The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFalpha2 transcription factor

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.     

Clinical and cytological features of acute myelogenous leukemia with 8; 21 chromosome translocation

Eight cases of acute myelogenous leukemia with (8; 21) translocation were reported. As recently reported, they showed following features: M2 morphology in FAB classification (all 8 patients), abnormal granulocyte maturation, i.e. large granules and pseudo Pelger-Huet forms (5), Auer rods (8), occasional eosinophilia (2), frequent loss of one sex chromosome (5), the low neutrophil alkaline phosphatase activity (5), and tumor formation (one). Both CD13 and CD33 antigens were expressed on smaller number of leukemic cells than the other AML (M2) cells, whereas CD34 and HLA-DR antigens were expressed on higher number of cells. Interestingly CD19 antigen was detected on a small to large population of tumor cells from four out of six patients. Despite the high remission rate, many of them relapsed within one year. More intensive postinduction and maintenance therapy should be considered for those patients.     

Bone marrow eosinophilia as a prognostic indicator in acute myelogenous leukemia with 8;21 translocation.

The pretreatment characteristics of nine acute myelogenous leukemia (AML) patients with 8;21 translocation were evaluated to assess their value as prognostic indicators. All patients had AML-M2 according to French-American-British classification. The percentage of eosinophils in the bone marrow (EO/BM) correlated negatively with the percentage of blast cells in the peripheral blood (BL/PB) (p less than 0.05) and positively with complete remission duration (p less than 0.01). Three of the six patients with EO/BM under 5% showed aggressive clinical courses complicated with extra marrow lesions, while the patient with the highest EO/BM was the only one who survived for 5 years. These findings suggest that bone marrow eosinophilia could be useful as a prognostic indicator in AML patients with t(8;21).     

The 8;21 chromosome translocation in acute myeloid leukemia is always detectable by molecular analysis using AML1

The AML1 gene was rearranged in leukemic cells with t(8;21)(q22;q22) or its variant, complex t(8;V;21) translocations from 33 acute myeloid leukemia (AML) patients. The AML1 rearrangement was also detected in three AML patients without t(8;21); two had a normal diploid karyotype, and one had a karyotype of 45,X, - X. The AML1 rearrangement in the t(8;21) breakpoint cluster region was not detected in leukemic cells with cytogenetic abnormalities other than t(8;21), or with normal diploidy obtained from 23 AML patients. Because leukemic cells of the five patients with complex t(8;V;21) translocations had a der(8)t(8;21) chromosome with a break in band 8q22 in common, the juxtaposition of the 5' side of AML1 to a predicted counterpart gene located in the breakpoint region of 8q22 may be an essential step in the leukemogenesis of AML with t(8;21). Our findings show that the 8;21 translocation, its variants, and the masked t(8;21) may all be detectable by the Southern hybridization method using the AML1 probes.     

Association of a chromosomal 3;21 translocation with the blast phase of chronic myelogenous leukemia

An identical reciprocal translocation between the long arms of chromosomes 3 and 21 with breakpoints in bands 3q26 and 21q22, t(3;21)(q26;q22), was found in three male patients with the blast phase of chronic myelogenous leukemia (CML). The abnormality was clonal in all three patients and was always accompanied by either a standard or variant 9;22 translocation resulting in a Philadelphia chromosome (Ph1). In two cases, the t(3;21) was the only abnormality other than a t(9;22) in the primary clone. Serial studies of one patient demonstrated that the t(3;21) occurred as a result of clonal evolution near the time of development of the blast phase. We have not observed the t(3;21) in greater than 500 patients with CML in the chronic phase. Thus, the t(3;21) is a new recurring cytogenetic abnormality associated with the blast phase of CML.     

A variant 8;21 translocation in acute myeloblastic leukemia

A three-way rearrangement involving chromosomes 1, 8, and 21 was observed in bone marrow cells from a female with acute myeloblastic leukemia, subtype M2 (AML-M2). The translocation break points in chromosomes 8 and 21 were identical to those found in the standard t(8;21) that is associated with this disease type. A review of this and four previously reported cases involving variant 8;21 translocations indicates that a consistent pattern of exchange may be involved in these complex rearrangements even though the third chromosome affected may differ.     

Isolation of a yeast artificial chromosome spanning the 8;21 translocation breakpoint t(8;21)(q22;q22.3) in acute myelogenous leukemia.

The 8;21 translocation is one of the most common specific rearrangements in acute myelogenous leukemia. We have identified markers (D21S65 and a Not I boundary clone, Not-42, referred to as probe B) flanking the chromosome 21 translocation breakpoint (21q22.3) that demonstrate physical linkage in normal genomic DNA, by using at least three restriction endonucleases (Not I, Sac II, and BssHII), and that are located not more than 250-280 kilobases apart. Pulsed-field gel analysis of DNA from somatic cell hybrids containing the 8;21 translocation chromosomes demonstrates rearrangement of these markers. A 470-kilobase yeast artificial chromosome, YAC-Not-42, has been isolated that contains both probes. Mapping of lambda subclones constructed from YAC-Not-42 suggests that greater than 95% (25/26 probes tested) of the yeast artificial chromosome DNA is located on the proximal (D21S65) side of the breakpoint. In situ hybridization studies using metaphase chromosomes from five acute myelogenous leukemia patients with the 8;21 translocation confirmed these results and demonstrated the translocation of probe B to the derivative chromosome 8. A chromosome walk of approximately 39 kilobases from probe B has allowed identification of the breakpoint in DNA from a somatic cell hybrid containing the derivative chromosome 8. Since probe B contains conserved DNA sequences and is in close proximity to the translocation breakpoint, it may represent a portion of the involved gene on chromosome 21.     

Hematologic and cytologic characterization of 8/21 translocation acute granulocytic leukemia.

Cytogenetic studies were performed in 546 patients with acute leukemia between 1968 and 1975. Two hundred thirty-four patients were aneuploid (42.9%), and 312 patients were diploid (57.1%). Among these, 32 patients were found to exhibit similar chromosomal alterations that appeared to involve specifically chromosomes 8 and 21. Banding studies in at least 15 of these patients confirmed the presence of a translocation between these two chromosomes. The cytogenetic findings were correlated with the hematologic and clinical data. It was found that each of these individuals had a typical picture of acute granulocytic leukemia with Auer rod-positive and peroxidase-positive cells. Ultrastructurally, the patients in this group also consistently demonstrated the presence of a nuclear bleb that has been positively associated with aneuploidy in acute leukemia. Clinically, they seemed to respond better to therapy than other adult patients with acute granulocytic leukemia. It is proposed that the 8/21 translocation acute leukemia represents a definite subgroup within the general category of acute granulocytic leukemia, with an incidence of approximately 7.3%.     

Recurrent extramedullary relapse of acute myelogenous leukemia after allogeneic hematopoietic stem cell transplantation in a patient with the chromosomal abnormality t(8;21) and CD56-positivity

Isolated extramedullary (EM) relapse of acute myelogenous leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is rare. Predisposing factors include CD56 expression and the chromosomal abnormality t(8;21). We describe an AML patient showing the chromosomal abnormality t(8;21) and CD56 expression who experienced a unique EM relapse after allo-HSCT. Approximately 10 months after allo-HSCT, he experienced relapse involving the femur and lumbar vertebrae and, subsequently, an EM relapse of the stomach. Although we administered only local radiotherapy and not systemic chemotherapy, he showed no bone marrow relapse on long-term follow-up after achieving complete hematological remission. These findings suggest that the graft-versus-leukemia effect may preferentially maintain marrow remission rather than prevent EM relapse. In addition, our findings show that extended survival is possible after EM relapse following allo-HSCT in patients with marrow hematopoiesis of donor origin, and that augmentation of the graft-versus-leukemia effect may be useful.     

In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia

Recent reports have established the prenatal origin of leukemia translocations and resultant fusion genes in some patients, including MLL-AF4 translocations in infants and TEL-AML1 translocations in children. We now report evidence for the prenatal origin of a translocation in childhood acute myeloid leukemia (AML). The t(8;21) AML1-ETO translocations were sequenced at the genomic level in 10 diagnostic leukemia samples from children with available neonatal Guthrie blood spots. Clonotypic genomic AML1-ETO sequences were detected in the Guthrie spots for 5 individuals, providing unambiguous evidence of prenatal origin in these cases. Two of these patients were older than 10 years of age at diagnosis, indicative of a protracted postnatal latency. Three of the patients were assessed for the persistence of genomic fusion sequences in complete clinical remission samples and were found to be positive. These data indicate that t(8;21) in childhood AML can arise in utero, possibly as an initiating event in childhood AML, and may establish a long-lived or stable parental clone that requires additional secondary genetic alterations to cause leukemia.     

Fibrinogen — proteolysis in acute myelogenous leukemia (AML)

Summary Fibrinopeptides were measured as direct indices of thrombin, plasmin and elastase in plasma samples obtained from patients with AML. Peptide patterns observed were consistent with spontaneous or drug induced plasmin-specific fibrinogenolysis (AML FAB M 1/3), elastase mediated proteolysis (AML FAB M 3/4) or DIC (AML FAB M 4/5). DIC was also observed in septic, agranulocytotic patients.Presented in part at the XX Congress of the International Society of Hematology, September 1–7, 1984, Buenos Aires, Argentina; Supported by Deutsche Forschungsgemeinschaft (grant Ec 58-3/2)This paper is dedicated to the memory of Dr. H. L. Nossel, who most generously supplied reagents for this studyThe doctoral thesis of Michael Koch is partly presented in this work     

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34⁺ cells, especially the LSC-enriched CD34⁺CD38⁻ subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34⁺ cells but not those from normal CD34⁺ cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide–down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.Acute myelogenous leukemia (AML) represents a group of clonal hematopoietic stem cell disorders, in which a small subpopulation of leukemia stem cells (LSCs) are responsible for the accumulation of large numbers of immature myeloblasts in the bone marrow of AML patients. In addition to their crucial roles in leukemia initiation and progression, LSCs are also responsible for the high frequency of relapse that is characteristic of current AML therapies. Of patients receiving treatment with curative intent, less than one-half will achieve long-term survival (1). Similar to normal hematopoietic stem cells (HSCs), LSCs exhibit stem cell-like characteristics such as the capacity for self-renewal, differentiation potential, and relative quiescence (2, 3). The quiescent feature renders LSCs resistant to conventional chemotherapeutic agents that predominantly target proliferating rather than quiescent cells (1). For this reason, it is not surprising that relapse occurs in the majority of cases; this is further supported by recent studies showing that AML patients with LSCs enrichment have worse clinical outcomes (4–7). It is therefore crucial that therapies be developed targeting the quiescent and drug-resistant LSCs.Despite the similarities shared by LSCs and HSCs, LSCs often possess several unique features as well, which may provide important hints for designing LSC-targeted therapy. For instance, LSCs are usually associated with the abnormal expression of CD markers (e.g., CD44, CD47, CD96, and CD123), constitutive activation of nuclear factor κB (NF-κB), active Wnt/β-catenin signaling, and elevated levels of interferon regulatory factor-1 (IRF-1) and death-associated protein kinase (DAPK) (8–12). Most recently, emerging evidence points to oxidative signaling as being a two-edged sword in AML: moderate levels of reactive oxygen species (ROS) are important for driving disease, whereas higher levels result in cell death (13–15). The dual roles of oxidative signaling suggest that LSCs, in comparison with normal HSCs, are more vulnerable to ROS-generating agents. Accordingly, pharmacological agents favoring the generation of ROS are worth exploring in LSC-targeted therapy. Indeed, ROS induction has been shown as a critical mechanism for the selective eradication of LSCs by several compounds, such as parthenolide (PTL), dimethyl-aminoparthenolide (DMAPT), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), and 4-hydroxynonenal (HNE) (16–19).Another promising agent that could be used in this regard is fenretinide, a synthetic retinoid that lacks a carboxyl functional group likely necessary for retinoid receptor activity (20). We and others have previously demonstrated that fenretinide, unlike classical retinoids that often induce differentiation, triggers apoptotic effects; it is largely achieved through the generation of ROS (21–24), enhanced cellular ceramide, and/or ganglioside D3 (25). Moreover, several key stem cell survival-associated signaling pathways, such as NF-κB, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), have been reported to be inactivated in the fenretinide-induced apoptosis in different cancer cell types (25, 26); this further suggests the therapeutic value of fenretinide in targeting cancer stem cells.Fenretinide has been used clinically for some time as an effective chemopreventive agent for various cancers (27). It can significantly reduce the risk of breast cancer and small cell lung cancer (28, 29), suggesting an ability to prevent the development of cancer and/or eliminate early-stage malignant cells (likely cancer-initiating cells). Furthermore, long-term clinical trials have demonstrated only minimal side effects in patients receiving fenretinide (28, 30–32). In particular, no significant hematopoietic toxicity has been observed in patients treated with fenretinide (28). To illustrate the potential value of fenretinide in AML therapies, in this study, we defined the fenretinide effects on primary AML CD34⁺ cells and LSC-enriched AML CD34⁺CD38⁻ cells.