sepantronium and Neuroblastoma

Amplification of the MYCN gene (MNA) is observed in approximately 25 to 35% of neuroblastoma patients, and is a well-recognized marker of tumor aggressiveness and poor outcome. Targeting MYCN is a novel therapy strategy to induce tumor regression. Here, we discovered that a BIRC5/Survivin inhibitor, YM155, specifically inhibits MNA neuroblastoma cell growth in vitro. We found that YM155 promotes MYCN degradation in MNA cells. Further, we found that YM155 inhibits USP7 deubiquitinase activity in vitro, using Ub-aminomethylcoumarin (Ub-AMC) as substrate. Results from in vivo studies further demonstrated that YM155 significantly inhibited the tumor growth in MNA neuroblastoma xenograft model. Our data support a novel mechanism of action of YM155 in inhibition of growth of cancer cells through inducing MYCN degradation by inibition of activity of deubiquitinase like USP7.

Topics: Cell Line, Tumor; Cell Proliferation; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Proteolysis; Ubiquitin-Specific Peptidase 7

Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells.. PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG. YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of -10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155.. YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.

Topics: Animals; Antibodies; Antineoplastic Agents; Cell Line, Tumor; Drug Compounding; Drug Liberation; Drug Stability; Female; Gangliosides; Half-Life; Humans; Hydrophobic and Hydrophilic Interactions; Imidazoles; Injections, Intravenous; Liposomes; Mice; Mice, Nude; Naphthoquinones; Neuroblastoma; Pilot Projects; Polyethylene Glycols; Survivin; Xenograft Model Antitumor Assays

Adverse side effects of cancer agents are of great concern in the context of childhood tumors where they can reduce the quality of life in young patients and cause life-long adverse effects. Synergistic drug combinations can lessen potential toxic side effects through lower dosing and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model.

Topics: Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Drug Synergism; Humans; Imidazoles; Lapatinib; N-Myc Proto-Oncogene Protein; Naphthoquinones; Neuroblastoma; Protein Kinase Inhibitors; Receptor, trkA; RNA Interference; Zebrafish

Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action.

Topics: ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Cell Survival; DNA Damage; Drug Resistance, Neoplasm; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Kinetics; Membrane Transport Proteins; Mutation; Naphthoquinones; Neuroblastoma; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Survivin; Tumor Suppressor Protein p53

Aggressive cell growth and chemoresistance are notorious obstacles in neuroblastoma therapy. Accumulating evidence suggests that survivin is preferentially expressed in cancer cells and plays a crucial role in cell division and apoptosis dysfunction. Thus, in the present study, we investigated whether silencing of survivin, using a novel small-molecule survivin suppressant, YM155 could suppress the proliferation and induce chemosensitization of neuroblastoma cells.. SH-SY5Y human neuroblastomas cells were treated with YM155 (10 to 500 mM) and/or chemotherapeutic agent cisplatin for 72 hours, and cell viability, apoptosis, mRNA and protein expression level were then evaluated. Furthermore, the efficacy of YM155 combined with cisplatin was further examined in established xenograft models.. YM155 suppressed expression of survivin, inhibited the proliferation and induced apoptosis in SH-SY5Y cells in a concentration-dependent manner. Reduced levels of survivin sensitized SH-SY5Y to the chemotherapeutic agent cisplatin. YM155 showed antiproliferative effects and induced tumor regression and apoptosis in established SH-SY5Y xenograft models. Cisplatin showed antitumor activity against SH-SY5Y cells, it did not induce survivin upregulation. Combination treatment of YM155 and cisplatin induced a greater rate of apoptosis than the sum of the single-treatment rates and promoted tumor regression without enhanced body weight loss in the SH-SY5Y xenograft models.. The concomitant combination of YM155 with cisplatin induced more intense apoptosis compared with each single treatment in vivo and in vitro. YM155 in combination with cisplatin is well tolerated and shows greater efficacy than either agent alone in mouse xenograft models.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Neuroblastoma; RNA, Messenger; Survivin; Xenograft Model Antitumor Assays

The BIRC5 (Survivin) gene is located at chromosome 17q in the region that is frequently gained in high risk neuroblastoma. BIRC5 is strongly over expressed in neuroblastoma tumour samples, which correlates to a poor prognosis. We recently validated BIRC5 as a potential therapeutic target by showing that targeted knock down with shRNA's triggers an apoptotic response through mitotic catastrophe. We now tested YM155, a novel small molecule selective BIRC5 suppressant that is currently in phase I/II clinical trials. Drug response curves showed IC50 values in the low nM range (median: 35 nM, range: 0.5-> 10,000 nM) in a panel of 23 neuroblastoma cell lines and four TIC-lines, which resulted from an apoptotic response. Nine out of 23 cell lines were relatively resistant to YM155 with IC50 values > 200 nM, although in the same cells shRNA mediated knock down of BIRC5 caused massive apoptosis. Analysis of differentially expressed genes between five most sensitive and five most resistant cell lines using Affymetrix mRNA expression data revealed ABCB1 (MDR1) as the most predictive gene for resistance to YM155. Inhibition of the multi-drug resistance pump ABCB1 with cyclosporine or knockdown with shRNA prior to treatment with YM155 demonstrated that cell lines with ABCB1 expression became 27-695 times more sensitive to YM155 treatment. We conclude that most neuroblastoma cell lines are sensitive to YM155 in the low nM range and that resistant cells can be sensitised by ABCB1 inhibitors. Therefore YM155 is a promising novel compound for treatment of neuroblastoma with low ABCB1 expression.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Silencing; HEK293 Cells; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Naphthoquinones; Neuroblastoma; RNA, Small Interfering; Survivin; Xenograft Model Antitumor Assays