Cytotoxicity In Multiple Myeloma Cells Research Articles

Novel Etodolac Analog SDX-308 (CEP-18082) Induces Cytotoxicity in Multiple Myeloma Cells Associated with Inhibition of Wnt/β-Catenin Pathway.

In a previous study we reported that R-enantiomer of etodolac (R-etodolac) which is under investigation in Phase II clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in both drug-sensitive and drug-resistant multiple myeloma (MM) cell lines, as well as in patient MM tumor cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against MM cell lines and tumor cells from patients with refractory MM. It also induces cytotoxicity against bortezomib-resistant tumor cells. The molecular mechanisms whereby SDX-308 triggers MM cell cytotoxicity were next delineated. SDX-308 induces apoptosis via caspase-8/-9/-3 activation and poly(ADP-ribose) polymerase PARP cleavage. It strongly inhibits Wnt/β-catenin pathway by blockade of nuclear translocation of β-catenin, followed by significant inhibition of transcription and expression of target proteins. These target proteins include cell cycle regulatory c-myc molecules and anti-apoptotic survivin molecules. Neither interleukin-6 nor insulin-like growth factor-1, which induce MM cell growth and abrogate Dex-induced apoptosis, protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 has more potent cytotoxicity in MM cells than R-etodolac even in the context with BM microenvironment, providing the preclinical rationale to conduct clinical trials of this agent to improve patient outcome in MM.

The Combination of the mTOR Inhibitor Rapamycin and Proteasome Inhibitor Bortezomib Is Synergistic In Vitro in Multiple Myeloma.

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

Akt Inhibitor Perifosine-Induced Cytotoxicity Is Associated with Significant Downregulation of Survivin in Human Multiple Myeloma (MM) Cells.

Perifosine (NSC 639966; Keryx Biopharmaceuticals, New York, NY) is a synthetic novel alkylphospholipid, a new class of anti-tumor agents which potently inhibits Akt (PKB) activity. Our previous studies have shown that Perifosine induces significant cytotoxicity in MM cells triggered by c-Jun NH2-terminal kinase (JNK) activation followed by caspase-8, caspase-9, and PARP cleavage even in the presence of cytokines (ie, IL-6 and IGF-1) or bone marrow stromal cell (BMSCs). Importantly, MEK inhibitor and bortezomib enhance Perifosine-induced cytotoxicity. It has also shown significant anti-tumor activity in a human MM cell xenograft mouse model (Hideshima et al. Blood 2006, 107:4053–4062). In this study, we further delineated molecular mechanisms whereby Perifosine triggers cytotoxicity as a single agent and in combination with bortezomib in MM cells. In most MM cell lines, the IC50 for Perifosine-induced cytotoxicity is 5–10 μM range assessed by MTT assay at 24h; however, apoptosis assessed by APO2.7 staining, varied in each cell line. Moreover, neither the degree of JNK phosphorylation nor caspase-8/9/PARP cleavage correlated with Perifosine-induced cytotoxicity. Therefore we further examined expression level of anti-apoptotic proteins in MM cell lines and found that survivin, which has a crucial role in regulation of caspase-3 activity, was markedly downregulated by Perifosine treatment in a time- and dose-dependent fashion, without affecting expression of other anti-apoptotic proteins (ie, cIAP, XIAP, Bcl-2, Bcl-xL). Since survivin is a known downstream protein of β-catenin/TCF-4 cascade, we next hypothesized that Perifosine may inhibit β-catenin activity. As expected, Perifosine significantly downregulated both phosphorylation and protein expression of β-catenin, associated with downregulation of survivin and enhanced caspase-3 cleavage. Real-time PCR confirmed that gene expression of survivin was suppressed 35% and 55% after 3h and 6h Perifosine treatment, respectively. Since β-catenin is a substrate of proteasomes, we further examined whether bortezomib could augment survivin expression by blocking its degradation. Importantly, bortezomib significantly upregulated β-catenin and survivin, which was blocked in the presence of Perifosine. These results suggest that inhibition of bortezomib-induced survivin expression, at least in part, accounts for enhanced bortezomib-induced cytotoxicity by Perifosine. Based upon these preclinical studies, a rational combination trial of bortezomib with Perifosine to treat relapsed refractory MM is currently ongoing.

A Multicenter Phase II Study of Perifosine (KRX-0401) Alone and in Combination with Dexamethasone (Dex) for Patients with Relapsed or Relapsed/Refractory Multiple Myeloma (MM).

Perifosine is an oral, novel synthetic alkylphospholipid, with multiple effects on signal transduction pathways, including inhibition of Akt and activation of JNK. Preclinical in vitro studies showed that perifosine induces significant cytotoxicity in both multiple myeloma(MM) cell lines and patient MM cells resistant to conventional therapies, and augments dexamethasone(dex), doxorubicin, melphalan and bortezomib-induced MM cell cytotoxicity. In vivo studies showed significant antitumor activity in a human plasmacytoma mouse model. PhaseI studies in solid tumors have shown that perifosine is well tolerated at a dose of up to150mg daily, with responses also seen. We report preliminary results of a PhaseII trial of perifosine, alone and in combination with dex, in patients(pts) with relapsed or relapsed/refractory MM. Pts received 150mg of perifosine daily for a 21-day(d) cycle, and were assessed by serum and/or urine electrophoresis. Eligible pts had relapsed or relapsed/refractory MM with measurable disease. Pts were permitted bisphosphonate treatment. Concomitant steroids(prednisone>10 mg/d), serum creatinine of >3.0 mg/dL, and hemoglobin<8.0g/dL within 14 d of enrollment were exclusion criteria. Progressing pts, documented on 2 occasions at least one week apart, had dex 20 mg twice per week added to perifosine. Toxicities were assessed by NCI-CTCAE, v3.0. 40 pts (22 men and 18 women, median age 61 y, range 38–78) have been treated to date. All had relapsed/refractory MM, with a median of 4 lines of prior treatment (range 1–9). Prior therapy included dex(100%), thalidomide(100%), bortezomib(73%), lenalidomide(28%) and stem cell transplant(73%). Among 25 pts currently evaluable for response, best response(EBMT criteria) to single agent perifosine after≥2 cycles was stable disease(<25% reduction in M-protein) in 6 pts(24%). Dex was added in 15 of 25 pts with PD, with 9 pts evaluable for response on the combination: 3 pts(33%) achieved MR and 2(22%) pts achieved SD. The most common adverse events included nausea (45%, 3% grade 3); vomiting (40%); diarrhea(40%); fatigue(24%, 3% grade 3), and increased creatinine(55%, 11% grade 3/4 in the context of PD and light chain nephropathy). 2 pts had G3 neutropenia which resolved. Dose reduction(150 to 100 mgs/d) was required in 11 pts and 4 pts discontinued treatment due to adverse events. Attributable toxicities otherwise proved manageable with appropriate supportive care and perifosine was generally well tolerated, with no peripheral neuropathy or DVT seen. Perifosine as monotherapy and in combination with dex has activity in pts with advanced, relapsed/refractory MM, achieving MR and/or stabilization of disease in 55% of evaluable pts to date. It was generally well tolerated, although caution in pts with renal dysfunction is warranted. PK, IHC and gene array studies are ongoing. Future studies evaluate perifosine at other dosing schedules and in combination with other agents including bortezomib.

In vivo and in vitro cytotoxicity of R‐etodolac with dexamethasone in glucocorticoid‐resistant multiple myeloma cells

Glucocorticoids have been widely used in the treatment of multiple myeloma (MM) both as single agents and in combination with other drugs. However, primary or acquired glucocorticoid resistance occurs in most cases. It was recently reported that R-etodolac induced in vitro cytotoxicity in MM cell lines and in primary MM cells, as well as synergistically enhanced dexamethasone (Dex)-induced apoptosis in Dex-sensitive MM.1S cells. This study examined the in vitro and in vivo effects of combination treatment with R-etodolac and Dex on Dex-resistant OPM1 cells. Treatment with R-etodolac and Dex was found to enhance cytotoxicity, inhibit nuclear factor kappaB activity via upregulation of IkappaBalpha, as well as enhance Dex-induced caspase activation and poly (ADP)-ribose polymerase cleavage in OPM1 cells. R-etodolac also enhanced Dex cytotoxicity in patient MM cells that were resistant to glucocorticoids. The in vivo anti-tumour effect of this combination on MM cells was evaluated by using severe combined immunodeficient mice engrafted with OPM1. Treatment with R-etodolac or Dex alone did not induce a significant reduction of tumour volume; in contrast, combination treatment with R-etodolac and Dex induced significant synergistic inhibition of tumour growth. These data indicate that R-etodolac overcomes resistance to Dex in glucocorticoid-resistant MM cells, providing the framework for clinical trials of R-etodolac combined with Dex, to improve patient outcome in MM.

Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agents which targets cell membranes and inhibits Akt activation. Here we show that baseline phosphorylation of Akt in multiple myeloma (MM) cells is completely inhibited by perifosine [octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate] in a time- and dose-dependent fashion, without inhibiting phosphoinositide-dependent protein kinase 1 phosphorylation. Perifosine induces significant cytotoxicity in both MM cell lines and patient MM cells resistant to conventional therapeutic agents. Perifosine does not induce cytotoxicity in peripheral blood mononuclear cells. Neither exogenous interleukin-6 (IL-6) nor insulinlike growth factor 1 (IGF-1) overcomes Perifosine-induced cytotoxicity. Importantly, Perifosine induces apoptosis even of MM cells adherent to bone marrow stromal cells. Perifosine triggers c-Jun N-terminal kinase (JNK) activation, followed by caspase-8/9 and poly (ADP)-ribose polymerase cleavage. Inhibition of JNK abrogates perifosine-induced cytotoxicity, suggesting that JNK plays an essential role in perifosine-induced apoptosis. Interestingly, phosphorylation of extracellular signal-related kinase (ERK) is increased by perifosine; conversely, MEK inhibitor synergistically enhances Perifosine-induced cytotoxicity in MM cells. Furthermore, perifosine augments dexamethasone, doxorubicin, melphalan, and bortezomib-induced MM cell cytotoxicity. Finally, perifosine demonstrates significant antitumor activity in a human plasmacytoma mouse model, associated with down-regulation of Akt phosphorylation in tumor cells. Taken together, our data provide the rationale for clinical trials of perifosine to improve patient outcome in MM.

Small Molecule Inhibition of Proteasome and Aggresome Function Induces Synergistic Anti-Tumor Activity in Multiple Myeloma.

Bortezomib (Velcade®) triggers significant anti-tumor activity in multiple myeloma (MM) both in preclinical models and in patients with relapsed refractory disease. However, 60–65% of patients did not respond to bortezomib, and mechanisms of resistance to bortezomib are not fully understood. Recent studies have revealed an alternative system to the proteasome for degradation of polyubiquitinated misfolded/unfolded proteins, termed the aggresome. Aggresome formation ultimately induces autophagic clearance, which terminates in lysosomal degradation. The aggresome pathway therefore likely provides a novel system for delivery of aggregated proteins from cytoplasm to lysosomes for degradation. In aggresomal protein degradation, histone deacetylase6 (HDAC6) has an essential role. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins, followed by significant cell stress and cytotoxicity in MM cells. We demonstrate that HDAC6 specific inhibitor Tubacin specifically triggers acetylation of α-tubulin, as a result of HDAC6 inhibition, in a dose- and time-dependent fashion. It induces cytotoxicity in MM cells, which is mediated via caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity via c-Jun NH2-terminal kinase (JNK)/caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow (BM). Adherence of MM cells to BM stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of tubacin and bortezomib triggers toxicity even in adherent MM cells (Hideshima et al. PNAS 102:8567–8572, 2005). In the present study, we further examined the biologic sequelae of inhibiting both proteasome and aggresome pathways in MM. Bortezomib induces aggresome formation, evidenced by immunohistochemical analyses using DAPI, ubiquitin and γ-tubulin Abs. Tubacin enhances 20S proteasome activity in a time-dependent fashion; conversely bortezomib significantly augments HDAC6 protein expression. Taken together, these results suggest that proteasome function could compensate for aggresome function and vise versa. Importantly, treatment with the combination of Tubacin and bortezomib induces caspase-12 cleavage and upregulation of Grp78 in MM cells suggesting endoplasmic reticulum (ER) stress, associated with increased apoptosis. Finally, heat shock protein (Hsp)90 is a chaperone for transport of ubiquitinated proteins to the proteasome. HDAC6 is also constitutively associated with Hsp90, and the HDAC6 inhibitor Tubacin augments Hsp90 acetylation. Ongoing studies will delineate the biologic role of HDAC6 in the chaperone function both in proteasome and aggresome protein degradation cascades. Our studies therefore demonstrate that Tubacin combined with bortezomib mediates significant anti-MM activity by blocking both the aggresome and proteasome, respectively, thereby providing the framework for clinical evaluation of combined therapy to improve patient outcome in MM.

Targeting the Protein Degradation Pathway in Multiple Myeloma with Synergistic, Selective Small Molecules.

The response of refractory multiple myeloma to the proteasome inhibitor bortezomib reveals an intriguing sensitivity of this incurable malignancy to perturbations of protein catabolism. However, an overall clinical response rate of approximately 30% as a single agent suggests the importance of chemoresistance mediated by compensatory mechanisms of protein degradation. With proteasome inhibition, juxtanuclear inclusion bodies accumulate. These “aggresomes” are specific cellular structures comprised of chaperones, misfolded proteins, and proteasome components. The cytoplasmic histone deacetylase inhibitor (HDAC6) is essential for aggresome formation. Recently, we have demonstrated robust cytotoxic synergy in multiple myeloma cells between bortezomib and the carboxy-terminal domain-selective inhibitor of HDAC6, tubacin (1). Tubacin is a hydroxamic acid member of a diversity-oriented synthetic chemical library realized and validated previously by members of our laboratory (2). In our study of bortezomib and tubacin in multiple myeloma, we noted the dose-dependent, significant association between cytotoxicity and the marked accumulation of polyubiquitinated proteins in sensitized cells. With an interest in further interruption of the misfolded protein response, we have explored the cytosolic chaperone protein, hsp90, as an additional target using a chemical biologic approach. 17-AAG is an analog of the benzoquinone ansamycin antibiotic geldanamycin, known to bind to the ADP/ATP pocket of hsp90. 17-AAG stabilizes a conformation of the chaperone favoring targeted degradation of its client proteins via recruitment of the hsp70 co-chaperone complex (3). Further supporting this strategy, collaborators have recently identified that HDAC6 binds to and principally mediates the deacetylation of hsp90 (4). Inhibition with non-selective HDAC inhibitors was shown to augment hsp90 acetylation and inhibit ATP binding, resulting in the promotion of protein degradation by polyubiquitination. With the support of these data, we investigated whether the addition of 17-AAG to bortezomib and tubacin results in increased cytotoxicity in multiple myeloma cells. Indeed, we demonstrate potent cytotoxicity in cultured myeloma cells at low concentrations of each small molecule. Focused study of the MM.1S cell line demonstrates that the addition of 17-AAG to tubacin and bortezomib markedly increases the burden of ubiquitinated, cytosolic cellular protein by 24 hours, correlating with enhanced cell killing. These data further credential the protein degradation pathway in multiple myeloma, demonstrate the contribution of targeted, combined approaches with active small molecules, and provide a blueprint for a rational therapeutic strategy.

R-Etodolac and a Novel Indole-Pyran Structural Analog, SDX-308, Induce Cytotoxicity and Overcome Drug Resistance in Multiple Myeloma.

SDX-101, the less toxic R-isomer of the commercially available non-steroidal inflammatory drug R,S-etodolac (Lodine®), lacks COX inhibitory activity and is being investigated in Phase II clinical trials in chronic lymphocytic leukemia. Recently, we reported that R-etodolac, at clinically relevant concentrations, induces potent in vitro cytotoxicity in drug-sensitive and conventional drug-resistant multiple myeloma (MM) cell lines, as well as in primary tumor cells from MM patients. R-etodolac triggers caspase/poly-ADP-ribose polymerase (PARP) cleavage and downregulates of cyclin D1 expression (Yasui et al. Blood 2005). Importantly, R-etodolac at sub-cytotoxic doses upregulates Mcl-1s and synergistically enhances dexamethasone (Dex)-induced caspase-dependent apoptosis in Dex-sensitive MM.1S cells. Combination of R-etodolac with Dex enhances cytotoxicity in Dex resistant OPM1 MM cells and in Dex-resistant patient MM cells in vitro. We further studied the in vivo anti-tumor effect of combined R-etodolac and Dex in SCID mice injected subcutaneously with OPM1 human MM cells. While oral treatment of SCID mice with R-etodolac alone (250 mg/kg/d) or Dex alone (1 mg/kg/d) did not induce any significant reduction of tumor volume compared with control (PBS), the combination of R-etodolac and Dex inhibited tumor growth synergistically (synergism quotient = 1.6) and significantly (p = 0.023), suggesting that R-etodolac may reverse Dex resistance in MM. Finally, we demonstrated that racemic SDX-308, a novel indole-pyran structural analog of etodolac, has 10-fold more potent cytotoxicity than R-etodolac in MM cell lines both sensitive and resistant to conventional therapies, as well as in patient's MM cells. Moreover, SDX-308, like R-etodolac, can overcome the viability and proliferative enhancing effects of exogeneous IL-6, IGF-1, or bone marrow stroma cells. These combined observations indicate that SDX-308 is a promising more potent second generation analog of R-etodolac for MM therapy. Our data suggest that R-etodolac and its novel analog SDX-308 overcome resistance to some conventional therapeutics used for MM, and provide preclinical rationale to conduct clinical trials of R-etodolac and SDX-308 to treat MM.

Honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis

AbstractHonokiol (HNK) is an active component purified from magnolia, a plant used in traditional Chinese and Japanese medicine. Here we show that HNK significantly induces cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM. Neither coculture with bone marrow stromal cells nor cytokines (interleukin-6 and insulin-like growth factor-1) protect against HNK-induced cytotoxicity. Although activation of caspases 3, 7, 8, and 9 is triggered by HNK, the pan-caspase inhibitor z-VAD-fmk does not abrogate HNK-induced apoptosis. Importantly, release of an executioner of caspase-independent apoptosis, apoptosis-inducing factor (AIF), from mitochondria is induced by HNK treatment. HNK induces apoptosis in the SU-DHL4 cell line, which has low levels of caspase 3 and 8 associated with resistance to both conventional and novel drugs. These results suggest that HNK induces apoptosis via both caspase-dependent and -independent pathways. Furthermore, HNK enhances MM cell cytotoxicity and apoptosis induced by bortezomib. In addition to its direct cytotoxicity to MM cells, HNK also represses tube formation by endothelial cells, suggesting that HNK inhibits neovascurization in the bone marrow microenvironment. Taken together, our results provide the preclinical rationale for clinical protocols of HNK to improve patient outcome in MM. (Blood. 2005;106:1794-1800)

SDX-101, the R-enantiomer of etodolac, induces cytotoxicity, overcomes drug resistance, and enhances the activity of dexamethasone in multiple myeloma

AbstractIn this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex. (Blood. 2005;106:706-712)

Adenoviral-mediated transfer of human wild-type p53, GM-CSF and B7-1 genes results in growth suppression and autologous anti-tumor cytotoxicity of multiple myeloma cells in vitro.

Multiple myeloma (MM) remains incurable despite the use of high-dose chemotherapy and stem cell transplantation. However, immunotherapy is expected to offer long-term disease control, or even possibly a cure. We have previously demonstrated the suppressive effect of a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor, and B7-1 genes (Ad-p53/GM-CSF/B7-1) on the growth of laryngeal cancer cells. In the present study, we evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis and to augment the immunogenicity of MM cells. Both MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. High expression levels of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. When wild-type p53, GM-CSF and B7-1 genes were introduced, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these three genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of two (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro. These results suggest that myeloma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a promising immunotherapeutic approach against MM.

Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.

Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma

We have shown that the proteasome inhibitor bortezomib (formerly known as PS-341) triggers significant antitumor activity in multiple myeloma (MM) in both preclinical models and patients with relapsed refractory disease. Recent studies have shown that unfolded and misfolded ubiquitinated proteins are degraded not only by proteasomes, but also by aggresomes, dependent on histone deacetylase 6 (HDAC6) activity. We therefore hypothesized that inhibition of both mechanisms of protein catabolism could induce accumulation of ubiquitinated proteins followed by significant cell stress and cytotoxicity in MM cells. To prove this hypothesis, we used bortezomib and tubacin to inhibit the proteasome and HDAC6, respectively. Tubacin specifically triggers acetylation of alpha-tubulin as a result of HDAC6 inhibition in a dose- and time-dependent fashion. It induces cytotoxicity in MM cells at 72 h with an IC50 of 5-20 microM, which is mediated by caspase-dependent apoptosis; no toxicity is observed in normal peripheral blood mononuclear cells. Tubacin inhibits the interaction of HDAC6 with dynein and induces marked accumulation of ubiquitinated proteins. It synergistically augments bortezomib-induced cytotoxicity by c-Jun NH2-terminal kinase/caspase activation. Importantly, this combination also induces significant cytotoxicity in plasma cells isolated from MM patient bone marrow. Finally, adherence of MM cells to bone marrow stromal cells confers growth and resistance to conventional treatments; in contrast, the combination of tubacin and bortezomib triggers toxicity even in adherent MM cells. Our studies therefore demonstrate that tubacin combined with bortezomib mediates significant anti-MM activity, providing the framework for clinical evaluation of combined therapy to improve patient outcome in MM.

Targeting IKK Inhibits Multiple Myeloma (MM) Cell Growth in the Bone Marrow Microenvironment.

NF-κB is a transcriptional factor promoting tumor cell growth and inhibition of apoptosis via regulating expression of proteins modulating cell cycle and anti-apoptosis. NF-κB is constitutively associated with an inhibitor (IκB), which is phosphorylated by IκB kinase (IKK) upon cell stimulation (ie, TNFα) and subsequently degraded by ubiquitin-proteasome pathway, thereby allowing NF-κB to translocate to nucleus. We have previously shown that an IKK inhibitor PS-1145 partially (20–50%) inhibits MM cell proliferation; however, it inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. Targeting IKKβ is therefore a possible therapeutic option for inhibition of MM cell growth in the bone marrow microenvironment by downregulating NF-κB activity.In this study, we further delineated the biologic significance of IKK inhibition in MM cells using IKKβ specific inhibitor MLN120B (Millennium Pharmaceuticals, Cambridge, MA). MLN120B induced 60–90% growth inhibition in cells from the MM cell lines RPMI8226, RPMI/Dox40, RPMI/LR5, U266, and INA-6; on the other hand, it induced only 25–30% inhibition in MM.1S and MM.1R cells, assessed by 72 h tritiated-thymidine uptake. Importantly, neither IL-6 nor IGF-1 overcomes the growth inhibitory effect of MLN120B in both MM.1S and U266 cells. Interestingly, MLN120B significantly augmented TNFα-induced cytotoxicity in MM.1S cells. We next examined whether MLN120B could enhance the cytotoxicity of other agents. MLN120B augmented growth inhibition triggered by doxorubicin and melphalan in RPMI8226 and IL-6 dependent INA-6 cell line. We therefore studied growth inhibitory effect of MLN120B in the presence of bone marrow stromal cells (BMSCs). MLN120B inhibited 70–80% of constitutive IL-6 secretion from BMSCs, without toxicity. Importantly, MLN120B almost completely blocked stimulation of MM.1S, U266 and INA-4 cell growth and IL-6 secretion from BMSCs induced by binding of tumor cells to BMSCs. Finally, MLN120B overcame the protective effect of BMSCs, cell adhesion mediated drug resistance, against dexamethasone in MM.1S cells. Taken together, our data demonstrate that an IKKβ inhibitor induces cytotoxicity in MM cells in the BM milieu, providing the framework for clinical trials of these novel agents to improve patient outcome in MM.

A Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Overcomes Conventional Drug-Resistance in Multiple Mueloma Cells in the Bone Marrow Microenvironment.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA) is a small molecule, selective, uncompetitive novel inhibitor directed against human IMPDH enzyme. IMPDH inhibitors have been demonstrated to induce growth arrest, and extensively investigated as immunosuppressants. Here we show that VX-944 inhibits growth of human multiple myeloma (MM) cell lines, including those resistant to conventional agents, via induction of apoptosis and S phase arrest in vitro. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs), do not protect against VX-944-induced MM cell growth inhibition. We next delineated the molecular mechanism of VX-944-induced MM cell death in the MM.1S human MM cell line. VX-944 induced apoptosis in MM.1S cells, confirmed by PARP cleavage as well as flow cytometric detection of the mitochondrial membrane protein 7A6 and TdT-mediated dUTP nick-end labelling (TUNEL) positive cells, without significant cleavage of caspases 3, 8 and 9. While the pan-caspase inhibitor z-VAD-fmk did not inhibit the VX-944-induced apoptosis and cell death suggesting that VX-944 triggers apoptosis in MM1.S cells primarily via caspase-independent pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin, melphalan and bortezomib, all of which activate caspases in MM cells and induce apoptosis, even in the presence of BMSCs. Taken together, our data demonstrate non-caspase-dependent apoptotic pathway triggered by VX-944 thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional and/or novel agents which trigger caspase activation. Our ongoing studies are delineating the mechanisms whereby VX-944 induces MM cell apoptosis.

Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341.

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.

Beta-lapachone, a novel plant product, overcomes drug resistance in human multiple myeloma cells.

To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.

Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells

AbstractMultiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.