scholarly journals Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1654-1664 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita Singh ◽  
Mohan Brahmandam ◽  
Klaus Podar ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Er Stress Response ◽  
Synergistic Cytotoxicity ◽  
Proteolytic Activities ◽  
Dose Combination ◽  
Inhibition Of Tumor Growth

AbstractOur recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib–induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-κB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib–treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

scholarly journals Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1669-1677 ◽  
Author(s):  
Klaus Podar ◽  
Marc S. Raab ◽  
Jing Zhang ◽  
Douglas McMillin ◽  
Iris Breitkreutz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Xenograft Model ◽  
Multidrug Resistant ◽  
The Novel ◽  
Pkc Inhibitor ◽  
Downstream Signaling ◽  
Synergistic Cytotoxicity ◽  
And Migration

Abstract In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCμ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

Combination of Proteasome Inhibitors Bortezomib and NPI-0052 Trigger In Vivo Synergistic Cytotoxicity in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2524-2524
Author(s):  
Dharminder Chauhan ◽  
Ajita Singh ◽  
Mohan Brahmandam ◽  
Klaus Podar ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Proteasome Inhibitor ◽  
Histone Deacetylase Inhibitors ◽  
Treatment Strategies ◽  
Proteasome Inhibition ◽  
Synergistic Cytotoxicity ◽  
Proteolytic Activities

Abstract Bortezomib (Velcade™), the first in class proteasome inhibitor is FDA approved drug for the treatment of relapsed and relapsed/refractory multiple myeloma (MM). However, as with other agents, dose-limiting toxicities and the development of resistance limits its long-term utility. Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in MM cells; and importantly, is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 + bortezomib-induced synergistic apoptosis is associated with: activation of caspase-8, caspase-9, caspase-3, and PARP; induction of ER-stress response and JNK; inhibition of migration of MM cells and angiogenesis; suppression of chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteolytic activities; and blockade of NF-kappa B signaling. Studies in a xenograft MM model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth. Importantly, analysis of resected xenografted tumors show that 30–40% proteasome inhibition of all three (CT-L, C-L and T-L) proteasomal activities is sufficient to trigger significant MM cell apoptosis, confirming both the sensitivity of MM cells to proteasome inhibition and the importance of inhibiting all three proteolytic activities to obtain maximum response. Immunohistochemical analysis of MM tumors excised from NPI-0052 + bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. The clinical observation that bortezomib therapy can be associated with toxicity and drug-resistance, coupled with our present preclinical findings demonstrating that low doses of bortezomib together with NPI-0052 trigger a potent anti-MM effect in vitro and in vivo, suggests the promise of combination treatment strategies to enhance anti-MM activity, reduce toxicity, overcome drug resistance, and improve outcome in MM patients. In addition to the above studies, data related to combination of NPI-0052 with lenalidomide (Revlimid™) and with histone deacetylase inhibitors such as MS-275, Tubacin or LBH589, will be presented.

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

scholarly journals Identification of a novel c-Myc inhibitor with antitumor effects on multiple myeloma cells

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Ruosi Yao ◽  
Xiaoyang Sun ◽  
Yu Xie ◽  
Xiaoshen Sun ◽  
Yao Yao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Xenograft Model ◽  
Molecular Compound ◽  
Severe Combined Immune Deficiency ◽  
Antitumor Effects ◽  
Mouse Xenograft Model ◽  
The Stability ◽  
Rpmi 8226

Increasing evidence shows that c-Myc oncoprotein is tightly associated with multiple myeloma (MM) progression. Herein, we identified compound 7594-0035, which is a novel inhibitor that specifically targets c-Myc. It was identified from the ChemDiv compound database by molecular docking-based, high-throughput virtual screening. Compound 7594-0035 inhibited MM cell proliferation in vitro, induced cell cycle G2-phase arrest, and triggered MM cell death by disturbing the stability of c-Myc protein. Additionally, we also found that compound 7594-0035 overcame bortezomib (BTZ) drug resistance and increased the killing effect on MM cells in combination with BTZ. The severe combined immune deficiency (SCID) mouse xenograft model revealed that compound 7594-0035 partially decreased the primary tumor growth of Roswell Park Memorial Institute (RPMI)-8226 cells in vivo. The novel small molecular compound 7594-0035 described in the present study that targets c-Myc protein is likely to be a promising therapeutic agent for relapsed/refractory MM.

Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid–based proteasome inhibitors

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 5927-5937 ◽  
Author(s):  
Encouse B. Golden ◽  
Philip Y. Lam ◽  
Adel Kardosh ◽  
Kevin J. Gaffney ◽  
Enrique Cadenas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Tumor Cell ◽  
Green Tea ◽  
Boronic Acid ◽  
Proteasome Inhibitors ◽  
Green Tea Polyphenols ◽  
Tumor Cell Death

Abstract The anticancer potency of green tea and its individual components is being intensely investigated, and some cancer patients already self-medicate with this “miracle herb” in hopes of augmenting the anticancer outcome of their chemotherapy. Bortezomib (BZM) is a proteasome inhibitor in clinical use for multiple myeloma. Here, we investigated whether the combination of these compounds would yield increased antitumor efficacy in multiple myeloma and glioblastoma cell lines in vitro and in vivo. Unexpectedly, we discovered that various green tea constituents, in particular (-)-epigallocatechin gallate (EGCG) and other polyphenols with 1,2-benzenediol moieties, effectively prevented tumor cell death induced by BZM in vitro and in vivo. This pronounced antagonistic function of EGCG was evident only with boronic acid–based proteasome inhibitors (BZM, MG-262, PS-IX), but not with several non–boronic acid proteasome inhibitors (MG-132, PS-I, nelfinavir). EGCG directly reacted with BZM and blocked its proteasome inhibitory function; as a consequence, BZM could not trigger endoplasmic reticulum stress or caspase-7 activation, and did not induce tumor cell death. Taken together, our results indicate that green tea polyphenols may have the potential to negate the therapeutic efficacy of BZM and suggest that consumption of green tea products may be contraindicated during cancer therapy with BZM.

MTOR Inhibitors Activate the AKT Kinase in Multiple Myeloma Cells by Upregulating the IGF-1/IRS-1/PI-3 Kinase Cascade.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3350-3350 ◽  
Author(s):  
Yijiang Shi ◽  
HuaJun Yan ◽  
Patrick Frost ◽  
Bao Hoang ◽  
Joseph Gera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
G1 Arrest ◽  
Akt Activation ◽  
Induced Apoptosis

Abstract MTOR inhibitors, such as rapamycin and CCI-779, have shown pre-clinical potential as therapy for multiple myeloma (MM). By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent phosphorylation of insulin receptor substrate-1 (IRS-1), they may alter its subcellular localization and/or prevent its degradation which could enhance IGF-1 signaling and downstream PI3-kinase/AKT activation. This may be a particular problem in MM where IGF-1-induced activation of AKT is an important anti-apoptotic cascade. We, thus, studied PI3-kinase/AKT activation in MM cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation and PI3-kinase activity in MM cell lines. Both PTEN-null as well as PTEN-wild type myeloma lines were similarly affected. Rapamycin also significantly prolonged activation of AKT induced by exogenous IGF-1. CCI-779, used in a xenograft model, also resulted in MM cell AKT activation in vivo. Blockade of IGF-1 receptor function prevented rapamycin’s activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1 and IRS-1 degradation. Though similarly blocking IRS-1 degradation, proteasome inhibitors did not activate MM cell AKT. Although rapamycin sensitized MM cells for dexamethasone-induced apoptosis, it protected against PS-341-induced apoptosis. Thus, mTOR inhibitors activate PI3-K/AKT in MM cells and activation depends on basal IGF-1/IGF-R signaling. As activated AKT may protect against apoptosis, future use of mTOR inhibitors in myeloma patients will have to carefully consider the types of anti-myeloma agents used in combination.

A Humanized Anti-Ganglioside GM2 Antibody, BIW-8962, Exhibits ADCC/CDC Activity against Multiple Myeloma Cells and Potent Anti- Tumor Activity in Mouse Xenograft Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1718-1718 ◽  
Author(s):  
Toshihiko Ishii ◽  
Asher Alban Chanan-Khan ◽  
Jazur Jafferjee ◽  
Noreen Ersing ◽  
Takeshi Takahashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Phase 1 ◽  
Xenograft Model ◽  
Surface Expression ◽  
Scid Mice ◽  
Subcutaneous Xenograft ◽  
Anti Tumor Activity

Abstract BIW-8962 is a humanized anti-ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hiroshi Ikeda ◽  
Teru Hideshima ◽  
Robert J. Lutz ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Growth Delay ◽  
Selective Cytotoxicity ◽  
Bystander Killing

Abstract CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

In-Vitro and in-Vivo Synergistic Effect of Melphalan and Dual DNA Repair Inhibition in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3301-3301
Author(s):  
Pritesh R. Patel ◽  
Annie L. Oh ◽  
Vitalyi Senyuk ◽  
Dolores Mahmud ◽  
Nadim Mahmud ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Combination Index ◽  
Fold Increase ◽  
High Dose ◽  
Myeloma Cells ◽  
Synergistic Cytotoxicity ◽  
Significant Difference

Abstract High dose melphalan is commonly used in patients with multiple myeloma (MM). Resistance to melphalan has been linked to the ability to repair DNA damage. To test whether DNA repair inhibitors overcome resistance to melphalan and and also have a direct anti-MM effect, we tested MM cell lines RPMI8226 and U266 in-vitro and in-vivo, using a NOD/SCID/ gamma null (NSG) xenograft model. RPMI8226 and U266 cells were initially treated in-vitro with the PARP inhibitor ABT-888. Using a proliferative assay, myeloma cells appeared sensitive to ABT-888 with low GI50 values (8.7μM for RPMI8226 cells, 49μM for U266 cells) and increased γH2AX foci, which persisted at 24 hours after treatment. This was confirmed in methycellulose colony assay where ABT-888 treatment reduced RPMI8226 colonies by 35% (p=0.002). Next we showed synergistic cytotoxicity between ABT-888 and melphalan. In both RPMI8226 and U266 cells strong synergy was displayed with a combination index (CI) less than 1 in proliferative assays (CI 0.5 and 0.3 at 50% proliferation respectively). Combination ABT-888 and melphalan treated cells underwent accelerated senescence compared to cells treated by melphalan alone (27% versus 51% βGal+ staining at 24 hours, p=0.02). This was confirmed by upregulation of senescence related genes p16 (1.6 fold increase) and p21 (1.5 fold increase). We did not find significant difference in apoptosis by Annexin V/ PI staining. Given that increased non-homologous end joining (NHEJ) activity has been shown to lead to resistance to melphalan, we tested whether an inhibitor of NHEJ could be synergistic with PARP inhibition and melphalan. Treatment with the DNA-PK inhibitor NU7026 at 10μM in addition to ABT-888 at 4μM resulted in 46% reduction in proliferation in RPMI8226 cells and 52% in U266 cells. When used in combination with melphalan chemotherapy, the dual DNA repair inhibitor therapy showed marked synergy in RPMI8226 cells with a combination index of 0.39. Finally we tested the ability of the combination of ABT-888 and melphalan to treat myeloma in-vivo. NSG mice were injected via tail vein with 5x106 RPMI8226 cells. Control (untreated) mice subsequently developed myeloma infiltrating the marrow, spleen and axial skeleton, with hind limb paralysis occurring at a median of 42 days. Treated mice received intraperitoneal injections of ABT-888 (3 times a week), or melphalan (weekly) or a combination of both agents starting on day 28 post-injection of MM cells for a total of 3 weeks. Using ABT-888, melphalan and a combination of both agents, median survival of mice was progressively prolonged (44 vs. 67 vs. 107 days, respectively) (p=0.02). Here we show that PARP and DNA-PK inhibition synergizes with melphalan in myeloma cells lines, providing a rationale for the addition of these agents to conditioning chemotherapy. In addition, we also show a direct anti-myeloma activity of these agents without the use of alkylator chemotherapy. Disclosures No relevant conflicts of interest to declare.

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