Abstract 3001: Accurins are endocytosed by KRAS-mutant cells via macropinocytosis
Authors: Cullis Jane E. , Taylor Laura , Low Susan , Bar-Sagi Dafna 
Published: July 2016

Abstract
Background:
BIND-014 is a novel prostate-specific membrane antigen (PSMA)-targeted Accurin (polymeric nanoparticle) encapsulating docetaxel. In a Phase 2 non-small cell lung cancer (NSCLC) trial, BIND-014 demonstrated promising anti-tumor activity in patients with tumors expressing KRAS mutations (mutated Kirsten ras oncogene homolog). KRAS mutations in NSCLC are generally associated with poor response to currently available drug therapy regimens, including docetaxel. Mutant KRAS stimulates an endocytic process known as macropinocytosis, in which extracellular fluid and its constituents are internalized non-selectively into cells. We hypothesized that Accurins preferentially accumulate in KRAS-mutant cells via macropinocytosis, potentially contributing to anti-tumor activity of BIND-014 in KRAS-mutant cancer.
Methods:
To determine whether BIND-014 is internalized in cells with high levels of macropinocytosis, we treated KRAS-mutant, macropinocytosis-high human NSCLC A549 cells and human bladder cancer T24 cells with PSMA targeted Accurins in which docetaxel was replaced by a fluorescent label, and visualized co-localization with fluorescently labeled markers of macropinocytosis, dextran and BSA, by confocal microscopy. In addition, we quantified the levels of labeled Accurin uptake in A549 cells by fluorescence-activated cell sorting (FACS) and compared them to labeled Accurin internalization in macropinocytosis-low, KRAS-wild-type BxPC3 cells. We further tested the macropinocytosis-dependence of labeled Accurin uptake by treating A549 and BxPC3 cells with the macropinocytosis inhibitor EIPA and measuring changes in Accurin internalization by FACS.
Results:
Confocal microscopy analysis of A549 and T24 cells co-treated with labeled Accurin and dextran or BSA showed substantial accumulation of Accurin in dextran- and BSA-positive macropinosomes. Moreover, FACS analysis of A549 cells treated with 0.1 ug and 1.0 ug of labeled Accurin revealed 60.4% and 81.8% uptake, respectively, that could be reduced to 3.6% and 3.7% with 50 uM EIPA. By contrast, BxPC3 cells showed 1.8% and 1.5% uptake of 0.1 ug and 1.0 ug labeled Accurin that was reduced to 1.3% and 0.9%, respectively, with 50 uM EIPA by FACS analysis.
Conclusions:
These data demonstrate that Accurins preferentially enter human cancer cells that display high levels of macropinocytosis and co-localize with several markers of macropinosomes. Moreover, Accurin internalization can be inhibited in macropinocytosis-positive cells with an established chemical inhibitor of macropinocytosis. BIND-014 may therefore preferentially accumulate in KRAS-mutant NSCLC tumors, thereby contributing to the clinical activity seen to date in this tumor type.
Citation Format: Jane E. Cullis, Laura Taylor, Susan Low, Dafna Bar-Sagi. Accurins are endocytosed by KRAS-mutant cells via macropinocytosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3001.