ABCB1 as predominant resistance mechanism in cells with acquired SNS-032 resistance.

The CDK inhibitor SNS-032 had previously exerted promising anti-neuroblastoma activity via CDK7 and 9 inhibition. ABCB1 expression was identified as major determinant of SNS-032 resistance. Here, we investigated the role of ABCB1 in acquired SNS-032 resistance. In contrast to ABCB1-expressing UKF-NB-3 sub-lines resistant to other ABCB1 substrates, SNS-032-adapted UKF-NB-3 (UKF-NB-3rSNS- 032300nM) cells remained sensitive to the non-ABCB1 substrate cisplatin and were completely re-sensitized to cytotoxic ABCB1 substrates by ABCB1 inhibition. Moreover, UKF-NB-3rSNS-032300nM cells remained similarly sensitive to CDK7 and 9 inhibition as UKF-NB-3 cells. In contrast, SHEPrSNS-0322000nM, the SNS-032-resistant sub-line of the neuroblastoma cell line SHEP, displayed low level SNS-032 resistance also when ABCB1 was inhibited. This discrepancy may be explained by the higher SNS-032 concentrations that were used to establish SHEPrSNS-0322000nM cells, since SHEP cells intrinsically express ABCB1 and are less sensitive to SNS-032 (IC50 912 nM) than UKF-NB-3 cells (IC50 153 nM). In conclusion, we show that ABCB1 expression represents the primary (sometimes exclusive) resistance mechanism in neuroblastoma cells with acquired resistance to SNS-032. Thus, ABCB1 inhibitors may increase the SNS-032 efficacy in ABCB1-expressing cells and prolong or avoid resistance formation.

Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status.

The PKCß inhibitor enzastaurin was tested in parental neuroblastoma and rhabdomyosarcoma cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53-depleted cells. Enzastaurin IC50s ranged from 3.3 to 9.5 µM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125 µM interfered with ABCB1-mediated drug transport. PKCα and PKCß may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations ≥ 1.25 µM. The investigated cell lines did not express PKCß. PKCα depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. These findings are important for the further development of therapies combining enzastaurin with ABC transporter substrates.

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen.

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.

Aurora kinases as targets in drug-resistant neuroblastoma cells.

Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs.

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3(r)CDDP(1000) in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.

The multi-targeted kinase inhibitor sorafenib inhibits human cytomegalovirus replication.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals. Here, non-toxic concentrations of the anti-cancer kinase inhibitor sorafenib were shown to inhibit replication of different HCMV strains (including a ganciclovir-resistant strain) in different cell types. In contrast to established anti-HCMV drugs, sorafenib inhibited HCMV major immediate early promoter activity and HCMV immediate early antigen (IEA) expression. Sorafenib is known to inhibit Raf. Comparison of sorafenib with the MEK inhibitor U0126 suggested that sorafenib inhibits HCMV IEA expression through inhibition of Raf but independently of signaling through the Raf downstream kinase MEK 1/2. In concordance, siRNA-mediated depletion of Raf but not of MEK-reduced IEA expression. In conclusion, sorafenib diminished HCMV replication in clinically relevant concentrations and inhibited HCMV IEA expression, a pathophysiologically relevant event that is not affected by established anti-HCMV drugs. Moreover, we demonstrated for the first time that Raf activation is involved in HCMV IEA expression.