Tomohiko Taki

Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children

We examined mRNA expression and internal tandem duplication of the Fms‐like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and genomic PCR followed by sequencing. By RT‐PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B‐precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B‐ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis

The mechanisms by which mixed-lineage leukemia (MLL) fusion products resulting from in utero translocations in 11q23 contribute to leukemogenesis and infant acute leukemia remain elusive. It is still controversial whether the MLL fusion protein is sufficient to induce acute leukemia without additional genetic alterations, although carcinogenesis in general is known to result from more than 1 genetic disorder accumulating during a lifetime. Here we demonstrate that the fusion partner-mediated homo-oligomerization of MLL-SEPT6 is essential to immortalize hematopoietic progenitors in vitro. MLL-SEPT6 induced myeloproliferative disease with long latency in mice, but not acute leukemia, implying that secondary genotoxic events are required to develop leukemia. We developed in vitro and in vivo model systems of leukemogenesis by MLL fusion proteins, where activated FMS-like receptor tyrosine kinase 3 (FLT3) together with MLL-SEPT6 not only transformed hematopoietic progenitors in vitro but also induced acute biphenotypic or myeloid leukemia with short latency in vivo. In these systems, MLL-ENL, another type of the fusion product that seems to act as a monomer, also induced the transformation in vitro and leukemogenesis in vivo in concert with activated FLT3. These findings show direct evidence for a multistep leukemogenesis mediated by MLL fusion proteins and may be applicable to development of direct MLL fusion-targeted therapy.

FBXW7 and NOTCH1 mutations in childhood T cell acute lymphoblastic leukaemia and T cell non-Hodgkin lymphoma

Mutation analysis of FBXW7 and NOTCH1 genes was performed in 55 T cell acute lymphoblastic leukaemia (T‐ALL) and 14 T cell non‐Hodgkin lymphoma (T‐NHL) patients who were treated on the Japan Association of Childhood Leukaemia Study (JACLS) protocols ALL‐97 and NHL‐98. FBXW7 and/or NOTCH1 mutations were found in 22 (40·0%) of 55 T‐ALL and 7 (50·0%) of 14 T‐NHL patients. FBXW7 mutations were found in 8 (14·6%) of 55 T‐ALL and 3 (21·4%) of 14 T‐NHL patients, and NOTCH1 mutations in 17 (30·9%) of 55 T‐ALL and 6 (42·9%) of 14 T‐NHL patients. Three (5·4%) T‐ALL and two (1·4%) T‐NHL patients had mutations in both FBXW7 and NOTCH1. FBXW7 mutations included one insertion, one deletion, one deletion/insertion and nine missense mutations. NOTCH1 mutations were detected in the heterodimerization domain (HD) in 15 cases, in the PEST domain in seven cases, and in both the HD and PEST domains in one case. Five‐year event‐free survival and overall survival for patients with FBXW7 and/or NOTCH1 mutations were 95·5% (95% CI, 71·9–99·4%) and 100% respectively, suggesting that T‐ALL patients with FBXW7 and/or NOTCH1 mutation represent a good prognosis compared to those without FBXW7 and/or NOTCH1 mutations (63·6%, P = 0·007 and 78·8%, P = 0·023, respectively).

Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers

Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer.

Galectin-3 (Gal-3) induced by leukemia microenvironment promotes drug resistance and bone marrow lodgment in chronic myelogenous leukemia

Bone marrow (BM) microenvironment (BMME) constitutes the sanctuary for leukemic cells. In this study, we investigated the molecular mechanisms for BMME-mediated drug resistance and BM lodgment in chronic myelogenous leukemia (CML). Gene-expression profile as well as signal pathway and protein analyses revealed that galectin-3 (Gal-3), a member of the β-gal–binding galectin family of proteins, was specifically induced by coculture with HS-5 cells, a BM stroma cell-derived cell line, in all five CML cell lines examined. It was also found that primary CML cells expressed high levels of Gal-3 in BM. Enforced expression of Gal-3 activated Akt and Erk, induced accumulation of Mcl-1, and promoted in vitro cell proliferation, multidrug resistance to tyrosine kinase inhibitors for Bcr-Abl and genotoxic agents as a result of impaired apoptosis induction, and chemotactic cell migration to HS-5–derived soluble factors in CML cell lines independently of Bcr-Abl tyrosine kinase. The conditioned medium from Gal-3–overexpressing CML cells promoted in vitro cell proliferation of CML cells and HS-5 cells more than did the conditioned medium from parental cells. Moreover, the in vivo study in a mice transplantation model showed that Gal-3 overexpression promoted the long-term BM lodgment of CML cells. These results demonstrate that leukemia microenvironment-specific Gal-3 expression supports molecular signaling pathways for disease maintenance in BM and resistance to therapy in CML. They also suggest that Gal-3 may be a candidate therapeutic target to help overcome BMME-mediated therapeutic resistance.

Mutations of the PTPN11 and RAS genes in rhabdomyosarcoma and pediatric hematological malignancies

PTPN11 has been identified as a causative gene in Noonan syndrome (NS), responsible for about 50% of cases of NS. Given the association between NS and an increased risk of some malignancies, notably leukemia and probably some solid tumors including neuroblastoma (NB) and rhabdomyosarcoma (RMS), recent studies have reported that gain‐of‐function somatic mutations in PTPN11 occur in some hematological malignancies, especially de novo juvenile myelomonocytic leukemia (JMML) and in some solid tumors such as NB, although at a low frequency. In a screen for mutations of PTPN11 in 7 cell lines and 30 fresh tumors of RMS and in 25 cell lines and 40 fresh tumors of NB, we identified a missense mutation (A72T) in an embryonal RMS patient. In the RMS samples, we also detected mutations of NRAS in 1 cell line and 1 patient; both mutations were in embryonal RMSs and had no PTPN11 mutations. No mutations of PTPN11 were detected in NB. In 95 leukemia cell lines and 261 fresh leukemia samples including 22 JMMLs, 9 kinds of missense mutations were detected in 17 leukemia samples, which included 11 (50.0%) mutations in JMML samples and lower frequencies in other hematological malignancies. Furthermore, we identified 4 (18.2%) NRAS mutations and 1 (4.5%) KRAS mutation in 5 JMML samples, 1 of which had a concomitant PTPN11 mutation. Our data suggest that mutations of PTPN11 as well as RAS play a role in the pathogenesis of not only myeloid hematological malignancies but also a subset of RMS malignancies.

Down-regulation of TCF8 is involved in the leukemogenesis of adult-T cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is caused by latent human T-lymphotropic virus-1 (HTLV-1) infection. To clarify the molecular mechanism underlying leukemogenesis after viral infection, we precisely mapped 605 chromosomal breakpoints in 61 ATLL cases by spectral karyotyping and identified frequent chromosomal breakpoints in 10p11, 14q11, and 14q32. Single nucleotide polymorphism (SNP) array-comparative genomic hybridization (CGH), genetic, and expression analyses of the genes mapped within a common breakpoint cluster region in 10p11.2 revealed that in ATLL cells, transcription factor 8 (TCF8) was frequently disrupted by several mechanisms, including mainly epigenetic dysregulation. TCF8 mutant mice frequently developed invasive CD4(+) T-cell lymphomas in the thymus or in ascitic fluid in vivo. Down-regulation of TCF8 expression in ATLL cells in vitro was associated with resistance to transforming growth factor beta1 (TGF-beta1), a well-known characteristic of ATLL cells, suggesting that escape from TGF-beta1-mediated growth inhibition is important in the pathogenesis of ATLL. These findings indicate that TCF8 has a tumor suppressor role in ATLL.

The chromosome translocation t(7;11)(p15;p15) in acute myeloid leukemia results in fusion of the NUP98 gene with a HOXA cluster gene, HOXA13, but not HOXA9

The nucleoporin gene NUP98 has been reported to be fused to 9 partner genes in hematologic malignancies with 11p15 translocations. The NUP98‐HOXA9 fusion gene has been identified in acute myeloid leukemia (AML) and chronic myelogenous leukemia with t(7;11)(p15;p15). We report here a novel NUP98 partner gene, HOXA13, in a patient with de novo AML having t(7;11)(p15;p15). The HOXA13 gene is part of the HOXA cluster genes and contains 2 exons, encoding a protein of 338 amino acids with a homeodomain. The NUP98‐HOXA13 fusion protein consists of the N‐terminal phenylalanine‐glycine repeat motif of NUP98 and the C‐terminal homeodomain of HOXA13, similar to the NUP98‐HOXA9 fusion protein. Reverse transcriptase–polymerase chain reaction (RT‐PCR) analysis in various leukemic cell lines showed that the HOXA13 gene was expressed significantly more frequently in acute monocytic leukemic cell lines than in other leukemic cell lines (P = 0.039). HOXA13 and three HOXA cluster genes (A9, A10, A11) located at the 5′ end of the HOXA9 gene were frequently expressed in myeloid leukemic cell lines. Our results revealed that t(7;11)(p15;p15) was not a single chromosomal abnormality at the molecular level. The protein encoded by the NUP98‐HOXA13 fusion gene is similar to that encoded by NUP98‐HOXA9, and the expression pattern of the HOXA13 gene in leukemic cell lines is similar to that of the HOXA9 gene, suggesting that the NUP98‐HOXA13 fusion protein may play a role in leukemogenesis through a mechanism similar to that of the NUP98‐HOXA9 fusion protein.

Consistent detection of CALM-AF10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts

The t(10;11)(p13‐14;q14‐21) is a rare but recurring translocation associated with acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Recently the CALM gene was cloned from the t(10;11) breakpoint of U937 and fused to AF10, a putative transcription factor, which had been identified as one of the fusion partners of the MLL gene. In order to define the involvement of these genes in primary leukaemias and cell lines with t(10;11), we analysed the expression of fusion transcripts by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in five patient samples including ALL, AML and lymphoblastic lymphoma, and three monocytic cell lines (P31/Fujioka, KP‐Mo‐TS and U937). The CALM‐AF10 fusion transcript was detected in all samples; however, the AF10‐CALM fusion was not detected in two patient samples and one cell line. In RT‐PCR analysis there were six isoforms of the CALM‐AF10 fusion transcripts and five of AF10‐CALM fusion transcripts. We also detected novel transcripts in U937. Sequence analysis revealed that all these isoforms had in‐frame junctions and that some of them resulted from alternative splicing at different exons of CALM and others from different breakpoints at CALM and/or AF10. There were at least two different breakpoints of CALM and three of AF10 gene. Our results suggest that the CALM‐AF10 fusion gene is a constant feature and is involved in the pathogenesis of haematological malignancies with t(10;11)(p13‐14;q14‐21), showing various and often multilineage phenotypes. Thus, t(10;11) needs to be investigated by RT‐PCR for identification of the genes involved.

AML1/RUNX1 mutations are infrequent, but related to AML-M0, acquired trisomy 21, and leukemic transformation in pediatric hematologic malignancies.

AML1/RUNX1, located on chromosome band 21q22, is one of the most important hematopoietic transcription factors. AML1 is frequently affected in leukemia and myelodysplastic syndrome with 21q22 translocations. Recently, AML1 mutations were found in adult hematologic malignancies, especially acute myeloid leukemia (AML)–M0 or leukemia with acquired trisomy 21, and familial platelet disorder with a predisposition toward AML. Through the use of polymerase chain reaction–single‐strand conformation polymorphism analysis, we examined the AML1 gene for mutations in 241 patients with pediatric hematologic malignancies, and we detected AML1 mutations in seven patients (2.9%). Deletion was found in one patient, and point mutations in four patients, including three missense mutations, two silent mutations, and one mutation within an intron resulting in an abnormal splice acceptor site. All of the mutations except for one were heterozygous. Mutations within the runt domain were found in six of seven patients. Six of seven patients with AML1 mutations were diagnosed with AML, and one had acute lymphoblastic leukemia. In three of these seven patients, AML evolved from other hematologic disorders. AML1 mutations were found in two of four AML‐M0 and two of three patients with acquired trisomy 21. Patients with AML1 mutations tended to be older children. Three of four patients with AML1 mutations who received stem cell transplantation (SCT) are alive, whereas the remaining three patients with mutations without SCT died. These results suggest that AML1 mutations in pediatric hematologic malignancies are infrequent, but are possibly related to AML‐M0, acquired trisomy 21, and leukemic transformation. These patients may have a poor clinical outcome.