RICTOR Amplification Defines a Novel Subset of Patients with Lung Cancer Who May Benefit from Treatment with mTORC1/2 Inhibitors

Abstract

We identified amplification of RICTOR, a key component of the mTOR complex 2 (mTORC2), as the sole actionable genomic alteration in an 18-year-old never-smoker with lung adenocarcinoma. Amplification of RICTOR occurs in 13% of lung cancers (1,016 cases) in The Cancer Genome Atlas and at a similar frequency in an independent cohort of 1,070 patients identified by genomic profiling. In the latter series, 11% of cases harbored RICTOR amplification as the only relevant genomic alteration. Its oncogenic roles were suggested by decreased lung cancer cell growth both in vitro and in vivo with RICTOR ablation, and the transforming capacity of RICTOR in a Ba/F3-cell system. The mTORC1/2 inhibitors were significantly more active against RICTOR-amplified lung cancer cells as compared with other agents targeting the PI3K-AKT-mTOR pathway. Moreover, an association between RICTOR amplification and sensitivities to mTORC1/2 inhibitors was observed. The index patient has been treated with mTORC1/2 inhibitors that led to tumor stabilization for more than 18 months.

Significance: RICTOR amplification may define a novel and unique molecular subset of patients with lung cancer who may benefit from treatment with mTORC1/2 inhibitors.

Figure 1

A. Time-line of diagnosis, treatment and responses of the index patient with RICTOR amplification. Insert western blot: CC223 therapy was associated with modest inhibition of p-AKT(S473) and p-4E-BP1 in patient's blood cells (PBMC). Dx: diagnosis. Dz: disease. Carbo: carboplatin, pem: pemetrexed,. PR: partial response. POD: progression of disease. NGS: next generation sequencing. ImmunoRx: immunotherapy. B. The tile plot showing the gene alterations in each individual with RICTOR-amplified tumors, focusing on the commonly assessed genomic alterations in lung cancer (RTK/RAS/RAF) and PI3K/AKT/mTOR alterations. For the full list, please refer to supplementary figure 3A and 3B. Note: The case of the index patient was sequenced in 2012 using FoundationOne 182 gene panel, and all 85 RICTOR amplified cases presented in figure 1B used FoundationOne 236 gene panel. Thus, the index patient is not a part of the 85 amplified series.

Figure 2

The effects of genetic knockdown of RICTOR and pharmacological inhibition on RICTOR-amplified lung cancer cells. A: FISH in lung cancer cell lines: Both H23 and H1703 cells have a ratio of RICTOR/control: 2, thus RICTOR amplified. Western blotting and colony formation assays: The effects of transient siRNA-mediated RICTOR knockdown on RICTOR-amplified H23 cells and H1703 cells. P<0.05. B: Western blot: The effects of inducible shRNA-mediated RICTOR knockdown on RICTOR-amplified H23 cells and H1703 cells. The addition of doxycycline led to RICTOR knockdown in cells. C: Colony formation assay: The addition of doxycycline led to RICTOR knockdown and was associated with decreased colony formation. D. xenograft mouse models: The effects of inducible shRNA-mediated RICTOR knockdown on H23 cells and H1703 xenografts. P<0.05. E. The effects of PI3K/AKT/mTOR inhibitors on RICTOR-amplified H23 cells. Among all the examined agents targeting the PI3K/AKT/mTOR pathways, BEZ235 (dual PI3K/mTOR inhibitor) and AZD2014 (dual mTOR1/2 inhibitor) were the most potent and showed similar inhibition of p-mTOR, p-AKT(S473) and p-S6.

Figure 3

A: FISH in lung cancer cell lines revealed that neither A549 nor HCC827 cells were RICTOR amplified. B. Western blotting and clonogenic assays: The effects of siRNA-mediated RICTOR knockdown on RICTOR expressing-but-not-amplified A549 cells and HCC827 cells. P<0.05. C. The in vivo effects of RICTOR knockdown in RICTOR-expressing but-not-amplified A549 and HCC827 xenograft mouse models. D. Western blot: RICTOR expression in parental Ba/F3 cells and inducible Ba/F3-RICTOR cells. Ba/F3 RICTOR were inducible Ba/F3 cells with RICTOR overexpression in the presence of doxycycline. IL-3 was withdrawn for 2 days for the parental Ba/F3 cells and was not added for 1 month for the rest. Of note, although the inducible Ba/F3-RICTOR system had some “leakage” of RICTOR expression in the absence of doxycycline, the addition of doxycycline caused higher IL-3 independent RICTOR expression and cell survival. E. Relative viable cells after withdrawal of IL-3. F. Modulation of IC₅₀ of two structurally different mTOR1/2 inhibitors, MLN0128 and AZD2014 when RICTOR was upregulated by the addition of doxycycline in Ba/F3-RICTOR cells. *=P<0.05 when comparing IC₅₀ of Ba/F3-RICTOR in the absence of doxycycline vs. IC₅₀ in the presence of doxycycline. It appears that higher levels of RICTOR rendered Ba/F3-RICTOR cells more sensitive to mTOR1/2 inhibitors.