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. 2015 Jan;21(1):71-5.
doi: 10.1038/nm.3751. Epub 2014 Dec 8.

Mutations in G protein β subunits promote transformation and kinase inhibitor resistance

Akinori Yoda  1 , Guillaume Adelmant  2 , Jerome Tamburini  1 , Bjoern Chapuy  1 , Nobuaki Shindoh  3 , Yuka Yoda  1 , Oliver Weigert  4 , Nadja Kopp  1 , Shuo-Chieh Wu  1 , Sunhee S Kim  1 , Huiyun Liu  1 , Trevor Tivey  1 , Amanda L Christie  1 , Kutlu G Elpek  5 , Joseph Card  6 , Kira Gritsman  1 , Jason Gotlib  7 , Michael W Deininger  8 , Hideki Makishima  9 , Shannon J Turley  10 , Nathalie Javidi-Sharifi  11 , Jaroslaw P Maciejewski  9 , Siddhartha Jaiswal  12 , Benjamin L Ebert  13 , Scott J Rodig  14 , Jeffrey W Tyner  11 , Jarrod A Marto  2 , David M Weinstock  15 , Andrew A Lane  1
Affiliations

Mutations in G protein β subunits promote transformation and kinase inhibitor resistance

Akinori Yoda et al. Nat Med. 2015 Jan.

Abstract

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

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Figures

Figure 1
Figure 1. Recurrent GNB1 and GNB2 mutations confer cytokine-independent growth
(a) Schematic representation of functional screening using patient-derived cDNA libraries and cytokine-dependent cells. (b) IL3-independent growth of BaF3-MYC cells expressing wild-type (WT) GNB1, GNB1 K89E or empty vector. * p < 0.05 vs wild-type; ** p < 0.01 vs wild-type; †† p < 0.01 vs empty by t-test; graphs represent mean ± SD of three replicates. (c) Mutations identified in GNB1 and GNB2 in human cancers. Tumor types are indicated for recurrent mutation sites with 3 or more missense alterations. Abbreviations: AML, acute myelogenous leukemia; aCML, atypical chronic myelogenous leukemia; PV, polycythemia vera; MDS, myelodysplastic syndrome; B-ALL, B-cell acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia; FL, follicular lymphoma; DLBCL, diffuse large B-cell lymphoma; BPDCN, blastic plasmacytoid dendritic cell neoplasm. (d) Cell counts of IL3-independent BaF3-MYC cells expressing GNB1 and GNB2 alleles or empty vector 14 days after cytokine withdrawal. Data is represented as mutant relative to wild-type for GNB1 or GNB2. * p < 0.05 and ** p < 0.01 vs wild-type by t-test; graphs represent mean ± SD of three replicates. (e) GM-CSF-independent growth of TF-1 cells, as in (d).
Figure 2
Figure 2. Mutant Gβ proteins lose interaction with Gα subunits and induce activation of PI3K/AKT/mTOR and MAPK pathways
(a) Molecular representation of a heterotrimeric G protein (Gα: blue, Gβ: red, Gγ: yellow) based on a crystal structure (PDB ID 1GP2; DOI:10.2210/pdb1gp2/pdb). Corresponding sites of multiple recurrent oncogenic mutations are indicated and side chains of the residues are shown in green. (b) Silver staining of anti-Flag immunoprecipitates (IP) from BaF3-MYC cells expressing wild-type GNB1 or GNB1 K89E. The band indicated by the arrow was analyzed by mass spectrometry and the three Gα subunits indicated were identified. (c) Unsupervised clustering analysis of proteins identified in TAP-MS analyses of GNB1, based on their relative association to wild-type and mutant GNB1. Combined extracts of SILAC-labeled TF-1 cells (Heavy: TF-1-Flag-HA-GNB1 mutants, Light: TF-1-Flag-HA-GNB1 WT) were used for tandem affinity purification (TAP) with Flag and HA antibodies followed by mass spectrometry. The ratio of wild type and mutant GNB1 in each TAP was used to normalize the abundance of all interacting proteins. Normalized abundance of G protein subunits and PDCL associated with mutant compared to wild type GNB1 is shown (red = more binding to mutant, blue = less binding to mutant compared to wild-type). (d) Western blotting of anti-Flag IP and whole cell lysates (WCL) from BaF3-MYC cells expressing Gβ alleles. (e) GSEA plots of the indicated gene sets in TF-1 cells transduced with GNB1 K89E that were isolated 12 hours after GM-CSF withdrawal (K89E starved, n = 3) compared to the combined data from TF-1 cells transduced with empty vector that were isolated after GM-CSF withdrawal (n = 3) and either cell line without cytokine withdrawal (n = 3 each; total of n = 9 controls). (f) Heatmap of leading edge genes in the AKT gene set from gene set enrichment analysis in TF-1 GNB1 K89E or empty vector-transduced cells with (+) or without (−) cytokine starvation. (g) Western blotting of TF-1 cells expressing GNB1 alleles or empty vector.
Figure 3
Figure 3. GNB1 mutants promote myeloid dendritic cell neoplasms in vivo
(a) Survival of recipient mice after transplantation of 5-FU-treated Cdkn2a−/− bone marrow (BM) transduced with GNB1 alleles or empty vector (n = 20 mice/group for GNB1 K89E or WT, n = 10 mice/group for others; * p = 0.048 vs empty, *** p < 0.0001 vs empty, curves compared by log-rank test). (b) Representative flow cytometry of neoplastic cells from spleens of GNB1 K89E recipients from panel A. (c) Western blotting of K562 cells expressing GNB1 or GNB1 K89E. Cells were starved of serum two hours followed by drug treatment (BEZ235 300 nM, AZD8055 300 nM, Rapamycin 10 nM, U0126 10 μM) for two hours. (d) Survival of recipient mice after secondary transplantation of splenocytes from mice in the GNB1 K89E group in panel A. Mice transplanted with two separate malignancies were treated with BEZ235 or vehicle from day 10–31 after transplantation (n = 6 mice/group; * p < 0.05 vs vehicle, curves compared by log-rank test). (e) Western blotting of splenocytes from secondary recipients of malignancies from panel A treated with DMSO or vehicle. Beginning 10 days after transplantation, recipient mice were treated with vehicle or BEZ235 for 3 days (1023) or 16 days (1026).
Figure 4
Figure 4. GNB1/2 mutations confer resistance to kinase inhibitors
(a) Dose-response of A375 cells transduced with GNB2, GNB2 K78E or empty vector and treated with vemurafenib for 48 hours. PC9 cells, which do not harbor a BRAF mutation, were used as a negative control for vemurafenib sensitivity. Data normalized to DMSO only and to maximal response in empty vector. ** p < 0.01 for pairwise comparison between GNB mutant and wild-type at a given dose level by t-test; graphs represent mean ± SD of three replicates. (b) Western blotting of K562 cells transduced with wild-type GNB1, GNB1 K89E or empty vector. Cells were starved of serum for two hours followed by two hours of drug treatment (C: control DMSO, N: nilotinib 100 nM, Be: BEZ235 300 nM, I: imatinib 1000 nM). (c) Dose-response of K562 cells transduced with GNB1, GNB1 K89E or empty vector and treated with nilotinib for 48 hours, analyzed as in panel (a). (d) Growth of BaF3-Myc cells transduced with BCR-ABL in combination with GNB1, GNB1 K89E or empty vector and cultured in the presence of nilotinib (100 nM). * p < 0.05 and ** p < 0.01 vs wild-type; † p < 0.01 vs empty by t-test; graphs represent mean ± SD of three replicates. (e) Dose-response of SET2 cells transduced with GNB1, GNB1 K89E or empty vector and treated with ruxolitinib for 48 hours, analyzed as in panel (a). (f) Growth of SET2 cells transduced with GNB1, GNB1 K89E or empty vector and cultured in the presence of ruxolitinib (1 μM), analyzed as in panel (d). (g) Growth of BaF3-Myc cells transduced with MPL P440L in combination with GNB1, GNB1 K89E or empty vector and cultured in the presence of ruxolitinib (1 μM), analyzed as in panel (d).
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