ALBERT

Description: Context: Akt plays a pivotal role in the survival and proliferation of multiple myeloma (MM) cells and functions as a central link between upstream signaling pathways, such as growth factor receptors (e.g. IL-6/IL-6R, IGF/IGF-1R) and the kinase PI-3K with their downstream signaling effectors, such as the multifunctional mTOR-p70S6K cascade. The pivotal role of Akt in these proliferative/anti-apoptotic cascades for MM has provided the impetus for development of small molecule inhibitors against the kinase activity of Akt. We show that the dual Akt/p70S6K kinase inhibitor EXEL-6075 has potent anti-MM activity in preclinical models. Methods/Results: We tested a panel of 15 human MM cell lines for in vitro response to EXEL-6075 using MTT colorimetric survival assays, which showed that the majority of MM cells responded to EXEL-6075 with IC50 values 1-log differential activity against the non-neoplastic tissues tested in our study. This remains an interesting strategy for the treatment of MM. Future in vivo studies will be needed to confirm these interesting in vitro results.

Description: Ras mutations occur in 40–60% of multiple myeloma (MM) patients and are implicated in progression to advanced MM (including plasma cell leukemia/extramedullary lesions). The small molecule PRLX (Prolexys Pharmaceuticals) was identified in the context of “synthetic lethal” chemical screening for genotype-selective cytotoxicity against cells transformed with forced expression of mutant Ras (but not against isogenic normal cell counterparts) and was tested for anti-MM activity. In MTT survival assays, 34 of 46 human MM cell lines (74%) responded to 48hr treatment with sub-μM PRLX concentrations (achievable in preclinical pharmacokinetic studies). (24 MM lines had IC50 values

Description: Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of 〈 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of 〈 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Description: Abstract 1722 Poster Board I-748 Conventional anti-cancer drug screening in vitro has traditionally been performed in the absence of accessory cells of the tumor microenvironment. These normal cells of the bone marrow milieu can profoundly alter anti-tumor drug activity. To address this major limitation of traditional in vitro models, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. In this platform, tumor cells (e.g. myeloma, leukemia and solid tumors) stably expressing bioluminescent reporters are co-cultured with non-malignant accessory cells (e.g. stromal cells) to selectively quantify tumor cell viability to treatments in presence vs. absence of accessory cells. We applied CS-BLI to test various chemical libraries and showed that this platform is high-throughput scalable. We also identified stroma-induced chemoresistance in diverse malignancies, including imatinib-resistance in leukemic cells, as well as MM cell resistance to certain investigational agents. The majority of compounds screened in our studies were less active against tumor cells in the presence of stromal cells compared to their absence. Most interestingly, however, we identified a fraction of compounds which were more active against tumor cells in the presence of stromal cells. For example, we identified reversine, a compound exhibiting this stroma-dependent synthetic lethality in vitro, which we further confirmed in vivo, as it is active in an orthotopic model of diffuse MM bone lesions, but not in conventional subcutaneous xenografts. Mechanistically, in vitro kinase activity assays showed that reversine exhibits a distinct pattern of inhibition against targets such as Auroras, JAK2, and SRC, but not against other important kinases for MM survival, such as AKT1, 2, 3, FGFR3, or GSK3. These observations are compatible with the role of SRC and JAK kinases as downstream regulators of IL-6/IL-6R signaling, a key cascade triggered by tumor-stromal interactions in MM. Further mechanistic evaluation of this interaction at the transcriptional level showed that a stromal-induced gene expression signature in MM tumor cells correlates with inferior overall survival in patients with advanced MM (APEX dataset) and includes enhanced amplitude of signatures for activated Akt, Ras, NF-κB, HIF-1á, myc, hTERT, and IRF4; as well as signatures for biological aggressiveness and stem cell self-renewal. This suggests that selective inhibitors which block the activity of these pathways may exhibit tumor specific stromal-dependent synthetic lethality. Historically, synthetic lethality has focused on how tumor cells harboring specific constitutive oncogenetic lesions are responsive to certain agents, but not in absence of these genetic events. Our study introduces the notion that a synthetic lethal phenotype, rather than being exclusively genotype-dependent, can also be driven by the extrinsic influences of the tumor microenvironment. Importantly, the CS-BLI system can probe both genetically- and microenvironment-related synthetic lethality in a high-throughput scalable manner. This allows the testing of a large number of permutations, including multiple candidate therapeutics, cell lines, and non-malignant accessory cells, thus enabling the previously intractable large-scale evaluation of how genetics and microenvironment play a role in modulating cancer cell response to treatment. Importantly, unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells when interacting with stroma. These agents, in the past, may have been discarded from further preclinical or clinical development. We now provide a system with which to evaluate the role of the tumor microenvironment and identify novel agents capable of overcoming its protective effects. Disclosures Munshi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

Description: Obesity and insulin resistance are associated with increased risk for several malignancies, including multiple myeloma (MM), possibly due to hyperinsulinemia and the resulting increased circulating free IGF-I levels that can have a growth-promoting and anti-apoptotic effect on MM cells. It has been proposed that improving insulin sensitivity, with a low-fat diet or with the use of the biguanide metformin, could suppress circulating insulin and free IGF-I levels and lower cancer risk. Metformin has been widely used as an antidiabetic agent since the 1950s and has an excellent safety profile. Interestingly, epidemiologic studies have suggested that patients with type 2 diabetes mellitus who are treated with metformin have lower cancer incidence and lower cancer-related mortality than diabetics who receive sulfonylurea monotherapy or insulin. Even more intriguing is the recent finding that metformin can have direct anticancer activity in vitro against prostate and breast carcinoma cell lines. In breast carcinoma cells, metformin inhibits mammalian target of rapamycin (mTOR) activity and suppresses translation initiation and global protein synthesis, via an AMP-activated protein kinase (AMPK)-dependent pathway. We investigated the direct effects of metformin in vitro in a panel of 17 MM cell lines and sublines using MTT, flow cytometry, annexin V-PI, as well as compartment-specific bioluminescence (CS-BLI) assays in the absence or presence of bone marrow stromal cells (BMSCs). We found that metformin suppressed the growth of all MM cell lines and sublines, including those that are resistant to conventional and several investigational anti-MM agents. The anti-MM activity of metformin was achieved with clinically relevant sub-mM or low mM levels, and was in the same order of magnitude or stronger than its activity reported in solid tumor cell line models. The anti-MM activity of metformin did not involve apoptotic/necrotic cell death, but inhibition of proliferation (G0/G1 arrest). Western blot analyses showed that metformin treatment of RPMI-8226/S MM cells led to increased phosphorylation of AMPK; suppression of p70S6K phosphorylation; and decreased cyclin D1 levels. These results are consistent with the known effect of metformin in triggering AMPK activity and thereby suppressing the function of its downstream targets mTOR and p70S6K. Co-culture with BMSCs did not abrogate the anti-MM effect of metformin, but further sensitized MM cell to this agent, at concentrations which did not affect BMSC viability. This sensitization was more prominent in MM cell lines that respond to BMSCs with increased proliferation/survival (e.g. MM-1S, MM-1R, KMS-11, L363) than in cell lines independent/unresponsive to stromal stimulation (e.g. OPM-2). To further probe the mechanistic basis for this stroma-induced sensitization to metformin, we studied the transcriptional signature of genes suppressed by mTOR inhibition, a downstream effect of metformin, in MM cells cultured alone vs. co-cultured with BMSCs. We observed that stroma triggers a significant increase in the average expression of mTOR-responsive genes in stroma responsive MM cell lines, but not in stroma unresponsive lines, in which this transcriptional signature is constitutively upregulated. This observation suggests that the increased metformin sensitivity of MM cells co-cultured with BMSCs may reflect the ability of metformin to counteract the upregulation of p70S6K/mTOR pathway activity triggered by stroma. Our studies demonstrate a direct, growth-suppressive effect of metformin in MM cells in vitro, which can be potentiated by interactions with the bone marrow microenvironment. Taken together with the beneficial effect of this antidiabetic agent on circulating insulin and free IGF-I levels and its favorable safety profile, these data support the clinical evaluation of metformin in patients with MM.

Description: Context: Novel therapeutic strategies targeting cell cycle regulation are attractive for multiple myeloma (MM) because of the increased proliferative index in advanced drug-resistant disease. Having previously studied the role of Cdk inhibition in MM, we have looked at the cell cycle-related polo kinases (PLKs), because their expression is associated with adverse prognosis in solid tumors. We report preclinical studies on the anti-MM activity of the PLK1/2/3 small molecule inhibitor BI2536. Methods/Results: We tested 39 human tumor cell lines by MTT colormetric assay, including MM (n=26), T-ALL (n=8), solid tumors (n=5), as well normal human tissues. BI2536 exhibited activity in the low nano-molar range with IC50 values

Description: Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p

Description: Abstract 451 Given the heterogeneity of human cancers, to comprehensively interrogate the anti-tumor cytotoxicity of immune effector cells for the identification of novel treatments, high-throughput platforms are vital for screening the multitude of cell types and experimental conditions and drugs. Conventional assays for anti-tumor immune effector cell activity have limited high-throughput scalability (e.g. use of radioactivity for Cr release) or measure indirect markers (e.g. IFN-γ levels, LDH release). In addition, conventional cell viability assays (e.g. MTT, Alamar Blue, CellTiterGlo) are suboptimal because the background viability signal from immune cells confounds the selective readout of tumor cell viability. To address these limitations, we adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique (McMillin et al, Nature Med 2010) to study tumor-immune system interactions. We first applied CS-BLI to quantify the anti-tumor activity of IL-2 pre-stimulated PBMCs on luciferase (Luc)-expressing MM.1S multiple myeloma (MM) cells or KU812F leukemic cells. Immediate CS-BLI bioluminescence readout showed significant linear correlation with viable tumor cell number, both in the presence (r=0.9996, p