Mouse Model Of Human Multiple Myeloma Research Articles

Vκ*MYC Mouse Model Recapitulates Key Genomic Defining Events in Multiple Myeloma

Background: The Vk*MYC transgenic model of multiple myeloma (MM) is based on the sporadic AID-induced activation of MYC in a single germinal center (GC) B-cell, in a mouse strain (C57Bl6) that spontaneously develops monoclonal gammopathy. With age, Vk*MYC mice universally develop a progressive expansion of class-switched, somatically hypermutated, monoclonal plasma cells. Myeloma defining events occur only after long latency, suggesting that additional mutations are required to induce malignancy. Here, we interrogated the genomic landscape of Vk*MYC MM to capture somatic mutations selected during progression. Methods: We interrogated whole genome (WGS) and exome sequencing (WES) from 55 purified plasma cell (PC) from Vκ*MYC transgenic mice tumors. Additional 27 and 12 Vκ*MYC mice were investigated with array comparative genomic hybridization (aCGH) and mate-pair low coverage WGS, respectively. Our analysis included 34 individual de novo tumors from aged Vk*MYC mice, 37 individual tumors from recipient mice transplanted with tumor cell lines established from de novo donors, and 13 tumors selected to propagate in vitro. Results: Tumor mutational burden was lowest in de novo samples (median 2.3 mutations/Mb, range 0.3-4.3 mutations/Mb) and highest in in vitro samples (median 5.8 mutations/Mb, range 1.2-12.7 mutations/Mb), similar to that of human MGUS and MM, respectively. To identify genes affected by nonsynonymous mutations more frequently than expected by chance (i.e., driver genes under positive selection), we ran dndscv. A total of 11 significant driver genes were detected, 7 of which are known driver genes in human MM: Dusp2, Trp53, Nfkbia, Pim1, Tent5c (Fam46c), Hist1h1c, Hist1h1e. Driver genes not previously described in MM were: Apobec3, Pten, Cd74, Cd79b. Additional nonsynonymous mutations were detected in driver genes frequently altered in MM including Kras, Nras, Dis3, as well as histone modifiers and NF-kB regulators. Differential mutation analysis revealed bi-allelic inactivation of Trp53 and Pten were more frequent in in vitro samples compared to de novo and transplanted tumor, in line with the almost universal p53 inactivation reported in human MM cell lines. As in human hyperdiploid MM, GISTIC and molecular time analyses revealed evidence of simultaneous multi-gain events of chromosomes 1, 9, 11, and 15. As reported previously, we observed frequent deletion of chromosome 5. There were numerous recurrent focal CNAs involving oncodrivers. These included focal deletion of tumor suppressors genes Cdkn2a, Cdkn2b, Cdh11, Ncor1, Rb1, Trp53, Kdm6a and Pten, as well as amplification of oncogenes Mdm4, Mcl1, and Kras. We detected several structural variants and complex events throughout the Vκ*MYC genome, similar to human MM. Importantly, we found chromothripsis in five (14%) cases. Additionally, in three Vκ*MYC mice we detected chromosomal translocations involving the immunoglobulin heavy or light chain loci that partnered and induced overexpression of MM associated genes such as Irf4, Map3k14 and Pou2af1. Overall, mutations activating the NF-kB and STAT3 pathways were the most common events, each present in more than half MM. Overall, the genomic landscape of the Vκ*MYC model resembles hyperdiploid MM in humans, with MYC translocations and enrichment of events affecting the NF-κB pathway. Finally, analysis of mutational signatures uncovered similar signatures present in human MM, including aging, APOBEC, and poly-eta in the GC, among others. In particular, APOBEC mutational activity was detected in more than half of Vκ*MYC MM, further supporting the similarities with human MM where APOBEC mutational activity is detectable in 80-90% of human MM and progressive precursor, but it is undetectable in the stable precursor conditions. Conclusions: Overall, these data reveal a progressive accumulation of genomic events from de novo, to transplant, to in vitro tumors that mimic the natural progression from newly diagnosed, to relapsed disease, to cell lines noted in human MM. Thus, our results highlight how the transformation pathways to malignant PC are conserved between mice and men. Given the long lead time between precursor diagnosis and malignant transformation to MM that limit the ability to investigate the earliest phases of tumorigenesis in human MM, the Vκ*MYC transgenic mouse model of human MM represents a unique opportunity to overcome this temporal limitation.

Laboratory Mice - A Driving Force in Immunopathology and Immunotherapy Studies of Human Multiple Myeloma.

Mouse models of human cancer provide an important research tool for elucidating the natural history of neoplastic growth and developing new treatment and prevention approaches. This is particularly true for multiple myeloma (MM), a common and largely incurable neoplasm of post-germinal center, immunoglobulin-producing B lymphocytes, called plasma cells, that reside in the hematopoietic bone marrow (BM) and cause osteolytic lesions and kidney failure among other forms of end-organ damage. The most widely used mouse models used to aid drug and immunotherapy development rely on in vivo propagation of human myeloma cells in immunodeficient hosts (xenografting) or myeloma-like mouse plasma cells in immunocompetent hosts (autografting). Both strategies have made and continue to make valuable contributions to preclinical myeloma, including immune research, yet are ill-suited for studies on tumor development (oncogenesis). Genetically engineered mouse models (GEMMs), such as the widely known Vκ*MYC, may overcome this shortcoming because plasma cell tumors (PCTs) develop de novo (spontaneously) in a highly predictable fashion and accurately recapitulate many hallmarks of human myeloma. Moreover, PCTs arise in an intact organism able to mount a complete innate and adaptive immune response and tumor development reproduces the natural course of human myelomagenesis, beginning with monoclonal gammopathy of undetermined significance (MGUS), progressing to smoldering myeloma (SMM), and eventually transitioning to frank neoplasia. Here we review the utility of transplantation-based and transgenic mouse models of human MM for research on immunopathology and -therapy of plasma cell malignancies, discuss strengths and weaknesses of different experimental approaches, and outline opportunities for closing knowledge gaps, improving the outcome of patients with myeloma, and working towards a cure.

RNA Regulator of Lipogenesis (RROL) Is a Novel Lncrna Mediating Protein-Protein Interaction at Gene Regulatory Loci Driving Lipogenic Programs in Multiple Myeloma

Long noncoding RNA (lncRNAs) are key epigenetic factors that drive the origin and progression of human cancers via mechanisms that are largely unknown. We have previuosly reported the clinical significance of a lncRNA signature in multiple myeloma (MM) as independent risk predictor for clinical outcome; and recently identified a lncRNA RROL (RNA Regulator of Lipogenesis) with impact on MM cell proliferation. Here we describe a unique regulatory function for RROL in the control of gene networks involved in the de novo lipogenesis (DNL) pathway, ultimately impacting MM cell growth and survival. Based on growth and survival impact of RROL depletion, we performed integrated transcriptomic analysis of RNA-seq data after RROL depletion in MM cell lines and CD138+ patient MM cells, and identified a set of significantly modulated metabolic genes including the acetyl Co-A Carboxylase 1 (ACC1) gene, encoding a rate-limiting enzyme of the DNL pathway. This metabolic pathway converts nutrients into fatty acids serving for energy storage or biosynthesis of membranes and signaling molecules. Consistent with the transcriptional control of ACC1, we have observed that RROL inhibition in cell lines and primary MM cells significantly decreased the incorporation of C14-radiolabeled glucose into de novo synthesized lipids. Importantly, supplementation with exogenous palmitate, the main downstream product of DNL pathway, rescued the growth inhibitory effect of RROL depletion on MM cells, further confirming the importance of the DNL pathway in the oncogenic activity of RROL in MM. To understand the molecular mechanism through which RROL regulates ACC1 expression and its metabolic axis, we evaluated the RROL interactome in MM cells. RNA-Protein Pull Down (RPPD) and in vivo RNA yeast three-hybrid (Y3H) assays led to the identification of MYC as relevant direct partner of RROL. These results were further validated by qRT-PCR analysis of MYC-bound RNA obtained through RNA immunoprecipitation (RIP) assay. Using experimental model of conditional MYC KD (P493-6), we found that RROL exerts regulatory activity on ACC1 only in the presence of MYC. Mapping of MYC genomic occupancy by ChIP-seq and gene expression after MYC KD in MM cells revealed that ACC1 is a direct transcriptional target of MYC in cells expressing RROL. These data indicate that RROLand MYC cooperate in the transcriptional control of ACC1. Moreover, we found that RROL itself is transcriptionally regulated by MYC, suggesting the existence of a feed-forward regulatory loop in which MYC enhances the expression of RROL that, in turn, drives MYC transcriptional activity to ACC1. We hypothesized that RROL may shape the protein interacting network of MYC to confer specificity for ACC1 promoter. To this end, we performed mass spectrometry analysis of MYC interactome in three MM cell lines in the presence or in the absence of RROL and identified the transcriptional modulator WDR82 as RROL-dependent MYC partner. Interestingly, WDR82 directly interacts with RROL as assessed in the RPPD and RNA Y3H assays, and transcriptionally regulates ACC1 in RROL-dependent manner. These data indicate that RROL catalyzes the interaction of MYC with the transcriptional modulator WDR82 to form a transcriptional ternary complex regulating ACC1 expression. To therapeutically antagonize the RROL lipogenic signaling we have pre-clinically tested small molecule inhibitors of ACC1 (ACC1i). We have observed significant anti-MM activity of ACC1i in vitro in a large panel of MM cell lines and primary MM cells from patients; and in vivo in mouse models of human MM including the localized subcutaneous model and the disseminated model that establish a more aggressive systemic disease. Importantly, we have now developed clinically applicable ASOs and small molecule-like compounds to directly target RROL in MM cells. These studies are ongoing and will be presented. In conclusion, we here report a unique regulatory function of a novel lncRNA supporting MM cell growth via its control of the lipogenic metabolic axis. The availability of oral inhibitors of ACC1 as well as the ongoing development of RROL inhibitors may allow clinical application of this unique targeted therapy in MM. Disclosures Fulciniti: NIH: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Long-term survival without graft-versus-host-disease following infusion of allogeneic myeloma-specific V\u03b2 T cell families

BackgroundDespite chemo-induction therapy and autologous stem cell transplantation (ASCT), the vast majority of patients with Multiple Myeloma (MM) relapse within 7 years and the disease remains incurable. Adoptive Allogeneic T-cell therapy (ATCT) might be curative for MM, however current ATCT protocols often lead to graft versus host disease (GvHD). Transplanting only tumor reactive donor T cells that mediate a graft-versus-myeloma (GvM) but not GvHD may overcome this problem.MethodsWe used an MHC-matched/miHA-disparate B10.D2 → Balb/c bone marrow transplantation (BMT) murine model and MOPC315.BM MM cells to develop an ATCT protocol consisting of total body irradiation, autologous-BMT and infusion of selective, myeloma-reactive lymphocytes of T cell receptor (TCR) Vβ 2, 3 and 8.3 families (MM-auto BMT ATCT).ResultsPre-stimulation ex vivo of allogeneic T cells by exposure to MOPC315.BM MM cells in the presence of IL-2, anti-CD3 and anti-CD28 resulted in expansion of the myeloma-reactive T cell TCRVβ 2, 3 and 8.3 subfamilies. Their isolation and infusion into MM-bearing mice resulted in a vigorous GvM response without induction GvHD and long-term survival. Repeated infusion of naïve myeloma-reactive T cell TCRVβ 2, 3 and 8.3 subfamilies was also effective.ConclusionsThese data demonstrate that a transplantation protocol involving only selective tumor-reactive donor T cell families is an effective immunotherapy and results in long-term survival in a mouse model of human MM. The results highlight the need to develop similar ATCT strategies for MM patients that result in enhanced survival without symptoms of GvHD.

Effective Targeting of Multiple B-Cell Maturation Antigen–Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

The orally available multikinase inhibitor regorafenib (BAY 73-4506) in multiple myeloma.

A promising approach to the treatment of multiple myeloma (MM) involves agents that target not only the myeloma cells directly, but also the tumor microenvironment which promotes tumor cell growth, angiogenesis, and MM bone disease. Here we investigate the orally available multikinase inhibitor, regorafenib (BAY 73-4506), for its therapeutic efficacy in MM. Regorafenib is a potent inhibitor of angiogenic (VEGFR 1-3, PDGFR-b) as well as oncogenic (c-KIT, RET, FGFR, Raf) kinases. We show that regorafenib induces apoptosis in all MM cell lines at below clinically achievable concentrations. Regorafenib overcomes the growth advantage conferred by a stroma cell MM and an endothelial cell MM, co-culture systems, and abrogates growth factor-stimulated MEK, ERK, and AKT phosphorylation at nanomolar to micromolar concentrations. Moreover, it inhibits endothelial cell growth and tubule formation, abrogates both VEGF secretion and VEGF-induced MM cell migration, inhibits osteoclastogenesis, and shows synergistic cytotoxicity with dexamethasone, the immunomodulatory drug pomalidomide, and the p110δ inhibitor idelalisib. Most importantly, regorafenib significantly delays tumor growth in a xenograft mouse model of human MM. These results provide the rationale for further clinical evaluation of regorafenib, alone and in combination, in the treatment of MM.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas.We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance.Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. DisclosuresChekmasova:bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

A Novel Mouse Model for Multiple Myeloma (MOPC315.BM) That Allows Noninvasive Spatiotemporal Detection of Osteolytic Disease

Multiple myeloma (MM) is a lethal human cancer characterized by a clonal expansion of malignant plasma cells in bone marrow. Mouse models of human MM are technically challenging and do not always recapitulate human disease. Therefore, new mouse models for MM are needed. Mineral-oil induced plasmacytomas (MOPC) develop in the peritoneal cavity of oil-injected BALB/c mice. However, MOPC typically grow extramedullary and are considered poor models of human MM. Here we describe an in vivo-selected MOPC315 variant, called MOPC315.BM, which can be maintained in vitro. When injected i.v. into BALB/c mice, MOPC315.BM cells exhibit tropism for bone marrow. As few as 104 MOPC315.BM cells injected i.v. induced paraplegia, a sign of spinal cord compression, in all mice within 3–4 weeks. MOPC315.BM cells were stably transfected with either firefly luciferase (MOPC315.BM.Luc) or DsRed (MOPC315.BM.DsRed) for studies using noninvasive imaging. MOPC315.BM.Luc cells were detected in the tibiofemoral region already 1 hour after i.v. injection. Bone foci developed progressively, and as of day 5, MM cells were detected in multiple sites in the axial skeleton. Additionally, the spleen (a hematopoietic organ in the mouse) was invariably affected. Luminescent signals correlated with serum myeloma protein concentration, allowing for easy tracking of tumor load with noninvasive imaging. Affected mice developed osteolytic lesions. The MOPC315.BM model employs a common strain of immunocompetent mice (BALB/c) and replicates many characteristics of human MM. The model should be suitable for studies of bone marrow tropism, development of osteolytic lesions, drug testing, and immunotherapy in MM.

Abstract 2852: Combination therapy: Galectin-3C and bortezomib potent anticancer activity and inhibition of angiogenesis, chemotaxis, and NF-kB activation in human multiple myeloma

Abstract Introduction Galectin-3 is a human lectin involved in 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). Methods We analyzed 9 human MM cell lines tested in vitro for cell proliferation/viability; chemotaxis. The human MM cell lines used in this study were RPMI-8226, U266 (American Type Culture Collection, VA, USA), ARP-1, ARK-B, (gifts from J. Epstein, University of Arkansas for Medical Sciences, Little Rock, AR, USA). The 8226/Dox and 8226/LR-5 cell lines were kindly provided by Dr. William S. Dalton of the H. Lee Moffitt Cancer Center and Research Institute in Tampa, Florida. All MM cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 °C. The 8226/Dox cells were cultured with 40 nM doxorubicin (Sigma-Aldrich, St. Louis, MO, USA). The 8226/LR-5 cells were cultured in media containing 5 µM mephalan (Sigma-Aldrich). 8226/Dox and 8226/LR-5 cells were maintained in drug-free medium for 1 week prior to drug sensitivity assays. Human umbilical cord vascular endothelial cells (HUVEC, American Type Culture Collection, VA, USA) were maintained in EGM-2 medium (Lonza, Houston, TX, USA) and were used within 10 passages. Furthermore we used U266 MM model in the NOD/SCID mice to evaluate the efficacy of galectin-3C in single or in combination with BOR. Results Galectin-3C exhibited modest anti-proliferative effects on MM cells 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. Bortezomib, a proteasome inhibitor approved by the F.D.A. for MM treatment, also inhibited the growth of the U266 tumors. Treatment with both agents was more effective than either alone, such that the average tumor volume of the combination group was just 14.0% of controls at day 35. We show that mechanisms by which galectin-3C facilitates the anticancer activity of BOR include inhibition of angiogenesis, chemotaxis, and NF-kB activation. Conclusion In conclusion, our study shows that in a mouse model of human MM, treatment with galectin-3C alone is efficacious, and that it enhances the anti-tumor activity of bortezomib. These findings provide the proof-of-principle to test galectin-3C clinically for human MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2852. doi:1538-7445.AM2012-2852

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.

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.

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.

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.

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.

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.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Selective Cytotoxicity Against CD138-Positive Multiple Myeloma CellsIn vitroandIn vivo

We investigated the antitumor effect of murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives against multiple myeloma (MM) cells in vitro and in vivo. We examined the growth inhibitory effect of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 against MM cell lines and primary tumor cells from MM patients. We also examined in vivo activity of these agents in murine MM cell xenograft model of human and severe combined immunodeficient (SCID) mice bearing implant bone chips injected with human MM cells (SCID-hu model). Anti-CD138 immunoconjugates significantly inhibited growth of MM cell lines and primary tumor cells from MM patients without cytotoxicity against peripheral blood mononuclear cells from healthy volunteers. In MM cells, they induced G(2)-M cell cycle arrest, followed by apoptosis associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. Nonconjugated nBT062 completely blocked cytotoxicity induced by nBT062-maytansinoid conjugate, confirming that specific binding is required for inducing cytotoxicity. Moreover, nBT062-maytansinoid conjugates blocked adhesion of MM cells to bone marrow stromal cells. The coculture of MM cells with bone marrow stromal cells protects against dexamethasone-induced death but had no effect on the cytotoxicity of immunoconjugates. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both the xenograft mouse models of human MM and SCID-hu mouse model. These results provide the preclinical framework supporting evaluation of nBT062-maytansinoid derivatives in clinical trials to improve patient outcome in MM.

AT7519, a Novel Small Molecule Multi-Cyclin Dependent Kinase Inhibitor, Induces Apoptosis in Multiple Myeloma VIA GSK3β

Cyclin dependent kinases (CDKs) and their cyclin complexes play a crucial role in cell cycle control and transcriptional regulation. In multiple myeloma (MM), the abnormal activation of different CDKs and their cyclin partners, especially CDK4/cyclin D1 and CDK6/Cyclin D2, mediate uncontrolled cell cycle progression. Therefore CDKs represent promising novel therapeutic targets for MM. Additionally the cytokine dependent PI3K/Akt signaling pathway mediates growth, survival, drug resistance, migration and cell cycle regulation in MM. Activated Akt in turn phosphorylates downstream target molecules like glycogen synthase kinase (GSK)-3 β impacting growth and survival. Here we investigated the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519 in MM. In vitro kinase assays demonstrated more potent inhibition of CDK 1, 2, 4, 5 and 9 compared to CDK 3, 6, and 7. AT7519 also demonstrated potent inhibitory activity against GSK-3 β. No significant inhibitory effects against other kinases were observed. We next investigated the growth inhibitory effect of AT7519 on MM cell lines. Maximal cytotoxicity was observed in 48 hour culture with IC50 values ranging from 0.5μM (MM.1S, U266) to 4 μM (MM1R). AT7519 was also effective against primary tumor cells from MM patients with no significant cytotoxicity noted in peripheral blood mononuclear cells from healthy volunteers. To delineate the underlying mechanism of cytotoxicity induced by AT7519, cell cycle analysis using PI staining in MM.1S cell line was performed. No significant accumulation of cells in a particular phase of cell cycle was noted; however, AT7519 showed an increased sub-G1 population, indicative of apoptosis, which was confirmed by Annexin V+PI+ staining and associated with caspase-8-9 and -3 cleavage. Importantly, we found that AT7519 markedly inhibited phosphorylation (serine 2 and serine 5 sites) of the carboxyl terminal domain of RNA polymerase II (RNA pol II) within 6 hours of treatment. Non-cell cycle CDKs including CDK9 are responsible for phosphorylation and activation of RNA pol II. Similarly, AT7519 also inhibited phosphorylation of GSK-3β while no significant effects on CDK expression levels were evident at early time points. To investigate whether there was a correlation between inhibition of phosphorylation of GSK-3β and RNA pol II, MM.1S cells were cultured with α-amanitin, a specific inhibitor of RNA pol II. Although phosphorylation of RNA pol II was significantly inhibited, phosphorylation of GSK-3β was not altered by amanitin (10 μM for up to 24 hours). These results suggest that GSK-3β and RNA pol II dephosphorylation at serine 2 and serine 5 may be two independent mechanisms by which AT7519 induces apoptosis in MM cells. Ongoing studies are confirming the role of GSK-3 β in AT7519 induced cytotoxicity of MM cells. Finally, the in vivo efficacy of AT7519 was examined using a xenograft mouse model of human MM. Mice treated with AT7519 demonstrated slower tumor growth compared to the control group without adverse effects. Moreover, AT7519 resulted in a significant prolongation in median overall survival in treated mice (40 days in the treatment group versus 27.5 days in the control cohort, p = 0.0324). In conclusion, these results show significant anti-MM activity of AT7519, and provide the rationale for its clinical evaluation in MM.

C-Myc- Dependent Stabilization of Hif-1alpha in MM: Therapeutic Implications

Bone marrow (BM) angiogenesis has been associated with multiple myeloma (MM) progression. Here we demonstrate that oncogenic c-Myc, also prominently associated with adverse prognosis in MM, regulates Hif-1α promoter activity, Hif-1alpha protein levels, and consequently the expression of pro-angiogenic target genes such as VEGF in MM cells. As observed with c-Myc knockdown, anti-MM agents with known anti-angiogenic activity including bortezomib, lenalidomide, and enzastaurin also downregulate Hif-1alpha and VEGF. Moreover, tyrophostin adaphostin induces similar effects, associated with anti-angiogenic activity, in a zebrafish model. The correlation between c-Myc, Hif-1alpha and tumor angiogenesis was further confirmed in a xenograft mouse model of human MM. Mechanistically, our in vitro data show that oncogenic c-Myc stabilizes caspase-8, and thereby protects Hif-1alpha against caspase-8- dependent degradation and attenuation of VEGF secretion. These sequelae also contribute to enhanced VEGF secretion triggered by MM cell-bone marrow stromal cell binding. Indeed, conditioned media from adaphostin- treated co-cultures inhibit endothelial cell growth and tubule formation. In addition, HIF1alpha gene demonstrated significant increased expression (p=0.02, Mann Whitney test) in tumor cells versus plasma cells derived from healthy donors. Importantly, when tested on a dataset of 559 patients, a significant link between HIF1alpha overexpression and poor prognosis was found (p=0,0069). These data further point to the possible relevance of HIF1alpha in the progression of a subset of MM patients. In summary, our data provide strong evidence of a key role of Hif-1alpha in MM angiogenesis and the therapeutic potential of specific Hif-1alpha and c-Myc inhibitors. Moreover they demonstrate additional biologic sequelae of novel MM therapies.

The BCL9 Oncogene Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic and Angiogenic Properties to Myeloma Cells

Wnt signaling plays an important role in tissue development and maintenance during embryogenesis, cell differentiation, and stem cell growth. Several components of the Wnt signaling cascade have been shown to function as either tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis. Deregulation of the canonical Wnt/b-catenin pathway has been implicated in numerous human epithelial malignancies as well as hematologic malignancies including multiple myeloma (MM), generating immense interest in these molecules as targets for cancer therapy. Activation of Wnt/b-catenin in cancer has been associated with mutations that enable b-catenin to escape degradation by the proteasome, thereby allowing its accumulation in the nucleus where it functions as a transcriptional regulator in conjunction with coactivators by constitutively activating target genes such as c-Myc and Cyclin D1. To date, however, no mutations in Wnt pathway have been documented in MM, suggesting that mechanisms other than gene mutation may contribute to Wnt pathway deregulation. BCL9, a key component of the Wnt pathway, is required for b-catenin transcriptional activity and resides on chromosome 1q21, a region frequently involved in secondary chromosomal aberrations associated with MM tumor progression. Here we provide evidence that dysregulation of BCL9 expression is a novel oncogenic mechanism of Wnt pathway activation in MM. Using in vitro and in vivo functional analyses, we demonstrate that BCL9 is a bonafide oncogene that is aberrantly expressed in MM and associated with survival. Using the TCF- specific luciferase reporter, we show that enforced expression of BCL9 in MM cells enhanced b-catenin mediated transcription by >12 fold, suggesting a possible role of BCL9 overexpression in the pathogenesis of MM. BCL9 enhanced proliferation (1.5 fold, P<0.02), migration (3.5 fold, P<0.0001) and the metastatic potential of MM cells. We also showed that BCL9 plays an important role in tumor progression by regulating Cyclin D1 and c-Myc mediated cell proliferation, CD44 mediated tumor metastasis, as well as VEGF mediated host angiogenesis. Importantly, BCL9 knockdown significantly increased the survival in a xenograft mouse model of human MM (P=0.001), associated with decreased tumor burden and host angiogenesis. In summary, we have demonstrated that BCL9 is a novel and potent oncogene of the Wnt pathway in MM, playing fundamental roles in tumor progression by regulating proliferation, migration, invasion, angiogenesis and the metastatic potential of tumor cells. The pleiotropic roles of BCL9 and its aberrant expression highlight its importance as an attractive and novel therapeutic target in the treatment of MM.