Mutations in TSC2 and MTOR

Figure 2. Figure 2. Mutations in TSC2 and MTOR in Anaplastic Thyroid Carcinoma Revealed by Whole-Exome Sequencing. Whole-exome sequencing of the tumor tissue before treatment and after the development of resistance to everolimus revealed a TSC2 nonsense mutation in both tumors (Panel A) and an F2108L mutation in MTOR in the resistant tumor that was undetectable in the pretreatment tumor (Panel B). Representative genome images from the Integrative Genomics Viewer (Broad Institute), along with the number of reads for the reference allele and the variant allele, are shown for each alteration. A comparison of the proportion of cancer cells harboring specific mutations in the pretreatment biopsy sample (Panel C, x axis) versus those in the post-resistance biopsy sample (y axis) is shown. Mutations in TSC2, TP53, and FLCN were present in all cancer cells from both biopsy samples (yellow triangle at upper right), whereas the MTOR mutation was detected in all cancer cells in the post-resistance biopsy sample but was not detected in the pretreatment biopsy sample (red triangle at upper left). Additional mutations were detected in all cancer cells in the pretreatment biopsy sample but in no cancer cells in the post-resistance biopsy sample (light blue triangle at lower right). Other mutations were predicted to occur in a subgroup of the cancer cells in the pretreatment biopsy but in none of the cancer cells in the post-resistance biopsy sample (dark blue) or in a subgroup of the cancer cells in the post-resistance biopsy sample but in none of the cancer cells in the pretreatment biopsy sample (dark red). Shaded areas denote Bayesian posterior probability distributions over cancer-cell fraction values for each mutation. Gray shading indicates mutations with cancer-cell fraction distributions having considerable uncertainty, with lighter shading indicating greater uncertainty.

The pretreatment tumor contained a somatic nonsense mutation in the tumor-suppressor gene TSC2 that inactivates the gene, allowing for activation of the mTOR pathway7,8 and resulting in sensitivity to mTOR inhibition in some cancers1,2,4,9 (Figure 2A). This truncating mutation (Q1178*) is known to inactivate TSC2 by eliminating the guanosine triphosphatase–activating protein domain near the C-terminal, a domain essential for inhibiting mTOR complex 1.8,10 Somatic mutations in TSC2 have been identified in many tumor types, including cancers of the kidney and bladder as well as hamartomas and malignant perivascular epithelioid-cell tumors in patients with tuberous sclerosis complex. TSC2 mutations have not previously been reported in any type of thyroid cancer. TSC2 Q1178* has been identified twice in the germline of patients with tuberous sclerosis complex,11 but somatic mutations at this locus have not been reported.

In addition to the TSC2 mutation, 317 somatic coding single-nucleotide variants and 44 coding indels were identified (Table S1 in Supplementary Appendix 2). These included a common mutation in TP53 (C135Y) and an N-terminal frame shift in FLCN (R17fs), a tumor-suppressor gene that encodes folliculin. Germline mutations in folliculin result in the Birt–Hogg–Dubé syndrome, an autosomal dominant disorder characterized by fibrofolliculomas, renal and pulmonary cysts, and renal cancer. Folliculin is involved in TSC2 and mTOR signaling12-15; in some studies, inactivation of FLCN has been shown to result in increased mTOR activity,14 raising the possibility that this mutation, along with the TSC2 mutation, may contribute to sensitivity to everolimus.

Genomewide copy-number analysis showed near-haploidization of the cancer genome with retention of chromosome 7, a feature that appears to be pathognomonic of oncocytic follicular thyroid carcinoma,16 This finding is consistent with the putative origin of this anaplastic thyroid cancer (Fig. S2 in Supplementary Appendix 1).

The TSC2 nonsense mutation persisted in the resistant tumor (Figure 2A). Immunohistochemical analysis of tissue from the resistant tumor revealed the absence of TSC2 and the presence of pS6, a downstream target of mTOR. These findings are indicative of persistent mTOR-pathway activity (Fig. S3 in Supplementary Appendix 1). Immunohistochemical analysis of the pretreatment tissue could not be completed for technical reasons. Mutations in TP53 and FLCN also persisted in the resistant tumor.

The resistant tumor also had a somatic mutation in MTOR (MTOR F2108L). This mutation was not detected in the pretreatment tumor despite robust sequence coverage of this locus (189×) (Figure 2B). To identify the relative change in the frequency of each genomic alteration from the pretreatment tumor to the resistant tumor, the fraction of tumor cells harboring a given alteration in each pair of samples was estimated (Figure 2C, and Table S1 in Supplementary Appendix 2). Although the TSC2, TP53, and FLCN alterations were estimated to be present in 98 to 100% of the cancer cells in both the pretreatment and resistant tumors, the estimated proportion of cancer cells with MTOR F2108L was 0% in the pretreatment tumor as compared with 96% in the resistant tumor (Figure 2C). To our knowledge, MTOR mutations have not previously been identified in patients with acquired resistance to everolimus, and this particular mutation has not been described in patients.17

Resistance to Allosteric mTOR Inhibitors Resulting from mTORF2108l

Although MTOR F2108L has not been described previously, the homologous mutation was characterized nearly 20 years ago in fission yeast.18 In a mutagenesis screen to identify mutations in tor2 (the fission yeast homologue of MTOR) that conferred resistance to rapamycin, tor2 F2049L was one of five rapamycin-resistant mutants identified. All five tor2 residues identified have been conserved in TOR proteins across species, including mTOR.18 In a yeast two-hybrid assay, tor2F2049L did not bind FKBP (FK506 binding protein)–rapamycin, suggesting a mechanism for rapamycin resistance in fission yeast.18

Figure 3. Figure 3. Effects of Nonmutant mTOR and mTORF2108L. Ribbon rendition of mTOR crystal structure (PDB 1FAP) for nonmutant mTOR (Panel A) and mTORF2108L (Panel B) shows the FKBP (FK506 binding protein)–rapamycin binding (FRB) domain, FKBP12, and rapamycin. Enlarged views show that the substitution of leucine for phenylalanine sterically hinders rapamycin binding (Panels A and B, right side). Ribbon rendition of mTOR crystal structure (PDB 4JSX) (Panel C) highlights the kinase domain (KD) C-terminal lobe, KD N-terminal lobe, FRB domain, and FAT domain. As shown, residue F2108 is not expected to be involved with binding of torin 2. Growth-inhibition curves are shown for rapamycin (Panel D) and torin 1 (Panel E) in human embryonic kidney (HEK) 293T cells stably expressing nonmutant mTOR or MTORF2108L. Constructs expressing mTORF2108L or nonmutant mTOR were expressed in HEK 293T cells (Panel F). The levels of phosphorylated and total S6 kinase 1 (S6K1), the downstream target of mTOR, are shown for the HEK 293T cells after treatment with 0 nM, 0.5 nM, 2.5 nM, or 12.5 nM of rapamycin or torin 1, as indicated. GAPDH denotes glyceraldehyde 3-phosphate dehydrogenase.

On the basis of these findings, we hypothesized that mTORF2108L causes resistance to allosteric mTOR inhibition by preventing the binding of the drug to the protein. Indeed, structural studies confirm that mTORF2108L occurs in the FKBP–rapamycin binding (FRB) domain, the region of the protein that is required to bind to rapamycin and its analogues. As shown in Figure 3A and 3B, the substitution of a leucine for a phenylalanine is predicted to prevent binding of the drug to the protein by means of steric hindrance.

To confirm that mTORF2108L confers resistance to allosteric mTOR inhibition, the F2108L mutation was introduced into nonmutant mTOR, and the mutant cDNA was stably expressed in human embryonic kidney (HEK) 293T cells. Cells expressing mTORF2108L were significantly more resistant to inhibition with rapamycin than were cells expressing nonmutant mTOR (Figure 3D). We next examined the effect of the mutant on the phosphorylation of endogenous S6 kinase 1 (S6K1), a downstream target of mTOR. At baseline, cells expressing mTORF2108L and those expressing nonmutant mTOR had similar levels of phosphorylated S6K1. Treatment with rapamycin, however, completely inhibited phosphorylation of S6K1 in cells expressing nonmutant mTOR but had virtually no effect in cells expressing mTORF2108L (Figure 3F).

Sensitivity of mTORF2108l to Direct TOR Kinase Inhibition

Because mTORF2108L occurs in the FRB domain rather than the active site, we hypothesized that this mutant protein should remain sensitive to inhibition by direct ATP-competitive TOR kinase inhibitors. As shown in the structural model in Figure 3C, there is no predicted effect of the F2108L mutation on binding of torin 1, a direct TOR inhibitor. In contrast to cells treated with rapamycin, cells expressing mTORF2108L and those expressing nonmutant mTOR were equally sensitive to treatment with torin 1 (Figure 3E). Similarly, inhibition of S6K1 phosphorylation by torin 1 was equivalent in cells expressing mTORF2108L and those expressing nonmutant mTOR (Figure 3F). Taken together, these results indicate that mTORF2108L remains sensitive to direct kinase inhibition.