K‐Ras mutation and amplification status is predictive of resistance and high basal pAKT is predictive of sensitivity to everolimus in biliary tract cancer cell lines

Abstract

Advanced biliary tract cancer (BTC) has a poor prognosis and limited treatment options. The PI3K/Akt/mTOR signalling pathway is hyperactivated in a subset of BTCs, and clinical activity of the mTOR inhibitor everolimus has been observed in some patients with BTC. The goal of this study was to identify biomarkers predictive of everolimus response. Twenty BTC cell lines were assessed for everolimus sensitivity with a spectrum of growth inhibitory responses observed. Molecular biomarkers of sensitivity and resistance were identified by interrogation of the activation status of the Ras/MAPK and PI3K/Akt/mTOR pathways. K‐Ras mutations and/or amplifications were identified in 45% of cell lines and were associated with resistance to everolimus. Activating mutations in PIK3CA or loss of PTEN was not predictive of everolimus response; however, high basal levels of pAKT were associated with sensitivity, independent of Ras/MAPK pathway activation status. Notably, everolimus inhibited mTOR signalling to a similar extent in sensitive and resistant cell lines, suggesting that relative dependence on the mTOR pathway rather than the magnitude of pathway inhibition determines everolimus response. Consistent with the known limitations of rapalogs, everolimus induced feedback‐mediated activation of AKT in BTC cell lines, which could be overcome by cotreatment with an AKT inhibitor or ATP‐competitive mTORC1/mTORC2 inhibitors. However, both approaches failed to induce greater apoptosis compared to everolimus, and mTORC1/mTORC2 kinase inhibitors induced compensatory activation of pERK, identifying an inherent limitation of these agents in BTC cell lines. These findings suggest that future trials of everolimus in BTC would benefit from preselecting patients based on their K‐Ras and PI3K/mTOR pathway activation status. The study also identifies strategies for enhancing inhibition of the PI3K/mTOR pathway in BTC cell lines.

Phase II study of everolimus (RAD001) monotherapy as first-line treatment in advanced biliary tract cancer with biomarker exploration: the RADiChol Study

“…In this regard, we recently observed that BTC cell lines with high basal p-AKT expression respond preferentially to everolimus in vitro. 28 Similarly, renal and gastric cancers harbouring higher basal activation of the PI3K/AKT/mTOR pathway have also been reported to show improved response to everolimus. 29,30 In comparison, in the current study we failed to observe an association between the levels of a number of IHC readouts of PI3K/AKT/mTOR pathway activity and benefit to everolimus treatment.…”

“…Analysis of KRAS amplification and mutation status. Mutations in KRAS have been associated with resistance to everolimus in BTC cell lines, 17 and in patients with colorectal cancer who are treated with this agent. 25 Sanger sequencing of KRAS (exons 2 and 3) identified only two patients harbouring KRAS mutations, of whom one was the patient with ampullary carcinoma with intestinal differentiation who also harboured a PIK3CA (E545K) mutation.…”

“…In this regard, we and others have recently demonstrated that mTOR inhibitors, including everolimus, inhibit the proliferation of BTC cell lines in vitro and in vivo. 9,[14][15][16][17] Biomarker studies in a panel of BTC cell lines revealed mutations and/or amplification of KRAS to be associated with resistance, and high basal p-AKT levels to be associated with response to everolimus. 17 Activating KRAS mutations are present in 16-22% of BTCs.…”

“…9,[14][15][16][17] Biomarker studies in a panel of BTC cell lines revealed mutations and/or amplification of KRAS to be associated with resistance, and high basal p-AKT levels to be associated with response to everolimus. 17 Activating KRAS mutations are present in 16-22% of BTCs. [18][19][20] mTOR inhibitors are approved for the treatment of renal cell, neuroendocrine and hormone-positive/HER-2-negative advanced breast cancer.…”

Emerging role of precision medicine in biliary tract cancers

“… 58 , 66 Mutations in PIK3CA are frequent in GBC (8–13%). 57 , 67 PI3K/AKT/mTOR often signals downstream of ErbB/HER and preclinical studies suggest activity of PI3K/AKT/mTOR inhibition in BTCs, 68 , 69 although clinical studies are scarce. PI3K inhibitors in clinical development such as BKM-120 and taselisib may be relevant to BTCs.…”