Mammalian cells contain potent activity for removal of 3-phosphoglycolates from single-stranded oligomers and from 3 overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a singlestranded 3 terminus to leave a 3-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3-phosphoglycolate on a 3-base 3 overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3 overhangs.Ionizing radiation, radiomimetic drugs, and to some extent all free radical-based genotoxins induce DNA double strand breaks (DSBs) 1 by oxidative fragmentation of DNA sugars (1-4). Most such breaks bear terminal 3Ј-phosphate or 3Ј-phosphoglycolate (PG) moieties (1, 2, 5) that must be removed to allow fill-in of gaps by DNA polymerase and final religation by DNA ligase. While the human apurinic/apyrimidinic endonuclease Ape1 can remove PGs, albeit inefficiently, from blunt and recessed 3Ј ends of DSBs (6), PGs on 3Ј overhangs are highly resistant to this enzyme (6, 7). Nevertheless, in mammalian cell extracts, PGs on 3Ј overhangs of DSBs are removed by an as yet unidentified activity, and a fraction of such DSBs are accurately rejoined by Ku-mediated end alignment, gap-filling, and ligation (8).The yeast tyrosyl-DNA phosphodiesterase scTdp1 was isolated from Saccharomyces cerevisiae as an activity that hydrolyzes the phosphodiester bond linking tyrosine to a 3Ј DNA end (9). Such linkages are formed as intermediates in DNA relaxation by topoisomerase I and in DNA cleavage and reunion by various recombinases. These intermediates can become trapped if the process is interrupted, for example, by a topoisomerase inhibitor or by collision with a replication fork. The hypersensitivity of tdp1⌬ yeast to topoisomerase I-mediated DNA damage (10) suggests a critical role for scTdp1 in repair of such lesions. A human homologue with similar 3Ј-phosphotyrosyl-processing activity, hTdp1, was cloned by homology to scTdp1 and was eventually shown to have some homology to the phospholipase D superfamily of phosphodiesterases (11). Yeast and human Tdp1 differ from other activities for processing blocked 3Ј ends in that they leave a 3Ј-phosphate rather than a 3Ј-hydroxyl. A 3Ј-phosphate could then be removed by polynucleotide kina...

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3-overhangs and a onebase gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3 3 5 resection before ligation, was formed. Similar results were seen for a substrate with 5-overhangs and recessed 3-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.

• Whole-transcriptome sequencing reveals NPM1-TYK2 gene fusion in cutaneous CD30-positve lymphoproliferative disorders.• NPM1-TYK2 activates STAT signaling and is a therapeutic target in a subset of cutaneous CD30-positive lymphoproliferative disorders.The spectrum of cutaneous CD30-positive lymphoproliferative disorders (LPDs) includes lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Chromosomal translocations targeting tyrosine kinases in CD30-positive LPDs have not been described. Using whole-transcriptome sequencing, we identified a chimeric fusion involving NPM1 (5q35) and TYK2 (19p13) that encodes an NPM1-TYK2 protein containing the oligomerization domain of NPM1 and an intact catalytic domain in TYK2. Fluorescence in situ hybridization revealed NPM1-TYK2 fusions in 2 of 47 (4%) primary cases of CD30-positive LPDs and was absent in other mature T-cell neoplasms (n 5 151). Functionally, NPM1-TYK2 induced constitutive TYK2, signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5 activation. Conversely, a kinase-defective NPM1-TYK2 mutant abrogated STAT1/3/5 signaling. Finally, short hairpin RNA-mediated silencing of TYK2 abrogated lymphoma cell growth. This is the first report of recurrent translocations involving TYK2, and it highlights the novel therapeutic opportunities in the treatment of CD30-positive LPDs with TYK2 translocations. (Blood. 2014;124(25):3768-3771) IntroductionRecurrent chromosomal translocations frequently underlie the pathogenesis of several hematopoietic malignancies and often define molecular subtypes with distinct biological behavior. 1,2 Frequently, these translocations target tyrosine kinases resulting in constitutive activation and promotion of oncogenesis.3 Cutaneous CD30-positive lymphoproliferative disorders (LPD) represents a clinicopathologic spectrum including lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large cell lymphoma (ALCL). 4 Gene fusions targeting tyrosine kinases underlying the pathogenesis of CD30-positive LPD have not been described. MethodsPatient biopsy samples were obtained with institutional review board approval. Complete description of methods and clinical samples are presented in the supplemental Methods (available on the Blood Web site). RNA was subjected to chimera analysis by producing paired-end libraries sequenced on the Illumina Genome Analyzer II. Sequencing data were analyzed using custom bioinformatics tools and Chimerascan software.5 Sequencing confirmation of NPM1-TYK2 fusion transcripts was achieved using SYBR Green-based quantitative real-time polymerase chain reaction (PCR) assays and Sanger sequencing of amplicons by reverse transcription PCR (RT-PCR (see supplemental "Materials"). Fluorescence in situ hybridization (FISH) was performed on tissue microarrays of primary patient samples using standard methods. TYK2 break-apart FISH and NPM1-TYK2 fusion FISH assays were designed to detect TYK2 rearrangements and NPM1-TYK2 fusions, respectively (see supplemental Methods). Immunohi...

The concomitant evaluation of cell of origin along with tumor microenvironment components identifies patients with DLBCL treated with R-CHOP chemotherapy portraying a worse prognosis.

No abstract

Serum thymidine kinase 1 (TK1) levels have been reported to have prognostic significance in patients with chronic lymphocytic leukemia (CLL). Until recently, serum TK1 levels were assessed using inconvenient radioenzyme assays. In this study, we used a novel chemiluminescence assay to assess serum TK1 levels in patients with CLL at the time of first examination. We show that high serum TK1 levels predict poorer overall survival and correlate with unmutated immunoglobulin variable region genes, CD38 and ZAP-70 expression, and subsequent risk of developing large B-cell lymphoma (Richter syndrome). Similar findings were observed in a subset of patients treated with current fludarabine-based chemotherapy regimens. We suggest that serum TK1 levels analyzed using this convenient chemiluminescence assay may be useful in the risk assessment of patients with CLL.

Purpose: Peripheral T-cell lymphomas are clinically aggressive and usually fatal, as few complete or durable remissions are achieved with currently available therapies. Recent evidence supports a critical role for lymphoma-associated macrophages during T-cell lymphoma progression, but the specific signals involved in the cross-talk between malignant T cells and their microenvironment are poorly understood. Colony-stimulator factor 1 receptor (CSF1R, CD115) is required for the homeostatic survival of tissue-resident macrophages. Interestingly, its aberrant expression has been reported in a subset of tumors. In this article, we evaluated its expression and oncogenic role in T-cell lymphomas.Experimental Design: Loss-of-function studies, including pharmacologic inhibition with a clinically available tyrosine kinase inhibitor, pexidartinib, were performed in multiple in vitro and in vivo models. In addition, proteomic and genomic screenings were performed to discover signaling pathways that are activated downstream of CSF1R signaling.Results: We observed that CSF1R is aberrantly expressed in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1), in an autocrine or paracrine-dependent manner, leads to CSF1R autophosphorylation and activation in malignant T cells. Furthermore, CSF1R signaling was associated with significant changes in gene expression and in the phosphoproteome, implicating PI3K/AKT/mTOR in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R in vivo and in vitro models is associated with decreased T-cell lymphoma growth.Conclusions: Collectively, these findings implicate CSF1R in T-cell lymphomagenesis and have significant therapeutic implications.

Nodal follicular helper T-cell-derived lymphoproliferations (specifically the less common peripheral T-cell lymphomas of follicular type) exhibit a spectrum of histologic features that may mimic reactive hyperplasia or Hodgkin lymphoma. Even though angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma of follicular type share a common biologic origin from follicular helper T-cells and their morphology has been well characterized, flow cytometry of peripheral T-cell lymphomas of follicular type has not been widely discussed as a tool for identifying this reactive hyperplasia/Hodgkin lymphoma mimic. We identified 10 peripheral T-cell lymphomas of follicular type with available flow cytometry data from five different institutions, including two cases with peripheral blood evaluation. For comparison, we examined flow cytometry data for 8 classical Hodgkin lymphomas (including 1 lymphocyte-rich classical Hodgkin lymphoma), 15 nodular lymphocyte predominant Hodgkin lymphomas, 15 angioimmunoblastic T-cell lymphomas, and 26 reactive nodes. Lymph node histology and flow cytometry data were reviewed, specifically for the presence of a CD3 − /dim CD4 + aberrant T-cell population (described in angioimmunoblastic T-cell lymphomas), besides other T-cell aberrancies. Nine of 10 (90%) peripheral T-cell lymphomas of follicular type showed a CD3 − /dim CD4 + T-cell population constituting 29.3% (range 7.9-62%) of all lymphocytes. Five of 10 (50%) had nodular lymphocyte predominant Hodgkin lymphoma or lymphocyte-rich classical Hodgkin lymphoma-like morphology with scattered Hodgkin-like cells that expressed CD20, CD30, CD15, and MUM1. Three cases had a nodular growth pattern and three others exhibited a perifollicular growth pattern without Hodgkin-like cells. Epstein-Barr virus was positive in 1 of 10 cases (10%). PCR analysis showed clonal T-cell receptor gamma gene rearrangement in all 10 peripheral T-cell lymphomas of follicular type. By flow cytometry, 11 of 15 (73.3%) angioimmunoblastic T-cell lymphomas showed the CD3 − /dim CD4 + population (mean: 19.5%, range: 3-71.8%). Using a threshold of 3% for CD3 − /dim CD4 + T cells, all 15 nodular lymphocyte predominant Hodgkin lymphoma controls and 8 classical Hodgkin lymphomas were negative (Mann-Whitney P = 0.01, F-PTCL vs Hodgkin lymphomas), as were 25 of 26 reactive lymph nodes. The