A novel SLC1A1‐RIC1 fusion sensitive to asparaginase‐based therapy in natural killer/T‐cell lymphoma

Natural killer/T-cell lymphoma (NKTCL) is the most common extranodal lymphoma with a highly aggressive clinical course.1 Recurrent somatic gene mutations, mainly including RNA helicase genes, tumour suppressors, JAK–STAT pathway molecules, epigenetic modifiers and RAS–MAPK pathway molecules, have been revealed as major molecular aberrations of NKTCL.2 However, chromosomal translocation frequently observed in leukaemia to underlie disease pathogenesis and define treatment responses3, 4 has never been reported in NKTCL. At the same time, as the introduction of treatment regimens containing asparaginase, clinical outcomes in patients with NKTCL have remarkably improved.5 In the era of asparaginase, high expression of excitaory amino acid transport 3 (EAAT3, SLC1A1-encoded protein) mediates aberrant glutamine metabolism and tumour progression, rendering therapeutic sensitivity and independently predictes superior progression-free and overall survival.6 We have recently reported low expression of LINC00486 in NKTCL, whose partner protein NKRF transcriptionally represses the expression of EAAT3.7 The better understanding of EAAT3 expression is important for the improvement of anti-metabolic therapy in NKTCL. To explore the molecular mechanism regulating EAAT3 expression, we first performed an integrated analysis to determine whether the LINC00486 is interplayed with EAAT3. As expected, we found that patients with high EAAT3 expression showed significantly lower expression level of LINC00486 (Figure 1A). We then divided patients into the LINC00486-high and LINC00486-low groups based on the previously reported cut-off value7 and confirmed its negative correlation with EAAT3 expression (Figure 1B). Among the full cohort (n = 82), 18.3% patients were LINC00486-high/EAAT3-low (designated as G1), 6.1% were LINC00486-low/EAAT3-low (G2), 29.3% were LINC00486-high/EAAT3-high (G3) and 46.3% were LINC00486-low/EAAT3-high (G4). Considering the dissociation of LINC00486 and EAAT3 in G2 and G3, we therefore profiled the mutation pattern in four groups (Figure 1C) and found that G2 were characterized by mutations involving epigenetic modifiers (Figure 1D). Notably, no featured mutations were identified in G3. Given that the genomic alteration is predominated in haematological malignancies,8 we further investigated the chromosomal rearrangement and identified a novel SLC1A1-RIC1 fusion in 2 out of 24 NKTCL patients in the G3 group. These two patients (T26m and T148m) were diagnosed as NKTCL at 49 and 42 years of age respectively. Histological diagnoses were established according to the World Health Organization classification9 and T-cell receptor gene rearrangement assays were negative corresponding to NK cell-derived subtype. Both were classified as Ann Arbor stage I–II and treated with an asparaginase-based MESA (methotrexate, etoposide, dexamethasone and pegaspargase) regimen sandwiched with local radiotherapy. In both cases, the patients remained in complete remission, after 3.9 and 3.7 years respectively. To validate the new SLC1A1-RIC1 fusion gene in NKTCL, we conducted RNA sequencing (RNA-seq) analysis (Figure 1E) and polymerase chain reaction assay and Sanger sequencing (Figure 1F), both suggesting that 5′ exons of SLC1A1 (exons 1–11) were fused out of frame to 3′ exons of RIC1 (exons 13–26 or exons 13–22 transcripts), introduced a stop codon and resulted in a nonsense fusion. Despite the deletion of 3′ open reading frame (ORF), the main functional domains of SLC1A1 (1–443AA) still retained in fusion transcript. In addition, an overexpression of EAAT3 protein containing the major ORF of SLC1A1 (1–443AA) was validated by immunohistochemistry in both patients (Figure 1G). SLC1A1 was associated with metastasis in osteosarcoma and breast cancers.10, 11 Therefore, it is deduced that SLC1A1-RIC1 fusion gene was an alternative molecular mechanism regulating SLC1A1 expression and probably involved in the metabolism reprogramming-mediated malignant transformation. To test this assumption, we retrieved differentially expressed genes driven by SLC1A1-RIC1 using RNA-seq data on SLC1A1-RIC1 overexpressed cells versus the control cells. A total of 245 upregulated genes and 1056 downregulated genes were found in SLC1A1-RIC1 overexpressed cells (Figure 2A). Enrichment analysis showed that the SLC1A1-RIC1-upregulated genes were mainly enriched with oncogenic pathways, such as nucleotide excision repair, DNA replication, cell cycle and homologous recombination, as well as metabolic pathways (Figure 2B). To further investigate the role of this new fusion in metabolism reprogramming during lymphoma progression, NK cell line NK-92 cells were transfected with vector control or SLC1A1-RIC1 fusion. SLC1A1-RIC1 fusion gene remarkably accelerated NK-92 cell proliferation (Figure 2C) and enhanced colony formation (Figure 2D), as compared to vector control. In zebrafish models, xenograft tumour formation rate was also significantly increased when injected with NK-92 cells expressing SLC1A1-RIC1 fusion (Figure 2E). As serum metabolomic profile was characterized by aberrant glutamine metabolism in NKTCL,6 we performed high-performance liquid chromatography with tandem mass spectrometry to quantify targeted amino acids in cytoplasm samples of NK-92 cells transfected with SLC1A1-RIC1 fusion or vector control. SLC1A1-RIC1 cells showed increased cellular glutamine (Figure 2F) and a phenotype of glutamine addiction, as demonstrated by remarkably reduced cell viability when cultured in medium lack of glutamine and interlinked asparagine (Minimum Essential Medium, no glutamine; Gibco) (Figure 2G, left panel). Cell growth inhibition was significantly rescued by the addition of glutamine to SLC1A1-RIC1 cells (Figure 2G, right panel), as compared to control cells. These data revealed that SLC1A1-RIC1 fusion gene promoted cell growth through reprogramming glutamine metabolism. Asparaginase was known as key anti-metabolic agent to treat NKTCL, exerting therapeutic effect through depleting extracellular asparagine and inhibiting glutamine-dependent tumour cell growth.12 Our experimental data suggested that SLC1A1-RIC1 cells were more sensitive to asparaginase than to control cells (Figure 2H), and zebrafish xenograft models injected with NK-92 cells transfected with SLC1A1-RIC1 fusion also presented longer survival time upon asparaginase treatment (Figure 2I). Taken together, NKTCL with SLC1A1-RIC1 fusion could be targeted by asparaginase treatment. In conclusions, our study reported a novel recurrent SLC1A1-RIC1 fusion as an alternative mechanism contributed to EAAT3 overexpression and provided its functional support for accelerating lymphoma cell proliferation and colony formation in a glutamine-dependent manner. In alignment with SLC1A1 as an important prognostic biomarker and therapeutic target,6 SLC1A1-RIC1 fusion renders sensitivity to asparaginase treatment. Our study highlighted the importance of comprehensive molecular analysis to characterize gene fusion in lymphomagenesis. This study was supported, in part, by research funding from the National Natural Science Foundation of China (82130004, 81830007 and 82270194), National Key Research and Development Program (2022YFC2502600), Chang Jiang Scholars Program, Shanghai Rising-Star Program (23QA1406100), Shanghai Municipal Commission of Science and Technology Project (23141903100), Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (20152206, 20152208 and 20161303), Clinical Research Plan of Shanghai Hospital Development Center (SHDC 2020CR1032B), Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine (DLY201601), Multi-center Hematology-Oncology Protocols Evaluation System (M-HOPES) network from China, Samuel Waxman Cancer Research Foundation and the Center for High Performance Computing at Shanghai Jiao Tong University. Jie Xiong performed experiments, gathered clinical information and carried out analysis. Wei-Li Zhao conceived the study, directed and supervised research. Jie Xiong and Wei-Li Zhao wrote the manuscript. The authors declared no competing financial interests. The study was approved by the ethics committee and institutional review board of Shanghai Ruijin Hospital. Informed consents were obtained from all patients in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.