Synergistic Anticancer Effects of the 9.2.27PE Immunotoxin and ABT-737 in Melanoma Karianne Risberg*, Øystein Fodstad, Yvonne Andersson Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Montebello, Oslo, Norway Abstract In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE) - based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78 and phosphorylated eIF2a protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA) enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination. Citation: Risberg K, Fodstad Ø, Andersson Y (2011) Synergistic Anticancer Effects of the 9.2.27PE Immunotoxin and ABT-737 in Melanoma. PLoS ONE 6(9): e24012. doi:10.1371/journal.pone.0024012 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received July 20, 2011; Accepted August 1, 2011; Published September 7, 2011 Copyright: 2011 Risberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Norwegian Extra Foundation for Health and Rehabilitation (project number 2008/2/0054) and South-Eastern Norway Regional Health Authority (32254/6051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Karianne.Risberg@rr-research.no Introduction Surgical treatment of primary melanoma is associated with high curative rate. However, if the melanoma has progressed to distant metastases, treatment failure is common due to high resistance to current treatment modalities [1,2]. The median survival rate of metastatic melanoma is 6 months, and less than 5% of the patients survive 5 years, making metastatic melanoma one of the most aggressive cancers in humans [1]. The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in approxi- mately 90% of all melanomas [3], and new drugs targeting this pathway, e.g inhibitors of mutated BRAF or MEK, initially showed promising effects in vitro. PLX4032, a selective BRAFV600E inhibitor, caused partial or complete response in early clinical trials [4,5], however the patients eventually develop resistance. The MEK inhibitors PD0325901 and AZD6244 have also been evaluated in early clinical trials, however, without improvement in the progression-free survival compared to Temozolomide [6], an oral form of the chemotherapeutic drug Dacarbazine used to treat melanoma patients. These results underscore the importance of developing new drugs with yet different mechanisms of action. An immunotoxin (IT) consists of an antibody linked to a toxin and is designed to specifically kill tumor cells, as the antibody targets cancer cell-associated antigens or antigens over expressed on cancer cells. ITs are taken up through endocytosis, processed within the cell, and cell death is caused by inhibition of protein synthesis through ADP-ribosylation of Elongation Factor 2 and by induction of apoptosis [7]. The 9.2.27PE immunotoxin consists of the Pseudomonas Exotoxin A (PE) linked to the 9.2.27 antibody that targets the high molecular weight-melanoma associated antigen, an antigen expressed on most melanomas and melanoma cell lines [8,9]. Previous studies show that other PE-based immunotoxins cause strong induction of apoptosis [7,10]. The 9.2.27PE, on the other hand, caused cell death in melanoma cells primarily through inhibition of protein synthesis, with minor signs of apoptosis [11]. This is possibly due to the high resistance to apoptosis in melanoma cells [2]. Apoptosis is commonly associated with activation of caspases, inactivation of the DNA repair enzyme PARP, chromatin condensation and fragmentation of DNA. It involves two major pathways, the intrinsic/mitochondrial and extrinsic/death receptor apoptotic pathway, which interlace at certain points and ultimately result in cell death [12]. Further- more, complex interplay between the pro- and anti-apoptotic members of the Bcl-2 family proteins ensures the homeostatic balance within the cell. The Bcl-2 family proteins have an important role in regulating the intrinsic apoptotic pathway, but have also been shown to be involved in maintaining the endoplasmic reticulum (ER) homeostasis, as well as being involved in ER stress signal transduction pathways [13]. The small-molecule inhibitor ABT-737 is developed to neutralize the pro-survival effects of Bcl-2 family proteins. It binds to and inhibits the pro-survival proteins Bcl-2, Bcl-XL and Bcl-W, but not Mcl-1 and A1 [14]. By binding to Bcl-2 and Bcl-XL, pro-apoptotic proteins such as BAX and BAK may be released from Bcl-2 and Bcl-XL and subsequently induce apoptosis as they are proteins of the intrinsic apoptotic pathway. Others have shown that combining ABT-737 with agents decreasing Mcl-1 protein expression act synergistically with ABT-737 [15,16], and depletion of Mcl-1 PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24012