Xiaozhou Hu

Activating mutations of STAT5B and STAT3 in lymphomas derived from γδ-T or NK cells

Lymphomas arising from NK or γδ-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n=51), γδ-T-cell lymphomas (n=43) and their cell lines (n=9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of γδ-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylated protein and a growth advantage to transduced cell lines or normal NK cells. Growth-promoting activity of the mutants can be partially inhibited by a JAK1/2 inhibitor. Molecular modelling and surface plasmon resonance measurements of the N642H mutant indicate a marked increase in binding affinity of the phosphotyrosine-Y699 with the mutant histidine. This is associated with the prolonged persistence of the mutant phosphoSTAT5B and marked increase of binding to target sites. Our findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy.

PRDM1 is a tumor suppressor gene in natural killer cell malignancies

Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma, and there is little insight into its pathogenesis. Here we show that PRDM1 is a tumor suppressor gene in NKCLs that is inactivated by a combination of monoallelic deletion and promoter CpG island hypermethylation. We observed monoallelic deletion of PRDM1 loci in 8 of 18 (44%) NKCL cases. The other allele showed significant promoter methylation in 12 of 17 (71%) cases. In support of its role as a tumor suppressor gene, the reconstitution of PRDM1 in PRDM1-null NK cell lines led to G2/M cell cycle arrest, increased apoptosis, and a strong negative selection pressure with progressive elimination of PRDM1-expressing cells, which was enhanced when IL-2 concentration is limiting. We observed a progressive increase in PRDM1 expression—in particular, PRDM1α—in normal NK cells in response to IL-2 and in normal NK cells activated with an engineered NK cell target, K562-Cl9-mb21, suggesting its role in NK cell homeostasis. In support of this role, knockdown of PRDM1 by shRNA in normal NK cells resulted in the positive selection of these cells. We identified MYC and 4-1BBL as targets of PRDM1 in NK cells. Disruption of homeostatic control by PRDM1 may be an important pathogenetic mechanism for NKCL.

MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma

Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, miR-708, miR-135b; 2 downregulated: miR-146a, miR-155) significantly associated with ALK(+) ALCL cases. In addition, we derived an 11-miRNA signature (4 upregulated: miR-210, miR-197, miR-191, miR-512-3p; 7 downregulated: miR-451, miR-146a, miR-22, miR-455-3p, miR-455-5p, miR-143, miR-494) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway.

HACE1 is a tumor suppressor gene candidate in natural killer cell neoplasms.

HACE1 is an E3 ubiquitin ligase located in 6q21, the genomic region frequently deleted in natural killer (NK) cell malignancies. Here, we report HACE1 as a candidate tumor suppressor gene silenced through a combination of deletion and cytosine phosphate guanine island hypermethylation. We detected deletion of HACE1 in malignant NK cell lines (6 of 9, 67%) and primary biopsies (5 of 15, 33%) by quantitative PCR, with most of the specimen showing cytosine phosphate guanine island hypermethylation in the remaining allele, leading to low mRNA transcription. The ectopic expression of HACE1 in an HACE1-null NK cell line led to apoptosis and G2/M cell cycle arrest. Moreover, HACE1 expression was up-regulated in IL-2-activated normal NK cells and NK cells cocultured with an engineered NK cell target, K562 Clone 9.mbIL21, suggesting its role in the regulation of NK cell homeostasis. In conclusion, HACE1 is another potent tumor suppressor gene located within the 6q21 region, and loss of function of multiple tumor suppressor genes within 6q21 may be a critical determinant of NK cell lymphomagenesis.

Clinicopathologic Characterization of Aggressive Natural Killer Cell Leukemia Involving Different Tissue Sites

Aggressive natural killer cell leukemia (ANKL) is a rare disease with an extremely aggressive clinical course. The etiology of ANKL is unclear with few genetic/epigenetic aberrations described to date. Moreover, misdiagnosis of ANKL is a frequent problem. Clinicopathologic characteristics of 35 retrospective cases of ANKL were investigated with the aim of improving diagnosis and to find the genetic/epigenetic aberrations associated with ANKL etiology. Because of the relatively low number of leukemic cells in the peripheral blood and bone marrow, diagnosis of ANKL can be missed; therefore, it is important to perform biopsy on solid tissues, if necessary. We describe the pathology of ANKL in the lymph nodes, bone marrow, spleen, liver, and skin, with focus on diagnosis and differentiated diagnosis. We observed young male predominance in our cohort, and the clinical course was more aggressive than reported previously. Low lactate dehydrogenase (<712 IU/L), chemotherapy or L-asparaginase administration were found to be associated with more favorable outcomes. SH2 domains of STAT5B and STAT3 also were screened for the presence of activating mutations. Moreover, CpG island methylation status of HACE1, a candidate tumor-suppressor gene, was determined in ANKL samples. We observed activating STAT5B mutations (1/5) and hypermethylation of HACE1 (3/4) in ANKL cases, suggesting that these aberrations may contribute to ANKL pathogenesis.

Lack of evidence that HACE1 is not a tumor suppressor gene in NKTCL: to the editor-in-chief.

Disclosures: None declared. We read with great interest the recent article by Sako et al about the role of HACE1 in natural killer/T-cell lymphomas (NKTCLs). However, we feel that many of the conclusions are overstated given the experimental results presented. Our concerns are outlined below. First, evidence for a potential role for HACE1 as a tumor suppressor gene in NKTCL has previously been reported in NK-cell lymphoma, but most of the NK cell lines used by Sako et al are not well characterized. In particular, the study did not include KAI3, the NK cell line in which HACE1 expression was previously shown to induce apoptosis and G2/Mcell cycle arrest.NKTCLprincipally originates fromNK cells and only rarely from T cells. Three of the cell lines used by Sako et al (SNT-8, SNT-15, and SNT-16) are g/d T-cell lines, and all three cell lines lack deletion of 6q21 and PRDM1, which is frequent in well-characterized NK cell lines and NKTCL cases. Second, we disagree with the use of IL-2eactivated peripheral blood lymphocytes (PBLs), which principally constitute a/b T cells, for protein detection in Figure 2A. Rather, IL-2eactivated NK cells should have been used. Third, in the experiments presented in Figure 5, Annexin V, which measures both early and late apoptotic cells, should have been used instead of 7AAD, which measures only late apoptotic cells. Surprisingly, and contrary to previous reports, SK-NEP1 cells (Wilms tumor cell line used as positive control) lacked an increase in 7AAD staining or in G2/M phase after TAT-HACE1 expression. In that study by Zhang et al, HACE1 re-expression decreased proliferation in SK-NEP1 cells, in striking contrast with the increase in S phase observed by Sako et al.

Generation of a Genetically Engineered Aggressive Nk-Cell Leukemia Cell Line with Stable IL2 Expression

Introduction: Aggressive NK-cell leukemia (ANKL) is a highly aggressive disease with extremely poor prognosis. A few malignant NK cell lines reflecting ANKL biology have been generated; however, these NK cell lines require the inclusion of exogeneous IL2 in the culture medium continuously, which increases the cost of cell culture significantly. Methods: IL2 coding sequenced was cloned into MSCV-IRES-YFP (PMIY) vector by directional PCR-cloning. IL2 was ectopically expressed in KHYG1 cell line through retroviral transduction. The transduced cells were cultured in limiting IL2 concentrations during which they were quantified with flow cytometry in regular time intervals to track the transduced population. IL2 transduced KHYG1 cells were then sorted to generate IL2-KHYG1 cells. PRDM1 was ectopically expressed in IL2-KHYG1 cells through retroviral transduction. Trypan blue count was performed to test proliferation of IL2-KYHG1 cells in the absence of IL2. Results: IL2-KHYG1 cells were enriched (from ~7% to ~16% YFP+ cells) during cell culture in limiting (6.25IU) IL2 concentrations. Complete removal of IL2 further enriched the transduced portion to 27% YFP-positivity, which did not increase with additional culturing. IL2 expressing KHYG1 cells were further enriched by sorting transduced IL2-KHYG1 cells with high level IL2 expression. IL2-KHYG1 cells survived and continued to grow without IL2. IL2-KHYG1 cells were transduced successfully using a PRDM1 construct having GFP as a marker under cell culture conditions having no IL2. Conclusion: IL2-KHYG1 cell line may decrease the cost associated with culturing ANKL cell lines, and it may facilitate in vitro investigation of the molecular basis of this malignancy.