combination treatment compared to single agent treatments, an effect that subsequently resulted in synergistic decrease in cell viability and strong induction of apoptosis. As ABT-737 leads to liver deterioration in Mcl-1 knockout mice, inhibition of Mcl-1 should be done in a tumor specific manner to induce apoptosis and tumor specific growth control [40]. We have previously shown that Mcl-1 protein levels rapidly is decreased in cancer cells when targeted with several of our Pseudomonas Exotoxin A based immunotoxins [7,11], of which one (the MOC31PE targeting the EpCAM antigen) is currently in clinical trial phase I/II (www.clinicaltrials.gov, assessed July 20, 2011). Furthermore, ABT-263/Navitoclax, the oral form of ABT- 737, is currently in phase I and phase II clinical trials (www. clincaltrials.gov, assessed July 20, 2011). From a clinical perspec- tive, 9.2.27PE+ABT-263 is therefore an interesting treatment combination for further investigations. Importantly, administra- tion of 9.2.27PE+ABT-737 caused growth delay effect in Melmet- 5 xenograft tumors, implying that this particular drug combination show the ability to kill melanoma cells in vivo. However, as only tumor growth delay was obtained, treatment of solid melanoma tumors might prove to be challenging. Immunotoxins and ABT- 737 as single agents have, nevertheless, been shown to be effective in hematological tumors, where in fact one immunoconjugate consisting of an IL-2 fused to a modified Diphteria toxin (OntakH) is approved by the Food and Drug Administration is an option for patient with cutaneous T-cell lymphoma. To circumvent the challenges related to treatment of solid metastatic melanoma tumors, targeting circulating melanoma cells or minimal residual disease which is present in patients treated with e.g. the new BRAF or MEK inhibitors (as these patients do relapse after some time), could be a viable option. As melanoma is a diverse disease, and the combination treatment with 9.2.27PE+ABT-737 was able to efficiently cause cell death in several melanoma cell lines tested in vitro, this approach could reach a broader patient group, not limited by specific mutations. Taken together, the combination of 9.2.27PE and the ABT-737 caused synergistic cell death in a panel of melanoma cells, and the cell death observed was associated with apoptosis involving the intrinsic apoptosis pathway. ER stress caused by ABT-737, and enhanced Ca2+ release caused by ABT-737 as a single agent or the 9.2.27PE+ABT-737 combination treatment, likely contributed to the cell cytotoxicity and the synergistic anti-melanoma activity. Importantly, cytostatic effect was obtained in a melanoma xenograft model, indicating that this combination is effective in vivo. Whether melanoma cells in vivo will recruit additional survival mechanisms after treatment with the oral form of ABT-737, the ABT-263, resulting in resistance to the drug, remains to be investigated. Lymphoma cells in vitro acquired resistance after long- term exposure ABT-737 due to up-regulation of Mcl-1 and A1 [41]. We do, however, know that long term exposure of low doses of 9.2.27PE causes cell cytotoxicity to all melanoma cells in vitro, due to the general inhibition of protein synthesis leading to cell death (data not published). Generation of immunotoxin-resistant cells is therefore not likely to occur. References 9.2.27PE and ABT-737 in Melanoma Supporting Information Figure S1 The effect of A-793844 on cell viability in melanoma cells. FEMX and MelRM cells were treated with the enantiomer A-793844 (the negative control of ABT-737) for the indicated time points. No significant decrease in cell viability was observed. The data represents the mean 6 SD. (TIF) Figure S2 Both 9.2.27PE and ABT-737 causes active caspase-3 in melanoma cells. FEMX and Melmet-5 cells were treated with 9.2.27PE and ABT-737 as indicated. Active caspase-3 was measured using the Caspase-Glo 3/7 from Promega. Caspase-3 activation could be inhibited by 45 min pre-incubation with the caspase-3 inhibitor Z-DEVD-FMK. Staurosporine (STS) was used as a positive control for caspase-3 activation, and CHX was used as a negative control. (TIF) Figure S3 Calcium release caused by 9.2.27PE6ABT-737 in melanoma cells. MelRM, MelRMshCtr and MelRMshMcl-1 were subjected to 9.2.27PE (100 ng/ml)+ABT-737 (10 mM). [Ca2+]i levels, and cell viability was measured after 24 h. Knockdown of Mcl-1 using shRNA enhanced the calcium release and decreased cell viability caused by ABT-737, an effect which could not be enhanced by 9.2.27PE. The data shown are the mean 6 SD. p9 = passage 9, the highest passage used for this experiment. (TIF) Figure S4 Body weight of nude mice treated with 9.2.27PE6ABT-737. Nude mice were treated with ABT-737 (100 mg/kg) day 1–5 and 15–19 and 9.2.27PE (31.25 mg/kg) on day 1 and 15 (left panel) or ABT-737 (50 mg/kg) day 1–5 and 15– 19 and 9.2.27PE (31.25 mg/kg) on day 1 and 15 (right panel). Body weight was measured throughout the experiment. (TIF) Table S1 9.2.27PE in combination with ABT-737 causes synergistic cell cytotoxicity in melanoma cells. Fractional inhibi- tion = fraction decreased cell viability after treatment, control cells were set to ‘‘1’’, CI = combination index. CI values below 0.9 indicate synergistic effect. ND = not done. (DOC) Acknowledgments We acknowledge the assistance of Mr. Alexander Kristian at the animal facility at the Oslo University Hospital Radiumhospitalet, and Ms. Amanda J Croft at the Oncology and Immunology Unit, Calvary Mater Newcastle Hospital, University of Newcastle, Newcastle, NSW, Australia for generating the MelRMshMcl-1 and MelRMshCtr cell lines. Author Contributions Conceived and designed the experiments: KR. Performed the experiments: KR. Analyzed the data: KR. Contributed reagents/materials/analysis tools: KR ØF YA. Wrote the paper: KR. Designed in vivo experiments: KR YA. Preparation of cells and drugs for in vivo experiments: YA. Obtained permission for use of cell lines: KR. Discussion of manuscript: KR ØF YA. 1. Cummins DL, Cummins JM, Pantle H, Silverman MA, Leonard AL, et al. (2006) Cutaneous malignant melanoma. Mayo Clinic proceedings 81: 500–507. 2. Soengas MS, Lowe SW (2003) Apoptosis and melanoma chemoresistance. Oncogene 22: 3138–3151. 3. 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