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Oncogenic RIT1 mutations in lung adenocarcinoma.

Berger AH, Imielinski M, Duke F, Wala J, Kaplan N, Shi GX, Andres DA, Meyerson M - Oncogene (2014)

Bottom Line: However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases.Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition.These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Program, The Broad Institute of Harvard and M.I.T., 7 Cambridge Center, Cambridge, MA, USA [2] Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

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Endogenous mutated RIT1 regulates MEK and PI3K in NCI-H2110 cells. (a) Sanger sequencing of RIT1 RT–PCR products generated from NCI-H1993 or NCI-H2110 cell line cDNA. Numbering in black bold refers to amino-acid positions and colored letters refer to nucleotide sequence. An arrow indicates the position of a heterozygous p.M90I mutation. (b) Western blot of lysates from NCI-H1299 and NCI-H2110 following expression of shRNA hairpins targeting RIT1 (shRIT1-1, -2 and -3) or non-targeting hairpin control (shlacZ). (c) Tumor volume of NCI-H2110 xenografts in nude mice. 2 × 106 cells were injected subcutaneously into the flanks of nude mice. When tumors reached ∼100 mm3, drug treatment was initiated (day 0). Mice were treated daily with 150 mg/kg GDC-0941 or vehicle control by oral gavage. *P<0.05. n=9 tumors per condition. (d) Weight of tumors from NCI-H2110 xenografts shown in b. At day 18, animals were euthanized and tumors excised and weighed.
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fig3: Endogenous mutated RIT1 regulates MEK and PI3K in NCI-H2110 cells. (a) Sanger sequencing of RIT1 RT–PCR products generated from NCI-H1993 or NCI-H2110 cell line cDNA. Numbering in black bold refers to amino-acid positions and colored letters refer to nucleotide sequence. An arrow indicates the position of a heterozygous p.M90I mutation. (b) Western blot of lysates from NCI-H1299 and NCI-H2110 following expression of shRNA hairpins targeting RIT1 (shRIT1-1, -2 and -3) or non-targeting hairpin control (shlacZ). (c) Tumor volume of NCI-H2110 xenografts in nude mice. 2 × 106 cells were injected subcutaneously into the flanks of nude mice. When tumors reached ∼100 mm3, drug treatment was initiated (day 0). Mice were treated daily with 150 mg/kg GDC-0941 or vehicle control by oral gavage. *P<0.05. n=9 tumors per condition. (d) Weight of tumors from NCI-H2110 xenografts shown in b. At day 18, animals were euthanized and tumors excised and weighed.

Mentions: Given the prevalence of RIT1 mutation in primary human lung adenocarcinomas, we hypothesized that human lung adenocarcinoma cell lines may also harbor mutations in RIT1 and that identification of these cell lines would facilitate study of RIT1 function in human cancer pathogenesis. On the basis of the pattern of RIT1 mutation mutual-exclusivity with driver mutations in primary samples, we focused our efforts on ‘oncogene-negative' human non-squamous non-small cell lung cancer cell lines. We curated the mutational status of all known lung adenocarcinoma driver genes from non-squamous non-small cell lung cancer lines in the Cancer Cell Line Encyclopedia.27 Among 35 ‘oncogene-negative' cell lines, NCI-H2110 showed the highest level of RIT1 mRNA expression (Supplementary Figure 4 and Supplementary Table 4). Next, we sequenced the switch II domain of RIT1 in cDNA from 19 ‘oncogene-negative' cell lines and 3 ‘oncogene-positive' cell lines (Supplementary Table 5). In all, 1/19 ‘oncogene-negative' and 0/3 ‘oncogene-positive cell lines' had a non-synonymous change in RIT1; NCI-H2110 cells harbored the p.M90I mutation also seen in human primary lung adenocarcinomas (Figure 3a).


Oncogenic RIT1 mutations in lung adenocarcinoma.

Berger AH, Imielinski M, Duke F, Wala J, Kaplan N, Shi GX, Andres DA, Meyerson M - Oncogene (2014)

Endogenous mutated RIT1 regulates MEK and PI3K in NCI-H2110 cells. (a) Sanger sequencing of RIT1 RT–PCR products generated from NCI-H1993 or NCI-H2110 cell line cDNA. Numbering in black bold refers to amino-acid positions and colored letters refer to nucleotide sequence. An arrow indicates the position of a heterozygous p.M90I mutation. (b) Western blot of lysates from NCI-H1299 and NCI-H2110 following expression of shRNA hairpins targeting RIT1 (shRIT1-1, -2 and -3) or non-targeting hairpin control (shlacZ). (c) Tumor volume of NCI-H2110 xenografts in nude mice. 2 × 106 cells were injected subcutaneously into the flanks of nude mice. When tumors reached ∼100 mm3, drug treatment was initiated (day 0). Mice were treated daily with 150 mg/kg GDC-0941 or vehicle control by oral gavage. *P<0.05. n=9 tumors per condition. (d) Weight of tumors from NCI-H2110 xenografts shown in b. At day 18, animals were euthanized and tumors excised and weighed.
© Copyright Policy - open-access
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fig3: Endogenous mutated RIT1 regulates MEK and PI3K in NCI-H2110 cells. (a) Sanger sequencing of RIT1 RT–PCR products generated from NCI-H1993 or NCI-H2110 cell line cDNA. Numbering in black bold refers to amino-acid positions and colored letters refer to nucleotide sequence. An arrow indicates the position of a heterozygous p.M90I mutation. (b) Western blot of lysates from NCI-H1299 and NCI-H2110 following expression of shRNA hairpins targeting RIT1 (shRIT1-1, -2 and -3) or non-targeting hairpin control (shlacZ). (c) Tumor volume of NCI-H2110 xenografts in nude mice. 2 × 106 cells were injected subcutaneously into the flanks of nude mice. When tumors reached ∼100 mm3, drug treatment was initiated (day 0). Mice were treated daily with 150 mg/kg GDC-0941 or vehicle control by oral gavage. *P<0.05. n=9 tumors per condition. (d) Weight of tumors from NCI-H2110 xenografts shown in b. At day 18, animals were euthanized and tumors excised and weighed.
Mentions: Given the prevalence of RIT1 mutation in primary human lung adenocarcinomas, we hypothesized that human lung adenocarcinoma cell lines may also harbor mutations in RIT1 and that identification of these cell lines would facilitate study of RIT1 function in human cancer pathogenesis. On the basis of the pattern of RIT1 mutation mutual-exclusivity with driver mutations in primary samples, we focused our efforts on ‘oncogene-negative' human non-squamous non-small cell lung cancer cell lines. We curated the mutational status of all known lung adenocarcinoma driver genes from non-squamous non-small cell lung cancer lines in the Cancer Cell Line Encyclopedia.27 Among 35 ‘oncogene-negative' cell lines, NCI-H2110 showed the highest level of RIT1 mRNA expression (Supplementary Figure 4 and Supplementary Table 4). Next, we sequenced the switch II domain of RIT1 in cDNA from 19 ‘oncogene-negative' cell lines and 3 ‘oncogene-positive' cell lines (Supplementary Table 5). In all, 1/19 ‘oncogene-negative' and 0/3 ‘oncogene-positive cell lines' had a non-synonymous change in RIT1; NCI-H2110 cells harbored the p.M90I mutation also seen in human primary lung adenocarcinomas (Figure 3a).

Bottom Line: However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases.Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition.These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

View Article: PubMed Central - PubMed

Affiliation: 1] Cancer Program, The Broad Institute of Harvard and M.I.T., 7 Cambridge Center, Cambridge, MA, USA [2] Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

Show MeSH
Related in: MedlinePlus