Blm helicase facilitates rapid replication of repetitive DNA sequences in early Drosophila development.

The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome, which results in developmental abnormalities, DNA repair defects, genomic instability, and a predisposition to cancer. In Drosophila melanogaster, the orthologous Blm protein is essential during early development when the embryo is under the control of maternal gene products. We show that lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality.

Sources and structures of mitotic crossovers that arise when BLM helicase is absent in Drosophila.

The Bloom syndrome helicase, BLM, has numerous functions that prevent mitotic crossovers. We used unique features of Drosophila melanogaster to investigate origins and properties of mitotic crossovers that occur when BLM is absent. Induction of lesions that block replication forks increased crossover frequencies, consistent with functions for BLM in responding to fork blockage.

Asynchronous replication, mono-allelic expression, and long range Cis-effects of ASAR6.

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously.

An autosomal locus that controls chromosome-wide replication timing and mono-allelic expression.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times, following a temporal replication program. Homologous alleles typically replicate synchronously; however, mono-allelically expressed genes such as imprinted genes, allelically excluded genes and genes on the female X chromosome replicate asynchronously. We have used a chromosome engineering strategy to identify a human autosomal locus that controls this replication timing program in cis.

High-throughput sequence analysis of the tyrosine kinome in acute myeloid leukemia.

To determine whether aberrantly activated tyrosine kinases other than FLT3 and c-KIT contribute to acute myeloid leukemia (AML) pathogenesis, we used high-throughput (HT) DNA sequence ana-lysis to screen exons encoding the activation loop and juxtamembrane domains of 85 tyrosine kinase genes in 188 AML patients without FLT3 or c-KIT mutations. The screen identified 30 nonsynonymous sequence variations in 22 different kinases not previously reported in single-nucleotide polymorphism (SNP) databases. These included a novel FLT3 activating allele and a previously described activating mutation in MET (METT1010I).

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib.

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays.

Activating alleles of JAK3 in acute megakaryoblastic leukemia.

Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK.

Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes.

STAT5 is constitutively phosphorylated in leukemic cells in approximately 70% of acute myeloid leukemia (AML) patients. To identify kinase candidates potentially responsible for STAT5 phosphorylation, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) mass spectrometry to detect phosphoproteins in AML cell lines. We established TEL-ARG and BCR-ABL fusion proteins as the mechanism underlying STAT5 phosphorylation in HT-93 and KBM-3 cells, respectively.

Combined Abl inhibitor therapy for minimizing drug resistance in chronic myeloid leukemia: Src/Abl inhibitors are compatible with imatinib.

Purpose: Chronic myeloid leukemia (CML) is effectively treated with imatinib. However, reactivation of Bcr-Abl via kinase domain mutations that reduce sensitivity to imatinib can cause relapse. As combination therapy is frequently used to prevent emergence of resistance, the combination of imatinib with an inhibitor of imatinib-resistant Bcr-Abl mutants (e.

The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia.

Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL).

In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants.

Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity.

High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy.

Mutations in the kinase domain (KD) of BCR-ABL are the leading cause of acquired imatinib resistance. In some cases, identical mutations were detected at relapse and in pretherapeutic specimens, consistent with selection of resistant clones in the presence of drug. However, the incidence of KD mutations in imatinibnaive patients, irrespective of response to therapy, is unknown.

RNAi-induced down-regulation of FLT3 expression in AML cell lines increases sensitivity to MLN518.

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in approximately 30% of acute myelogenous leukemia (AML) patients and appears to confer an adverse prognosis. Thus, development of inhibitors and/or antibodies that specifically target FLT3 has been of substantial interest. In this regard, phase 1 and 2 trials involving FLT3 inhibitors have recently reported FLT3 inhibition and leukemic blast reduction in some patients.

Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: implications for CML.

The deregulated, oncogenic tyrosine kinase Bcr-Abl causes chronic myeloid leukemia (CML). Imatinib mesylate (Gleevec, STI571), a Bcr-Abl kinase inhibitor, selectively inhibits proliferation and promotes apoptosis of CML cells. Despite the success of imatinib mesylate in the treatment of CML, resistance is observed, particularly in advanced disease.

No correlation between the proliferative status of Bcr-Abl positive cell lines and the proapoptotic activity of imatinib mesylate (Gleevec/Glivec).

Imatinib mesylate (Gleevec, formerly STI571) has been shown to be a safe and effective treatment for chronic myelogenous leukemia (CML). However, despite high rates of hematologic and cytogenetic remissions, molecular remissions are rare. Recent work has revealed the existence of a population of Bcr-Abl-positive, quiescent hematopoietic CML stem cells that are insensitive to induction of apoptosis by imatinib ex vivo.

In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl-positive cell lines.

Imatinib mesylate (Gleevec, formerly STI571) is an effective therapy for all stages of chronic myelogenous leukemia (CML). While responses in chronic-phase CML are generally durable, resistance develops in many patients with advanced disease. We evaluated novel antileukemic agents for their potential to overcome resistance in various imatinib-resistant cell lines.

Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib.

Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse.

Activity of the Bcr-Abl kinase inhibitor PD180970 against clinically relevant Bcr-Abl isoforms that cause resistance to imatinib mesylate (Gleevec, STI571).

Imatinib mesylate, a selective inhibitor of the Abl tyrosine kinase, is effective as a single-agent therapy for chronic myelogenous leukemia. However, resistance has been reported, particularly in patients with advanced-stage disease. Mutations within the Abl kinase domain are a major cause of resistance, demonstrating that Bcr-Abl remains a critical drug target.