214. Combination Therapy with Novel Nanoparticle, SNS01-T, and Bortezomib Results in Synergistic Cytotoxicity In Vitro and In Vivo in Multiple Myeloma

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood-2007-08-105601 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1654-1664 ◽

Cited By ~ 138

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.

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

Blood ◽

10.1182/blood.v128.22.3301.3301 ◽

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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Efficacy of Panobinostat (LBH589) in Multiple Myeloma Cell Lines and In Vivo Mouse Model: Tumor-Specific Cytotoxicity and Protection of Bone Integrity in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.1510.1510 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1510-1510 ◽

Cited By ~ 2

Author(s):

Joseph D. Growney ◽

Peter Atadja ◽

Wenlin Shao ◽

Youzhen Wang ◽

Minying Pu ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Bone Density ◽

Mouse Model ◽

Cortical Bone ◽

Cell Lines ◽

Single Agent ◽

Synergistic Cytotoxicity

Abstract Panobinostat (LBH589) is a highly potent oral pan-deacetylase (DAC) inhibitor currently undergoing clinical development in hematologic and solid malignancies. Here we report the effects of panobinostat on multiple myeloma (MM) cells in vitro and in a murine xenograft model in vivo. Panobinostat exhibited potent cytotoxic activity (IC50 <10 nM) against 8 MM cell lines (KMS-12PE, KMS-18, LP-1, NCI H929, KMS-11, RPMI8226, OPM-2, and U266). Panobinostat has been shown to affect signals involved in MM cell-cycle arrest and cell death, and to induce apoptosis via mitochondrial perturbation. In addition, panobinostat has been shown to selectively induce cell death of plasma cells isolated from MM patients without toxicity to normal lymphocytes or granulocytes. To investigate the effect of panobinostat in vivo, a disseminated luciferized MM.1S xenograft mouse model was treated with vehicle or panobinostat 15 mg/kg by intraperitoneal (i.p.) administration qd×5 for 3 weeks. Panobinostat treatment reduced the burden of MM.1S tumor cells to 22% treated over control (T/C) relative to vehicle-treated animals. In addition, MM.1S tumor-bearing mice treated with panobinostat displayed reduced trabecular and cortical bone damage relative to vehicle-treated animals. The mean ± SEM trabecular bone density and cortical bone density (% Bone Volume/Total Volume) of panobinostat-treated animals was 14.5% ± 2.0 and 98.1% ± 0.4, respectively, compared with 2.2% ± 0.3 and 89.1% ± 1.5 in vehicle-treated animals. In combination with the proteosome inhibitor bortezomib (BZ), panobinostat displayed significant synergistic cytotoxicity without additional toxicity to normal bone marrow stromal cells in vitro. In the MM.1S-luciferase tumor mouse model, combined treatment with panobinostat at 10 mg/kg i.p. qd×5 for 4 weeks and BZ at 0.2 mg/kg intravenously 1qw for 4 weeks reduced tumor burden to 7% T/C relative to vehicle, panobinostat alone (31% T/C), or BZ alone (44% T/C). Disease progression, measured as median time to endpoint (TTE) was improved from 37 to 54 days (P<0.05) by panobinostat and to 46 days by BZ (P<0.05). The combination treatment further improved clinical outcome relative to both single-agent treatment groups (P<0.05), extending the TTE to 73 days. In contrast to BZ, the immunomodulatory drug thalidomide (TH) had no significant single-agent activity at 150 mg/kg p.o. qd for 4 weeks. However, combination activity (18% T/C) was observed when TH was combined with a sub-efficacious dose of panobinostat (5 mg/kg, 64% T/C). Combination of panobinostat and TH increased the TTE to 50 days, compared with 37.5, 43, and 39.5 days (P<0.05), respectively, for the vehicle, panobinostat, or TH as single agents. These data demonstrate that panobinostat exhibits significant anti-proliferative and anti-tumor activities on MM cells both in vitro and in vivo. Panobinostat, as a single agent or in combination with BZ or TH, is a promising therapy for MM, and these studies may provide the rationale for clinical evaluation of panobinostat and BZ combination in the treatment of MM.

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STK405759 as a combination therapy with bortezomib or dexamethasone, in in vitro and in vivo multiple myeloma models

Oncotarget ◽

10.18632/oncotarget.25825 ◽

2018 ◽

Vol 9 (59) ◽

pp. 31367-31379 ◽

Cited By ~ 4

Author(s):

Gabriela Rozic ◽

Lena Paukov ◽

Ziv Cohen ◽

Irit Shapira ◽

Adrian Duek ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy

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Dasatinib Inhibits Multiple Myeloma Growth by Blocking PDGF-Rb and c-Src Activity in Patient-Derived Tumor and Endothelial Cells.

Blood ◽

10.1182/blood.v110.11.550.550 ◽

2007 ◽

Vol 110 (11) ◽

pp. 550-550

Author(s):

Addolorata M.L. Coluccia ◽

Teresa Cirulli ◽

Paola Neri ◽

Franco Dammacco ◽

Pierfrancesco Tassone ◽

...

Keyword(s):

Multiple Myeloma ◽

Endothelial Cells ◽

Growth Factor ◽

Plasma Cells ◽

High Dose ◽

Tumor Vessel ◽

Angiogenic Switch ◽

Synergistic Cytotoxicity

Abstract Multiple myeloma (MM) is characterized by a clonal proliferation of immunoglobulin-secreting plasma cells in the bone-marrow (BM) and remains an incurable disease, despite the use of high-dose chemotherapies. Since a marked (BM)-angiogenesis is the hallmark of MM, but not of monoclonal gammopathies of undetermined significance (MGUS), validation of novel agents targeting MM tumor cells and their permissive BM-stroma is crucial to improve patient outcome. Patients fulfilling the International Myeloma Working Group diagnostic criteria for MM (n = 21) and MGUS (n = 14) were studied. Healthy donors or patients with benign anemia (due to vitamin B12 deficiency) were also incuded as controls. In plasma cells and endothelial cells (ECs) isolated from BM-aspirates by anti-CD138 and Ulex Europaeus agglutinin-1 (UEA-1) coated-beads, we dissected the contribution of activity against individual targets such as platelet-derived growth factor (PDGF)-receptor beta (PDGF-Rb) and c-Src tyrosine kinases (TKs), to the anti-tumor/vessel efficacy of dasatinib (BMS-354825), a novel orally bioavailable TK inhibitor. The PDGF-BB/PDGF-Rb kinase-axis was found constitutively activated in plasma cells from patients with MM but not with MGUS or benign anemias, thus supporting its pathophysiological role in MM. PDGF-Rb activated, independently of vascular endothelial growth factor (VEGF)-receptors (VEGF-R1 and VEGF-R2), the mitogen-activated protein kinases (ERK1/2) and the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent cascade, thereby increasing MM plasma cell growth. Expression of PDGF-Rb, at both mRNA and protein levels, was also increased in MMECs compared to MGECs, correlating with AKT phosphorylation. Exposure to recombinant PDGF-BB or conditioned media from MM plasma cells triggered PDGF-Rb phosphorylation and MMEC migration and spontaneous sprouting in vitro (both being mandatory for angiogenesis). Dasatinib abrogated PDGF-elicited tumor/vessel growth and impaired VEGF-signaling via c-Src TK-inhibition (IC50=25–100nM) in both MM-patient tumor and ECs. The use of small-interfering (si)-RNAs validated c-Src as a key VEGF-downstream effector of MMEC proliferation, migration and capillarogenesis in vitro. Nevertheless, the inhibitory effect elicited by siSrc was partially rescued by recombinant PDGF-BB which sustained the expression of pro-angiogenic factors such as VEGF, interleukin (IL)-8, basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in MMECs. Dasatinib reversed all these transcriptional effects, thereby abrogating MMEC angiogenesis in the CAM assay as well as the neovascularization and tumor growth of MM-xenografts in vivo. More importantly, low-dose dasatinib showed synergistic cytotoxicity in vitro when tested in combination with conventional MM drugs (i.e. bortezomib and thalidomide), thereby increasing therapeutic efficacy and overcoming drug resistance. These findings indicate that: the PDGF-BB/PDGF-Rb kinase-axis elicits direct effects on MM plasma cells and could promote the MM “angiogenic switch”, hence disease progression; the inhibition of this pathway could provide the rationale for clinical trials with dasatinib which interferes with shared growth-signaling cascades in MM-patient isolated plasma cells and ECs, involving PDGF-Rb and cytosolic c-Src TKs.

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Combination Therapy with Novel Nanoparticle, SNS01-T, and Lenalidomide Triggers Synergistic Cytotoxicity in Vitro and in Vivo in Multiple Myeloma and Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v120.21.4223.4223 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4223-4223

Author(s):

Catherine A Taylor ◽

Terence Tang ◽

Zhongda Liu ◽

Sarah Francis ◽

Zheng Qifa ◽

...

Keyword(s):

Multiple Myeloma ◽

Combination Therapy ◽

Tumor Growth ◽

B Cell ◽

Cell Lymphoma ◽

Xenograft Model ◽

Significant Activity ◽

Rpmi 8226

Abstract Abstract 4223 SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and B-cell lymphomas. SNS01-T is comprised of a plasmid encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an siRNA that inhibits expression of the pro-survival hypusine-eIF5A protein, and a polymer that serves to assemble the nucleic acids into a nanoparticle. SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in subjects with relapsed or refractory multiple myeloma (MM). SNS01-T and its preclinical precursors have been studied extensively in multiple myeloma and B cell lymphoma tumor models. In this study we tested the in vitro and in vivo anti-cancer activity of SNS01-T in combination with the immunomodulatory drug lenalidomide. The combination of low doses of SNS01-T and lenalidomide synergistically reduced viability of RPMI 8226 MM cells and induced apoptosis to a greater degree than either drug alone. To determine whether SNS01-T treatment increases the anti-myeloma activity of lenalidomide in vivo, 0.375 mg/kg SNS01-T was combined with either 15 or 50 mg/kg lenalidomide in a RPMI 8226 xenograft model of multiple myeloma. Mice were dosed for two cycles of treatment for a total of 11 weeks of dosing. Mice with no measurable tumor at the end of the first cycle of treatment did not receive treatment in the second cycle but were monitored closely for tumor recurrence. A two-week observation period at the end of the study allowed monitoring of tumor growth after the cessation of the second cycle of treatment. At the end of the second cycle of dosing, tumor growth was inhibited by 84 % (p < 0.0001), 34 % (p = 0.05), and 98.1 % (p << 0.0001) in animals treated with SNS01-T, 50 mg/kg lenalidomide, and SNS01-T plus 50 mg/kg lenalidomide, respectively. Complete tumor regression (undetectable tumor) was achieved in 40% of mice treated with SNS01-T, 0% of mice treated with 50 mg/kg lenalidomide, and 83% of mice treated with the combination therapy of SNS01-T and 50 mg/kg lenalidomide. Complete regression of tumors treated with the combination therapy was maintained for more than 8 weeks without treatment until the end of the study in 4 of 6 (67%) of treated mice. Combining SNS01-T treatment with 50 mg/kg lenalidomide inhibited tumor growth more effectively than either drug alone and prolonged survival with 100% of mice surviving to the end of the 102-day study. Combination therapy with SNS01-T and 15 mg/kg lenalidomide also demonstrated significant activity in a murine JMV-2 mantle cell lymphoma (MCL) xenograft model. Treatment of mice with the drug combination of SNS01-T and lenalidomide resulted in a statistically significant increase in survival compared to either SNS01-T (p = 0.002; logrank test) or lenalidomide (p = 0.007) alone. Collectively, these preclinical studies indicate that the combination therapy of SNS01-T and lenalidomide is well tolerated, has significant activity against MM and MCL, and provides a strong rationale to evaluate SNS01-T and lenalidomide combination therapy to improve patient outcome in MM and B cell lymphomas. Disclosures: Taylor: Senesco Technologies Inc.: stock options Other. Dondero:Senesco Technologies Inc.: Employment. Thompson:Senesco Technologies Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽

10.1182/blood-2006-08-042747 ◽

2006 ◽

Vol 109 (4) ◽

pp. 1669-1677 ◽

Cited By ~ 81

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.

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Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽

10.1182/blood-2009-03-213009 ◽

2010 ◽

Vol 115 (4) ◽

pp. 834-845 ◽

Cited By ~ 81

Author(s):

Dharminder Chauhan ◽

Ajita V. Singh ◽

Bryan Ciccarelli ◽

Paul G. Richardson ◽

Michael A. Palladino ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitor ◽

Tumor Model ◽

Caspase 8 ◽

Caspase 9 ◽

Model Studies ◽

Synergistic Cytotoxicity ◽

Induced Apoptosis

Abstract Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

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Targeting PKC in Multiple Myeloma: In Vitro and In Vivo Effects of the Novel, Orally Available Small-Molecule Inhibitor Enzastaurin (LY317615.HCl).

Blood ◽

10.1182/blood.v108.11.3466.3466 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3466-3466

Author(s):

Marc S. Raab ◽

Klaus Podar ◽

Jing Zhang ◽

Douglas McMillin ◽

Iris Breitkreutz ◽

...

Keyword(s):

Multiple Myeloma ◽

Endothelial Cells ◽

Signaling Pathways ◽

Signaling Molecules ◽

Extracellular Matrix Protein ◽

The Novel ◽

Pkc Inhibitor ◽

Synergistic Cytotoxicity

Abstract In multiple myeloma (MM), protein kinase C (PKC) overexpression has been reported including the prognostic adverse patient group with t (4;14) translocation. Importantly, PKC signaling pathways have been implicated in MM cell proliferation, apoptosis, and migration. Here, we investigated the novel, orally available PKC inhibitor Enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits membrane, cytosolic, and nuclear phosphorylation of homologous PKC isoform residues, as well as associated kinase activity, induced by the major PKC activator TPA (Tumor-promoting phorbol ester). Consequently, it also abrogates TPA-induced phosphorylation of signaling molecules downstream of PKC including MARCKS and PKCm. In MM, Enzastaurin inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs); co-stimulation with the extracellular matrix protein fibronectin, VEGF or IL-6; as well as MM patient serum. Phosphorylation of downstream signaling molecules was also abrogated, including cytoplasmic and nuclear ERK, JNK, ribosomal protein S6 and GSK3b as well as nuclear cMyc. Enzastaurin inhibits both proliferation and survival of MM cell lines and MM cells isolated from multidrug- resistant patients; as well as overcomes MM cell growth triggered by tumor cell binding to BMSCs and endothelial cells at a low micromolar range equivalent to the concentrations achieved in patient plasma during clinical trials. Importantly, synergistic cytotoxicity is observed when Enzastaurin is combined with bortezomib. Besides proliferation and survival, Enzastaurin inhibits MM cell adhesion, as well as VEGF- and IGF-1-triggered MM cell migration. It also blocks VEGF- triggered signaling pathways in endothelial cells, thereby inhibiting tubule formation. Finally and most importantly, 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 patient outcome in MM.

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