Martina Podleschny

A BCR-JAK2 fusion gene as the result of a t(9;22)(p24;q11.2) translocation in a patient with a clinically typical chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by the presence of a t(9;22)(q34;q11.2), which leads to the well‐known BCR–ABL1 fusion protein. We describe a patient who was diagnosed clinically with a typical CML but on cytogenetic analysis was found to have a t(9;22)(p24;q11.2). Chromosomal fluorescence in situ hybridization showed that the BCR gene locus spanned the breakpoint at band 22q11.2 but that the ABL1 gene was not rearranged. By means of a candidate gene approach, the JAK2 gene, at 9p24, was identified as the fusion partner of BCR in this case. The BCR–JAK2 fusion protein contains the coiled‐coil dimerization domain of BCR and the protein tyrosine kinase domain (JH1) of JAK2. The patients disease did not respond to Imatinib, and this unresponsiveness was most likely a result of the BCR–JAK2 fusion protein.

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling.

Wnt signalling is an evolutionarily conserved pathway that directs cell‐fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co‐precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non‐canonical Wnt signalling by turning off the canonical signalling branch.

Identification of an ETV6–ABL2 fusion transcript in combination with an ETV6 point mutation in a T‐cell acute lymphoblastic leukaemia cell line

Summary. ETV6, a member of the Ets family of transcription factors, is frequently rearranged to various translocation partners in human leukaemias. We previously described a CD3+/TCRα/β+ mature T‐cell acute lymphoblastic leukaemia (T‐ALL) cell line, MT‐ALL, carrying a t(1;10;12)(q25; p13;p13) with cytokine‐inducible lineage switch into the myeloid lineage. Using reverse transcription polymerase chain reaction with primers complementary to ETV6 and ABL2, two ETV6–ABL2 fusion transcripts were identified in MT‐ALL which resulted from alternative splicing of an ABL2 exon. The fusion transcripts code for putative ETV6–ABL2 fusion proteins containing the pointed domain of ETV6 and almost the complete ABL2 protein, including the SH2, SH3 domains and the protein tyrosine kinase domain (PTK). Identical ETV6–ABL2 fusion transcripts have been reported in an acute myeloid leukaemia (AML) M3 cell line, carrying both a t(15;17)(q22;q21) and a t(1;12)(q25;p13) with unusual inducible differentiation to eosinophils, and in a patient with AML‐M4eo. Interestingly, the non‐rearranged allele of ETV6 in the MT‐ALL cell line carries an arginine to histidine (R399H) mutation which affects a conserved amino acid in the ets DNA binding domain.

Therapy‐associated genetic aberrations in patients treated for non‐Hodgkin lymphoma

Therapy‐associated myelodysplastic syndromes and acute myeloid leukaemia (t‐AML/MDS) following high dose chemotherapy are significant problems, with a cumulative incidence of 20% or more in myeloablative treatment regimen. Retrospective findings indicated that t‐AML/MDS associated genetic aberrations can be observed directly after exposure to chemotherapy and can precede t‐AML by several months. To determine the incidence of post‐therapeutic aberrations and their predictive value, we prospectively investigated 316 samples of 95 patients with non‐Hodgkin lymphoma (NHL) who were treated with intermediate and high dose chemotherapy (Arm A and B of the megaCHOEP (cyclophosphamide, doxorubicin, etoposide, vincristine, prednisolone) trial of the German High Grade NHL study group). Molecular aberrations (RUNX1/RUNX1T1, PML‐RARA, CBFB‐MYH11, MLL‐MLLT1, BCR‐ABL1) were observed in 33·3% (Arm A) and 55·4% (Arm B) of patients and in 14·9% and 28·7% of respective samples. Cytogenetic analysis of 53 NHL patients after high dose therapy showed frequent chromosomal breakage. Clonal aberrations were found in three patients. None of these patients developed a t‐AML/MDS during a 3‐year clinical follow up period. We concluded that the high incidence of genetic aberrations reflected a dose‐dependent, transient therapy‐induced genetic damage which is not predictive of a t‐AML/MDS.

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration.

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.

17β-Estradiol induces supernumerary primordial germ cells in embryos of the polychaete Platynereis dumerilii

In the polychaete Platynereis dumerilii exactly four primordial germ cells (PGCs) arise in early development and are subject to a transient mitotic arrest until the animals enter gametogenesis. In order to unravel the mechanisms controlling the number of PGCs in Platynereis, we tested whether the steroid 17β-estradiol (E2) is able to induce PGC proliferation, as it had been described in other species. Our data provide strong support for such a mechanism, showing that E2 significantly increases the occurrence of larvae with supernumerary PGCs in Platynereis in a dose dependent manner. E2 responsiveness is restricted to early developmental stages, when the PGCs are specified. During these stages, embryos exhibit high expression levels of the estradiol receptor (ER). The ER transcript localizes to the yolk-free cytoplasm of unfertilized eggs and segregates into the micromeres during cleavage stages. Nuclear ER protein is found asymmetrically distributed between daughter cells. Neither transcript nor protein is detectable in PGCs at larval stages. Addition of the specific estradiol receptor inhibitor ICI-182,780 (ICI) abolishes the proliferative effect of E2, suggesting that it is mediated by ER signaling. Our study reports for the first time an ER mediated proliferative effect of E2 on PGCs in an invertebrate organism.

PTK7 localization and protein stability is affected by canonical Wnt ligands

ABSTRACT Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved transmembrane receptor with important roles in embryonic development and disease. Originally identified as a gene upregulated in colon cancer, it was later shown to regulate planar cell polarity (PCP) and directional cell movement. PTK7 is a Wnt co-receptor; however, its role in Wnt signaling remains controversial. Here, we find evidence that places PTK7 at the intersection of canonical and non-canonical Wnt signaling pathways. In presence of canonical Wnt ligands PTK7 is subject to caveolin-mediated endocytosis, while it is unaffected by non-canonical Wnt ligands. PTK7 endocytosis is dependent on the presence of the PTK7 co-receptor Fz7 (also known as Fzd7) and results in lysosomal degradation of PTK7. As we previously observed that PTK7 activates non-canonical PCP Wnt signaling but inhibits canonical Wnt signaling, our data suggest a mutual inhibition of canonical and PTK7 Wnt signaling. PTK7 likely suppresses canonical Wnt signaling by binding canonical Wnt ligands thereby preventing their interaction with Wnt receptors that would otherwise support canonical Wnt signaling. Conversely, if canonical Wnt proteins interact with the PTK7 receptor, they induce its internalization and degradation. Summary: In the presence of canonical Wnt proteins, the regulator of planar cell polarity PTK7 is endocytosed and degraded in lysosomes. By contrast, PTK7 inhibits canonical Wnt signaling suggesting a mechanism of mutual inhibition.

MLL rearrangements emerge during spontaneous apoptosis of clinical blood samples.

The MLL gene (11q23) is frequently involved in genetic aberrations in de novo and therapy-related acute myeloid leukemia (t-AML). In t-AML, MLL translocations are detectable in up to 10% of all cases. Typically, these t-AML are characterized by a short latency period of up to 2 years and are associated with previous topoisomerase inhibitor treatment. The initiating event is thought to be a breakage at the topoisomerase binding site within the breakpoint cluster region (bcr; exons 5–11) of the MLL gene. In more than 70% of MLLpositive t-AML, the MLL breakpoint is localized in the telomeric region of the bcr, whereas in de novo AML, it is in the centromeric region. MLL aberrations (deletions, insertions, translocations) can be induced in normal hematopoietic progenitors in vitro by exposure to topoisomerase II inhibitors, and by retrospective analysis, t-AML-associated MLL aberrations have been backtracked up to a few months after topoisomerase II inhibitor therapy in clinical samples. As the incidence of t-AML may be up to 10% in high-dose, myeloablative treatment schedules, it is of certain interest to identify patients who are at an increased risk to attract this complication. Recently, Ng et al. investigated clinical samples of children who were treated with topoisomerase inhibitors/ anthracyclines by Southern blot and panhandle PCR and observed MLL aberrations (MLL breakage and illegitimate recombination) in 7% (5/71) of the patients. We sought to investigate prospectively whether MLL aberrations are detectable in adult patients with aggressive non-Hodgkin’s lymphoma treated with a myeloablative topoisomerase II inhibitor-containing regimen (MegaChoep trial of the German high-grade lymphoma study group, DSNHL). Samples, collected after informed consent and as approved by the Ethics Committee of the University of Goettingen, were subjected to the isolation of mononuclear cells, DNA isolation by DNAeasy System (Quiagen, Hilden, Germany), Xba1 digest and inverse genomic MLL-PCR (igPCR) for the therapyassociated MLL breakpoint cluster/topoisomerase binding site region as described. Processing of samples was carried out according to GLP guidelines for PCR. The mononuclear cells count was 5 10–5 10 and the DNA yield ranged between 4.5 and 300 mg. Four microgram was used for igPCR. We first studied a control group of adult (peripheral blood, n1⁄4 8) and newborn (cord blood, n1⁄4 10) individuals not exposed to chemotherapy and 18 peripheral blood samples of patients who underwent therapy. Surprisingly, aberrant amplification products (deletions, insertions), indicating MLL breakage and illegitimate recombination, were observed in mononuclear cells of the adult (5/8) and newborn cohort (8/10). The incidence of MLL aberrations in this healthy control cohort (13/18) was comparable to that of the patients (12/18) (see Figure 1). As the samples were processed up to 30 h after being taken, we supposed that MLL breakage and religation might occur during spontaneous apoptosis. We then performed a sequential investigation of 10 peripheral blood samples of healthy adults. After being taken, aliquots were either submitted directly to the isolation of mononuclear cells and further processing or were stored as whole blood at room temperature. The incidence of aberrant amplification products increased depending on the time interval between sample taking and sample processing. At 0 h, 0/10 samples, at 24 h, 5/10 samples and at 48 h, 7/10 samples exhibited aberrant amplification products (see Figure 2) parallel to the increase of apoptotic cells as determined by the annexin binding assay (data not shown). Sequencing of the aberrant amplification products in the control and patients cohort showed internal rearrangements in the MLL bcr (deletions and insertions), which were not MLL partial tandem duplications. No translocations have been observed so far. We conclude that MLL breakage and religation in the topoisomerase-sensitive bcr, leading to MLL rearrangements (deletions, insertions) in clinical samples, may occur not only after exposure to topoisomerase II inhibitors but also during spontaneous apoptosis of clinical samples. The MLL gene is sensitive to site-specific cleavage in the t-AML-associated bcr during the initial stages of apoptosis owing to a colocalized scaffold attachment region. Religation of cleaved MLL,