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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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Mutated RIT1 induces cellular transformation via activation of MEK and PI3K. (a) Soft agar transformation assay in NIH3T3 cells. Cells were transduced with retrovirus to ectopically express wild-type (WT) or mutated RIT1 constructs or empty vector (control), then plated in soft agar (Methods). Colonies were visualized at 14 days and quantified using CellProfiler. Top panel, data shown are mean+s.e.m. of triplicate wells. Data shown are representative of at least three independent experiments. *P<0.05 by two-tailed t-test. Bottom panels, western blot showing expression of RIT1 or vinculin (loading control). ‘INS', T76_insTLDT. (b) Tumor growth of xenografts of NIH3T3 cells with or without expression of RIT1. Data shown is mean+s.e.m. of nine replicates per construct. *P<0.01 by two-tailed t-test. Data shown are representative of at least two independent experiments per construct. (c) Western blot of PC6 cell lysates following transfection of wild-type or mutant RIT1 or vector control (‘Emp'). Data shown is representative of at least three independent experiments. (d) Western blot of PC6 lysates following transfection of FLAG-RIT1 constructs or vector control (‘Emp') in the presence or absence of 10 μM PD98059. Cells were serum starved for 5 h prior to lysis. (e) Western blot of PC6 cell lysates generated after transfection of FLAG-RIT1 mutant constructs in the presence or absence of 10 μM LY294002. Cells were serum starved for 5 h prior to lysis. (f) Soft agar colony formation of NIH3T3 cells stably expressing RIT1 M90I or RIT1 Q79L in the presence or absence of 1 μM erlotinib, GDC-0941, AZD-6244 or GDC-0941/AZD-6244 or vehicle control (dimethylsulfoxide). 5 × 103 cells were suspended in a top agar solution together with each respective drug to a final concentration of 1 μM in triplicate. After 15 days, colonies were photographed and quantified using CellProfiler. *P<0.05.
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fig2: Mutated RIT1 induces cellular transformation via activation of MEK and PI3K. (a) Soft agar transformation assay in NIH3T3 cells. Cells were transduced with retrovirus to ectopically express wild-type (WT) or mutated RIT1 constructs or empty vector (control), then plated in soft agar (Methods). Colonies were visualized at 14 days and quantified using CellProfiler. Top panel, data shown are mean+s.e.m. of triplicate wells. Data shown are representative of at least three independent experiments. *P<0.05 by two-tailed t-test. Bottom panels, western blot showing expression of RIT1 or vinculin (loading control). ‘INS', T76_insTLDT. (b) Tumor growth of xenografts of NIH3T3 cells with or without expression of RIT1. Data shown is mean+s.e.m. of nine replicates per construct. *P<0.01 by two-tailed t-test. Data shown are representative of at least two independent experiments per construct. (c) Western blot of PC6 cell lysates following transfection of wild-type or mutant RIT1 or vector control (‘Emp'). Data shown is representative of at least three independent experiments. (d) Western blot of PC6 lysates following transfection of FLAG-RIT1 constructs or vector control (‘Emp') in the presence or absence of 10 μM PD98059. Cells were serum starved for 5 h prior to lysis. (e) Western blot of PC6 cell lysates generated after transfection of FLAG-RIT1 mutant constructs in the presence or absence of 10 μM LY294002. Cells were serum starved for 5 h prior to lysis. (f) Soft agar colony formation of NIH3T3 cells stably expressing RIT1 M90I or RIT1 Q79L in the presence or absence of 1 μM erlotinib, GDC-0941, AZD-6244 or GDC-0941/AZD-6244 or vehicle control (dimethylsulfoxide). 5 × 103 cells were suspended in a top agar solution together with each respective drug to a final concentration of 1 μM in triplicate. After 15 days, colonies were photographed and quantified using CellProfiler. *P<0.05.

Mentions: To test whether mutated RIT1 is capable of inducing cellular transformation, we expressed wild-type or mutated RIT1 cDNA constructs in NIH3T3 cells and assayed the ability of these cells to form colonies in soft agar. A constitutively active form of RIT1, RIT1 Q79L,24 was used as a positive control. With the exception of RIT1 Q40L, all other RIT1 mutations robustly induced colony formation of NIH3T3 cells in soft agar (Figure 2a). To confirm that these cells were indeed transformed, we injected RIT1-transduced cells subcutaneously into nude mice and assessed tumor-forming capability. Six of six switch II domain mutations induced significant tumor formation by 3 weeks post-injection, comparable to transformation induced by KRAS G12V or EGFR L858R (Figure 2b and Supplementary Figure 3), whereas transformation capability of non-switch II domain mutations varied. RIT1 Q40L, although consistently less active than other RIT1 variants, showed intermediate transforming capability in the xenograft assay (Supplementary Figure 3). It remains possible that RIT1 Q40L is weakly activating, and should be noted that RIT1 Q40 is homologous to KRAS Q22, which is found mutated in Noonan syndrome25 and rarely in somatic cancers.


Oncogenic RIT1 mutations in lung adenocarcinoma.

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

Mutated RIT1 induces cellular transformation via activation of MEK and PI3K. (a) Soft agar transformation assay in NIH3T3 cells. Cells were transduced with retrovirus to ectopically express wild-type (WT) or mutated RIT1 constructs or empty vector (control), then plated in soft agar (Methods). Colonies were visualized at 14 days and quantified using CellProfiler. Top panel, data shown are mean+s.e.m. of triplicate wells. Data shown are representative of at least three independent experiments. *P<0.05 by two-tailed t-test. Bottom panels, western blot showing expression of RIT1 or vinculin (loading control). ‘INS', T76_insTLDT. (b) Tumor growth of xenografts of NIH3T3 cells with or without expression of RIT1. Data shown is mean+s.e.m. of nine replicates per construct. *P<0.01 by two-tailed t-test. Data shown are representative of at least two independent experiments per construct. (c) Western blot of PC6 cell lysates following transfection of wild-type or mutant RIT1 or vector control (‘Emp'). Data shown is representative of at least three independent experiments. (d) Western blot of PC6 lysates following transfection of FLAG-RIT1 constructs or vector control (‘Emp') in the presence or absence of 10 μM PD98059. Cells were serum starved for 5 h prior to lysis. (e) Western blot of PC6 cell lysates generated after transfection of FLAG-RIT1 mutant constructs in the presence or absence of 10 μM LY294002. Cells were serum starved for 5 h prior to lysis. (f) Soft agar colony formation of NIH3T3 cells stably expressing RIT1 M90I or RIT1 Q79L in the presence or absence of 1 μM erlotinib, GDC-0941, AZD-6244 or GDC-0941/AZD-6244 or vehicle control (dimethylsulfoxide). 5 × 103 cells were suspended in a top agar solution together with each respective drug to a final concentration of 1 μM in triplicate. After 15 days, colonies were photographed and quantified using CellProfiler. *P<0.05.
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fig2: Mutated RIT1 induces cellular transformation via activation of MEK and PI3K. (a) Soft agar transformation assay in NIH3T3 cells. Cells were transduced with retrovirus to ectopically express wild-type (WT) or mutated RIT1 constructs or empty vector (control), then plated in soft agar (Methods). Colonies were visualized at 14 days and quantified using CellProfiler. Top panel, data shown are mean+s.e.m. of triplicate wells. Data shown are representative of at least three independent experiments. *P<0.05 by two-tailed t-test. Bottom panels, western blot showing expression of RIT1 or vinculin (loading control). ‘INS', T76_insTLDT. (b) Tumor growth of xenografts of NIH3T3 cells with or without expression of RIT1. Data shown is mean+s.e.m. of nine replicates per construct. *P<0.01 by two-tailed t-test. Data shown are representative of at least two independent experiments per construct. (c) Western blot of PC6 cell lysates following transfection of wild-type or mutant RIT1 or vector control (‘Emp'). Data shown is representative of at least three independent experiments. (d) Western blot of PC6 lysates following transfection of FLAG-RIT1 constructs or vector control (‘Emp') in the presence or absence of 10 μM PD98059. Cells were serum starved for 5 h prior to lysis. (e) Western blot of PC6 cell lysates generated after transfection of FLAG-RIT1 mutant constructs in the presence or absence of 10 μM LY294002. Cells were serum starved for 5 h prior to lysis. (f) Soft agar colony formation of NIH3T3 cells stably expressing RIT1 M90I or RIT1 Q79L in the presence or absence of 1 μM erlotinib, GDC-0941, AZD-6244 or GDC-0941/AZD-6244 or vehicle control (dimethylsulfoxide). 5 × 103 cells were suspended in a top agar solution together with each respective drug to a final concentration of 1 μM in triplicate. After 15 days, colonies were photographed and quantified using CellProfiler. *P<0.05.
Mentions: To test whether mutated RIT1 is capable of inducing cellular transformation, we expressed wild-type or mutated RIT1 cDNA constructs in NIH3T3 cells and assayed the ability of these cells to form colonies in soft agar. A constitutively active form of RIT1, RIT1 Q79L,24 was used as a positive control. With the exception of RIT1 Q40L, all other RIT1 mutations robustly induced colony formation of NIH3T3 cells in soft agar (Figure 2a). To confirm that these cells were indeed transformed, we injected RIT1-transduced cells subcutaneously into nude mice and assessed tumor-forming capability. Six of six switch II domain mutations induced significant tumor formation by 3 weeks post-injection, comparable to transformation induced by KRAS G12V or EGFR L858R (Figure 2b and Supplementary Figure 3), whereas transformation capability of non-switch II domain mutations varied. RIT1 Q40L, although consistently less active than other RIT1 variants, showed intermediate transforming capability in the xenograft assay (Supplementary Figure 3). It remains possible that RIT1 Q40L is weakly activating, and should be noted that RIT1 Q40 is homologous to KRAS Q22, which is found mutated in Noonan syndrome25 and rarely in somatic cancers.

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