Abstract 3936: Frequent gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATLL)
Authors: Nakagawa Masao , Schmitz Roland , Xiao Wenming , Goldman Carolyn K. , Xu Weihong , Yang Yandan , Yu Xin , Waldmann Thomas A. , Staudt Louis M. 
Published: August 2015

Abstract
High expression of CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATLL, an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking. CCR4 ligands, CCL17 and CCL22, were produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATLL cases express surface CCR4 (90%) and infiltrate to lymph nodes and skin. These observations suggest that CCR4 could have a role in ATLL biology, but it is still unclear whether dysregulation of CCR4 function contributes to ATLL pathogenesis.
We performed RNA-Seq for two primary ATLL cases and discovered recurrent non-sense mutations in CCR4. Though an extended analysis using Sanger sequencing, 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. In 5 cases for which paired normal DNA was available, three different CCR4 mutations were detected only in the ATLL cells (Q330*; Q330 frameshift; Y331*), demonstrating that they were acquired somatically during malignant transformation or progression. Chemotaxic assay using 32Dβ, a mouse myeloid cell line, and ED40515(+), an ATLL cell line, clarified that the ectopic expression of CCR4-Q330* enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22 rather than CCR4-WT transduced cells. To understand the mechanism of this enhanced chemotactic ability, we studied the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515(+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATLL CCR4 mutants impair desensitization by ligand, which likely contributes to the enhanced chemotaxis of cells bearing these mutants.
We explored the influence of the ATLL CCR4 mutants on PI3K/AKT signaling by immunoblot analysis and phosphor-flow analysis. CCR4-Q330*-reconstituted ED40515(+) showed strong activation of AKT with CCL22 ligation compared with CCR4-WT-reconstituted cell. Furthermore, we tested whether the acquisition of CCR4 mutations by ATLL cells imparts a selective growth advantage relative to cells with wild type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting at least in part the hypothesis that CCR4 mutation are able to provide the affected cells a positive selection pressure through CCL22 ligation and contributes to ATLL pathogenesis.
Our 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.
Citation Format: Masao Nakagawa, Roland Schmitz, Wenming Xiao, Carolyn K. Goldman, Weihong Xu, Yandan Yang, Xin Yu, Thomas A. Waldmann, Louis M. Staudt. Frequent gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATLL). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3936. doi:10.1158/1538-7445.AM2015-3936