Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma

Abstract

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemokine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense isoform was gain-of-function because it increased cell migration toward the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and conferred a growth advantage in long-term in vitro cultures. These findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy.

Figure 1.

CCR4 mutations in ATLL. (A) Amino acid residues in the carboxy-terminal region of CCR4. (B) Schematic of CCR4 mutant isoforms in ATLL. (C) DNA sequence of the CCR4-Y331* mutation in the TW14R ATLL biopsy sample by Sanger sequencing (bottom). The WT sequence of normal DNA obtained from the same patient is shown in the top.

Figure 2.

CCR4 mutant isoforms enhance chemotaxis and impair receptor internalization. (A) CCL22-mediated chemotaxis of mouse 32Dβ cells ectopically expressing CCR4-WT or CCR4-Q330*. In these Transwell assays, the lower chamber contained the indicated amount of CCL22. After a 2-h incubation, the number of cells migrating from the upper to lower chamber was determined and plotted as a percentage of the input cell number. (B) Surface CCR4 expression levels of 32Dβ cells ectopically expressing CCR4-WT or CCR4-Q330* analyzed by FACS. (C) Chemotactic ability of CCR4-WT– or CCR4-Q330*–reconstituted ED40515(+) ATLL cells toward CCL17 and CCL22. (D) Surface CCR4 expression levels in CCR4-WT– or CCR4-Q330*–reconstituted ED40515(+) ATLL cells analyzed by FACS. (E) Surface CCR4 expression levels analyzed by FACS in ED40515(+) ATLL cells ectopically expressing CCR4-WT and/or CCR4-Q330*. (B, D, and E) Isotype control IgG staining is indicated in gray. (F) CCL22-induced chemotaxis of ED40515(+) ATLL cells ectopically expressing CCR4-WT and/or CCR4-Q330*. (G) Time course of surface CCR4 levels after CCL22 exposure in CCR4-WT– or CCR4-Q330*–reconstituted ED40515(+) ATLL cells. Surface CCR4 levels were analyzed by FACS and normalized to the levels at time 0. Data in all panels are presented as mean ± SEM of technical duplicates representative of at least two biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for a comparison between CCR4-WT and CCR4-Q330*.

Figure 3.

Enhanced PI3K/AKT activation by mutated CCR4. (A) Immunoblot analysis for P-AKT levels in ED40515(+) and KOB ATLL cells after CCL22 exposure (50 ng/ml). Blots using P-AKT (S473) antibody are shown with short and long exposure times. Relative scanning densitometry estimates of P-AKT levels are depicted under the panel showing the short exposure. The pan-PI3K inhibitor BKM120 was used at 1 µM. (B and C) Time course experiment of AKT activation after CCL22 exposure (50 ng/ml) using CCR4-reconstituted ED40515(+) cells (B) and KOB cells (C), transduced as indicated with shRNA and cDNA expression vectors. P-AKT (S473) levels were analyzed by FACS at the indicated times after CCL22 exposure. MFI, mean fluorescence intensity. (D) Surface CCR4 expression levels in CCR4-WT– or CCR4-Q330*–reconstituted KOB cells. Isotype control IgG staining is indicated in gray. (E) Time course experiment of AKT activation after CCL22 exposure (50 ng/ml) in ED40515(+) ATLL cells ectopically expressing CCR4-WT and/or CCR4-Q330*. (F) P-AKT (S473) levels of ED40515(+) treated with inhibitors. Cells were pretreated with 100 ng/ml PTX for 16 h or the indicated amount of the pan-PI3K inhibitor BKM120 for 1 h and then incubated with 50 ng/ml CCL22 for 5 min. P-AKT (S473) was analyzed by FACS. (G) Chemotaxis of ED40515(+) cells treated with inhibitors. Cells were pretreated with PTX or BKM120 as in F and then used in a CCL22-mediated chemotaxis assay. Data in all panels are presented as mean ± SEM of technical duplicates representative of at least two biological replicates. *, P < 0.05; **, P < 0.01 for a comparison between CCR4-WT and CCR4-Q330*.

Figure 4.

Growth advantage of CCR4-Q330*–reconstituted ATLL cells. (A) Schematic of the competitive growth assay. ED40515(+) cells depleted of endogenous CCR4 expression by RNA interference were transduced with the indicated CCR4-expressing vectors or mock vectors. After puromycin selection, two transduced populations, one expressed GFP and the other did not, were mixed equally and co-cultured for 12 d in vitro. The ratio of the two populations was determined by FACS every 4 d. (B) CCR4-Q330*–transduced cells have a competitive growth advantage. CCL22 was added every 2 d at 50 ng/ml. The ratios of the two populations were normalized to the value at day 0. Growth curves represent the mean of eight replicates obtained from four biologically independent experiments ± SEM. *, P < 0.05; ***, P < 0.001.