Tumor genetic analyses of patients with metastatic renal cell carcinoma and extended benefit from mTOR inhibitor therapy

Abstract

Purpose: Rapalogs are allosteric mTOR inhibitors and approved agents for advanced kidney cancer. Reports of clonal heterogeneity in this disease challenge the concept of targeted monotherapy, yet a small subset of patients derives extended benefit. Our aim was to analyze such outliers and explore the genomic background of extreme rapalog sensitivity in the context of intratumor heterogeneity.

Experimental design: We analyzed archived tumor tissue of 5 patients with renal cell carcinoma, who previously achieved durable disease control with rapalogs (median duration, 28 months). DNA was extracted from spatially separate areas of primary tumors and metastases. Custom target capture and ultradeep sequencing was used to identify alterations across 230 target genes. Whole-exome sequence analysis was added to investigate genes beyond this original target list.

Results: Five long-term responders contributed 14 specimens to explore clonal heterogeneity. Genomic alterations with activating effect on mTOR signaling were detected in 11 of 14 specimens, offering plausible explanation for exceptional treatment response through alterations in two genes (TSC1 and MTOR). In two subjects, distinct yet functionally convergent alterations activated the mTOR pathway in spatially separate sites. In 1 patient, concurrent genomic events occurred in two separate pathway components across different tumor regions.

Conclusions: Analysis of outlier cases can facilitate identification of potential biomarkers for targeted agents, and we implicate two genes as candidates for further study in this class of drugs. The previously reported phenomenon of clonal convergence can occur within a targetable pathway which might have implications for biomarker development beyond this disease and this class of agents.

Figure 1

Genomic alterations along the core mTORC1 pathway are identified in patients with exceptional rapalog response using the IMPACT assay. (A, B) Integrated Genomics Viewer (IGV) snapshots of region 1 (R1) of the primary tumors and matched adjacent normal tissues illustrate the c.932delC (P311fs*4) and the c.1738delAT (I580fs*7) frameshift mutations of TSC1 in patients #1 (A) and #2 (B), respectively. Number of reads carrying the mutation is noted. (C) IGV snapshots of R1 and adjacent normal in patient #3 illustrate the mTOR Q2223K missense kinase domain mutation. (D) Copy number plots of patients #1 to #5 with notations on pertinent chromosomal alterations. TSC1 and mTOR reside on chromosome bands 9q34 and 1p36, respectively. (E) A diagram of the central mTORC1 signaling pathway illustrates mutations identified in the core components from rapalog (everolimus and temsirolimus) responders.

Figure 2

The Q2223K mutation of mTOR causes hyperactivation of mTORC1. (A) The mTOR Q2223K mutant induces more phosphorylation of endogenous S6K at threonine 389 (T389) than wild-type mTOR. HEK293T cells, transfected with the indicated Flag-mTOR constructs for 24 hours, were serum-deprived overnight and then exposed to 1% serum-containing medium for 1 hour. Cellular lysates were subjected to immunoblot analysis using the indicated antibodies. Levels of Flag-mTOR and β-actin indicate equivalent transfection and protein loading, respectively. n.s. denotes non-specific bands. (B) Immunoblots of S6K (T389) and S6 (Serine 235/236; S235/236) phosphorylation demonstrate the hyperactivity of Q2223K mTORC1 over a range of serum concentrations. HEK293T cells, transfected with the indicated Flag-mTOR constructs for 24 hours, were washed with serum free medium, exposed to medium containing the indicated serum concentrations for 1 hour, and analyzed by immunoblots using the indicated antibodies. (C) The hyperactivity of Q2223K can be inhibited by rapamycin. The Q2223K mTORC1 is sensitive to rapamycin as wild-type mTORC1, determined by the phosphorylation of S6K (T389). Experiments were performed as in (B), except with addition of the indicated concentrations (nM) of rapamycin in medium containing 10% serum in the final hour prior to harvest. (D, E) Cells in (D) and (E) were treated similarly to (B) and (C), respectively, except with the co-transfection of Myc-tagged S6K. (F) Structural modeling of the mTOR kinase active site, based on the solved structure of the PI3K kinase domain, illustrates the position of glutamine 2,223. Q2223, shown in blue sticks, is localized on a loop in close proximity to the ATP binding site (an ATP molecule is modeled based on its position in PI3K and is shown as colored: light blue - carbon; red - oxygen; dark blue - nitrogen; orange – phosphorus). The kinase activation and catalytic loops are colored red and green, respectively.