Synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with histone deacetylase inhibitors,melphalan, or doxorubicin in multiple myeloma

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP‐AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM‐2, RPMI‐8226, U‐266, LP‐1, MM1.S, and primary myeloma cells were exposed to NVP‐AUY922 and one of the combination partners histone deacetylase inhibitor NVP‐LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP‐AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP‐AUY922 with SAHA, NVP‐LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI‐8226 cell line and relative resistance of some primary myeloma cells against NVP‐AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with melphalan, doxorubicin, NVP‐LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.     

Inhibition of heat shock protein 90 (HSP90) as a therapeutic strategy for the treatment of myeloma and other cancers

Heat shock protein 90 (HSP90) is a molecular chaperone that is induced in response to cellular stress and stabilizes client proteins involved in cell cycle control and proliferative/anti-apoptotic signalling. HSP90 is overexpressed in a range of cancers, and may contribute to tumour cell survival by stabilizing aberrant signalling proteins and by interfering with apoptosis. Tanespimycin, an HSP90 inhibitor, reduces tumour cell survival in vitro. In multiple myeloma (MM), HSP90 inhibition affects multiple client proteins that contribute to tumour cell survival, including the IGF1 receptor and the IL-6 receptor, and elements of the PI3/Akt, STAT3, and MAPK signalling pathways. HSP90 inhibition also abrogates the protective effect of bone marrow stromal cells and inhibits angiogenesis and osteoclastogenesis. Tanespimycin acts synergistically with the proteasome inhibitor bortezomib in MM cells and tumour explants, possibly reducing their ability to resist bortezomib-induced stress to the endoplasmic reticulum. The combination of tanespimycin and bortezomib has demonstrated significant and durable responses with acceptable toxicity in a phase I/II study in patients with relapsed and relapsed/refractory MM. HSP90 inhibition is a promising strategy in MM especially in combination with bortezomib; additional studies will further evaluate optimal dosings of candidate drugs and schedules, as well as confirm efficacy in comparative phase III trials.     

Baicalein, a component of Scutellaria radix from Huang-Lian-Jie-Du-Tang (HLJDT), leads to suppression of proliferation and induction of apoptosis in human myeloma cells

In the search for a more effective adjuvant therapy to treat multiple myeloma (MM), we investigated the effects of the traditional Chinese herbal medicines Huang-Lian-Jie-Du-Tang (HLJDT), Gui-Zhi-Fu-Ling-Wan (GZFLW), and Huang-Lian-Tang (HLT) on the proliferation and apoptosis of myeloma cells. HLJDT inhibited the proliferation of myeloma cell lines and the survival of primary myeloma cells, especially MPC-1- immature myeloma cells, and induced apoptosis in myeloma cell lines via a mitochondria-mediated pathway by reducing mitochondrial membrane potential and activating caspase-9 and caspase-3. Further experiments confirmed that Scutellaria radix was responsible for the suppressive effect of HLJDT on myeloma cell proliferation, and the baicalein in Scutellaria radix showed strong growth inhibition and induction of apoptosis in comparison with baicalin or wogonin. Baicalein as well as baicalin suppressed the survival in vitro of MPC-1- immature myeloma cells rather than MPC-1+ myeloma cells from myeloma patients. Baicalein inhibited the phosphorylation of IkB-alpha, which was followed by decreased expression of the IL-6 and XIAP genes and activation of caspase-9 and caspase-3. Therefore, HLJDT and Scutellaria radix have an antiproliferative effect on myeloma cells, especially MPC-1- immature myeloma cells, and baicalein may be responsible for the suppressive effect of Scutellaria radix by blocking IkB-alpha degradation.     

Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells

Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G(2)/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients.     

HSP70 inhibition reverses cell adhesion mediated and acquired drug resistance in multiple myeloma

Heat shock proteins (HSPs) are a super family of highly conserved molecular chaperone proteins, which are induced in response to stress. HSP70 has been demonstrated to inhibit apoptosis induced by a number of chemotherapeutic agents. Previous investigations have suggested the development of drug resistance in multiple myeloma (MM) cells after adhesion to stroma. This study used MM cell lines and primary plasma cells to determine if HSP70 had a role in development of chemo resistance. Adhesion of MM cells to either bone marrow stromal cells or fibronectin (FN) enhanced HSP70 expression. Inhibition of the HSP70 expression decreased 8226 cell adhesion to stroma or FN and induced more apoptosis in FN-adhered 8226 cells than in suspension cultures at 24 h. Further, HSP70 inhibitors enhanced melphalan-induced apoptosis and reversed melphalan-induced cell adhesion-mediated drug resistance (CAM-DR) phenotype. In addition, compared to parental cells, KNK-437, a heat shock factor inhibitor caused more apoptosis in melphalan-resistant 8226/LR5 cells and sensitized them to melphalan. Primary CD138 positive cells showed high expression of HSPA4 mRNA, and KNK-437 caused apoptosis in these cells. In conclusion, our data suggest inhibition of HSP70, reduced adhesion and caused apoptosis of both acquired and de novo drug resistant MM cells.     

Targeting NF-kappaB pathway with an IKK2 inhibitor induces inhibition of multiple myeloma cell growth

The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IkappaB kinase/nuclear factor-kappaB (IKK/NF-kappaB). The present study aimed at investigating the effect of a pharmaceutical IKK2 inhibitor, the anilinopyrimidine derivative AS602868, on the in vitro growth of 14 human MM cell lines (HMCL) and primary cells from 13 patients. AS602868 induced a clear dose-dependent inhibition of MM cell growth on both HMCL and primary MM cells, which was the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Combination of AS602868 with suboptimal doses of melphalan or Velcade showed an additive effect in growth inhibition of HMCL. AS602868 also induced apoptosis of primary myeloma cells. Importantly, AS602868 did not alter the survival of other bone marrow mononuclear cells (CD138(-)) co-cultured with primary MM (CD138(+)) cells, except for CD34(+) haematopoietic stem cells. The results demonstrate the important role of NF-kappaB in maintaining the survival of MM cells and suggest that a pharmacological inhibition of the NF-kappaB pathway by the IKK2 inhibitor AS602868 can efficiently kill HMCL and primary myeloma cells and therefore might represent an innovative approach for treating MM patients.     

Inhibitors of protein kinase C sensitise multiple myeloma cells to common genotoxic drugs

Objectives:  Despite high dose treatment regimes multiple myeloma (MM) disease is still not curable. Patients become resistant to cytotoxic drugs and die of disease progression. Therefore, besides new cytotoxic compounds drug sensitisers are urgently needed.
Methods:  The MM cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the common anti-myeloma drugs like melphalan together with protein kinase C (PKC) inhibitors. Growth inhibition was measured using the water-soluble tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay), and apoptosis was determined by flow cytometry after staining with fluorescein isothiocyanate-labeled annexin V (annexin-V-FITC) and propidium iodide. Intracellular signalling was shown by western blotting.
Results:  In this study we show that the combination of melphalan or doxorubicin with a PKC inhibitor, Gö6976 or enzastaurin, strongly increases cell toxicity. Increase of cytotoxicity is shown to be due to increased induction of apoptosis. Furthermore, we show that the protective effect of human bone marrow stromal cells (hBMSC) is abrogated by the PKC inhibitors. Finally, western blotting experiments revealed that incubation of myeloma cells with cytotoxic drugs like melphalan or doxorubicin leads to increased phosphorylation and therefore degradation of inhibitor of nuclear factor kappa B (IκB) and release of nuclear factor kappa B (NFκB). In contrast, enzastaurin inhibits phosphorylation of IκB.
Conclusions:  We conclude that the combination of conventional drugs and PKC inhibitors might be very effective and represents a new strategy in the treatment of MM.     

Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)-kappaB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.     

Targeting mitochondrial factor Smac/DIABLO as therapy for multiple myeloma (MM)

Second mitochondria-derived activator of caspases (Smac) promotes apoptosis via activation of caspases. Here we show that a low-molecular-weight Smac mimetic LBW242 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Examination of purified patient MM cells demonstrated similar results, without significant cytotoxicity against normal lymphocytes and bone marrow stromal cells (BMSCs). Importantly, LBW242 abrogates paracrine MM cell growth triggered by their adherence to BMSCs and overcomes MM cell growth and drug-resistance conferred by interleukin-6 or insulinlike growth factor-1. Overexpression of Bcl-2 similarly does not affect LBW242-induced cytotoxicity. Mechanistic studies show that LBW242-induced apoptosis in MM cells is associated with activation of caspase-8, caspase-9, and caspase-3, followed by PARP cleavage. In human MM xenograft mouse models, LBW242 is well tolerated, inhibits tumor growth, and prolongs survival. Importantly, combining LBW242 with novel agents, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the proteasome inhibitors bortezomib and NPI-0052, as well as with the conventional anti-MM agent melphalan, induces additive/synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating LBW242, alone and together with other anti-MM agents, to improve patient outcome in MM.     

SNX-2112, a selective Hsp90 inhibitor, potently inhibits tumor cell growth, angiogenesis, and osteoclastogenesis in multiple myeloma and other hematologic tumors by abrogating signaling via Akt and ERK

Heat-shock protein 90 (Hsp90) acts as a molecular chaperone required for maintaining the conformational stability of client proteins regulating cell proliferation, survival, and apoptosis. Here we investigate the biologic significance of Hsp90 inhibition in multiple myeloma (MM) and other hematologic tumors using an orally available novel small molecule inhibitor SNX-2112, which exhibits unique activities relative to 17-allyamino-17-demethoxy-geldanamycin (17-AAG). SNX-2112 triggers growth inhibition and is more potent than 17-AAG against MM and other malignancies. It induces apoptosis via caspase-8, -9, -3, and poly (ADP-ribose) polymerase cleavage. SNX-2112 inhibits cytokine-induced Akt and extracellular signal-related kinase (ERK) activation and also overcomes the growth advantages conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cells. Importantly, SNX-2112 inhibits tube formation by human umbilical vein endothelial cells via abrogation of eNOS/Akt pathway and markedly inhibits osteoclast formation via down-regulation of ERK/c-fos and PU.1. Finally, SNX-2112, delivered by its prodrug SNX-5422, inhibits MM cell growth and prolongs survival in a xenograft murine model. Our results indicate that blockade of Hsp90 by SNX-2112 not only inhibits MM cell growth but also acts in the bone marrow microenvironment to block angiogenesis and osteoclastogenesis. Taken together, our data provide the framework for clinical studies of SNX-2112 to improve patient outcome in MM and other hematologic malignancies.     

The histone deacetylase inhibitor, PXD101, potentiates bortezomib-induced anti-multiple myeloma effect by induction of oxidative stress and DNA damage

Clinical trials have shown the high anti-myeloma activity of the proteasome inhibitor bortezomib. The present study examined the activity of bortezomib combined with PXD101, a histone deacetylase inhibitor, against multiple myeloma (MM) and osteoclastogenesis. Treatment of myeloma cell lines with combinations of bortezomib and PXD101 led to synergistic inhibition of proliferation and induction of cell death. The combination significantly decreased the viability of primary human CD138(+) myeloma cells but not of bone marrow mononuclear cells. Further studies showed a dose-dependent activation of caspases-3, -8 and -9 and nuclear fragmentation in myeloma cells. Bortezomib/PXD101 treatment markedly triggered reactive oxygen species (ROS) generation that was accompanied by p53, H2A.X and p38-mitogen-activated protein kinase phosphorylation. ROS generation could be blocked by the free radical scavenger N-acetyl-L-cysteine. The combination of bortezomib and PXD101 also resulted in synergistic inhibition of osteoclast formation. In conclusion, bortezomib and PXD101 have different molecular targets. The combination induces cell death in myeloma cells via ROS-mediated DNA damage and also inhibits osteoclastogenesis. Therefore, this study provides the rationale for the clinical evaluation of bortezomib combined with PXD101 in patients with MM.     

Analysis of histone deacetylase inhibitor, depsipeptide (FR901228), effect on multiple myeloma

Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)-6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL-2 family proteins (BCL-2, BCL-XL, BAX and MCL-1) were also investigated. In addition, the RPMI 8226 cell line (IL-6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time- and dose-dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 micromol/l). BCL-2, BCL-XL and MCL-1 showed decreased expression in depsipeptide-treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.     

Anti-myeloma effect of homoharringtonine with concomitant targeting of the myeloma-promoting molecules, Mcl-1, XIAP, and β-catenin

Since a variety of cell intrinsic and extrinsic molecular abnormalities cooperatively promote tumor formation in multiple myeloma (MM), therapeutic approaches that concomitantly target more than one molecule are increasingly attractive. We herein demonstrate the anti-myeloma effect of a cephalotaxus alkaloid, homoharringtonine (HHT), an inhibitor of protein synthesis, through the induction of apoptosis. HHT significantly reduced Mcl-1, a crucial protein involved in myeloma cell survival, in all three myeloma cell lines examined, whereas certain BH3-only proteins, such as Bim, Bik, and Puma, remained unchanged following HHT treatment, and their expression levels depended on the cell type. HHT also reduced the levels of c-FLIP(L/S), activated caspase-8, and induced active truncated-Bid. Thus, HHT-induced apoptosis appears to be mediated via both intrinsic and extrinsic apoptosis pathways, and the resultant imbalance between BH3-only proteins and Mcl-1 may be pivotal for apoptosis by HHT. In addition, HHT treatment resulted in reduced levels of beta-catenin and XIAP proteins, which also contribute to disease progression and resistance to chemotherapy in MM. In combination, HHT enhanced the effects of melphalan, bortezomib, and ABT-737. These results suggest that HHT could constitute an attractive option for MM treatment though its ability to simultaneously target multiple tumor-promoting molecules.     

TG101209, a novel JAK2 inhibitor,has significant in vitro activity in multiple myeloma and displays preferential cytotoxicity for CD45+ myeloma cells

Interaction of myeloma cells with the bone marrow microenvironment is mediated in large part through different cytokines, especially VEGF and IL6. These cytokines, especially IL6, leads to upregulation of the JAK/STAT pathway in myeloma cell, contributing to increased proliferation, decreased apoptosis, and acquired drug resistance. Here, we examined the preclinical activity of a novel JAK2 inhibitor TG101209. TG101209 induced dose‐ and time‐dependent cytotoxicity in a variety of multiple myeloma (MM) cell lines. The induction of cytotoxicity was associated with inhibition of cell cycle progression and induction of apoptosis in myeloma cell lines and patient‐derived plasma cells. Evaluation of U266 cell lines and patient cells, which have a mix of CD45 positive and negative cells, demonstrated more profound cytotoxicity and antiproliferative activity of the drug on the CD45+ population relative to the CD45? cells. Exploring the mechanism of action of TG101209 indicated downregulation of pJak2, pStat3, and Bcl‐xl levels with upregulation of pErk and pAkt levels indicating cross talk between signaling pathways. TG101209, when used in combination with the PI3K inhibitor LY294002, demonstrated synergistic cytotoxicity against myeloma cells. Our results provide the rationale for clinical evaluation of TG101209 alone or in combination with PI3K/Akt inhibitors in MM. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.