Review of clinical, cytogenetic, and molecular aspects of Ph-negative CML

Abstract Between 1985 and 1989, many cases of Philadelphia (Ph) chromosome negative chronic myelogenous leukemia (CML) were reported. For this review, the following selection criteria were used: the original articles on Ph-negative cases should provide clinical, hematologic, cytogenetic as well as molecular data. In addition, eight unpublished cases of Ph-negative CML are included that were studied in our institute during the last two years. Our purpose was to correlate presence or absence of the Ph rearrangement with the clinical features in an attempt to test whether the entity “Ph-negative CML” really exists and to identify the pathologic characteristics, frequency of occurrence, prognosis for survival, and underlying molecular mechanisms. Data on Ph-negative CML patients were compared with data on Ph-positive CML, atypical CML (aCML), and chronic myelomonocytic leukemia (CMMoL), reported in the same papers as the Ph negative patients. Essential for comparison of data from the different investigators appeared to be a clear description of criteria they used to establish the diagnosis CML, or alternatively a complete presentation of data for all patients reported in the articles. In most cases. Ph-negative CML was distinguishable from CMMoL and aCML, using simple criteria, e.g., differential count of peripheral blood and absence of dysplasia in the bone marrow. Cytogenetic analysis showed normal karyotype in most cases of Ph-negative CML. Interestingly, in cases with abnormal karyotype, chromosome 9 band q34 was relatively frequently involved in translocations with other chromosomes than chromosome 22, suggesting a variant Ph translocation not visible by cytogenetic techniques. This assumption was confirmed by molecular analysis, demonstrating bcr-abl rearrangement in 9 out of 10 of the latter cases. Results of cytogenetic and molecular investigations in 136 cases of Ph-negative CML reviewed in this article clearly indicated that molecular techniques are valuable tools for identification of bcr-abl rearrangements, indicative for the Ph translocation. The different mechanisms responsible for bcr-abl rearrangement in Ph-negative CML patients are discussed. The question remains whether all Ph-negative CML patients will have bcr-abl rearrangements, or whether alternative mechanisms will be identified that are responsible for this disease

The restricted expression pattern of the POU factor Oct-6 during early development of the mouse nervous system

Oce-6 is a POU transcription factor that is thought to play a role in the differentiation of cells of neuroectodermal origin. To investigate whether the Oct-6 protein could play a role in the establishment of neuroectoderm in vivo we studied the expression of the Oct-6 protein during early mouse development. Expression is first observed in the primitive ectoderm of the egg cylinder stage embryo. In gastrulating embryos, Oct-6 protein is found in the extra-embryonic ectoderm of the chorion and the anterior ectoderm of the embryo proper. As development proceeds, Oct-6 expression becomes more restricted to the anterior medial part of the embryo until Oct-6 positive cells are observed only in the neural groove of the headfold stage embryo. In the late headfold stage embryo, Oct-6 expression is detected in the neuroepithelium of the entire brain and later is restricted to a more ventral and anterior position. As the anterior neuropore closes, Oct-6 protein is detected in a segment-like pattern in the mid- and forebrain. Thus, the expression pattern of the Oct-6 gene agrees with a role for the Oct-6 protein in the establishment and regional specification of the neuroectoderm in vivo. The two waves of widespread induction of the Oct-6 gene, one in the primitive ectoderm and another in the primitive brain, both followed by a progressive restriction in the expression patterns suggest a mechanism for the regulation of the gene

AML1, the Target of Multiple Chromosomal Translocations in Human Leukemia, Is Essential for Normal Fetal Liver Hematopoiesis

AbstractThe AML1–CBFβ transcription factor is the most frequent target of chromosomal rearrangements in human leukemia. To investigate its normal function, we generated mice lacking AML1. Embryos with homozygous mutations in AML1 showed normal morphogenesis and yolk sac–derived erythropoiesis, but lacked fetal liver hematopoiesis and died around E12.5. Sequentially targeted AML1−/− ES cells retained their capacity to differentiate into primitive erythroid cells in vitro; however, no myeloid or erythroid progenitors of definitive hematopoietic origin were detected in either the yolk sac or fetal livers of mutant embryos. Moreover, this hematopoietic defect was intrinsic to the stem cells in that AML1−/− ES cells failed to contribute to hematopoiesis in chimeric animals. These results suggest that AML1-regulated target genes are essential for definitive hematopoiesis of all lineages

FOXO1a acts as a selective tumor suppressor in alveolar rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common pediatric soft-tissue sarcoma, has two major histological subtypes: embryonal RMS (ERMS), which has a favorable prognosis, and alveolar RMS (ARMS), which has a poor outcome. Although both forms of RMS express muscle cell–specific markers, only ARMS cells express PAX3-FOXO1a or PAX7-FOXO1a chimeric proteins. In mice, Pax3 and Pax7 play key roles in muscle cell development and differentiation, and FoxO1a regulates myoblast differentiation and fusion; thus, the aberrant regulation of these proteins may contribute to the development of ARMS. In this paper, we report that FOXO1a is not expressed in primary ARMS tumors or ARMS-derived tumor cell lines and that restoration of FOXO1a expression in ARMS cells is sufficient to induce cell cycle arrest and apoptosis. Strikingly, the effects of FOXO1a are selective, as enforced expression of FOXO1a in ERMS-derived tumor cell lines had no effect. Furthermore, FOXO1a induced apoptosis in ARMS by directly activating the transcription of caspase-3. We conclude that FOXO1a is a potent and specific tumor suppressor in ARMS, suggesting that agents that restore or augment FOXO1a activity may be effective as ARMS therapeutics

Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.[Author Summary]: Many cancers carry recurrent chromosome translocations, which often result in the formation of fusion genes that are directly involved in the tumorigenic process. Alveolar rhabdomyosarcoma, a muscle tumor in children, is typified by a translocation that fuses the PAX3 gene on chromosome 2 to the FOXO1 gene on chromosome 13. For translocation to occur both genes need to break and the disparate ends need to fuse via a process called non-homologous end joining. We determined that physical proximity of Pax3 and Foxo1 in mouse muscle progenitor cells (myoblasts) facilitates fusion gene formation. Because Pax3 and Foxo1 in the mouse are in an opposite orientation, we used a chromosome engineering strategy to invert the orientation of Foxo1 so that upon translocation a productive Pax3-Foxo1 fusion gene is created. Co-localization of the Pax3 and Foxo1 loci is higher in fore limb than in hind limb myoblasts. Simultaneous induction of a targeted double strand DNA break in each gene by CRISPR-Cas9 nuclease generated more fusion genes in fore limb than in hind limb myoblasts. Thus, gene proximity facilitates fusion gene formation. We propose that CRISPR-Cas9 nuclease can be used for the precise modeling of chromosome translocations of human cancer in mice.This work was supported by the Van Vleet foundation of Memphis and ALSAC of St Jude Children’s Research Hospital and the Cancer Center Core Grant 5P30CA021765-34. BVB and JJC were supported by grant C1178/A4520 from Cancer Research UK.Peer Reviewe

Translocation of BCR to chromosome 9: A new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia

Leukemic cells from two patients with Philadelphia-negative chronic myeloid leukemia (CML) were investigated: I) Cytogenetics showed a normal 46.XY karyotype in both cases, 2) molecular studies revealed rearrangement of the M-BCR region and formation of BCR-ABL fusion mRNA with b2a2 (patient I) or b3a2 (patient 2) configuration, and 3) fluorescence in situ hybridization (FISH) demonstrated relocation of the 5′ BCR sequences from one chromosome 22 to one chromosome 9. The ABL probe hybridized to both chromosomes 9 at band q34, while two other probes which map centromeric and telomeric of BCR on 22q 11 hybridized solely with chromosome 22. For the first time, a BCR-ABL rearrangement is shown to take place on 9q34 instead of in the usual location on 22q 11. A rearrangement in the latter site is found in all Ph-positive CML and in almost all investigated CML with variant Ph or Ph-negative, BCR-positive cases. The few aberrant chromosomal localizations of BCR-ABL recombinant genes found previously were apparently the result of complex and successive changes. Furthermore in patient 2, both chromosomes 9 showed positive FISH signals with both ABL and BCR probes. Restriction fragment length polymorphism (RFLP) analysis indicated that mitotic recombination had occurred on the long arm of chromosome 9 and that the rearranged chromosome 9 was of paternal origin. The leukemic cells of this patient showed a duplication of the BCR-ABL gene, analogous to duplication of the Ph chromosome in classic CML. In addition they had lost the maternal alleles of the 9q34 chromosomal region. The lymphocytes of patient 2 carried the maternal chromosome 9 alleles and were Ph-negative as evidenced by RFLP and FISH analyses, respectively. © 1993 Wiley-Liss, Inc

Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.

The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein

The Leukemia-Associated Fusion Protein MN1-TEL Blocks TEL-Specific Recognition Sequences

The leukemia-associated fusion protein MN1-TEL combines the transcription-activating domains of MN1 with the DNA-binding domain of the transcriptional repressor TEL. Quantitative photobleaching experiments revealed that ~20% of GFP-tagged MN1 and TEL is transiently immobilised, likely due to indirect or direct DNA binding, since transcription inhibition abolished immobilisation. Interestingly, ~50% of the MN1-TEL fusion protein was immobile with much longer binding times than unfused MN1 and TEL. MN1-TEL immobilisation was not observed when the TEL DNA-binding domain was disrupted, suggesting that MN1-TEL stably occupies TEL recognition sequences, preventing binding of factors required for proper transcription regulation, which may contribute to leukemogenesis