Rational application of targeted therapeutics in mucinous colon/appendix cancers with positive predictive factors.

Ashokkumar Dilly,Brendon D Honick,David L Bartlett,Yong J Lee,Haroon A Choudry

doi:10.1002/cam4.2847

Abstract

Molecular‐targeted therapies have demonstrated disappointing results against most advanced solid cancers. This may largey be attributed to irrational drug use against unselected cancers. We investigated the efficacy of dual MEK‐PI3K drug therapy against KRAS mutated mucin 2 (MUC2)‐secreting LS174T cells and patient‐derived ex vivo and in vivo models of KRAS mutated mucinous colon/appendix cancers. These tumors demonstrate unique phenotypic and genotypic features that likely predict sensitivity to this targeted co‐therapy. Co‐treatment with MEK inhibitor (trametinib) and PI3K inhibitor (pictilisib)‐induced synergistic cytotoxicity and intrinsic mitochondrial‐mediated apoptosis in LS174T cells and tumor explants in vitro. Dual drug therapy also induced endoplasmic reticulum stress (ERS)‐associated proteins (GRP78/BiP, ATF4, and CHOP). However, CHOP knock‐down assays demonstrated that mitochondrial‐mediated apoptosis in LS174T cells was not ERS‐dependent. Dual drug therapy also significantly decreased MUC2 expression, MUC2 post‐translational modification (palmitoylation) and secretion in LS174T cells, suggesting a simultaneous cytotoxic and mucin suppressive mechanism of action. We also demonstrated effective mucinous tumor growth suppression in ex vivo epithelial organoid (colonoid) cultures and in in vivo intraperitoneal patient‐derived xenograft models derived from mucinous colon/appendix cancer. These promising preclinical data support a role for dual MEK‐PI3K inhibitor therapy in mucinous colon/appendix cancers. We postulate that mucinous KRAS mutated cancers are especially vulnerable to this co‐treatment based on their unique phenotypic and genotypic characteristics.

Highlights

Tremendous preclinical and clinical researches have been conducted with targeted therapies against mutationally activated oncogenes
We investigated the efficacy of dual MEKPI3K inhibition in established KRAS mutated MUC2secreting colon cancer cells (LS174T), and patient-derived in vitro and in vivo models derived from KRAS mutated mucinous colon/ appendix cancers
We found that LS174T cells co-treated with trametinib (12 μmol/L) and pictilisib (8 μmol/L) for 24 hours demonstrated significantly higher levels of upregulated protein response (UPR) proteins, including glucose-regulated protein 78 kDa (GRP78)/binding immunoglobulin protein (BiP), and CCAAT-enhancerbinding protein homologous protein (CHOP), suggesting endoplasmic reticulum stress (ERS) aggravation (Figure 2A-C)

Summary

| INTRODUCTION

Tremendous preclinical and clinical researches have been conducted with targeted therapies against mutationally activated oncogenes. We tested the efficacy of dual MEK-PI3K drug therapy against mucinous colon/appendix cancers since these tumors demonstrate unique phenotypic and genotypic features that are likely to be predictive of sensitivity to this combination therapy. These tumors frequently demonstrate KRAS mutations, resulting in downstream activation of MAPK and PI3K signaling pathways.[9,10,11,12,13,14,15,16,17,18,19] Phenotypically, mucinous tumors demonstrate high basal endoplasmic reticulum stress (ERS), and associated signaling pathways known as the upregulated protein response (UPR), likely due to high mucin 2 (MUC2) protein turnover.[20,21,22,23] they are likely to be vulnerable to aggravated ERS-associated apoptosis. We investigated the efficacy of dual MEKPI3K inhibition in established KRAS mutated MUC2secreting colon cancer cells (LS174T), and patient-derived in vitro and in vivo models (tumor explants and epithelial organoid [colonoid] cultures, and intraperitoneal [IP] murine xenografts) derived from KRAS mutated mucinous colon/ appendix cancers

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION