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O'Hare T.,Oregon Health And Science University | O'Hare T.,Howard Hughes Medical Institute | O'Hare T.,University of Utah | Zabriskie M.S.,Oregon Health And Science University | And 16 more authors.
Blood | Year: 2011

Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutation-mediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/truncation mutant, BCR-ABL35INS, in which structural integrity of the kinase domain is compromised and all ABL sequence beyond the kinase domain is eliminated. Although we speculated that BCR-ABL35INSis kinase-inactive, recent reports propose this mutant contributes to ABL TKI resistance.We present cell-based and biochemical evidence establishing that BCR-ABL35INS is kinase-inactive and does not contribute to TKI resistance, and we find that detection of BCR-ABL35INS does not consistently track with or explain resistance in clinical samples from chronic myeloid leukemia patients. © 2011 by The American Society of Hematology. Source

Theruvathu J.A.,University of Texas Medical Branch | Darwanto A.,University of Texas Medical Branch | Darwanto A.,MolecularMD | Hsu C.W.,University of Texas Medical Branch | Sowers L.C.,University of Texas Medical Branch
Nucleic Acids Research | Year: 2014

Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA-protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. Source

Poritz M.A.,Idaho Technology Inc. | Blaschke A.J.,University of Utah | Byington C.L.,University of Utah | Meyers L.,Idaho Technology Inc. | And 13 more authors.
PLoS ONE | Year: 2011

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon. © 2011 Poritz et al. Source

Deininger M.W.,University of Utah | Hodgson J.G.,ARIAD Pharmaceuticals Inc. | Shah N.P.,University of California at San Francisco | Cortes J.E.,University of Houston | And 19 more authors.
Blood | Year: 2016

BCR-ABL1 kinase domain mutations can confer resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). In preclinical studies, clinically achievable concentrations of the third-generation BCR-ABL1 TKI ponatinib inhibit T315I and all other single BCR-ABL1mutants except T315M, which generates a single amino acid exchange, but requires 2 sequential nucleotide exchanges. In addition, certain compound mutants (containing ‡2 mutations in cis) confer resistance. Initial analyses based largely on conventional Sanger sequencing (SS) have suggested that the preclinical relationship betweenBCR-ABL1mutation status and ponatinib efficacy is generally recapitulated in patients receiving therapy. Thus far, however, such analyses have been limited by the inabilityofSStodefinitively identify compound mutations or mutations representing less than ∼20%of total alleles (referred to as "low-level mutations"), aswell as limited patient follow-up. Here we used next-generation sequencing (NGS) to define the baseline BCR-ABL1 mutation status of 267 heavily pretreated chronic phase (CP)-CML patients from the PACE trial, and used SS to identify clonally dominant mutants that may have developed on ponatinib therapy (30.1 months median follow-up). Durable cytogenetic and molecular responses were observed irrespective of baseline mutation status and included patients with compound mutations. No single or compoundmutation was identified that consistently conferred primary and/or secondary resistance to ponatinib in CP-CML patients. Ponatinib is effective in CP-CML irrespective of baseline mutation status. © 2016 by The American Society of Hematology. Source

White H.E.,National Genetics Reference Laboratory Wessex | Matejtschuk P.,Public Health England | Rigsby P.,Public Health England | Gabert J.,Biochemistry and Molecular Laboratory | And 32 more authors.
Blood | Year: 2010

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freezedried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials. © 2010 by The American Society of Hematology. Source

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