Key Points• PTPRK binds to STAT3 and directly dephosphorylates phospho-STAT3 at Tyr705.• Loss of PTPRK, located in the deleted 6q region, leads to STAT3 activation and contributes to nasal-type NK/ T-cell lymphoma pathogenesis. . Restoration of PTPRK inhibited tumor cell growth and reduced the migration and invasion ability of NKTCL cells. Monoallelic deletion and promoter hypermethylation caused underexpression of PTPRK messenger RNA in NKTCL, and methylation of the PTPRK promoter significantly correlated with inferior overall survival (P 5 .049) in NKTCL patients treated with the steroid-dexamethasone, methotrexate, ifosfamide, L-asparaginase, and etoposide regimen. Altogether, our findings show that PTPRK underexpression leads to STAT3 activation and contributes to NKTCL pathogenesis. (Blood. 2015;125(10):1589-1600

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Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHS were detected in follicular lymphomas and exclusively in germinal center IntroductionMature B-cell non-Hodgkin lymphomas (NHLs) are often associated with chromosomal translocations involving IGH at chromosome band 14q32. 1-5 V(D)J rearrangement and class switch recombination (CSR) are IG rearrangement processes that occur during B-cell development. [6][7][8][9] In CSR, DNA breakage is mediated by activation-induced cytidine deaminase in the germinal centers (GCs) of secondary follicles. 10,11 Any aberrant rearrangement during the DNA breakage and repair processes composed of V(D)J rearrangement and CSR may consequently generate a chromosomal translocation. 12,13 IGH translocations contribute to the pathogenesis of B-cell lymphomas via deregulated expression of the genes located at the partner chromosome locus partly because of the presence of potent B cell-specific transcriptional enhancers within the IGH gene locus. [1][2][3][4][5] Gastric B-cell lymphomas usually exhibit chromosomal translocations involving IGH. 14-16 Molecular cloning of IGH translocation breakpoints has been successfully used to identify novel cancerrelated genes in B-cell lymphomas. 17,18 In this study, we used inverse polymerase chain reaction (PCR) 18 to identify a novel translocation involving the 5Ј S region of the IGH gene (IGHS) and CD44 (located at chromosome 11p13) in gastric as well as other mature B-cell NHLs. Functional studies suggest a possible pathogenic role of this IGHS/CD44 translocation in mature B-cell malignancies. Methods Detection of translocations involving the IGHS by inverse PCRGenomic DNA extracted from frozen sections of tumor specimens from gastric 19 and other mature B-cell NHL cases (supplemental Table 1, available on the Blood website; see the Supplemental Materials link at the top of the online article) was digested with HindIII, and long-distance inverse PCR was performed to detect translocations involving the IGHS region using the primers SAE/JXE, followed by SAI/JXI 18 (supplemental Table 2). Overexpression of CD44⌬Ex1-GFP tagged protein in transfected BJAB cellsCD44⌬Ex1 (CD44 variant mRNA lacking exon 1) and CD44s (standard form; wild-type) cDNAs were amplified by reverse-transcribed (RT)-PCR (supplemental Table 2) from total RNA extracted from a case of gastric lymphoma with the IGHS/CD44 translocation (GL47) and from the peripheral blood lymphocytes of a healthy volunteer; they were then cloned For personal use only. on April 29, 2019. by guest www.bloodjournal.org From in-frame with green fluorescent protein (GFP) at the C-terminus of the pmaxFP-Green-N vector (Amaxa) and transfected into the CD44 Ϫ GC B cell-like (GCB)-diffuse large B-cell lymphoma (DLBCL) cell line BJAB by nucleofection (Amaxa). The subcellular loca...

1378 Tumorigenesis is a multi-step process and involves the silencing of tumor suppressor genes (TSGs) by genetic and/or epigenetic mechanisms. Aberrant hypermethylation of gene promoters is a major epigenetic mechanism associated with TSG silencing in cancer. To identify putative TSGs that might be epigenetically silenced in extranodal NK/T-cell lymphoma, nasal type (ENKL), a genome-wide screening was performed in a commonly deleted region 6q22.33-q23.2. PTPRK (protein tyrosine phosphatase, receptor type, kappa) was identified as the only gene out of 77 genes mapped to the 6q22.33-q23.2 region that was upregulated in four of five ENKL cell lines after treatment with the demethylation agent 5-aza-2' deoxycytidine (5-aza-dC). Further analysis by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) confirmed that the CpG island surrounding the transcriptional start site of PTPRK was methylated in ENKL cell lines not expressing PTPRK. Similarly, a significant correlation between methylation of the PTPRK promoter and downregulation of PTPRK mRNA and protein expression (p=0.019 and p=0.048, respectively) was detected in 39 primary ENKLs. PTPRK gene allelic loss was detected in 80% of cell lines and 47% of primary ENKLs. Functional analyses by in vitro assays showed that the re-expression of PTPRK by retroviral transduction in the PTPRK non-expressing NKYS cell line suppressed the size and number of colonies formed, led to a remarkable increase in the apoptotic cell population and cell cycle arrest at G0/G1 phase. Moreover, PTPRK re-expression substantially reduced the migration and invasion of NKYS cells. Conversely, the inhibitory effect of PTPRK was significantly decreased by partial shRNA knockdown of PTPRK expression in the PTPRK-expressing SNK-6 cell line. The re-expression of PTPRK in another PTPRK non-expressing YT cell line suppressed tumor growth and metastasis in a nude mouse xenograft model. Consistent with these in vitro and in vivo findings, clinicopathological correlation analysis showed that PTPRK silencing was detected mostly in the ENKL patients with advanced and metastatic disease. Examination of the PTPRK protein sequence revealed that the cytoplasmic domain possesses a consensus STAT3 binding domain (YXXQ). Re-expression of PTPRK in NKYS cells resulted in a significant decrease in the level of phospho-STAT3 (Tyr705), whereas PTPRK knockdown in SNK-6 cells resulted in an increased level of phospho-STAT3 (Tyr705). These data suggested that PTPRK dephosphorylates and regulates the oncoprotein STAT3. Overall, this study shows that PTPRK is a putative TSG in the 6q22.33-q23.2 region that is frequently deleted and epigenetically silenced in ENKL, and the loss of PTPRK expression promotes tumor growth via the aberrant constitutive activation of STAT3 in ENKL. Specific therapies aimed at targeting STAT3 warrants further exploration. Disclosures: No relevant conflicts of interest to declare.