Selective Eradication of Colon Cancer Cells Harboring PI3K and/or MAPK Pathway Mutations in 3D Culture by Combined PI3K/AKT/mTOR Pathway and MEK Inhibition

Colorectal cancer (CRC) is the second deadliest cancer worldwide. In addition to APC and p53 mutations, the PI3K/AKT/MTOR and MAPK pathways are frequently altered in CRC. However, no targeted therapies for these pathways have yet been implemented in routine clinical practice for CRC patients. In this study, we systematically evaluated the response of CRC cells to combinations of small-molecule inhibitors targeting the PI3K and MAPK pathways. We conducted drug response experiments using CRC cells in 2D cultures, 3D spheroids, collagen gels, and freshly isolated organoids. Drug effectiveness was assessed by measuring spheroid and organoid growth, spheroid outgrowth, metabolic activity, and through Western blotting and immunofluorescence.

Our results showed significant tumor cell destruction when using a PKI-587 combination of Torin 1 (an mTOR inhibitor), MK2206 (an AKT inhibitor), and selumetinib (a MEK inhibitor) in 3D models, but not in 2D cultures. The induced cell death was driven by apoptosis, confirmed by Western blot analysis. Notably, Gedatolisib, a dual PI3K/mTOR inhibitor, could replace the combination of Torin 1 and MK2206 with comparable efficacy. The presence of PI3K and/or RAS-RAF-MAPK mutations correlated with treatment sensitivity. This study highlights a novel and effective therapeutic strategy that halts cell proliferation and induces tumor destruction in vitro, tailored to the genetic background of the cancer. These promising preclinical findings warrant further in vivo testing in mice and could pave the way for mutation-specific targeted therapies for CRC patients.