, cellular proliferation, and apoptosis was examined. The in vivo effect of FOH on the growth of human MM xenograft tumors alone and in combination with bortezomib (Bor) in athymic nu/nu female mice was also investigated. We found that FOH suppressed both constitutive and inducible STAT3 activation at Tyr705 in MM cells. The suppression of STAT3 was mediated through the inhibition of activation of upstream JAK1, JAK2, and c-Src kinases. Also, treatment with the protein tyrosine phosphatase (PTP) inhibitor, pervanadate treatment reversed the FOH-induced down-regulation of STAT3, possibly indicating the involvement of a PTP. Indeed, we found that FOH treatment induces the increased expression of SHP-2 protein and knockdown of the SHP-2 gene by small interfering RNA suppressed the ability of FOH to inhibit STAT3 activation. FOH inhibited proliferation and significantly potentiated the apoptotic effects of bortezomib (Bor) in U266 cells. When administered intraperitoneally, FOH enhanced Bor-induced growth suppression of human MM xenograft tumors in athymic nu/nu female mice. Our results suggest that FOH is a novel blocker of STAT3 signaling pathway and exerts both anti-proliferative and apoptotic activities in MM in vitro and in vivo.

Abstract 4600: P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells

Abstract Despite proteasome inhibitor (e.g., bortezomib/btz and carfilzomib/cfz) success in multiple myeloma (MM), drug resistance remains a key problem. MM cells commonly over-express Mcl-1, a protein implicated in survival and drug-resistance. Mcl-1 exhibits rapid protein turnover (half-life &amp;lt; 1h), suggesting that efficient transcriptional machinery for de novo synthesis may be required for its constitutive expression in MM. Eukaryotic protein-coding gene transcription is controlled by RNA polymerase II (RNAPII), which is regulated by negative (N-TEF) and positive elongation factors (P-TEF). P-TEFb, a holoenzyme Cdk9/cyclin T complex, activates RNAPII by phosphorylating the S2 site within the carboxy-terminal domain (CTD). Here, the role of P-TEFb in Mcl-1 maintenance was investigated in MM cells. Anti-apoptotic Bcl-2 family members profiling revealed high Mcl-1 protein levels in virtually all MM lines tested and up-regulation in drug-resistant lines (e.g., btz-resistant PS-R cells). Similar patterns occurred in human MM xenograft mouse models and primary CD138+ MM cells. Ectopic Mcl-1 over-expression strikingly diminished btz lethality, arguing that Mcl-1 is a critical determinant of btz-resistance. All tested MM cell lines, primary MM specimens, and murine xenograft samples displayed constitutive P-TEFb activation i.e., high CTD (S2) phosphorylation, which correlated with a) up-regulation of P-TEFb subunits i.e., Cdk9 (42 and 55kD isoforms) and its partner cyclin T1 (but not T2a/b or K); b) marked 42kD T186 phosphorylation, while drug-resistant cells also displayed sharply increased 55kD T186 phosphorylation; and c) constitutive P-TEFb complex assembly and RNAPII interactions, but little inhibitory association with HEXIM which binds to P-TEFb via 7SK snRNP. The function of P-TEFb in Mcl-1 regulation was then validated by genetic and pharmacological strategies. First, Cdk9 or cyclin T1 shRNA knock-down dramatically inhibited CTD S2 phosphorylation and down-regulated Mcl-1. Second, pan-Cdk inhibitors e.g., dinaciclib rapidly inhibited P-TEFb and down-regulated Mcl-1, leading to apoptosis prior to cell cycle perturbations. These events primarily reflected P-TEFb complex disassembly, but not levels of P-TEFb subunits or T186 phosphorylation. Third, like Mcl-1 knock-down, Cdk9 shRNA or a specific Cdk9 inhibitor significantly restored susceptibility of btz-resistant cells to btz or cfz, and also sensitized them to the BH3-mimetic ABT-737 which does not target Mcl-1. Finally, pan-Cdk inhibitors reduced tumor burden and eliminated human CD138+ cells in the bone marrow of murine models bearing drug-naïve or btz-resistant MM cells. Collectively, these findings implicate constitutive activation of P-TEFb in maintenance of high Mcl-1 levels in MM, and argue that P-TEFb may serve as a rational target to overcome btz-resistance. This study was supported by the MD Anderson SPORE in Myeloma. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Robert Z. Orlowski, Steven Grant. P-TEFb is a therapeutic target in human bortezomib-resistant multiple myeloma cells. [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 4600. doi:10.1158/1538-7445.AM2014-4600

Genetic Modification of T Cells Redirected toward CS1 Enhances Eradication of Myeloma Cells

Our goal is to test whether CS1 could be targeted by chimeric antigen receptor (CAR) T cells to treat multiple myeloma (MM). We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1-CAR T cells to eradicate human MM tumor cells in vitro, ex vivo, and in vivo using orthotopic MM xenograft mouse models. In vitro, compared with mock-transduced T cells, upon recognizing CS1-positive MM cells, CS1-CAR-transduced T cells secreted more IFN-γ as well as interleukin (IL)-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1-CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1-CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival. CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM.

Thymoquinone overcomes chemoresistance and enhances the anticancer effects of bortezomib through abrogation of NF-κB regulated gene products in multiple myeloma xenograft mouse model

Multiple myeloma (MM) is a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow. With the advent of novel targeted agents, the median survival rate has increased to 5 -7 years. However, majority of patients with myeloma suffer relapse or develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of MM. Thus in the present study, we investigated whether thymoquinone (TQ), a bioactive constituent of black seed oil, could suppress the proliferation and induce chemosensitization in human myeloma cells and xenograft mouse model. Our results show that TQ inhibited the proliferation of MM cells irrespective of their sensitivity to doxorubicin, melphalan or bortezomib. Interestingly, TQ treatment also resulted in a significant inhibition in the proliferation of CD138+ cells isolated from MM patient samples in a concentration dependent manner. TQ also potentiated the apoptotic effects of bortezomib in various MM cell lines through the activation of caspase-3, resulting in the cleavage of PARP. TQ treatment also inhibited chemotaxis and invasion induced by CXCL12 in MM cells. Furthermore, in a xenograft mouse model, TQ potentiated the antitumor effects of bortezomib (p<0.05, vehicle versus bortezomib + TQ; p<0.05, bortezomib versus bortezomib + TQ), and this correlated with modulation of various markers for survival and angiogenesis, such as Ki-67, vascular endothelial growth factor (VEGF), Bcl-2 and p65 expression. Overall, our results demonstrate that TQ can enhance the anticancer activity of bortezomib in vitro and in vivo and may have a substantial potential in the treatment of MM.

CS1-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells and T Cells Enhance In Vitro and In Vivo Anti-Tumor Activity Against Human Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by the aberrant clonal expansion of plasma cells (PCs) within the bone marrow (BM). Despite the use of proteasome inhibitors and immune-modulating drugs, which have improved overall survival, MM remains an incurable malignancy for which novel therapeutic approaches are urgently needed. Immunotherapy that specifically targets antigens expressed by MM would be a promising approach to treat MM patients refractory to any current treatments. Chimeric antigen receptors (CARs) are engineered fusion proteins containing tumor antigen-recognition moieties and immune cell activation domains. CAR-expressing T cells have been demonstrated successful in the clinic to treat chronic lymphocytic leukemia (CLL) and acute lymphoid leukemia (ALL). However, the potential utility of antigen-specific CAR-engineered natural killer (NK) cells or T cells to target MM-expressed CS1 to treat MM has not been previously explored, and is the focus of our study.CS1 is a surface glycoprotein and represents an ideal target for the treatment of MM. CS1 is highly, and nearly ubiquitously, expressed on MM cells, while expression remains very low on NK cells, some T-cell subsets, and normal B cells, and also it is almost undetectable on myeloid cells. In addition, monoclonal antibody directed against CS1, elotuzumab, has already been proven safe in phase 1 and 2 clinical trials, and phase 3 trials are ongoing. Therefore, it should be safe to target CS1 for the treatment of MM.We successfully generated a specific CS1-CAR construct with a lentiviral vector backbone, sequentially containing a signal peptide (SP), a heavy chain variable region (VH), a linker, a light chain variable region (VL), a hinge, CD28 and CD3ζ. Flow cytometry analysis with an antibody against VH and VL regions indicated that CS1-CAR was successfully expressed on the surface of NK cells and T cells transduced with the CAR construct. In vitro, CS1-CAR NK cells and T cells displayed enhanced MM cytolysis (detected by Cr51 release assay) and augmented production of cytokines (determined by enzyme-linked immunosorbent assay, ELISA), such as IFN-g for NK and T cells and IL-2 for T cells, when co-cultured with CS1-expressing MM cell lines. These effects relied on CS1-dependent recognition of MM cells because CS1-CAR NK or T cells possessed higher activity when they were co-cultured with CS1-expressing cells, but remained much lower activity when they were co-cultured with CS1-negative cells. However, CS1-CAR NK or T cells launched significantly higher killing of MM cells and secrete more abundant cytokines when the CS1-negative MM cells ectopically overexpressed CS1 and were co-cultured with the CAR cells. Ex vivo, compared to NK or T cells transduced with the empty vector, NK or T cells transduced with CS1-CAR also showed significantly enhanced effector functions when responding to purified primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, when compared to untreated mice or mice treated with empty vector-tranduced NK or T cells, adoptive transfer of 5 × 106 NK or T cells expressing CS1-CAR once every five days efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged survival of mice bearing IM9 MM cells. Our efforts to translate these findings into clinical trials are ongoing.In summary, CS1 is a promising target for using CAR NK or T cells for MM treatment, and we have generated a CAR that recognizes CS1. We demonstrate that NK cells or T cells armed with this CS1-CAR can recognize and eradicate myeloma cells in vitro and in vivo. Autologous or allogeneic transplantation of these CS1-specific CAR NK cells or CAR T cells may be a promising strategy to treat MM. Disclosures:Caligiuri:Innate Pharma: Membership on an entity's Board of Directors or advisory committees. Hofmeister:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Chimeric antigen receptor (CAR)-expressing T cells have been demonstrated successful in the clinic to treat B-lymphoid malignancies. However, the potential utility of antigen-specific CAR-engineered natural killer (NK) cells to treat MM has not been explored. In this study, we determined whether CS1, a surface protein that is highly expressed on MM cells, can be targeted by CAR NK cells to treat MM. We successfully generated a viral construct of a CS1-specific CAR and expressed it in human NK cells. In vitro, CS1-CAR NK cells displayed enhanced MM cytolysis and IFN-γ production, and showed a specific CS1-dependent recognition of MM cells. Ex vivo, CS1-CAR NK cells also showed similarly enhanced activities when responding to primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, adoptive transfer of NK-92 cells expressing CS1-CAR efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged mouse survival. Thus, CS1 represents a viable target for CAR-expressing immune cells, and autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat MM.

Inhibitory effects of gamma secretase inhibitor on human multiple myeloma xenograft mouse model

To explore the tumor growth inhibition of gamma secretase inhibitor MRK003 on human multiple myeloma xenograft mice by inhibition of AKT and Notch1 expression. NOD/SCID mice were injected with human multiple myeloma cell lines RPMI8226 to establish a xenograft mouse model. Mice were randomized into two groups：the experimental group were injected with MRK003 at a dose of 5 mg× kg⁻¹×d⁻¹ for 14 days; the inhibitor was replaced by an equal saline in the control group. Mice were sacrificed by cervical dislocation on the next day after the last injection and tumor tissue was removed to detect the expression of Notch1 and AKT by immunohistochemistry. After subcutaneous injection with RPMI8226, mice had tumor formation in 5-7 days and the largest tumor block in 10-12 days. Before RPMI8226 injection, the mean sizes of tumor block in the experimental and the control groups were 509.2 mm³, 511.2 mm³(P>0.05). 9 days after injection, the mean sizes of tumor tissue in the experimental and the control groups were 636.6 mm³, 691.2 mm³(P<0.01). On the next day after the last injection, the tumor sizes of the experimental and the control groups were 683.5 mm³ and 1798.7 mm³(P<0.01). The size of tumor block in the experimental group was significantly smaller than that of the control group(P<0.01). Immunohistochemistry staining showed that the positive expression rates of Notch1(11.1%, P<0.01) and AKT(13.3%, P<0.01) in experimental group were significantly decreased compared with the control group(Notch1: 95.6%; AKT: 93.3%). Western blot results showed that Notch1 and AKT protein in experimental group were significantly lower than those in the control group. MRK003 could inhibit the tumor growth of human multiple myeloma xenograft mice by downregulated expression of Notch1 signaling pathway.

Galectin-3C Inhibits Tumor Growth and Increases the Anticancer Activity of Bortezomib in a Murine Model of Human Multiple Myeloma

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM.

An Antibody-based Multifaceted Approach Targeting the Human Transferrin Receptor for the Treatment of B-cell Malignancies

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, also known as CD71), which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q, suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition, in 2 disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant antitumor activity, including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies.

Antimyeloma Activity of a Multitargeted Kinase Inhibitor, AT9283, via Potent Aurora Kinase and STAT3 Inhibition Either Alone or in Combination with Lenalidomide

Aurora kinases, whose expression is linked to genetic instability and cellular proliferation, are being investigated as novel therapeutic targets in multiple myeloma (MM). In this study, we investigated the preclinical activity of a small-molecule multitargeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A, Aurora kinase B, and Janus kinase 2/3. We evaluated the in vitro antimyeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a xenograft mouse model of human MM. Our data showed that AT9283 induced cell-growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both Aurora kinase A and Aurora kinase B inhibition, such as increase of cells with polyploid DNA content, decrease in phospho-histone H3, and decrease in phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, Aurora kinase A, or Aurora kinase B showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies showed decreased MM cell growth and prolonged survival in AT9283-treated mice compared with controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells. Enhanced cytotoxicity was associated with increased inhibition of phosphorylated STAT3 and phosphorylated extracellular signal-regulated kinase. Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination therapy for treating patients with MM.

Abstract 3622: An antibody-based multifaceted approach targeting the human transferrin receptor for the treatment of multiple myeloma

Abstract We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1, CD71) that demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is due to its ability to decrease TfR1 levels leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fc gamma receptors and the complement component C1q, suggesting that is capable of eliciting Fc-mediated effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). Additionally, in two disseminated multiple myeloma xenograft mouse models, we show that a single dose of ch128.1Av results in significant anti-tumor activity including long-term survival. Interestingly, the parental antibody without avidin (ch128.1) also shows remarkable in vivo anti-cancer activity despite its lack of in vitro cytotoxicity. Finally, we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the Long-Term Cell-Initiating Culture (LTC-IC) assay suggesting that these important progenitors would be preserved in different therapeutic approaches, including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2011-3622

AT9283, a Small Molecule Multi-Targeted Kinase Inhibitor with Potent Activity Against Aurora Kinase and STAT3 In Combination with Lenalidomide Results In Synergistic Anti-Myeloma Activity

AbstractAbstract 2994Despite recent advances with new drugs such as bortezomib, thalidomide and lenalidomide, multiple myeloma (MM) remains an incurable disease. Used as single agents, these compounds have shown marked antitumor activity, but the number of patients with relapsed and refractory disease remains high. Combination of these agents with other classes of novel drugs would offer great promise to improve patient outcome. AT9283 (Astex therapeutics, Cambridge UK) is a multi-targeted kinase inhibitor that inhibits Aurora A (AURKA), Aurora B (AURKB) and Janus Kinase (JAKs). AURKA and AURKB expression has been correlated with genetic instability and cellular proliferation in MM; therefore, Aurora kinases represent an attractive therapeutic target in MM. In addition the JAK/STAT pathway plays an important role in the survival and proliferation of MM cells. Blocking this pathway may therefore be critical for the survival of MM cells. AT9283 decreased both phospho-Histone H3 and the phosphorylation of Aurora A at Thr 288 in Nocodazole treated cells, suggesting the dual activity of AT9283 against AURKA and AURKB. Importantly, besides Aurora kinase inhibition, we observed that AT9283 inhibited STAT3 tyrosine phosphorylation within 30 minutes of treatment. The effect of AT9283 on pSTAT3 inhibition was further investigated by using U3A cells stably expressing a luciferase reporter gene under the control of a STAT-dependent promoter. AT9283 inhibited STAT3-dependent luciferase activity with an EC50 of approximately 0.125 μ M. Consistent with AT9283 induced cytotoxicity, genetic depletion of STAT3, AURKA or AURKB showed growth inhibition of MM cells, suggesting that AT9283-induced inhibition of these molecules is in part the underlying mechanism of MM cell growth inhibition. In vivo data using a xenograft mouse model of human MM show that mice treated with AT9283 demonstrated slower tumor growth compared to the control group (p=0.018) and prolongation in median overall survival (32 days in treated group versus 18 days in control group; p < 0.0001) without adverse effects. We next evaluated the activity of AT9283 in combination with established MM drugs and strong synergistic effect was found when AT9283 was combined with lenalidomide (Selleck Chemicals LLC, TX, USA) (Combination Index < 0.9). We hypothesized that the synergistic effect of this combination is due to the fact that the two drugs target different pathways and different phases of the cell cycle, thus augmenting their individual anti-myeloma activity. We examined MM cell cytotoxicity of the combination by using AT9283 and lenalidomide at concentrations lower than their maximal cytotoxic concentrations. Increasing doses of AT9283 (0 -0.125 μ M) were added to lenalidomide (0-2μ M) and a significant decrease in viability (as measured by MTT and cell growth as determined by 3H-TdR at 48 h) was observed with combined therapy compared to either agent alone. A significant increase (55.7%) in early and late apoptosis occurred after 72 hours of exposure of cells to combined therapy with associated caspase-8 and PARP cleavage. Combination treatment resulted in downregulation of pSTAT3 and pERK following 4 hours of treatment. Considering the role that the BM microenvironment plays in growth and survival of MM cells, we examined whether the combination of low dose AT9283 plus lenalidomide induced MM cell death in the context of the BM microenvironment. MM.1S cells were cultured with or without BMSCs in the presence or absence of AT9283, lenalidomide or in combination regimen. Combined therapy inhibited 3H-TdR uptake of MM.1S cells cultured in the presence of BMSCs. Interestingly, we observed that AT9283 plus lenalidomide downregulated the expression of the p-STAT3 and p-ERK when MM.1S cells were cultured with BMSCs, highlighting the role of this drug combination in overcoming the protective effect of BMSCs. These results provide the rationale for the clinical evaluation of AT9283 in combination with lenalidomide in MM patients. Disclosures:Squires:3Astex Therapeutics Ltd: Employment. Anderson:MILLENNIUM: Consultancy; CELGENE: Consultancy; NOVARTIS: Consultancy; MERCK: Consultancy; ONYX: Consultancy; BMS: Consultancy. Raje:novartis: Consultancy; celgene: Consultancy; astra zeneca: Research Funding; acetylon: Research Funding.

AT9283, a Small Molecule Multi-Targeted Kinase Inhibitor Induces Antimyeloma Activity Via Potent Aurora Kinase and STAT3 Inhibition.

Abstract 3833Poster Board III-769Aurora kinases, a family of mitotic regulators, whose expression has been recently linked to genetic instability and cellular proliferation in several cancers including multiple myeloma (MM), are being studied as novel mitotic therapeutic targets. Aurora A plays a crucial role in centrosome separation and spindle assembly and is required for mitosis and bipolar mitotic spindle formation. Aurora B, a member of the chromosomal passenger complex, is required for chromosome segregation, spindle assembly checkpoint and cytokinesis. Both aurora kinase A and B are significantly overexpressed in MM cells prompting the investigation of aurora kinase inhibitors as a therapeutic strategy in MM. Here, we investigated the preclinical activity of a small molecule multi-targeted inhibitor, AT9283, with potent in vitro kinase activity against aurora A and B kinases (3 nM), JAK2 and 3 (at 1.2 and 1.1 nM, respectively) and Abl T315I (at 4 nM). Growth inhibitory effects of AT9283 on MM cell lines and patient derived cells was observed with IC50 values of 0.25μM -0.5 μM at 48 hours using a [3H]-thymidine incorporation assay. Cell cycle analysis following AT9283 treatment resulted in increased G2/M phase and polyploidy consistent with failed cytokinesis (associated with aurora kinase B inhibition) confirmed by immunofluorescence assay. This was followed by induction of apoptosis assessed by Annexin V+PI+ staining peaking at 48 - 72 hours with associated caspase-8/-9 cleavage. The cellular inhibition of aurora kinase activity by AT9283 was confirmed by evaluating the phosphorylation of histone H3 at serine-10, a direct downstream substrate of aurora B kinase. Pretreatment of MM.1S cells with nocodazole, known to induce maximal phosphorylation of histone H3 by causing an M-phase block, resulted in decreased levels of phosphorylated histone H3 after AT9283 treatment suggesting the role of aurora B kinase inhibition by AT9283. Importantly, in addition to aurora kinase inhibition, we observed that AT9283 also inhibited signal transducer and activator of transcription (STAT3) tyrosine phosphorylation in MM cells within 30 minutes of treatment. Janus Kinase (JAK)2/STAT3 pathway is one of the major signaling cascades activated by gp130 family member cytokines that promotes MM cell survival. The effect of AT9283 on pSTAT3 inhibition was further investigated by using U3A cells stably expressing a luciferase reporter gene under the control of a STAT-dependent promoter. AT9283 inhibited STAT3-dependent luciferase activity with an EC50 of approximately 0.125 μM suggesting that STAT3 was functionally inhibited by AT9283. Since MM cell lines with the constitutive STAT3 tyrosine phosphorylation were more sensitive to AT9283, ongoing studies are aimed at understanding whether AT9283-induced effects on the JAK/STAT pathway enhances the efficacy of the aurora kinase inhibition in the context of MM. Finally, in vivo data using a xenograft mouse model of human MM show that mice treated with AT9283 demonstrated slower tumor growth compared to the control group without adverse effects. Our results show pleiotropic effects of AT9283 in MM and warrant further study to determine its suitability for clinical evaluation in MM. Disclosures:Squires:Astex Therapeutics, Ldt: Employment. Yule:Astex Therapeutics Ldt: Employment. Anderson:Novartis, Millennium, Celgene: Consultancy, Honoraria, Research Funding. Raje:Celgene, Norvartis, Astrazeneca: Research Funding.

A Novel Heat Shock Protein (HSP) 90 Inhibitor KW-2478 shows Activity in B-Cell Malignancies in Vitro and in Vivo.

Background: HSP90 plays an important role in chaperoning key proteins implicated in malignant disease and is a promising therapeutic target. We now report the in vitro and in vivo activity of a novel HSP90 inhibitor of non-ansamycin, non-purine analogue class, KW-2478, (Kyowa Hakko Kirin) in B-cell malignancies including multiple myeloma (MM), B-cell lymphoma (BCL) and mantle cell lymphoma (MCL) cells, and in primary tumour cells from MM and BCL patients.Procedures: The binding affinity of KW-2478 to HSP90 was examined using immobilised human HSP90a and a biotinylated HSP90 binding agent, radicicol (bRD). The effect of KW-2478 on cell viability, cell growth and apoptosis induction were evaluated in cell lines, with KW-2478 induced changes in major HSP90 client proteins studied by Western blotting analysis. The in vivo anti-tumour activity of KW-2478 was evaluated in a human MM xenograft mouse model,. Primary MM cells were studied using a co-culture system with the HS-5 bone marrow stromal cell line (BMSCs), while primary BCL samples were cultured on CHO cells stably transfected to produce CD40L.Results: KW-2478 inhibited the binding of bRD to HSP90α in concentration-dependent manner with an IC50 value of 3.8 nM. KW-2478 clearly inhibited cancer cell growth in all cell lines, with EC50 values from 101–252 nM in BCL, 81.4–91.4 nM in MCL and 120–622 nM in MM. The drug also exhibited potent growth inhibitory activity in primary CLL (n=3) and NHL (n=2) cells with EC50 values of 40–170 nM and 200–400 nM, respectively. In 2 of 4 human primary myeloma cells, KW-2478 at 2 μM inhibited cell growth by at least 50%. The presence of BMSCs did not affect drug activity against primary MM cells and importantly there was little or no effect on cell number or viability of normal BMSCs at up to 20 μM KW-2478. Exposure of MM and BCL cell lines to KW-2478 for 24 hours resulted in the degradation of HSP90 client proteins (IGF-1Rβ and Raf-1) and the induction of HSP70. KW-2478 also induced PARP cleavage and dephosphorylated Erk1/2 in NCI-H929 cells. Further studies in selected cell lines showed that exposure to 1 μM KW-2478 or lower resulted in the depletion of p53 and Akt proteins, a reduction in nuclear NFKB, and the cleavage of caspase-3. In the NCI-H929 xenograft model, KW-2478 (qd×5, i.v.) showed a statistically significant suppression of tumour growth at the doses of 25, 50, 100 and 200 mg/kg. Moreover, tumour regressions were observed at doses of 100 and 200 mg/kg, with a significant decrease in serum M protein concentration at doses of 50, 100 and 200 mg/kg. No severe KW-2478 toxicity was observed as assessed by treatment-related mortality and body weight change.Conclusions: The novel HSP90 inhibitor KW-2478 showed a potent anti-tumour activity both in vitro and in vivo, including activity in primary patient samples. The agent retained its activity in primary myeloma cells in the presence of BMSCs, suggesting that KW-2478 can overcome the protective effect of the bone marrow microenvironment. Additional pharmacokinetic and safety data support the further development of KW-2478 and the drug is currently undergoing clinical evaluation in a phase I trial.