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Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and 2 novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood. We cloned coding sequences of AIF1L-ETV6 and ABL1-AIF1L in-frame fusion transcripts from the patient's leukemic blasts and we show that the chimeric protein containing the DNA binding domain of ETV6 is expressed from the AIF1L-ETV6 transcript and localized in both the cytoplasm and nucleus of transfected HEK293T cells. Transcriptomic and genomic profiling of the diagnostic bone marrow sample revealed Ph-like gene expression signature and loss of the IKZF1 and CDKN2A/B genes, the typical genetic lesions accompanying ETV6-ABL1-positive ALL. The prenatal origin of the rearrangement confirms that ETV6-ABL1 is not sufficient to cause overt leukemia, even when combined with the 2 novel fusions. We did not find the AIF1L-ETV6 and ABL1-AIF1L fusions in other ETV6-ABL1-positive ALL. Nevertheless, functional studies would be needed to establish the biological role of AIF1L-ETV6 and ABL1-AIF1L and to determine whether they contribute to leukemogenesis and/or to the final leukemia phenotype.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL–SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL–SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL–SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs and widespread vasculopathy. Current therapies for SSc focus on treating specific symptoms, but disease-modifying agents targeting the underlying pathogenesis are lacking. The pathogenesis of SSc involves activation of profibrotic pathways, with over-expression of the cytokines transforming growth factor (TGF)-β and platelet derived growth factor (PDGF). A recent report showed that SSc patients have autoantibodies against the PDGF receptor, which stimulate the production of reactive oxygen species and type I collagen expression(1). PDGF receptors are upregulated in the skin and bronchoalveolar lavage fluid of patients with SSc, and when activated, lead to fibroblast and myofibroblast proliferation(2, 3). PDGF participates in smooth muscle cell recruitment and mitogenic signaling that underlie the vasculopathy associated with pulmonary arterial hypertension (PAH), a complication of SSc associated with high mortality(4). In addition, stimulation of the TGF-β profibrotic pathway involves activation of c-Abl(2). Thus, the PDGF and TGF-β pathways are thought to contribute to the fibrotic and vascular complications in SSc. Imatinib mesylate (Gleevec, Novartis, East Hanover, New Jersey) is a small molecule that antagonizes specific tyrosine kinases that mediate fibrotic pathways, including c-Abl, a downstream mediator of TGF-β(2) and PDGF receptors(5). Imatinib has been shown to inhibit lung and dermal fibrosis in bleomycin-induced mouse models(6, 7), and the proliferation of synovial fibroblasts derived from patients with rheumatoid arthritis(8). Imatinib has also been reported to provide benefit in the treatment of refractory idiopathic PAH through its effects on vascular remodeling(9). We now describe two patients with early diffuse SSc who experienced clinical improvement in response to imatinib therapy and provide evidence that both c-Abl and PDGFR are targets of imatinib in scleroderma skin. Finally, we show that an imatinib-responsive gene signature is present in most cases of diffuse SSc.

We have investigated two patients with acquired chromosomal rearrangements, a male presenting with a t(1;9)(p34;q34) and B cell progenitor acute lymphoid leukemia and a female presenting with a t(8;12)(p11;q15) and the 8p11 myeloproliferative syndrome. We determined that the t(1;9) fused ABL to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing factor. The t(8;12) fused CPSF6, a cleavage and polyadenylation specificity factor, to FGFR1. The fusions were confirmed by amplification of the genomic breakpoints and RT-PCR. The predicted oncogenic products of these fusions, SFPQ-ABL and CPSF6-FGFR1, are in-frame and encode the N-terminal domain of the partner protein and the entire tyrosine kinase domain and C-terminal sequences of ABL and FGFR1. SFPQ interacts with two FGFR1 fusion partners, ZNF198 and CPSF6, that are functionally related to the recurrent PDGFRalpha partner FIP1L1. Our findings thus identify a group of proteins that are important for pre-mRNA processing as fusion partners for tyrosine kinases in hematological malignancies.

N-acetyl-cysteine (NAC) has been reported to have anticancer properties such as counteractions against mutagens and prevention of tumor progression by scavenging reactive oxygen species (ROS). However, here we report that NAC can enhance the anchorage-independent growth of cells transformed by activated ABL tyrosine kinases or Ras. This effect was not dependent on loss of focal adhesion kinase activation. NAC rescued cell growth that was suppressed by heat shock protein (Hsp) 90 inhibitors possibly by chemical modification of their quinone moiety. NAC rendered Rat1/BCR-ABL cells resistance to a Ras inhibitor manumycin in soft agar colony formation. In the absence of Hsp90 inhibitors, NAC stimulated the activation of MAP kinase in BCR-ABL-transformed but not in the parental Rat1 cells. We propose that NAC should be used carefully in cancer treatment. (Cancer Sci 2005; 96: 240 –244)

We have molecularly cloned TPP36, a novel 36 kDa protein with 281 amino acids that was identified as a protein phosphorylated in B progenitor cells following stimulation with pervanadate/H2O2. Analysis with anti-TPP36 antiserum revealed that TPP36 was expressed ubiquitously and had an isoform with 236 amino acids, designated TPP32. TPP36/32 were localized mainly in cytoplasm despite the presence of a typical nuclear localization signal sequence. These proteins were phosphorylated preferentially by Abl among a panel of tyrosine kinases examined. Phosphorylation of tyrosine 120 in TPP36/32 led to an apparent mobility shift in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting conformational change in the phosphorylated protein. Thus, TPP36/32 appear to be novel substrates of Abl tyrosine kinase.

WAVE proteins are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. We identified WAVE-1 in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin as the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, we propose a previously unrecognized function for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl.

Summary BCR-ABL1 transcript numbers were monitored in 161 patients who started treatment with imatinib early after diagnosis of chronic myeloid leukaemia in chronic phase and achieved complete cytogenetic responses (CCyR). A confirmed doubling in BCR-ABL1/ABL1 transcript levels was found to be a significant factor for predicting loss of CCyR [relative risk (RR) 8·3, P

Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is regulated synergistically by the B-cell antigen receptor, CD40L and interleukin signaling pathways. Consistent with the requirement for multiple T cell-dependent signals to induce OCA-B, we find that OCA-B protein is highly expressed in germinal center B cells. Accordingly, germinal center formation is blocked completely in the absence of OCA-B expression in B cells, whereas the helper functions of OCA-B-deficient T cells are indistinguishable from controls. The requirement for OCA-B expression in B cells is germinal center specific since the development of primary B cell follicles, the marginal zone and plasma cells are all intact. Thus, OCA-B is the first example of a transcriptional coactivator that is both synergistically induced by and required for integration of signals that mediate cell fate decisions.

v-cbl is the transforming gene of a murine retrovirus which induces pre-B cell lymphomas and myelogenous leukaemias. It encodes 40 kDa of a gag fusion protein which is localized in the cytoplasm and nucleus of infected cells. The c-cbl oncogene encodes a 120 kDa cytoplasmic protein and its overexpression is not associated with tumorigenesis. The c-cbl sequence has shown that v-cbl was generated by a truncation that removed 60% of the C-terminus. In this study, we carried out experiments to identify the position within cbl where the transition occurs between non-tumorigenic and tumorigenic forms. These experiments focused attention on a region of 17 amino acids which is deleted from cbl in the 70Z/3 pre-B lymphoma due to a splice acceptor site mutation. This mutation activates cbl's tumorigenic potential and induces its tyrosine phosphorylation. We also show that the expression of the v-abl and bcr-abl oncogenes results in the induction of cbl tyrosine phosphorylation, and that abl and cbl associate in vivo. These findings demonstrate that tyrosine-phosphorylated cbl promotes tumorigenesis and that cbl is a downstream target of the bcr-abl and v-abl kinases.

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.