BCR/ABL Regulates the Expression and Interacts with Werner Syndrome Helicase/Exonuclease To Modulate Its Biochemical Properties.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2873-2873
Author(s):  
Artur Slupianek ◽  
Stanislaw Jozwiakowski ◽  
Ewa Gurdek ◽  
Michal O. Nowicki ◽  
Tomasz Skorski
Keyword(s):  
Werner Syndrome ◽  
Genotoxic Stress ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Premature Aging ◽  
Leukemia Cells ◽  
Dna Double Strand Breaks ◽  
Abl Kinase ◽  
A Genome ◽  
Transformed Cell Lines

Abstract A genome-wide screen suggested that BCR/ABL kinase might stimulate WRN, a member of the RecQ-like DNA helicases family. The Werner syndrome protein (WRN) exerts DNA helicase and 3′-5′ exonuclease activities. Inactivating mutations in the WRN gene causes Werner syndrome, characterized by premature aging, genomic instability and cancer predisposition. The WRN helicase unwinds unusual DNA structures, which can occur physiologically, or can be accidentally generated during DNA repair (double-stranded DNA with mismatched tails, bimolecular G4 quartets and Holliday junctions). In addition, WRN physically interacts with components of two major systems for DNA double-strand breaks (DSBs) repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here we demonstrated that BCR/ABL regulates the expression of WRN mRNA and protein in CML primary cells and BCR/ABL-transformed cell lines. BCR/ABL kinase-induced WRN expression is mediated by c-MYC, but not STAT5 - dependent transcription as well as by inhibition of caspases-dependent cleavage. In addition, immunoprecipitation and pull-down studies indicated that BCR/ABL interacts directly with WRN resulting in its tyrosine phosphorylation. Mutation analysis revealed that multiple domains/amino acid residues of BCR/ABL and WRN are involved in the interaction. BCR/ABL-positive leukemia cells exerted an enhanced WRN-dependent helicase activity. In addition, immunoprecipitation and double-immunofluorescence co-localization studies demonstrated an elevated interaction between WRN and RAD51 in BCR/ABL cells undergoing genotoxic stress in comparison to parental counterparts. Altogether, it is likely that WRN is involved in DSBs repair by HR in leukemia cells. More detailed studies are underway to pinpoint the role of WRN in DNA damage response in BCR/ABL-transformed cells.

scholarly journals Human WRN is an intrinsic inhibitor of progerin, abnormal splicing product of lamin A

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
So-mi Kang ◽  
Min-Ho Yoon ◽  
Su-Jin Lee ◽  
Jinsook Ahn ◽  
Sang Ah Yi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Life Span ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Werner Syndrome ◽  
Genetic Disorder ◽  
Dna Helicase ◽  
Premature Aging ◽  
Lamin A ◽  
Short Life Span

AbstractWerner syndrome (WRN) is a rare progressive genetic disorder, caused by functional defects in WRN protein and RecQ4L DNA helicase. Acceleration of the aging process is initiated at puberty and the expected life span is approximately the late 50 s. However, a Wrn-deficient mouse model does not show premature aging phenotypes or a short life span, implying that aging processes differ greatly between humans and mice. Gene expression analysis of WRN cells reveals very similar results to gene expression analysis of Hutchinson Gilford progeria syndrome (HGPS) cells, suggesting that these human progeroid syndromes share a common pathological mechanism. Here we show that WRN cells also express progerin, an abnormal variant of the lamin A protein. In addition, we reveal that duplicated sequences of human WRN (hWRN) from exon 9 to exon 10, which differ from the sequence of mouse WRN (mWRN), are a natural inhibitor of progerin. Overexpression of hWRN reduced progerin expression and aging features in HGPS cells. Furthermore, the elimination of progerin by siRNA or a progerin-inhibitor (SLC-D011 also called progerinin) can ameliorate senescence phenotypes in WRN fibroblasts and cardiomyocytes, derived from WRN-iPSCs. These results suggest that progerin, which easily accumulates under WRN-deficient conditions, can lead to premature aging in WRN and that this effect can be prevented by SLC-D011.

A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence

2021 ◽  
Vol 13 (575) ◽  
pp. eabd2655
Author(s):  
Wei Wang ◽  
Yuxuan Zheng ◽  
Shuhui Sun ◽  
Wei Li ◽  
Moshi Song ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Werner Syndrome ◽  
Embryonic Stem ◽  
Accelerated Aging ◽  
Premature Aging ◽  
Mesenchymal Precursor Cells ◽  
Genome Wide ◽  
A Genome ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome

Understanding the genetic and epigenetic bases of cellular senescence is instrumental in developing interventions to slow aging. We performed genome-wide CRISPR-Cas9–based screens using two types of human mesenchymal precursor cells (hMPCs) exhibiting accelerated senescence. The hMPCs were derived from human embryonic stem cells carrying the pathogenic mutations that cause the accelerated aging diseases Werner syndrome and Hutchinson-Gilford progeria syndrome. Genes whose deficiency alleviated cellular senescence were identified, including KAT7, a histone acetyltransferase, which ranked as a top hit in both progeroid hMPC models. Inactivation of KAT7 decreased histone H3 lysine 14 acetylation, repressed p15INK4b transcription, and alleviated hMPC senescence. Moreover, lentiviral vectors encoding Cas9/sg-Kat7, given intravenously, alleviated hepatocyte senescence and liver aging and extended life span in physiologically aged mice as well as progeroid Zmpste24−/− mice that exhibit a premature aging phenotype. CRISPR-Cas9–based genetic screening is a robust method for systematically uncovering senescence genes such as KAT7, which may represent a therapeutic target for developing aging interventions.

scholarly journals Werner Syndrome Protein and DNA Replication

2018 ◽  
Vol 19 (11) ◽  
pp. 3442 ◽  
Author(s):  
Shibani Mukherjee ◽  
Debapriya Sinha ◽  
Souparno Bhattacharya ◽  
Kalayarasan Srinivasan ◽  
Salim Abdisalaam ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ataxia Telangiectasia ◽  
Replication Fork ◽  
Genome Stability ◽  
Werner Syndrome ◽  
Genotoxic Stress ◽  
Premature Aging ◽  
Replication Forks ◽  
Werner Syndrome Protein ◽  
Syndrome Protein

Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 3′ to 5′ helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN’s contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN’s molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS.

scholarly journals Evolution of the RECQ Family of Helicases: A Drosophila Homolog, Dmblm, Is Similar to the Human Bloom Syndrome Gene

Genetics ◽  
1999 ◽  
Vol 151 (3) ◽  
pp. 1027-1039
Author(s):  
Kohji Kusano ◽  
Mark E Berres ◽  
William R Engels
Keyword(s):  
Drosophila Melanogaster ◽  
Homo Sapiens ◽  
Werner Syndrome ◽  
Bloom Syndrome ◽  
Dna Helicase ◽  
Functional Similarity ◽  
Premature Aging ◽  
Model Systems ◽  
C Elegans ◽  
Blm Gene

Abstract Several eukaryotic homologs of the Escherichia coli RecQ DNA helicase have been found. These include the human BLM gene, whose mutation results in Bloom syndrome, and the human WRN gene, whose mutation leads to Werner syndrome resembling premature aging. We cloned a Drosophila melanogaster homolog of the RECQ helicase family, Dmblm (Drosophila melanogaster Bloom), which encodes a putative 1487-amino-acid protein. Phylogenetic and dot plot analyses for the RECQ family, including 10 eukaryotic and 3 prokaryotic genes, indicate Dmblm is most closely related to the Homo sapiens BLM gene, suggesting functional similarity. Also, we found that Dmblm cDNA partially rescued the sensitivity to methyl methanesulfonate of Saccharomyces cerevisiae sgs1 mutant, demonstrating the presence of a functional similarity between Dmblm and SGS1. Our analyses identify four possible subfamilies in the RECQ family: (1) the BLM subgroup (H. sapiens Bloom, D. melanogaster Dmblm, and Caenorhabditis elegans T04A11.6); (2) the yeast RECQ subgroup (S. cerevisiae SGS1 and Schizosaccharomyces pombe rqh1/rad12); (3) the RECQL/Q1 subgroup (H. sapiens RECQL/Q1 and C. elegans K02F3.1); and (4) the WRN subgroup (H. sapiens Werner and C. elegans F18C5.2). This result may indicate that metazoans hold at least three RECQ genes, each of which may have a different function, and that multiple RECQ genes diverged with the generation of multicellular organisms. We propose that invertebrates such as nematodes and insects are useful as model systems of human genetic diseases.

Enhanced Phosphorylation of Nbs1, a Member of DNA Repair/Checkpoint Complex RAD50-Mre11-Nbs1, Can Be Targeted Simultaneously with BCR/ABL Kinase To Eliminate Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2127-2127 ◽  
Author(s):  
Lori Rink ◽  
Tomasz Stoklosa ◽  
Margaret Nieborowska-Skorska ◽  
Artur Slupianek ◽  
Ilona Seferynska ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Kinase Activity ◽  
S Phase ◽  
Leukemia Cells ◽  
Abl Kinase ◽  
Transformed Cell Lines ◽  
S Phase Checkpoint

Abstract Growing evidence indicate that ABL kinase inhibitors may need partner drugs to cure BCR/ABL-positive leukemias. Genotoxic drugs have been successfully combined with imatinib mesylate to increase its anti-leukemia activity in vitro. Although BCR/ABL-positive cells may accumulate even higher levels of DNA damage in comparison to their normal counterparts the former cells repair the lesions more proficiently and eventually survive. Therefore, targeting the mechanisms responsible for survival of leukemia cells after genotoxic treatment may increase the chances to eradicate BCR/ABL-positive leukemias. Nbs1, a member of the Rad50/Mre11/Nbs1 complex, is phosphorylated by ATM on Serine 343 (S343) in response to DNA double strand breaks (DSBs) to regulate intra-S and G2/M cell cycle checkpoints and DNA repair. Here we show that BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC, BCR/FGFR, and NPM/ALK, stimulate Nbs1 expression by protection from caspase-dependent degradation and induction of c-Myc-dependent transactivation. Downregulation of Nbs1 in BCR/ABL positive cells using siRNA increased their sensitivity to mitomycin C (MMC). Enhanced phosphorylation of Nbs1 on S343 (pNbs1) was detected by Western analysis in BCR/ABL-positive leukemia cells (CD34+ CML patient cells and leukemic cell lines) treated with various cytotoxic drugs (MMC, hydroxyurea = HU, cisplatin - CPL) in comparison to normal counterparts. This effect is associated with increased ATM kinase activity in BCR/ABL cells treated with MMC. In addition, immunofluoresence studies demonstrated an increase of the pNbs1 nuclear foci in BCR/ABL cells after MMC treatment in comparison to parental counterparts. DNA damage-dependent enhancement of pNbs1 appears to be a broad phenomenon because it was also detected in MMC-treated tumor cells expressing other FTKs. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells and BCR/ABL-transformed cell lines displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase, indicating an intact intra-S phase checkpoint. Expression of the Nbs1-S343A phosphorylation-less mutant downregulated pNbs1 and disrupted intra-S phase checkpoint resulting in reduced accumulation of BCR/ABL leukemia cells in S phase after MMC treatment. This effect was associated with an increase of the sensitivity of leukemia cells to genotoxic treatment (MMC, HU, CPL). A combinatorial strategy was employed targeting enhanced Nbs1 phosphorylation and the deregulated BCR/ABL tyrosine kinase activity, using the Nbs1-S343A phosphorylation-less mutant and a sub-optimal concentration of STI571 eliminating ~50% of leukemia cells, respectively. Targeting both BCR/ABL kinase activity and Nbs1 phosphorylation in combination significantly sensitizes B/A-positive cells to MMC treatment, nearly eradicating all leukemia cells.

BCR/ABL Stimulates WRN Helicase Activity To Facilitate DNA Double-Strand Breaks Repair.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1024-1024
Author(s):  
Artur Slupianek ◽  
Stanislaw Jozwiakowski ◽  
Dariusz Pytel ◽  
Tomasz Poplawski ◽  
Michal Nowicki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Werner Syndrome ◽  
Genotoxic Stress ◽  
Double Strand Breaks ◽  
Leukemia Cells ◽  
Helicase Activity ◽  
Strand Breaks ◽  
Increased Sensitivity ◽  
Repair Pathways ◽  
Wrn Helicase

Abstract DNA damage and defects in DNA repair pathways may severely predispose to genomic instability which is one of the major factors associated with the formation and progression of chronic myelogenous leukemia (CML). However, leukemia cells transfected by BCR/ABL seem to be better equipped to survive DNA damage generated by reactive oxygen species (ROS) and external factors through activation of double-strand breaks (DSBs) repair by homologous recombination (HR) and non-homologous end-joining (NHEJ). A genome-wide screen was performed to identify genes regulated by BCR/ABL kinase and involved in DSBs repair. Werner syndrome protein (WRN), which exhibits helicase and exonuclease activity, was upregulated in CML cells. WRN is capable of unwinding various DNA structures associated with progressing replication forks as well as promoting Holliday junctions formed as intermediates in DNA recombination. Moreover, the helicase can directly interact with a variety of proteins involved in DSBs repair including Ku complex (NHEJ), and RAD51 (HR). Lack of WRN protein in Werner syndrome is characterized by accumulation of DSBs, genomic instability and a high incidence of cancer. Here we present evidence that BCR/ABL induced the expression of WRN mRNA and protein by activation of c-MYC transcription and inhibition of caspase-dependent cleavage, respectively. Immunoprecipitation and pull-down studies indicated that WRN is phosphorylated by BCR/ABL, and that BCR/ABL SH2 domain interacts directly with phospho-Y1346 of WRN. Drug sensitivity assays performed after downregulation of WRN expression by shWRN in BCR/ABL-positive cells have demonstrated an increased sensitivity to genotoxic stress induced by cisplatin and oxidative stress caused by H2O2. Experiments using TUR90010 lymphoblast cell line established from a Werner syndrome patient (3724C>T) and transfected with BCR/ABL confirmed that WRN plays an important role in response to DNA damage in CML cells. Further studies revealed that BCR/ABL-positive leukemia cells exert an enhanced WRN-mediated helicase activity. Bone marrow cells derived from transgenic mice expressing the helicase-defective WRN mutant (K577M) and transfected with BCR/ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. The role of WRN in BCR/ABL-induced DSBs repair pathways, HR and NHEJ, was examined. NHEJ activity was measured in nuclear cell lysates of BCR/ABL-positive leukemia cells using linearized double-stranded plasmid as a substrate. Removal of WRN by immunoprecipitation did not affect the efficacy of NHEJ reaction. HR was assessed using cells containing one copy of the modified gene for GFP containing a unique I-SceI restriction site with two stop codons as a recombination reporter and a truncated fragment of the GFP gene as a template for homologous repair. A HR event restores functional GFP expression. Downregulation of WRN protein by shRNA abrogated HR activity induced by BCR/ABL. Therefore BCR/ABL-dependent overexpression of WRN helicase seemed to be important for HR, but not NHEJ. Finally, an enhanced interaction between WRN and RAD51 upon DNA damage in BCR/ABL-positive cells supported that conclusion. In summary, BCR/ABL-mediated overexpression and enhanced activation of WRN helicase played an essential role in response of CML cells to elevated numbers of DBSs induced by oxidative and genotoxic stress.

Targeting BCR/ABL-RAD51 Interaction to Prevent Unfaithful Homeologous Recombination Repair.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3272-3272
Author(s):  
Artur Slupianek ◽  
Yashodhara Dasgupta ◽  
Shuyue Ren ◽  
Kimberly Cramer ◽  
Tomasz Skorski
Keyword(s):  
Amino Acid ◽  
Chromosomal Aberrations ◽  
Genotoxic Stress ◽  
Leukemia Cells ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Abl Kinase ◽  
Recombination Repair ◽  
Resistant Mutants ◽  
Homeologous Recombination

Abstract Abstract 3272 Poster Board III-1 CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) and cell lines transformed by non-mutated BCR/ABL kinase or the tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress. In addition CD34+CD38- CML-CP and CD34+ CML-BC stem cell-enriched populations seem to display more DSBs than normal counterparts. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS- and radiation- induced DSBs induce chromosomal instability implicating enhanced, but unfaithful repair in BCR/ABL-positive leukemias [Leukemia, 2008]. We show here that BCR/ABL kinase (non-mutated and TKI-resistant mutants) facilitate recombination repair (RR) of DSBs. Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease-;inducible DSB site and a repair template displaying 19% divergence sequence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells and BCR/ABL-positive counterparts. BCR/ABL kinase caused about 3-fold increase in HomeoRR activity implicating its role in accumulation of chromosomal aberrations in CML cells. RAD51, a key regulator of recombination repair, forms a complex with BCR/ABL which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL. SH3+SH2-CD domains of BCR/ABL form a pocket binding the PP regions of RAD51. Single amino acid substitutions in the BCR/ABL SH3+SH2-CD pocket, which disrupted binding to the RAD51 PP regions reduced complex formation with RAD51. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3+SH2-CD pocket mutant displayed slow proliferation rate and responded poorly to genotoxic stress despite intact kinase activity. On the other hand, disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) prevented its interaction with BCR/ABL SH3+SH2-CD pocket. Interestingly, expression of RAD51 PP-LL mutant abrogated the clonogenic capability of BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. BCR/ABL-RAD51 complex results in direct phosphorylation of RAD51 on Y315, which is located in the vicinity of PP motifs in the C-terminal portion (aa 271–339) of RAD51. C-terminal Y315F mutant formed more abundant complex with BCR/ABL that the wild-type form, but it did not restore the lost interaction of the PP/LL mutant. Thus, BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for disassembly of RAD51 from BCR/ABL. In concordance, RAD51[Y315F] mutant remained mostly in the cytoplasm, while the wild-type protein accumulated in the nucleus in BCR/ABL-positive cells in response to DSBs induced by genotoxic treatment. In addition to the regulation of BCR/ABL-RAD51 interaction, phospho-Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. To test this hypothesis we employed a peptide aptamer strategy targeting phospho-Y315 of RAD51. Peptides corresponding to the RAD51 fragment containing phospho-Y315, but not these with Y315F substitution reduced HomeoRR activity by approximately 2-fold in BCR/ABL-positive leukemia cells. Altogether, it appears that PP-regions of RAD51 interact with SH3+SH2-CD niche of BCR/ABL, which leads to phosphorylation of RAD51 on Y315 and disassembly of the complex. Phospho-Y315 stimulates abundant nuclear localization of RAD51 on DSBs, which disrupts the mechanisms responsible for preventing recombination using divergent templates resulting in unfaithful HomeoRR in BCR/ABL-positive leukemia cells. In summary, BCR/ABL-RAD51 interaction promotes survival and accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR/ABL kinase. We hypothesize that targeting BCR/ABL-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BC progression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals NAD+ augmentation restores mitophagy and limits accelerated aging in Werner syndrome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Evandro F. Fang ◽  
Yujun Hou ◽  
Sofie Lautrup ◽  
Martin Borch Jensen ◽  
Beimeng Yang ◽  
...  
Keyword(s):  
Werner Syndrome ◽  
Accelerated Aging ◽  
Dna Helicase ◽  
Premature Aging ◽  
Metabolic Dysfunction ◽  
Gene Encoding ◽  
Metabolic Phenotypes ◽  
Cell Dysfunction ◽  
Underlying Mechanisms ◽  
Invertebrate Models

AbstractMetabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.

Selective Anti-Leukemia Targeting of the Interaction Between BCR/ABL and Mammalian RecA Homologs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 195-195
Author(s):  
Artur Slupianek ◽  
Shuyue Ren ◽  
Tomasz Skorski
Keyword(s):  
Amino Acid ◽  
Chromosomal Instability ◽  
Chronic Phase ◽  
Direct Interaction ◽  
Genotoxic Stress ◽  
Single Amino Acid ◽  
Leukemia Cells ◽  
Amino Acid Substitutions ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract We showed before that cells transformed by BCR/ABL and other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2 and TEL/PDGFR, inducing chronic myeloproliferative disorders (MPDs), and CD34+ chronic myeloid leukemia (CML) stem/ progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain an excess of DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress [Blood, 2005; Cell Cycle, 2006; DNA Repair, 2006; Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cellenriched populations seem to display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Elevated levels of DSBs were also observed in leukemia cells expressing imatinib-resistant BCR/ABL kinase mutants. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. Numerous ROS- and radiation- induced DSBs are not lethal for BCR/ABL-positive leukemia cells; instead, they induce chromosomal instability implicating enhanced, but unfaithful repair [Leukemia, 2008]. The previous report [Mol. Cell, 2001] and ongoing studies demonstrated that BCR/ABL kinase (non-mutated and imatinib-resistant mutants) modulates expression of the mammalian RecA homologs RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, which are responsible for homologous recombination repair (HRR) of DSBs. RAD51 plays a key role in HRR in cells transformed by BCR/ ABL and other FTKs [Mol. Cell, 2001; Mol. Cell. Biol., 2002]. BCR/ABL stimulates the expression of, interacts with and phosphorylates RAD51 on Y315, which is located in a critical fragment of RAD51 essential for its filament formation on DNA. Accordingly, our recent results indicated that BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for nuclear RAD51 foci formation in response to DNA damage. In addition to RAD51, BCR/ABL interacts directly with and phosphorylates RAD51B and XRCC2, but not other RecA homologs. Altogether, it appears that BCR/ABL can deregulate the expression and phosphorylation of some RecA homologs, which may have a significant impact on the efficiency and fidelity of DSB repair resulting in protection from apoptosis and chromosomal instability. Therefore, disassembly of BCR/ABL from RecA homologs should reduce the capability of CML cells to repair numerous ROS-induced DSBs and eventually trigger apoptosis. Based on this hypothesis we investigated the mechanisms of association between BCR/ABL and RecA homologs. Interactions between BCR/ ABL and RAD51 or RAD51B depend on the proline- rich (PP) regions of RAD51 and RAD51B, and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL, which form a pocket binding the PP regions. Disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) abrogated direct interaction with the BCR/ABL SH3-SH2-CD pocket. On the other hand, single amino acid substitutions in the BCR/ABL SH3-SH2-CD pocket, which eliminated its capability of binding the PP regions, prevented complex formation with RAD51 and RAD51B. In addition, RAD51 and RAD51B may interact with members of the BCR/ABL proteome such as Grb2 and Shc (RAD51 and RAD51B), and c-CrkL (only RAD51B), but not Gab2 and c-Cbl. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3-SH2-CD pocket mutant, where single amino acid substitutions disrupted its direct interaction with RAD51, displayed a slower proliferation rate and responded poorly to genotoxic stress despite intact kinase activity in comparison to cells transformed with non-mutated BCR/ABL. Interestingly, expression of RAD51 PP-LL mutant eliminated BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. These results suggest that the interaction between BCR/ABL and RAD51 may be targeted for selective elimination of leukemia cells and/or suppression of genomic instability. To test this hypothesis we are employing the peptide aptamer strategy targeting RAD51 PP regions in CD34+ cells obtained from imatinib-sensitive and imatinib-resistant CML patients and healthy volunteers in vivo and in vitro. In summary, we hypothesize that mechanisms regulating the association of BCR/ABL with RAD51 and other mammalian RecA homologs may be explored for the planning of more effective anti-tumor modalities.

Mechanisms Generating free Radicals in CML Stem/Progenitor Cell Populations Causing DNA Damage and Genomic Instability

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 192-192 ◽  
Author(s):  
Margaret Nieborowska-Skorska ◽  
Mateusz Koptyra ◽  
Grazyna Hoser ◽  
Regina Ray ◽  
Danielle Ngaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Free Radicals ◽  
Genomic Instability ◽  
Chromosomal Aberrations ◽  
Point Mutations ◽  
Leukemia Cells ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Transformed Cell Lines

Abstract BCR/ABL kinase is the founding member of a family of oncogenic tyrosine kinases (OTKs) also including TEL/JAK2, TEL/PDGFR, TEL/ABL, and JAK2V617F, which induce myeloproliferative disorders (MPDs). BCR/ABL transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but progenitor-driven disease. In CML-CP, leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) reside in the CD34+CD38- and CD34+CD38+ populations, respectively, whereas in CML-BC, LSCs are also found in the CD34+CD38+ population. In addition, CD34+ CML cells belong to either proliferative or quiescent populations; the latter of which responds poorly to the ABL kinase inhibitors. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations in the kinase domain and additional chromosomal aberrations associated with progression to CML-BC (Oncogene, 2007). Since genomic instability usually results from enhanced DNA damage, we investigated the mechanisms responsible for “spontaneous” DNA damage in cells transformed by BCR/ ABL and other OTKs. Much endogenous DNA damage arises from free radicals such as reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). We showed that CD34+ stem/progenitor CML cells contain higher levels of ROS (superoxide anion = ·O2−, hydrogen peroxide = H2O2 and hydroxyl radical = ·OH) and RNS (nitric oxide = NO·) than CD34+ cells from normal donors (CML-BC>CML-CP>Normal). Moreover, ROS levels were elevated in CD34+CD38- and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to the corresponding cells from healthy donor. In addition, both proliferative and quiescent CD34+ CML cell sub-populations contained more ROS than their normal counterparts. Interaction with the stromal cells further elevated ROS levels in BCR/ABL-positive cells. Higher ROS/RNS levels induced more oxidative/nitrative DNA lesions, such as 8-oxoG and DNA double-strand breaks (DSBs), in CML-CP cells resulting in induction of point mutations in BCR/ABL kinase causing imatinib resistance and accumulation of chromosomal aberrations characteristic of CML-BC. In addition, cells transformed by other OTKs also displayed elevated ROS/ RNS and oxidative/nitrative DNA damage, implicating their role in malignant progression of MPDs. Our previous studies showed that elevated levels of oxidative DNA damage in OTK-transformed cells could be diminished by scavenging of ROS with N-acetyl-cysteine and vitamin E, which reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells cultured in vitro and growing in SCID mice (Blood, 2006; Leukemia, 2008). These studies highlighted the importance of identification of the sources of free radicals in CML and other MPDs. We found that elevated levels of ROS in BCR/ABL-transformed cell lines and CD34+ CML cells were generated by three major mechanisms: NADPH oxidase (NOX) complexes containing NOX1 and/or NOX2, complex III of the mitochondrial respiratory chain (MRC), and 5-lipoxygenase (LOX). In addition, inducible nitric oxygen synthase (iNOS) produced RNS in leukemia cells. Using selective inhibitors of NOX, MRC, LOX and iNOS we estimated the contribution of these pathways to accumulation of free radicals causing oxidative/nitrative DNA damage in CML cells. In summary, BCR/ABL kinase-dependent elevation of ROS/RNS depends on several mechanisms, which are now targeted to determine their actual role in genomic instability in CML.

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