Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

Direct observation of DNA threading in flap endonuclease complexes

Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted Vshaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “flycasting, thread, bend and barb” mechanis

The Murchison Widefield Array Transients Survey (MWATS). A search for low-frequency variability in a bright Southern hemisphere sample

We report on a search for low-frequency radio variability in 944 bright (>4 Jy at 154 MHz) unresolved, extragalactic radio sources monitored monthly for several years with the Murchison Widefield Array. In the majority of sources, we find very low levels of variability with typical modulation indices 2.8 yr) with time-averaged modulation indices M¯¯¯¯¯=3.1−7.1M¯=3.1−7.1 per cent. With 7/15 of these variable sources having peaked spectral energy distributions, and only 5.7 per cent of the overall sample having peaked spectra, we find an increase in the prevalence of variability in this spectral class. We conclude that the variability seen in this survey is most probably a consequence of refractive interstellar scintillation and that these objects must have the majority of their flux density contained within angular diameters less than 50 milliarcsec (which we support with multiwavelength data). At 154 MHz, we demonstrate that interstellar scintillation time-scales become long (∼decades) and have low modulation indices, while synchrotron-driven variability can only produce dynamic changes on time-scales of hundreds of years, with flux density changes less than one milli-jansky (without relativistic boosting). From this work, we infer that the low-frequency extragalactic southern sky, as seen by SKA-Low, will be non-variable on time-scales shorter than 1 yr

A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice

The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62DOK, and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62DOK and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant's ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites

A phase I study of the oral gamma secretase inhibitor R04929097 in combination with gemcitabine in patients with advanced solid tumors (PHL-078/CTEP 8575)

PURPOSE: To establish the recommended phase II dose of the oral γ-secretase inhibitor RO4929097 (RO) in combination with gemcitabine; secondary objectives include the evaluation of safety, tolerability, pharmacokinetics, biomarkers of Notch signaling and preliminary anti-tumor activity. METHODS: Patients with advanced solid tumors were enrolled in cohorts of escalating RO dose levels (DLs). Tested RO DLs were 20 mg, 30 mg, 45 mg and 90 mg. RO was administered orally, once daily on days 1-3, 8-10, 15-17, 22-24. Gemcitabine was administered at 1,000 mg/m(2) on d1, 8, and 15 in 28 d cycles. Dose limiting toxicities (DLTs) were assessed by CTCAE v4. Serial plasma was collected for RO (total and unbound) and gemcitabine pharmacokinetic analysis. Biomarkers of Notch signaling were assessed by immunohistochemistry in archival tissue. Antitumor activity was evaluated (RECIST 1.1). RESULTS: A total of 18 patients were enrolled to establish the recommended phase II dose. Of these, 3 patients received 20 mg RO, 7 patients received 30 mg RO, 6 patients received 45 mg RO and 2 patients received 90 mg RO. DLTs were grade 3 transaminitis (30 mg RO), grade 3 transaminitis and maculopapular rash (45 mg RO), and grade 3 transaminitis and failure to receive 75 % of planned RO doses secondary to prolonged neutropenia (90 mg); all were reversible. The maximum tolerated dose was exceeded at 90 mg RO. Pharmacokinetic analysis of both total and free RO confirmed the presence of autoinduction at 45 and 90 mg. Median levels of Notch3 staining were higher in individuals who received fewer than 4 cycles (p = 0.029). Circulating angiogenic factor levels did not correlate with time to progression or ≥ grade 3 adverse events. Best response (RECIST 1.1) was partial response (nasopharyngeal cancer) and stable disease > 4 months was observed in 3 patients (pancreas, tracheal, and breast primary cancers). CONCLUSIONS: RO and gemcitabine can be safely combined. The recommended phase II dose of RO was 30 mg in combination with gemcitabine 1,000 mg/m(2). Although RO exposure was limited by the presence of autoinduction, RO levels achieved exceeded the area under the concentration-time curve for 0-24 h (AUC(0-24)) predicted for efficacy in preclinical models using daily dosing. Evidence of clinical antitumor activity and prolonged stable disease were identified

Melanoma Cell Expression of CD200 Inhibits Tumor Formation and Lung Metastasis via Inhibition of Myeloid Cell Functions

CD200 is a cell surface glycoprotein that functions through engaging CD200 receptor on cells of the myeloid lineage and inhibits their functions. Expression of CD200 has been implicated in a variety of human cancer cells including melanoma cells and has been thought to play a protumor role. To investigate the role of cancer cell expression of CD200 in tumor formation and metastasis, we generated CD200-positive and CD200-negative B16 melanoma cells. Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1−/−C57BL/6 mice. However, i.v. injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci formation in the lungs of both C57BL/6 and Rag1−/−C57BL6 mice. Flow cytometry analysis revealed higher expression of CD200R in Gr1+ myeloid cells in the lung than in peripheral myeloid cells. Depletion of Gr1+ cells or stimulation of CD200R with an agonistic antibody in vivo dramatically inhibited tumor foci formation in the lungs. In addition, treatment with tumor antigen specific CD4 or CD8 T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Taken together, we have revealed a novel role for CD200-CD200R interaction in inhibiting tumor formation and metastasis. Targeting CD200R may represent a novel approach for cancer immunotherapy

Translating microarray data for diagnostic testing in childhood leukaemia

BACKGROUND: Recent findings from microarray studies have raised the prospect of a standardized diagnostic gene expression platform to enhance accurate diagnosis and risk stratification in paediatric acute lymphoblastic leukaemia (ALL). However, the robustness as well as the format for such a diagnostic test remains to be determined. As a step towards clinical application of these findings, we have systematically analyzed a published ALL microarray data set using Robust Multi-array Analysis (RMA) and Random Forest (RF). METHODS: We examined published microarray data from 104 ALL patients specimens, that represent six different subgroups defined by cytogenetic features and immunophenotypes. Using the decision-tree based supervised learning algorithm Random Forest (RF), we determined a small set of genes for optimal subgroup distinction and subsequently validated their predictive power in an independent patient cohort. RESULTS: We achieved very high overall ALL subgroup prediction accuracies of about 98%, and were able to verify the robustness of these genes in an independent panel of 68 specimens obtained from a different institution and processed in a different laboratory. Our study established that the selection of discriminating genes is strongly dependent on the analysis method. This may have profound implications for clinical use, particularly when the classifier is reduced to a small set of genes. We have demonstrated that as few as 26 genes yield accurate class prediction and importantly, almost 70% of these genes have not been previously identified as essential for class distinction of the six ALL subgroups. CONCLUSION: Our finding supports the feasibility of qRT-PCR technology for standardized diagnostic testing in paediatric ALL and should, in conjunction with conventional cytogenetics lead to a more accurate classification of the disease. In addition, we have demonstrated that microarray findings from one study can be confirmed in an independent study, using an entirely independent patient cohort and with microarray experiments being performed by a different research team

Hormone-like (endocrine) Fgfs: their evolutionary history and roles in development, metabolism, and disease

Fibroblast growth factors (Fgfs) are proteins with diverse functions in development, repair, and metabolism. The human Fgf gene family with 22 members can be classified into three groups, canonical, intracellular, and hormone-like Fgf genes. In contrast to canonical and intracellular Fgfs identified in invertebrates and vertebrates, hormone-like Fgfs, Fgf15/19, Fgf21, and Fgf23, are vertebrate-specific. The ancestral gene of hormone-like Fgfs was generated from the ancestral gene of canonical Fgfs by gene duplication early in vertebrate evolution. Later, Fgf15/19, Fgf21, and Fgf23 were generated from the ancestral gene by genome duplication events. Canonical Fgfs act as autocrine/paracrine factors in an Fgf receptor (Fgfr)-dependent manner. In contrast, hormone-like Fgfs act as endocrine factors in an Fgfr-dependent manner. Canonical Fgfs have a heparin-binding site necessary for the stable binding of Fgfrs and local signaling. In contrast, hormone-like Fgfs acquired endocrine functions by reducing their heparin-binding affinity during their evolution. Fgf15/19 and Fgf23 require βKlotho and αKlotho as cofactors, respectively. However, Fgf21 might physiologically require neither. Hormone-like Fgfs play roles in metabolism at postnatal stages, although they also play roles in development at embryonic stages. Fgf15/19 regulates bile acid metabolism in the liver. Fgf21 regulates lipid metabolism in the white adipose tissue. Fgf23 regulates serum phosphate and active vitamin D levels. Fgf23 signaling disorders caused by hereditary diseases or tumors result in metabolic disorders. In addition, serum Fgf19 or Fgf21 levels are significantly increased by metabolic disorders. Hormone-like Fgfs are newly emerging and quite unique in their evolution and function