Abstract A67: The mTOR inhibitor, everolimus, enhances the cytotoxicity of pseudomonas-based immunotoxins: Mechanistic insights

Abstract

Abstract Pseudomonas exotoxin (PE)-based immunotoxins (antibody-toxin fusion proteins) have produced frequent complete remissions in patients with hairy cell leukemia but far fewer responses in other cancers. PE and PE-immunotoxins kill cells via a pathway that includes endocytosis, proteolytic processing, transport to the ER and translocation of an enzymatically active toxin fragment from the ER to the cytosol. In the cytosol the toxin ADP-ribosylates EF2 which results in the cessation of protein synthesis. Using two cell lines (KB or Nalm6) that exhibit moderate resistance to immunotoxin killing, we screened 500 compounds from the MIPE-3 library seeking compounds that enhanced the cytotoxicity of immunotoxins targeting either mesothelin or CD22. Results indicated that as few as 20 compounds could enhance cytotoxicity and only a few appeared potentially useful in a clinical setting. One compound of particular interest was the mammalian target of rapamycin (mTOR) inhibitor, everolimus. The mTOR pathway plays a pivotal role in cell signaling and proliferation and ultimately tumorigenesis. Moreover, protein synthesis is the best characterized process controlled by the mTORC1 pathway, making everolimus an excellent candidate to enhance the cytotoxic activity of PE-immunotoxins. We tested combinations of PE-immunotoxins and everolimus on various tumor cell lines (Nalm-6, Daudi, MD-MBA-468 and KB) using different concentration of both compounds. In all cell lines, the cytotoxicity of the combination was greater than either compound alone. As could be anticipated, the increased cytotoxicity was associated with enhanced reductions in cellular protein synthesis: confirming synergy between the immunotoxin and everolimus. To define the mechanism for the observed synergy, we evaluated changes in the levels of the principal constituents of the mTORC1 pathway. We noted that in 24 hours the immunotoxin drastically reduced the level of the p70S6 kinase in a dose dependent manner. No significant changes were seen in the levels of the TSC1, TSC2, FKBP12, mTOR, 4EBP1 or S6 proteins. Reduced levels of p70S6 kinase resulted in the absence of phospho-S6 protein from cells treated with everolimus and immunotoxin compared to the reduced phosphorylation observed with everolimus alone. We suggest that the lack of the p70S6K caused by the immunotoxin-mediated inhibition of protein synthesis is an essential element of the synergy with everolimus-mediated inhibition of mTORC1. Further, results of synergy in vitro were replicated in xenograft models where combinations exhibited enhanced anti-tumor action compared to either agent alone. Citation Format: Antonella Antignani, Lesley Mathews-Griner, Mark Ferrer, Nathan Simon, Evan Angelus, Manjie Huang, Ira Pastan, Craig Thomas, David J. FitzGerald. The mTOR inhibitor, everolimus, enhances the cytotoxicity of pseudomonas-based immunotoxins: Mechanistic insights. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A67.