Model Of Human Multiple Myeloma Research Articles

The effect of selective inhibition of HDAC6 with ACY1215 on bortezomib activity in multiple myeloma (MM).

e18569 Background: HDACs are being studied as novel therapeutic targets in MM. Here, we investigated the preclinical activity of an HDAC6 selective inhibitor ACY1215 in MM, alone and in combination with bortezomib (BZ). Methods: In vitro enzyme assays were used to evaluate the selectivity of ACY1215. The cytotoxicity of ACY1215 versus pan-HDAC inhibitor (e.g. SAHA) were compared in MM cell lines, MM patient cells, MM osteoblasts (OB) and osteoclasts (OC), as well as peripheral blood mononuclear cells (PBMCs) from healthy donors. Western blotting and RT-PCR were used to identify the mechanisms of cell death. The activity of ACY1215 and BZ was confirmed in vivo using two different murine xenograft models of human MM, plasmacytoma model and disseminated MM model. Results: ACY1215 showed more potent inhibitory activity against HDAC6 (EC50 0.0054 µM) than SAHA. Like SAHA, ACY1215 triggered potent MM cytotoxicity but showed less toxicity than SAHA against PBMCs and activated T-cells. Low doses of ACY1215 with BZ showed synergistic anti MM activity, resulting in apoptosis via caspase-8,-9,-3 and PARP activation. Moreover, ACY1215 increased BZ-induced ER stress in MM cells, evidenced by induction of UPR components including PERK, CHOP and p-IRE1α. ACY1215, alone and with BZ, enhanced OB differentiation (increased alkaline phosphatase enzyme activity) and increased mRNA expression of β-catenin, osteocalcin, and Runx2 (OB markers). Importantly, both treatments inhibited OC differentiation (reduced number of multinucleated cells staining for TRAP). In vivo studies confirmed decreased human MM cell growth and prolonged host survival after combination ACY1215 and BZ therapy versus controls (34 vs 22 days, n=7, p<0.0011, in plasmacytoma model and 40 vs 17 days, n=12, p<0.0001, in disseminated model), without significant adverse effects. PK/PD studies showed ACY1215 plasma levels (2133 ng/ml) at 1 hr following treatment. Immunohistochemistry on excised human MM demonstrated increased in acetylated-α tubulin, a biomarker of HDAC6 inhibition, stained cells 4 hrs after ACY1215 treatment. Conclusions: These results provide the rationale for clinical evaluation of ACY1215, alone and together with BZ, in the treatment of MM.

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 660: Inhibition of centrosomal clustering suppresses tumor growth in vivo

Abstract Tumor cells frequently contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles (centrosomal clustering). We recently described a phenotype-based small molecule screening strategy directed to induce tumor cells with supernumerary centrosomes to undergo multipolar mitoses, thereby resulting in apoptotic cell death (Rebacz et al., Cancer Res 2007; 67: 6342-6350). We here describe the novel small molecule GF-15, a derivative of griseofulvin and a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis. We tested more than 25 cancer cell lines from hematologic malignancies (including acute and chronic leukemias, lymphomas), solid tumors (including glioblastoma, colon, cervix, and pancreatic cancers) and a wide array of myeloma cell lines. We found mean inhibitory concentrations (IC50) for proliferation and survival in the range of 1-5μM GF-15, associated with activation of caspases 8, 9, and 3. As expected, tumor cell lines displaying only limited centrosomal aberrations or microsatellite instability were less sensitive to treatment with GF-15. Importantly, non-malignant cells without supernumerary centrosomes including PBMCs, immortalized hepatocytes, and bone marrow stromal cells, did not reach their IC50 even at 30μM GF-15. Specifically, cell cycle analysis of synchronized myeloma cells showed marked G2/M arrest, followed by a dramatic increase of the sub-G1 fraction, after treatment with GF-15. In addition, treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered myeloma cell migration. Pharmakodynamic studies in vivo revealed rapid renal clearance of GF-15 and metabolites within 6 hours p.i. No acute or chronic toxicity was observed. Finally, both intraperitoneal and oral GF-15 treatment of murine xenograft models of human multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival associated with significant increase of aberrant mitoses in vivo. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of the first in class small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and strongly support its clinical evaluation to improve patient outcome in both hematologic malignancies and solid tumors. 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 660. doi:10.1158/1538-7445.AM2011-660

Highly specific and reliable biomarker of human MM cells in NOD/SCID animal models through the cancer/testis antigen AKAP-4: new opportunities for pharmacological studies (165.17)

Abstract Despite recent findings for the treatment of multiple myeloma (MM) it still remains a deadly disease. In the effort to develop new and more effective therapies for human tumors, murine animal models have been proven critical for a variety of neoplasias. Unfortunately, although recent studies have described different animal models of human MM, none is available for tracking the tumor progression with a specific biomarker employing human MM cells. This lack hampers the development of new approaches for treating MM. Here we describe a human MM model in NOD/SCID mice that allows for highly sensitive and specific monitoring of disease progression through the endogenously-expressed tumor specific cancer/testis antigen AKAP-4. The human MM cells U266 were subcutaneously injected in NOD/SCID animals. AKAP-4 expression resulted to be a reliable and sensitive antigen to monitor tumor growth and spread by different and independent techniques, namely flow-cytometry, E.L.I.S.A., Western blot and RT-PCR. The relevance of our findings stems from the suitability of this innovative model for a thorough pre-clinical evaluation of experimental pharmacological strategies for the treatment of human MM.

Synergistic Anti-Myeloma Effects of the Lyn Kinase Inhibitor INNO-406 In Combination with Doxorubicin, Melphalan and Bortezomib

AbstractAbstract 5015Lyn is a member of the Src kinase (SFK) family and controls the activation threshold of multiple signaling pathways in different types of cells, including B cells (Yamanashi et al., 1989, PNAS). Multiple myeloma (MM) is a hematological malignancy of plasma cells. In MM, Lyn is involved in IL-6-induced cell proliferation (Hallek et al., 1997, Exp Hematol). As one of the most important growth and anti-apoptotic factors for MM cells, both the IL-6/STAT3 and IL-6/ERK pathways have been shown to promote MM cell proliferation and prevent apoptosis. However, STAT3 and ERK activation is not sufficient for IL-6-induced proliferation, which further requires the activation of SFK (Ishikawa et al., 2002, Blood). IL-6-induced proliferation of U266 myeloma cells has been reported to be significantly suppressed following exposure to Lyn-specific antisense oligonucleotides or a Src kinase inhibitor (Ishikawa et al., 2002, Blood). Therefore, a Lyn specific inhibitor, either alone or in combination with other anti-MM drugs, may be a more effective regimen for the treatment of MM. INNO-406 is a novel inhibitor of ABL as well as Lyn. In laboratory-based studies, it has shown anti-tumor activity in hematological malignancies, especially in leukemia cells harboring BCR-ABL mutations (Kimura et al., 2005, Blood). Since Lyn is involved in MM cell proliferation, targeting Lyn may be an alternative therapeutic approach and improve the efficacy of other anti-MM agents. Thus, we conducted this study in order to determine the anti-MM effects of INNO-406. First, Western blot analyses demonstrated that Lyn was expressed in all MM cells analyzed, including bone marrow (BM) mononuclear cells derived from MM patients and MM cells from LAGκ1A, LAGκ1B and LAGκ2 xenografts, three of our unique in vivo SCID-hu models of human MM, which were originally developed from MM patient BM biopsies and have been maintained in SCID mice. However, Lyn activation, as demonstrated by phosphorylation of its downstream molecule HS1, was infrequently found in MM cells. INNO-406 inhibited the growth of the three MM cell lines tested including RPMI8226, U266 and MM1S. Interestingly, the U266 cell line is most resistant to INNO-406 among the three MM cell lines tested although its Lyn activation and expression level is the highest. Apoptosis analysis using flow cytometric analysis following Annexin V staining demonstrated that INNO-406 induced apoptosis in MM cells. When combined with melphalan, doxorubicin or bortezomib, INNO-406 synergistically inhibited MM cell growth. We are currently studying the molecular mechanisms of INNO-406's anti-MM effect, either alone or in combination treatment, and validating our in vitro findings using our unique in vivo mouse models of human MM. Updated data from these additional studies will be reported at the meeting. Disclosures:No relevant conflicts of interest to declare.

Preclinical Development of Small-Molecule CRM1 Inhibitors as Novel Therapy for the Treatment of Myeloma and Other Hematological Malignancies

AbstractAbstract 3012 Background:CRM1 (XPO1) is a key nuclear export protein which controls multiple tumor suppressor proteins (TSP) and cell proliferation pathways including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Further, mislocalization of proteins can abrogate TSP functions and render chemotherapies ineffective. For example, multiple myeloma (MM) cells that are grown at high densities (to mimic the in vivo situation) become resistant to topoisomerase 2 (topo2) inhibitors (e.g., doxorubicin) simply because topo2α is exported from the nucleus. Forcing the nuclear expression of chemotherapy targets, TSP and growth regulatory proteins by CRM1 inhibition can restore drug sensitivity and restore checkpoint control/genome surveying functions. These events lead to apoptosis or autophagy in cancer cells while sparing normal cells. Methods:CRM1 inhibitors were synthesized and nuclear distribution studies were performed in U2OS cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in multiple myeloma (MM), leukemia and lymphoma cell lines and in human peripheral blood mononuclear cells (PBMCs). Toxicology studies were performed in several mouse strains. Antitumor activity is assessed in a xenograft model of human MM.1 cells growing in scid-mice. Results:The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ~300 nM. KPT-0127 is selectively cytotoxic to various hematological cell lines with EC50s in the 0.02–1.0 μM range, and shows limited cytotoxicity in similar studies in normal cell lines (NIH-3T3, MRC5, HUVECs) and PBMCs (EC50 >5-20 μM). KPT-0127 increases the nuclear localization of IkB in HUT-78 leukemia cells and human PBMCs, and inhibits TNF-α secretion in LPS stimulated U937 macrophage derived cells, likely through inhibition of NF-kB signaling. In MM cells grown at high densities which actively export topo2 to the cytoplasm via CRM1, blocking CRM1-dependent transport strongly enhanced topo2 nuclear localization and augmented the apoptotic effects of doxorubicin in <24 hours showing clear synergy between KPT-0127 and the anthracycline. Combination of sublethal concentrations of bortezomib plus KPT-0127 in MM1.S and MM.1R myeloma cells (as well as in Jurkat and HS-Sultan) induced synergistic cytotoxicities. In mice, KPT-0127 given by subcutaneous (SC) injection of 30–100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours. In 5 day repeated dose toxicology studies, SC administration of 100 mg/kg KPT-0127 (QD × 5) was well tolerated in mice with no cutaneous or obvious systemic clinical findings. Modest neutrophilia and mild lymphopenia were seen and no neurologic signs resulted from treatment with KPT-0127. Administration of KPT-0127 SC to mice bearing HCT-116 colon cancer results in dose-dependent antitumor activity. Xenograft studies with MM.1 myeloma cells are underway and will be presented at the meeting. Conclusions:The sensitivity of tumors with multiple TSP and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, to killing with KPT-0127 likely reflects the ability to affect multiple critical and non-redundant regulatory pathways. CRM1 inhibition also forces topo 2 to remain in the nucleus, and increases levels of nuclear IkB antagonizing NF-kB function, thereby reducing the likelihood of resistance development. These results support the development of small molecule, drug-like CRM1 inhibitors for the treatment of MM and other hematological cancers. IND-enabling CMC and toxicology work are expected to begin in early 2011. Disclosures:Shacham:Karyopharm: Employment, Equity Ownership, Patents & Royalties. Nir:Karyopharm: Consultancy. Draetta:Karyopharm: Consultancy. Sandanayaka:Karyopharm: Employment. Shechter:Karyopharm: Employment. Kauffman:Karyopharm: Consultancy, Equity Ownership, Patents & Royalties.

SCID-Synth-Hu: a Novel Multiple Myeloma Model for In Vivo Expansion of Primary Cells

AbstractAbstract 452The critical role of the human bone marrow microenvironment (HuBMM) in the pathogenesis of Multiple Myeloma (MM) has recently allowed the design of novel therapeutical approaches targeting not only MM cells but also their specific HuBMM. However, the lack of adequate mouse models, capable to recapitulate a HuBMM, has restrained large scale in vivo screening of investigational drugs. In fact, only the SCID-hu model, in which human MM cells are grown in human fetal bone chips previously implanted in SCID mice, addresses this specific requirement. However the poor availability of human fetal bone chips, the allogeneic nature of the fetal BM milieu versus MM cells and the heterogeneity of implanted human bone chips are important restrains of this system. Here we report the development of a novel in vivo model of human MM (SCID-synth-hu), which is based on the implant of a three-dimensional (3-D) poly-ε-caprolactone polymeric scaffold (PCLS) into SCID mice as recipient to allow growth of MM cells in a reconstituted HuBMM. This biosynthetic scaffold has been designed to resemble the micro-architecture of a normal human adult bone and was characterized by 3-D interconnected large and small pores suitable for engraftment of bone marrow-derived cells. Human bone marrow stromal cells (BMSCs) were collected from BM aspirates from MM patients and firstly used for coating 3D internal surface of PCLSs. We performed in vitro dynamic seeding of BMSCs into PCLSs using a suspension of 106 cells in 500 μl of growth medium. Before implantation, PCLSs were incubated in complete medium at 37°C, in 5% CO2 for 24 hours, in order to allow cell adhesion on 3D surfaces. Then, PCLSs were implanted subcutaneously into SCID mice. CD138+ immune-selected primary MM cells, obtained by MM patient with a different disease status, were directly injected into PCLSs, which have been previously coated with allogeneic BMSCs 2–3 weeks after the in vivo implant. By this experimental approach, we achieved engraftment of primary MM cells in PCLSs within a non autologous bone marrow milieu. In a subsequent series of experiments, bone marrow mononuclear cells (BMMCs), obtained by Ficoll gradient separation and containing primary unselected CD138+ and their autologous BMSCs, were directly seeded in vitro into PCLSs which were implanted in SCID mice after 24 hours of incubation. At different time points, H&E and CD138 or κ/λ staining demonstrated engraftment and filling of 3-D spaces by primary MM cells within the autologous bone marrow milieu in PCLSs retrieved from SCID-synth-hu mice. Neo-synthesized extracellular matrix and angiogenesis were also shown by H&E and immune histochemical staining in retrieved PCLSs. Angiogenesis mostly occurred within areas of MM infiltration, suggesting its role in our system. In vivo MM growth was monitored by ELISA measuring of human monotypic immunoglobulins (Igs) in mouse sera 4 to 10 weeks after cell injection. To demonstrate the usefulness of our SCID-synth-hu model as an experimental platform for in vivo testing of investigational drugs, mice bearing human MM implants were treated intraperitoneally with bortezomib plus dexamethasone (Bort+Dex). As expected, SCID-synth-hu mice treated with Bort+Dex exhibited a significant decrease of monotypic light chains in mice sera and induction of apoptosis of MM cells in retrieved PCLSs, as compared to untreated control mice. Our experimental findings demonstrate that the SCID-synth-hu is the first experimental system which allows the in vivo expansion of human primary MM cells within their autologous adult HuBMM. The unlimited availability and the low cost of PCLSs, as well as the potential for dissecting the biological events within the HuBMM, represent a clear improvement over the available preclinical in vivo models of MM. We conclude that the SCID-synth-hu is a unique tool for large scale in vivo preclinical screening of novel agents targeting MM in its autologous HuBMM, and a novel resource for translational research in the experimental treatment of this still incurable disease. Disclosures:No relevant conflicts of interest to declare.

Bortezomib Induced BRCAness Sensitizes Multiple Myeloma Cells to PARP Inhibitors

AbstractAbstract 789 Background:Poly-ADP-ribose-polymerase (PARP) inhibitors are cytotoxic to tumor cells with impaired DNA damage repair machinery (DRR), in particular those with a deficient homology directed repair (HR) of DNA double stranded breaks (DSB). Multiple Myeloma (MM) cells are characterized by a highly unstable genome and while the exact mechanisms for this karyotypic instability is largely unknown, their DDR machinery is thought to be highly stressed. The ubiquitin-proteasome system (UPS) is involved in the regulation of several cellular functions including DDR and in particular HR. In addition proteasome inhibitors are reported to induce an unfolded protein response (UPR) in MM cells resulting in their apoptotic death. We have postulated that inhibition of the 26S proteasome also alters the DNA-DSB repair machinery leading to a BRCAness state in MM cells, sensitizing them to PARP inhibitors. Methods and results:In order to biochemically inhibit PARP in MM cells, we used a novel selective inhibitor of PARP1 and PARP2, 2-(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide or ABT-888. We first demonstrated inhibition of PARP activity as measured by a reduction in poly-ADP-ribose (PAR) polymer levels (western blotting) in human MM cell lines (MM1S, U266, H929, RPMI8226, KMS-11, OPM2, INA-6) treated with ABT-888 (5 μM). PARP inhibition and the reduction of PAR levels resulted in DNA damage as evidenced by ATM phosphorylation and induced DNA-DSBs with increased γH2AX (phospho-Ser139-H2AX) levels within 6–12 hours of MM cells treatment with ABT-888. Increased γH2AX foci formation was also detected by immunofluorescent staining within 6–12 hours of ABT-888 treatment and nearly fully resolved by 24 hours, consistent with repair of resultant DNA-DSBs. As expected treatment with ABT-888 alone had no effect on the viability of MM cells consistent with their ability to repair DNA-DSBs resulting from PARP inhibition.We then examined the effect of bortezomib on HR-mediated repair of DNA-DSBs, in particular on the BRCA/FA pathway. A significant reduction of MM cells' FANCD2, BRCA1, BRCA2 and RAD51 mRNA levels (qRT-PCR) was observed within 6–12 hours of bortezomib treatment (10 nM). Similar results were observed at the protein level indicating that bortezomib impedes homology-directed DNA-DSBs repair and results in an operational BRCAness state in MM cells. Therefore, we next tested whether this bortezomib-induced BRCAness was sufficient to sensitize MM cells to PARP inhibition with ABT-888. Consistent with our hypothesis, we observed that co-treatment of MM cell lines with bortezomib and ABT-888 lead to persistent and increased γH2AX foci at 24 hours compared to treatment with ABT-888 alone. Co-treatment also significantly potentiated cell death (Annexin V/PI staining) compared to treatment with bortezomib alone. Similar results were observed in CD138+ primary MM cells (n=8) with strong synergistic effect (CI < 1) between bortezomib and ABT-888. Importantly, no impaired viability (Annexin/PI staining) or function (colony forming unit assay) was noted for CD138− cells or CD34+ peripheral blood stem cells after bortezomib and ABT-888 co-treatment. Mechanistic studies have also shown that apoptotic events (caspase 3, caspase 8 and PARP cleavage) are markedly enhanced by this combination.Based on our in vitro data, we evaluated in vivo the activity of ABT-888 in combination with bortezomib in a Scid murine xenograft model of human MM. Significant inhibition of tumour growth (p<0.005) was noted in mice treated with the combination of bortezomib and ABT-888 compared to bortezomib alone or control-treated mice. This tumour growth inhibition also resulted in a significant increase in survival (p<0.05) of the animals. No toxicity (e.g. weight loss, ruffled coats, paralysis, etc.) was observed in mice treated with the combination. Induction of DNA-DSBs was also confirmed in vivo as shown by an increase in 53BP1 and γH2AX foci formation in tumors of mice treated with the combination compared to bortezomib alone. Conclusion:Our studies indicate that bortezomib induces a BRCAness state in MM cells by impairing HR-mediated repair of DNA-DSBs and results in a contextual synthetic lethality when combined with the PARP inhibitor ABT-888. These data provide the scientific basis for the future clinical testing of PARP inhibitors in combination with proteasome inhibitors for the treatment of MM. Disclosures:No relevant conflicts of interest to declare.

Multiple Myeloma (MM) Highly Expresses CGEN-928 and the Anti-CGEN-928 TM21 Antibody Markedly Inhibits the Growth of MM Cells and Enhances the Anti-Myeloma Effects of Dexamethasone, Melphalan and Bortezomib

AbstractAbstract 4067Although patients with multiple myeloma (MM) initially respond to current treatment modalities, it remains an incurable disease. Many new therapeutic options have become available during the past several years but nearly all patients develop resistance to currently available therapeutic options. In addition, there is no tumor marker that is uniformly expressed in all MM cells although CD138 is considered to be present on the surface of tumor cells in most cases of MM but generally is only present in a subset of the patients' tumor population and may be absent in the most resistant part of the tumor clone. In order to address these unmet clinical needs, we queried Compugen's MED Platform, an expression database which covers over 40,000 microarray experiments, for genes overexpressed in B cell-derived malignancies including MM and that exhibit low expression levels in normal cells and tissues. One of the most prominent candidates was CGEN-928, which was validated as over-expressed in MM at the mRNA level using an independent panel of both hematological malignancies and normal tissues. In this study, we first investigated whether the previously unidentified membrane antigen CGEN-928 is expressed in cells from human MM cell lines, human MM xenografts and fresh bone marrow (BM) aspirates derived from MM patients using flow cytometric analysis and immunohistochemical staining with the anti-CGEN-928 TM21 polyclonal antibody (Compugen Ltd, Tel Aviv, Israel). Using this antibody, we found that CGEN-928 was highly expressed in cells from the MM1s, U266 and RPMI8226 MM cell lines. Next, we examined CGEN-928 antigen expression in fresh tumor cells from BM aspirates from 17 MM patients and also showed high expression of CGEN-928. Notably, expression of this antigen was not only found on CD138+ MM cells but also on MM tumor cells lacking CD138 expression. We also examined the expression of CGEN-928 using our human MM xenograft models LAGκ-1A (bortezomib-sensitive), LAGκ-1B (bortezomib-resistant) and LAGλ-1 (melphalan-resistant). The bortezomib-sensitive MM tumor LAGκ-1A expresses CD138 whereas the bortezomib-resistant version LAGκ-1B developed from the same patient after the patient developed bortezomib reisistance does not express CD138. Cells from all three tumor types showed high levels of reactivity with the TM21 antibody. Similar to the fresh MM BM samples, CGEN-928 expression was not only found on CD138+ MM cells but also on CD138- tumor cells derived from these human MM xenografts. Because this molecule is highly expressed on MM cells, we hypothesized that the anti-CGEN-928 antibody may show anti-MM effects and enhance the anti-MM effects of other anti-MM drugs. To evaluate this, we examined the effect of the TM21 antibody alone and in combination with dexamethasone, melphalan and bortezomib in vitro using cell proliferation MTT assays. Anti-TM21 polyclonal antibody (100 mg/ml) decreased MM tumor cell proliferation and increased apoptosis in cells from the MM1s, RPMI8226 and U266 cell lines. Next, we determined the effects of combining the anti-CGEN-928 antibody with bortezomib, melphalan or dexamethasone on MM1s cells. Cell proliferation assays demonstrated marked enhanced anti-proliferative effects when CGEN-928 antibody at concentrations of 5, 10, 50 and 100 mg/ml was combined with bortezomib, melphalan or dexamethasone. Further investigations are defining the mechanisms and signal transduction pathways that produce the anti-MM effects of CGEN-928. These preliminary studies suggest that the CGEN-928 antigen is highly expressed in MM and treatment with an anti-CGEN-928 polyclonal antibody produces anti-MM effects alone and in combination with other anti-MM agents; and thus, this antigen may be a target for the treatment of multiple myeloma. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures:Levy:Compugen Ltd.: Employment. Dassa:Compugen Ltd.: Employment. Cojocaru:Compugen Ltd.: Employment. Berenson:Compugen Ltd.: Research Funding. Levine:Compugen Ltd.: Employment.

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.

PI3K/p110δ is a novel therapeutic target in multiple myeloma

In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110delta signaling in multiple myeloma (MM). Knockdown of p110delta by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110delta specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110delta potently induced autophagy. The in vivo inhibition of p110delta with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110delta is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.

Elevated IL-17 produced by Th17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma

AbstractElevated cytokines in bone marrow (BM) micro-environment (interleukin-6 [IL-6], transforming growth factor-beta [TGF-β], and IL-1β) may play an important role in observed immune dysfunction in multiple myeloma (MM). As IL-6 and TGF-β are important for the generation of T-helper 17 (TH17) cells, we evaluated and observed a significantly elevated baseline and induced frequency of Th17 cells in peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells (BMMCs) from MM patients compared with healthy donors. We observed significant increase in levels of serum IL-17, IL-21, IL-22, and IL-23 in blood and BM in MM compared with healthy donors. We also observed that myeloma PBMCs after TH17 polarization significantly induced IL-1α, IL-13, IL-17, and IL-23 production compared with healthy donor PBMCs. We next observed that IL-17 promotes myeloma cell growth and colony formation via IL-17 receptor, adhesion to bone marrow stromal cells (BMSCs) as well as increased growth in vivo in murine xenograft model of human MM. Additionally, we have observed that combination of IL-17 and IL-22 significantly inhibited the production of TH1-mediated cytokines, including interferon-γ (IFN-γ), by healthy donor PBMCs. In conclusion, IL-17–producing Th17 cells play an important role in MM pathobiology and may be an important therapeutic target for anti-MM activity and to improve immune function.

Systemic Therapy of Disseminated Myeloma in Passively Immunized Mice Using Measles Virus-infected Cell Carriers

Multiple myeloma (MM) is bone marrow plasma cell malignancy. A clinical trial utilizing intravenous administration of oncolytic measles virus (MV) encoding the human sodium-iodide symporter (MV-NIS) is ongoing in myeloma patients. However, intravenously administered MV-NIS is rapidly neutralized by antiviral antibodies. Because myeloma cell lines retain bone marrow tropism, they may be ideal as carriers for delivery of MV-NIS to myeloma deposits. A disseminated human myeloma (KAS 6/1) model was established. Biodistribution of MM1, a myeloma cell line, was determined after intravenous infusion. MM1 cells were found in the spine, femurs, and mandibles of tumor-bearing mice. Lethally irradiated MM1 cells remained susceptible to measles infection and transferred MV to KAS 6/1 cells in the presence of measles immune sera. Mice-bearing disseminated myeloma and passively immunized with measles immune serum were given MV-NIS or lethally irradiated MV-NIS-infected MM1 carriers. The antitumor activity of MV-NIS was evident only in measles naive mice and not in passively immunized mice. In contrast, survivals of both measles naive and immune mice were extended using MV-NIS-infected MM1 cell carriers. Hence, we demonstrate for the first time that systemically administered cells can serve as MV carriers and prolonged survival of mice with pre-existing antimeasles antibodies.

The Novel Proteasome Inhibitor MLN9708 Demonstrates Efficacy in a Genetically-Engineered Mouse Model of DeNovo Plasma Cell Malignancy.

Abstract 3849Poster Board III-785 IntroductionThe first-in-class proteasome inhibitor VELCADË (bortezomib) is a critical component of chemotherapeutic strategy in treating multiple myeloma (MM), a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic lesions. However, patients eventually develop bortezomib resistance, highlighting the continuous challenge to further improve myeloma therapy. We have recently identified a novel proteasome inhibitor MLN9708 that shows greater antitumor activity than bortezomib in a number of preclinical xenograft models. Here we describe antitumor activity of MLN9708 in a genetically engineered mouse model of human MM. In this model, neoplastic plasma cell development is driven by deregulated expression of Myc (c-myc, myelocytomatosis oncogene) and Bcl-x (official gene name: Bcl2l1; encodes the anti-apoptotic Bcl-XL oncoprotein). This is the first preclinical in vivo study of proteasome inhibitors employing a transgenic mouse model of human MM in which plasma cell neoplasms develop de novo. MaterialsUpon exposure to aqueous solutions or plasma, MLN9708 immediately hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all preclinical studies described below. MethodsWe have previously demonstrated that intercrossing C57BL/6 mice carrying the iMycCa transgene (insertion of Myc in the immunoglobulin heavy-chain locus, Igh) with FVB/N mice carrying the 3'KE-Bcl-XL transgene (enforced expression of Bcl-x driven by the immunoglobulin light-chain 3' k enhancer and Vk21 promoter) produces double transgenic F1 hybrid iMycCa/Bcl-XL mice that develop plasma cell malignances with short onset (135 days on average) and full penetrance (100% tumor incidence; J. Clin. Invest. 113:1763-1773, 2004). These double transgenic iMycCa/Bcl-XL mice develop hypergammaglobulinemia and tissue plasmacytosis by 6-8 weeks of age and rapidly succumb to malignant plasma cell tumors by less than 200 days of age. Preliminary studies based on 11 tumor-bearing iMycCa/Bcl-XL mice showed this form of plasma cell malignancy recapitulates important features of human MM, including serum paraproteins, infiltration of bone marrow with malignant plasma cells, osteolytic lesions and myeloma-like global gene expression profiles (Cancer Res. 67:4069-4078, 2007). Here we used the iMycCa/Bcl-XL mouse model of human MM to assess the antitumor activity of bortezomib and MLN9708 in a preclinical setting. ResultsUntreated double transgenic iMycCa/Bcl-XL mice (n=30) invariably developed plasma cell tumors with short onset (median tumor-free survival = 112 days) and showed marked elevations in plasma immunoglobulin IgM, IgG1, IgG2a and IgG2b levels. To assess antitumor activity of bortezomib and MLN2238, 9-week-old iMycCa/Bcl-XL mice were treated intravenously (IV) twice per week (BIW) with bortezomib (1.2 mg/kg) or MLN2238 (18 mg/kg) for 6 consecutive weeks. These doses represent the maximum tolerated dose (MTD) for each drug as previously determined in normal C57BL/6 x FVB/N F1 hybrid mice. Treatment of iMycCa/Bcl-XL mice with bortezomib (n=30) significantly prolonged tumor-free survival compared to untreated controls (median tumor-fee survival = 139 days; hazard ratio = 0.116; 95% CI = 0.057 to 0.235; p<0.0001). Importantly, treatment of iMycCa/Bcl-XL mice with MLN2238 (n=30) also significantly prolonged tumor-free survival relative to untreated controls (median tumor-free survival = 148 days; hazard ratio = 0.144; 95% CI = 0.071 to 0.289; p<0.0001). Immunoglobulin levels, overall tumor burden, osteolytic lesions (mCT) and biochemical parameters of myeloma bone disease after treatment with bortezomib and MLN2238 will be presented. ConclusionThe novel proteasome inhibitor MLN9708 demonstrates striking antitumor activity in a genetically engineered mouse model of human MM, significantly prolonging tumor-free survival of double transgenic iMycCa/Bcl-XL mice. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures:Janz:Millennium – The Takeda Oncology Company: Research Funding. Van Ness:Millennium: Research Funding; Scientific Advisory Board, International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees. Liu:Milllennium: Employment, Equity Ownership. Pickard:Milllennium: Employment. Terkelsen:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.

Centrosomal Clustering – a Novel Therapeutic Target for Multiple Myeloma.

Abstract 300The lack of tumor-specific targets that would allow for the selective eradication of malignant cells without affecting healthy tissues is a major challenge in the development of novel therapies for multiple myeloma (MM). In contrast to normal cells, malignant plasma cells frequently contain multiple centrosomes, associated with the transient formation of multipolar mitotic spindles that lead to segregation defects and chromosomal instability. As in most tumor types, mitotic stability in these cells is maintained by coalescence of multiple centrosomes into two functional spindle poles, termed “centrosomal clustering”. As we have recently shown, this mechanism is an attractive therapeutic target with specificity for tumor cells. To identify potent and selective inhibitors of centrosomal clustering, we performed a phenotype-based small molecule screen in order to force tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. We here describe the characterization of a novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis in MM cells. We tested a wide array of MM cell lines, including those resistant to conventional chemotherapeutic agents, and primary patient cells. We found mean inhibitory concentrations (IC50) of proliferation and survival in the range of 1-5 mM, associated with annexin V conversion and activation of caspases 8, 9, and 3. Importantly, GF-15 also overcomes the tumor cell growth advantage conferred by both bone marrow stromal cell-MM, and endothelial cell-MM, co-culture systems. Moreover, non-malignant cells without supernumerary centrosomes like activated PBMCs, immortalized hepatocytes, and bone marrow stromal cells did not reach their IC50 at doses of up to 50 mM. To further demonstrate the specificity of GF-15, we generated resistant MM cell lines by long-term culture with sub-IC50 doses of GF-15. In resistant cell lines, therapeutic doses of GF-15 no longer induce multipolar spindles, consistent with a significant loss of centrosomal aberrations in these cells, as observed by immunoflourescence microscopy. Mechanistically, cell cycle analysis of synchronized MM cells showed marked G2/M arrest within 12-16h followed by a dramatic increase of the sub-G1 fraction after treatment with GF-15. In addition, short term treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered MM cell migration. Co-treatment assays to assess potential partners for therapeutic combinations revealed at least additive effects for GF-15 together with bortezomib and marked synergism with paclitaxel at very low doses (1-5 nM), while the combination with melphalan resulted in antagonistic effects due to the S-phase arrest of tumor cells induced by melphalan. Finally and most importantly, i.p. as well as oral treatment of murine xenograft models of human MM resulted in tumor growth inhibition and significantly prolonged survival in vivo. Growth inhibition of xenograft tumor samples was associated with a dramatic increase of mitotic aberrations and multipolarity, as assessed by immunohistochemistry. In vivo biodistribution studies are ongoing. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a prototype small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and therefore strongly support its further evaluation and development as a lead compound of a new class of therapeutics for human malignancies. Disclosures:No relevant conflicts of interest to declare.

PR-924, a Selective Inhibitor of the Immunoproteasome Subunit LMP-7 Blocks Multiple Myeloma Cell Growth Both in Vitro and In Vivo.

Abstract 612 Background:Therapeutic targeting of Ubiquitin-Proteasome pathway is exemplified by the recent FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of multiple myeloma (MM). As with other agents, dose-limiting toxicities and the development of drug resistance limit its long-term utility. The β5, β1 and β2 catalytic subunits within the 26S proteasome mediate chymotrypsin-like, caspase-like, and trypsin-like activities, respectively. Importantly, these catalytic subunits have corresponding immunoproteasome components LMP-7, LMP-2 and multicatalytic endopeptidase complex subunit-1 (MECL-1), which regulate immune cell function and cytokine production; however, the role of the immunoproteasome in MM cells is still unclear. Recent studies have therefore focused on the discovery and development of small molecule inhibitors of the immunoproteasomes, which will both delineate the function of immunproteasomes and allow for specific therapeutic targeting of the UPS in order to reduce off-target activities and associated toxicities. Here, we examined PR-924, an LMP-7-selective peptide-ketoepoxide proteasome inhibitor related to carfilzomib. PR-924, like carfilzomib, contains a ketopoxide pharmacophore that covalently modifies proteasomal N-terminal threonine active sites. We examined the effects of PR-924 in MM cell lines and primary patient cells in vitro. To determine the in vivo efficacy of PR-924, we utilized two xenograft models of human MM in SCID mice, a subcutaneous tumor plasmacytoma model and the SCID-hu model, which best reflects the human MM-BM microenvironment in vivo. Methods and Model:We utilized MM.1S, MM.1R, RPMI-8226, U266, DOX40, KMS12, LR-5, OPM1, OPM2 and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability and apoptosis assays were performed using Trypan blue, MTT and Annexin V staining. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, caspase-7, PARP, Bcl-2, BID, or GAPDH. For tumor xenograft studies, CB-17 SCID male mice (n = 10; 5 mice/each group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum-free RPMI-1640 medium. When tumors were measurable (∼150 mm3) 2-3 weeks after MM cell injection, mice were injected IV with either PR-924 (6 mg/kg BW) or vehicle twice weekly. Mice were sacrificed when their tumors reached >2 cm3. In the SCID-hu model, 2 × 106 INA-6 cells were injected directly into human bone chips implanted subcutaneously in SCID mice (n=10: 5 mice/EA group), and MM cell growth was assessed by serial measurements of circulating levels of soluble human interleukin-6 receptor (shulIL6R) in mouse serum. Statistical significance of differences observed in PR-924 vs. vehicle treated mice was determined using a Student t test. Results:PR-924 significantly inhibits growth of all the MM cell lines in a time- and dose-dependent manner (IC50 range: 3-5 μM; P <0.005 for all cell lineIt alsos. reduced the viability of primary patient cells (P < 0.05; n=5), without significant effects on normal peripheral mononuclear cells. The PR-924-triggered decrease in MM cell viability is due to apoptosis, as evidenced by Annexin V/PI staining. Moreover, PR-924-induced apoptosis in MM.1S and MM.1R MM cells is associated with activation of caspase-3, caspase-8, caspase-9, caspase-7, BID and PARP. In vivo PR-924 triggered significant tumor growth inhibition in tumor plasmacytoma xenografts (2.3 fold decrease in tumor volume in mice receiving PR-924 versus mice injected with vehicle alone; P value = 0.01). Similarly, a significant reduction in the shuIL6R levels (3.4 fold decrease; P value = 0.02) was observed in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated, as evidenced by the lack of weight loss even after three weeks of treatment. Importantly, treatment of tumor bearing mice with PR-924, but not vehicle alone, significantly prolonged survival (P < 0.005). Conclusion:Our preclinical findings establish immunoproteasome LMP-7 as a novel therapeutic target in MM. Disclosures:Aujay:Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership.

Pterostilbene: A Novel Histone Deacetylase 1 Inhibitor (HDACi1) Demonstrating Efficacy in Multiple Myeloma.

Abstract 3844Poster Board III-780Pterostilbene (3,5-Dimethoxy-4'-hydroxy-trans-stilbene) is a key compound found predominantly in blueberries and grapes. Pterostilbene has been shown to exhibit potential anti-cancer characteristics, anti-hypercholesterolemia and anti-hypertriglyceridemia properties. However, the mechanism of anti-cancer effects has not been elucidated and this compound has not been previously evaluated in multiple myeloma (MM). In this study, we first examined the HDAC binding ability of pterostilbene in the RPMI8226 multiple myeloma (MM) cell line and 293 HEK cells by using a novel HDAC screening assay. Briefly, RPMI8226 cells or 293 HEK cells were lysed with M-PER supplemented with protease and phosphatase inhibitors. The lysates were diluted and incubated with pterostilbene in concentrations ranging from 1 to 200 μM or without drug. Proteolysis was performed using thermolysin and aliquots were removed every 5 minutes and western blot analysis completed using antibodies against different HDACs or GAPDH. The results showed that pterostilbene prevents specifically HDAC1 digestion by thermolysin in both RPMI8226 and 293 cells. Next, we examined H4 acetylation of lysine residues in RPMI8226 cells following treatment with pterostilbene using western blot analysis. Increases in histone acetylation in RPMI8226 cells following exposure to pterostilbene treatment occurred in a concentration-dependent manner. Specifically, 1 to10μM of the pterostilbene markedly induced histone acetylation in RPMI8226 cells within 24 hrs. We also analyzed pterostilbene's effect on MM tumor cell proliferation using the MTS assay and apoptosis with flow cytometric analysis following Annexin V staining, in cells from the RPMI8226 and MM1S MM cell lines and freshly obtained tumor cells from MM patients. Pterostilbene alone showed a 50% growth inhibition (IC50) of cells from the RPMI8226 and MM1S lines as well as fresh bone marrow tumor cells from MM patients treated for 48 hours at approximately the same concentration (5 μM) as showing anti-MM effects in the MTS assay. Notably, the combination of pterostilbene and melphalan or the proteasome inhibitor bortezomib showed markedly increased inhibition of MM cell growth compared to treatment of the cells with the drugs alone. Since this effect may have resulted from decreased cell proliferation due to cell cycle arrest or increased cell death, we further determined apoptosis in cells from RPMI8226, U266, and MM1S cell lines as well as fresh tumor cells from MM patients following treatment with pterostilbene. Our data demonstrated that pterostilbene induced apoptotic cell death in a concentration dependent fashion. We also evaluated the combination of this compound with bortezomib (1-6 nM) and showed marked increases in apoptosis using this combination compared to either drug alone. These studies establish pterostilbene as a novel HDACi with potent anti-MM activity alone and also show its ability to enhance the anti-MM effects of chemotherapeutic agents and bortezomib. Currently, we are evaluating pterostilbene alone and in combination treatments using our SCID-hu murine models of human MM. Disclosures:No relevant conflicts of interest to declare.

Multiple Myeloma-Specific Targeted Therapy: Use of the Tumor Specific Immunoglobulin Gene Sequence to Activate Drugs Only within Tumor Cells.

Abstract 1851Poster Board I-877Currently available drugs for multiple myeloma (MM) treatment show very little ability to distinguish MM cells from normal cells. This limits the dose of drugs with anti-MM activity that can be administered safely; and, thus, reduces their efficacy in eliminating the malignant plasma cell population. Therefore, novel strategies are needed that allow concentrations of higher levels of active drugs within tumor cells than in other healthy cells. One approach is to allow release of active drugs only within the tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells in these patients. We have explored the possibility of using a unique sequence from this tumor specific transcript, the complementarity determining region (CDR) gene sequence, to direct drug release only within MM cells. First, we determined whether this type of tumor specific oligonucleotide could specifically recognize the tumor cell population. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe (designated as molecular beacon [MB] 8226), we demonstrated that this oligonucleotide specifically distinguished the RPMI8226 from other cell lines, including U266, another MM cell line. Second, in order to translate this approach into potentially therapeutically active anti-MM agents, we synthesized naphthyridine-modified (N) vorinostat, a FDA-approved histone deacetylase inhibitor, and N-melphalan. The modification allowed us to conjugate vorinostat or melphalan, two drugs currently used in MM treatment, with MB8226. Histone acetylation analysis demonstrated that the modification of vorinostat with naphthyridine did not change its function compared to the unmodified compound. Moreover, using cell viability and apoptosis assays, there was no reduction in the cytotoxic effects of N-vorinostat or N-melphalan compared to the parent drugs. In addition, induction of cell death occurred in a cell specific matter with N-melphalan conjugated to its tumor specific oligonucleotide. Upon transfection with N-melphalan-conjugated MB8226, only RPMI8226 but not U266 cells showed cytotoxic effects from exposure to this tumor specific cytotoxic construct. We are currently evaluating the specific efficacy of N-vorinostat-conjugated MB8226 as well as an oligonucleotide that specifically recognizes the CDR3 sequence of U266 cells conjugated with N-vorinostat or N-melphalan. We are also determining the efficacy of this approach in vivo using our severe combined immunodeficiency-hu mouse models of human MM. Our studies provide a novel targeted therapeutic approach for the treatment MM as well as other B cell malignancies that should specifically be active only within the malignant cell population and not impact nonmalignant cells. This type of treatment should allow delivery of higher concentrations of drugs that will be active only within tumor cells ultimately leading to both much more effective and better tolerated therapies for patients with MM and other B-cell malignancies. Disclosures:No relevant conflicts of interest to declare.

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 Phase II Study of Pegylated Liposomal Doxorubicin, Bortezomib and Dexamethasone (DVD) for Patients with Previously Untreated Multiple Myeloma (MM).

Abstract 4936Despite recent advances in the treatment of MM, the disease remains incurable and many of the most effective, newer combination therapies are accompanied by significant side effects that have a major negative impact on the patient's quality of life. Pegylated liposomal doxorubicin (PLD) and bortezomib have shown anti-MM efficacy in the laboratory and for the treatment of previously treated MM patients, leading to FDA approval for patients who have failed one prior therapy. Using our severe combined immunodeficiency-hu murine models of human MM, we have previously demonstrated that lower doses of PLD administered daily are more effective and better tolerated than higher amounts given weekly. Moreover, the combination of bortezomib and dexamethasone has been shown to be effective for previously untreated MM patients. Prior studies by our group have shown that combining chemotherapy including PLD with bortezomib administered at 1.0 mg/m2 on days 1, 4, 8, and 11 of a 28-day cycle rather than the standard 1.3 mg/m2 on the same days of a 21-day schedule is effective for MM patients with relapsed or refractory disease and associated with a reduction in the incidence and severity of peripheral neuropathy. Thus, we conducted a single-arm multi-center phase II study for previously untreated MM patients to evaluate the combination of intravenously administered dexamethasone, bortezomib and PLD (DVD). The treatment consisted of intravenous administration of 40 mg dexamethasone followed by 1.0 mg/m2 bortezomib and finally 5.0 mg/m2 PLD on days 1, 4, 8, and 11 of a 28-day cycle. Patients were treated to a maximum response plus two additional cycles or completed a maximum of eight cycles of therapy without disease progression. To date, 22 (of 35 planned) patients have been enrolled with a median age of 64 years (range, 42-79 years). The majority of those on study (68 %) were diagnosed with International Staging System II or III MM. Four patients are too early to assess for response. To date, among the 18 evaluable patients, 16 (89%) have shown objective responses to the DVD regimen, including 2 complete responses (11%), 8 partial responses (44%) and 6 minimal responses (33%). The other 2 patients (11%) had stable disease, with one of these subjects showing a continuing reduction in M-protein after 2 cycles of therapy to date. Thus, disease control was achieved in all patients. To date, no patient has shown progressive disease after 2+ - 12+ months of follow-up. Six patients experienced grade 3 adverse events and one patient with a prior history of pulmonary interstitial fibrosis developed a grade 4 toxicity (shortness of breath). Grade 3 adverse events in three of the six patients were judged not to be related to the study treatment. The most common grade 3 adverse event was reversible neutropenia (n=2). To date, only 2 patients (9%) have developed peripheral neuropathy (grade 1). Notably, there have been no cases of stomatitis or hand-foot syndrome. Thus, these results suggest that the DVD regimen using a modified schedule and doses of the combination of intravenous dexamethasone, bortezomib and PLD is a well tolerated treatment that produces high response rates for previously untreated patients with multiple myeloma. DisclosuresBerenson:Millennium Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Centocor Ortho Biotech: Consultancy, Speakers Bureau. Hilger:Millennium Pharmaceutcals: Employment.