Targeting KRAS Mutant Cancers with Polyisoprenylated Cysteinyl Amide Inhibitors

Abstract

Abnormalities of the MAPK pathway play vital roles in cancer initiation and progression. RAS GTPases that are key upstream mediators of the pathway are mutated in 20%–30% of human cancers. The estimated annual number of newly diagnosed mutant RAS cancer cases in the US is about 260,000, accounting for 19% of cancer patients. Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed as potential targeted therapies against RAS-driven cancers. These agents mimic the essential posttranslational modifications of G-proteins. The hypothesized anticancer mechanism for the PCAIs is that they disrupt the polyisoprenylation-dependent functional interactions of the G‐Proteins. The current study reports on the optimization of the PCAIs and the determination of their mechanisms of action in KRAS-mutant cancer cells. The new analogs display ClogP values ranging from 3.01 to 6.35, suppressing the viabilities of KRAS-mutant MDA-MB-231, A549, MIA PaCa-2, and NCI-H1299 cells in 2D and 3D cultures with EC50 values of 2.2 to 6.8, 2.2 to 7.6, 2.3 to 6.5, and 5.0 to 14 µM, respectively. When A549 cells were treated with the PCAIs, NSL-YHJ-2-27, for 48 h, no significant difference was observed in the levels of total or phosphorylated B- and C-Raf proteins. However, at 5 µM, it stimulated the phosphorylation of MEK1/2, ERK1/2, and p90RSK by 84%, 59%, and 160%, respectively, relative to controls. A non-farnesylated analog, NSL-YHJ-2-62, did not elicit similar effects. These data reveal that effects on the RAS-MAPK signaling axis most likely contribute to the anticancer effects of the PCAIs, possibly through the apoptotic isoforms of p90RSK. The effects of the PCAIs on the levels of RAS and related monomeric G-proteins were also determined. Following 48 h exposure, significant decreases in the levels of KRAS, RhoA, Rac1, and Cdc42 ranging from 20 to 66% after treatment with 5 μM of NSL-YHJ-2-27 were observed in A549, NCI-H1299, MDA-MB-231, and MDA-MB-468 cell lines tested. However, no significant difference was observed in the levels of the doubly geranylgeranylated Rab5A protein. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, the PCAIs inhibited A549 cells migration and invasion by 72 and 70 %, respectively. Furthermore, the vinculin and fascin levels in A549 cells were depleted by 33 and 43%, respectively. These findings indicate direct actions on monomeric G-proteins in the anticancer effects of the PCAIs. The PCAIs may thus have the potential to serve the unmet therapeutic needs of patients with aberrant hyperactive G-protein signaling.