A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an intractable T cell malignancy with regulatory T cell phenotype, which caused by long-term infection of human T-cell leukemia virus type-1 (HTLV-1). In addition to HTLV-1-derived oncogenic proteins such as Tax and HBZ, genomic aberrations including gain-of-function alterations in genes related to T-cell receptor (TCR) signaling pathway have been implicated in the pathogenesis of ATL. RLTPR is essential for CD28 co-stimulation in human T cells and loss-of-function mutations in RLTPR is reported to reduce proportions of regulatory T cells. The RLTPR p.Q575E has been reported as a recurrent mutation in cutaneous T cell lymphoma¹. Here, we show that RLTPR p.Q575E is a novel recurrent gain-of-function mutation related to T-cell receptor co-stimulatory pathway in patients with acute type ATL.

Methods

To elucidate the genomic basis of ATL, we performed whole exome sequence (WES) in peripheral blood mononuclear cells (CD4/CD25 positive cells were sorted if ATL cell were not abundant) from 47 patients with acute-type ATL. Sequences were aligned against the reference genome (GRCh37/hg19) by using TMAP Alignment. Sanger sequencing was performed to confirm the variant obtained after WES genotyping for some genes including RLTPR. Clinical information regarding ATL cell phenotype, treatment and clinical course were also collected. To analyze the function of RLTPR Q575E, the human RLTPR isoform 3 wild type (WT) (most abundant isotype in lymphoid cells, RLTPR-WT hereafter) and RLTPR isoform 3 Q575E (RLTPR Q575E hereafter) expression retroviral vector were constructed. RLTPR isoform 1 WT and RLTPR isoform 1 Q575E were used as control. RLPTR WT or RLTPR Q575E transduced Jurkat cells were generated by retroviral transduction. NFAT, NF- κB or AP-1 promoter activity was measured as luciferase activity. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin calcium salt for TCR stimulation and CD86 Fc chimera for CD28 stimulation. IL-2 mRNA was quantitated by using Taqman Gene Expression Assays.

Results

Exome analysis from 47 acute-type ATL samples revealed gene alterations precipitated in TCR signaling pathway (CARD11;30.0%, PLCG1;27.7%, PRKCB;21.3%, STAT3;21.3%, VAV1;19.1%, NOTCH1;19.1%, RELA;12.8%, CNSK1A1;8.5%, IRF4;6.4%, FYN;2.1%, CBLB;2.1%, IKBKB;2.1%). In addition to these previously reported driver genes, a novel mutation, RLTPR Q575E was discovered in 4 out of 47 acute-type ATL samples (8.5%) with median variant allele frequency 0.52 (range 0.11-0.68). Although RLTPR Q575E has been reported in cutaneous T cell lymphoma, 3 out of 4 ATL patients carrying RLTPR Q575E mutation have no skin involvement by ATL. ATL patients carrying RLTPR Q575E were also harboring CARD11 (75%), PLCG1 (25%), PRKCB (25%), or IKBKB (25%) mutations, which are related to TCR/NF-κB signaling pathway. We next performed luciferase reporter assay to evaluate NF-κB activity in transfected 293T cells with RLTPR Q575E cDNA to explore its function. 293T cells transfected with RLTPR Q575E cDNA has higher NF-κB activity than those transfected with RLTPR wild type cDNA. No significant increased promoter activity of AP-1 or NFAT was observed with RLTPR Q575E. IL-2 mRNA was significantly increased in RLTPR Q575E transduced Jurkat cells under PMA/ionomycin and CD86 co-stimulation. We immunoprecipitated FLAG-tagged RLTPR WT or RLTPR Q575E and blotted for CARD11. We found that the RLTPR Q575E increases interaction of RLTPR with CARD11. Previously identified gene alterations such as CARD11 and PRKCB in ATL have been reported as Tax interactome, thus we further analyzed the interaction of RLTPR with Tax. We immunoprecipitated FLAG-tagged WT RLTPR or RLTPR Q575E and blotted for Tax. We found that the RLTPR WT and Q575E increases interaction of RLTPR with Tax.

Conclusions

In patients with acute-type ATL, we have identified RLTPR Q575E, a novel gain of function mutation, and functionally validated this mutation. This mutation has minimal effect without TCR co-stimulation, but patients with this mutation harboring gain-of-function mutations in TCR signaling molecules. We also found direct interaction between Tax and RLTPR. In the presence of TCR activation by Tax or gain-of-function mutations in this signaling pathway, RLTPR Q575E mutation selectively upregulates the NF-κB signaling pathway and confers the oncogenic effect on the pathogenesis of ATL.

Reference

¹ Joonhee Park, Jingyi Yang, Alexander T. Wenzel, Akshaya Ramachandran, Wung J. Lee, Jay C. Daniels, Juhyun Kim, Estela Martinez-Escala, Nduka Amankulor, Barbara Pro, Joan Guitart, Marc L. Mendillo, Jeffrey N. Savas, Titus J. Boggon, Jaehyuk Choi; Genomic analysis of 220 CTCLs identifies a novel recurrent gain-of-function alteration in RLTPR (p.Q575E). Blood 2017; 130 (12): 1430-1440. doi: https://doi.org/10.1182/blood-2017-02-768234