- Record: found
- Abstract: found
- Article: found
Safety, tolerability, pharmacokinetics and pharmacodynamics of AZD8055 in advanced solid tumours and lymphoma
research-article
Author(s):
A Naing
1
,
* ,
C Aghajanian
2 ,
E Raymond
3 ,
D Olmos
4 ,
G Schwartz
2 ,
E Oelmann
5 ,
L Grinsted
5 ,
W Burke
5 ,
R Taylor
5 ,
S Kaye
4 ,
R Kurzrock
1 ,
U Banerji
4
,
*
Publication date (Electronic):
30 August 2012
Journal:
British Journal of Cancer
Publisher:
Nature Publishing Group
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Abstract
Background:
This study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of the first-in-class dual mammalian target of rapamycin complex (mTORC)1/mTORC2 inhibitor, AZD8055.
Methods:
Patients with advanced solid malignancies or lymphomas were recruited into this phase I, open-label, dose-escalation study of AZD8055 starting at 10 mg twice-daily oral dosing (BID).
Results:
Forty-nine patients received AZD8055. Dose-limiting toxicities were reported at 40 mg ( n=1), 90 mg ( n=1) and 120 mg ( n=3) BID; all were grade 3 rises in transaminases, reversible in all patients, apart from one who had liver metastases. The maximum tolerated dose was defined as 90 mg BID. The most frequent adverse events assessed to be related to AZD8055 were increased alanine aminotransferase (22%), increased aspartate aminotransferase (22%) and fatigue (16%). AZD8055 was rapidly absorbed (median t max ∼0.5 h) and exposure increased with increasing doses. Seven patients had stable disease for ⩾4 months. Partial metabolic responses, assessed by fluorodeoxyglucose positron emission tomography, were observed at ⩾40 mg BID ( n=8 at day 35).
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Most cited references12
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- Abstract: found
- Article: not found
Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2
Morris E. Feldman, Beth Apsel, Aino Uotila … (2009)
Introduction The mammalian target of rapamycin (mTOR) is a serine-threonine kinase related to the lipid kinases of the phosphoinositide 3-kinase (PI3K) family. mTOR exists in two complexes, mTORC1 [1,2] and mTORC2 [3,4], which are differentially regulated, have distinct substrate specificities, and are differentially sensitive to rapamycin. mTORC1 integrates signals from growth factor receptors with cellular nutritional status and controls the level of cap-dependent mRNA translation by modulating the activity of key translational components such as the cap-binding protein and oncogene eIF4E [5]. mTORC2 is insensitive to rapamycin, and selective inhibitors of this complex have not been described. Partly because acute pharmacological inhibition of mTORC2 has not been possible, the functions of mTORC2 are less well understood than those of mTORC1. mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt [3,6] and SGK [7] although other kinases, including DNA-PK and Ilk, have also been implicated in Akt hydrophobic motif phosphorylation [8–11]. Growth factor stimulation of PI3K causes activation of Akt by phosphorylation at two key sites: the activation loop (T308) and the C-terminal hydrophobic motif (S473). Active Akt promotes cell survival in many ways, including suppressing apoptosis, promoting glucose uptake, and modifying cellular metabolism [12]; consequently, there is significant interest in identifying the kinase(s) responsible for each activating phosphorylation, the relationship between these phosphorylation sites, and the role of differential Akt phosphorylation on Akt substrate phosphorylation. Of the two phosphorylation sites on Akt, activation loop phosphorylation at T308, which is mediated by PDK1, is indispensable for kinase activity, whereas hydrophobic motif phosphorylation at S473 enhances Akt kinase activity by approximately 5-fold [13]. The disruption of mTORC2 by different genetic and pharmacological approaches has variable effects on Akt phosphorylation. Targeting mTORC2 by RNA interference (RNAi) [6,14], homologous recombination [15–17], or long-term rapamycin treatment [18] results in loss of Akt hydrophobic motif phosphorylation (S473), strongly implicating mTORC2 as the kinase responsible for phosphorylation of this site. RNAi targeting mTORC2 and long-term rapamycin result in loss of Akt phosphorylation on its activation loop (T308), but this phosphorylation remains intact in mouse embryonic fibroblasts (MEFs) lacking the critical mTORC2 component SIN1. It cannot be inferred from this genetic data whether acute pharmacological inhibition of mTORC2 would block the phosphorylation of Akt only at S473, resulting in partial Akt deactivation, or also disrupt phosphorylation at T308, resulting complete Akt inhibition. Several small molecules have been identified that directly inhibit mTOR by targeting the ATP binding site; these include LY294002, PI-103, and NVP-BEZ235 [19–22]. These molecules were originally discovered as inhibitors of PI3Ks and later shown to also target mTOR. Because all of these molecules inhibit PI3Ks and mTOR with similar potency, they cannot be used to selectively inhibit mTOR or PI3Ks in cells. Indeed, because mTORC1 and mTORC2 function downstream of PI3Ks in most settings, it is unclear to what extent the ability of these molecules to block the activation of signaling proteins such as Akt reflects PI3K versus mTOR inhibition. It is possible that some of the functions attributed to PI3Ks using the classical inhibitor LY294002 are a consequence of mTOR inhibition [19,23], but it is has not been possible address this, because small molecules that inhibit mTOR without inhibiting PI3Ks have not been available. We recently reported the synthesis of pyrazolopyrimidines that inhibit members of the PI3K family, including mTOR [24]. Two of these molecules, PP242 and PP30, are the first potent, selective, and ATP-competitive inhibitors of mTOR. Unlike rapamycin, these molecules inhibit both mTORC1 and mTORC2, and, unlike PI3K family inhibitors such as LY294002, these molecules inhibit mTOR with a high degree of selectivity relative to PI3Ks and protein kinases. To distinguish these molecules from the allosteric mTORC1 inhibitor rapamycin, we are calling them “TORKinibs” for TOR kinase domain inhibitors. The dual role of mTOR within the PI3K→Akt→mTOR pathway as both an upstream activator of Akt and the downstream effector of pathway activity on cell growth and proliferation has excited interest in active-site inhibitors of mTOR [25–30]. We describe here the biological activity of these molecules. Another small-molecule ATP-competitive mTOR inhibitor called Torin1 was reported while our manuscript was in the process of publication [56]. Results Specific Active-Site Inhibition of mTOR by the TORKinibs PP242 and PP30 PP242 and PP30 inhibit mTOR in vitro with half-maximal inhibitory concentrations (IC50 values) of 8 nM and 80 nM, respectively. As expected for active-site inhibitors, PP242 and PP30 inhibit mTOR in both mTORC1 and mTORC2 (Table S1). Both compounds are selective within the PI3K family, inhibiting other PI3Ks only at substantially higher concentrations (Figure 1). Testing of PP242 against 219 purified protein kinases at a concentration 100-fold higher than its mTOR IC50 value revealed exceptional selectivity with respect to the protein kinome; most protein kinases were unaffected by this drug, and only four—PKC-alpha, PKC-beta, RET, and JAK2 (V617F)—were inhibited more than 80% [24]. We determined IC50 values for PP242 against these kinases in vitro using purified proteins. In these assays, PP242 was relatively inactive against PKC-beta, RET, or JAK2 but inhibited PKC-alpha with an in vitro IC50 of 50 nM (Figure 1). Importantly, PP30 showed no activity against PKC-alpha or PKC-beta in the same assay (Figure 1). These data indicate that PP242 is a highly selective inhibitor of mTOR and that PP30 can be used to confirm that the effects of PP242 are due to inhibition of mTOR and not PKC-alpha. The availability of a second structurally dissimilar mTOR inhibitor—PP30—provides additional control for unanticipated off-targets of PP242. Figure 1 In Vitro IC50 Values for PP242 and PP30 Determined in the Presence of 10 μM ATP Inhibition of mTORC2 and Akt Phosphorylation by TORKinibs We characterized the effect of PP242 on the PI3K→Akt→mTOR pathway. PP242 and PP30 both inhibited insulin-stimulated phosphorylation of Akt at S473, confirming that mTOR kinase activity is required for hydrophobic motif phosphorylation (Figure 2A). The inhibition of mTOR by PP242 and PP30 also resulted in loss of Akt phosphorylation at T308, but significantly higher doses of PP242 and PP30 were required to inhibit T308 as compared with S473 (Figure 2A and 2B). PP242 inhibited S473-P and T308-P at both early and late time points after insulin stimulation, indicating that the differential sensitivity of these sites to PP242 does not reflect differing kinetics of phosphorylation (Figure S1). By comparison, the PI3K inhibitor PIK-90, which does not inhibit mTOR, inhibited the phosphorylation of both Akt sites equipotently (Figure 2B), as observed previously [21]. Figure 2 Inhibition of mTORC2 by TORKinibs Affects pS473 and pT308 of Akt (A) Serum-starved L6 myotubes were pre-treated with kinase inhibitors prior to stimulation with insulin for 3 min. Lysates were analyzed by Western blotting. (B) PP242 inhibits pS473 (red) of Akt more potently than pT308 (gray). Serum-starved L6 myotubes were treated with kinase inhibitors prior to stimulation with insulin for 10 min. Akt phosphorylation was measured by in-cell Western and is shown relative to serum starvation and insulin stimulation (n = 3 for each inhibitor dose). EC50 values from the best fit curves are plotted. ***p < 0.001, F test. EC50 values for PIK-90 on pS473 and pT308 were not significantly different (p = 0.2, F test). We sought to confirm that the loss of T308-P caused by PP242 and PP30 results from inhibition of mTOR-mediated phosphorylation of S473, rather than from inhibition of an off-target kinase, or from an effect of mTOR inhibition unrelated to S473-P. To do this, we examined the effect of PP242 on T308 phosphorylation in two situations in which Akt could not be phosphorylated on S473. First, we overexpressed S473A mutant Akt and stimulated these cells with insulin (Figure 3A). S473A Akt was phosphorylated on T308 to a similar level as wild-type, yet in contrast to the wild-type, T308-P on S473A Akt was not inhibited by PP242. The lack of effect of PP242 on S473A Akt confirms that PP242 inhibition of pT308 requires S473 and also that PP242 does not inhibit PDK1 in cells, as was suggested by direct testing of PDK1 in vitro (Figure 1). Figure 3 PP242 Does Not Directly Inhibit Phosphorylation of Akt at T308 (A) pT308 is not inhibited by PP242 in cells overexpressing S473A Akt. HEK293 cells were transfected with wild-type Akt, S473A Akt, or not transfected (Mock) and were treated with 2.5 μM PP242 or 625 nM PIK-90 as indicated prior to insulin stimulation. Lysates were analyzed by Western blotting. Quantitation of pT308 relative to insulin treated cells overexpressing wild-type Akt (lane 2) is shown below that blot. Data are representative of two independent experiments. (B) pT308 is not inhibited by PP242 in SIN1−/− MEFs, which lack pS473. Primary wild-type (WT) and SIN1−/− MEFs were pre-treated with 625 nM PIK-90, 10 μM BX-795, or 100 nM rapamycin for 24 h, 100 nM rapamycin for 30 min, or the indicated concentrations of PP242 prior to stimulation with insulin. Lysates were analyzed by Western blotting. As a further test of the specificity of PP242 and the requirement for functional S473 phosphorylation in order for PP242 to inhibit T308-P, we examined the effect of PP242 on the phosphorylation of Akt in primary MEFs from embryos that lack SIN1 [16] (Figure 3B). SIN1 is a component of mTORC2, and knockout of SIN1 compromises the physical integrity of mTORC2 leading to a complete loss of Akt phosphorylation at S473 without affecting its phosphorylation at T308. Consistent with our results from L6 cells, PP242 inhibited the phosphorylation of Akt at both S473 and T308 in wild-type MEFs. By contrast, PP242 had no effect on the phosphorylation of T308 in SIN1−/− MEFs that lack mTORC2. Furthermore, PP242 had no effect on the constitutive phosphorylation of the turn motif of Akt at T450 [16,31]. As a further comparison, we examined the effect of long-term rapamycin, which is known to block the assembly of mTORC2 is some cell lines [18]. Similar to PP242, long-term rapamycin treatment of wild-type MEFs inhibited S473-P and reduced the phosphorylation of T308-P, as was seen previously [18]. Importantly, the PI3K inhibitor PIK-90 and the PDK1 inhibitor BX-795 [32] blocked phosphorylation of T308 in SIN1−/− MEFs, indicating that the failure of PP242 to block T308 in SIN1−/− MEFs does not reflect a general resistance of T308 to dephosphorylation in cells that lack mTORC2. From these data, we conclude that PP242′s effect on T308-P is dependent on its inhibition of Akt phosphorylation by mTOR at S473. It remains unclear why mTORC2 knockout cells, but not cells treated with RNAi or pharmacological inhibitors of mTORC2, are able to retain T308 phosphorylation in the absence of phosphorylation at S473. However, there are a growing number of examples in which genetic deletion of a kinase results in compensatory changes that mask relevant phenotypes observed with the corresponding small molecule inhibitor [33]. Akt Substrate Phosphorylation Is Only Modestly Inhibited by PP242 Akt requires phosphorylation at both S473 and T308 for full biochemical activity in vitro [13], but it is unclear whether all of the cellular functions of Akt require it to be dually phosphorylated. Singly phosphorylated (T308-P) Akt from SIN1−/− MEFs is competent to phosphorylate the cytoplasmic Akt substrates GSK3 and TSC2, but not the nuclear target FoxO [16]. Because low concentrations of PP242 inhibit the phosphorylation of S473 and higher concentrations partially inhibit T308-P in addition to S473-P, we used PP242 to examine whether some substrates of Akt are especially sensitive to loss of S473-P (Figure 4). We compared PP242 to the PI3K inhibitor PIK-90 and the allosteric Akt inhibitor Akti-1/2 [34], which inhibit the phosphorylation of Akt at both sites. In contrast to PIK-90 and Akti-1/2, which completely inhibited the phosphorylation of Akt and its direct substrates, PP242 only partially inhibited the phosphorylation of cytoplasmic and nuclear substrates of Akt. This suggests that phosphorylation of the Akt substrates we examined is only modestly sensitive to loss of S473-P. A caveat of comparing Akt substrates in Sin1−/− MEFs with PP242-treated cells is the different turn motif (T450-P) status in these two conditions (Figure 3B). Figure 4 Phosphorylation of the Akt Substrates GSK3α/β, TSC2, and FoxO1/O3a Is Not Potently Inhibited by PP242 Lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin were analyzed by Western blotting. Quantitation of pAkt and pTSC2 relative to the insulin control (lane 2) is show below these blots. In contrast to Akt, which maintains T308-P, SGK activity is completely inhibited by genetic disruption of mTORC2 [7]. Because SGK can phosphorylate FoxO and its activity is completely inhibited by disruption of mTORC2, it was suggested that the loss of FoxO phosphorylation in SIN1−/− MEFs indicates that FoxO is primarily phosphorylated by SGK rather than Akt [7]. Because Akti-1/2 does not inhibit SGK [34] but inhibits FoxO1/O3a phosphorylation at T24/T32 in L6 myotubes (Figure 4), our data suggests that the major kinase for T24/T32 of FoxO1/O3a in L6 myotubes is Akt and not SGK. PP242 Does Not Have an Obvious Effect on Actin Stress Fibers TORC2 is required for the generation of a polarized actin cytoskeleton in yeast [35]. Previous analysis of mTORC2 function using RNAi revealed a role for mTORC2 in the control of the actin cytoskeleton [3,4], yet these findings were not confirmed in primary MEFs lacking mTORC2 [15,17]. We examined actin stress fibers in NIH 3T3 cells (Figure 5) and in primary MEFs (unpublished data) treated with PP242. After 8 h of treatment with PP242, we found no obvious effect on the morphology or abundance of actin stress fibers (Figure 5), suggesting that mTORC2 activity is not required for the maintenance of actin stress fibers in these cells. That PP242 didn't obviously affect the morphology or abundance of actin stress fibers, does not rule out a role for mTOR in the control of the actin cytoskeleton, but it does show that pharmacological inhibition of mTORC2 does not affect the obvious changes in actin structure seen with RNAi. Figure 5 PP242 Inhibits Proliferation without Affecting Actin Stress Fibers (A) NIH 3T3 cells were stained for actin with Alexa 488-phalloidin (green) and for DNA with DAPI (blue). Images are representative of greater that 100 cells. (B) Differential inhibition of cell proliferation by PP242 and rapamycin does not require mTORC2. Proliferation of primary MEFs cultured for 3 d in the presence of kinase inhibitors was assayed by resazurin fluorescence (RF) and is shown in arbitrary units. PP242 Inhibits Proliferation More Completely Than Rapamycin We next measured the effect of dual mTORC1/mTORC2 inhibition by PP242 on the proliferation of primary MEFs (Figure 5B). For this analysis, we compared PP242 to selective mTORC1 inhibition by rapamycin. Rapamycin was tested at concentrations above its mTOR IC50, and at all concentrations tested, it inhibited growth to the same extent. By contrast, PP242 had a dose-dependent effect on proliferation and at higher doses was much more effective than rapamycin at blocking cell proliferation. The ability of PP242 to block cell proliferation more efficiently than rapamycin could be a result of its ability to inhibit mTORC1 and mTORC2, because rapamycin can only inhibit mTORC1. To test this possibility, we measured the effects of both compounds on the proliferation of SIN1−/− MEFs, which lack mTORC2. In SIN1−/− MEFs, rapamycin was also less effective at blocking cell proliferation than PP242. That PP242 and rapamycin exhibit very different anti-proliferative effects in SIN1−/− MEFs suggests that the two compounds differentially affect mTORC1. Rapamycin-Resistant mTORC1 mTORC1 regulates protein synthesis by phosphorylating the hydrophobic motif of p70S6-Kinase (S6K) at T389 and the eIF4E-binding-protein, 4EBP1, at multiple sites. Our proliferation experiments suggest that rapamycin and PP242 have distinct effects on mTORC1. We compared the effects of acute treatment with rapamycin and PP242 on S6K, ribosomal protein S6 (S6), and 4EBP1 phosphorylation (Figure 6A) to see if these inhibitors differentially affect the phosphorylation of these canonical substrates of mTORC1. Both rapamycin and PP242 inhibited the phosphorylation of S6K and its substrate S6, and neither rapamycin nor PP242 affected the phosphorylation of 4EBP1 on T70 (Figure S2A). In contrast, PP242 fully inhibited the phosphorylation of 4EBP1 at T36/45 and S65, whereas rapamycin only had a modest affect on these same phosphorylations. Treatment of cells with PP30 was also effective at reducing the phosphorylation of 4EBP1 at T36/45 (Figure S3), indicating that the block of T36/45 phosphorylation by PP242 is due to its inhibition of mTOR and not PKC-alpha. PIK-90 did not reduce the phosphorylation of 4EBP1 at T36/45, demonstrating that inhibition of PI3K and Akt activation alone is not sufficient to block the phosphorylation of 4EBP1 at T36/45 (Figure S3). Figure 6 PP242 Inhibits Rapamycin-Resistant mTORC1 (A) Western blots of lysates from L6 myotubes treated with kinase inhibitors and stimulated with insulin for 10 min. (B) Western blots of lysates from Figure 3B. Actin loading control is repeated here for clarity. The enhanced dephosphorylation of 4EBP1 caused by PP242 as compared with rapamycin could be due to incomplete inhibition of mTORC1 by rapamycin or involvement of mTORC2 in the phosphorylation of 4EBP1. To examine these alternatives, we analyzed the effect of PP242 and rapamycin on the phosphorylation of 4EBP1 in SIN1−/− MEFs that lack mTORC2 (Figure 6B). SIN1−/− MEFS showed higher levels of p4EBP1, suggesting that due to the lack of mTORC2, these cells have more mTORC1 activity, although stronger S6K phosphorylation in wild-type cells contradicts this simple interpretation. Despite an increase in p4EBP1 in SIN1−/− compared with wild-type MEFs, shorter exposures of the p4EBP1 blots (Figure S2B) show that PP242 inhibits p4EBP1 with the same potency in both cells. The fuller inhibition of p4EBP1 by PP242 than by rapamycin in wild-type and SIN1−/− MEFs indicates that the presence of mTORC2 is not required for rapamycin and PP242 to have distinct effects on 4EBP1 phosphorylation, and suggests that PP242 is a more complete inhibitor of mTORC1 than rapamycin. Inhibition of Translation by TORKinibs While the precise role of S6K in translation control is still poorly understood, it is known that the hypophosphorylated 4EBP1 protein acts as negative regulator of the major cap-binding protein eIF4E. We directly assessed the effect of PP242 on cap-dependent translation downstream of mTOR activation. The phosphorylation of 4EBP1 by mTOR in response to growth factor and nutrient status causes it to dissociate from eIF4E allowing eIF4G and associated factors to bind to the 5' cap, recruit the 40S subunit of the ribosome, and scan the mRNA for the start codon to initiate translation. The phosphorylation of 4EBP1 by mTOR is complicated in that it occurs at multiple sites, and not all sites are equally effective at causing dissociation of 4EBP1 from eIF4E [36]. Furthermore, a hierarchy is thought to exist whereby the N-terminal threonine phosphorylations at 36/45 precede and are required for the C-terminal phosphorylations at S65 and T70 [37,38]. Phosphorylation at S65 causes the greatest decrease in affinity of 4EBP1 for eIF4E [39,40], and S65 is probably the most important site in cells for dissociation of 4EBP1 from eIF4E [41], but other sites are also important [36,42]. We examined the effect of PP242 on the active eIF4E initiation complex of translation by using a cap-binding assay. eIF4E binds tightly to beads coated with the cap analogue 7-methyl GTP (m7GTP), allowing proteins bound to eIF4E to be examined. Rapamycin caused partial inhibition of the insulin-stimulated release of 4EPB1 from eIF4E (Figure 7A), consistent with its partial inhibition of S65 phosphorylation (Figure 6A). The rapamycin-induced retention of 4EBP1 was accompanied by a loss of recovery of eIF4G, because the binding of 4EBP1 and eIF4G to eIF4E are mutually exclusive. In contrast, treatment with PP242 caused a much larger retention of 4EBP1, raising the retention of 4EBP1 above the level seen in unstimulated serum-starved cells, which are known to have low levels of protein translation [43]. Figure 7 PP242 Inhibits Cap-Dependent Translation (A) Cap-binding proteins in lysates from Figure 6A were purified by 7-methyl GTP (m7GTP) affinity and analyzed by Western blotting. (B) Primary MEFs were transfected with a bicistronic reporter vector. The ratio of renilla (cap-dependent) to firefly (IRES-dependent) luciferase activity was measured after incubation overnight in either 10% serum (steady state) or with the indicated inhibitors in the presence of 10% serum (n = 3). *p < 0.05, **p < 0.01, ANOVA with Tukey's post test. (C) Primary MEFs were incubated overnight as in (B) prior to labeling new protein synthesis with 35S. Newly synthesized proteins were separated by SDS-PAGE, transferred to nitrocellulose and visualized by autoradiography. (D) Newly synthesized protein from three experiments as in (C) was quantified. *p < 0.05, ANOVA with Tukey's post test. Translation initiation depending on eIF4E activity is the rate-limiting step in cap-dependent protein translation [44]. PP242 caused a higher level of binding between 4EBP1 and eIF4E than rapamycin (Figure 6A), suggesting that cap-dependent translation will be more highly suppressed by PP242 than by rapamycin. To quantify the efficiency of cap-dependent translation in the presence of PP242 and rapamycin, we used the well-established bicistronic reporter assay where translation initiation of the first cistron is dependent on the 5′ cap, whereas initiation of the second cistron depends on a viral internal ribosome entry site (IRES) that bypasses the need for cap-binding proteins such as eIF4E [45]. PP242 caused a significant decrease in cap-dependent, but not IRES-dependent (Figure S4), translation, whereas rapamycin did not have a statistically significant effect on cap-dependent translation (Figure 7B), consistent with the modest effect of rapamycin on 4EBP1 phosphorylation (Figure 6A). Based on this assay, inhibition of mTOR and p4EBP1 reduces cap-dependent translation by about 30%, suggesting that cap-dependent translation is only partially inhibited by hypophosphorylated 4EBP1. The majority of protein synthesis is thought to be cap-dependent [44], and consistent with this we find that PP242 also reduces total protein synthesis by about 30%, whereas rapamycin does not have a significant effect (Figure 7C and 7D). Inhibition of mTORC1 and mTORC2 In Vivo Mouse knock-outs of mTORC1 or mTORC2 result in embryonic lethality and thus it has been difficult to examine the effects of loss of mTOR in animals. To begin to explore the tissue specific roles of mTORC1 and mTORC2 and confirm the pathway analysis from cell culture experiments, we treated mice with PP242 and rapamycin and examined the acute effect of these drugs on insulin signaling in fat, skeletal muscle, and liver tissue (Figure 8). In fat and liver, PP242 was able to completely inhibit the phosphorylation of Akt at S473 and T308, consistent with its effect on these phosphorylation sites observed in cell culture. Surprisingly, PP242 was only partially able to inhibit the phosphorylation of Akt in skeletal muscle and was more effective at inhibiting the phosphorylation of T308 than S473, despite it's ability to fully inhibit the phosphorylation of 4EBP1 and S6. These results will be confirmed by in vivo dose-response experiments, but, consistent with the partial effect of PP242 on pAkt in skeletal muscle, a muscle-specific knockout of the integral mTORC2 component rictor resulted in only a partial loss of Akt phosphorylation at S473 [46]. These results suggest that a kinase other than mTOR, such as DNA-PK [8,9], may contribute to phosphorylation of Akt in muscle. Figure 8 PP242 Inhibits mTORC2 and Rapamycin-Resistant mTORC1 In Vivo Rapamycin (5 mg/kg), PP242 (20 mg/kg), or vehicle were injected into the intraperitoneal (IP) cavity of mice, followed by IP injection of 250 mU insulin or saline. Lysates were prepared from perigenital fat, leg muscle, and liver and analyzed by Western blotting. Rapamycin often stimulates the phosphorylation of Akt [47,48], probably by relieving feedback inhibition from S6K to the insulin receptor substrate 1 (IRS1) [49], a key signaling molecule that links activation of the insulin receptor to PI3K activation. In all tissues examined, and especially in fat and muscle, acute rapamycin treatment activated the phosphorylation of Akt at S473 and T308 (Figure 8). In contrast to rapamycin, by inhibiting both mTORC2 and mTORC1, PP242 suppresses rather than enhances Akt activation. As was seen in cell culture, rapamycin and PP242 also differentially affect the mTORC1 substrates S6K and 4EBP1 in vivo. S6 phosphorylation was fully inhibited by rapamycin and PP242 in all tissues examined. While PP242 was effective at blocking the phosphorylation of 4EBP1 on both T36/45 and S65 in all tissues examined, rapamycin did not block 4EBP1 phosphorylation as completely as PP242. Further experiments will be required to identify the mechanism by which 4EBP1 phosphorylation is partially resistant to rapamycin. Discussion Rapamycin has been a powerful pharmacological tool allowing the discovery of mTOR's role in the control of protein synthesis. Since the discovery of a rapamycin-insensitive mTOR complex, there has been a significant effort to develop pharmacological tools for studying this complex. We have used two structurally distinct compounds to pharmacologically dissect the effects of mTOR kinase inhibition toward mTORC1 and mTORC2 activity. We have shown through the use of these inhibitors that the inhibition of mTOR kinase activity is sufficient to prevent the phosphorylation of Akt at S473, providing further evidence that mTORC2 is the kinase responsible for Akt hydrophobic motif phosphorylation upon insulin stimulation. We also find that phosphorylation at T308 is linked to phosphorylation at S473, as had been observed in experiments where mTORC2 was disabled by RNAi and long-term rapamycin, but not homologous recombination. Surprisingly however, inhibition of mTORC2 does not result in a complete block of Akt signaling, as T308P is partially maintained and Akt substrate phosphorylation is only modestly affected when S473 is not phosphorylated. Despite its modest effect on Akt substrate phosphorylation, PP242 was a strikingly more effective anti-proliferative agent than rapamycin. These results were reproduced even in cells lacking mTORC2 (SIN1−/−), suggesting that downstream mTORC1 substrates might be responsible for PP242′s strong anti-proliferative effects. Interestingly, we observe that phosphorylation of the mTORC1 substrate 4EBP1 is partially resistant to rapamycin treatment at concentrations that fully inhibit S6K, whereas PP242 completely inhibits both S6K and 4EBP1. Because rapamycin can only partially inhibit the phosphorylation of 4EBP1, but it can fully in inhibit the phosphorylation of S6K, rapamycin appears to be a substrate-selective inhibitor of mTORC1. Consistent with this finding, experiments with purified proteins have shown that rapamycin/FKBP12 only partially inhibits the in vitro phosphorylation of 4EBP1 at Ser 65 by mTOR but can fully inhibit the in vitro phosphorylation of S6K [50]. By contrast, LY294002, a direct inhibitor of many PI3K family members including mTOR, was equally effective at inhibiting the phosphorylation of S6K and 4EBP1 by mTOR in vitro [50] and in cells [23], although this finding is complicated by LY294002′s inhibition of multiple lipid and protein kinases [51] including PIM, a kinase potentially upstream of 4EBP1 phosphorylation [52,53]. These results argue that PP242, in addition to being useful for investigating mTORC2, can reveal rapamycin-resistant components of mTORC1 function. Indeed, proliferation of SIN1−/− MEFs is more sensitive to PP242 than rapamycin (Figure 5B), suggesting that rapamycin-resistant functions of mTORC1, including the aspects of translation initiation highlighted in Figure 7, are key to the anti-proliferative effects of PP242. Furthermore, our findings suggest that the inhibition of translational control and the anti-proliferative effects of PP242 require inhibition of 4EBP1 phosphorylation and eIF4E activity. Using TORKinibs to acutely inhibit mTOR has surprisingly led to the identification of outputs from mTORC1 that are rapamycin-resistant. These observations should motivate further studies aimed at understanding how rapamycin is able to selectively affect different outputs downstream of mTORC1. As active site inhibitors of mTOR join rapamycin and its analogs in the clinic [22,27,30], it will be important to understand the distinct effects of these pharmacological agents on cellular and organismal physiology and to evaluate their efficacy in the treatment of disease and cancer caused by hyperactivation of the PI3K→Akt→TOR pathway. Materials and Methods Ethics statement. Mice were handled in accordance with protocols approved by the committee for animal research at the University of California San Francisco, United States of America. Cell culture. Cells were grown in DMEM supplemented with 10% FBS, glutamine, and penicillin/streptomycin. Confluent L6 myoblasts were differentiated into myotubes by culturing them for 5 d in medium containing 2% FBS. L6 myotubes were maintained in medium containing 2% FBS until use. Primary wild-type MEFs used in Figure 7 were isolated at embryonic day 13.5 as previously described [54]. Primary SIN1−/− MEFs and matching wild-type controls were provided by B. Su and isolated as previously described [16]. Cell lysis and Western blotting. Except where indicated otherwise, cells were serum starved overnight and incubated with inhibitors or 0.1% DMSO for 30 min prior to stimulation with 100 nM insulin for 10 min. All inhibitors were either synthesized as previously described [21,24,55] or were from Calbiochem (rapamycin and Akti-1/2). Cells were lysed by scraping into ice cold lysis buffer followed by brief sonication. Lysates were cleared by centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies from Cell Signaling Technology. Unless otherwise indicated, cells were lysed in 300 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.02% NaN3, 20 nM microcystin (Calbiochem), Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail, and 2 mM PMSF. For Figures 6A and 7A, and Figure S2A, cells were lysed in cap lysis buffer (140 mM KCl, 10 mM Tris pH 7.5, 1 mM EDTA, 4 mM MgCl2, 1 mM DTT, 1% NP-40, 20 nM microcystin, Sigma phosphatase inhibitor cocktails 1 and 2, Roche protease inhibitor cocktail without EDTA and 2 mM PMSF). Cap pull-down assay. L6 myotubes from one well of a six-well plate were lysed in 300 μl of cap lysis buffer as described above. 50 μl of detergent-free cap lysis buffer and 20 μl of pre-washed cap beads were added to 150 μl of cleared lysate and incubated at 4 °C overnight with tumbling. The beads were washed twice with 400 μl of cap wash buffer (cap lysis buffer with 0.5% NP-40 instead of 1% NP-40) and twice with 500 μl of PBS. The beads were boiled in SDS-PAGE sample buffer and the retained proteins analyzed by Western blot. All antibodies were from Cell Signaling Technologies except for the anti-eIF4E antibody, which was from BD Biosciences. Kinase assays. Phosphorylation of histone H1 (4 μM) by PKC was assayed in a buffer containing 200 ng/ml recombinant kinase, 25 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM ß-glycerol phosphate, 0.05 mg/ml phosphatidylserine, 0.03% Triton X-100, 0.5 mg/ml BSA, 2.5 mM DTT, 100 μM CaCl2, 1 μM PMA, 10 μM ATP, and 15 μCi/ml of γ-32P-ATP. Inhibitors were tested in a four-fold dilution series from 10 μM to 600 pM, and four measurements were made at each concentration. The kinase reaction was terminated by spotting onto nitrocellulose, which was washed 5 times with 1 M NaCl/1% phosphoric acid. The radioactivity remaining on the nitrocellulose sheet was quantified by phosphorimaging, and IC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package. PDK1, mTORC1, and mTORC2 were assayed as previously described [21]. In-cell Western. L6 myotubes were grown and differentiated in 96-well plates. The outside wells of the plate were not used for the experiment, but were kept filled with media. Following stimulation, cells were fixed for 15 min with 4% formaldehyde in PBS with Ca++ and Mg++. The cells were washed three times with PBS and the blocked and permeabilized with 5% goat serum in PBS with 0.3% Triton X-100 (PBS-GS-TX). Primary antibodies to S473 (Cell Signaling #4060) and T308 (Cell Signaling #2965) were added at 1:1000 and 1:500, respectively, in PBS-GS-TX, and the plates were incubated at 4 °C overnight. The plates were then washed three times with PBS, and goat anti-rabbit secondary antibody (Pierce Biotechnology) was added at 0.01 μg/ml in PBS-GS-TX. After 1 h at room temperature, plates were washed three times with PBS. ELISA chemiluminescent reagent (Femto, Pierce Biotechnology) was added to each well and after 1 min, the plate was read in a luminescence plate reader using a 100-ms integration time. The pAkt signal from pT308 and pS473 was normalized to control wells, so that 0 represents the level of pAkt in serum starved cells and 1 represents the level upon insulin stimulation. EC50 values were determined by fitting the data to a sigmoidal dose-response curve using the Prism software package. The significance of differences between EC50 values was evaluated using the F test. Akt transfection. Akt was transfected into HEK293 cells using Lipofectamine 2000 according the manufacturers protocol. Two days after transfection, cells were serum starved overnight and the next day they were treated with inhibitors and processed for western blotting as described above. Actin cytoskeleton staining. NIH 3T3 cells were plated on poly-lysine coated coverslips at 30% confluence the day before the experiment. Following treatment with PP242 or 0.1% DMSO for 8 h in 10% serum growth medium, the actin cytoskeleton was stained as previously described [24]. Bicistronic reporter assay. Primary MEFs were transfected with a bicistronic reporter [54] containing a viral IRES using Lipofectamine 2000 according to the manufacturers protocol. At 2 d post transfection, cells were treated overnight with compounds as indicated or starved of serum. The next day, Renilla and Firefly luciferase activity were measured using the Dual-Luciferase kit (Promega). Differences in the ratio of Renilla to Firefly luciferase signals were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package. 35S labeling of new protein synthesis. Primary MEFs grown to 70% confluence in six-well plates were incubated overnight in either 10% Serum (Steady State), kinase inhibitors in 10% serum, or 0.1% serum (starved). Cells were then washed once with DMEM lacking cysteine and methionine (DMEM-noS), and the medium was replaced with DMEM-noS including dialyzed serum and kinase inhibitors as indicated. After incubation for 1 h, 50 μCi of Expre35S35S (NEN) was added to each well and the cells were labeled for 4 h. Cells were washed once with ice-cold PBS, and lysed as described above for Western blotting. Following separation by SDS-PAGE, and transfer to nitrocellulose, 35S-labeled proteins were visualized by autoradiography with film. For quantitation, the membrane was exposed to a phosphorimager screen and the resulting image was quantified in ImageJ. Differences in 35S incorporation were analyzed for statistical significance by one-way ANOVA with Tukey's post test using the Prism software package. In vivo drug treatment and Western blotting. Drugs were prepared in 100 μl of vehicle containing 20% DMSO, 40% PEG-400, and 40% saline. Six-wk-old male C57BL/6 mice were fasted overnight prior to drug treatment. PP242 (0.4 mg), rapamycin (0.1 mg), or vehicle alone was injected IP. After 30 min for the rapamycin-treated mouse or 10 min for the PP242 and vehicle-treated mice, 250 mU of insulin in 100 μl of saline was injected IP. 15 min after the insulin injection, the mice were killed by CO2 asphyxiation followed by cervical dislocation. Tissues were harvested and frozen on liquid nitrogen in 200 μl of cap lysis buffer. The frozen tissue was thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue-homogenizer (Fisher PowerGen 125 with Omni-Tip probe). Protein concentration of the cleared lysate was measured by Bradford assay and 5–10 μg of protein was analyzed by Western blot as described above. Cell proliferation assay. Wild-type and SIN1−/− MEFs were plated in 96-well plates at approximately 30% confluence and left overnight to adhere. The following day cells were treated with PP242, rapamycin, or vehicle (0.1% DMSO). After 72 h of treatment, 10 μl of 440 μM resazurin sodium salt (Sigma) was added to each well, and after 18 h, the florescence intensity in each well was measured using a top-reading florescent plate reader with excitation at 530 nm and emission at 590 nm. Supporting Information Figure S1 PP242 Inhibits Akt Phosphorylation over the Course of 1 h L6 myotubes were pre-treated with PP242 or DMSO for 30 min and stimulated with insulin for the indicated times prior to lysis and analysis by Western blotting. (4.31 MB AI). Click here for additional data file. Figure S2 Additional Analysis of 4EBP1 Phosphorylation (A) 4EBP1 phosphorylation at T70 is not inhibited by either PP242 or rapamycin. L6 myotube lysates from Figure 6A were analyzed by Western blotting. (B) 4EBP1 phosphorylation is inhibited by PP242 with similar potency in SIN1−/− and wild-type (WT) MEFs. Western blotting from Figure 6B is shown with shorter exposures of p4EBP1. (4.97 MB AI). Click here for additional data file. Figure S3 Rapamycin-Resistant Phosphorylation of 4EBP1 Is Sensitive to the TORKinibs PP30 and PP242, but Not the PI3K Inhibitor PIK-90 L6 myotube lysates from Figure 2A were analyzed by Western blotting. (2.44 MB AI). Click here for additional data file. Figure S4 PP242 Inhibits Cap, but Not IRES-Dependent, Translation (A) Renilla (cap-dependent) luciferase activity from samples in Figure 7B. (B) Firefly (IRES-dependent) luciferase activity from samples in Figure 7B. Firefly luciferase activity of the PP242 treated sample is not significantly different from control (p = 0.4, ANOVA). (1.55 MB AI). Click here for additional data file. Table S1 In Vitro IC50 Determinations Using Three Forms of mTOR (19 KB DOC) Click here for additional data file.
- Record: found
- Abstract: found
- Article: not found
Measurement of clinical and subclinical tumour response using [18F]-fluorodeoxyglucose and positron emission tomography: review and 1999 EORTC recommendations. European Organization for Research and Treatment of Cancer (EORTC) PET Study Group.
H. Young, R. Baum, U Cremerius … (1999)
[18F]-fluorodeoxyglucose ([18F]-FDG) uptake is enhanced in most malignant tumours which in turn can be measured using positron emission tomography (PET). A number of small clinical trials have indicated that quantification of the change in tumour [18F]-FDG uptake may provide an early, sensitive, pharmacodynamic marker of the tumoricidal effect of anticancer drugs. This may allow for the introduction of subclinical response for anticancer drug evaluation in early clinical trials and improvements in patient management. For comparison of results from smaller clinical trials and larger-scale multicentre trials a consensus is desirable for: (i) common measurement criteria; and (ii) reporting of alterations in [18F]-FDG uptake with treatment. This paper summarises the current status of the technique and recommendations on the measurement of [18F]-FDG uptake for tumour response monitoring from a consensus meeting of the European Organization for Research and Treatment of Cancer (EORTC) PET study group held in Brussels in February 1998 and confirmed at a subsequent meeting in March 1999.
- Record: found
- Abstract: found
- Article: not found
Antitumor Activity of Rapamycin in a Phase I Trial for Patients with Recurrent PTEN-Deficient Glioblastoma
Tim F Cloughesy, Koji Yoshimoto, Phioanh Nghiemphu … (2008)
Introduction When a new cancer drug first enters the clinic, its development typically proceeds empirically by defining the maximum tolerated dose, then assessing clinical activity across a range of diseases. In the era of molecularly targeted cancer therapy, this approach has been questioned, because it is anticipated that these agents will be effective primarily in those patients whose tumors are dependent on the molecular lesion that is specifically targeted by the new agent [1–3]. However, target-focused clinical development is challenging, because clearly defined, validated molecular criteria to select patients for clinical trials must be established. Inability to access tumor tissue in most patients with solid tumors presents further difficulties. One approach is to conduct small pilot studies in which the targeted agent is administered to patients prior to a scheduled tumor resection to ensure access to tissue during treatment. Such neoadjuvant studies have been successfully implemented with hormonal agents alone or in combination with kinase inhibitors in breast cancer [4,5]. Current technologies permit analyses of gene copy number, mutation status, and mRNA and protein expression from small tissue samples, thereby allowing for the collection of high–molecular content datasets that can guide further clinical development. We have used this approach to study the targeted agent rapamycin in a molecularly defined subset of patients with recurrent glioblastoma. Inhibitors of the mammalian target of rapamycin (mTOR) have received regulatory approval as immunosuppressive agents for the treatment of allograft rejection and as antitumor agents for kidney cancer [6,7]. Rapamycin and its analogs (CCI-779, RAD001) have shown antitumor activity across a variety of human cancers in clinical trials, but molecular determinants of drug response are currently unknown [8]. Previous work by our group [9] and others [10–15] demonstrated that mutational activation of the phosphatidyl-inositol-3-kinase (PI3K) pathway through loss of PTEN (phosphatase and tensin homolog deleted on Chromosome 10) or activation of the serine/threonine kinase Akt sensitizes tumor cells to the antiproliferative activity of mTOR inhibitors in preclinical models. These findings provided the rationale to explore the antitumor activity of mTOR inhibitors in patients with PTEN-deficient tumors. Glioblastoma is one model disease to address this question, because PTEN inactivation occurs in ∼40% of patients. Furthermore, salvage surgical resection is often part of the clinical management of patients who relapse after standard up-front therapy (which typically consists of surgical resection followed by adjuvant radiation and chemotherapy). This second resection is an opportunity to collect tumor tissue to assess the molecular effects of treatment administered pre-operatively. Indeed, others have used this salvage surgery to define the dose of O6-benzylguanine required to deplete the DNA-repair protein AGT, which is associated with resistance to temozolamide [16]. Importantly, the antitumor effects of mTOR inhibition in many preclinical models are cytostatic, raising the possibility that traditional radiographic clinical endpoints of tumor shrinkage may not be observed. Glioblastoma may be suitable for assessing cytostatic activity, because these tumors are highly proliferative. Therefore, short-term effects of treatment on growth kinetics could be detectable by immunohistochemical analysis. Finally, clinical benefit can be assessed by measuring time-to-tumor progression after surgery. For these reasons, we conducted a neoadjuvant clinical trial of rapamycin in patients with relapsed, PTEN-negative glioblastoma undergoing salvage resection, with the primary goals of defining a dose required for mTOR target inhibition and assessing potential antiproliferative effects on tumor cells. Methods Participants This Phase I trial was registered with http://www.ClinicalTrials.gov (#NCT00047073) (see also http://www.cancer.gov/search/ViewClinicalTrials.aspx?cdrid=257255&version=patient&protocolsearchid=3718462). The clinical trial protocol (#02-03-078–11) was approved by the Institutional Review Board of the University of California Los Angeles. Enrollment was restricted to patients with a histological diagnosis of glioblastoma (GBM), radiographic evidence for disease recurrence after standard GBM therapy (surgery, radiation, temozolamide), evidence for PTEN loss in tumor tissue (see below), and no previous mTOR inhibitor therapy. Other enrollment criteria included age > 18 y, Karnofsky performance score (KPS) ≥ 60, life expectancy ≥ 8 wk, adequate bone marrow function (white blood cell [WBC] > 3,000/μl, absolute neutrophil count [ANC] > 2,000/μl, platelets > 100,000/μl, hemoglobin > 10 gm/dl), adequate liver and renal function (serum glutamic oxaloacetic transaminase [SGOT] and bilirubin 50% inhibition of S6 phosphorylation for at least one of the two examined phosphosites (p 20% of tumor cells) [37]. Discussion Rapamycin and other mTOR inhibitors have shown great promise as anticancer drugs in a spectrum of preclinical models, but it has been difficult to demonstrate convincing clinical activity in single-agent trials using conventional radiographic and clinical criteria for response [38]. Potential explanations include the largely cytostatic action of these drugs in the laboratory, uncertainty over dose and schedule, and lack of studies to evaluate the drug in subsets of patients most likely to respond based on molecular phenotypes defined preclinically. The goal of this study was to evaluate directly rapamycin in patients whose tumors have defects in PTEN, based on preclinical findings originally generated by our group and others showing mTOR dependence in such models [9–15]. In designing the clinical experiment, we sought to validate the use of a PTEN assay for patient selection, document mTOR inhibition in tumor tissue (of particular importance for brain cancers), and gain preliminary evidence of antitumor activity. Glioblastoma was selected based on the high frequency of PTEN loss (∼40%), the clinical opportunity to collect tumor tissue at the time of salvage surgical resection, and the high proliferative index of these tumors, providing a robust endpoint for assessing antitumor effect. The intent was to generate information that could be used for more focused hypothesis testing in subsequent trials. In the present study 165 patients were screened for PTEN status after initial surgical resection, then followed until relapse. Fifteen patients whose initial surgical samples stained negative for PTEN by immunohistochemistry were treated with rapamycin for about 1 wk before a planned salvage surgical resection. Short-term effects of rapamycin on mTOR inhibition in tumor cells and on the tumor proliferation index were determined by comparing immunohistochemical measures of these indices in the initial surgical sample (surgery 1 or S1) to the salvage resection sample (surgery 2 or S2). Rapamycin treatment led to substantial inhibition of tumor cell proliferation in seven of 14 patients, which correlated with the greatest magnitude of mTOR inhibition in tumor tissue. As predicted from preclinical studies [27,28], rapamycin also led to the activation of Akt in some cases, and this activation was significantly correlated with shorter time-to-tumor progression. The primary findings from this neoadjuvant rapamycin trial are evidence of antitumor activity using a short-term endpoint, novel insights into the importance of achieving sufficient target inhibition, and clinical evidence for evaluating combination PI3-kinase/mTOR therapy to address negative feedback. All three findings should guide future clinical development of mTOR inhibitors in this disease. The Ki-67 response data demonstrate that rapamycin has clear antitumor activity in a subset of patients with PTEN loss. In addition to effects on tumor cell proliferation, two patients also had radiographic evidence of response. Patient 8 received an extended course of neoadjuvant rapamycin (25 d) due to an intercurrent upper respiratory infection and had >50% tumor regression by magnetic resonance imaging prior to surgery (Figure S8A). Patient 11 showed continued radiographic improvement during the postoperative phase of rapamycin treatment and died without evidence of tumor recurrence 538 d after starting rapamycin (Figure S8B). The experience with patient 8 might justify a longer neoadjuvant treatment period to gain radiographic response data on all patients in subsequent trials. While our trial was underway, a single-arm phase II study of the mTOR inhibitor CCI-779 reported that 20 of 65 patients with recurrent glioblastoma (36%) had radiographic improvement [39]. Of note, these patients were not evaluated prospectively for PTEN status (no molecular selection criteria), and CCI-779 was delivered weekly rather than daily based on a phase I experience that defined a maximum tolerated dose using this schedule [40,41]. In light of our findings about the magnitude of mTOR inhibition required for response (discussed below), this schedule raises concerns about the presumed lack of target coverage during nontreatment days. Nonetheless, the fact that both trials showed evidence of antitumor activity provides confidence that further investigation of mTOR inhibitors is warranted. The role of PTEN loss in defining sensitivity could be determined using a trial design in which all patients are initially eligible but sufficient numbers of PTEN negative versus PTEN positive are accrued to allow subset analysis. Although intuitive, the correlation we found between the magnitude of mTOR inhibition and Ki-67 response was not anticipated from preclinical studies. Nearly complete inhibition of S6 phosphorylation is typically achieved with rapamycin treatment in xenografts and other mouse model systems; therefore, most studies of response have focused on defining genetic lesions (Pten, Akt, Tsc, Vhl, etc.) that affect mTOR dependence of tumor cells [38,42]. The surprising finding in this trial is that despite using doses of rapamycin sufficient to give low nM intratumoral levels, such doses do not translate into mTOR inhibition in all patients. Through ex vivo analysis of tumor cells isolated at salvage surgery, we established that resistance in these patients is not cell intrinsic. Consistent with an extrinsic mechanism of rapamycin resistance, our genomic survey of S2 tumor samples failed to identify significant copy-number alterations within genes in the mTOR pathway (FKBP12, S6 kinase 1, RAPTOR, RHEB, Akt) that might explain the observed rapamycin resistance in vivo. This result contrasts with mechanisms of resistance to other kinase inhibitors (in chronic myeloid leukemia, gastrointestinal stromal tumors, and EGFR-dependent lung cancer), which often occurs through point mutations in the kinase target in tumor cells [43] and raises the possibility that a larger fraction of PTEN null glioblastomas could be rapamycin-sensitive if more significant mTOR inhibition could be achieved. The more challenging question is whether strategies can be developed to improve delivery of rapamycin directly to tumor cells and maximize mTOR inhibition broadly across all patients. Oral delivery of significantly higher daily doses is an unlikely solution due to problems with tolerability (mucositis, thrombocytopenia) seen in other diseases. Invasive approaches such as convection-enhanced delivery or implantation of drug-impregnated wafers have been used to treat glioblastoma patients with chemotherapeutic agents and may be considered. Alternatively, a better understanding of the reason underlying the failure to achieve mTOR inhibition in selected patients could point to a solution. For example, if rapamycin in these patients is sequestered in red cells due to enhanced tumor vascularity, antiangiogenic agents such as bevacizumab (already known to have activity in glioblastoma) [44] may prevent sequestration and allow more efficient drug delivery. Evaluation of all of these approaches requires quantitative assessment of mTOR activity and highlights the need to develop broadly useful clinical tools for quantitative analysis of target inhibition. In the short term, it may be possible to identify the early Ki-67 responders using PET tracers such as 3′-deoxy-3′-18F-fluorothymidine (FLT) that can read out proliferation noninvasively [45]. Although such identification would not itself improve rapamycin delivery to the tumor cells, it could at least identify the subset of tumors in which rapamycin delivery appears to be problematic. Success here would also obviate the need for salvage surgery and could greatly expand eligibility of patients for larger trials. There seems little doubt from the time-to-progression curves reported here and in the CCI-779 study that combination therapy is required for significant clinical impact. The challenge, of course, lies in choosing the most promising second drug from an almost infinite number of possibilities. Based on earlier work from us and others, combined EGFR/mTOR blockade is one logical choice, because PTEN loss predicts for resistance to EGFR inhibitors in patients with the mutant EGFRviii variant [18,46–48]. Another possibility is combined PI3K/mTOR blockade to prevent rapamyin-induced activation of Akt caused by loss of negative feedback [27,28]. The time-to-progression analysis in our study suggests that the prognosis of these patients is worse, therefore inhibitors that act upstream of Akt may be useful to prevent this complication. Indeed, one dual PI3K/mTOR inhibitor has shown superiority to a pure mTOR inhibitor in preclinical models [49]. Although the findings reported here are directly relevant to mTOR inhibitors in glioblastoma, the implication is that these drugs will have activity in a broad range of cancers with PI3K/Akt pathway dysregulation—through PTEN loss, PI3K p110α mutation, AKT gene amplification, or other mechanisms. Recently, mTOR inhibitors have shown clinical activity in metastatic kidney cancer, where the frequency of PTEN loss is low [50]. The molecular basis for sensitivity in this disease is unknown, but loss of the von Hippel-Lindau (VHL) tumor suppressor and subsequent mTOR-dependent HIF-1α expression is one postulated mechanism [51]. For reasons similar to those articulated above for glioblastoma, mTOR-based combination therapies are also under consideration in kidney cancer. The neoadjuvant clinical trial design described here should be easily exportable to other cancers in which experimental drug delivery can be timed prior to a planned surgical excision of tumor, and such an approach is consistent with recent national efforts to speed clinical development through novel trial designs [52]. Supporting Information Figure S1 Digital Scoring of Immunohistochemical Stains Adjacent tissue sections from each tumor were stained with antibodies against Ki-67, phospho S6 ribosomal protein (S6RP), phospho-PRAS40, and PTEN (unpublished data). Five areas per slide, each representing approximately 500–1,000 tumor cells, were selected for digital scoring. Image conversion and scoring was performed using Soft Imaging System Software. The distribution of immunoreactivity within these 2,500–5,000 cells was graphed for each sample as a box plot. The “fold change” (F.C.) in S6 immunoreactivity (Table 2 and Figure S2) was calculated for each tumor as the ratio between median staining score in the S2 and the median staining score in the S1 sample. (135 KB PDF) Click here for additional data file. Figure S2 S6 Phosphorylation at Ser 235/236 in Matched S1/S2 Tumor Tissue Pairs (A) Representative IHC staining results for pSer 235/236 S6. Shown are examples for a tumor with biochemical mTOR inhibitor resistance (patient 2) compared to a tumor with marked mTOR inhibition in response to rapamycin (patient 8). (B and C) Quantification of S6 phosphorylation at Ser 235/236 in matched S1/S2 tumor samples from 14 patients in the rapamycin clinical trial cohort (B) and nine glioblastoma patients who did not receive rapamycin prior to S2 (C). For additional information regarding IHC scoring methodology, see Figure S1 and Text S1. (1.2 MB PPT) Click here for additional data file. Figure S3 S6 Phosphorylation at Ser 240/244 in Matched S1/S2 Tumor Tissue Pairs IHC-based quantification of S6 phosphorylation at Ser 240/244 in (A) matched S1/S2 tumor samples from 14 patients in the rapamycin clinical trial cohort and (B) matched S1/S2 tumor samples from nine glioblastoma patients who did not receive rapamycin prior to S2. (206 KB PPT) Click here for additional data file. Figure S4 CD31 Immunostaining of Representative Tumor Tissue Sections from Rapamycin-Treated Tumors with Low (Patients 1 and 3) Versus High (Patients 5 and 15) Intratumoral Rapamycin Concentrations Arrows indicate CD31 IHC-positive intratumoral vasculature. (159 KB PDF) Click here for additional data file. Figure S5 Frequency of Genomic Aberrations in S2 Samples by Array-CGH Genomic DNA microdissected from fresh frozen tumor samples (S2) was subjected to oligonucleotide microarray-based CGH (aCGH) analysis. Aberrations were scored using CGH Analytics Software (Agilent) using the ADM1 algorithm (parameters listed in Text S1), and filtered to retain only aberrations with a log2 ratio less than −0.3 or greater than 0.3. The frequency of DNA copy-number increases (red, right of the zero axis for each chromosome) and losses (green, left of the axis) within the genome for the samples profiled is plotted in terms of percentage of the samples analyzed. (56 KB PDF) Click here for additional data file. Figure S6 Gene Copy-Number Alterations Stratified by pPRAS40 Response CGH data for biopsies following rapamycin treatment were plotted using Cluster and TreeView Software (available at http://rana.lbl.gov/EisenSoftware.htm). DNA copy-number losses are represented in green, while gains are represented in red. Patient samples are stratified by the presence or absence of significant induction of pPRAS40 following rapamycin therapy as determined by IHC (see Figure 4B and Table 2). (53 KB PDF) Click here for additional data file. Figure S7 IHC Scoring of PTEN Expression in Clinical Tumor Samples from Rapamycin Study Patients Compared to a Group of 44 Archival Glioblastoma Tissue Samples (A) Comparison of two PTEN IHC scoring methods (x-axis: manual score; y-axis: image-guided digital score) in 44 archival glioblastoma samples. The manual score compares PTEN expression in tumor cells to PTEN expression in adjacent endothelial cells and assigns scores of 0 (absent in tumor cells), 1 (reduced in tumor cells relative to endothelial cells), and 2 (tumor cells staining similar to endothelial cells). Digital PTEN scores are based on absolute values of PTEN immunoreactivity as determined through an image-guided software scoring system (Figure S1). Digital PTEN scores were determined by an independent reviewer without knowledge of the manual PTEN score. (B) Mean digital PTEN IHC score of tumors from the rapamycin study group before (S1) and during (S2) rapamycin therapy. (18 KB PDF) Click here for additional data file. Figure S8 Radiographic Response to Rapamycin in 2/15 Study Patients Magnetic resonance imaging results for two patients who experienced a clinical response to single-agent rapamycin. (A) Patient 8 received an extended preoperative course of rapamycin (25 d) due to an intercurrent upper respiratory infection and experienced >50% tumor regression by magnetic resonance imaging. (B) Patient 11 showed radiographically stable disease for >16 mo postoperatively and died without evidence for tumor recurrence. (72 KB PDF) Click here for additional data file. Table S1 Rapamycin-Related Adverse Events (11 KB PDF) Click here for additional data file. Table S2 PTEN Copy Number (Log2 Ratio) and Missense Mutations in Tumor Tissue Genomic DNA was extracted from microdissected tumor cells. N/A: no fresh frozen tumor aliquot available for analysis. Log2 ratios ≤ −0.3 are consistent with DNA copy-number loss. (13 KB PDF) Click here for additional data file. Text S1 Supplementary Methods (42 KB DOC) Click here for additional data file. Text S2 Protocol (254 KB DOC) Click here for additional data file. Text S3 CONSORT Checklist (59 KB DOC) Click here for additional data file.
Author and article information
Journal
Journal ID (nlm-ta): Br J Cancer
Journal ID (iso-abbrev): Br. J. Cancer
Title:
British Journal of Cancer
Publisher:
Nature Publishing Group
ISSN
(Print):
0007-0920
ISSN
(Electronic):
1532-1827
Publication date
(Print):
25
September
2012
Publication date
(Electronic):
30
August
2012
Volume: 107
Issue: 7
Pages: 1093-1099
Affiliations
[1
]The University of Texas MD Anderson Cancer Center , 1515 Holcombe Boulevard, Houston, TX 77030, USA
Author notes
Article
Publisher Item ID: bjc2012368DOI: 10.1038/bjc.2012.368
PMC ID: 3461162
PubMed ID: 22935583
SO-VID: e70ee5ec-9ebc-4ad6-b4ad-ae22d6e20101
Copyright © Copyright © 2012 Cancer Research UK
License:
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
History
Date
received
: 24
July
2012
Date
revision received
: 27
April
2012
Date
accepted
: 26
July
2012
Categories
Subject:
Translational Therapeutics
ScienceOpen disciplines: Oncology & Radiotherapy