Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma.

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Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

Nature Communications ◽

10.1038/s41467-021-24109-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

A. Rose Brannon ◽

Gowtham Jayakumaran ◽

Monica Diosdado ◽

Juber Patel ◽

Anna Razumova ◽

...

Keyword(s):

Cancer Patients ◽

De Novo ◽

Low Frequency ◽

High Sensitivity ◽

Clinical Analysis ◽

De Novo Mutation ◽

Cell Free Dna ◽

Free Dna ◽

Somatic Alterations ◽

Mutation Profiling

AbstractCirculating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

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Contributions of the structural domains of filensin in polymer formation and filament distribution

Journal of Cell Science ◽

10.1242/jcs.109.2.447 ◽

1996 ◽

Vol 109 (2) ◽

pp. 447-456

Author(s):

G. Goulielmos ◽

S. Remington ◽

F. Schwesinger ◽

S.D. Georgatos ◽

F. Gounari

Keyword(s):

De Novo ◽

Chimeric Proteins ◽

Tail Domain ◽

Structural Domains ◽

Filament Assembly ◽

Polymer Formation ◽

Filament Bundles ◽

Specific Contribution ◽

Mcf 7

Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.

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Long-term outcomes of patients with advanced-stage cutaneous T-cell lymphoma and large cell transformation

Blood ◽

10.1182/blood-2008-05-154609 ◽

2008 ◽

Vol 112 (8) ◽

pp. 3082-3087 ◽

Cited By ~ 103

Author(s):

Suzanne O. Arulogun ◽

H. Miles Prince ◽

Jonathan Ng ◽

Stephen Lade ◽

Gail F. Ryan ◽

...

Keyword(s):

De Novo ◽

Cell Transformation ◽

Early Stage ◽

Large Cell ◽

Tumor Stage ◽

Advanced Stage ◽

Long Term Outcomes ◽

Stage Disease ◽

Large Cell Transformation

Abstract Although mycosis fungoides (MF) is typically an indolent disease, patients with advanced-stage disease (stages IIB-IVB), including Sézary syndrome (SS), often have a poor outcome. A 31-year, retrospective analysis of our cutaneous lymphoma database, of 297 patients with MF and SS, was undertaken to study long-term outcomes and identify clinical predictors of outcome in patients with advanced-stage disease (ASD, n = 92) and large cell transformation (LCT, n = 22). Two-thirds of patients with ASD presented with de novo ASD. The median overall survival (OS) for ASD was 5 years with a 10-year predicted OS of 32%. Age at initial diagnosis (P = .01), tumor stage (P = .01), and clinical stage (P = .001) were found to be significant predictors of outcome. Patients who presented with de novo ASD demonstrated better outcomes that were not statistically significant than those with a prior diagnosis of early-stage MF (P = .25). Transformation developed in 22 of the 297 MF/SS patients (7.4%), with a transformation rate of only 1.4% in patients with early-stage disease, compared with stage IIB (27%) and stage IV (56%-67%) disease. The median OS from diagnosis of LCT was 2 years. We confirm that the incidence of LCT is strongly dependent on tumor stage at diagnosis, and we demonstrate a much lower overall risk of LCT than previously reported.

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Low frequency of parental mosaicism in de novo COL4A5 mutations in X‐linked Alport syndrome

Molecular Genetics & Genomic Medicine ◽

10.1002/mgg3.1452 ◽

2020 ◽

Vol 8 (10) ◽

Author(s):

Ole Magnus Bjorgaas Helle ◽

Torkild Høieggen Pedersen ◽

Lilian Bomme Ousager ◽

Mads Thomassen ◽

Jens Michael Hertz

Keyword(s):

Alport Syndrome ◽

De Novo ◽

Low Frequency

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Aberrant Somatic Hypermutation in Extranodal Marginal Zone B-Cell Lymphoma of MALT Type.

Blood ◽

10.1182/blood.v106.11.417.417 ◽

2005 ◽

Vol 106 (11) ◽

pp. 417-417 ◽

Cited By ~ 3

Author(s):

Alexander Deutsch ◽

Ariane Aigelsreiter ◽

Christine Beham-Schmid ◽

Alfred Beham ◽

Werner Linkesch ◽

...

Keyword(s):

B Cell ◽

Malt Lymphoma ◽

Marginal Zone ◽

De Novo ◽

Cell Lymphoma ◽

Somatic Hypermutation ◽

B Cell Lymphoma ◽

Chromosomal Translocations ◽

Genome Wide ◽

Balanced Translocations

Abstract Extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma) accounts for approximately 7% to 8% of all non-Hodgkin lymphomas (NHLs) being the third most frequent histological subtype. The gastrointestinal tract - particularly the stomach - is the most common site of MALT lymphoma comprising 50% of all cases, but virtually every organ may be affected by this type of lymphoma. Transformation (or de novo emergence at extranodal sites) in diffuse large B-cell lymphoma (DLBCL) occurs but - according to the WHO criteria - is considered as separate entity. The understanding of the molecular biology of MALT lymphoma has significantly improved following the recent cloning of recurrent balanced translocations such as t(11;18) or t(14;18), but a mechanism for genome-wide instability during MALT lymphomagenesis has not been described. We have reported that the somatic hypermutation process (SHM) physiologically aimed at mutating the immunoglobulin variable gene (IgV) aberrantly targets multiple proto-oncogenes in >50% of DLCBL (Pasqualucci et al., Nature412:341, 2001). Consequently, multiple mutations are introduced in the 5′ region of genes including known proto-oncogenes such as PIM-1, PAX-5, Rho/TTF and c-MYC. To further investigate whether aberrant somatic hypermutation (ASHM) also occurs in MALT lymphoma, we studied the mutation profile of these genes in 17 MALT lymphomas (6 of gastric- and 11 of nongastric origin) and 18 extranodal DLBCL (10 gastric, 8 nongastric). Mutations in one or more genes were detected in 15 of 17 (88.2%) cases of MALT lymphoma and in all of 18 (100%) cases of extranodal DLBCL. 7 of 17 (41.2%) and 15 of 18 (83.3%) carried mutations in two or more genes in the MALT- and DLBC-lymphoma group, respectively. Overall, mutations in PIM-1 occurred in 5 of 17 (29.4%) cases with MALT lymphoma and in 10 of 18 (55.5%) in extranodal DLBCL cases. For PAX-5, the distribution of mutated cases between MALT- and DLBC-lymphoma was 6 of 17 (35.3%) and 10 of 18 (55.5%), for Rho/TTF 3 of 17 (17.6%) and 8 of 18 (44.4%) and for c-MYC 9 of 17 (52.9%) and 12 of 18 (66.6%), respectively. A total of 99 sequence variants were found in 35 cases, 29 in the MALT lymphomas and 70 in extranodal DLBCL. Although the mutations were almost exclusively single base pair substitutions (n=98 ), an insertion was also present (n=1). Mutations were of somatic origins, occur independent of chromosomal translocations to the Ig loci and share features of the IgV SHM process including bias for transition over transversion, preferential hotspot (RGYW/WRCY) targeting and restriction to the first 1–2Kb from the promoter. The mean mutation frequency in mutated MALT lymphomas was with 0.045 x10−2/bp 1.7 fold lower compared to 0.08 x10−2/bp in mutated extranodal DLBCL. Further in PIM-1, PAX-5 and c-MYC some of the mutations were found to affect coding exons, leading to amino acid exchanges, thus potentially altering gene function. These data indicate that aberrant SHM is associated with extranodal DLBCL and MALT lymphoma, likewise. By mutating regulatory and coding sequences of the targeted genes and by possibly favouring chromosomal translocations ASHM may represent a major contributor to their pathogenesis. ASHM may further support a model of MALT lymphomagenesis leading from an antigen driven lesion to transformed MALT lymphoma finally evolving to overt DLBCL.

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Exisulind Selectively Induces Apoptosis Via C-Jun Kinase Activation in MDS and sAML/MDS.

Blood ◽

10.1182/blood.v106.11.4902.4902 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4902-4902

Author(s):

Akos Czibere ◽

Wolf C. Prall ◽

Sabine Knipp ◽

Andrea Kuendgen ◽

Luiz F. Zerbini ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

De Novo ◽

Bone Marrow Cells ◽

Kinase Activation ◽

Jun Kinase ◽

Secondary Acute Myeloid Leukemia ◽

M Phase ◽

De Novo Aml ◽

Induced Apoptosis

Abstract Treatment of patients suffering from myelodysplastic syndromes (MDS) is difficult and frustrating, particularly when in progression or already progressed to secondary acute myeloid leukemia (sAML). In these cases, the bone marrow contains an increasing number of immature cells, which are characterized by reduced apoptotic and increased proliferative activity. Therefore, pro-apoptosis has recently been proposed as a novel therapeutic approach. Exisulind is a potentially pro-apoptotic agent, and therefore, we investigated its influence on proliferation, differentiation, cell cycle and apoptosis in peripheral blood-derived CD34+ stem cells (PBSC) obtained from 10 patients suffering from refractory anaemia with excess of blasts in transformation (RAEB-T), two sAML/MDS cell lines, one de-novo AML cell line and healthy CD34+ bone marrow cells. Incubation of sAML/MDS cells with Exisulind clearly inhibited colony formation in the CFU-assays. Interestingly, Exisulind did not alter the percentages of sAML/MDS cells in G1-, S-, G2-, M-phase, but reduced proliferation and induced apoptosis in this cell type. Likewise, in 8 out of 10 RAEB-T samples Exisulind significantly inhibited proliferation and increased the rate of apoptotic cells. Exisulind had no effect on de-novo AML or normal CD34+ cells. We detected increased c-Jun NH2-terminal kinase activity in sAML/MDS cells treated with Exisulind. Adding a specific JNK-inhibitor to Exisulind-treated sAML/MDS and RAEB-T cells partly abrogated apoptosis, thus proving that Exisulind-mediated apoptosis in sAML/MDS and RAEB-T cells is dependent on JNK activation. We conclude that JNK is one mediator of apoptosis in MDS cells treated with Exisulind. Moreover, our data strongly suggests to explore the potential use of Exisulind as a novel, pro-apoptotic therapy for patients with RAEB-T and sAML/MDS.

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Prognostic significance of leukopenia at the time of diagnosis in acute myeloid leukemia (AML)

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.7070 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 7070-7070

Author(s):

M. L. Arellano ◽

E. Winton ◽

L. Pan ◽

L. Souza ◽

S. Sunay ◽

...

Keyword(s):

Cell Surface ◽

Adhesion Molecules ◽

De Novo ◽

Risk Group ◽

Statistical Significance ◽

Univariate Analysis ◽

Prognostic Significance ◽

Surface Expression ◽

Cell Surface Expression ◽

Surface Adhesion

7070 Background: In contrast to the poor prognosis associated with hyperleukocytosis, the prognostic significance of leukopenia at the time of diagnosis of AML is unknown. Methods: Single institution retrospective analysis of 225 consecutive, newly diagnosed AML patients (pts), homogeneously treated between July 1996 and February 2005; and divided into 2 groups based on presenting WBC: < 2,000/uL (30) and > 2,000/uL (195). Simultaneously obtained peripheral blood and marrow blasts were analyzed for cell surface expression of CD34, cKit, CXCR4, PCAM, VLA-2, VLA-3, VLA-4, VLA-5, and FLT3 using flow cytometry. Results: Patients’ characteristics (gender, secondary vs. de novo, and cytogenetic [CTG] risk) were comparable between the 2 groups. Leukopenic AML pts were older (median 56 vs. 53 years, p = 0.02), and had lower induction complete remission [CR] rates: 63% vs. 81% (p = 0.03) by univariate analysis. Induction mortality was 0% for leukopenic and 5% for non-leukopenic pts. In primary refractory pts, median survival was longer for leukopenic (11) vs. non-leukopenic (34) pts: 137 vs. 81 d (p = 0.026). Median follow-up was 22 mos. Event-free (EFS), disease-free (DFS), and overall survivals (OS) were lower in the leukopenic group: 12 vs. 14; 14 vs. 17; and 17 vs. 19 mos, respectively; but did not reach statistical significance. By multivariate analysis, age (p < 0.0001) and CTG risk group (p < 0.0001) were independent predictors of OS, while CTG risk group predicted RFS (p < 0.0001). The level of expression of cell surface adhesion molecules on blood and marrow blasts was comparable for the 2 groups. Conclusions: AML pts presenting with leukopenia have comparable outcomes to those presenting with normal or high WBC despite a lower likelihood of achieving remission. Leukopenic AML did not have over-expression of cell surface adhesion molecules. No significant financial relationships to disclose.

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Frequency and Heritability of WT1 Mutations in Nonsyndromic Wilms' Tumor Patients: A UK Children’s Cancer Study Group Study

Journal of Clinical Oncology ◽

10.1200/jco.2004.02.136 ◽

2004 ◽

Vol 22 (20) ◽

pp. 4140-4146 ◽

Cited By ~ 71

Author(s):

Suzanne E. Little ◽

Sandra P. Hanks ◽

Linda King-Underwood ◽

Chris Jones ◽

Elizabeth A. Rapley ◽

...

Keyword(s):

De Novo ◽

Age Of Onset ◽

Wilms Tumor ◽

Low Frequency ◽

Heteroduplex Analysis ◽

Study Group ◽

Histologic Subtype ◽

Cancer Study ◽

Cancer Study Group ◽

Wt1 Mutation

Purpose Constitutional WT1 mutations in patients with Wilms' tumor (WT) have specifically been associated with genitourinary abnormalities, such as cryptorchidism and hypospadias. We sought to ascertain the frequency and heritability of constitutional WT1 mutations in nonsyndromic WT patients. Patients and Methods Constitutional DNA from 282 patients treated at seven United Kingdom Children's Cancer Study Group centers was screened for WT1 mutations using heteroduplex analysis. Bidirectional sequencing was used to confirm the mutation and to analyze the corresponding parental DNA samples. Results Five different constitutional WT1 mutations were identified in six children. Mutations in four patients were confirmed to be de novo, and all five mutations are predicted to produce truncated protein. The WT1 mutation group had a young median age at diagnosis of 13.8 months, compared with 34.9 months in the group in whom no WT1 mutations were found; four were female and two were male; and all tumors were of favorable histology. The three tumors with known histologic subtype were stromal-predominant. Contrary to expectation, four of six mutations occurred in children with unilateral tumors without any associated genitourinary abnormality. Conclusion Constitutional WT1 mutations occur with a low frequency (2.1%; 95% CI, 0.8% to 4.6%) in nonsyndromic WT patients. Most mutations occurred in children with unilateral WT without associated genitourinary abnormalities, creating difficulties in identifying individuals with germline mutations on phenotype alone. Two factors that may indicate that an individual is carrying a germline WT1 mutation are an early age of onset and stromal-predominant histology of the WT.

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Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs

Zygote ◽

10.1017/s0967199400003543 ◽

1997 ◽

Vol 5 (1) ◽

pp. 35-46 ◽

Cited By ~ 12

Author(s):

Noriko Usui ◽

Atsuo Ogura ◽

Yasuyuki Kimura ◽

Ryuzo Yanagimachi

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

De Novo ◽

Germinal Vesicle ◽

Mature Oocyte ◽

Sperm Nucleus ◽

Sperm Chromatin ◽

Early Prophase ◽

Nuclear Envelope Breakdown ◽

M Phase

SummaryDuring fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entery into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been apoint of dispute. To resolve this issue, the fate of the germinal vesicle stage(GV oocytes) or fertilised eggs at the pronuclear stage(PN eggs). The SINEs disappeared quikly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelops around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle. i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase Promoting factor (MPF) is absent from the ooplasm.

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