Abstract 5482: Combining the anti-mesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cell lines.
Authors: Hollevoet Kevin , Antignani Antonella , Fitzgerald David J. , Pastan Ira 
Published: August 2014

Background

Pancreatic ductal adenocarcinoma (PDAC) responds poorly to most chemotherapeutic agents, and alternative treatments are needed. Our laboratory focuses on the development of recombinant immunotoxins (RITs) for cancer treatment. RITs are antibody-toxin fusion proteins composed of an antigen-binding Fv fused to a 38-kDa portion of Pseudomonas exotoxin A (PE38). SS1P (SS1(dsFv)-PE38) is a mesothelin-targeting RIT that is in clinical trials for mesothelioma. Mesothelin is also highly expressed in PDAC. We previously reported that mesothelin-expressing PDAC cell lines were resistant to SS1P. This finding fuelled efforts to combine RITs with drugs that can tip the balance in favor of cell death. ABT-737, a BH3-mimitic, has a high affinity for Bcl-2, Bcl-xl and Blc-w, but little affinity for Mcl-1. High Mcl-1 expression therefore is a source of resistance to ABT-737. Since RITs inhibit protein synthesis, thereby downregulating Mcl-1, we examined whether combining SS1P and ABT-737 could induce cell death in PDAC.

Methods

PDAC cell lines KLM-1, BxPc-3 and Panc 3.014 were studied. Mesothelin expression was evaluated with FACS. Cell proliferation was evaluated with Cell Counting Kit-8 WST-8 assay, and cell death with 7-AAD and Annexin V-PE staining. Protein synthesis inhibition was quantified via 3H-leucine incorporation.

Results

KLM-1 had 92.6 x 103 mesothelin-binding sites/cell, respectively about 4- and 42-fold higher than Panc 3.014 (24.9 x 103) and BxPc-3 (2.2 x 103). In all three cell lines, 100 ng/ml SS1P and 10 uM ABT-737 separately did not induce substantial apoptosis as compared to the untreated controls. Combining both compounds, however, led to a significant increase in cell death. After 48hrs of treatment, apoptosis was observed in 92% of the KLM-1 cells, 55% of the BxPc-3 cells, and 23% of the Panc 3.014 cells. Combining 100 ng/ml SS1P and 10 uM ABT-737 also significantly enhanced the protein synthesis inhibition after 8hrs of treatment. Again, this increase was most pronounced in KLM-1 (42% inhibition versus 18% by SS1P), followed by BxPc-3 (30% inhibition versus 12% by SS1P), and Panc 3.014 (7% inhibition versus 3% by SS1P).

Conclusion

Combining SS1P with ABT-737 induced cell death in PDAC cell lines that are resistant to both compounds alone. The efficacy of this combo varied greatly among PDAC cell lines, and correlated with the extent to which protein synthesis was inhibited, but not with the amount of cell-surface mesothelin expression. These findings further support the therapeutic potential of combining a RIT and a BH3-mimetic. Further study is needed to elucidate differences in sensitivity among cell lines. This can improve the efficacy of this approach, discover novel targets, and eventually identify patients most likely to benefit from such a combination therapy.

Citation Format: Kevin Hollevoet, Antonella Antignani, David J. Fitzgerald, Ira Pastan. Combining the anti-mesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2013-5482