Abstract
Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.
Highlights
The active site mammalian target of rapamycin (mTOR) inhibitor pp242 overcomes feedback activation of AKT, it may still be complicated by feedback ERK activation
In a prior study [5], we demonstrated the ability of pp242 to inhibit TORC1 and TORC2 in MM cells and induce a significantly greater anti-MM effect when compared with rapamycin
PP242 Activates ERK More Potently Than Rapamycin—Initially, 8226 and MM1.S MM cell lines were exposed to increasing concentrations of either rapamycin or the TORC1/ TORC2 active kinase inhibitor pp242 for 30 min
Summary
The active site mTOR inhibitor pp242 overcomes feedback activation of AKT, it may still be complicated by feedback ERK activation. Targeting mTOR therapeutically with rapamycin or related rapalogs, can be complicated by several feedback loops whereby compensatory pathways are activated These drugs primarily target the TORC1 complex and inhibit by an allosteric mechanism. The de-repression of IRS-1 hyperactivates the insulin growth factor-1(IGF-1) receptor/PI3-K/AKT signaling pathway and up-regulation of AKT activity limits anti-tumor effects of rapalog mTOR inhibitors This occurs in multiple myeloma (MM) cells [2] as well as in other tumor models [3, 4]. Newer 2nd generation TORC1/TORC2 inhibitors may overcome the PI3-K/AKT feedback loop by inhibiting AKT phosphorylation, a second potentially complicating compensatory pathway is the ERK MAPK cascade, which can be activated subsequent to mTOR inhibition. Our results surprisingly demonstrate pp242 is considerably more effective than rapamycin on a molar basis as an ERK activator, that ERK activation was regulated by effects on 4E-BP1/eIF4E and RAF and that such activation restricts pp242’s anti-MM efficacy
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