Differential Transcriptional Reprogramming by Wild Type and Lymphoma-Associated Mutant MYC Proteins as B-Cells Convert to a Lymphoma Phenotype

The MYC transcription factor regulates a vast number of genes and is implicated in many human malignancies. In some hematological malignancies, MYC is frequently subject to missense mutations that enhance its transformation activity. Here, we use a novel murine cell system to (i) characterize the transcriptional effects of progressively increasing MYC levels as normal primary B-cells transform to lymphoma cells and (ii) determine how this gene regulation program is modified by lymphoma-associated MYC mutations (T58A and T58I) that enhance its transformation activity. Unlike many previous studies, the cell system exploits primary B-cells that are transduced to allow regulated MYC expression under circumstances where apoptosis and senescence pathways are abrogated by the over-expression of the Bcl-xL and BMI1 proteins. In such cells, transition from a normal to a lymphoma phenotype is directly dependent on the MYC expression level, without a requirement for secondary events that are normally required during MYC-driven oncogenic transformation. A generalized linear model approach allowed an integrated analysis of RNA sequencing data to identify regulated genes in relation to both progressively increasing MYC level and wild type or mutant status. Using this design, a total of 7569 regulated genes were identified, of which the majority (n = 7263) were regulated in response to progressively increased levels of wild type MYC, while a smaller number of genes (n = 917) were differentially regulated, compared to wild type MYC, in T58A MYC- and/or T58I MYC-expressing cells. Unlike most genes that are similarly regulated by both wild type and mutant MYC genes, the set of 917 genes did not significantly overlap with known lipopolysaccharide regulated genes, which represent genes regulated by MYC in normal B cells. The genes that were differently regulated in cells expressing mutant MYC proteins were significantly enriched in DNA replication and G2 phase to mitosis transition genes. Thus, mutants affecting MYC proteins may augment quantitative oncogenic effects on the expression of normal MYC-target genes with qualitative oncogenic effects, by which sets of cell cycle genes are abnormally targeted by MYC as B cells transition into lymphoma cells. The T58A and T58I mutations augment MYC-driven transformation by distinct mechanisms.

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Role of FOXO1 in Germinal Center Development and Lymphomagenesis

Blood ◽

10.1182/blood.v124.21.3536.3536 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3536-3536 ◽

Cited By ~ 2

Author(s):

David Dominguez-Sola ◽

Jennifer Kung ◽

Victoria A Wells ◽

Antony B Holmes ◽

Laura Pasqualucci ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Nuclear Localization ◽

Germinal Center ◽

Target Genes ◽

Cell Receptor ◽

Missense Mutations ◽

Wild Type ◽

Foxo1 Gene

Abstract A significant fraction of B cell non-Hodgkin lymphomas (B-NHL) of germinal center origin carry heterozygous missense mutations in FOXO1, a member of the FOXO family of transcription factors. FOXO1 is a central component of the PI3K signaling cascade engaged by the B cell receptor and is essential for B cell homeostasis and survival (Dengler et al, Nat Immunol 2008; Srinivasan et al, Cell 2009; Lin et al, Nat Immunol 2010). In response to PI3K activation, AKT phosphorylates FOXO1 leading to its nuclear-cytoplasmic translocation and inactivation. Missense mutations of the FOXO1 gene are detectable in germinal center (GC)-derived B-NHL, including ~12% of Burkitt Lymphoma (BL) and ~9% of Diffuse Large B Cell Lymphoma (DLBCL) cases (Schmitz et al, Nature 2012; Trinh et al, Blood 2013; Pasqualucci et al, Cell Rep 2014). The role of FOXO1 in normal GC development as well as the contribution of its mutations to lymphomagenesis is unclear. We show that FOXO1 expression is restricted to the dark zone of GCs, where its nuclear localization is detectable in most B cells. Mice carrying the conditional inactivation of FOXO1 in GC B cells display normal GC in number and size. However, these GCs lack phenotypically defined (CXCR4hi/CD86lo) dark zones and are entirely composed by light zone B cells (CXCR4lo/CD86hi). FOXO1-/- GC B cells express AICDA and carry a normal number of mutations in their immunonoglobulin genes, but do not undergo affinity maturation, resulting in severely impaired antigen responses. In order to identify the biological program controlled by FOXO1 in GC B cells, we identified candidate transcriptional target genes by integrating ChIP-seq and gene expression data. These analyses showed that that the establishment of the dark zone fate relies on a FOXO1-dependent transcriptional network that is enriched for genes involved in immune signaling cascades triggered by the B cell receptor and by a variety of cytokines controlling GC polarity. Notably, a majority of these target genes are co-bound and co-regulated, in a FOXO1-dependent manner, by BCL6, a well characterized GC master regulator. To assess the role of BL- and DLBCL-associated mutations, we first investigated the subcellular localization of FOXO1 mutant proteins by transfecting wild type and mutant GFP-tagged FOXO1 alleles into HeLa cells. As previously shown (Trinh et al, Blood 2013), this analysis showed that mutant FOXO1 proteins, but not the wild-type one, readily localize in the nucleus. Analogously, immunofluorescence analysis of BL and DLBCL samples showed the presence of nuclear FOXO1 in all tumors carrying mutations in the FOXO1 gene. However, nuclear localization was also detectable in virtually all cases carrying normal FOXO1 genes. Accordingly, in vitro experiments testing the ability of normal and mutated FOXO1 proteins to respond to various signals activating the PI3K pathway in multiple BL and DLBCL cell lines, failed to display a correlation between the presence of mutations and responsiveness to these signals. Taken together, these results suggest that other mechanisms in addition to direct gene mutation are responsible for the constitutive nuclear localization of FOXO1 in tumors. We are now examining the consequences of FOXO1 missense mutations in vivo, by reconstituting FOXO1-/- GC B cells with FOXO1 mutants using bone marrow chimeras. Disclosures No relevant conflicts of interest to declare.

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BCL6, p53 and Molecular Targeted Therapy in B-Cell Lymphomas.

Blood ◽

10.1182/blood.v106.11.1484.1484 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1484-1484 ◽

Cited By ~ 2

Author(s):

Leandro C.A. Cerchietti ◽

Jose M. Polo ◽

Gustavo F. Da Silva ◽

Steve M. Dowdy ◽

Catoretti M. Giorgio ◽

...

Keyword(s):

Targeted Therapy ◽

B Cell ◽

Target Gene ◽

Target Genes ◽

Wild Type ◽

B Cell Lymphomas ◽

Lymphoma Cells ◽

Sequential Administration ◽

Wild Type P53

Abstract The BCL6 transcriptional repressor is an oncogene often constitutively expressed in diffuse large B-cell lymphomas (DLBCL). The oncogenic mechanism of action of BCL6 presumably involves repression of its direct target genes. We recently developed a targeted therapy agent (called BPI - BCL6 peptide inhibitor) that specifically blocks transcriptional repression by BCL6, and which causes apoptosis in lymphoma cells in vitro and in vivo. We present here potent and stable derivatives of BPI able to specifically eradicate lymphoma cells after a single dose in vitro. Expression array studies of BCL6 target genes reactivated by BPI revealed that one such gene is the p53 tumor suppressor. p53 was also recently shown to be BCL6 target gene by Phan et. al., Nature 2004. We find that BCL6 represses p53 in DLBCL cells through recruitment of the SMRT and N-CoR corepressors, which explains how BPI, which blocks recruitment of these corepressors, reactivates p53. We next wished to determine the contribution of BCL6-mediated repression of p53 to lymphomagenesis, and how p53 modulation might affect BCL6 targeted therapy strategies for DLBCL. We found that BPI could induce p53 target gene expression in DLBCL cells with wild-type p53 and that small molecules or peptides that block p53 rescue apoptosis induced by BPI. In contrast, although BPI also induces p53 in DLCBL cells with mutant p53, there was no activation of p53 target genes and no rescue by p53 blocking molecules. However BPI causes apoptosis of DLBCL cells regardless of p53 status indicating the BCL6 mediates its oncogenic actions through both p53 dependent and independent pathways. p53 is usually wild-type in DLBCL and our analysis of >100 patients show that p53 protein is, surprisingly, still expressed in these tumors. These data suggest that p53 is not fully active in DLBCL cells, consistent with the fact that we found that BCL6 also directly represses upstream activators of p53 such as Chk1 and ATR. BCL6 blockade thus can fully restore activity of p53, both by increasing its expression levels and by enhancing its activation by upstream mediators. Accordingly, sequential administration of p53 activating molecules that enhance p53 activity, potently synergizes with BPI in killing lymphoma cells. BPI also synergizes with chemotherapy drugs that act in part through p53, such as doxorubicin. From these studies we conclude that i) BCL6 mediates lymphomagenesis by direct repression of p53 and upstream target gene pathways; ii) BCL6 positive lymphomas are dependent on BCL6 for their survival regardless of whether p53 is wild type or mutated; iii) Sequential targeting of BCL6 and p53 with BPI and a p53 activating molecule or doxorubicin is likely to be a highly effective therapeutic regimen for patients with DLBCL, especially for the majority who have wild-type p53; iv) The new BPI derivatives are sufficiently potent and stable to be tested in the clinical setting.

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MDM2-Dependent Inhibition of p53 Is Required for Epstein-Barr Virus B-Cell Growth Transformation and Infected-Cell Survival

Journal of Virology ◽

10.1128/jvi.01681-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2491-2499 ◽

Cited By ~ 40

Author(s):

Eleonora Forte ◽

Micah A. Luftig

Keyword(s):

B Cells ◽

Epstein Barr Virus ◽

Target Genes ◽

Wild Type ◽

P53 Pathway ◽

Protein Levels ◽

Barr Virus ◽

Growth Transformation ◽

Wild Type P53 ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) growth transformation of primary B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs) depends on the concerted activities of a subset of viral proteins expressed during latency. EBV drives quiescent B cells into S phase, and consequently, a host response is activated that includes expression of p53 and its target genes. Since LCLs retain wild-type p53, it was of interest to determine what contribution the p53 pathway may have in controlling established LCL growth and EBV-mediated transformation of primary B cells. We found that liberation of p53 through chemical antagonism of one of its major ubiquitin ligases, MDM2, using the small-molecule Nutlin-3 led to apoptosis of established LCLs and suppressed EBV-mediated transformation of primary B cells. The activation of latent p53 induced target genes associated with apoptosis. Furthermore, MDM2 antagonism synergized with NF-κB inhibition in killing LCLs. NF-κB was important to increase steady-state MDM2 protein levels rather than in affecting p53-dependent transcription, suggesting a unique mechanism by which LCLs survive in the presence of a primed p53 pathway. Nutlin sensitivity of EBV-infected cells provides a novel system for studying the pathways that dictate LCL survival and regulate EBV transformation. Finally, MDM2 antagonists may be considered for therapeutic intervention in EBV-associated malignancies expressing wild-type p53.

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Regulation of Hox Genes by Polycomb Group Protein EZH2 Plays a Critical Role in Diffuse Large B-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v110.11.1562.1562 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1562-1562

Author(s):

Irina Velichutina ◽

Ari Melnick

Keyword(s):

Gene Expression ◽

B Cells ◽

Target Genes ◽

Hox Genes ◽

Critical Role ◽

Polycomb Group ◽

Lymphoma Cells ◽

Self Renewal ◽

Hox Gene ◽

Large B Cell

Abstract Coordinated regulation of Hox gene expression during hematopoiesis is epigenetically controlled via chromatin modification by Polycomb group (PcG) and Trithorax (MLL) protein complexes. Whereas the oncogenic potential of certain HOX genes in leukemia has already been defined, little is known about their role in Diffuse Large B-cell Lymphomas (DLBCL). The primary focus of our studies is to determine the contribution of PcG-mediated repression of HOX and other genes to DLBCL pathogenesis. The PcG protein, Ezh2, is vital for maintaining both pluripotency of stem cells and identity of differentiated cells. Ezh2 tri-methylates lysine K27 of histone 3 (H3K27me3), a histone modification associated with gene silencing. Importantly, Ezh2 is frequently overexpressed in DLBCLs suggesting a role for EZH2 in lymphomagenesis. In support to this notion we discovered that Ezh2 is essential for DLBCL cell survival. By depleting Ezh2 level using RNAi, we found that loss of Ezh2 triggers cell cycle arrest and death of DLBCL cells. This finding prompted us to initiate functional studies aimed at uncovering Ezh2 target genes that mediate the observed cellular response in DLBCL cells. We first focused on a potential role of Ezh2 in regulation of HOX genes. We compared and contrasted Ezh2 targets in both normal Germinal Center (GC) B-cells and GC-derived DLBCLs to determine the normal and pathologic function of EZH2. We employed a tiling ChIP-chip approach covering the four human HOX clusters and mapped Ezh2 and H3K27m3 within HOX gene clusters. We further verified gene expression status of a subset of Hox genes by QPCR. These data indicated that Ezh2 and its cognate H3K27m3 mark are present at promoters of HoxC genes in both mature GC B-cells and GC-derived lymphoma cells, thereby driving the HoxC locus silent, suggesting that both rapidly dividing GC cells and GC-derived lymphoma cells require epigenetic silencing of this locus in order to maintain their phenotype. Both Ezh2 and the corresponding H3K27m3 transcription repression mark are absent within the promoter region of HoxA9 gene. HoxA9 promotes stem cell self-renewal and it is aberrantly activated in AML cells. This observation is especially striking as the HoxA9 is embedded into the Ezh2-sealed region in DLBCL cells, suggesting an Ezh2-independent mode of regulation. We are in the process of testing functional significance of this finding for lymphoma pathogenesis. we found that HoxB genes that are differentially expressed in progenitor vs. lineage committed cells are silent in DLBCL cells according to H3K27m3/Ezh2 pattern and gene expression analysis. Intriguingly, the early progenitor specific gene, HoxB3, is uniquely not bound by EZH2 nor H3K27 methylated and was highly expressed in lymphoma cells. This finding underscores a potential functional significance of re-expression of genes that control cell self-renewal in malignances that derive from mature B cells. We also examined transcriptional programming by EZH2 at the genomic level by ChIP-on-chip using NimbleGen 24,000 promoter arrays. EZH2 was bound to ∼1700 promoters in DLBCL cells and a similar number of genes displayed H3K27 methylation. Gain and loss of function studies are underway to identify the contribution of the most likely EZH2 direct targets genes to the DLBCL survival including both HOX genes and other genomic direct target genes. Taken together, our data suggest a critical role for EZH2 mediated epigenetic silencing of HOX and other genes in DLBCL - and implicate aberrant HOX gene expression in DLBCL pathogenesis.

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KZF-L162R Mutation Affects Splenic Mature B Cell Development and Alters Expression of Aiolos Target Genes

Blood ◽

10.1182/blood-2018-99-115483 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 668-668

Author(s):

Gregory Lazarian ◽

Shanye Yin ◽

Alba Font-tello ◽

Elisa Ten Hacken ◽

Tomasz Sevastianik ◽

...

Keyword(s):

B Cells ◽

Dna Binding ◽

B Cell ◽

Target Genes ◽

Cell Development ◽

B Cell Development ◽

Wild Type ◽

Bcr Signaling ◽

Average Proportion ◽

Follicular B Cells

Abstract Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL. To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT). Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3WTor Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161RHetmice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHetmice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161RHomomice are ongoing. By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161RHetmice (9 mice/group; p<0.05). Since the mutation localizes to a DNA binding domain, we hypothesized that it modifies the ability of Aiolos to control expression of its target genes. We therefore performed CHIP-seq in Ikzf3WTsplenic B cells, and identified Aiolos-associated high confidence peaks (fold change (FC) enrichment compared to input > 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC>10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos. Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161RHomoB cells (p<0.05) but not in Ikzf3-L161RHetB cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease. In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner. Disclosures Wu: Neon Therapeutics: Equity Ownership.

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The Role of p53 Signaling in Colorectal Cancer

Cancers ◽

10.3390/cancers13092125 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2125

Author(s):

Magdalena C. Liebl ◽

Thomas G. Hofmann

Keyword(s):

Colorectal Cancer ◽

Cancer Progression ◽

Target Genes ◽

Tp53 Gene ◽

Cellular Responses ◽

Missense Mutations ◽

Loss Of Function ◽

Wild Type ◽

P53 Signaling ◽

Stress Signals

The transcription factor p53 functions as a critical tumor suppressor by orchestrating a plethora of cellular responses such as DNA repair, cell cycle arrest, cellular senescence, cell death, cell differentiation, and metabolism. In unstressed cells, p53 levels are kept low due to its polyubiquitination by the E3 ubiquitin ligase MDM2. In response to various stress signals, including DNA damage and aberrant growth signals, the interaction between p53 and MDM2 is blocked and p53 becomes stabilized, allowing p53 to regulate a diverse set of cellular responses mainly through the transactivation of its target genes. The outcome of p53 activation is controlled by its dynamics, its interactions with other proteins, and post-translational modifications. Due to its involvement in several tumor-suppressing pathways, p53 function is frequently impaired in human cancers. In colorectal cancer (CRC), the TP53 gene is mutated in 43% of tumors, and the remaining tumors often have compromised p53 functioning because of alterations in the genes encoding proteins involved in p53 regulation, such as ATM (13%) or DNA-PKcs (11%). TP53 mutations in CRC are usually missense mutations that impair wild-type p53 function (loss-of-function) and that even might provide neo-morphic (gain-of-function) activities such as promoting cancer cell stemness, cell proliferation, invasion, and metastasis, thereby promoting cancer progression. Although the first compounds targeting p53 are in clinical trials, a better understanding of wild-type and mutant p53 functions will likely pave the way for novel CRC therapies.

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Differential transcriptional reprogramming by wild type and lymphoma-associated mutant MYC proteins as B-cells convert to lymphoma-like cells

10.1101/2020.07.13.200238 ◽

2020 ◽

Author(s):

Amir Mahani ◽

Gustav Arvidsson ◽

Laia Sadeghi ◽

Alf Grandien ◽

Anthony P. Wright

Keyword(s):

B Cells ◽

Poor Prognosis ◽

Ribosome Biogenesis ◽

Cell Model ◽

Human Cancer ◽

Hot Spot ◽

Transactivation Domain ◽

Wild Type ◽

Vast Number ◽

Wide Range

AbstractThe transcription factor MYC regulates the expression of a vast number of genes and is implicated in various human malignancies, for which it’s deregulation by genomic events such as translocation or amplification can be either disease-defining or associated with poor prognosis. In hematological malignancies MYC is frequently subject to missense mutations and one such hot spot where mutations have led to increased protein stability and elevated transformation activity exists within its transactivation domain. Here we present and characterize a model system for studying the effects of gradually increasing MYC levels as B-cells progress to lymphoma-like cells. Inclusion of two frequent lymphoma-associated MYC mutants (T58A and T58I) allowed for discrimination of changes in the MYC regulatory program according to mutation status. Progressive increase in MYC levels significantly altered the transcript levels of 7569 genes and subsets of these were regulated differently in mutant MYC proteins compared to WT MYC or between the mutant MYC proteins. Functional classification of the differentially regulated genes based on expression levels across different MYC levels confirmed previously found MYC regulated functions such as ribosome biogenesis and purine metabolism while other functional groups such as the downregulation of genes involved in B-cell differentiation and chemotaxis were novel. Gene sets that were differently regulated in cells overexpressing mutant MYC proteins contained an over-representation of genes involved in DNA Replication and transition from the G2 phase to mitosis. The cell model presented here mimics changes seen during lymphoma development in the Eμ-Myc mouse model as well as MYC-dependent events associated with poor prognosis in a wide range of human cancer types and therefore constitutes a relevant cell model for in vitro mechanistic studies of wild type and mutant MYC proteins in relation to lymphoma development.

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Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614241 ◽

1998 ◽

Vol 79 (01) ◽

pp. 211-216 ◽

Cited By ~ 13

Author(s):

Lysiane Hilbert ◽

Claudine Mazurier ◽

Christophe de Romeuf

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Von Willebrand Disease ◽

Platelet Glycoprotein ◽

Missense Mutations ◽

Inhibit Platelet Aggregation ◽

Wild Type ◽

A1 Domain ◽

Von Willebrand ◽

Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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Putative transmembrane transporter modulates higher-level aggression in Drosophila

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618354114 ◽

2017 ◽

Vol 114 (9) ◽

pp. 2373-2378 ◽

Cited By ~ 3

Author(s):

Budhaditya Chowdhury ◽

Yick-Bun Chan ◽

Edward A. Kravitz

Keyword(s):

Causal Effect ◽

Nerve Terminals ◽

Rna Seq ◽

Wild Type ◽

Temperature Dependent ◽

Number Of Genes ◽

Interesting Family ◽

Solute Carrier ◽

Transmembrane Transporter ◽

Selection Of

By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

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Mutated p53 in HGSC—From a Common Mutation to a Target for Therapy

Cancers ◽

10.3390/cancers13143465 ◽

2021 ◽

Vol 13 (14) ◽

pp. 3465

Author(s):

Aya Saleh ◽

Ruth Perets

Keyword(s):

Cancer Progression ◽

Target Genes ◽

P53 Protein ◽

Genetic Alterations ◽

Common Mutation ◽

Missense Mutations ◽

P53 Expression ◽

Histologic Subtype ◽

Tp53 Mutations

Mutations in tumor suppressor gene TP53, encoding for the p53 protein, are the most ubiquitous genetic variation in human ovarian HGSC, the most prevalent and lethal histologic subtype of epithelial ovarian cancer (EOC). The majority of TP53 mutations are missense mutations, leading to loss of tumor suppressive function of p53 and gain of new oncogenic functions. This review presents the clinical relevance of TP53 mutations in HGSC, elaborating on several recently identified upstream regulators of mutant p53 that control its expression and downstream target genes that mediate its roles in the disease. TP53 mutations are the earliest genetic alterations during HGSC pathogenesis, and we summarize current information related to p53 function in the pathogenesis of HGSC. The role of p53 is cell autonomous, and in the interaction between cancer cells and its microenvironment. We discuss the reduction in p53 expression levels in tumor associated fibroblasts that promotes cancer progression, and the role of mutated p53 in the interaction between the tumor and its microenvironment. Lastly, we discuss the potential of TP53 mutations to serve as diagnostic biomarkers and detail some more advanced efforts to use mutated p53 as a therapeutic target in HGSC.

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