Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS) and IKZF3 (AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer.     

Novel chimaeric protein expressed in Philadelphia positive acute lymphoblastic leukaemia

Cytogenic changes are becoming increasingly important in understanding the pathogenesis of human malignancies. The t(9;22) (q34;q11) translocation is one of the most consistent and generates the Philadelphia chromosome (Ph1) (ref. 1) in chronic myeloid leukaemia (CML); it has also been observed in some acute lymphoblastic leukaemias (ALL) (ref. 2). In CML the breakpoints occur on chromosome 22 in the region designated bcr (ref. 3) and result in the expression of a bcr-abl fusion product of relative molecular mass (MT) 210,000 (210K) with associated in vitro tyrosine kinase activity (P210bcr-abl). In some cases of Ph1-positive ALL, a novel abl-related protein (P190all-abl) of 190K has been shown to have tyrosine kinase activity. In this report we demonstrate that the P190all-abl protein has a bcr determinant from the amino-terminal region, but is lacking a bcr determinant normally found in the P210bcr-abl near the bcr-abl junction. The chimaeric nature of the P190all-abl was confirmed by sequential immunoprecipitation with antisera against abl and bcr peptides.     

A novel c-abl protein product in Philadelphia-positive acute lymphoblastic leukaemia

Activation of cellular proto-oncogenes as a result of chromosomal abnormalities has been implicated in the development of some human malignancies. Perhaps one of the most striking examples of this association occurs in chronic myelogenous leukaemia, where the Philadelphia (Ph) translocation results in substitution of the 5' end of the c-abl proto-oncogene with bcr gene sequences. A unique hybrid bcr-abl message is produced. As the Ph translocation is also present in some patients with acute lymphoblastic leukaemia, we initiated studies to determine if similar genomic events occur in these two different forms of Ph-positive leukaemia. Here we report that the Ph translocation in acute lymphoblastic leukaemia can result in production of a novel aberrant c-abl protein that is distinct from the bcr-abl protein found in Ph-positive chronic myelogenous leukaemia. Our observations suggest that alternative mechanisms of activation of c-abl exist, and may be important in the development of human acute lymphoid rather than chronic myeloid malignancies.     

A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia

The Philadelphia (Ph) chromosome breakpoints in chronic myelocytic leukaemia are clustered on chromosome 22 band q11 in a 5.8-kilobase (kb) region designated bcr. The c-abl protooncogene is translocated from chromosome 9 band q34 into bcr and the biochemical consequence of this molecular rearrangement is the production of an abnormal fusion protein bcr-abl p210 with enhanced protein-tyrosine kinase activity compared to the normal p145 c-abl protein. The Ph chromosome translocation is also seen in some acute lymphoblastic leukaemias with B-cell precursor phenotypes some of which have bcr rearrangement (bcr+) and some do not (bcr-). We present evidence that the Ph+, bcr- leukaemias are associated with a novel p190 abl kinase. We propose that acute lymphoblastic leukaemias that are bcr+, p210+ are probably lymphoid blast crises following a clinically silent chronic phase of chronic myelocytic leukaemia arising in multipotential stem cells whereas bcr-, p190+ cases are de novo acute lymphoblastic leukaemias arising in more restricted precursors.     

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.     

Regulated progression of a cultured pre-B-cell line to the B-cell stage

The variable (V) regions of heavy and light immunoglobulin chains are encoded by multiple germline DNA elements which are assembled into complete variable-region genes in precursor(pre-) B lymphocytes. The heavy-chain V region (VH) is assembled from three separate germline DNA elements, the variable (VH), diversity (D) and joining (JH) segments; whereas light-chain variable regions of either the kappa or lambda type are assembled from two elements, the VL and JL. Analysis of tumour cell lines or sorted cell populations which represent early and late pre-B cells has suggested that heavy-chain assembly and expression generally precedes that of light chains; but, primarily because of the lack of appropriate model systems to study the phenomenon, the mechanism and significance of this apparently orderly differentiation process are much debated. Here we describe for the first time a transformed cell line, 300-19, which sequentially undergoes all of the immunoglobulin gene rearrangement and expression events associated with the differentiation of pre-B cells to surface immunoglobulin-positive B lymphocytes. Analysis of the in vitro differentiation of 300-19 cells provides direct evidence for distinct differentiation phases of first VH and subsequently VL assembly during B-cell differentiation. Furthermore, these analyses suggest that the mu heavy chain, resulting from a productive VHDJH rearrangement, has both a positive and a negative regulatory role in mediating this ordered differentiation process, that is, signalling the cessation of VH gene assembly and simultaneously signalling the onset of VL assembly.     

Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines

The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.     

Interleukin-3 supports growth of mouse pre-B-cell clones in vitro

Study of the differentiation of immunoglobulin-producing B lymphocytes has been hampered by the inability to maintain homogeneous populations of precursor cells in vitro. We describe here that interleukin-3 supports the growth of freshly isolated fetal liver pre-B cells and the long-term culture of interleukin-3 dependent pre-B-cell clones that can be induced to mature into antibody secreting cells in vitro.     

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.     

Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing

Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.