Janet D. Rowley

Rearrangement of the MLL Gene in Acute Lymphoblastic and Acute Myeloid Leukemias with 11q23 Chromosomal Translocations

BACKGROUND Translocations involving chromosome band 11q23 are very frequent in both acute lymphoblastic and acute myeloid leukemias and are the most common genetic alteration in infants with leukemia. In all age groups and all phenotypes of leukemia, an 11q23 translocation carries a poor prognosis. A major question has been whether one or several genes on band 11q23 are implicated in these leukemias. Previously, we identified the chromosomal breakpoint region in leukemias with the common 11q23 translocations and subsequently cloned a gene named MLL that spans the 11q23 breakpoint. METHODS We isolated a 0.74-kb BamHI fragment from a complementary DAN (cDNA) clone of the MLL gene. To determine the incidence of MLL rearrangements in patients with 11q23 abnormalities, we analyzed DNA from 61 patients with acute leukemia, 3 cell lines derived from such patients, and 20 patients with non-Hodgkins lymphoma and 11q23 aberrations. RESULTS The 0.74-kb cDNA probe detected DNA rearrangements in the MLL gene in 58 of the patients with leukemia, in the 3 cell lines, and in 3 of the patients with lymphoma. All the breaks occurred in an 8.3-kb breakpoint cluster region within the MLL gene. The probe identified DNA rearrangements in all 48 patients with the five common 11q23 translocations involving chromosomes 4, 6, 9, and 19, as well as in 16 patients with uncommon 11q23 aberrations. Twenty-one different chromosomal breakpoints involving the MLL gene were detected. CONCLUSIONS MLL gene rearrangements were detected with a single probe and a single restriction-enzyme digest in all DNA samples from patients with the common 11q23 translocations as well as in 16 patients or cell lines with other 11q23 anomalies. The ability to detect an MLL gene rearrangement rapidly and reliably, especially in patients with limited material for cytogenetic analysis, should make it possible to identify patients who have a poor prognosis and therefore require aggressive chemotherapy or marrow transplantation.

Association of an inversion of chromosome 16 with abnormal marrow eosinophils in acute myelomonocytic leukemia. A unique cytogenetic-clinicopathological association.

We identified 18 patients with an inversion of chromosome 16, inv(16)(p13q22), among 308 patients with newly diagnosed acute nonlymphocytic leukemia. Each of these 18 patients had acute myelomonocytic leukemia (M4 subtype) and eosinophils with distinctly abnormal morphology, cytochemical staining, and ultrastructure. These eosinophils constituted from 1 to 33 per cent of the nucleated marrow cells. In our series, every patient with acute myelomonocytic leukemia and abnormal eosinophils also had an abnormal chromosome 16. This subgroup of M4 patients had a good response to intensive therapy designed to induce remission; 13 of 17 treated patients entered a complete remission, and 10 remain in first remission. Thus, patients with an inversion of chromosome 16 appear to represent a unique cytogenetic-clinicopathological subtype of acute nonlymphocytic leukemia with a favorable prognosis.

The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.

The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUPP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98–HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.

MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, whereas acute myeloid leukemia (AML) is the most common acute leukemia in adults. In general, ALL has a better prognosis than AML. To understand the distinct mechanisms in leukemogenesis between ALL and AML and to identify markers for diagnosis and treatment, we performed a large-scale genome-wide microRNA (miRNA, miR) expression profiling assay and identified 27 miRNAs that are differentially expressed between ALL and AML. Among them, miR-128a and -128b are significantly overexpressed, whereas let-7b and miR-223 are significantly down-regulated in ALL compared with AML. They are the most discriminatory miRNAs between ALL and AML. Using the expression signatures of a minimum of two of these miRNAs resulted in an accuracy rate of >95% in the diagnosis of ALL and AML. The differential expression patterns of these four miRNAs were validated further through large-scale real-time PCR on 98 acute leukemia samples covering most of the common cytogenetic subtypes, along with 10 normal control samples. Furthermore, we found that overexpression of miR-128 in ALL was at least partly associated with promoter hypomethylation and not with an amplification of its genomic locus. Taken together, we showed that expression signatures of as few as two miRNAs could accurately discriminate ALL from AML, and that epigenetic regulation might play an important role in the regulation of expression of miRNAs in acute leukemias.

Evidence for a 15:17 translocation in every patient with acute promyelocytic leukemia

Cytogenetic specimens were obtained from blood or bone marrow in 27 patients with acute promyelocytic leukemia, including four with the microgranular variant. A 15;17 translocation was identified in 21 to 100 percent of metaphase cells from all 27 patients. The structural rearrangement was identical in every case, and the breakpoints were assigned to 15q22 and 17q21.1. Twelve patients had complete remission, and two (both with the microgranular variant) have had unmaintained continuous remission longer than four years. These data indicate that the 15;17 translocation may be found in every patient with acute promyelocytic leukemia if optimal chromosome analysis is performed.

Distinct microRNA expression profiles in acute myeloid leukemia with common translocations

MicroRNAs (miRNAs) are postulated to be important regulators in cancers. Here, we report a genome-wide miRNA expression analysis in 52 acute myeloid leukemia (AML) samples with common translocations, including t(8;21)/AML1(RUNX1)-ETO(RUNX1T1), inv(16)/CBFB-MYH11, t(15;17)/PML-RARA, and MLL rearrangements. Distinct miRNA expression patterns were observed for t(15;17), MLL rearrangements, and core-binding factor (CBF) AMLs including both t(8;21) and inv(16) samples. Expression signatures of a minimum of two (i.e., miR-126/126*), three (i.e., miR-224, miR-368, and miR-382), and seven (miR-17–5p and miR-20a, plus the aforementioned five) miRNAs could accurately discriminate CBF, t(15;17), and MLL-rearrangement AMLs, respectively, from each other. We further showed that the elevated expression of miR-126/126* in CBF AMLs was associated with promoter demethylation but not with amplification or mutation of the genomic locus. Our gain- and loss-of-function experiments showed that miR-126/126* inhibited apoptosis and increased the viability of AML cells and enhanced the colony-forming ability of mouse normal bone marrow progenitor cells alone and particularly, in cooperation with AML1-ETO, likely through targeting Polo-like kinase 2 (PLK2), a tumor suppressor. Our results demonstrate that specific alterations in miRNA expression distinguish AMLs with common translocations and imply that the deregulation of specific miRNAs may play a role in the development of leukemia with these associated genetic rearrangements.

Chromosome translocations: dangerous liaisons revisited

Although it has been clear for more than a century that the chromosomes in human tumour cells are often wildly abnormal, there has been controversy as to whether these changes are primary events or are merely secondary epiphenomena that reflect the genomic instability of these cells. The prevailing view for most of this period was that chromosome changes were secondary events. What happened to change this view?

The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute leukemia. This translocation creates a fusion protein consisting of the acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor (AML1–ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and immortalizes hematopoietic progenitor cells. We have identified the p14ARF tumor suppressor, a mediator of the p53 oncogene checkpoint, as a direct transcriptional target of AML1–ETO. AML1–ETO repressed the p14ARF promoter and reduced endogenous levels of p14ARF expression in multiple cell types. In contrast, AML1 stimulated p14ARF expression and induced phenotypes consistent with cellular senescence. Chromatin immunoprecipitation assays demonstrated that AML1–ETO was specifically bound to the p14ARF promoter. In acute myeloid leukemia samples containing the t(8;21), levels of p14ARF mRNA were markedly lower when compared with other acute myeloid leukemias lacking this translocation. Repression of p14ARF may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14ARF is an important tumor suppressor in a large number of human leukemias.

Leukaemogenesis: more than mutant genes

Acute leukaemias are characterized by recurring chromosomal aberrations and gene mutations that are crucial to disease pathogenesis. It is now evident that epigenetic modifications, including DNA methylation and histone modifications, substantially contribute to the phenotype of leukaemia cells. An additional layer of epigenetic complexity is the pathogenetic role of microRNAs in leukaemias, and their key role in the transcriptional regulation of tumour suppressor genes and oncogenes. The genetic heterogeneity of acute leukaemias poses therapeutic challenges, but pharmacological agents that target components of the epigenetic machinery are promising as a component of the therapeutic arsenal for this group of diseases.

A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis

The t(8;21)(q22;q22) translocation is one of the most common genetic abnormalities in acute myeloid leukemia (AML), identified in 15% of all cases of AML, including 40–50% of FAB M2 subtype and rare cases of M0, M1 and M4 subtypes. The most commonly known AML1-ETO fusion protein (full-length AML1-ETO) from this translocation has 752 amino acids and contains the N-terminal portion of RUNX1 (also known as AML1, CBFα2 or PEBP2αB), including its DNA binding domain, and almost the entire RUNX1T1 (also known as MTG8 or ETO) protein. Although alterations of gene expression and hematopoietic cell proliferation have been reported in the presence of AML1-ETO, its expression does not lead to the development of leukemia. Here, we report the identification of a previously unknown alternatively spliced isoform of the AML1-ETO transcript, AML1-ETO9a, that includes an extra exon, exon 9a, of the ETO gene. AML1-ETO9a encodes a C-terminally truncated AML1-ETO protein of 575 amino acids. Expression of AML1-ETO9a leads to rapid development of leukemia in a mouse retroviral transduction–transplantation model. More importantly, coexpression of AML1-ETO and AML1-ETO9a results in the substantially earlier onset of AML and blocks myeloid cell differentiation at a more immature stage. These results indicate that fusion proteins from alternatively spliced isoforms of a chromosomal translocation may work together to induce cancer development.