Search

Protein tyrosine kinases of the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, survival, and differentiation. In multiple myeloma, JAKs may be persistently activated due to a constant stimulation by interleukin (IL)-6, which is produced in the bone marrow environment. INCB20 is a synthetic molecule that potently inhibits all members of the JAK family with a 100- to 1,000-fold selectivity for JAKs over >70 other kinases. Treatment of multiple myeloma cell lines and patient tumor cells with INCB20 resulted in a significant and dose-dependent inhibition of spontaneous as well as IL-6-induced cell growth. Importantly, multiple myeloma cell growth was inhibited in the presence of bone marrow stromal cells. The IL-6 dependent cell line INA-6 was particularly sensitive to the drug (IC50<1 micromol/L). Growth suppression of INA-6 correlated with an increase in the percentage of apoptotic cells and inhibition of signal transducer and activator of transcription 3 phosphorylation. INCB20 also abrogated the protective effect of IL-6 against dexamethasone by blocking phosphorylation of SHP-2 and AKT. In contrast, AKT phosphorylation induced by insulin-like growth factor-I remained unchanged, showing selectivity of the compound. In a s.c. severe combined immunodeficient mouse model with INA-6, INCB20 significantly delayed INA-6 tumor growth. Our studies show that disruption of JAKs and downstream signaling pathways may both inhibit multiple myeloma cell growth and survival and overcome cytokine-mediated drug resistance, thereby providing the preclinical rationale for the use of JAK inhibitors as a novel therapeutic approach in multiple myeloma.

We show that multiple myeloma (MM), the second most commonly diagnosed hematologic malignancy, is responsive to hsp90 inhibitors in vitro and in a clinically relevant orthotopic in vivo model, even though this disease does not depend on HER2/neu, bcr/abl, androgen or estrogen receptors, or other hsp90 chaperoning clients which are hallmarks of tumor types traditionally viewed as attractive clinical settings for use of hsp90 inhibitors, such as the geldanamycin analog 17-AAG. This class of agents simultaneously suppresses in MM cells the expression and/or function of multiple levels of insulin-like growth factor receptor (IGF-1R) and interleukin-6 receptor (IL-6R) signaling (eg, IKK/NF-kappaB, PI-3K/Akt, and Raf/MAPK) and downstream effectors (eg, proteasome, telomerase, and HIF-1alpha activities). These pleiotropic proapoptotic effects allow hsp90 inhibitors to abrogate bone marrow stromal cell-derived protection on MM tumor cells, and sensitize them to other anticancer agents, including cytotoxic chemotherapy and the proteasome inhibitor bortezomib. These results indicate that hsp90 can be targeted therapeutically in neoplasias that may not express or depend on molecules previously considered to be the main hsp90 client proteins. This suggests a more general role for hsp90 in chaperoning tumor- or tissue-type-specific constellations of client proteins with critical involvement in proliferative and antiapoptotic cellular responses, and paves the way for more extensive future therapeutic applications of hsp90 inhibition in diverse neoplasias, including MM.

Multiple myeloma is a B-cell malignancy for which no curative therapies exist to date, despite enormous research efforts. The remarkable activity of the proteasome inhibitor bortezomib (PS-341, Velcade) observed in clinical trials of patients with relapsed refractory myeloma has led to investigations of the role of the ubiquitin-proteasome pathway in the pathogenesis of myeloma. Here we report a biochemical analysis of proteasome activity and composition in myeloma cells exposed to PS-341 in the presence or absence of cytokines present in the bone marrow milieu. We observed that the myeloma cell lines MM1.S, RPMI8226, and U266 contain active immunoproteasomes, the amount of which is enhanced by IFN-gamma and tumor necrosis factor-alpha. Using a radiolabeled active site-directed probe specific for proteasome catalytic subunits, we show that PS-341 targets the beta5 and beta1 subunits in a concentration-dependent manner. Furthermore, PS-341 also targeted the corresponding catalytic subunits of the immunoproteasome, beta5i and beta1i, respectively. These data suggest that PS-341 targets both normal and immunoproteasome species to a similar extent in myeloma cells.

We tested the in vitro and in vivo antitumor activity of the maytansinoid DM1 (N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine), a potent antimicrotubule agent, covalently linked to the murine monoclonal antibody (mAb) B-B4 targeting syndecan-1 (CD138). We evaluated the in vitro activity of B-B4-DM1 against a panel of CD138(+) and CD138(-) cell lines, as well as CD138(+) patient multiple myeloma (MM) cells. Treatment with B-B4-DM1 selectively decreased growth and survival of MM cell lines, patient MM cells, and MM cells adherent to bone marrow stromal cells. We further examined the activity of B-B4-DM1 in 3 human MM models in mice: (1) severe combined immunodeficient (SCID) mice bearing subcutaneous xenografts; (2) SCID mice bearing green fluorescent protein-positive (GFP(+)) xenografts; and (3) SCID mice implanted with human fetal bone (SCID-hu) and subsequently injected with patient MM cells. Tumor regression and inhibition of tumor growth, improvement in overall survival, and reduction in levels of circulating human paraprotein were observed in mice treated with B-B4-DM1. Although immunohistochemical analysis demonstrates restricted CD138 expression in human tissues, the lack of B-B4 reactivity with mouse tissues precludes evaluation of its toxicity in these models. In conclusion, B-B4-DM1 is a potent anti-MM agent that kills cells in an antigen-dependent manner in vitro and mediates in vivo antitumor activity at doses that are well tolerated, providing the rationale for clinical trials of this immunoconjugate in MM.

Topoisomerase I inhibitors are effective anticancer therapies and have shown activity in hematologic malignancies. Here we show for the first time that SN38, the potent active metabolite of irinotecan, induces c-Jun NH(2)-terminal kinase activation, Fas up-regulation, and caspase 8-mediated apoptosis in multiple myeloma (MM) cells. Proteasomal degradation of nuclear topoisomerase I has been proposed as a resistance mechanism in solid malignancies. SN38-induced proteasomal degradation of topoisomerase I was observed during SN38-mediated cytotoxicity against MM.1S myeloma cell line but occurred after c-Jun NH(2)-terminal kinase activation, Fas up-regulation, and poly(ADP-ribose) polymerase cleavage and failed to protect cells from apoptosis. Differential toxicity was observed against MM cells versus bone marrow stromal cells, and SN38 inhibited adhesion-induced up-regulation of MM cell proliferation when MM cells adhere to bone marrow stromal cells. In addition, SN38 directly inhibited constitutive and inducible interleukin 6 and vascular endothelial growth factor secretion by bone marrow stromal cells. Synergy was observed when SN38 was used in combination with doxorubicin, bortezomib, as well as poly(ADP-ribose) polymerase inhibitor NU1025 and Fas-activator CH11. These findings have clinical significance, because identification of downstream apoptotic signaling after topoisomerase I inhibition will both elucidate mechanisms of resistance and optimize future combination chemotherapy against MM.

Previous studies have shown that the multiple myeloma (MM) cell line and MM patient cells express high-affinity vascular endothelial growth factor (VEGF) receptor-1 or Fms-like tyrosine kinase-1 (Flt-1) but not VEGF receptor-2 or Flk-1/kinase insert domain-containing receptor (Flk-1/KDR) and that VEGF triggers MM cell proliferation through a mitogen-activated protein kinase (MAPK)-dependent pathway and migration through a protein kinase C (PKC)-dependent pathway. The present study evaluates the efficacy of the small molecule tyrosine-kinase inhibitor GW654652, which inhibits all 3 VEGF receptors with similar potency. We show that GW654652 acts directly on MM cells and in the bone marrow microenvironment. Specifically, GW654652 (1-10 microg/mL) inhibits, in a dose-dependent fashion, VEGF-triggered migrational activity and cell proliferation of MM cell lines that are sensitive and resistant to conventional therapy. As expected from our previous studies of VEGF-induced signaling and sequelae in MM cells, GW654652 blocked VEGF-induced Flt-1 phosphorylation and downstream activation of AKT-1 and MAPK-signaling cascades. Importantly, GW654652 also inhibits interleukin-6 and VEGF secretion and proliferation of MM cells induced by tumor cell binding to bone marrow (BM) stromal cells. The activity of a pan-VEGF receptor inhibitor against MM cells in the BM milieu, coupled with its lack of major toxicity in preclinical mouse models, provides the framework for clinical trials of this drug class to improve patient outcome in MM.

The ubiquitin-conjugating enzyme CDC34 (UBC3) is linked to cell cycle progression in diverse cell types; however, its role in multiple myeloma (MM) pathogenesis is unclear. Here, we show that CDC34 is highly expressed in patient MM cells and MM cell lines versus normal cells. Blocking CDC34 using a dominant-negative strategy enhances the anti-MM activity of Bortezomib/Proteasome inhibitor PS-341, dexamethasone (Dex) and 2-Methoxyestradiol (2ME2). The expression of wild-type CDC34 reduces Dex-induced cytotoxicity in MM cells. Moreover, inhibition of CDC34 enzymatic activity abrogates interleukin-6-induced protection against Dex-induced apoptosis. Together, these findings provide evidence that (1) CDC34 expression is associated with growth and survival of MM cells and (2) blocking CDC34 activity not only enhances anti-MM activity of Bortezomib and 2ME2 but also overcomes IL-6-triggered Dex-resistance.

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid (CDDO) induces apoptosis in leukemic cells. Here we show that CDDO and its new derivative CDDO-imidazolide (CDDO-Im) trigger apoptosis in multiple myeloma (MM) cells resistant to conventional therapies including melphalan (LR-5), doxorubicin (Dox-40), and dexamethasone (MM.1R, U266, RPMI 8226) without affecting the viability of normal cells. CDDO-IM also triggers apoptosis in bone marrow stromal cells (BMSCs) and decreases interleukin-6 (IL-6) secretion induced by MM cell adhesion to BMSCs. Moreover, CDDO-Im-induced apoptosis in MM cells is not blocked by IL-6 or insulin growth factor-1 (IGF-1). Importantly, CDDO-Im and bortezomib/proteasome inhibitor PS-341 trigger synergistic apoptosis in MM cells associated with loss of mitochondrial membrane potential, superoxide generation, release of mitochondrial proteins cytochrome c/second mitochondria-derived activator of caspases (cytochrome c/Smac), and activation of caspase-8, -9, and -3. Conversely, the pancaspase inhibitor Z-VAD-fmk abrogates the CDDO-Im + bortezomib-induced apoptosis. Low doses of CDDO-Im and bortezomib overcome the cytoprotective effects of antiapoptotic proteins Bcl2 and heat shock protein-27 (Hsp27) as well as nuclear factor-kappa B (NF-kappaB)-mediated growth/survival and drug resistance. Finally, combining CDDO-Im and bortezomib induces apoptosis even in bortezomib-resistant MM patient cells. Together, these findings provide the framework for clinical evaluation of CDDO-Im, either alone or in combination with bortezomib, to overcome drug resistance and improve patient outcome in MM.

Histone deacetylases (HDACs) affect cell growth at the transcriptional level by regulating the acetylation status of nucleosomal histones. HDAC inhibition induces differentiation and/or apoptosis in transformed cells. We recently showed that HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), potently induce apoptosis of human multiple myeloma (MM) cells. In this study, we focused on MM as a model to study the transcriptional profile of HDAC inhibitor treatment on tumor cells and to address their pathophysiological implications with confirmatory mechanistic and functional assays. We found that MM cells are irreversibly committed to cell death within few hours of incubation with SAHA. The hallmark molecular profile of MM cells before their commitment to SAHA-induced cell death is a constellation of antiproliferative and/or proapoptotic molecular events, including down-regulation of transcripts for members of the insulin-like growth factor (IGF)/IGF-1 receptor (IGF-1R) and IL-6 receptor (IL-6R) signaling cascades, antiapoptotic molecules (e.g., caspase inhibitors), oncogenic kinases, DNA synthesis/repair enzymes, and transcription factors (e.g., XBP-1, E2F-1) implicated in MM pathophysiology. Importantly, SAHA treatment suppresses the activity of the proteasome and expression of its subunits, and enhances MM cell sensitivity to proteasome inhibition by bortezomib (PS-341). SAHA also enhances the anti-MM activity of other proapoptotic agents, including dexamethasone, cytotoxic chemotherapy, and thalidomide analogs. These findings highlight the pleiotropic antitumor effects of HDAC inhibition, and provide the framework for future clinical applications of SAHA to improve patient outcome in MM.

Cytokines of the gp130 family, particularly interleukin 6 (IL-6), play a central role in the growth and survival of malignant plasma cells. Recently, novel neurotrophin-1 (NNT-1)/B cell-stimulating factor-3 (BSF-3), also reported as cardiotrophin-like cytokine (CLC), was identified as a cytokine belonging to the gp130 family. BSF-3, similar to IL-6, exerts regulatory effects on normal B cell functions, but its functional significance in haematological malignancies has not been defined. The purpose of this study was to evaluate the biological effects and signalling pathways that are induced by BSF-3 in malignant plasma cells. Recombinant human BSF-3 was found to have growth stimulatory activity on plasmacytoma cell lines and primary tumour cells. In addition, BSF-3 was able to protect from Dexamethasone (Dex)-induced apoptosis. BSF-3 stimulated cell growth could not be inhibited by neutralizing anti-IL-6 or anti-IL-6 receptor antibodies, but was abrogated by anti-gp130 antibodies. In INA-6.Tu11 cells, a subline of the IL-6-dependent human plasma cell line INA-6 expressing gp130 and the receptor for leukaemia inhibitory factor (LIF), stimulation with BSF-3 induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3). AG490, an inhibitor of Janus kinases, decreased BSF-3 induced cell growth in a dose-dependent manner. This correlated with a reduction of STAT3 phosphorylation levels, while p44/42 mitogen-activated protein kinase (MAPK) phosphorylation was not affected. In conclusion, BSF-3 is a novel myeloma growth and survival factor with a potential role in the pathophysiology of the disease.

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Heat shock protein 27 (Hsp27) negatively regulates another mitochondrial protein, cytochrome c, during apoptosis; however, the role of Hsp27 in modulating Smac release is unknown. Here we show that Hsp27 is overexpressed in both dexamethasone (Dex)-resistant multiple myeloma (MM) cell lines (MM.1R, U266, RPMI-8226) and primary patient cells. Blocking Hsp27 by an antisense (AS) strategy restores the apoptotic response to Dex in Dex-resistant MM cells by triggering the release of mitochondrial protein Smac, followed by activation of caspase-9 and caspase-3. Moreover, AS-Hsp27 overcomes interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis. These data demonstrate that Hsp27 inhibits the release of Smac, and thereby confers Dex resistance in MM cells.

Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematologic malignancies. Here we show that NVP-LAQ824, a novel hydroxamic acid derivative, induces apoptosis at physiologically achievable concentrations (median inhibitory concentration [IC50] of 100 nM at 24 hours) in multiple myeloma (MM) cell lines resistant to conventional therapies. MM.1S myeloma cell proliferation was also inhibited when cocultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Importantly, NVP-LAQ824 also inhibited patient MM cell growth in a dose- and time-dependent manner. NVP-LAQ824-induced apoptotic signaling includes up-regulation of p21, caspase cascade activation, and poly (adenosine diphosphate [ADP]) ribose (PARP) cleavage. Apoptosis was confirmed with cell cycle analysis and annexin-propidium iodide staining. Interestingly, treatment of MM cells with NVPLAQ824 also led to proteasome inhibition, as determined by reduced proteasome chymotrypsin-like activity and increased levels of cellular polyubiquitin conjugates. Finally, a study using NVP-LAQ824 in a preclinical murine myeloma model provides in vivo relevance to our in vitro studies. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in MM.

Insulin-like growth factor-1 (IGF-I) is a growth and survival factor in human multiple myeloma (MM) cells. Here we examine the effect of IGF-I on MM cell adhesion and migration, and define the role of beta1 integrin in these processes. IGF-I increases adhesion of MM.1S and OPM6 MM cells to fibronectin (FN) in a time- and dose-dependent manner, as a consequence of IGF-IR activation. Conversely, blocking anti-beta1 integrin monoclonal antibody, RGD peptide, and cytochalasin D inhibit IGF-I-induced cell adhesion to FN. IGF-I rapidly and transiently induces association of IGF-IR and beta1 integrin, with phosphorylation of IGF-IR, IRS-1, and p85(PI3-K). IGF-I also triggers phosphorylation of AKT and ERK significantly. Both IGF-IR and beta1 integrin colocalize to lipid rafts on the plasma membrane after IGF-I stimulation. In addition, IGF-I triggers polymerization of F-actin, induces phosphorylation of p125(FAK) and paxillin, and enhances beta1 integrin interaction with these focal adhesion proteins. Importantly, using pharmacological inhibitors of phosphatidylinositol 3'-kinase (PI3-K) (LY294002 and wortmannin) and extracellular signal-regulated kinase (PD98059), we demonstrate that IGF-I-induced MM cell adhesion to FN is achieved only when PI3-K/AKT is activated. IGF-I induces a 1.7-2.2 (MM.1S) and 2-2.5-fold (OPM6) increase in migration, whereas blocking anti-IGF-I and anti-beta1 integrin monoclonal antibodies, PI3-K inhibitors, as well as cytochalasin D abrogate IGF-I-induced MM cell transmigration. Finally, IGF-I induces adhesion of CD138+ patient MM cells. Therefore, these studies suggest a role for IGF-I in trafficking and localization of MM cells in the bone marrow microenvironment. Moreover, they define the functional association of IGF-IR and beta1 integrin in mediating MM cell homing, providing the preclinical rationale for novel treatment strategies targeting IGF-I/IGF-IR in MM.

The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM). Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To elucidate mechanisms of bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant SUDHL-4 and bortezomib-sensitive SUDHL-6 diffuse large B-cell lymphoma lines in response to bortezomib. At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in SUDHL-6 cells, but not in SUDHL-4 cells. We showed that overexpression of activating transcription factor 3 (ATF3), ATF4, ATF5, c-Jun, JunD and caspase-3 is associated with sensitivity to bortezomib-induced apoptosis, whereas overexpression of heat shock protein (HSP)27, HSP70, HSP90 and T-cell factor 4 is associated with bortezomib resistance. [ABSTRACT FROM AUTHOR]