Inhibition of Growth of TSC2-Null Cells by a PI3K/mTOR Inhibitor but Not by a Selective MNK1/2 Inhibitor

Abstract

Lymphangioleiomyomatosis (LAM) is a rare metastatic cystic lung disease due to a mutation in a TSC tumor suppressor, resulting in hyperactive mTOR growth pathways. Sirolimus (rapamycin), an allosteric mTORC1 inhibitor, is a therapeutic option for women with LAM but it only maintains lung volume during treatment and does not provide benefit for all LAM patients. The two major mTORC1 protein synthesis pathways are via S6K/S6 or 4E-BP/eIF4E activation. We aimed to investigate rapamycin in combination with compounds that target associated growth pathways, with the potential to be additive to rapamycin. In this study we demonstrated that rapamycin, at a clinically tolerable concentration (10 nM), inhibited the phosphorylation of S6, but not the critical eIF4E releasing Thr 37/46 phosphorylation sites of 4E-BP1 in TSC2-deficient LAM-derived cells. We also characterized the abundant protein expression of peIF4E within LAM lesions. A selective MNK1/2 inhibitor eFT508 inhibited the phosphorylation of eIF4E but did not reduce TSC2-null cell growth. In contrast, a PI3K/mTOR inhibitor omipalisib blocked the phosphorylation of Akt and both S6K/S6 and 4E-BP/eIF4E branches, and additively decreased the growth of TSC2-null cells with rapamycin. Omipalisib, or another inhibitor of both major mTORC1 growth pathways and pAkt, might provide therapeutic options for TSC2-deficient cancers including, but not limited to, LAM.

Sorafenib inhibits invasion of multicellular organoids that mimic Lymphangioleiomyomatosis nodules

“…This data supports observations that multi-kinase inhibitors are able to block tumor cell growth and tumor associated angiogenesis simultaneously improving patient outcome and survival. Sorafenib is an FDA-approved multikinase inhibitor (37)(38)(39) that is known to inhibit RAF/MEK/ERK signalling, leading to increased tumor cell apoptosis, decreased micro-vessel density, and decreased metastatic shedding of tumor cells (37)(38)(39)(40) and inhibit the phosphorylation of eIF4E, eukaryotic translation initiation factor, which is upregulated in LAM, downstream of mTOR (3,(39)(40)(41). Our data supports the potential for the use of multikinase inhibitors as a therapeutic option in LAM.…”

“…Sorafenib, an FDA-approved multi-kinase inhibitor, inhibits multiple pathways in the tumor microenvironment and has been shown to improve patient outcomes by decreasing tumor cell growth, tumor-associated-angiogenesis, and metastasis (37)(38)(39). Sorafenib also has potential therapeutic benefits by inhibiting eIF4E, eukaryotic translation initiation factor, which is upregulated in LAM downstream of mTOR activation and blocking VEGFR-2 and VEGFR-3 to prevent LAM-associated lymphangiogenesis (3,(39)(40)(41).…”

Lost in translation: a neglected mTOR target for lymphangioleiomyomatosis

“…However, in a study of 30 LAM lung biopsies, the distribution of p4E-BP1 versus pS6K1 and pS6 demonstrated that there was high expression of all three phosphorylated proteins in LAM bronchi and lung parenchyma [ 29 ]. In four LAM lung fibroblast lines grown in vitro , we showed that pS6, but not p4E-BP1 Thr37/46 , was potently inhibited by 10 nM rapamycin [ 30 ]. In contrast, the dual PI3K/mTOR kinase inhibitor omipalisib inhibited both axes, albeit much more potently (∼100-fold) for pS6 than p4E-BP1 [ 30 ].…”

“…In four LAM lung fibroblast lines grown in vitro , we showed that pS6, but not p4E-BP1 Thr37/46 , was potently inhibited by 10 nM rapamycin [ 30 ]. In contrast, the dual PI3K/mTOR kinase inhibitor omipalisib inhibited both axes, albeit much more potently (∼100-fold) for pS6 than p4E-BP1 [ 30 ].…”

CrossTORC and WNTegration in Disease: Focus on Lymphangioleiomyomatosis

“…An example of a promising PI3K/mTOR inhibitor with phase 1 clinical data in idiopathic pulmonary fibrosis is GSK2126458 (omipalisib) [ 53 ]. In preclinical in vitro studies in Tsc2 -null cells, we showed that omipalisib inhibited phosphorylation of Akt and both protein synthesis arms downstream of mTORC1 activation, namely S6K and 4EBP1 [ 54 ]. It is not widely appreciated that rapamycin, and other allosteric rapalogs, inhibit phosphorylation of S6K at much lower concentrations than they inhibit phosphorylation of 4E-BP1 [ 55 ].…”