Biomarker Development for the Clinical Activity of the mTOR Inhibitor Everolimus (RAD001): Processes, Limitations, and Further Proposals

Abstract

The mTOR inhibitor everolimus (RAD001, Afinitor) is an orally active anticancer agent. Everolimus demonstrates growth-inhibitory activity against a broad range of tumor cell histotypes in vitro and has the capacity to retard tumor growth in preclinical tumor models in vivo through mechanisms directed against both the tumor cell and the solid tumor stroma components. These properties have rendered it to be a clinically active drug, with subsequent registration in renal cell carcinoma (Motzer et al. [2008]. Lancet372, 449-456) as well as showing strong potential as a combination partner (André F et al. [2008]. J Clin Oncol26. Abstract 1003). Although everolimus has a high specificity for its molecular target, the ubiquitous nature of mTOR and the multifactorial influence that mTOR signaling has on cell physiology have made studies difficult on the identification and validation of a biomarker set to predict and monitor drug sensitivity for clinical use. In this review, a summary of the preclinical and clinical data relevant to biomarker development for everolimus is presented, and the advantages and problems of current biomarkers are reviewed. In addition, alternative approaches to biomarker development are proposed on the basis of examples of a combination of markers and functional noninvasive imaging. In particular, we show how basal levels of pAKT and pS6 together could, in principle, be used to stratify patients for likely response to an mTOR inhibitor.

Figure 1

Mechanism of action and structure of everolimus. (A) Everolimus (yellow-red) binds to the FK506-binding protein-12 immunophilin, FKBP-12 (white), and this complex interacts with the FRB domain (blue) of the large protein mTOR. (B) PI3K/AKT/mTOR pathway: the FKBP-12-RAD001 complex inhibits mTORC1 function. 4E-BP1 indicates eIF-4E (eukaryotic initiation factor 4E) binding protein 1; HIF-1, hypoxia-inducible factor 1 or 2; mTORC1/2, mammalian target of rapamycin complex 1 or 2; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homologue, dual-specificity lipid/protein phosphatase; S6K, 40S ribosomal S6 kinase; TSC1/2, tuberous sclerosis tumor suppressor genes; VHL, von Hippel-Lindau protein. Tumor suppressors are in brown and underlined. (C) Molecular structure of everolimus (RAD001).

Figure 2

In vitro activity of everolimus: examples of IC₅₀ determination. Results show the dose-dependent inhibition of cell growth by everolimus as a percentage of the control determined using methylene blue staining after 96 hours of incubation in four different human tumor cell lines, which can be regarded as sensitive (HCT-15, A549) and insensitive (KB-31 and HCT-116).

Figure 3

In vitro activity of everolimus against mammalian cell lines and PK in mice and rats. Everolimus in vitro activity in 48 different cell lines assessed by IC₅₀ values in ascending values (A) and ranked (B), showing cutoffs at 100 nM (red) or 350 nM (blue). Everolimus (RAD001) was administered once at 5 mg/kg per os (p.o.) to (C) female BALB/c athymic nude mice bearing s.c. KB-31 tumors or (D) Lewis rats bearing s.c. CA20498 tumors. Plasma/blood, tumors, and tissues were obtained at various time points after administration, and everolimus levels were determined using high-performance liquid chromatography/mass spectrometry. Data are expressed as mean ± SEM, n = 4. Horizontal lines represent the in vitro IC₅₀ values shown in panels A and B, and the IC₅₀ values for endothelial cells are from Lane et al. [9].

Figure 4

Efficacy and tolerability of everolimus in mice bearing s.c. human tumor xenografts. (A and B) When tumors reached 100 mm3, everolimus was administered daily at 0.1, 0.5, or 2.5 mg/kg p.o. to mice bearing NCI-H596 human lung tumor xenografts. Alternatively, mice received 5 mg/kg cisplatin intravenously once perweek. Results show the mean ± SEM (n = 8) for tumor volume(A) and bodyweight (B). Only the 2.5-mg/kg per day regimen of everolimus produced a statistically significant reduction in tumor volume (Dunnett's test vs vehicle controls). (C and D) When HCT-116 tumors reached 100 mm³, everolimus was administered daily at 2.5 or 5 mg/kg from day 7 to day 25 (C) or (D) until day 35 in comparison to only day 7 to day 25. Alternatively, mice received 75 mg/kg 5-FU once per week. Results show the mean ± SEM (n = 7) for tumor volume, where *P < .05 versus controls by Dunnett's one-way ANOVA.

Figure 5

Duration (A, B, C) and recovery (D) of the effects of everolimus on S6K1 activity in CA20498 tumors, rat skin, and PBMCs after a single dose (5 mg/kg). Lewis rats bearing CA20498 tumors were treated with a single dose of 5 mg/kg everolimus or vehicle. At the times indicated, tumor (A) and skin (B) extracts from three rats were prepared separately, and PBMC (C) extracts were prepared from pooled blood samples. p70^S6K1 activity was measured using 40S ribosomal subunits as an in vitro substrate. In each case, autoradiographs of [³²P]phosphate incorporation into S6 protein are shown. Graphs represent PhosphorImager quantification of the autoradiographs, where *P < .05 versus untreated controls (Dunnett's test); from Boulay et al. [13]. (D) S6 kinase activity was determined in circulating PMBCs from rats treated with 0.5, 1, 2, and 5 mg/kg everolimus once per day. The PK profiles were based on a single administration of everolimus at 5 mg/kg, and the everolimus concentrations obtained with doses were estimated assuming a linear PK profile. Inspection of the data suggests that 0.03 ng/ml blood seems to be a minimal value below which recovery of S6K activity begins. Back extrapolation of the S6 phosphorylation curves suggests that, by the first measured time point, the recovery process of S6K has already begun with the 0.5mg/kg dose, with the estimated rate of recovery being 0.425, and the estimated blood everolimus (RAD001) level was approximately 0.05 ng/ml. However, considering the other dose groups, the recovery of S6K activity seems to occur when the drug level is approximately 0.03 ng/ml. The recovery after 1, 2, or 5 mg/kg everolimus began at 24, 72, and approximately 125 hours after everolimus administration, respectively. The S6K recovery rate was 0.408, 0.284, and 0.244 for 1, 2, and 5 mg/kg everolimus. The time where blood levels drop below 0.03 ng/kg everolimus was correlated to the S6K recovery rate (setting the initiation of recovery at 12 hours for the 0.5-mg/kg dose) (r = -0.968, P = .0316). The total amount of S6K inhibition (AUC) was correlated to the estimated everolimus AUC at each dose (r = 0.96, P = .038) and the everolimus AUC was also correlated to the rate of S6K recovery (r = 0.95, P = .046).

Figure 6

Data in panels (A) and (B) are from the patient group dosed with 5 mg of everolimus as described by O'Donnell et al. [28]. (A) Everolimus blood PK (open symbols) with the PD readout of S6K activity (closed symbols) in circulating PBMCs from treated patients. (B) Time-dependent PBMC S6K levels in four individual patients. (C) Recovery of S6K acitivity in PBMC from patients receiving various doses (5–30 mg) of everolimus [28].

Figure 7

Role of PTEN in sensitivity of tumor cell lines in vitro to everolimus. Results show the individual IC₅₀ values for different human tumor cell lines defined as glioblastoma multiforme (GBM) or not, according to presence of PTEN mutation (P values from a 2-tailed t test).

Figure 8

Recursive partitioning of the sensitivity of the 21 tumor cell lines to everolimus activity in vitro. H, high level expression; L, low level expression.

Figure 9

Segregation of 21 tumor cell lines according to levels (A) or categories (B, C) of p-AKT and the ratio of p235-S6/total S6. L,H, low pAKT expression and high p235 S6/total S6 expression; H,H, high pAKT expression and high p235 S6/total S6 expression; H,L, high pAKT expression and low p235 S6/total S6 expression; L,L, low pAKT expression and low p235 S6/total S6 expression. Sensitive (S): IC₅₀ < 100 nM; insensitive (IS): IC₅₀ > 100 nM.

Figure 10

ROC curve of pAKT and p235-S6/total S6 levels as predictors of sensitivity of 21 tumor cell lines to everolimus. Nominal logistic regression was performed using sensitivity to everolimus at the 100 nM (A) and 4 nM (B) cutoff limits and pAKT and p235 S6/total S6 expression categories (see legend to Figure 9) and the ROCs derived from the regression are presented.