Vanessa Rodrik-Outmezguine

AKT Inhibition Relieves Feedback Suppression of Receptor Tyrosine Kinase Expression and Activity

Activation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone.

mTOR Kinase Inhibition Causes Feedback-Dependent Biphasic Regulation of AKT Signaling

UNLABELLED mTOR kinase inhibitors block mTORC1 and mTORC2 and thus do not cause the mTORC2 activation of AKT observed with rapamycin. We now show, however, that these drugs have a biphasic effect on AKT. Inhibition of mTORC2 leads to AKT serine 473 (S473) dephosphorylation and a rapid but transient inhibition of AKT T308 phosphorylation and AKT signaling. However, inhibition of mTOR kinase also relieves feedback inhibition of receptor tyrosine kinases (RTK), leading to subsequent phosphoinositide 3-kinase activation and rephosphorylation of AKT T308 sufficient to reactivate AKT activity and signaling. Thus, catalytic inhibition of mTOR kinase leads to a new steady state characterized by profound suppression of mTORC1 and accumulation of activated AKT phosphorylated on T308, but not S473. Combined inhibition of mTOR kinase and the induced RTKs fully abolishes AKT signaling and results in substantial cell death and tumor regression in vivo. These findings reveal the adaptive capabilities of oncogenic signaling networks and the limitations of monotherapy for inhibiting feedback-regulated pathways. SIGNIFICANCE The results of this study show the adaptive capabilities of oncogenic signaling networks, as AKT signaling becomes reactivated through a feedback-induced AKT species phosphorylated on T308 but lacking S473. The addition of RTK inhibitors can prevent this reactivation of AKT signaling and cause profound cell death and tumor regression in vivo, highlighting the possible need for combinatorial approaches to block feedback-regulated pathways.

mTORC1 Inhibition Is Required for Sensitivity to PI3K p110α Inhibitors in PIK3CA-Mutant Breast Cancer

Persistent mTORC1 signaling correlates with resistance to PI3K p110α inhibition in breast cancer, which can be overcome by inhibiting mTORC1. Caveat mTOR In recent years, numerous new drugs have been developed to take advantage of specific molecular changes in cancer cells. Unfortunately, tumors are often a step ahead of the scientists, becoming resistant to these targeted drugs just when they seem to be working perfectly. Now, two groups of researchers have developed rational combination treatments that block resistance to targeted cancer drugs by inhibiting mTOR. Elkabets and coauthors were working on breast cancer, where the PIK3CA gene is frequently mutated. Inhibitors of PI3K (the protein product of PIK3CA) are currently in clinical trials, but some of the cancers are resistant to these drugs. The authors have discovered that breast cancers resistant to the PI3K inhibitor BYL719 had persistently active mTOR signaling, both in cultured cell lines and in patient tumors. Adding an mTOR inhibitor to the treatment regimen could reverse the resistance and kill the tumor cells. Corcoran et al. found a similar mTOR-dependent drug resistance mechanism to be active in melanoma as well. BRAF-mutant melanomas, the most common type, are frequently treated with RAF and MEK inhibitors, but only with mixed results, because melanomas quickly develop resistance to these drugs. Now, the authors have shown that drug-resistant melanomas also have persistent activation of mTOR, and adding an mTOR inhibitor to the treatment regimen can block drug resistance and kill the cancer cells. In both studies, the activation of mTOR in drug-resistant tumors has been confirmed in human patients, but the combination treatments have only been tested in cells and in mouse models thus far. Thus, the next critical step would be to confirm that adding mTOR inhibition to treatment regimens for these cancers is effective in the clinical setting as well. Some mTOR inhibitors are already available for use in patients, so hopefully soon mTOR activation will not be something to beware of, but something to monitor and target with specific drugs. Activating mutations of the PIK3CA gene occur frequently in breast cancer, and inhibitors that are specific for phosphatidylinositol 3-kinase (PI3K) p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly, in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as insulin-like growth factor 1 and neuregulin 1 can activate mammalian target of rapamycin (mTOR) and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α-targeted drugs and delay the appearance of resistance.

Feedback Suppression of PI3Kα Signaling in PTEN-Mutated Tumors Is Relieved by Selective Inhibition of PI3Kβ

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor

Precision medicines exert selective pressure on tumor cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next generation therapies to treat the evolving cancer. The PIK3CA/AKT/mTOR pathway is one of the most commonly activated pathways in human cancers1, which has led to the development of small molecule inhibitors that target various nodes in the pathway. Among these agents, first generation mTOR inhibitors (rapalogs) have caused responses in so-called “N-of-1” cases and second generation mTOR kinase inhibitors (TORKi) are currently in clinical trials2–4. We sought to delineate the likely resistance mechanisms to existing mTOR inhibitors as a guide for next generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active site mutation interfering with drug binding. Indeed, the identical drug resistant mutations have been also identified in drug-naïve patients4, suggesting that tumors with activating mTOR mutations will be intrinsically resistant to second generation mTOR inhibitors. Here, we report the development of a new class of mTOR inhibitors which overcomes resistance to existing first and second generation inhibitors. The third generation mTOR inhibitor exploits the unique juxtaposition of two drug binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS-ERK signaling

The effects of selective phosphoinositide 3-kinase (PI3K) and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of receptor tyrosine kinases, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild-type RAS and of RAF-MEK-ERK signaling. Inhibition of RAS-ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death, and in murine models of HER2(+) cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regression. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nano-molar concentrations, however at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyper-phosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants—those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS–GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

Mechanistically distinct cancer-associated mTOR activation clusters predict sensitivity to rapamycin

Genomic studies have linked mTORC1 pathway-activating mutations with exceptional response to treatment with allosteric inhibitors of mTORC1 called rapalogs. Rapalogs are approved for selected cancer types, including kidney and breast cancers. Here, we used sequencing data from 22 human kidney cancer cases to identify the activating mechanisms conferred by mTOR mutations observed in human cancers and advance precision therapeutics. mTOR mutations that clustered in focal adhesion kinase targeting domain (FAT) and kinase domains enhanced mTORC1 kinase activity, decreased nutrient reliance, and increased cell size. We identified 3 distinct mechanisms of hyperactivation, including reduced binding to DEP domain-containing MTOR-interacting protein (DEPTOR), resistance to regulatory associated protein of mTOR-mediated (RAPTOR-mediated) suppression, and altered kinase kinetics. Of the 28 mTOR double mutants, activating mutations could be divided into 6 complementation groups, resulting in synergistic Rag- and Ras homolog enriched in brain-independent (RHEB-independent) mTORC1 activation. mTOR mutants were resistant to DNA damage-inducible transcript 1-mediated (REDD1-mediated) inhibition, confirming that activating mutations can bypass the negative feedback pathway formed between HIF1 and mTORC1 in the absence of von Hippel-Lindau (VHL) tumor suppressor expression. Moreover, VHL-deficient cells that expressed activating mTOR mutants grew tumors that were sensitive to rapamycin treatment. These data may explain the high incidence of mTOR mutations observed in clear cell kidney cancer, where VHL loss and HIF activation is pathognomonic. Our study provides mechanistic and therapeutic insights concerning mTOR mutations in human diseases.

Abstract 4774: The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling

The PI3K pathway is dysregulated in many cancers via selective activation of class 1 isoforms. In tumors with deficient PTEN function, signaling is driven by PI3K beta. We show here that a selective inhibitor of PI3K beta inhibits the AKT/mTOR pathway in tumors with defective PTEN function, but is ineffective in those where the pathway is driven by receptor tyrosine kinases. However, inhibition of PI3K signaling by PI3K beta inhibitors is limited by relief of AKT/mTOR dependent feedback and reactivation of IGF1R and other receptors. This results in activation of PI3K alpha and a rebound of PI3K-AKT signaling. This rebound is suppressed and tumor cell inhibition is enhanced with combined inhibition of PI3K alpha and beta. Combined administration of isoform selective PI3K inhibitors may more effectively inhibit the pathway than pan-PI3K inhibitors because of the greater selectivity and decreased off-target toxicity of the former. In PTEN deficient models of prostate cancer, triple therapy with PI3K alpha and beta selective inhibitors combined with a potent androgen receptor inhibitor suppresses the reciprocal feedback activation of both pathways and results in marked (complete) eradication of tumors in vivo. Citation Format: Sarit Schwartz, Brett S. Carver, John Wongvipat, Vanessa Rodrik-Outmezguine, Elisa De Stanchina, Cath Trigwell, Simon Barry, Jose Baselga, Sarat Chandarlapaty, Howard I. Scher, Charles L. Sawyers, Neal Rosen. The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4774. doi:10.1158/1538-7445.AM2014-4774