Abstract A018: Synergy between IAP inhibitors and a cytotoxic antibody-based chimeric protein in pancreatic cancer cells (2019)

Abstract Background: immunotoxins are cytotoxic antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. Immunotoxins derived from Pseudomonas exotoxin inhibit protein synthesis via the ADP- ribosylation of the eukaryotic elongation factor (eEF2). Clear clinical benefit has been achieved in patients treated with an immunotoxin targeting CD22 expressed on B-cell malignancies and the immunotoxin has recently been approved by FDA. However, success has not been universal and, when treating solid tumors, immunotoxins have performed less well in producing complete responses, due to poor apoptotic responses. Screening for enhancing compounds revealed IAP inhibitors as potentially useful for improving immunotoxin killing. The immunotoxin, LMB-100, targets surface mesothelin on pancreatic tumor cells but when tested on two pancreatic adenocarcinoma cell lines, AsPC1 and BxPc3, there was no detectable reduction in cell viability. Upon further investigation, we found that birinapant, an IAP inhibitor, strongly synergized with this immunotoxin against AsPC1 but not against BxPC3 cells. We sought to understand the different responses in each cell line. Materials and Methods: viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison WI). TNFalpha in the cell medium was detected using the hTNFa QuantiGlo ELISA (R&D System). The level of the major cell-associated apoptotic proteins was assessed using the Human apoptosis Array kit (R&D System). The spots on the arrays were quantified using ImageJ. Results: both AsPC1 and BxPC3 cells were resistant to the immunotoxin LMB-100. In the case of AsPC1, the immunotoxin caused only a modest cytostatic response at high immunotoxin concentrations at 72h whereas, in combination of birinapant, there was a strong cytotoxic effect, starting at 48h. BxPC3 cells remained resistant to the immunotoxin even in the presence of birinapant: the combination with the highest concentrations of both compounds reduced cell viability only to 50% after 72 hours. From the evaluation of the level of the major apoptotic proteins, we found that the most significative difference between the two cell lines was the phosphorylated status of p53, in particular the S392 hyper-phosphorylation in BxPC3 which has been associated with a poor prognosis, advanced tumor stage and grade in p53 positive cancers. AsPC1 cells also had a higher level of Bax, making them more predisposed to apoptosis. No difference in TNFalpha secreted in the medium or TNFR1 protein level was detected between the two cell lines. Conclusion: knowing the level of key proteins involved in the cell death pathway can explain the efficacy of drug combinations and could be used to predict responses. Citation Format: Antonella Antignani, David J FitzGerald. Synergy between IAP inhibitors and a cytotoxic antibody-based chimeric protein in pancreatic cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A018. doi:10.1158/1535-7163.TARG-19-A018