C-abl and bcr are rearranged in a Ph1-negative CML patient.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.     

Lysis of fresh leukemic blasts by interferon-activated human natural killer cells

In a comprehensive study of 30 leukemia patients, it was found that a measurable fraction of fresh leukemic blasts from 8 of 8 adult patients with chronic myelogenous leukemia (CML) in blast crisis and 10 of 11 pediatric patients with childhood acute lymphocytic leukemia (ALL) were efficiently lysed by human peripheral blood natural killer (NK) cells as measured in 4-hour chromium release assays. The observed lysis of these fresh, noncultured, neoplastic blasts was mediated by a population of interferon-augmentable, FcR-positive, non-adherent large granular lymphoid cells from normal donors, which were also able to kill the 'standard' NK target K562. It was of further interest that all 8 of the patients with blast crisis CML exhibited myeloid type morphology. Furthermore, neoplastic lymphoblasts from 9 of 10 patients with NK-susceptible childhood ALL lacked easily detectable B or T cell markers and were of 'null' cell type. In marked contrast to the lytic susceptibility of fresh leukemic blasts from patients with ALL and CML in blast crisis, fresh neoplastic granulocytes from 5 patients with chronic phase CML (2 of which eventually progressed to myeloid type blast crisis), as well as leukemic blasts from 8 patients with acute myeloid leukemias (AML, AMMoL, and AMoL) were resistant to lysis as mediated by human NK cells from normal donors. The clinical implications of these findings are discussed.     

Influence of BCR/ABL fusion proteins on the course of Ph leukemias

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.     

Unique fusion of bcr and c-abl genes in Philadelphia chromosome positive acute lymphoblastic leukemia

The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is translocated to the Ph chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a new 190 kd c-abl protein has been identified, no breakpoints are observed in the bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome 22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb mRNA, encoding a novel fusion protein.     

Philadelphia chromosome-positive acute lymphoblastic leukemia- current concepts and future perspectives

Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) is diagnosed rarely in children, but constitutes the most frequent cytogenetic abnormality in adults with ALL. In contrast to chronic myeloid leukemia (CML), patients with Ph-positive ALL usually demonstrate expression of a truncated version of the BCR-ABL protein called p190bcr-abl. Irrespective of age and breakpoint location, Ph-positive ALL carries a poor prognosis. Although remission rates are identical to those of Ph-negative ALL, relapse is almost universal and long-term survival remains rare. Given the poor outcome with current chemotherapy consolidation programs, stem cell transplantation is usually recommended for these patients in first remission or as soon as feasible. Even with transplantation the impact on outcome is limited and new therapeutic concepts are urgently needed. One of the most promising developments in recent years has been the introduction of the tyrosine kinase inhibitors such as STI571. An overview of current treatment modalities in Ph-positive ALL will be provided and the rationale for new therapies will be discussed.     

Investigations on karyotype evolution in patients with chronic myeloid leukemia (CML)

In an attempt to relate karyotype evolution to clinical and hematological data serial chromosomal analyses were performed in 31 patients with chronic myeloid leukemia (CML), both in chronic and acute phases. Our results in Philadelphia chromosome (Ph1)-positive CML are in line with karyotype profiles described in the literature. In addition, we report on chromosomal findings in 4 cases of Ph1-negative disease, one presenting with an iso17q chromosome in the positive CML. The same chromosomal abnormality was observed in a small population of Ph1-negative cells present in one of two patients with mixed Ph1-positive/Ph1-negative CML. The first case of a female patient with the loss of a sex chromosome in Ph1-positive cells is reported. Two patients with unusually long and mild chronic phases despite the presence of trisomy 8 in their karyotypes are described. Our findings suggest that the order of appearance of additional chromosomal changes of CML is of prognostic significance for the progression and the clinical picture of the disease.     

BCR-ABL1-induced leukemogenesis and autophagic targeting by arsenic trioxide

We have recently shown that arsenic trioxide (As₂O₃) is a potent inducer of autophagic degradation of the BCR-ABL1 oncoprotein, which is the cause of chronic myeloid leukemia (CML) and Ph+ acute lymphoid leukemia (Ph+ ALL). Our recently published work has shown that pharmacological inhibition of autophagy or molecularly targeting of elements of the autophagic machinery partially reverses the suppressive effects of As₂O₃ on primitive leukemic precursors from CML patients. Altogether, our studies have provided direct evidence that arsenic-induced, autophagy-mediated, degradation of BCR-ABL1 is an important mechanism for the generation of the effects of As₂O₃ on BCR-ABL1 transformed leukemic progenitors. These studies raise the potential of future clinical-translational efforts employing combinations of arsenic trioxide with autophagy-modulating agents to promote elimination of early leukemic progenitors and, possibly, leukemia-initiating stem cells.     

Immunoglobulin gene organisation and expression in haemopoietic stem cell leukaemia

We have analysed the organisation and expression of mu genes in the granulocytic phase and in the lymphoid and myeloid blast crises of Philadelphia chromosome (Ph1) chronic granulocytic leukaemia (CGL), a leukaemia which is known to arise in multipotential stem cells. We find that mu chain gene rearrangement occurs exclusively in lymphoid blast crisis leading in some, but not all, cases to the synthesis of small amounts of cytoplasmic mu chains characteristic of early pre-B lymphocytes. In Southern blots, only one or two rearranged mu chain genes are seen, suggesting that a clonal event leading to blast crisis can occur in a committed B cell precursor rather than in the multipotential stem cell precursor, in which the Ph1 chromosome originated. The pattern of mu gene rearrangement observed in Ph1 CGL blast crisis is compared with that in normal B cells, other B lineage malignancies, myeloid leukaemias and T cell leukaemias.     

Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene

In order to elucidate molecular events in BCR/ABL-induced transformation, we adopted a polymerase chain reaction (PCR)-based technique of differential display and compared mRNA expression in human factor-dependent cells, TF-1, with that in factor-independent cells, ID-1, which were established from TF-1 cells by transfection of BCR/ABL. Cloning and sequencing of a gene which was upregulated in ID-1 cells revealed that the gene was identical to a melanoma antigen, PRAME. Our present study demonstrated that PRAME was markedly expressed in primary leukemic cells with chronic myeloid leukemia (CML) in blastic crisis and Philadelphia (Ph)+-acute lymphoblastic leukemia (ALL), in which BCR/ABL played an important role as a pathogenic gene. Moreover, comparison of PRAME expression among CD34+ cells with CML in blastic, accelerated, and chronic phases revealed a higher expression in CML in advanced phases. Thus PRAME was considered to be a good candidate for a marker of Ph+-leukemic blast cells as well as a new target antigen of leukemic blast cells that cytotoxic T cells can recognize.     

A 41-kilodalton protein is a potential substrate for the p210bcr-abl protein-tyrosine kinase in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.     

Fine mapping of chromosome 22 breakpoints within the breakpoint cluster region (bcr) implies a role for bcr exon 3 in determining disease duration in chronic myeloid leukemia.

The chromosomal translocation that fuses the phl gene with the c-abl proto-oncogene appears to be a pivotal step in the pathogenesis of some leukemias. In chronic myeloid leukemia (CML) the breakage within the phl gene is largely confined to a 5.8-kb segment referred to as the breakpoint cluster region (bcr). To determine whether the presence of specific bcr exons on the Philadelphia chromosome has any clinical significance, we have analyzed the bcr breakpoints in 134 patients with CML. As many as five probes were used in this analysis, including a synthetic oligonucleotide probe homologous to the bcr exon 3 (phl exon 14) region. The distribution of breakpoints indicates that, in fact, breakage is largely confined to a 3.1-kb segment lying between bcr exon 2 and exon 4 (phl exons 13-15). In 61 CML patients analyzed within 1 year of diagnosis, the distribution of breakpoints appeared to be random within the 3.1-kb region. However, a significant excess of 5' breakpoints was observed in the total population studied, consistent with previous data showing that patients with 3' breakpoints have shorter disease durations. Analysis using the bcr exon 3 sequence probe indicated it was probably the presence or absence of bcr exon 3 on the Philadelphia chromosome that accounts for some of the variability in disease duration seen in CML. The data suggest that the phl/abl protein product may influence the timing of the onset of blast crisis and imply a continuing role for this protein during the evolution of the disease.     

Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Structural alterations of the BCR and ABL genes in Ph1 positive acute leukemias with rearrangements in the BCR gene first intron: further evidence implicating Alu sequences in the chromosome translocation.

In the Philadelphia positive bcr negative acute leukemias (Ph1+bcr- AL), the chromosomal breakpoints on chromosome 22 have been shown clustered within 10.8kb (bcr2) and 5kb (bcr3) fragments of the first intron of the BCR gene. We previously reported that the breakpoints were localized in Alu repeats on chromosomes 9 and 22 in a Ph1+bcr- acute lymphoblastic leukemia with a rearrangement involving bcr2. Molecular data of two other Ph1 translocations, one a Ph1+bcr- acute myeloblastic leukemia in the bcr2 region, and the other an acute lymphoblastic leukemia in the bcr3 region are presented. In the former, the breakpoints on chromosomes 9 and 22 are localized in Alu repeats, in regions with two inverted Alu sequences, as in our previously reported case. In the second leukemia, the breakpoints are not located in Alu sequences, but such repeats are found in their vicinity. The implications of these findings are discussed.     

bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

The chromosome translocation forming the hybrid bcr-abl gene is thought to be the initiating event in chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. To assess the impact of bcr-abl upon haemopoiesis, lethally irradiated mice were reconstituted with bone marrow cells enriched for cycling stem cells and infected with a bcr-abl bearing retrovirus. The mice developed several fatal diseases with abnormal accumulations of macrophage, erythroid, mast and lymphoid cells, and marked strain differences in disease distribution and kinetics. Some mice exhibited more than one neoplastic cell type and, in some instances, these were clonally related, indicating that a progenitor or stem cell had been transformed. While classical CML was not observed, the macrophage tumours were accompanied by a mild CML-like syndrome, probably due to myeloid growth factor production by tumour cells. The erythroid and mast cell diseases were rarely transplantable, in contrast to the macrophage tumours and lymphomas, but all disease types displayed limited clonality. These results establish that bcr-abl confers a proliferative advantage on diverse haemopoietic cells but complete transformation probably involves additional genetic changes.     

Chromosomal rearrangements with a common breakpoint at 6p23 in five cases of myeloid leukemia

Rearrangement of the short arm of chromosome 6 with a breakpoint at 6p23 was found in five patients with myeloid leukemia. Three of them had different morphological variants of AML (M2, M3, M4) and two blastic crisis of Ph1 negative and Ph1 positive CML. Identical translocation, t(6;9)(p23;q34), was revealed in two patients. One of them had AML (M2), the other blastic crisis of Ph1 negative CML. The blast cells of the last patient were morphologically similar to those in the M2 variant of AML. Translocation (6;9)(p23;q34) was also detected in two AML patients of Rowley and Potter (1976). The role of the breakpoint at 6p23 in myeloid malignancies needs further investigation.