Minenori Eguchi-Ishimae

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

Molecular Pathogenesis of MLL-Associated Leukemias

Chromosome translocations disrupting theMLL gene are associated with various hematologic malignancies but are particularly common in infant and secondary therapy-related acute leukemias. The normal MLL-encoded protein is an essential component of a supercomplex with chromatin-modulating activity conferred by histone acetylase and methyltransferase activities, and the protein plays a key role in the developmental regulation of gene expression, includingHox gene expression. In leukemia, this function is subverted by breakage, recombination, and the formation of chimeric fusion with one of many alternative partners. SuchMLL translocations result in the replacement of the C-terminal functional domains of MLL with those of a fusion partner, yielding a newly formed MLL chimeric protein with an altered function that endows hematopoietic progenitors with self-renewing and leukemogenic activity. This potent impact of the MLL chimera can be attributed to one of 2 kinds of activity of the fusion partner: direct transcriptional transactivation or dimerization/oligomerization. Key unresolved issues currently being addressed include the set of target genes forMLL fusions, the stem cell of origin for the leukemias, the role of additional secondary mutations, and the origins or etiology of theMLL gene fusions themselves. Further elaboration of the biology ofMLL gene-associated leukemia should lead to novel and specific therapeutic strategies.

The role of the MLL gene in infant leukemia.

TheMLL gene is a major player in leukemia, particularly in infant leukemia and in secondary, therapy-related acute leukemia. The normalMLL gene plays a key role in developmental regulation of gene expression (includingHOX genes), and in leukemia this function is subverted by breakage, recombination, and chimeric fusion with one of 40 or more alternative partner genes. In infant leukemias, the chromosome translocations involvingMLL arise during fetal hematopoiesis, possibly in a primitive lymphomyeloid stem cell. In general, these leukemias have a very poor prognosis. The malignancy of these leukemias is all the more dramatic considering their very short preclinical natural history or latency. These data raise fundamental issues of how such divergentMLL chimeric genes transform cells, why they so rapidly evolve to a malignant status, and what alternative or novel therapeutic strategies might be considered. We review here progress in tackling these questions.

MLL chimeric protein activation renders cells vulnerable to chromosomal damage: An explanation for the very short latency of infant leukemia

MLL fusion genes are a predominant feature of acute leukemias in infants and in secondary acute myeloid leukemia (AML) associated with prior chemotherapy with topo‐II poisons. The former is considered to possibly arise in utero via transplacental chemical exposure. A striking feature of these leukemias is their malignancy and remarkably brief latencies implying the rapid acquisition of any necessary additional mutations. We have suggested that these coupled features might be explained if MLL fusion gene encoded proteins rendered cells more vulnerable to further DNA damage and mutation in the presence of chronic exposure to the agent(s) that induced the MLL fusion itself. We have tested this idea by exploiting a hormone regulated MLL‐ENL (MLLT1) activation system and show that MLL‐ENL function in normal murine progenitor cells substantially increases the incidence of chromosomal abnormalities in proliferating cells that survive exposure to etoposide VP‐16. This phenotype is associated with an altered pattern of cell cycle arrest and/or apoptosis.

Suppression of the let-7b microRNA pathway by DNA hypermethylation in infant acute lymphoblastic leukemia with MLL gene rearrangements

MicroRNAs (miRNAs) regulate cell proliferation and differentiation by controlling the expression of proteins involved in many signaling pathways. Recent studies have shown that dysregulation of miRNA expression is associated with increased tumorigenicity and a poor prognosis in several types of cancers. The miRNA let-7b is one of the severely downregulated miRNAs in mixed-lineage leukemia (MLL)-rearranged acute lymphoblastic leukemia (ALL) patients. In vitro transfection of leukemogenic MLL fusion genes into human embryonic kidney-293 cells suppressed let-7b expression. In leukemic cells with an MLL fusion gene, the regulatory region for let-7b expression was hypermethylated, and its expression was partially recovered after culturing the cells with the demethylating agent 5-azacitidine. These results suggest that loss of let-7b expression may be one of the consequences of oncogenic MLL fusion proteins, and contributes to leukemogenesis possibly through the upregulation of let-7b-regulated target genes with leukemogenic potential in hematopoietic cells. The enforced expression of let-7b in ALL cell lines with an MLL fusion gene inhibited their growth, indicating the possible use of let-7b as a new therapeutic tool for refractory infant ALL with an MLL fusion gene.

Chronic idiopathic myelofibrosis expressing a novel type of TEL - PDGFRB chimaera responded to imatinib mesylate therapy

Chronic idiopathic myelofibrosis expressing a novel type of TEL - PDGFRB chimaera responded to imatinib mesylate therapy

Recent advances in the treatment of infant acute myeloid leukemia

Infant acute myeloid leukemia (AML) of less than 12 months old is generally characterized by a high incidence of acute monoblastic or myelomonoblastic leukemia with hyperleukocytosis and extramedullary involvement. Most of the leukemic cells have 11q23 translocations, which lead to the MLL gene rearrangements. The MLL gene rearrangements occur at a high frequency in monoblastic subtype, hyperleukocytosis or young age in infant AML. Compared with acute lymphoblastic leukemia, however, it remains unknown whether prenatal origin exists in the pathogenesis of infant AML. Recently, the treatment outcome of infant AML has been clarified by two study groups, which confirmed the effect of intensive chemotherapy including repeated cycles of cytarabine and anthracyclines for infant AML. Presence of the MLL gene rearrangements, gender, age and white blood cell count showed no influence on the outcome of infant AML. The allogeneic hematopoietic stem cell transplantation (HSCT) remains the treatment of choice for infant AML when a matched related donor is available. Monitoring of minimal residual disease by real-time PCR is a useful technique to predict the outcome or efficacy of the treatment in infant AML. Although intensive chemotherapy and/or allogeneic HSCT have cured most AML infants, some still relapse and ultimately die. A need remains for future development by exploiting the unusual biologic properties of leukemic progenitor cells expressing the abnormal MLL gene product.

In Vitro Cleavage of the MLL Gene by Topoisomerase II Inhibitor (Etoposide) in Normal Cord and Peripheral Blood Mononuclear Cells

The correlation between infant leukemia and in utero exposure to topoisomerase II (topo-II) inhibitor has been clarified. We examined the in vitro effect of topo-II inhibitor (etoposide) on cleavage of theMLL gene in cord and peripheral blood mononuclear cells (MNCs). Southern blot analysis showed cleavage of theMLL gene in peripheral blood MNCs of infants when the MNCs were exposed to etoposide. MNCs were incubated with etoposide at various concentrations (1 to 50 µM), and a ligation-mediated polymerase chain reaction (LM-PCR) was used to detect double strand breaks (DSBs) of DNA in intron 8 of theMLL breakpoint cluster region. PCR products obtained with LM-PCR were subcloned and sequenced to identify the breakpoint in theMLL gene. The PCR products indicated DSBs of theMLL gene were obtained without any difference in the incidence between 3 different samples (cord and peripheral blood from infants and children). Sequencing analysis showed that the DSBs occurred on the telomeric side of intron 8 and near exon 9. There was no evidence that the cord blood was more susceptible toMLL DNA breakage by topo-II inhibitor than were other cells. Instability of the partner gene during the fetal period could be associated with the pathogenesis of infant leukemia.

Novel dominant-negative mutant of GATA3 in HDR syndrome.

HDR syndrome is an autosomal dominant disorder characterized by hypoparathyroidism, sensorineural deafness, and renal anomaly caused by mutation of the GATA3 gene located at chromosome 10p15. We report the case of a neonate with HDR syndrome and a novel GATA3 mutation. We performed genetic and functional analysis of GATA3 in this patient and identified a novel heterozygous 1516G> C missense mutation in exon 5, resulting in a cysteine-to-serine substitution at codon 321 (Cys321Ser). Mutated and wild-type GATA3 proteins were expressed at a similar level in vitro, indicating that the mutated GATA3 protein was stable. Luciferase assay revealed that the Cys321Ser-mutated GATA3 lacked transactivation activity due to loss of DNA-binding activity as confirmed by gel shift assay. Moreover, mutated GATA3 exerted a dominant-negative effect over the transactivation activity of wild-type GATA3. These findings indicate that not only haploinsufficiency of GATA3 but also the dominant-negative effect of Cys321Ser-mutated GATA3 might have been responsible for the HDR syndrome phenotype of our patient.

ETV6–ARNT fusion in a patient with childhood T lymphoblastic leukemia

The ETS variant gene 6 (ETV6) gene is located at 12p13, and is frequently involved in translocations in various human neoplasms, resulting in the expression of fusion proteins consisting of the amino-terminal part of ETV6 and unrelated transcription factors or protein tyrosine kinases. Leukemia with t(1;12)(q21;p13) was previously described in a 5-year-old boy with acute myeloblastic leukemia (AML-M2) who exhibited a novel ETV6-aryl hydrocarbon receptor nuclear translocator (ARNT) fusion protein. We herein report the case of a 2-year-old boy with T-cell lymphoblastic leukemia (T-ALL) harboring t(1;12)(q21;p13). Fluorescence in situ hybridization (FISH) with a ETV6 dual-color DNA probe revealed that the split signals of the ETV6 gene in 96.7% of bone marrow cells, indicating rearrangement of the ETV6 gene. Therefore, we performed a FISH analysis with bacterial artificial chromosome (BAC) probes containing the ARNT, BCL9, and MLLT11 genes located at 1q21, and these results indicated that the ARNT gene might be involved in the t(1;12)(q21;p13). Reverse transcriptase-polymerase chain reaction analysis disclosed the existence of a ETV6-ARNT fusion gene. To our knowledge, the current report is novel in its report of the ETV6-ARNT fusion in childhood T-ALL. The ETV6-ARNT fusion is associated not only with AML but also with T-ALL.