Human MM Research Articles

The Vk*MYC Mouse Model of Myeloma Accurately Reflects Clinical Efficacy of Established and Novel Drugs.

Abstract 1849Poster Board I-875With the increasing availability of novel compounds, a number of drugs that have shown promising anti-myeloma activity in extensive pre-clinical studies failed to demonstrate clear efficacy in subsequent clinical trials. One reason for that may be an intrinsic inadequacy of pre-clinical models to predict activity, since for the most part they do not represent the complexity of human MM in an endogenous micro-environment. Another reason may be that to be able to test novel compounds, only relapsed and refractory patients are studied, at a stage in their disease that is unlikely to respond to any drug. One solution to this problem is to treat patients earlier in the course of their disease, which has been done by using the novel drug in combination with an approved drug. Unfortunately, without a large randomized trial, it is often difficult to interpret the response rates observed in these patients, and determine the contribution of the novel compound to the response observed to the combination treatment.The immuno-competent Vk*MYC mouse model of myeloma has already demonstrated high biological fidelity to the human disease, making it a better model to study the behavior of myeloma cells in the context of a native microenvironment. Specifically, the Vk*MYC myeloma is indolent (with a very low proliferative index), confined to the bone marrow, and secretes a monoclonal protein detected by serum protein electrophoresis. The M-spike represents a very useful marker to monitor tumor burden by periodic bleeding, as is done in patients. We have re-evaluated the anti-myeloma activity of novel compounds that have passed extensive pre-clinical characterization and are currently in clinical trials. We have utilized “naïve‘, untreated Vk*MYC mice at early stages of myeloma disease. Response rates were calculated by comparing the M-spike levels after treatment to the levels observed at d0. We found that, in addition to the demonstrated single agent clinical activity of alkylating agents (melphalan), cortico-steroids (dexamethasone) and proteasome inhibitors (bortezomib and carfilzomib), the histone deacetylase (HDACs) inhibitors vorinistat and panobinostat showed strong anti-myeloma activity, inducing a dramatic reduction of the M-spike after only two weeks of treatment. In contrast, only a minor response was observed to a CDK inhibitor (SNS-032) or the AKT inhibitor perifosine. No response was observed to the HSP-90 inhibitor AUY-922, or to the PI-3K inhibitors BEZ-235 and GDC-0941, or statins. The summary of the results with all tested agents is given in Table 1. [Display omitted] Although the modest activity of SNS-032 and perifosine in the Vk*MYC mice mirrors the results of phase-I and –II trials, where disease stabilization was observed, the efficacy of HDACs inhibitors in the Vk*MYC mice is somewhat surprising, since no clear single agent activity had been reported in advanced MM patients. With all the limitations of a mouse model, our data, however, suggest that untreated, earlier stage myeloma patients may be more sensitive to HDAC inhibition.As more mice are treated and more compounds are screened to further validate the clinical predictive value of the Vk*MYC mice, we would like to propose a new approach to the introduction of novel compounds in the treatment of MM, in which they are tested alone in the Vk*MYC model, and the ones that clearly induce a complete response are then prioritized for testing in a small cohort of earlier stage/untreated patients for one cycle, accelerating the evaluation, and limiting the toxicity. For those compounds that generate a partial response, combination studies can be easily performed in the mouse to assess drug pair efficacy. Finally, drugs that do not show any response in the Vk*MYC mouse should be given a lower priority for further clinical development in MM. Disclosures:No relevant conflicts of interest to declare.

Constitutive Activation of p38 MAPK in Myeloma Cells Contributes to Myeloma-Induced Osteolytic Bone Lesions.

Abstract 740Bone destruction is common in osteolytic tumors such as multiple myeloma. Myeloma cells that reside in the bone cause osteolysis, which is characterized by severe bone pain, multiple fractures, and hypercalcemia in patients and thereby, compromises patient quality of life. While myeloma cells have been proposed to activate osteoclasts and/or inhibit osteoblasts leading to bone resorption, the mechanism underlying myeloma-induced osteolytic bone lesions is still poorly elucidated. As p38 mitogen-activated protein kinase (MAPK) is constitutively activated in myeloma, we hypothesized that p38 MAPK activation in myeloma cells might be responsible for myeloma-induced osteolytic bone lesions. Human myeloma cell lines ARP-1 and MM.1S caused bone lesions in SCID mice after intravenous injection. However, once p38 MAPK was knocked down by small hairpin RNAs (shRNAs) in these cell lines, myeloma was established but failed to cause bone lesions in SCID mice, whereas the wild-type or vector control myeloma cells established myeloma (with similar tumor burdens as myeloma cells with knocked-down p38 MAPK) and caused bone lesions in tibial bones, as measured by radiograph and peripheral quantitative m-computed tomography and further confirmed by histological examination. In vivo examination revealed that the number of bone surface-covering osteoclasts, osteoclast size, cellular nuclear numbers in per osteoclast, and levels of circulating collagen type I (bone resorption marker) and TRAP5b (active osteoclast marker) were all reduced in mice injected with myeloma cells with knocked-down p38 MAPK as compared with controls. Consistently, we found that tumor p38 MAPK had active effects on in vitro osteoclast differentiation and bone resorption, and inhibited osteoblast differentiation and function, suggesting that constitutive activation of p38 MAPK in myeloma cells induces osteoclastogenesis. By functional studies and protein array analysis, we showed that monocyte chemotactic protein-1 (MCP-1) and dickkopf-1 (DKK-1) are upregulated downstream of p38 MAPK activation in myeloma cells and are responsible for enhanced osteoclastogenesis. Our results showed that p38 MAPK activation in myeloma cells upregulate MCP-1 and DKK-1 expression and production, which are released into the microenvironment. MCP-1 enhanced RANK expression on osteoclast precursors and DKK-1 increased RANKL secretion from stromal cells, all of which led to activation of NF-kB and MAPK signaling pathways in osteoclasts. In vivo blockade of MCP-1 and DKK-1 by specific antibodies significantly abrogated tumor p38 MAPK-induced osteoclast activation and bone lesions in established myeloma-SCID mice. Thus, our results have elucidated a novel mechanism that p38 MAPK activity in myeloma cells contributes to myeloma-induced osteolytic bone lesions. This study indicates that disruption of tumor p38 MAPK may be a new therapeutic approach to treat osteolytic bone lesions in myeloma. Disclosures:No relevant conflicts of interest to declare.

ADAM10 Is the Constitutive Functional Sheddase of CD44 in Human Melanoma Cells

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

Potential role for serum soluble CD200 in human Chronic Lymphocytic Leukemia (88.15)

Abstract CD200, a transmembrane protein expressed on multiple cell types, regulates anti-tumor immunity in animal models. Its overexpression is associated with a poor prognosis in human AML, MM, and melanoma. Blockade of CD200 expression on CD200-expressing tumor cells using CD200-specific monoclonal antibodies or siRNA enhanced anti-tumor immunity against tumor cells both in vitro and in vivo. A soluble variant of CD200 (sCD200) is detectable in human serum, and patients with Chronic lymphocytic leukemia show elevated serum levels. We investigated the correlation between serum sCD200 levels and clinical disease in CLL patients. sCD200 levels are associated with Rai disease stage and lymphocyte-doubling time. Shedding of cell surface CD200 from CLL cells in vitro can be induced by PMA and TLR7 agonists, with patient-to-patient variability. Shedding of CD200 is correlated with patient serum sCD200 levels, suggesting that inherent differences of CLL cells in responses to shedding stimuli may have clinical significance. sCD200 levels in serum may provide prognostic and/or diagnostic information in CLL, and may contribute to the efficacy of treatments targeting cell-surface CD200.

Antitumor Effects of JBD57 in Human Multiple Myeloma Cells in Vitro

JBD57 is a nucleoside/nucleotide analogue that in human cells causes depletion of mitochondrial DNA by disrupting oxidative phosphorylation pathways leading to toxic accumulation of nonesterified fatty acids, dicarboxylic acids and free radicals. Human 26S proteasome is also a target for JBD57. Here we evaluated JBD57 citotoxicity in several human tumor cell lines in vitro. Human MM cell line RPMI 8226/S (CCL-155), human T-cell lymphoblastic-like (Jurkat) and human T-cell leukemia (1301) were grown in RPMI 1640 medium; uterine sarcoma (MES-S (CRL-1976) cells were grown in McCoy medium; HUV-EC-C (CRL-1730) cells were grown in 199/EBSS medium. Media were supplemented with 10 % FBS. Cells were incubated at 37°C in a water-jacketed incubator with 5 % CO2. To evaluate JBD57 citotoxicity in RPMI 8226/S, MES-S, Jurkat, 1301 and HUV-EC-C cells, 104cells/well were grown in flat-bottomed 96-well tissue culture plates for 24, 48 and 72 hr; JBD57 was added to the media in several concentrations (0μM, 32.25μM, 62.5μM, 125μM, 250μM and 500μM). At the end of the experimental periods, cell viability was determined by the MTT method. JBD57 inhibited the growth of MM cell line RPMI 8226/S in a dose- and time-dependent manner. Cell viability decreased progressively with increasing concentrations of JBD57 as well as with increasing time periods. The IC50 (inhibitory concentration at 50%) was 125 μM at 72 hr. The viability of the MM cells after 72 hr incubation with JBD57 500μM was 33%, whereas 100% viability was observed when no drug was added. On the other hand, JBD57 did not affect cell viability of any of the other studied cell lines (uterine sarcoma, Jurkat, 1301 and HUVEC-C). JBD57 promotes a significant human MM cell death in a dose and time dependent manner but do not affect neither normal cell HUV-EC-C nor the tumoral cells MES-S, Jurkat and 1301, at least in the studied conditions. These results suggest that the potent antitumoral activity of JBD57 observed against MM cells could be potentially useful in the treatment of multiple myeloma.

ZKSCAN3, a Novel Zinc Finger Transcription Factor, Regulates Cyclin D2 Expression in Multiple Myeloma (MM) Cells

Background: Dysregulation of cyclin D has been proposed to represent an early oncogenic event in MM, and often occurs as a result of chromosomal translocations. Cyclin D2 can be induced as a result of translocations involving c-maf, mafB, and FGFR3/MMSET, but its expression is also increased in hyperdiploid and nonhyperdiploid cells without these translocations through unknown mechanisms. We previously identified a novel zinc finger transcription factor, ZKSCAN3, as a new “driver” of colon cancer progression, and found that the cyclin D2 promoter harbored several ZKSCAN3 binding sites, prompting us to investigate a possible role of ZKSCAN3 in MM pathogenesis.Methods: ZKSCAN3 expression was studied in patient-derived primary tumors and normal cells, as well as in human MM cell lines.Results: Tissue microarray studies showed that ZKSCAN3 was strongly expressed in the neoplastic cells of 7/10 MM patients by immunohistochemistry, while in all samples the surrounding normal bone marrow cells showed only background staining. Real-time polymerase chain reaction (qPCR) analysis revealed some level of ZKSCAN mRNA expression in 5/5 MM cell lines studied (RPMI 8226, U266, KAS-6/1, INA-6, ANBL-6), but not in pooled CD19+ normal B-cells. By Western blotting, ZKSCAN3 was most highly expressed in RPMI 8226 and KAS-6/1 cells, which notably do not bear cyclin D dysregulating translocations, while U266 and ANBL-6, which have such translocations, showed the lowest levels of ZKSCAN3 expression. In RPMI 8226 cells, but not the other MM lines, six copies of the ZKSCAN3 gene were detected, suggesting the presence of a previous amplification event. cisRED analysis predicted that ZKSCAN3 may be transcriptionally regulated by paired box gene 5 (Pax5), an essential factor for maintaining the commitment of the B cell lineage which is inactivated during plasma cell differentiation. Consistent with a role for Pax5 in controlling ZKSCAN3 level, a reporter assay showed that Pax5 inhibited ZKSCAN3 promoter activity, and Pax5 expression negatively correlated with ZKSCAN3 expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed direct binding of ZKSCAN3 to the endogenous cyclin D2 promoter in these MM cell lines. Knockdown of ZKSCAN3 using short hairpin RNAs reduced Cyclin D2 mRNA and protein expression in RPMI 8226 and KAS-6/1 cells, and also decreased their proliferative rates.Conclusions: These findings support the possibility that ZKSCAN3 contributes to myeloma pathogenesis by dysregulation of cyclin D2, and that events impacting upon ZKSCAN3 may provide MM cells with an alternative pathway for induction of Cyclin D2 in the absence of an activating translocation. ZKSCAN3 may therefore be an attractive candidate for MM therapy.

The Novel, Orally Available Multi-Kinase Inhibitor BAY 73-4506 in Multiple Myeloma

Agents targeting not only myeloma cells directly but also bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) that cause enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for multiple myeloma. Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506 (BAY), currently in phase II clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1–3, PDGFR-β), as well as oncogenic, (cKIT, RET, FGFR, Raf) kinases We first tested the ability of BAY to suppress proliferation and survival in a wide array of MM cell lines, including those resistant to conventional chemotherapeutics. Our data show that BAY, in a low micromolar range that is well below concentrations achieved in patient plasma during the first clinical trial in solid tumors, induces apoptosis by caspase-9 and caspase-3 activation in all cell lines tested. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, co-culture system. BAY treatment abrogates growth factor-stimulated MEK, ERK and AKT phosphorylation at sub-micromolar concentrations. Since the VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1–3, we examined its anti-angiogenic properties. BAY inhibits endothelial cell growth and endothelial cell tubule formation in vitro at concentrations less than 1μM; moreover, it also markedly inhibited VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; and importantly, BAY also inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts. Finally, BAY significantly delays tumor growth and abrogates blood vessel formation in vivo in a xenograft mouse model of human MM. These in vitro and in vivo results provide the basis for further clinical evaluation of BAY to improve patient outcome in MM.

Sulforaphane and PEITC Augment Activity of Conventional and Novel Anti-Myeloma Drugs

Isothiocyanates (ITCs) represent a family of phytochemicals found in cruciferous vegetables. Several epidemiological studies indicated that high intake of diet-derived ITCs may provide chemopreventive effect associated with a reduced risk of renal, prostate, pancreatic and colorectal carcinoma. ITCs have also been reported to enhance the chemosensitivity of diverse types of tumor cells. We therefore evaluated the cytotoxic effect of ITC, sulforaphane (SFN) and phenylethyl isothiocyanate (PEITC) on a panel of human MM cell lines, including cells resistant to doxorubicin (RPMI-Dox40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5), and dexamethasone (MM.1R, OPM1 and OPM2); as well as cells with low sensitivity to thalidomide derivatives (RPMI 8226-S) and sensitive cell lines (MM.1S). We evaluated the anti-MM activity of these compounds using both MTT and flow cytometric assays. Our results suggest that all tested MM cell lines are susceptible to the cytotoxic effect of both ITCs at concentrations in the same order of magnitude as those achieved in vivo by dietary consumption of cruciferous vegetables. PEITC (IC50 of 3.5–8.2 μM) was more potent than SFN (IC50 of 5–15 mM) at 48 h. ITCs induce apoptotic death of MM cells, evidenced by Annexin V-FITC staining, increased sub-G1 population measured by flow cytometry, and cleavage of PARP and caspase-3 by western blot analysis, ITCs also induced G2/M cell cycle arrest and depletion of mitochondrial potential in JC-1 probed MM cell lines. Multiplex analysis of phoshorylation signaling pathways, using the Luminex system and confirmed by conventional western blot analysis, revealed that PEITC at 2hrs triggers MAPK activation (MEK1 (4-fold), ERK1/2 (4.4-fold), JNK (25.4-fold) and p38MAPK (6-fold)) at 2h, which likely are stress responses of cells to PEITC treatment. SFN induced less pronounced but a more sustained MAPK activation (up to 24 h) than PEITC. Concentration-dependent increases in phosphorylation of GSK3α/β, c-jun, p70S6 kinase, and p90RSK were also observed at early time-points after ITCs-treatment. In contrast, decreased phoshorylation of Akt was observed in MM cells treated with SFN at 2 h and PEITC at 24 h. Chou-Talalay analysis of the effects of combinations of ITCs with anti-MM drugs (dexamethasone, melphalan and bortezomib) revealed all combinations to have synergistic MM cytotoxicity. Importantly, ITCs treatment, both alone and in combination with the aforementioned agents, also significantly suppressed proliferation of CFSE-labeled MM cell lines co-cultured with the human bone stromal cell line HS-5. These results indicate that SFN and PEITC suppress survival and proliferation of MM cells, both alone and in combination, and suggest their therapeutic potential in MM.

Oncoprotein 18 (Op18) : A Differentially Expressed Gene Is a Novel Therapeutic Target in Multiple Myeloma

Stathmin(Op18) is a ubiquitous cytosolic 18Kda protein regulating microtubule (MT) dynamics via binding and stabilizing activities. The activity of Op18 is down regulated during cell cycle by phosphorylation at four Ser residues. The phospho-Op18 is unable to bind to tubulin allowing the progression of the cell cycle. Two additional Op18-binding proteins, KIS prevents Op18 dephosphorylation and activation while iASPP (inhibitor of apoptosis stimulatory protein phosphatase) binds to WT-p53 and inhibits p53-related apoptosis. We have identidfied Op18 as a differentially expressed gene by suppression subtractive hybridization in myeloma. We have also confirmed differential expression of KIS in myeloma cells compared to normal plasma cells. Overexpression of Op18 was confirmed at both RNA and at protein level by RT –PCR and Western blotting in human myeloma cell lines as well as primary samples compared to normal plasma cells and normal human fibroblasts. In contrast, none of the tonsillar CD138+ plasma cells and normal bone marrow mononuclear cells showed Op18 overexpression. To establish the role of Op18 overexpression in MM cell transformation, the human MM cell lines were treated with antisense Op18 oligodeoxynucleotides (ODN). The growth rate of the ODN treated human myeloma cells was significantly reduced compared to the control cells along with cell cycle arrest in G2/M phase and increase in apoptosis as measured by immunohistochemical staining. Additionally, we have silenced Op18 by transfecting Op18-specific siRNA in MM cells and observed that KIS is translocated to cytoplasm, from nucleus as well as MM cells are arrested in G2/M. Importantly, Op18 silencing increased sensitivity of MM cells to microtubule drugs suggesting possible combination approach for therapeutic application. Its role in networking cellular signal transduction pathways in myeloma is under investigation. Recent publication identifying Op18 as one of the 15 most relevant genes determining outcome in myeloma (Avet-loiseau et al, JCO 2008) adds to the validity of Op18 as a molecule playing important role in myeloma cell growth and survival and warrant investigation as a novel therapeutic target.

Decreased Thymosin Beta 4 Expression Results in Poor Prognosis and Decreased Survival in Multiple Myeloma.

Thymosin beta 4 (Tβ4) is a small cytoplasmic and nuclear protein involved in different cellular processes including differentiation, migration and proliferation. It is overexpressed in several malignant cells, resulting in an increased angiogenic respons and metastatic potential of tumor cells. In the current study we wanted to evaluate the expression of Tβ4 in primary human multiple myeloma cells (MM) and murine 5TMM cells. Microarray analysis and qRT-PCR of 171 MM patients treated with high dose therapy and autologous stem cell transplantation, showed that MM cells display a decreased Tβ4 expression compared to plasma cells from healthy individuals. We investigated whether Tβ4 expression in MM cells influences the event free (EFS) and overall survival (OAS) of these patients. As Tβ4 was expressed in all MM cells, we compared survival of patients with Tβ4 expression in MM cells in the upper thirtile (Tβ4high MMC) against the lowest thirtile (Tβ4low MMC). Patients with Tβ4high MMC had an increased EFS (P< 0.05) and also their OAS tended to be longer. A similar observation was made in the 5TMM model where qRT-PCR and ELISA revealed a decreased expression of Tβ4 in BM samples from 5T33MM and 5T2MM diseased mice compared to control mice. To study the functionality of Tβ4, we overexpressed Tβ4 in 5T33MMvitro (5T33MMvt) cells by lentiviral transduction. These 5T33MMvt-Tβ4+ cells demonstrated a significantly decreased proliferative capacitity and an increased sensitivity to different anti-myeloma agents (bortezomib, melphalan, dexamethasone) compared to control 5T33MMvt cells. Furthermore, injection of 5T33MMvt-Tβ4+ cells in C57BlKaLwRij mice resulted in a significant decreased tumor formation (paraprotein concentration was 1.92 g/dl versus 3.74 g/dl for untransduced cells, p<0.05) and a prolonged survival compared to mice injected with untransduced 5T33MMvt cells (respectively 65.9 days versus 88.9 days, p< 0.05). In conclusion, we found a decreased Tβ4 expression in human and murine MM cells compared to normal plasma cells. In our cohort of MM patients treated with high-dose chemotherapy, low expression of TB4 identifies a group of patients with adverse prognosis while in the murine 5TMM33vt model, overexpression of Tβ4 resulted in an inhibitory effect on tumor formation suggesting a role of Tβ4 as a tumor suppressor gene in MM.

Activity of New Heat Shock Protein 90 (hsp90) Inhibitor NVP-AUY922 Against Myeloma Cells Sensitive and Resistant to Conventional Agents.

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 <12.5 nM for 31 of 35 cell lines, which were also more sensitive than all non-malignant cells tested with NVP-AUY922, including bone marrow stromal cells (BMSCs), immunortalized human hepatocytes and normal donor PBMCs, indicating a differential selectivity of NVP-AUY922 against neoplastic tissues vs. non-malignant cells. Importantly, MM cell lines or primary MM tumor cells resistant to dexamethasone (Dex), melphalan, immunomodulatory thalidomide derivatives (IMIDs), bortezomib or TRAIL were sensitive to NVP-AUY922. In addition, MM-1S cells constitutively over-expressing Akt were equally sensitive to NVP-AUY922 compared to their parental cell line. These anti-MM effects were rapid, as MM cells were committed to cell death within 16 hrs of exposure to only 25 nM of NVP-AUY922. NVP-AUY922 overcame the protective effects conferred to MM cells by exogenous IL-6 and IGF-1, while BMSC co-culture attenuated NVP-AUY922 activity only at doses <20 nM, as evidenced by compartment-specific bioluminescence imaging (CS-BLI). Mechanistic studies of NVP-AUY922 treatment of MM-1S cells showed early G2/M arrest followed by increased cell death, accompanied by caspase-3 and -8 cleavage (as early as 16 hrs). These events were preceded by decreased levels of Akt, B-Raf, phospho-MEK, cIAP2, and XIAP; and compensatory upregulation of hsp27 and hsp70. Given the highly single-agent activity of NVP-AUY922 at even low nM concentrations, formal statistical documentation of synergy was not observed in NVP-AUY922 combinations with conventional (e.g. Dex, doxorubicin) or novel (e.g. bortezomib) anti-MM agents. Encouragingly, no evidence of antagonism with any of these combinations was observed, indicating that NVP-AUY922 can be combined with current anti-MM agents in clinical settings.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.

The Tyrophostin Adaphostin (NSC680410) Inhibits Multiple Myeloma Bone Marrow Angiogenesis In Vitro and In Vivo.

Our own and other previous studies demonstrate marked anti-proliferative activity of the tyrophostin adaphostin (NSC680410) in a variety of hematologic malignancies including chronic myelocytic leukemia (CML), chronic lymphcytic leukemia (CLL), acute myelocytic leukemia (AML), and Multiple Myeloma. Here we show that adaphostin (NSC680410), similar to bortezomib, additionally inhibits tumor angiogenesis within the MM bone marrow (BM) microenvironment. This effect is elicited both indirectly by inhibition of VEGF production and secretion in MM cells, as well as directly by abrogation of endothelial cell growth. Specifically, adaphostin triggers marked downregulation of nuclear c-Myc expression in MM cells. Both adaphostin, as well as specific downregulation of c-Myc using siRNA, lead to a decrease in cobalt chloride- induced Hif-1alpha- expression and Hif-1alpha activity, as evidenced by western blot analysis and expression of Hif-1alpha- driven luciferase, respectively. Indeed secretion of the Hif-1alpha target gene VEGF is markedly inhibited in a dose- and time- dependent manner. Importantly, neither knockdown of c-Abl expression nor exogenous overexpression of caspase- cleavage- induced c-Abl fragment abrogates drug- induced Hif-1alpha downregulation or inhibition of its activity. Taken together, these results indicate the existence of a c-Myc/ Hif-1alpha- dependent, but c-Abl- independent, pathway modulating MM cell production and secretion of VEGF. In contrast, we demonstrate a direct antiangiogenic effect of adaphostin on endothelial cells, similar to H2O2, is mediated via c-Jun upregulation, inhibition of cell proliferation, and the induction of cell apoptosis. Moreover, our data further demonstrate activity of adaphostin within the BM microenvironment. Adaphostin, similar to bortezomib, significantly inhibits VEGF secretion triggered by adhesion of MM cells to BMSCs and endothelial cells. Consequently, conditioned medium derived from adaphostin- treated co-cultures markedly inhibits endothelial cell growth and tubule formation in a dose- dependent manner. Finally, we confirmed these in vitro results using an in vivo xenograft mouse model of human MM. Specifically, western blot analysis, as well as immunohistochemistry, demonstrate marked downregulation of both Hif-1alpha and CD31 in tumors isolated from adaphostin- treated animals versus control animals, confirming the in vivo antiangiogenic effect of adaphostin. Similar effects were obtained using a SCIDhu mouse model as well as a significant decrease of MM- related bone disease, due to anti- VEGF activity of adaphostin. Taken together, these data provide the rationale for the clinical evaluation of adaphostin to target both MM cells and the BM milieu to improve patient outcome in Multiple Myeloma.

The BAFF Inhibitor AMG523 Blocks Adhesion and Survival of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment: Clinical Implication.

We previously identified a role of B-cell activating factor (BAFF), a member of the tumor necrosis factor superfamily, in localization and survival of MM cells in the BM microenvironment (Cancer Res 2006, 66:6675–82). In the present study, we examined the potential therapeutic utility of the BAFF inhibitor, AMG523, for treating human MM using MM lines, either sensitive or resistant to conventional chemotherapy, as well as freshly isolated patient MM cells, in the presence or absence of bone marrow stromal cells (BMSCs). AMG523 induces modest cytotoxicity in MM cell lines and patient MM cells, suggesting a minor role of autocrine mechanism of BAFF for MM growth and survival. In the presence of BMSCs, AMG523 significantly decreased growth and survival in dexamethasone (Dex)-sensitive MM1S, Dex-resistant MM1R, INA6 MM cells and in patient MM cells (n=7), in a dose-dependent manner (0.1–10 μg/ml). BAFF-augmented MM adhesion to BMSCs is also blocked by AMG523 at 0.1 mg/ml in MM lines (MM1S, 28PE, INA6), as well as in freshly isolated patient MM cells (n=4). BAFF protects MM cells against dex- and lenalidomide-induced cytotoxicity; conversely, AMG523 blocks BAFF-induced protection against drug-induced apoptosis. Importantly, pretreatment of AMG523 blocks BAFF-induced activation of AKT, nuclear factor kB, and ERK in MM cells, confirming its inhibitory effect on BAFF-mediated adhesion and survival. We next asked whether AMG523 enhances Dex-, bortezomib-, Lenalidomide-induced MM cell cytotoxicity. AMG523 augments the inhibitory effect of Dex and lenalidomide in patient MM cells in the presence of BMSCs. Since osteoclasts (OCLs) secrete BAFF in the bone marrow microenvironment, we further asked whether AMG523 inhibits protection by MM-OCL interaction. OCLs were derived from peripheral blood mononuclear cells from MM patients after 2-week culture with M-CSF and RANKL, and MM cells were added in the presence or absence of AMG523. OCLs significantly increased MM cell survival, evidenced by annexin V and PI staining followed by flow cytometric analysis; conversely, AMG523 blocked MM cell survival by coculture with OCLs. Taken together, our data demonstrate that the novel therapeutic AMG523 blocks the interaction between BAFF and its receptors in human MM, thereby providing the rationale for clinical trials of AMG523 to improve patient outcome in MM.

Role of BAFF in Adhesion and Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment.

Recent studies have underscored the role of B-cell activating factor (BAFF), a member of the tumor necrosis factor superfamily, in promoting the survival of malignant B cells, including human MM. Since the tumor bone marrow microenvironment plays a crucial role in MM cell growth and survival, we here characterize the functional significance of BAFF in the interaction between MM and bone marrow stromal cells (BMSCs), and further defined molecular mechanisms regulating these processes. We first confirmed the expression of BAFF and its receptors (BCMA, TACI, BAFF-R) on majority of MM cell lines and CD138-purified patient MM cells. Significantly, gene expression profiling revealed increased BCMA expression on newly diagnosed and relapsed MM versus normal plasma cells (p<0.0001, student T test, Abstract # 552872). Although BAFF and its 3 receptors are expressed on CD138+patient MM cells (n=10) by RT-PCR analysis, the pattern of expression of TACI and BAFF-R receptors was heterogeneous, assessed by flow cytometric analysis. We next examined BAFF expression in BMSC lines (n=4) and BMSCs derived from patient BM (n=5). Baff expression is easily detected in BMSCs, and Baff levels are approximately 3–10-fold higher in supernatants of BMSCs than equivalent numbers of MM cells. Thus BMSCs are the main source of BAFF in MM patients. We then asked whether MM adhesion to BMSCs further upregulates BAFF secretion from BMSCs. Adhesion of 5 MM cell lines to BMSCs augments BAFF secretion by 2–5 fold, using BAFF ELISA. Immunoblotting using anti-BAFF Ab confirmed markedly higher expression of BAFF in BMSCs than MCCAR MM cells, as well as greater BAFF upregulation induced by MM adhesion. Since NF-kappaB (NF-κB) is crucial for MM adhesion-induced cytokine secretion from BMSCs and the BAFF gene promoter contains at least six NF-κB-binding sites, we next transfected KM104 BMSC line with a luciferase reporter vector carrying the BAFF gene promoter (BAFF-LUC) and then allowed MM cells to adhere to BMSCs for 24 hr, followed by measurement of luciferase activity. NF-κB-LUC reporter was used as positive and pGL3 plasmid as a negative control. Adhesion of MCCAR and MM1S MM lines to KM103 BMSC line with BAFF-LUC and NF-κB-LUC, but not control reporters, significantly enhanced luciferase activity, suggesting that NF-κB activation induced by MM adhesion to BMSCs mediates BAFF upregulation. In parallel, we also asked whether BAFF induces MM adhesion to BMSCs. BAFF (0–100 ng/ml) increases adhesion of 5 MM lines to BMSCs in a dose-dependent manner; conversely, TACI-Ig or blocking anti-BCMA Ab inhibited BAFF stimulation, indicating increased adhesion specific triggered by BAFF. Using adenoviruses expressing dominant-negative and constitutively expressed AKT as well as IkappaB kinase (IKK) inhibitor (PS-1145), we further showed that BAFF-induced MM cell adhesion is primarily mediated via activation of AKT and NF-κB signaling. Finally, BAFF significantly increased adhesion of CD138-expressing patient MM cells to BMSCs. These studies suggest a role for BAFF in localization and growth of MM cells in the BM microenvironment and strongly support novel important therapeutics targeting the interaction between BAFF and its receptors in human MM.

Targeting p62ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation.

Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62ZIP, has been identified. p62ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62ZIP−/− mice to determine the effects of deleting p62ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.

Up-Regulation of c-Jun contributes to the Induction of Apoptosis by Adaphostin in Human Multiple Myeloma Cells.

Adaphostin (NCI, Bethesda, MD) has been examined as an alternative to the 2-phenylaminopyrimidine derivate imatinib mesylate, with remarkable efficacy in the treatment of chronic myeloic leukemia (CML) including (1) Bcr/Abl+ cells; (2) imatinib mesylate resistant cells; and (3) Bcr/ Abl - cells. However exact functional mechanisms by which adaphostin induces cell apoptosis are unknown. Previously we have compared effects of adaphostin, a small molecule of the tyrophostin family versus imatinib mesylate in human MM cells: significant concentration - dependent antiproliferative the effects were observed after treatment with adaphostin, but not with imatinib mesylate, on MM cells alone and adherent to BMSCs. Here, we used gene expression microarray to identify signaling molecules modulated by adaphostin. As confirmed by protein expression, several proteins, most prominently c-Jun, were upregulated in a dose- and time- dependent manner. In contrast, c-Jun upregulation was not observed in MM cells treated with imatinib mesylate. Although PARP cleavage was induced, c-Jun upregulation was also not observed after treatment with dexamethasone (0.5 and 1mM), bortezomib (5 and 20nM) or melphalan (5 and 20mM). The functional relevance of adaphostin- induced c-Jun- upregulation was next investigated using small- interfering RNAs targeting c-Jun mRNA. Adaphostin- induced caspase- cleavage is abrogated in MM cells transfected with cJun- specific RNAi, but not with mock-RNAi. Consequently, a decrease of thymidine incorporation induced by adaphostin was only observed in MM cells transfected with mockRNAi, but not c-Jun- specific RNAi. Moreover, our results also demonstrate that adaphostin- induced caspase cleavage is dependent on the expression of c-Jun, but not visa versa. These studies for the first time provide evidence for a specific adaphostin- induced c-Jun upregulation and related induction of apoptosis in MM cells. Based on these findings, ongoing studies are investigating the role of c-Jun in MM pathogenesis.

Enhanced Levels of Apoptosis by Combination of Farnesyl Transferase Inhibition (Tipifarnib) and Proteasome Inhibition (Bortezomib) in Myeloma Cell Lines and Primary Myeloma Cells and Its Mechanistic Effects on Akt and Caspase Pathways.

Introduction:Despite significant advances in the treatment of myeloma, patients invariably become resistant to therapy. Therefore, novel treatment strategies are needed to overcome resistance. Overexpression of the anti-apoptotic protein Akt has been associated with resistance to bortezomib induced apoptosis. We have previously shown that treatment with farnesyl transferase inhibitors (FTI) is associated with synergisitic myeloma cell apoptosis when combined with bortezomib. In this study we explored the mechanism of action of the combination of bortezomib with tipifarnib, a FTI with previously demonstrated clinical activity in patients with hematologic malignancies. Our hypothesis is that the combination of bortezomib and tipifarnib will result in synergistic cell death by overcoming the anti-apoptotic effects of Akt.Methods:MM.1S, MM.1R, RPMI8226 and U266 cell lines were used in addition to fresh unmanipulated human myeloma cells from patients with relapsed MM. Cell proliferation was measured using the MTT assay. Cell death was measured by flow cytometric analysis of Annexin V and propidium iodide staining in the presence or absence of both agents and the broad spectrum caspase inhibitor Z-VAD-FMK (ZVAD). Caspase activity was assessed by Western blot ananlysis of cleaved caspases. Transient transfection of cell lines of using activated Akt, wild type Akt and BCL2 was also performed.Results:Dose escalation in vitro demonstrated that 8nM was a subtherapeutic dose of bortezomib, and 20nm bortezomib was an effective dose as a single agent. Doses of tipifarnib alone up to 5μM had modest effects on MM cell death. When 8nM or 20nM of bortezomib are combined with tipifarnib at doses of 5μM, cell death increases significantly in MM cell lines. Combination resulted in increased caspase 3, 8, and 9 activities in MM cell lines. Inhibition of caspase activities were confirmed with the broad spectrum caspase inhibitor ZVAD. Individual caspase inhibitor studies after 18 hours of combination treatment suggested that the inhibition of apoptosis is mainly mediated through caspase 8 and caspase 6 as measured by Annexin-V staining in MM.1S cells. Additionally, similar studies with the pan-caspase inhibitor ZVAD also suggested that there are caspase independent pathways resulting in inducing apoptosis of MM.1S cells. Combination therapy significantly reduces phos-Akt as early as 24hrs in MM cells, although, complete inhibition of phos-473-Akt varies between cell lines. Overexpression of activated Akt or wild type Akt and the anti-apoptotic protein Bcl2 in MM.1S did not abrogate the effect of combination on apoptosis. Primary human MM cells also demonstrated synergistic cell death when exposed to the combination at clinically achievable levels.Conclusion:The combination of tipifarnib with bortezomib is associated with greater cell death than either agent alone in both myeloma cell lines and patient myeloma cells. Therefore, we propose that the use of combined tipifarnib and bortezomib represents a novel and potentially active approach to MM therapy. The synergistic mechanism involved in the combination warrants further investigation.

Transdifferentiation of Monocytes into Endothelial Cells by Pleiotrophin.

Pleiotrophin (PTN) is a secreted angiogenic protein. We have recently shown that malignant plasma cells express high amounts of PTN and that it is elevated in MM patient serum. In this study, we show that this protein leads to angiogenesis through a novel mechanism - transdifferentiation of monocytes into endothelial cells. First, we isolated human monocytes (CD14+ cells) from peripheral blood using immunomagnetic bead isolation. We excluded the presence of endothelial cells in these CD14-expressing cells using RT-PCR on 106 monocytes with primers to genes expressed by endothelial cells including Flk-1, Tie2, CD144, and von Willebrand factor (vWF). We cultured these purified monocytes on collagen I for one week in the presence of PTN, mCSF and VEGF. Cells cultured with the combination of mCSF and PTN developed Flk-1-expressing tube-like structures, and the addition of VEGF increased tube formation. Next, we performed RT-PCR analysis with primers to these endothelial genes on monocytes cultured with PTN, mCSF and VEGF following serial dilution in cells (T or B lymphocytes) that lack monocyte or endothelial cell gene expression. Many of the cells expressed Tie2 RNA (>10%), and a smaller proportion (0.1–1%) also expressed Flk-1, CD144 and vWF RNA. In contrast, purified monocytes incubated with mCSF, VEGF, or PTN alone or the combinations of mCSF and VEGF or PTN and VEGF lacked Flk-1 staining, did not form tubes and failed to express endothelial cell RNA. We also assessed these monocytes in three dimensional matrices using Matrigel. Cells treated with mCSF and PTN invaded the matrix and began to form tube-like structures in the three dimensional gels as early as 7 days following culture whereas monocytes treated with mCSF, VEGF, or PTN alone did not form these structures. Next, we transduced human monocytic THP-1 cells that lack PTN expression with PTN-sense or -anti-sense constructs. Using RT-PCR, THP-1 cells transduced with PTN expressed endothelial cell genes and lost expression of the monocyte genes c-fms and CD68. In contrast, endothelial cell RNA was not detected in either THP-1 cells infected with anti-sense or the GFP control vectors. We used Transwell plates to co-culture THP-1 monocytes with human MM RPMI8226 or U266 cells or cell lines lacking PTN expression. We also added serum from MM patients with high levels of PTN or normal controls lacking PTN to THP-1 cells. THP-1 cells cultured with the MM cell lines or MM serum expressed endothelial genes and lost expression of monocyte RNA. Endothelial gene expression was blocked by the addition of an anti-PTN antibody but not by a control antibody. Control serum and cell lines lacking PTN did not induce endothelial gene expression or changes in monocyte RNA expression in the THP-1 cells. These experiments define a previously unrecognized novel mechanism leading to angiogenesis in cancer patients - the transdifferentiation of monocytes into endothelial cells by a factor highly produced by the malignant cells in MM. These findings also suggest a potential new specific target, PTN, to inhibit angiogenesis in cancer patients and should have profound clinical implications.

Targeting β2-Microglobulin for Induction of Tumor Apoptosis in Human Hematological Malignancies.

We discovered that monoclonal antibodies (mAbs) specific to human β2-microglobulin (β2M) induce programmed death of myeloma and other hematological tumor cells. The mAbs exhibited potent in vitro tumoricidal activity on all 14 β2M/HLA-ABC-bearing myeloma, lymphoma, and leukemia cell lines and primary myeloma cells isolated from eight patients. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms. Although the expression of β2M on normal hematopoietic cells is a potential safety concern, the mAbs seem to be selective to tumor-transformed cells and did not induce apoptosis of normal cells, including T and B lymphocytes and CD34+ bone marrow stem cells. Furthermore, the mAbs were able to selectively kill myeloma cells without damaging normal stromal cells in their cocultures. After binding to cell surface, the mAbs mediated internalization and down-modulation of surface β2M and HLA-ABC molecules. The mAbs induced cell death via inhibiting PI3K/Akt and ERK, activating JNK, upregulating Bad and Bax protein expression, inducing phosphorylation of Bcl-2, and decreasing phosphorylation of Bad, all of which compromised mitochondrial integrity, leading to cytochrome c release into cytosol and activation of the caspase-9-dependent cascade. Inhibitors to pan-caspases or caspase-9, but not to caspase-8, or to JNK, and knockdown of surface β2M and HLA-ABC expression by β2M-specific siRNA, but not control siRNA, abrogated apoptosis of tumor cells induced by the mAbs. Furthermore, anti-β2M mAbs were also active and therapeutic in vivo; After administration subcutaneous or intraperitoneal, the mAbs substantially reduced tumor burdens and retarded tumor growth in SCID mice subcutaneously implanted with human myeloma cell lines ARP-1 or MM.1S. Therefore, such mAbs offer the potential for a novel therapeutic approach to hematological malignancies and possibly to other cancers that express surface β2M. This study also suggests that differential expression of β2M/MHC class I molecules by normal and cancer cells might be a crucial point in the sensitivity and selectivity of anti-β2M mAb-induced apoptosis.

Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) Inhibits Myeloma in Vivo.

Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) is an orally-bioavailable cationic amphiphilic compound which significantly inhibits inflammation in rat arthritis and multiple sclerosis models. It acts, at least in part, by significantly inhibiting production of interleukin (IL)-6. Since IL-6 mediates MM cell growth, survival and drug resistance in the bone marrow (BM) microenvironment, we in this study characterized the effect of Atiprimod on human MM cells. We first demonstrated that Atiprimod significantly inhibited growth (p<0.05) in MM.1S, U266, and RPMI8226 MM cell lines in a time- and dose-dependent fashion, with IC50s of 0.5–1.25 mM. It also induced cytotoxicity in dexamethasone (Dex)-, doxorubicin (Dox)-, and melphalan (Mel)-resistant MM cell lines; as well as in patient MM cells, but not in normal PBMCs. Atiprimod triggered MM cell apoptosis via activation of caspase-8 and caspase-3, followed by PARP cleavage. Importantly, neither IL-6 nor insulin-like growth factor (IGF)-1, which completely abrogate Dex-induced apoptosis, protects against Atiprimod-induced apoptosis in MM.1S cells. In combination treatment studies, both conventional (Dex, Dox, Mel) and novel (As2O3) agents augment MM cell apoptosis induced by Atiprimod. Atiprimod inhibits STAT3 and Akt, but not ERK1/2, phosphorylation triggered by IL-6, BAX, Bcl-xl, and Mcl-1 in MM.1S cells. Importantly Atiprimod inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BM stromal cells (BMSCs) triggered by MM cell adherence to BMSCs, as well as associated MM cell growth. Finally, Atiprimod inhibits tumor OPM1 MM cell growth in vivo and prolongs survival in a SCID mouse model. Our data therefore demonstrate that Atiprimod both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for ongoing clinical trials of this agent to improve patient outcome in MM.