Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation

The t(8;21)(q22;q22) translocation, which fuses the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO), is one of the most frequent cytogenetic abnormalities associated with acute myelogenous leukemia (AML). It is seen in approximately 12 to 15% of AML cases and is present in about 40% of AML cases with a French-American-British classified M2 phenotype. We have generated a murine model of the t(8;21) translocation by retroviral expression of AML1-ETO in purified hematopoietic stem cells (HSC). Animals reconstituted with AML1-ETO-expressing cells recapitulate the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21) translocation. Primitive myeloblasts were increased to approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. Accumulation of late-stage metamyelocytes was also observed in bone marrow along with an increase in immature eosinophilic myelocytes that showed abnormal basophilic granulation. HSC numbers in the bone marrow of 10-month-posttransplant animals were 29-fold greater than in transplant-matched control mice, suggesting that AML1-ETO expression overrides the normal genetic control of HSC pool size. In summary, AMLI-ETO-expressing animals recapitulate many (and perhaps all) of the developmental abnormalities seen in human patients with the t(8;21) translocation, although the animals do not develop leukemia or disseminated disease in peripheral tissues like the liver or spleen. This suggests that the principal contribution of AML1-ETO to acute myeloid leukemia is the inhibition of multiple developmental pathways.     

NPM1 Deletion Is Associated with Gross Chromosomal Rearrangements in Leukemia

Background

NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated.

Methodology and Principal Findings

Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the “5q- syndrome” but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p = 0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion.

Conclusions and Significance

NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.     

Cell surface marker phenotypes and gene expression profiles of murine radiation-induced acute myeloid leukemia stem cells are similar to those of common myeloid progenitors

Radiation exposure induces acute myeloid leukemia (AML) in humans and mice. Recent studies postulated that AML stem cells of spontaneous human AML arise from hematopoietic stem cells. However, other studies support the possibility that short-lived committed progenitors transform into AML stem cells, accompanied by a particular gene mutation. It remains unclear whether AML stem cells are present in radiation-induced AML, and information regarding AML-initiating cells is lacking. In this study, we identified and analyzed AML stem cells of mice with radiation-induced AML. The AML stem cells were identified by transplanting 100 bone marrow cells from mice with radiation-induced AML. We injected 100 cells of each of seven cell populations corresponding to different stages of hematopoietic cell differentiation and compared the latencies of AMLs induced in recipient mice. The identified radiation-induced AML stem cells frequently displayed similarities in both CD antigen and gene expression profiles with normal common myeloid progenitors. The number of common myeloid progenitor-like AML stem cells was significantly increased in mice with radiation-induced AML, but the progeny of common myeloid progenitors was decreased. In addition, analysis of radiation effects on the hematopoietic system showed that common myeloid progenitor cells were extremely radiosensitive and that their numbers remained at low levels for more than 2?months after radiation exposure. Our results suggest that murine radiation-induced AML stem cells arise from radiosensitive cells at a common myeloid progenitor stage.     

miR-345-5p regulates proliferation,cell cycle,and apoptosis of acute myeloid leukemia cells by targeting AKT2

Acute myeloid leukemia (AML) is a malignant clonal hematopoietic disease, which is caused by hematopoietic stem cell abnormalities. Epigenetic regulation, especially of microRNAs (miRNAs), mostly results from external or environmental effects and is critical to AML. In this study, for the first time, we report that decreased expression of miR-345-5p facilitates the proliferation of leukemia cells in AML. Further study demonstrated that AKT1/2 was the target of miR-345-5p and was responsible for the dysregulation of leukemia cell proliferation and apoptosis. Inhibition of AKT1/2 ameliorated this malignant effect, which provides new insight into AML diagnosis, treatment, prognosis, and next-step translational investigations.     

Stem cell transplantation for leukemias following myelodysplastic syndromes or secondary to cytotoxic therapy

Two main forms of therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) have been recognized. The most frequent type, occurring after treatment with alkylating agents, is characterized by abnormalities of chromosomes 5 and/or 7 and t-MDS/AML following treatment with topoisomerase II inhibitors and is associated with molecular aberrations of MLL (11q23) and AML-1 (21q22). Individuals with certain polymorphisms associated with impaired detoxification of cytotoxic agents have an increased risk of developing MDS or AML after treatment of unrelated cancers. Multidrug chemotherapy is less effective for patients with MDS, or AML following MDS, or t-MDS/AML when compared with primary AML, and results in lower complete remission (CR) rates and lower long-term survival. Patients with good risk cytogenetic features, such as t(15; 17), t(8; 21) and inversion 16 are an exception as their treatment outcome is comparable with primary AML patients. Patients who attain a polyclonal and/or a cytogenetic CR may be candidates for autologous stem cell transplantation. For the remaining patients, the only curative option is allogeneic stem cell transplantation with stem cells from a histocompatible sibling or an alternative donor. Reduced intensity conditioning regimens may be considered for patients older than 50 years or patients with comorbidities. The advice is to treat patients early after diagnosis and preferably before progression as these patients have the highest chance of a favorable outcome.     

Advances in the treatment of acute myeloid leukemia: from chromosomal aberrations to biologically targeted therapy

We describe several recent advances in our understanding and treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) including the use of cytogenetics to classify these diseases and to identify therapies that are specific for the abnormalities. Cell lines have provided readily available and very relevant models to understand these diseases. The two clear successes include the use of retinoic acid for acute promyelocytic leukemia and tyrosine kinase inhibitors (e.g., imatinib) for chronic myelogenous leukemia. Very recent results suggest a particular activity of lenalidomide, an analogue of thalidomide, in MDS patients with deletions of the long arm of chromosome 5 (so-called 5q minus syndrome), and notable activity of azanucleoside DNA demethylating agents in MDS with loss of chromosome 7. However, for the vast majority of cytogenetic abnormalities found in AML/MDS, no specific therapies have been identified. The use of a variety of molecular biology techniques have identified a large number of genomic abnormalities; the challenge of the next several decades is to identify specific therapies for these molecular defects.     

Identification of a common microdeletion cluster in 7q21.3 subband among patients with myeloid leukemia and myelodysplastic syndrome

Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (−7/7q−) is a common nonrandom chromosomal abnormality found frequently in myeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML). Using a short probe-based microarray comparative genomic hybridization (mCGH) technology, we identified a common microdeletion cluster in 7q21.3 subband, which is adjacent to ‘hot deletion region’ thus far identified by conventional methods. This common microdeletion cluster contains three poorly characterized genes; Samd9, Samd9L, and a putative gene LOC253012, which we named Miki. Gene copy number assessment of three genes by real-time PCR revealed heterozygous deletion of these three genes in adult patients with AML and MDS at high frequency, in addition to JMML patients. Miki locates to mitotic spindles and centrosomes and downregulation of Miki by RNA interference induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. In addition, a recent report indicated Samd9 as a tumor suppressor. These findings indicate the usefulness of the short probe-based CGH to detect microdeletions. The three genes located to 7q21.3 would be candidates for myeloid tumor-suppressor genes on 7q.     

Integrated Genomic Analysis Implicates Haploinsufficiency of Multiple Chromosome 5q31.2 Genes in De Novo Myelodysplastic Syndromes Pathogenesis

Deletions spanning chromosome 5q31.2 are among the most common recurring cytogenetic abnormalities detectable in myelodysplastic syndromes (MDS). Prior genomic studies have suggested that haploinsufficiency of multiple 5q31.2 genes may contribute to MDS pathogenesis. However, this hypothesis has never been formally tested. Therefore, we designed this study to systematically and comprehensively evaluate all 28 chromosome 5q31.2 genes and directly test whether haploinsufficiency of a single 5q31.2 gene may result from a heterozygous nucleotide mutation or microdeletion. We selected paired tumor (bone marrow) and germline (skin) DNA samples from 46 de novo MDS patients (37 without a cytogenetic 5q31.2 deletion) and performed total exonic gene resequencing (479 amplicons) and array comparative genomic hybridization (CGH). We found no somatic nucleotide changes in the 46 MDS samples, and no cytogenetically silent 5q31.2 deletions in 20/20 samples analyzed by array CGH. Twelve novel single nucleotide polymorphisms were discovered. The mRNA levels of 7 genes in the commonly deleted interval were reduced by 50% in CD34+ cells from del(5q) MDS samples, and no gene showed complete loss of expression. Taken together, these data show that small deletions and/or point mutations in individual 5q31.2 genes are not common events in MDS, and implicate haploinsufficiency of multiple genes as the relevant genetic consequence of this common deletion.     

Ring chromosome 8 and translocation t(8;21) in a patient with acute myeloblastic leukemia

Recurrent translocation t(8;21)(q22;q22) acute myeloid leukemia (AML) is often associated with secondary chromosome changes of which the clinical significance is not clear since they do not seem to impair the prognosis. Uncommon chromosome changes may lead to the identification of leukemogenetic factors associated with t(8;21) since the AML1/RUNX1-ETO fusion gene resulting from the translocation is thought to be unable alone to induce leukemia. We here report a patient with AML, t(8;21) and ring chromosome 8 resulting in partial chromosome 8 deletion. Another patient with partial 8q deletion has been previously reported. It is suggested that more attention be paid to the genes located in distal 8q in relation to leukemogenesis.     

Knockdown of SALL4 Protein Enhances All-trans Retinoic Acid-induced Cellular Differentiation in Acute Myeloid Leukemia Cells

All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy.     

TIM-3 as a therapeutic target for malignant stem cells in acute myelogenous leukemia

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Recent studies have shown that many AML LSC-specific surface antigens could be such candidates. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In mouse models reconstituted with human AML LSCs or human hematopoietic stem cells, a human TIM-3 mouse IgG2a antibody with complement-dependent and antibody-dependent cellular cytotoxic activities eradicates AML LSCs in vivo but does not affect normal human hematopoiesis. Thus, TIM-3 is one of the promising targets to eradicate AML LSCs.     

Extra copy of 20q deletion, acute myelomonocytic leukemia (M4) and Niemann-Pick disease

A 59-year-old hypertensive white male was diagnosed with acute myelogenous leukemia (AML), M4. A bone marrow aspirate showed a karyotype of 46,XY,del(20)(q11.2q13.3)[12]/ 47,XY,del(20)(q11.2q13.3)x2[8]. The majority of cases with 20q deletion are associated with myeloid disorders; however, an extra copy of the 20q deletion has rarely been reported. The patient expired seven days after admission to the hospital. At autopsy hepatosplenomegaly was present. Many foamy macrophages with bubbling cytoplasm in the spleen, liver, bone marrow and lymph nodes were suggestive of Niemann-Pick disease, type E. AML has not previously been reported with Niemann-Pick disease.     

Assessment of the CTNNA3 gene encoding human alpha T-catenin regarding its involvement in dilated cardiomyopathy

Alpha T-catenin is a novel member of the alpha-catenin family, which shows most abundant expression in cardiomyocytes and in peritubular myoid cells of the testis, pointing to a specific function for alpha T-catenin in particular muscle tissues. Like other alpha-catenins, alpha T-catenin provides an indispensable link between the cadherin-based cell-cell adhesion complex and the cytoskeleton, to mediate cell-cell adhesion. By isolating genomic clones, combined with database sequence analysis, we have been able to determine the structure of the CTNNA3 and Ctnna3 genes, encoding human and mouse alpha T-catenin, respectively. The positions of the exon-exon boundaries are completely conserved in CTNNA3, Ctnna3, and the alpha N-catenin encoding CTNNA2 gene. They overlap largely with the boundaries of the CTNNA1 and CTNNAL1 genes encoding alpha E-catenin and alpha-catulin, respectively. This emphasizes that these alpha-catenin genes evolved from the same ancestor gene. Nevertheless, the introns of CTNNA3 and Ctnna3 are remarkably large (often more than 100 kb) compared with introns of other CTNNA genes. The CTNNA3 gene was mapped to chromosome band 10q21 by both fluorescence in situ hybridization and polymerase-chain-reaction-based hybrid mapping. This region encodes a gene for autosomal dominant familial dilated cardiomyopathy (DCM), a common cause of morbidity and mortality. As alpha T-catenin is highly expressed in healthy heart tissue, we have considered CTNNA3 as a candidate disease gene in a family showing DCM linkage to the 10q21-q23 locus. Mutation screening of all 18 exons of the CTNNA3 gene in this family has, however, not detected any DCM-linked CTNNA3 mutations.     

AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ～40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.     

Somatic cell hybrid mapping of human chromosome band 5q31: a region important to hematopoiesis.

As a means of characterizing the distal long arm of chromosome 5, in particular, the region spanning 5q23-->q31, we analyzed somatic cell hybrids prepared from cells with overlapping chromosomal rearrangements. In one hybrid, the derivative chromosome 5 from a patient with acute myeloid leukemia (AML) de novo, whose bone marrow cells had a balanced translocation, t(5;7)(q31;q22), involving chromosome band 5q31, was isolated in a somatic cell hybrid (B294). In addition, we prepared somatic cell hybrids from a lymphoblastoid cell line (CC) derived from a patient who has a constitutional interstitial deletion of chromosome 5 spanning 5q23.1-->q31.1. By a combination of Southern hybridization analysis and fluorescent in situ hybridization, we constructed a map dividing 5q23-->q31 into four regions. We can assign genes to these regions and relate them to anonymous RFLP markers that have been genetically mapped.     

Autophagy in the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia

Autophagy is a conserved cellular pathway responsible for the sequestration of spent organelles and protein aggregates from the cytoplasm and their delivery into lysosomes for degradation. Autophagy plays an important role in adaptation to starvation, in cell survival, immunity, development and cancer. Recent evidence in mice suggests that autophagic defects in hematopoietic stem cells (HSCs) may be implicated in leukemia. Indeed, mice lacking Atg7 in HSCs develop an atypical myeloproliferation resembling human myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML). Studies suggest that accumulation of damaged mitochondria and reactive oxygen species result in cell death of the majority of progenitor cells and, possibly, concomitant transformation of some surviving ones. Interestingly, bone marrow cells from MDS patients are characterized by mitochondrial abnormalities and increased cell death. A role for autophagy in the transformation to cancer has been proposed in other cancer types. This review focuses on autophagy in human MDS development and progression to AML within the context of the role of mitochondria, apoptosis and reactive oxygen species (ROS) in its pathogenesis.