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Summary Jasmonates, plant stress hormones, have been demonstrated to be effective in killing various types of cancer cells. We therefore tested if methyljasmonate (MJ) has activity against multiple myeloma (MM) in vitro and in vivo. MM cell lines and primary MM tumour cells responded to MJ in vitro at concentrations that did not significantly affect normal haematopoietic cells, without stroma-mediated resistance. Brief MJ exposures of MM cells caused release of Hexokinase 2 (HK2) from mitochondria, rapid ATP depletion, perturbation of major intracellular signalling pathways, and ensuing mainly apoptotic cell death. Sensitivity to MJ correlated with lower cellular glucose consumption and lactate production, as well as lower intracellular protein levels of HK2, phosphorylated Voltage-dependent anion channel 2/3 (pVDAC2/3) and Aldo-keto reductase family 1 member C1 (AKR1C1), which represent potential biomarkers of responsiveness to MJ treatment, especially as AKR1C1 transcript levels also correlate with clinical outcome in bortezomib- or dexamethasone-treated MM patients. Interestingly, MJ synergized with bortezomib in vitro and prolonged survival of immunocompromised mice harbouring diffuse lesions of MM.1S cells compared to vehicle-treated mice (P = 0·0046). These studies indicate that jasmonates represent a new, promising strategy to treat MM.

Background Isothiocyanates, a family of phytochemicals found in cruciferous vegetables, have cytotoxic effects against several types of tumor cells. Multiple myeloma is a fatal disease characterized by clonal proliferation of plasma cells in the bone marrow. The growing body of preclinical information on the anti-cancer activity of isothiocyanates led us to investigate their anti-myeloma properties.Design and Methods We evaluated the anti-myeloma activity of the isothiocyanates, sulforaphane and phenethyl isothiocyanate, on a panel of human myeloma cell lines as well as primary myeloma tumor cells. Cell viability, apoptosis, cell cycle alterations and cell proliferation were then analyzed in vitro and in a xenograft mouse model in vivo. The molecular sequelae of isothiocyanate treatment in multiple myeloma cells were evaluated by multiplex analyses using bead arrays and western blotting.Results We observed that sulforaphane and phenylethyl isothiocyanate have activity against myeloma cell lines and patients‘ myeloma cells both in vitro and in vivo using a myeloma xenograft mouse model. Isothiocyanates induced apoptotic death of myeloma cells; depletion of mitochondrial membrane potential; cleavage of PARP and caspases-3 and -9; as well as down-regulation of anti-apoptotic proteins including Mcl-1, X-IAP, c-IAP and survivin. Isothiocyanates induced G2/M cell cycle arrest accompanied by mitotic phosphorylation of histone H3. Multiplex analysis of phosphorylation of diverse components of signaling cascades revealed changes in MAPK activation; increased phosphorylation of c-jun and HSP27; as well as changes in the phosphorylation of Akt, and GSK3α/β and p53. Isothiocyanates suppressed proliferation of myeloma cells alone and when co-cultured with HS-5 stromal cells. Sulforaphane and phenylethyl isothiocyanate enhanced the in vitro anti-myeloma activity of several conventional and novel therapies used in multiple myeloma.Conclusions Our study shows that isothiocyanates have potent anti-myeloma activities and may enhance the activity of other anti-multiple myeloma agents. These results indicate that isothiocyanates may have therapeutic potential in multiple myeloma and provide the preclinical framework for future clinical studies of isothiocyanates in multiple myeloma.

Conventional anti-cancer drug screening is typically performed in the absence of accessory cells of the tumor microenvironment, which can profoundly alter anti-tumor drug activity. To address this major limitation, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. Tumor cells (e.g. myeloma, leukemia and solid tumors) stably expressing luciferase are co-cultured with non-malignant accessory cells (e.g. stromal cells) for selective quantification of tumor cell viability, in presence vs. absence of stromal cells or drug treatment. CS-BLI is high-throughput scalable and identifies stroma-induced chemoresistance in diverse malignancies, including imatinib-resistance in leukemic cells. A stromal-induced signature in tumor cells correlates with adverse clinical prognosis and includes signatures for activated Akt, Ras, NF-κB, HIF-1α, myc, hTERT, and IRF4; signatures for biological aggressiveness and for self-renewal. Unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells interacting with stroma. One such compound, reversine, exhibits more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. Use of CS-BLI, therefore, enables refined screening of candidate anti-cancer agents to enrich preclinical pipelines with potential therapeutics that overcome stroma-mediated drug resistance and can act in a synthetic lethal manner in the context of tumor-stromal interactions.

We investigated the roles of specific subsets of donor APCs purified from bone marrow in donor T cell activation and graft-vs-leukemia (GvL) activity in murine models of hemopoietic stem cell transplantation. Lineage−CD11c+ APC precursors were separated from donor bone marrow based on expression of CD11b. Transplanting lineage−CD11c+CD11b− APC (CD11b− APC) in combination with c-kit+Sca-1+lineage− hemopoietic stem cells (HSC) and congenic donor T cells led to increased donor CD4+ and CD8+ T cell proliferation and higher donor T cell chimerism than with transplanting grafts containing HSC, T cells, and lineage−CD11c+CD11b+ APCs (CD11b+ APC), or grafts containing only HSC and T cells. Transplanting CD11b− APCs induced Th1/type 1 cytotoxic T lymphocyte donor T cell immune polarization and enhanced GvL activity of donor T cells without increased graft-vs-host disease in both MHC- and minor histocompatibility Ag-mismatched murine hemopoietic stem cell transplantation models, whereas CD11b+ APCs led to Th2/type 2 cytotoxic T lymphocyte donor T cell immune polarization. Donor CD11b− APCs were plasmacytoid dendritic cell progenitors (>90% CD317; PDCA-1+) and up-regulated CD80, CD86, and IL-12 during alloantigen presentation, whereas CD11b+ APCs expressed Gr-1 and up-regulated expression of programmed death ligands-1 and 2 after activation. These results are the first to show that manipulation of the content of donor APCs in allogeneic HSC grafts can regulate donor T cell immunity and enhance GvL without increasing graft-vs-host disease activity.

Purpose: p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM). Experimental Design: We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3. Results: The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells. Conclusions: This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy. (Clin Cancer Res 2009;15(23):7153–60)

The successful clinical development of thalidomide, bortezomib, and lenalidomide not only transformed the therapeutic management of multiple myeloma (MM) but also catalyzed a renewed interest in the development of additional classes of novel agents for this disease. This review focuses on a series of new therapeutics that have shown promising preclinical results, as well as encouraging safety profiles and early evidence of anti-MM activity in clinical studies, either alone or in combination with other, conventional or novel, anti-MM treatments. These agents include second-generation proteasome inhibitors and immunomodulatory agents, as well as members of other therapeutic classes, such as histone deacetylase inhibitors (HDAC), heat shock protein 90 (Hsp90) inhibitors, and the alkylphospholipid Akt inhibitor perifosine.

Clinical studies of patients with chronic myeloid leukemia revealed that a common pattern of response is a dramatic fall in the circulating population of blast cells, with a minimal or delayed decrease in marrow blasts, suggesting a protective environment. These observations suggest that a greater understanding of the interaction of stromal cells with leukemic cells is essential. Here, we present an in vivo system for monitoring relative tumor accumulation in leukemic mice and residual disease in leukemic mice treated with a tyrosine kinase inhibitor and an in vitro system for identifying integral factors involved in stromal-mediated cytoprotection. Using the in vivo model, we observed high tumor burden/residual disease in tissues characterized as significant sources of hematopoiesis-promoting stroma, with bone marrow stroma most frequently showing the highest accumulation of leukemia in untreated and nilotinib-treated mice as well as partial protection of leukemic cells from the inhibitory effects of nilotinib. These studies, which showed a pattern of leukemia distribution consistent with what is observed in imatinib- and nilotinib-treated chronic myeloid leukemia patients, were followed by a more in-depth analysis of stroma-leukemia cell interactions that lead to protection of leukemia cells from nilotinib-induced cytotoxicity. For the latter, we used the human BCR-ABL-positive cell line, KU812F, and the human bone marrow stroma cell line, HS-5, to more closely approximate the bone marrow–associated cytoprotection observed in drug-treated leukemia patients. This in vitro system helped to elucidate stromal-secreted viability factors that may play a role in stromal-mediated cytoprotection of tyrosine kinase inhibitor-treated leukemia cells. [Mol Cancer Ther 2008;7(5):1121–9]

Context: The Bcl-2 family of proteins regulates apoptosis in various models and may represent a promising therapeutic target in human malignancies. Objective/Methods: We evaluated the sensitivity of thyroid carcinoma cell lines (two papillary, one follicular, two anaplastic, three medullary) in vitro to BH3I-1 and BH3I-2′, two cell-permeable inhibitors of the Bcl-2 homology (BH)-3 domain-mediated interaction between proapoptotic and antiapoptotic Bcl-2 family members. The thyroid carcinoma cell line FRO was stably transfected with cDNA for Bcl-2 or constitutively active Akt and evaluated for sensitivity to BH3-domain inhibition. Results: BH3-domain inhibition disrupted the mitochondrial membrane potential in thyroid carcinoma cells, induced caspase-dependent apoptosis, and potently sensitized them to sublethal concentrations of doxorubicin and the proteasome inhibitor bortezomib (Velcade). Overexpression of constitutively active Akt suppressed BH3I-1-induced cell death. Bcl-2-overexpressing FRO cells were more resistant to conventional chemotherapeutic agents (such as doxorubicin) but significantly more sensitive to BH3I-1 than control cells and were found to overexpress caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and express less A1, BRaf, and FLIP transcripts. Conclusions: Bcl-2 expression protects thyroid carcinomas against chemotherapy-induced apoptosis. Nevertheless, overexpression of Bcl-2 may result in “oncogene addiction” of the cancer cell, which can be exploited by using BH3-domain inhibitors alone or in combination with other agents, including conventional chemotherapeutics (such as doxorubicin) or novel targeted therapies (such as the proteasome inhibitor bortezomib), for the treatment of aggressive thyroid cancer, including the medullary and anaplastic types.

Members of the inhibitor of apoptosis protein (IAP) family play a role in mediating apoptosis. Studies suggest that these proteins may be a viable target in leukemia because they have been found to be variably expressed in acute leukemias and are associated with chemosensitivity, chemoresistance, disease progression, remission, and patient survival. Another promising therapeutic target, FLT3, is mutated in about one third of acute myelogenous leukemia (AML) patients; promising results have recently been achieved in clinical trials investigating the effects of the protein tyrosine kinase inhibitor PKC412 on AML patients harboring mutations in the FLT3 protein. Of growing concern, however, is the development of drug resistance resulting from the emergence of point mutations in targeted tyrosine kinases used for treatment of acute leukemia patients. One approach to overriding resistance is to combine structurally unrelated inhibitors and/or inhibitors of different signaling pathways. The proapoptotic IAP inhibitor, LBW242, was shown in proliferation studies done in vitro to enhance the killing of PKC412-sensitive and PKC412-resistant cell lines expressing mutant FLT3 when combined with either PKC412 or standard cytotoxic agents (doxorubicin and Ara-c). In addition, in an in vivo imaging assay using bioluminescence as a measure of tumor burden, a total of 12 male NCr-nude mice were treated for 10 days with p.o. administration of vehicle, LBW242 (50 mg/kg/day), PKC412 (40 mg/kg/day), or a combination of LBW242 and PKC412; the lowest tumor burden was observed in the drug combination group. Finally, the combination of LBW242 and PKC412 was sufficient to override stromal-mediated viability signaling conferring resistance to PKC412. [Mol Cancer Ther 2007;6(7):1951–61]

B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAFV600E specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAFV600E patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-RafV600E induced a comparable reduction of viability in both wild-type and BRAFV600E mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAFV600E-harboring cells than wild-type ones, possibly because of better inhibitory activity against B-RafV600E. We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-RafV600E may provide clinical benefit for patients with thyroid cancer. [Mol Cancer Ther 2007;6(3):1070–8]

Purpose: The death receptor Fas is present in thyroid carcinomas, yet fails to trigger apoptosis. Interestingly, Fas has been reported to be actually overexpressed in papillary thyroid carcinomas, suggesting that it may confer a survival advantage. Experimental Design: We investigated the expression and activation status of Fas pathway mediators in thyroid carcinoma cell lines and tumor specimens. Results: All cell lines tested express Fas-associated death domain, procaspase-8, procaspase-9, and procaspase-3; resistance to Fas-mediated apoptosis could not be attributed to lack of any of these apoptosis mediators. Moreover, Fas death domain mutations were not found in our study. The proteasome inhibitors MG132 and PS-341 (bortezomib, Velcade), which lead to accumulation of the nuclear factor κB (NF-κB) inhibitor IκB, did not sensitize SW579 cells to Fas-mediated apoptosis, suggesting that resistance to Fas-mediated apoptosis is not due to proteasome or NF-κB activity. Cross-linking of Fas in vitro induced recruitment of Fas-associated death domain–like interleukin-1β–converting enzyme inhibitory protein (FLIP) instead of procaspase-8. Inhibition of FLIP expression with a FLIP antisense oligonucleotide resulted in significant sensitization to Fas-mediated apoptosis. Fas cross-linking promoted BrdUrd incorporation; activated the mitogen-activated protein kinase/extracellular signal–regulated kinase kinase/extracellular signal–regulated kinase, NF-κB, and activator protein-1 pathways in thyroid carcinoma cells in vitro; and protected cells from tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. We also found that good prognosis papillary thyroid carcinoma specimens exhibited higher immunoreactivity for cleaved (activated) caspase-8 than poor prognosis tumors. Conclusions: In thyroid carcinomas, the proteolytic cleavage and activation of caspase-8 depends on the balance between expression levels for procaspase-8 and FLIP and correlates with favorable clinical prognosis. Fas may actually stimulate proliferation and confer a survival advantage to thyroid cancer cells.

Using the cationic polymer, Polybrene, and the anionic polymer, chondroitin sulfate C, we concentrated recombinant retrovirus pseudotyped with an ecotropic envelope, which is susceptible to inactivation by high-speed concentration methods. To evaluate gene marking, murine bone marrow was harvested from C3H mice, transduced with polymer-concentrated GFP virus, and transplanted into lethally irradiated recipients. Total gene marking in mice averaged 30-35% at 8 weeks post-transplant and transgene expression remained stable for over 16 weeks. Using the polymer concentration method, a second retroviral vector encoding the drug resistant variant of dihydrofolate reductase (L22Y-DHFR) was concentrated and tested. Approximately 40% of transduced murine bone marrow progenitor cells were protected against trimetrexate concentrations that completely eliminated the growth of non-modified cells. These results show that anionic and cationic polymers can be combined to rapidly concentrate viruses that are normally difficult to concentrate, and the concentrated virus efficiently transduces hematopoietic stem cells.

BTK is a member of the TEC family of non-receptor tyrosine kinases whose deregulation has been implicated in a variety of B-cell-related diseases. We have used structure-based drug design in conjunction with kinome profiling and cellular assays to develop a potent, selective, and irreversible BTK kinase inhibitor, QL47, which covalently modifies Cys481. QL47 inhibits BTK kinase activity with an IC50 of 7 nM, inhibits autophosphorylation of BTK on Tyr223 in cells with an EC50 of 475 nM, and inhibits phosphorylation of a downstream effector PLCγ2 (Tyr759) with an EC50 of 318 nM. In Ramos cells QL47 induces a G1 cell cycle arrest that is associated with pronounced degradation of BTK protein. QL47 inhibits the proliferation of B-cell lymphoma cancer cell lines at submicromolar concentrations.

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4+ or CD8+ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Results: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell–cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR. Clin Cancer Res; 19(20); 5591–601. ©2013 AACR.

Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.

CR1642D, an endophytic isolate of Penicillium sp. collected from a Costa Rican rainforest, was identified through a high-throughput approach to identify natural products with enhanced antitumor activity in the context of tumor-stromal interactions. Bioassay-guided separation led to the identification of five xanthones (1-5) from CR1642D. The structures of the xanthone dimer penexanthone A (1) and monomer penexanthone B (2) were elucidated on the basis of spectroscopic analyses, including 2D NMR experiments. All of the compounds were tested against a panel of tumor cell lines in the presence and absence of bone marrow stromal cells. Compound 3 was the most active, with IC(50) values of 1-17 μM, and its activity was enhanced 2-fold against tumor cell line RPMI8226 in the presence of stromal cells (IC(50) 1.2 μM, but 2.4 μM without stromal cells).

Conventional assays evaluating antitumor activity of immune effector cells have limitations that preclude their high-throughput application. We adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique to perform high-throughput quantification of innate antitumor activity and to show how pharmacologic agents (eg, lenalidomide, pomalidomide, bortezomib, and dexamethasone) and autologous BM stromal cells modulate that activity. CS-BLI–based screening allowed us to identify agents that enhance or inhibit innate antitumor cytotoxicity. Specifically, we identified compounds that stimulate immune effector cells against some tumor targets but suppressed their activity against other tumor cells. CS-BLI offers rapid, simplified, and specific evaluation of multiple conditions, including drug treatments and/or cocultures with stromal cells and highlights that immunomodulatory pharmacologic responses can be heterogeneous across different types of tumor cells. This study provides a framework to identify novel immunomodulatory agents and to prioritize compounds for clinical development on the basis of their effect on antitumor immunity.

The NEDD8-activating enzyme is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases that regulate degradation of a distinct subset of proteins. The more targeted impact of NEDD8-activating enzyme on protein degradation prompted us to study MLN4924, an investigational NEDD8-activating enzyme inhibitor, in preclinical multiple myeloma models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability (EC50 = 25–150 nmol/L) in a panel of human multiple myeloma cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its bortezomib-sensitive parental cells. MLN4924 had submicromolar activity (EC50 values

Using a combination high-throughput screening technology, multiple classes of drugs and targeted agents were identified that synergize with dexamethasone (Dex) in multiple myeloma (MM) cells. Performing combination screening with these enhancers, we discovered an unexpected synergistic interaction between adenosine receptor agonists and phosphodiesterase (PDE) inhibitors that displays substantial activity in a panel of MM and diffuse large B-cell lymphoma (DLBCL) cell lines and tumor cells from MM patients. We have used selective adenosine receptor agonists, antagonists, and PDE inhibitors as well as small interfering RNAs targeting specific molecular isoforms of these proteins to dissect the molecular mechanism of this synergy. The adenosine A2A receptor and PDE2, 3, 4, and 7 are important for activity. Drug combinations induce cyclic AMP (cAMP) accumulation and up-regulate PDE4B. We also observe rigorous mathematical synergy in 3-way combinations containing A2A agonists, PDE inhibitors, and Dex at multiple concentrations and ratios. Taken together, these data suggest that A2A agonist/PDE inhibitor combinations may be attractive as an adjunctive to clinical glucocorticoid containing regiments for patients with MM or DLBCL and confer benefit in both glucocorticoid-sensitive and -resistant populations.

This study characterized the preclinical anti-myeloma activity of VE465, a low molecular weight pan-Aurora kinase inhibitor. After 96-h drug exposure, several multiple myeloma (MM) cell lines were more sensitive to VE465 compared to non-malignant cells. The anti-MM activity of VE465 was maintained in the presence of interleukin-6 and, interestingly, enhanced by co-culture with stromal cells. However, primary MM cells were less responsive than cell lines. Combinations with dexamethasone (Dex), doxorubicin (Doxo), and bortezomib showed no antagonism. Our study highlights the potential role of the tumor micro-environment in modulating the activity of this drug class.

The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway mediates proliferation, survival, and drug resistance in multiple myeloma (MM) cells. Here, we tested the anti-MM activity of NVP-BEZ235 (BEZ235), which inhibits PI3K/Akt/mTOR signaling at the levels of PI3K and mTOR. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assays showed that MM cell lines exhibited dose- and time-dependent decreased viability after exposure to BEZ235 (IC50, 25–800 nmol/L for 48 hours). MM cells highly sensitive (IC50, 800 nmol/L) than the majority of MM cell lines tested, suggesting a favorable therapeutic index. In addition, BEZ235 was able to target MM cells in the presence of exogenous interleukin-6, insulin-like growth factor-1, stromal cells, or osteoclasts, which are known to protect against various anti-MM agents. Molecular profiling revealed that BEZ235 treatment decreased the amplitude of transcriptional signatures previously associated with myc, ribosome, and proteasome function, as well as high-risk MM and undifferentiated human embryonic stem cells. In vivo xenograft studies revealed significant reduction in tumor burden (P = 0.011) and survival (P = 0.028) in BEZ235-treated human MM tumor-bearing mice. Combinations of BEZ235 with conventional (e.g., dexamethasone and doxorubicin) or novel (e.g., bortezomib) anti-MM agents showed lack of antagonism. These results indicate that BEZ235 merits clinical testing, alone and in combination with other agents, in MM. [Cancer Res 2009;69(14):5835–42]

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. We demonstrate up-regulated Akt activity in WM, and that Akt down-regulation by Akt knockdown and the inhibitor perifosine leads to significant inhibition of proliferation and induction of apoptosis in WM cells in vitro, but not in normal donor peripheral blood and hematopoietic progenitors. Importantly, down-regulation of Akt induced cytotoxicity of WM cells in the bone marrow microenvironment (BMM) context. Perifosine induced significant reduction in WM tumor growth in vivo in a subcutaneous xenograft model through inhibition of Akt phosphorylation and downstream targets. We also demonstrated that Akt pathway down-regulation inhibited migration and adhesion in vitro and homing of WM tumor cells to the BMM in vivo. Proteomic analysis identified other signaling pathways modulated by perifosine, such as activation of ERK MAPK pathway, which induces survival of tumor cells. Interestingly, MEK inhibitor significantly enhanced perifosine-induced cytotoxicity in WM cells. Using Akt knockdown experiments and specific Akt and PI3K inhibitors, we demonstrated that ERK activation is through a direct effect, rather than feedback activation, of perifosine upstream ERK pathway. These results provide understanding of biological effects of Akt pathway in WM and provide the framework for clinical evaluation of perifosine in WM patients.

Most conventional methods to sensitively quantify tumor cell proliferation and viability in vitro involve processing of cells in ways that preclude continuation of the respective experiment or prevent the longitudinal collection of data. This common technical feature of conventional assays limits their ability to provide detailed insight into the kinetics of tumor cell responses to treatment(s). Additionally, these limitations hinder the use of these assays to monitor how the kinetics of treatment response can be altered by nonmalignant "accessory" cells of the tumor microenvironment (e.g. bone marrow stromal cells [BMSCs] for hematologic malignancies or bone metastases of solid tumors). To address these obstacles, we modified our previously developed tumor cell compartment-specific bioluminescence imaging (CS-BLI) platform (McMillin et al. Nat Med. 2010), to enable longitudinal assessment of tumor cell response to diverse experimental conditions; we cultured luciferase-expressing tumor cells, with or without stromal cells, in the presence of bioluminescent substrates, using optimized conditions which provide detectable bioluminescent signal even after several days of culture, while having no adverse effect on the viability of tumor or non-malignant cells in this system. This modified approach (time-lapse CSBLI, [TL-CSBLI]) preserved the linear correlation of bioluminescent signal with tumor cell viability. Furthermore, results obtained at the end of the experiment and during interim time-points are consistent with those generated using either non-time-lapse applications of CS-BLI or conventional techniques. We applied TL-CSBLI to delineate, in high-throughput manner, the temporal dynamics of the responses of tumor cells (e.g. multiple myeloma (MM) and other hematologic malignancies) to diverse treatments (e.g. conventional chemotherapeutics, glucocorticoids; proteasome inhibitors (PIs, bortezomib or carfilzomib), and kinase inhibitors). Using the time-lapse capabilities of this assay, we evaluated tumor cell responses in the presence vs. absence of stromal cells. We observed that the kinetics of tumor cell response to diverse therapeutic classes are heterogeneous, even within the same tumor type: for instance, tumor cells with pronounced responses at the end of drug incubation (e.g. 24, 48, 72, hrs after initiation of treatment with PIs, DNA-damaging chemotherapeutics, or dexamethasone respectively), can have different magnitude of responses at intermediate time points. This suggests that TL-CSBLI data can further stratify treatment-responsive tumor cells into those with early vs. late kinetics of response. We also observed that the kinetics of the proliferative / anti-apoptotic effect conferred by stromal cells on tumor cells are highly variable between different cell lines, even within the same tumor type. For instance, the time between initiation of coculture and maximum stimulation of tumor cell viability by stromal cells was variable between cell lines and did not correlate with the magnitude of stimulation by stromal cells. Importantly, TL-CSBLI identified that the response of diverse types of tumor cells to treatments can be delayed in the presence of stromal cells, compared to conventional tumor cell monocultures: this initial delay in treatment response of tumor cells in stromal co-cultures may be observed even in cases where similar cytoreductive responses are eventually observed at later time-points in both the presence and absence of stromal cells. This observation suggests that a more expansive definition of stroma-induced resistance to a given treatment may be warranted, to specifically incorporate the ability of stromal cells to delay the tumor cell response to such treatment. In summary, TL-CSBLI enables detailed characterization of the kinetics of tumor cell responses to diverse experimental conditions. Its use can provide insight into the underappreciated impact that cell-autonomous variations or stroma-induced changes in the kinetics of tumor cell response to a given anti-tumor therapy can have on determining its efficacy. This is particularly consequential for agents (e.g. PIs) which have clinical pharmacokinetic profiles associated with transient peak exposure. Disclosures McMillin: Axios Biosciences: Equity Ownership; DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Negri:DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

Steady advances have been made with respect to genetically modifying hematopoietic stem cells (HSCs) using recombinant retroviruses, but there is still the problematic need of generating high viral titers. Using ecotropic pseudotyped recombinant retrovirus, which is typically inactivated during concentration methods employing centrifugation, we show that recombinant virus can be rapidly concentrated using a combination of polymers, and HSCs are readily transduced using concentrated virus. In addition, HSCs transduced with polymer-concentrated virus at very low MOIs can be expanded in vivo using an antifolate regimen if HSCs are induced to cycle prior to selection. Recombinant ecotropic pseudotyped virus encoding eGFP was generated by transient transfection of GP2-293 producer cells, and viral supernatants were collected over 72 hours. Viral stocks were concentrated by incubating the retroviral supernatant with equal concentrations of polybrene and chondroitin sulfate C followed by low-speed centrifugation. The resulting pellet contains competent ecotropic virus and was resuspended in cell growth media. To determine if polymer-concentrated virus can be used to transduced HSCs, bone marrow was isolated from C3H mice, transduced with a single application of concentrated virus (MOI~1) and transplanted into lethally irradiated recipients. Total gene marking in transplanted animals averaged 30–35%, as measured by flow at 8 weeks post transplant, and stable integration is observed for over 16 weeks in all hematopoietic lineages. In addition, retroviral vectors encoding a drug resistant variant of dihydrofolate reductase (L22YDHFR) was concentrated and tested using in vitro progenitor assays. Approximately 40% of murine bone marrow progenitor cells were protected against concentrations of trimetrexate that completely eliminated growth of non-modified cells. Bone marrow transduced with concentrated virus at a low MOI (~0.5) and transplanted into lethally irradiated recipients results in 5–10% gene marking 8 weeks post transplant, as measured by real-time PCR. Mathematical models were generated to calculate the efficiency of in vivo selection using variants of DHFR and antifolate selection under conditions of steady-state hematopoiesis, which predicted multiple rounds of selection are required to achieve only modest in vivo selection. However, if HSCs are induced to cycle, it is predicted that significant selection can be achieved with only a few rounds of selection. Using 5-FU to induce cycling, we show that >6-fold increase in circulating gene-modified cells can be achieved with a single round of antifolate selection. These results show that by incorporating basic retroviral processing steps, HSCs can be efficiently transduced using polymer-concentrated ecotropic virus, resulting in high level stable expression. In addition, HSCs transduced with variants of DHFR can be selectively enriched if stem cells are induced to cycle prior to antifolate selection.

Chemotherapy remains a central approach for the treatment of cancer but is limited by severe toxic side effects, particularly on the hematopoietic system. Various immunotherapy strategies are less toxic, such as tumor vaccines and cytokine therapy, and are emerging areas of cancer treatment. Although promising, these methods have limited effectiveness on established tumors. Combining chemotherapy agents with immunotherapy has proven difficult because development of an immune response can be significantly impaired in a chemotherapy-induced immunosuppressive environment. Therefore, we are investigating strategies that will allow cytotoxic chemotherapy and immunotherapy treatments to be used concurrently. Our aim is to generate drug resistant immunocompetent T-lymphocytes using retroviral gene transfer of cDNA sequences that confer drug resistance. Our drug resistant immunotherapy approach entails generating tumor-specific genetically engineered immunocompetent cells, such as T and NK cells, by transducing hematopoietic stem cells (HSC) with a retroviral vector encoding the L22Y variant of dihydrofolate reductase (L22YDHFR). This construct confers >10-fold resistance to the cytotoxic agent trimetrexate (TMTX) compared to non-modified hematopoietic cells. Drug resistant lymphocytes are generated by transplanting drug resistant bone marrow into irradiated recipient mice. Upon engraftment immunocompetent cells encoding the drug resistant marker are generated. To examine the effectiveness of drug resistance immunotherapy, C3H mice reconstituted with drug resistant bone marrow were inoculated with AG104 sarcoma cells and treated with either an agonistic anti-4-1BB antibody alone, TMTX alone, or the combination. Approximately 60% of animals transplanted with gene-modified cells and treated with anti-4-1BB, which induces CD8+ and NK anti-tumor responses, achieved complete tumor regression within 30 days. Although TMTX treatment alone regressed tumor size transiently, there was no effect on tumor clearance. However, animals treated with the combination of TMTX and anti-4-1BB antibody completely eliminated tumors in 100% of mice. Also important, compared to animals transplanted with non-modified marrow, hematopoiesis of animals transplanted with drug resistant marrow is protected against chemotherapy-induced toxicities. These results show that drug resistant immunocompetent cells can be conserved after a chemotherapy challenge and appear to retain their anti-tumor activity. This study supports our hypothesis that, in the context of drug resistant immunocompentent cells, chemotherapy can be used to control tumor growth and provide the critical time needed for the establishment of an effective anti-tumor immune response.

Abstract 2469 Introduction: Kinases are integral components of diverse signaling cascades which can mediate microenvironment-derived resistance of tumor cells to apoptosis. We thus hypothesized that tumor cell-microenvironment interactions alter the anti-tumor activity of multi-targeted kinase inhibitors and could contibute to discordant efficacy results in conventional preclinical models which do not account for these interactions vs. in clinical studies. Methods: We evaluated 6 multi-targeted kinase inhibitors initially developed to target IGF1R (XL-228), Akt/p70S6K (EXEL-6075), B-/C-RAF(EXEL-0094), CHK1/2 (XL844), met/VEGFR2(EXEL-6323) and JAK2 (XL019). Compounds were tested (0–20 μM, 48 hrs) against luciferase-expressing human multiple myeloma (MM) (n=8), mantle cell lymphoma (n=2) and breast cancer (n=1) cell lines. For each of these treatments, tumor cells were cultured in isolation or co-cultured with immortalized human bone marrow stromal cells (BMSCs, n=2); lung fibroblasts; hepatocytes; or brain astrocytes. Tumor cell viability was quantified by tumor cell compartment-specific bioluminescence imaging (CS-BLI). For each compound and tumor cell line, the area under the dose-response curve (AUC) was calculated as % of the AUC that corresponds to 100% tumor cell viability at all doses, to provide cumulative measure of drug activity across the range of tested drug concentrations. Differences in % AUCs in presence vs. absence of accessory cells were compared by 2-tailed paired t-test. The average value and skewness of distribution for log(10)IC50 of enzymatic activity for each kinase and inhibitor tested were calculated for quantitative comparison of multi-targeted nature of these compounds. Results: Co-culture of most tumor cell lines with accessory cells triggered resistance for 5 of 6 compounds tested, as evidenced by statistically significant (p Conclusions: Accessory cells from different tissues significantly modulated tumor cell responses to diverse multi-targeted kinase inhibitors. Co-cultures enhanced the anti-tumor activity of JAK2 inhibitor, but decreased the activity of the other compounds tested. These different responses did not correlate with how multitargeted each inhibitor was. The sensitization to the JAK inhibitor may specifically reflect increased dependency on JAK signaling for tumor cells when they interact with accessory cells. Accessory cell-induced resistance to some kinase inhibitors (e.g. IGF1R, Akt, CHK1–2) may account for observed differences in their preclinical vs. clinical efficacy. These results provide a rationale for preclinical testing of compounds against large panels of tumor cell lines and clinically-relevant accessory cell types to help develop therapeutics against tumors localized in specific tissue microenvironmental niches. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

Abstract 2923 Midostaurin (PKC412; Novartis Pharmaceuticals) is a multi-targeted kinase inhibitor currently being evaluated in clinical trials in acute myelogenous leukemia (AML), because of its potent activity in cells expressing mutant FLT3. Prior preclinical studies from our groups have shown that PKC412 has FLT3-independent anti-MM activity, and the effects on AML cells is suppressed by the presence of conditioned media from bone marrow stromal cells (BMSCs), such as the immortalized BMSC line HS-5 (Weisberg et al. Mol Cancer Ther 2007). In this study, we evaluated whether the microenvironment-dependent drug resistance to PKC412 applies to not only AML cells, but also to cells from MM and other FLT3-negative malignant cells. We tested a panel of cells from MM (n=8), FLT-ITDneg AML (n=1), CML (n=2) and breast cancer (n=2) for their response to PKC412 in the presence or absence of BMSCs and other non-malignant accessory cells using tumor cell compartment-specific bioluminescence imaging (CS-BLI), as in our antecedent studies (McMillin et al. Nat Medicine 2010). We also compared the PKC412 response of the aforementioned neoplastic cells when cultured in vitro in the presence or absence of conditioned media (CM) from different types of BMSCs known to confer PKC412 resistance in FLT3-mutant AML cells. Consistent with our previous studies of PKC412 treatment in conventional cultures of MM cells in isolation, we observed that PKC412 exhibits an anti-proliferative effect within the first 24 hrs of treatment, with major reduction of the numbers of viable cells at 48 and 72 hrs. At sub-micromolar doses that did not significantly affect the viability of non-malignant accessory cells tested, PKC412 had similar (or for some MM cell lines had more pronounced) activity against the MM cells, both in the presence and absence of the non-malignant accessory cells tested (HS-5, HS-27a, NIH-3T3 cells with or without transfection with human CD40L, etc.). In contrast, under the same experimental conditions, coculture with either BMSCs or exposure to their conditional media, decreased the response of MM cells to dexamethasone. These results suggested in contrast to the impact on FLT3mut AML, that the anti-MM activity of PKC412 is preserved (and in some cases slightly enhanced) when the MM cells interact with microenvironmental accessory cells and/or their secreted growth/survival factors. To obtain insight on possible mechanistic foundations of these observations, we examined the pattern of kinases inhibited by PKC412 at sub-μM concentrations (using FLT3 and FGFR3, known targets of PKC412 as positive controls). The results of in vitro kinase activity assays showed that PKC412 potently suppresses the aforementioned positive controls, but also exerts >50% inhibitory effect on the in vitro activity of additional kinases such as Akt2, Pim1, GSK3a, PDK1, p70S6K, SRC and Aurora A. Many of these kinases are known to participate in proliferative/anti-apoptotic signaling cascades downstream of cytokine/growth factor receptors or cell adhesion-mediated events triggered during MM–stromal interactions. We therefore conclude that the influence of the tumor microenvironment on the anti-neoplastic effects of PKC412 may be tumor-type dependent. The anti-MM activity of PKC412 is not subject to drug resistance triggered by non-malignant accessory cells, and conversely is occasionally moderately enhanced by these MM-stromal interactions. Mechanistically, this observation may be attributed in part to the multi-targeted nature of this inhibitor and, in particular, its aggregate impact on several kinases known to mediate stroma-induced proliferative and antipoptotic signaling in MM. Disclosures: Griffin: Novartis Pharmaceuticals: Consultancy, Research Funding. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mitsiades:EMD Serono: Research Funding; AVEO Pharma: Research Funding; Amgen: Research Funding; OSI Pharmaceuticals: Research Funding; PharmaMar: licensing royalties; Amnis Therapeutics: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

Abstract 2988 Background: Multiple myeloma (MM) and Waldenström Macroglobulinemia (WM) have both shown clinical responses to Bortezomib therapy which blocks the elimination of ubiquitin tagged regulatory proteins by the proteasome. The NEDD8 activating enzyme (NAE)-inhibitor MLN4924 is a novel agent which demonstrates selective inhibition of the proteins for degradation in the ubiquitin pathway and may offer benefits to MM and WM patients through the more targeted approach. Methods: A panel of human MM and WM cell lines were tested for their in vitro response to MLN4924 using MTT colorimetric survival assays. MM and WM cell lines tested exhibited dose and time dependent decrease of their viability upon exposure to MLN4924 (IC50=25-150 nM). In addition, miRNA and gene expression studies in response to MLN4924 were compared to treatment of the same cells with bortezomib. In vivo safety studies were performed in mice and animal efficacy studies are ongoing in both MM and WM engrafted mice. Results: A panel of MM and WM cells were treated with MLN4924 for 72hrs and compared to the colon carcinoma line HCT116 and normal cell lines HS-5 (stroma) and THLE-3 (hepatocytes). In addition, a longitudinal assessment of viability of MM1S (MM) and BCWM1 (WM) cells during a 72hr incubation with MLN4924 (500nM) showed commitment to death 1000 nM) than the MM or WM cell lines tested, suggest that this compound exhibits a rapid, tumor-selective effect at clinically relevant conditions. We also evaluated primary MM (CD138+) and WM (CD19+) patient bone marrow cells and observed sub-μ M activity by MLN4924. In addition, we tested a series of combinations of MLN4924 with dexamethasone, doxorubicin and bortezomib in both MM1S and BCWM1 cells lines and observed additive activity or greater with MLN4924. Gene expression profiling revealed distinct signatures, in MM1S and BCWM1 lines, as well as distinct patterns of gene expression changes which were induced by MLN4924 vs. bortezomib. For instance, while bortezomib potently induces a compensatory upregulation of transcripts for ubiquitin/proteasome and heat shock protein genes which, in MM1S or BCWM1 cells, were not observed in response to MLN4924 treatment. Additional studies with the proteasome inhibitor MLN9708 revealed similar patterns of expression as bortezomib. These results indicate that MLN4924 does not induce pronounced proteotoxic stress in MM or WM cells, highlighting the distinct effect of MLN4924 on the ubiquitin/proteasome pathway compared to inhibitors which target the 20S proteasome subunit. Longitudinal miRNA profiling revealed a distinct pattern of miRNA expression in MLN4924-treated vs. bortezomib-treated MM and WM cells. Lastly, animal safety studies showed that MLN4924 was tolerated at doses up to 60mg/kg 2x daily for 1 week. Efficacy studies in MM and WM are ongoing. Conclusions: MLN4924 induces cell killing at sub-μ M concentrations for both MM and WM cells with higher sensitivity of tumor cells compared to normal tissues, exhibits selective gene expression and miRNA regulation and can be safely administered to mice. These studies provide the framework for the clinical investigation of MLN4924 in MM and WM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Smith:Millennium: Employment. Birner:Millennium: Employment. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Abstract 1729 Poster Board I-755 Background Multidrug transporters are energy-dependent transmembrane proteins which can efflux a broad range of anticancer drugs and thereby play a role in resistance to the actions of substrate agents. Classically, three transporters, p-glycoprotein (Pgp; MDR-1; ABCB1), multidrug resistant protein-1 (MRP-1; ABCC1) and breast cancer resistance protein (BCRP; MXR; ABCG2), have been found to have the broadest substrate specificity and a strong correlation with drug resistance in vitro and in vivo in many models and forms of cancer. We have sought to characterize the interaction of bortezomib with these transporters and thereby explore the potential for these agents to play a role in resistance. Bortezomib is a novel proteosome inhibitor with significant activity in multiple myeloma, although subsets of patients remain refractory to the activity of the drug. Hence, better characterization of the interactions of this drug with classical resistance mechanisms may identify improved treatment applications. Methods and Results We investigated the role of these transporters by using isogenic cell line models which are resistant due to overexpression of a particular transporter: DLKP lung cancer cell line that overexpresses MRP-1; DLKP-A which overexpresses Pgp; and DLKP-SQ-Mitox which overexpresses BCRP. DLKP-A cells exhibited a 4.6-fold decrease in responsiveness to bortezomib compared to parental DLKP cells. In DLKP-SQ-Mitox, bortezomib-induced cytotoxicity was comparable to DLKP. When bortezomib was combined with elacridar, a Pgp and BCRP inhibitor, significant synergy was evident in DLKP-A (100% viable cells with single agent treatment versus 11% with the combination), but not DLKP-SQ-Mitox. Sulindac, an MRP-1 inhibitor, combined with bortezomib failed to produce any synergy in MRP-1 positive DLKP cells. Conversely, combination assays of Pgp substrate cytotoxics such as doxorubicin with Bortezomib were largely additive in nature. This indicates that bortezomib has little, if any, direct Pgp inhibitory activity, as combinations of a traditional Pgp inhibitor (such as elacridar) and doxorubicin would show marked synergy rather than just an additive effect in Pgp positive cells. To further characterize the extent of this interaction with Pgp, we conducted cytotoxicity assays in cell lines with varying levels of Pgp overexpression. NCI/Adr-res (ovarian cancer, high Pgp overexpression), RPMI-Dox40 (multiple myeloma, moderate Pgp overexpression) and A549-taxol (lung cancer, low Pgp overexpression). The combination of bortezomib and elacridar that produced the most synergy was in cell lines expressing moderate to high levels of Pgp expression. Cell lines with lower Pgp expression produced an additive cytotoxicity. We next examined whether bortezomib had any direct effect on Pgp expression. In RPMI-Dox40 cells, Pgp expression is reduced in a time-dependent manner with bortezomib treatment. Conclusions Our studies therefore show that bortezomib is a substrate for Pgp but not the other drug efflux pumps. In tumor cells expressing high levels of Pgp, the efficacy of bortezomib is synergistically enhanced by combinations with a Pgp inhibitor, while bortezomib treatment itself can reduce the expression of Pgp. This study suggests that in the subset of patients with advanced multiple myeloma or solid tumors which express high levels of Pgp, inhibition of its function could contribute to enhanced responsiveness to bortezomib. Disclosures Richardson: millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; celgene: Membership on an entity's Board of Directors or advisory committees, speakers bureau up to 7/1/09; MLNM: speakers bureau up to 7/1/09. Mitsiades:Millennium Pharmaceuticals : Consultancy, Honoraria; Novartis Pharmaceuticals : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: licensing royalties ; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding. Anderson:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Biotest AG: Consultancy, Research Funding.

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 Conclusion: Proteins pivotal for cell cycle progression represent promising targets for treating highly proliferating tumors. Treatment of MM with a PLK inhibitor provides evidence that polo kinases are promising targets for MM therapy. Importantly, BI2536 activity was enhanced in the presence of stromal cells, providing evidence that this class of compounds will be active in the tumor microenvironment.

The “guardian of the genome” p53 is inactivated in most human cancers via missense mutations in its DNA binding core domain or via overexpression of the human homolog of Mdm2 (Hdm2), an E3 ubiquitin ligase that binds and ubiquitinates p53 thereby leading to its degradation via the ubiquitin/proteasome pathway. Direct inhibition of Mdm2 function could potentially stabilize p53 and activate the p53 apoptotic pathway, which could be a useful non-genotoxic approach for the treatment of cancer. Nutlin-3, a cis-imidazoline small molecule with affinity for the p53-binding pocket of Mdm2 and capable of disrupting the p53-Mdm2 interaction, can activate p53 and induce apoptosis in vitro in many malignancies, including multiple myeloma (MM) cells. We hypothesized that suppression of Mdm2-mediated p53 ubiquitination may synergize with accumulation of p53 triggered by bortezomib. To address this question, we evaluated the response of MM cells vs. select models of epithelial cancers to bortezomib and its combination with nutlin-3. We observed that breast (MDA-MB-231-luc); prostate (DU145); thyroid (SW579, FRO, WRO, TT) and colon (ARO/HT-29) cancer cell lines exhibited synergistic apoptotic response to the combination of sublethal concentrations of bortezomib plus nutlin-3. This synergistic killing effect was associated with synergistic increase in the expression of p53, p21, Mdm2, Bax, Noxa, PUMA and the cleavage of caspase-3, caspase-9 and PARP. In contrast, MM cell lines (MM-1S, MM-1R, NCI-H929, KMS-11) exhibited mostly an additive effect when treated with the combination of bortezomib plus nutlin-3. To further probe the biological relevance of these findings in the context of the bone microenvironment in MM, we compared the transcriptional profile of p53 target genes as well as the response to nutlin-3 in MM cells cultured alone vs. in the presence of bone marrow stromal cells (BMSCs). We observed that co-culture with BMSCs attenuates the response of MM cells to single-agent nutlin-3 and is associated, in p53 wild-type cells, with increased amplitude of the transcriptional signature of genes suppressed by activated p53, suggesting that BMSCs suppress p53 activity in MM cells. Overall, these results suggest a complex correlation between the Mdm2/p53 and proteasome pathways: many p53 mutant epithelial cancer models can become more bortezomib-responsive by Mdm2 inhibition, while MM cells, with higher baseline responsiveness to bortezomib and nutlin-3, typically do not exhibit significant increases in their response to the combination of these 2 drug classes compared to the single-agent treatments. These observations suggest that concurrent Mdm2 inhibition may extend the spectrum of bortezomib applications to tumor types with currently limited single-agent response to proteasome inhibition. In MM, p53 mutations have been historically considered to be present in only late stage disease (e.g. plasma cell leukemia/extramedullary MM), but their prevalence may increase in the future as more patients with advanced MM survive longer thanks to recently introduced drug classes (thalidomide, bortezomib, lenalidomide). In those MM patients who harbor p53 pathway lesions and have developed bortezomib-resistance/refractoriness, combinations of bortezomib with Mdm2 inhibitors may also represent in the future an intriguing potential therapeutic option that merits further preclinical and clinical evaluation.

Using a combination high throughput screening technology, we have discovered an unexpected synergistic interaction between adenosine A2A receptor (A2A) agonism and phosphodiesterase (PDE) inhibition that displays substantial activity in preclinical Multiple Myeloma (MM) models. High throughput combination screening allows the systematic testing of combinations of approved drugs and other biologically active molecules in cell based assays of tumor cell proliferation and viability. In this approach we generate a dose matrix for each chemical combination, capturing the combined activity of two compounds over a broad range of single agent concentrations. Quantitative scoring of dose response matrices using models of drug interactions allows insight into the biological mechanism of action of drug combinations and the discovery of novel therapeutic applications. Using a panel of 4 MM cell lines (H929, MM.1S, MM.1R and RPMI-8226) we evaluated a defined set of combinations of approved drugs and molecular probes. A total of 2841 unique combinations were evaluated, 648 of which were assayed in all 4 cell lines. Multiple combinations were identified that exceeded the Loewe additivity model including enhancers of known anti-cancer drugs and combinations of targets not previously know in multiple myeloma. Multiple classes of drugs and targeted agents were identified that synergize with dexamethasone. Backcrosses of these active agents revealed multiple dexamethasone enhancers that also synergized with each other. Two of these classes, A2A agonists and PDE inhibitors demonstrated high levels of synergy and good breadth of activity across several MM cell lines. Interestingly, while the majority of the combinations screened had little or no synergistic effect in the glucocorticoid resistant MM.1R cell line, combinations of PDE inhibitors and A2A agonists demonstrated substantial efficacy (>90% inhibition of proliferation) and striking synergy (combination indices

We have previously demonstrated that a consistent feature of malignant plasma cells of multiple myeloma (MM) is the aberrant expression of genes important in patterning and development, such as members of Hedghehog (Hh) pathway (FE Davies et al, Blood 2003). These findings suggest that overexpression of genes of this pathway, already involved in many solid tumors and recently implicated in maintaining a proposed MM stem cell compartment (CD Peacock et al, PNAS 2007), might be one of the mechanism through which Hh-signaling contributes to tumorigenesis in MM. Therefore, several small molecule modulators of Hh-pathway, which work as agonists and antagonists, are currently under development. We evaluated, by microarray analysis, the expression of Hh pathway genes in MM cell lines and primary MM cells vs. plasma cells from healthy donors. We found that primary MM cells overexpress Sonic (Shh), Smoothened (Smo), Patched (Ptc), Gli-1 and Gli-3 (relative expression ratios ranging from +1.8 to +5.0). Overexpression of Patched was also observed in most of the MM cell lines analyzed (+5.0 ratio in 5 of 6 MM cell lines). Additionally, we confirmed the expression of Shh and of Gli-1, by flow cytometry and western blotting respectively, in a large panel of MM cell lines. These data suggest an activation of the Hh-pathway in MM that, in some cell lines, is Shh-dependent. Therefore, we investigated the therapeutic potential of Hh-inhibitors in MM. We assayed the cell viability and proliferation, by MTT and Thymidine uptake respectively, in 8 MM cell lines after 72 hours of treatment with the small molecule Smo-inhibitor CUR-0199691 (Genentech). We observed a reduction in MM cell viability, with IC50 values ranging between 4.5–9.5 μM in these 8 cell lines and an inhibition of MM cell proliferation with IC50 values ranging between 0.5 and 2.5μM in the same cell lines. MM cell sensitivity to this compound appears to be related to the level of expression of Gli-1, since the cell lines with lower level of expression of Gli-1 were more sensitive. The treatment of these MM cell lines with Cyclopamine, another Hh-inhibitor, showed an IC50 between 7.5μM and 10μM after at least 96 hours of treatment in 4 of the MM cell lines tested. CUR-0199691 is also active in primary MM cells, triggering inhibition of proliferation by 50% at 5μM after only 24h of treatment, while cyclopamine reduces MM cell proliferation (normalized to the effect of tomatidine, its inactive analog) by 30% at 20μM after a 48 hour treatment. Annexin V-PI staining of Hh inhibitor-treated KMS11 cells, which are one of the most sensitive MM cell lines, showed induction of apoptosis, evidenced by detection of 12 and 15% of MM cells being Annexin V+/PI- after 48h and 72h respectively with 5μM of CUR-0199691. These results, taken together, show that the Hh-pathway is fuctionally active in MM and that the novel Hh pathway inhibitor CUR-0199691 is 4–5 times more effective than cyclopamine in both MM cell lines and primary MM cells. These studies provide the framework for further preclinical evaluation of CUR-0199691 in MM models towards possible future clinical trials.

Context: The molecular chaperone hsp90 is a major anti-cancer therapeutic target because it regulates the function of proteins with pivotal roles in tumor cell proliferation, survival and drug resistance, including mutated/chimeric oncoproteins or oncogenic kinases/receptors. Our preclinical studies on the ansamycin hsp90 inhibitor tanespimycin (17-AAG) provided the rationale for clinical trials, either alone or in combination with the proteasome inhibitor bortezomib, for treatment of relapsed/refractory MM. In this study, we report preclinical studies of the new, non-ansamycin, hsp90 inhibitor NVP-AUY922. Methods/Results: We tested 36 human MM cell lines and observed with MTT colorimetric survival assays potent time- and dose-dependent anti-MM activity of NVP-AUY922. IC50 values were Conclusion: The new hsp90 inhibitor NVP-AUY922 has potent in vitro activity against MM cells resistant to conventional therapeutics, with selectivity for malignant compared to normal cells. Ongoing in vivo experiments and studies to identify biomarkers of pronounced sensitivity to NVP-AUY922 will help provide a framework for potential clinical trials of NVP-AUY922 in MM and other neoplasias.

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 5 μM), further underscoring its selectivity towards neoplastic cells. EXEL-6075-responsive cells included MM cells resistant to diverse conventional and novel agents, such as dexamethasone, bortezomib, and immunomodulatory thalidomide derivatives, suggesting that dual Akt/p70S6K inhibition may overcome mechanisms of constitutive resistance to other currently available therapeutics. EXEL-6075 was also able to overcome the protective effects of IGF-1 and IL-6 on MM.1S cells. In addition, there was modest, if any, protection conferred to MM cells by BMSCs at low drug doses. Interestingly, when we compared the in vitro activity of EXEL-6075 against cell lines from MM vs. other neoplasias (including 11 epithelial cancer cell lines which also exhibited dose-dependent response to this kinase inhibitor), EXEL-6075 was significantly more active against MM cells, as evidenced by the distribution of IC50 values and AUCs of the dose-response curves for each group of cell lines (p=0.028, t-test). Ongoing studies are utilizing gene expression profiles of MM cells highly- vs. moderately-responsive to EXEL-6075, in order to identify putative markers of sensitivity vs. resistance to this kinase inhibitor Conclusion: Targeting the Akt-mTOR-p70S6K pathway is an attractive strategy for the treatment of MM due to the central role Akt plays in proliferation and survival of MM. Inhibition of Akt using the small molecule inhibitor EXEL-6075 resulted in sub-μM killing of MM cell lines with > 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.

Context: Conceptually, targeting proteins involved in cell cycle regulation is an appealing therapeutic strategy for multiple myeloma (MM), given the increased proliferative rate of biologically aggressive multi-drug resistant MM cells compared to not only their normal plasma cell counterparts, but also MM cells from earlier stages of the disease. Within the intricate network of signaling cascades and regulatory checkpoints involved in cell cycle progression and survival of neoplastic cells, cyclin-dependent kinases (CDKs) have emerged as intriguing therapeutic targets. We report the results of preclinical studies on the anti-MM activity of the selective CDK1/2 small molecule inhibitor NVP-LCQ195/AT9311 (Novartis Pharma/Astex Therapeutics). Methods/Results: In MTT colorimetric survival assays against a panel of 28 human MM cell lines, NVP-LCQ195 exhibited dose-dependent anti-MM activity, with IC50 values at or below ~0.5 μM (a pharmacologically relevant concentration) for 12 of 28 cell lines many of which are resistant to conventional or novel anti-MM therapies (e.g. dexamethasone, melphalan, thalidomide derivatives). NVP-LCQ195 was also active against primary MM cells isolated from heavily-pretreated/drug-resistant cases of MM. In marked contrast, the IC50 values of NVP-LCQ195 against non-malignant cells such as bone marrow stromal cells (BMSCs) and immortalized hepatocyte cells was >4 μM. In addition, both PHA-stimulated and unstimulated normal donor PBMCs were less sensitive than the majority of MM cell lines of our panel. Treatment with NVP-LCQ195 was able to overcome the protective effects conferred on MM cells by exogenous IL-6 (10ng/mL) or IGF-1 (50ng/mL), as well as by co-culture of MM cells with bone marrow stromal cells (BMSCs). NVP-LCQ195 was observed to trigger a distinct pattern of S-phase arrest, followed by eventual induction of apoptotic cell death. Mechanistic studies revealed that hsp90, B-raf, cyclin D1, and cyclin E2 levels decrease in response to NVP-LCQ195 treatment, while caspases- 3, -8 and PARP are cleaved within 16 and 24 hrs of drug treatment. We evaluated a series of combinations of this agent with conventional (e.g. dexamethasone, doxorubicin) and novel (e.g. bortezomib) anti-MM agents. No evidence of antagonism with any of these anti-MM agents was observed, indicating that combinations of NVP-LCQ195 with the aforementioned anti-MM agents can be feasible in clinical settings. Conclusion: Functional inhibition of proteins involved in cell cycle regulation remains an attractive approach for the treatment of MM. Treatment of MM cells with the CDK1/2 inhibitor NVP-LCQ195 induces, in a subset of MM cell lines, a distinct pattern of initial cell cycle arrest, anti-proliferative/pro-apoptotic molecular sequleae and eventual induction of cell death. Our results determine intriguing potential combination regimens for MM treatment. Further in vitro and in vivo studies are attempting to delineate molecular markers of MM cell responsiveness vs. resistance to NVP-LCQ195 and provide a framework for individualized treatment of select MM patients with this interesting class of compounds.

PURPOSE. To evaluate the potential of proteasome inhibitors, a novel class of antitumor agents, for the treatment of retinoblastoma. The proteasome inhibitor bortezomib (PS-341, Velcade; Millennium Pharmaceuticals, Cambridge, MA), approved by the US Food and Drug Administration for the treatment of multiple myeloma, is being studied for the treatment of several other malignancies. Among other effects, it inactivates the transcription factor nuclear factor-B (NF-B) by blocking the degradation of its inhibitor, IB. NF-B, which is constitutively active in human retinoblastoma cells and promotes their survival, represents a therapeutic target for patients with this malignancy. METHODS. The authors evaluated the effect of bortezomib on the retinoblastoma cell lines Y79 and WERI-Rb1 in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry with propidium iodide, gene expression profiling, RT-PCR, and immunoblotting. RESULTS. Bortezomib induced caspase-dependent apoptosis in both retinoblastoma cell lines at clinically achievable concentrations. Bortezomib upregulated heat-shock proteins, other stress-response proteins, proapoptotic molecules, cell-cycle regulators, transcription factors, cytokines, and several proteasome subunits and solute carrier proteins, whereas it downregulated antiapoptotic and adhesion molecules. Bortezomib also induced cleavage of caspases, Bid and poly(ADP-ribose) polymerase (PARP), and sensitized retinoblastoma cells to doxorubicin. CONCLUSIONS. Bortezomib induces a stress response and triggers caspase-dependent apoptosis in human retinoblastoma cells at clinically achievable concentrations. This study provides insight into the molecular mechanism(s) of the antitumor activity of bortezomib and a basis for future preclinical studies leading to clinical trials of bortezomib, alone or in combination with conventional chemotherapy, to improve patient outcomes in retinoblastoma. (Invest Ophthalmol Vis Sci. 2007;48:4706‐4719)

Background: The PI3K and NFk-B/proteasome pathways are major regulators of survival in Multiple Myeloma (MM). Previous studies have demonstrated clinical efficacy of bortezomib in MM; however, not all patients responded to this agent. mTOR inhibitors have demonstrated significant in vitro and in vivo activity in MM, specifically clinical trials with the mTOR inhibitor CCI-779 (Wyeth) in MM. Therefore, we examined whether inhibition of the PI3K pathway by mTOR and proteasome inhibitors may lead to synergistic activity in MM. Methods: MM cell lines (MM.1S, RPMI, U266, OPM2) were treated with rapamycin 1–5nM (Sigma Aldrich), bortezomib 2.5–10nM (Millenium, MA), or the combination. Cytotoxicity was measured by the MTT assay at 48 hrs; DNA synthesis was measured using thymidine uptake assay; apoptosis was studied using Apo2.7 by flow cytometry, and cell cycle regulation was determined using flow cytometry. To determine whether these agents can overcome the growth advantage conferred by bone marrow stromal cells (BMSCs), we co-cultured cell lines with stromal cells. Normal peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with synergistic activity determined as a combination index (CI) of Results: Rapamycin induced dose-dependent cytotoxicity from 0.1nM to 1nM, with an IC50 of 5nM in MM.1S and OPM2. Interestingly, higher doses did not induce further cytotoxicity, confirming that low doses of rapamycin are as effective as higher doses. RPMI and U266 MM cell lines were less sensitive to rapamycin, with 5nM inducing 40% and 20% decrease in survival, respectively. Bortezomib induced significant inhibition of survival in all MM cell lines with an IC50 of 2.5nM, as previously reported. The combination of agents induced significant inhibition of proliferation as compared to each agent alone, specifically with the combination of 5nM rapamycin with 5nM of bortezomib. In the DNA synthesis assay, the combination of bortezomib and rapamycin was significantly cytotoxic compared to each agent alone, specifically at the dose of 5nM rapamycin and bortezomib 2.5nM. The combination of rapamycin 1 to 5 nM and bortezomib 5 to 10 nM were synergistic with a CI index less than 1.0, as in RPMI (CI=0.4) and U266 (CI=0.2) cell lines. The combination of rapamycin and bortezomib at serial concentrations did not trigger cytotoxicity in PBMCs from normal volunteers, indicating significant cytotoxicity in malignant cells, with lack of toxicity in normal PBMCs and suggesting a therapeutic index. The combination of bortezomib and rapamycin demonstrated a significant inhibitory effect on the growth of MM cell lines even in coculture with stromal cells. Cell cycle analysis demonstrated G1 arrest at 24 and 48 hrs in MM.1S cells. Similar results were obtained using primary CD138+ myeloma cells from patients. Conclusion: The combination of rapamycin and bortezomib resulted in synergistic in vitro cytotoxicity in MM cells. These results provide the framework for clinical trials evaluating the combination of CCI-779 and bortezomib in MM.

Multiple Myeloma (MM) remains an incurable plasma cell neoplasia, despite recent additions in the therapeutic arsenal for its management. Aurora kinases play integral roles in the orchestration of chromosomes and cytoskeletal mobility during the process of cell division. Aurora kinase activity has been implicated in several tumor types, including ovarian, colon, and prostate cancers. To determine whether inhibition of Aurora kinase activity could attenuate myeloma cell survival, we performed studies of the Aurora kinase inhibitor VE465 (Vertex Pharmaceuticals / Merck & Co., Inc.). VE465 inhibits all 3 Aurora isoforms (Aur A, B and C) with approximate Ki values of 1, 26, and 8.7 nM respectively. MTT colormetric survival assays (72–96hrs exposure) showed that VE465 is active against a wide panel of human MM cell lines: 26 of 38 MM cell lines had IC50 values at or < 100 nM, which are significantly lower than IC50 values for normal hematopoietic cells, e.g. unstimulated or PHA-stimatuled PBMCs. Importantly, VE465 was active in vitro against MM cell lines and/or primary MM tumor cells resistant to various anti-MM therapeutics, including dexamethasone, alkylating agents, anthracyclines, the proteasome inhibitor bortezomib, and/or immunomodulatory thalidomide derivatives (IMiDs). Moreover, VE465 maintained its activity despite the presence of protective bone marrow-derived cytokines (e.g. IL-6). PI cell cycle analyses showed that VE465 causes (even within 8 hrs of treatment) caused pronounced G2 arrest, followed by significant shift of MM cells to sub-G1 gate, consistent with cell death. Immunoblotting analyses confirmed that VE465 treatment induces cleavage of PARP, as well as cleavage of caspases-8 and -9, without significant changes in the expression levels of several key molecular effectors (e.g. Mcl-1, Bax, p53, hsp70, hsp90, hs27) which have been previously implicated in the mechanism of anti-MM activity of diverse other therapeutics. Screening of VE465-based combination regimens with other anti-MM agents showed additive effects of VE465 with the histone deacetylase inhibitor Vorinostat (SAHA) (Merck & Co., Inc). Ongoing studies in our Center are addressing the identification of specific molecular markers correlating with the degree of sensitivity of MM cells to VE465. Our in vitro evidence for induction of MM cell death and therapeutic window for the anti-MM effect of VE465, its ability to overcome protective effect of BM-derived cytokines, and the clearly distinct pattern of molecular sequelae of VE465 compared to several other agents in our current anti-MM therapeutic armamentarium, all suggest that Aurora kinase inhibition represents an intriguing novel targeted treatment strategy in MM. Importantly, these studies, particularly the identification of a sizeable subset of MM cell lines with higher sensitivity to VE465 than normal cells, provide the framework for in vivo VE465 studies in progress, alone and in combination with other anti-MM agents, to inform the design of potential clinical trials of this class of agents for MM.

Multiple myeloma (MM) is proposed to be driven by a CD138− CD20+ population of “MM stem cells” (MMSCs) which give rise to the more “differentiated” CD138+ malignant plasma cells (PCs), which have historically been the main target of anti-MM therapeutics. We hypothesized that inhibition of “differentiation” of these MMSCs to CD138+ derivative MM PCs could allow for expansion of the MMSC compartment and facilitate its cellular and molecular characterization. To test this hypothesis, we performed in vitro studies of MM cells (cell lines and primary MM cells) with reversine (2-(4-Morpholinoanilino)-6-cyclohexylaminopurine), a small molecule known to suppress differentiation (e.g. of myoblasts to myocytes) and/or cause de-differentiation of lineage-committed cells. We observed no reversine-induced expansion in CD138− populations deemed to represent MMSCs and no de-differentiation of CD138+ CD20− MM PCs to more “immature” CD138− CD20+ phenotype. Instead, sub-uM doses of reversine (as low as 0.1 uM, and significantly lower than the >5 uM required for its de-differentiation effects in other tissue type models) triggered major increases in MM cell death, including cells resistant to conventional (e.g. Dex, alkylating agents, anthracyclines, thalidomide, bortezomib, immunomodulatory thalidomide derivatives) and/or investigational agents (e.g. Apo2L/TRAIL) anti-MM agents. PBMCs, stromal cells, and immortalized hepatocytes were less sensitive to reversine than MM cells. Reversine was also active against a broad panel of neoplastic cells, e.g. various leukemia and lymphoma cell lines, suggesting a potentially broader spectrum of anti-neoplastic properties. Reversine was active against MM cells which are independent from or unresponsive to bone marrow stromal cell (BMSC) and was also able to overcome the effect of cytokine- (e.g.IL-6, IGF-1) or stroma-derived protection to BMSCs-responsive MM cells. The anti-MM effect of reversine involved G2 cell cycle arrest and commitment to cell death by drug exposure as short as 8hrs. Cleavage of PARP and caspase 3 were observed, and protein levels for diverse signaling effectors in the B-Raf and Akt cascades were decreased. Reversine enhanced responses of primary MM cells to other anti-MM agents, e.g. Dex, and had additive effects with cytotoxic chemotherapeutics or bortezomib. Reversine affected the molecular profiles of MM cells in a manner distinct from other anti-MM agents studied to date. Unlike bortezomib, reversine did not decrease NF-κB transcriptional activity, or affect the levels of several Bcl-2 family members, inhibitors of apoptosis, or heat shock proteins. These data suggest that reversine kills MM cells in an NF-κB independent manner, and also supports a broader conclusion that reversine has distinct profiles of molecular effects and anti-MM activity. Differential transcriptional profiling of MM cells with >1 vs.