Brent W. Weston

Tyrosine Kinase Inhibitor Therapy Induces Remission in a Patient With Refractory EBF1-PDGFRB–Positive Acute Lymphoblastic Leukemia

Introduction Although more than 80% of children who are diagnosed with acute lymphoblastic leukemia (ALL) experience favorable clinical outcomes, a substantial number of children have high-risk disease with an increased probability of relapse and poor prognosis. Genetic alterations, including chromosomal rearrangements and deletions, are important determinants of leukemogenesis and responsiveness to therapy. The ability to identify high-risk patients at the time of diagnosis would enable clinicians to select more targeted therapies and improve survival. For example, patients with BCR-ABL1–positive ALL generally respond poorly to conventional chemotherapy, but outcomes can be improved with the addition of firstand second-generation tyrosine kinase inhibitors (TKIs). Recent genomic analyses have identified a new high-risk subtype of BCR-ABL1–negative ALL with a gene expression profile that is similar to that of BCR-ABL1–positive ALL, deletion of IKZF1 and/or other lymphoid transcriptional regulators, and poor outcome (BCRABL1–like ALL). Approximately 40% of patients with BCR-ABL1– like disease harbor rearrangements that result in aberrant expression of cytokine receptor–like factor 2, and half of these patients also demonstrate activating Janus kinase (JAK1/2) mutations. Recent transcriptome and whole-genome sequencing of BCR-ABL1–like ALL identified a range of genetic alterations that activate kinase signaling, including a recurrent fusion of the B-cell lymphoid transcription factor early B-cell factor 1 to the receptor tyrosine kinase plateletderived growth factor receptor (PDGFRB) on chromosome 5q. It is predicted that the DNA dimerization domain of EBF1 facilitates autophosphorylation and constitutive activation of PDGFRB. The potential for treatment of EBF1-PDGFRB–positive leukemia has not been directly examined in vivo. Here we present a child with EBF1PDGFRB–positive ALL whose disease was refractory to conventional induction chemotherapy but who responded to the addition of imatinib to remission induction therapy, highlighting the potential for TKI therapy to improve the currently poor outcome of patients with BCR-ABL1–like ALL harboring kinase-activating alterations.

Diboronic acids as fluorescent probes for cells expressing sialyl lewis X

A series of fluorescent diboronic acids was synthesized in nine steps as potential sensors for sialyl Lewis X (sLex). The fluorescent binding studies of these sensors with sLex were carried out in a mixed aqueous solution. Compound 7e was found to show the strongest fluorescence enhancement upon binding with sLex. Using cell cultures, 7e was shown to label sLex-expressing HEPG2 cells at 1 microM, while non-sLex-expressing cells were not labeled.

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of alpha-(1, 3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and alpha-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organisms tropism for leukocytes.

Expression of human chromosome 19p α(1,3)-fucosyltransferase genes in normal tissues: Alternative splicing, polyadenylation, and isoforms

The human α(1,3)-fucosyltransferase genes FUT3, FUT5, and FUT6 form a cluster on chromosome 19p13.3. Expression was studied using reverse transcriptase-polymerase chain reaction, rapid amplification of cDNA ends, and Northern analyses. FUT3 and FUT6 were expressed at high levels, while FUT5 expression was lower and restricted to fewer cell types. Alternatively spliced transcripts were identified for FUT3 and FUT6 in kidney, liver, and colon. A 2.37-kilobase pair (kb) FUT3 transcript, detected at high levels in kidney and colon, was absent in liver. FUT6 expression was characterized by a 3.5-kb transcript present in kidney and liver, and a 2.5-kb transcript in colon and liver. Two polyadenylation sites were shown for FUT5, but absence of consensus sequences suggests reduced efficiency for cleavage and polyadenylation. Two polyadenylation sites were also shown for FUT6, with the alternatively spliced downstream signal in tissues expressing high levels of FUT6. In these tissues, additional splicing results in isoforms with catalytic domain deletions. No detectable α(1,3)- or α(1,4)-fucosyltransferase activity was found in assays of cells transfected with FUT6 isoform cDNAs. Thus, tissue-specific post-transcriptional modifications are associated with expression patterns of FUT3, FUT5, and FUT6.

Physical maps of human α(1,3)fucosyltransferase genes FUT3–FUT6 on chromosomes 19p13.3 and 11q21

Sialyl Lewis x and related fucosylated glycans are differentially expressed in human cells and form ligands for selectin adhesion receptors. alpha(1,3)Fucosyltransferases (FUTs) that complete their biosynthesis also show tissue specificity. We have established physical maps of the FUT3-6 loci to study regulation of this gene family. FUT4 has previously been localized to chromosome 11q21; FUT3, FUT6, and now FUT5 are localized to chromosome 19p13.3. Conventional and pulsed-field gel electrophoresis mapping of total genomic DNA and large genomic clones were used to generate a fine map of both loci, defining the order, orientation, and distances between FUTs. A P1 clone with all three 19p FUT genes in tandem orientation was isolated and used to study regions flanking FUT3, -5, and -6. Our studies provide preliminary information to study regulation of human FUT genes.

Transfection of α(1,3)fucosyltransferase antisense sequences impairs the proliferative and tumorigenic ability of human colon carcinoma cells

Sialyl Lewis x and sialyl Lewis a are oncodevelopmental antigens involved in the pathogenesis of colon adenocarcinoma. Biosynthesis of these glycans is controlled by α(1,3/1,4)fucosyltransferases. We report the disruption of sialyl Lewis x/a biosynthesis and inhibition of colon carcinoma cell proliferation by stable transfection of antisense sequences directed at the human Lewis α(1,3/1,4)fucosyltransferase gene, FUT3, and the plasma α(1,3)fucosyltransferase gene, FUT6. COLO‐205 cells expressed high levels of sialyl Lewis x/a, α(1,3)fucosyltransferase activity, and FUT3/6 transcripts, but COLO

Familial deficiency of vitamin K-dependent clotting factors.

Summary.  Combined deficiency of vitamin K‐dependent clotting factors II, VII, IX and X (and proteins C, S, and Z) is usually an acquired clinical problem, often resulting from liver disease, malabsorption, or warfarin overdose. A rare inherited form of defective γ‐carboxylation resulting in early onset of bleeding was first described by McMillan and Roberts in 1966 and subsequently has been termed ‘vitamin K‐dependent clotting factor deficiency’ (VKCFD). Biochemical and molecular studies identify two variants of this autosomal recessive disorder: VKCFD1, which is associated with point mutations in the γ‐glutamylcarboxylase gene (GGCX), and VKCFD2, which results from point mutations in the vitamin K epoxide reductase gene (VKOR). Bleeding ranges in severity from mild to severe. Therapy includes high oral doses of vitamin K for prophylaxis, usually resulting in partial correction of factor deficiency, and episodic use of plasma infusions or prothrombin complex concentrate. Recent molecular studies have the potential to further our understanding of vitamin K metabolism, γ‐carboxylation, and the functional role this post‐translational modification has for other proteins. The results may also provide potential targets for molecular therapeutics and pharmacogenetics.

Histo-blood group gene polymorphisms as potential genetic modifiers of infection and cystic fibrosis lung disease severity.

Background The pulmonary phenotype in cystic fibrosis (CF) is variable; thus, environmental and genetic factors likely contribute to clinical heterogeneity. We hypothesized that genetically determined ABO histo-blood group antigen (ABH) differences in glycosylation may lead to differences in microbial binding by airway mucus, and thus predispose to early lung infection and more severe lung disease in a subset of patients with CF. Methods and Principal Findings Clinical information and DNA was collected on >800 patients with the ΔF508/ΔF508 genotype. Patients in the most severe and mildest quartiles for lung phenotype were enrolled. Blood samples underwent lymphocyte transformation and DNA extraction using standard methods. PCR and sequencing were performed using standard techniques to identify the 9 SNPs required to determine ABO blood type, and to identify the four SNPs that account for 90–95% of Lewis status in Caucasians. Allele identification of the one nonsynonymous SNP in FUT2 that accounts for >95% of the incidence of nonsecretor phenotype in Caucasians was completed using an ABI Taqman assay. The overall prevalence of ABO types, and of FUT2 (secretor) and FUT 3 (Lewis) alleles was consistent with that found in the Caucasian population. There was no difference in distribution of ABH type in the severe versus mild patients, or the age of onset of Pseudomonas aeruginosa infection in the severe or mild groups. Multivariate analyses of other clinical phenotypes, including gender, asthma, and meconium ileus demonstrated no differences between groups based on ABH type. Conclusions and Significance Polymorphisms in the genes encoding ABO blood type, secretor or Lewis genotypes were not shown to associate with severity of CF lung disease, or age of onset of P. aeruginosa infection, nor was there any association with other clinical phenotypes in a group of 808 patients homozygous for the ΔF508 mutation.

Glucose-6-phosphatase mutation G188R confers an atypical glycogen storage disease type 1b phenotype.

Glycogen storage disease type 1a (GSD 1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). A variant (GSD 1b) is caused by a defect in the transport of glucose-6-phosphate (G6P) into the microsome and is associated with chronic neutropenia and neutrophil dysfunction. Mutually exclusive mutations in the G6Pase gene and the G6P transport gene establish GSD 1a and GSD 1b as independent molecular processes and are consistent with a multicomponent translocase catalytic model. A modified translocase/catalytic unit model based on biochemical data in a G6Pase knockout mouse has also been proposed for G6Pase catalysis. This model suggests coupling of G6Pase activity and G6P transport. A 5-mo-old girl with hypoglycemia, hepatomegaly, and lactic acidemia was diagnosed with GSD 1a. She also developed neutropenia, neutrophil dysfunction, and recurrent infections characteristic of GSD 1b. Homozygous G188R mutations of the G6Pase gene were identified, but no mutations in the G6P translocase gene were found. We have subsequently identified a sibling and two unrelated patients with similar genotypic/phenotypic characteristics. The unusual association of neutrophil abnormalities in patients with homozygous G188R mutations in the G6Pase gene supports a modified translocase/catalytic unit model.

Fluorescent conjugate of sLex-selective bisboronic acid for imaging application

Carbohydrate-based biomarkers such as sialyl Lewis X are known to correlate with cancer formation and progression. By targeting sialyl Lewis X, we have developed a boronolectin-fluorophore conjugate, which was able to selectively label and image xenograft (sc) tumor. This represents the very first example that a small molecule capable of recognizing a carbohydrate biomarker was used for optical imaging application.