Akira Kitanaka

Integrated molecular analysis of adult T cell leukemia/lymphoma

Adult T cell leukemia/lymphoma (ATL) is a peripheral T cell neoplasm of largely unknown genetic basis, associated with human T cell leukemia virus type-1 (HTLV-1) infection. Here we describe an integrated molecular study in which we performed whole-genome, exome, transcriptome and targeted resequencing, as well as array-based copy number and methylation analyses, in a total of 426 ATL cases. The identified alterations overlap significantly with the HTLV-1 Tax interactome and are highly enriched for T cell receptor–NF-κB signaling, T cell trafficking and other T cell–related pathways as well as immunosurveillance. Other notable features include a predominance of activating mutations (in PLCG1, PRKCB, CARD11, VAV1, IRF4, FYN, CCR4 and CCR7) and gene fusions (CTLA4-CD28 and ICOS-CD28). We also discovered frequent intragenic deletions involving IKZF2, CARD11 and TP73 and mutations in GATA3, HNRNPA2B1, GPR183, CSNK2A1, CSNK2B and CSNK1A1. Our findings not only provide unique insights into key molecules in T cell signaling but will also guide the development of new diagnostics and therapeutics in this intractable tumor.

The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo

Recent studies suggest that metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, but the mechanisms by which metformin affects various cancers, including gastric cancer, remains unknown. The goal of the present study was to evaluate the effects of metformin on human gastric cancer cell proliferation in vitro and in vivo and to study microRNAs (miRNA) associated with antitumor effect of metformin. We used MKN1, MKN45, and MKN74 human gastric cancer cell lines to study the effects of metformin on human gastric cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 4 weeks, and the expression of cell-cycle-related proteins was determined. In addition, we used miRNA array tips to explore the differences among miRNAs in MKN74 cells bearing xenograft tumors treated with or without metformin in vitro and in vivo. Metformin inhibited the proliferation of MKN1, MKN45, and MKN74 in vitro. Metformin blocked the cell cycle in G0–G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially in cyclin D1, cyclin-dependent kinase (Cdk) 4, Cdk6 and by a decrease in retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor-1 receptor in vitro and in vivo. The miRNA expression was markedly altered with the treatment of metformin in vitro and in vivo. Various miRNAs altered by metformin also may contribute to tumor growth in vitro and in vivo. Mol Cancer Ther; 11(3); 549–60. ©2012 AACR.

Src directly tyrosine-phosphorylates STAT5 on its activation site and is involved in erythropoietin-induced signaling pathway

Signal transducers and activators of transcription (STAT) proteins are transcription factors activated by phosphorylation on tyrosine residues after cytokine stimulation. In erythropoietin receptor (EPOR)-mediated signaling, STAT5 is tyrosine-phosphorylated by EPO stimulation. Although Janus Kinase 2 (JAK2) is reported to play a crucial role in EPO-induced activation of STAT5, it is unclear whether JAK2 alone can tyrosine-phosphorylate STAT5 after EPO stimulation. Several studies indicate that STAT activation is caused by members of other families of protein tyrosine kinases such as the Src family. We previously reported that reduction of Src by induction of antisense src RNA expression suppressed EPO-promoted erythroid differentiation in K562 cells. In the present study, we explored the function of Src downstream of the EPOR-initiated signaling. Reduction of Src diminished tyrosine phosphorylation of STAT5 in K562 cells regardless of EPO treatment. The tyrosine phosphorylation level of STAT5 induced by EPO in F-36P cells was reduced in the presence of PP1 or PP2 selective Src inhibitor. In addition, the expression of dominant negative Src in F-36P cells reduced the tyrosine phosphorylation of STAT5. When Src and STAT5 were co-expressed in COS7 cells, tyrosine phosphorylation of STAT5 was observed, and tyrosine residue 694 (Tyr 694) of STAT5A was identified as the major phosphorylation site by Src. In vitro kinase assay revealed that GST-STAT5 fusion protein with the conserved C-terminal, but not the C-terminal-truncated mutant which lacks Tyr 694, was tyrosine-phosphorylated by Src. Src can thus directly tyrosine-phosphorylate the activation site of STAT5 (Tyr 694 in STAT5A), and Src may contribute to EPO-induced signal transduction via STAT5.

Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase.

In this study, we examined the molecular mechanism of erythropoietin‐initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3‐kinase (PI3‐kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin‐dependent colonies derived from human bone marrow cells and erythropoietin‐induced differentiation of K562 human erythroleukaemia cells. Antisense p85α oligonucleotide or LY294002, a selective inhibitor of PI3‐kinase, independently inhibited the formation of erythropoietin‐dependent colonies. In K562 cells, Src associated with PI3‐kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin‐induced activation of PI3‐kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin‐induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3‐kinase in F‐36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine‐phosphorylated erythropoietin receptor, and associated with PI3‐kinase. In vitro binding experiments proved that glutathione S‐transferase–p85α N‐ or C‐terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine‐phosphorylated by Src. Taken together, Src transduces the erythropoietin‐induced erythroid differentiation signals by regulating PI3‐kinase activity.

TET2 is essential for survival and hematopoietic stem cell homeostasis.

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2trap) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2trap/trap mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2trap/trap HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2trap/trap HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

T-cell immunotherapy with a chimeric receptor against CD38 is effective in eliminating myeloma cells.

T-cell immunotherapy with a chimeric receptor against CD38 is effective in eliminating myeloma cells

Loss-of-TET2 has dual roles in murine myeloproliferative neoplasms: disease sustainer and disease accelerator

Acquired mutations of JAK2 and TET2 are frequent in myeloproliferative neoplasms (MPNs). We examined the individual and cooperative effects of these mutations on MPN development. Recipients of JAK2V617F cells developed primary myelofibrosis-like features; the addition of loss of TET2 worsened this JAK2V617F-induced disease, causing prolonged leukocytosis, splenomegaly, extramedullary hematopoiesis, and modestly shorter survival. Double-mutant (JAK2V617F plus loss of TET2) myeloid cells were more likely to be in a proliferative state than JAK2V617F single-mutant myeloid cells. In a serial competitive transplantation assay, JAK2V617F cells resulted in decreased chimerism in the second recipients, which did not develop MPNs. In marked contrast, cooperation between JAK2V617F and loss of TET2 developed and maintained MPNs in the second recipients by compensating for impaired hematopoietic stem cell (HSC) functioning. In-vitro sequential colony formation assays also supported the observation that JAK2V617F did not maintain HSC functioning over the long-term, but concurrent loss of TET2 mutation restored it. Transcriptional profiling revealed that loss of TET2 affected the expression of many HSC signature genes. We conclude that loss of TET2 has two different roles in MPNs: disease accelerator and disease initiator and sustainer in combination with JAK2V617F.

Growth Hormone Prevents Fas-Induced Apoptosis in Lymphocytes through Modulation of Bcl-2 and Caspase-3

Objective: Growth hormone (GH) has been reported to have a potent effect on the immune system. However, the detailed mechanism of the effect of GH on the immune system has not yet been clarified. This study was designed to investigate the nature of this mechanism. Methods: In the present study, we investigated the effects of GH on the susceptibility of both human CEM/C7 lymphocytes and human IM-9 lymphocytes to Fas-induced apoptosis. Results: Both cell lines expressed GH receptor mRNA. GH rescued Fas-induced suppression of [3H]-thymidine incorporation into each cell line. GH prevented Fas-induced apoptosis in each cell line without changing Fas antigen expression. We next investigated the mechanisms of the prevention of Fas-induced apoptosis, by focusing on intracellular molecules related to the apoptotic signal. Bcl-2 expression was increased by GH treatment in both CEM/C7 and IM-9 lymphocytes. GH also downregulated caspase-3 expression and inhibited activation of caspase-3 in both cell lines. Conclusion: These findings suggest that GH regulates the human immune system through inhibition of Fas-induced apoptosis in activated T and B lymphocytes.

Variegated RHOA mutations in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

Mediation by the Protein-tyrosine Kinase Tec of Signaling between the B Cell Antigen Receptor and Dok-1

A variety of growth factor receptors induce the tyrosine phosphorylation of a nonreceptor protein-tyrosine kinase Tec as well as that of a Tec-binding protein of 62 kDa. Given the similarity in properties between this 62-kDa protein and p62Dok-1, the possibility that these two proteins are identical was investigated. Overexpression of a constitutively active form of Tec in a pro-B cell line induced the hyperphosphorylation of endogenous Dok-1. Tec also associated with Dok-1 in a phosphorylation-dependent manner in 293 cells. Tec mediated marked phosphorylation of Dok-1 both in vivo and in vitro, and this effect required both the Tec homology and Src homology 2 domains of Tec in addition to its kinase activity. Expression of Dok-1 in 293 cells induced inhibition of Ras activity, suggesting that Dok-1 is a negative regulator of Ras. In the immature B cell line Ramos, cross-linking of the B cell antigen receptor (BCR) resulted in tyrosine phosphorylation of Dok-1, and this effect was markedly inhibited by expression of dominant negative mutants of Tec. Furthermore, overexpression of Dok-1 inhibited activation of the c-fos promoter induced by stimulation of the BCR. These results suggest that Tec is an important mediator of signaling from the BCR to Dok-1.