Differential Effects of the Oncogenic BRAF Inhibitor PLX4032 (Vemurafenib) and its Progenitor PLX4720 on ABCB1 Function

PURPOSE The clinically approved oncogenic BRAF inhibitor PLX4032 (vemurafenib) was shown to be a substrate of the ATP-binding cassette (ABC) transporter ABCB1. Here, we compared PLX4032 and its structurally closely related precursor compound PLX4720 for their interference with ABCB1 and the ABCB1-mediated compound transport using docking and cell culture experiments. METHODS For the docking study of PLX4032 and PLX4720 with ABCB1, we analysed binding of both compounds to mouse Abcb1a and to human ABCB1 using a homology model of human ABCB1 based on the 3D structure of Abcb1a. Naturally ABCB1 expressing cells including V600E BRAF-mutated and BRAF wild-type melanoma cells and cells transduced with a lentiviral vector encoding for ABCB1 were used as cell culture models. ABCB1 expression and function were studied by the use of fluorescent and cytotoxic ABCB1 substrates in combination with ABCB1 inhibitors. RESULTS Docking experiments predicted PLX4032 to interact stronger with ABCB1 than PLX4720. Experimental studies using different cellular models and structurally different ABCB1 substrates confirmed that PLX4032 interfered stronger with ABCB1 function than PLX4720. For example, PLX4032 (20µM) induced a 4-fold enhanced rhodamine 123 accumulation compared to PLX4720 (20µM) in ABCB1-transduced UKF-NB-3 cells and reduced the IC50 for the cytotoxic ABCB1 substrate vincristine in this model by 21-fold in contrast to a 9-fold decrease induced by PLX4720. CONCLUSIONS PLX4032 exerted stronger effects on ABCB1-mediated drug transport than PLX4720.  This indicates that small changes in a molecule can substantially modify its interaction with ABCB1, a promiscuous transporter that transports structurally different compounds.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page. PURPOSE The clinically approved oncogenic BRAF inhibitor PLX4032 (vemurafenib) was shown to be a substrate of the ATP-binding cassette (ABC) transporter ABCB1. Here, we compared PLX4032 and its structurally closely related precursor compound PLX4720 for their interference with ABCB1 and the ABCB1-mediated compound transport using docking and cell culture experiments. METHODS For the docking study of PLX4032 and PLX4720 with ABCB1, we analysed binding of both compounds to mouse Abcb1a and to human ABCB1 using a homology model of human ABCB1 based on the 3D structure of Abcb1a. Naturally ABCB1 expressing cells including V600E BRAF-mutated and BRAF wild-type melanoma cells and cells transduced with a lentiviral vector encoding for ABCB1 were used as cell culture models. ABCB1 expression and function were studied by the use of fluorescent and cytotoxic ABCB1 substrates in combination with ABCB1 inhibitors. RESULTS Docking experiments predicted PLX4032 to interact stronger with ABCB1 than PLX4720. Experimental studies using different cellular models and structurally different ABCB1 substrates confirmed that PLX4032 interfered stronger with ABCB1 function than PLX4720. For example, PLX4032 (20µM) induced a 4-fold enhanced rhodamine 123 accumulation compared to PLX4720 (20µM) in ABCB1-transduced UKF-NB-3 cells and reduced the IC50 for the cytotoxic ABCB1 substrate vincristine in this model by 21-fold in contrast to a 9-fold decrease induced by PLX4720. CONCLUSIONS PLX4032 exerted stronger effects on ABCB1-mediated drug transport than PLX4720.  This indicates that small changes in a molecule can substantially modify its interaction with ABCB1, a promiscuous transporter that transports structurally different compounds. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells.

Tumor resistance to lysis by resting natural killer (NK) cells may be overcome by priming of NK cells with cytokines or by binding of NK activating receptors to ligands expressed on target cells. In this study, major histocompatibility complex class I (MHC-I)-negative LNCaP and MHC-I-positive DU145 cells were infected with genetically modified influenza A virus lacking the non-structural gene 1 (NS1 IAV). The cells were used to investigate the influence of NS1 IAV infection on NK cell lysis of tumor cells as well as to prime NK cells for lysis of LNCaP and DU145 cells. While LNCaP cells infected with DeltaNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% +/- 1.47 vs. 52% +/- 0.74), both mock-infected and DeltaNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with DeltaNS1 IAV-infected LNCaP/DU145 cells effectively lysed resistant DU145 and sensitive LNCaP cells to a greater extent than NK cells primed with mock-infected LNCaP/DU145 or non-primed NK cells. Also, NK cell priming with DeltaNS1 IAV-infected tumor cells enhanced extracellular signal-regulated kinase phosphorylation and increased granule release in NK cells. The increased granule release was specifically mediated by NKp46, which eventually potentiated NK cells primed with DeltaNS1 IAV-infected tumor cells to overcome the inhibitory effects posed by MHC-I expression on DU145 cells. These findings show that in addition to direct lytic activity of NK cells, DeltaNS1 IAV may influence anti-tumoral responses by priming NK cells

Expression of biological active interleukin-15.

<p><b>A</b>) The cell lines were infected with delNS1 or delNS1-IL-15 at MOI 1 or non-infected (mock). Supernatants were analysed for interleukin-15 by ELISA after the indicated incubation periods. <b>B</b>) Induction of natural killer (NK) cell mediated tumour cell lysis. Primary human NK-cells were incubated with cell culture supernatants of mock, delNS1-, or delNS1-IL-15-infected Colo-679 cells. After four days of incubation, NK-cells were analysed for cytotoxicity against non-infected tumour target cells. NK-cells incubated without or with 100 U recombinant IL-15 served as control.</p

Cell signaling pathways activated by oncolytic influenza A viruses in melanoma cells.

<p><b>A</b>) Colo-679 cells were infected with IVR-116, delNS1 or delNS1-IL-15 respectively (MOI 1). Protein levels of ERK1/2, phosphorylated ERK1/2 (pERK1/2), AKT, phosphorylated AKT (pAKT) and β-Actin were determined 24 hpi by Western blot. <b>B–D</b>) The role of ERK1/2 phosphorylation in melanoma cell oncolysis by delNS1. Colo-679 cells were transfected with siRNA directed against ERK1, ERK2, or non-target siRNA and infected at MOI 1 with delNS1 48 hpi. <b>B</b>) Western blot analysis of ERK1 and ERK2 levels. Western blot extracts were taken 24 hpi. <b>C</b>) The cell viability was assesed 48 hpi by MTT assay. <b>D</b>) Virus titres were determined as TCID₅₀/mL 24 hpi.</p

Apoptosis induction in influenza A virus-infected melanoma cells.

<p>Cells were infected at MOI 1. 10 µg/mL Cisplatin (CDDP) were used as a control for apoptosis induction. <b>A</b>) The percentage of cells in sub-G1-phase was determined 48 hpi. <b>B–E</b>) Caspase 3/7, 8, or 9 activity was determined 24 hpi. *significantly different compared to mock, P<0.01, **significantly different compared to delNS1 infection, P<0.01.</p

Infection rate and replication kinetics of influenza A viruses.

<p><b>A</b>) Immune staining of infected cells. Cells were infected at MOI 1, 0.1 or 0.01 respectively. Staining for influenza A virus NP antigen expression was performed 24 hours post infection. <b>B</b>) Cells were infected with IVR-116, delNS1, or delNS1-IL-15 (MOI 0.1). At the given time points, aliquots of the supernatants were taken and the TCID₅₀/mL was determined.</p

The role of apoptosis in melanoma cell oncolysis.

<p><b>A</b>) Determination of Colo-679 cells in sub-G1-phase was performed 48 hpi (MOI 1) by flow cytometry in the absence or presence of the pan-caspase inhibitor Z-VAD-fmk (80 µM) or the caspase 3 inhibitor Z-DEVD-fmk (80 µM). Z-VAD-fmk or Z-DEVD were added to the cell cultures 30 min prior to virus infection. UV-inactivated viruses served as controls. <b>B</b>) Influence of Z-VAD-fmk (80 µM), Z-DEVD-fmk (80 µM) or cisplatin (CDDP) on delNS1 replication in Colo-679 cells. Colo-679 cells were treated with the caspase inhibitors 30 min prior to delNS1 (MOI 0.1) infection or treated simultaneously with 0.5 µg/mL cisplatin. Viral titres were determined 24 hpi. <b>C</b>) Cells were infected with delNS1 MOI 0.1 and treated with 0.5 µg/mL CDDP. Cell viability was determined 48 hpi by MTT-assay and <b>D</b>) Caspase 3/7 was measured 24 hpi. w/o = without treatment. *significantly different compared to non treated P<0.05; **significantly different than delNS1-infection and CDDP-treatment alone P<0.05.</p

Influence of influenza A virus infection on melanoma cell viability.

<p>Cells were infected at a MOI of 1 with IVR-116 (expressing WT NS1), delNS1 or delNS1-IL-15. MTT-assay was performed after 24, 48, and 72 hours post infection (left panel) or cells were infected at MOIs ranging from 1 to 0.01 and MTT-Assay was performed 72 hours post infection (right panel). All data represent mean ± SD of three independent experiments. *significantly different compared to non-infected control (mock), P<0.05.</p