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1.
Pharmacogenomics ; 23(7): 415-430, 2022 05.
Article in English | MEDLINE | ID: mdl-35485735

ABSTRACT

Aims: To investigate the role of MYBBP1A gene and rs3809849 in pancreatic cancer (PANC1) and lymphoblastic leukemia (NALM6) cell lines and their response to asparaginase treatment. Materials & methods: The authors applied CRISPR-Cas9 to produce MYBBP1A knock-out (KO) and rs3809849 knock-in (KI) cell lines. The authors also interrogated rs3809849's impact on PANC1 cells through allele-specific overexpression. Results: PANC1 MYBBP1A KO cells exhibited lower proliferation capacity (p ≤ 0.05), higher asparaginase sensitivity (p = 0.01), reduced colony-forming potential (p = 0.001), cell cycle blockage in S phase, induction of apoptosis and remarkable morphology changes suggestive of an epithelial-mesenchymal transition. Overexpression of the wild-type (but not the mutant) allele of MYBBP1A-rs3809849 in PANC1 cells increased asparaginase sensitivity. NALM6 MYBBP1A KO displayed resistance to asparaginase (p < 0.0001), whereas no effect for rs3809849 KI was noted. Conclusions:MYBBP1A is important for regulating various cellular functions, and it plays, along with its rs3809849 polymorphism, a tissue-specific role in asparaginase treatment response.


Subject(s)
Pancreatic Neoplasms , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Alleles , Asparaginase/genetics , Asparaginase/pharmacology , Asparaginase/therapeutic use , DNA-Binding Proteins/genetics , Humans , Pancreatic Neoplasms/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA-Binding Proteins/genetics , Transcription Factors/genetics , Pancreatic Neoplasms
2.
Oncotarget ; 8(46): 80645-80650, 2017 Oct 06.
Article in English | MEDLINE | ID: mdl-29113332

ABSTRACT

Childhood acute lymphoblastic leukemia (cALL) accounts for 25% of pediatric cancers and is one of the leading causes of disease-related death in children. Although long non-coding RNAs (lncRNAs) have been implicated in cALL etiology, progression, and treatment response, little is known about their exact functional role. We had previously sequenced the whole transcriptome of 56 cALL patients and identified lncRNA transcripts specifically silenced in tumoral cells. Here we investigated the impact of restoring the expression of three of these (RP11-624C23.1, RP11-203E8, and RP11-446E9) in leukemic cell lines had dramatic impact on cancer hallmark cellular phenotypes such as apoptosis, cell proliferation and migration, and DNA damage response. Interestingly, both RP11-624C23.1 and RP11-203E8 had very similar impacts on DNA damage response, specifically displaying lower γ-H2A.X and higher apoptosis levels than control cells in response to genotoxic stress. These results indicate that silencing RP11-624C23.1 or RP11-203E8 could provide a selective advantage to leukemic cells by increasing resistance to genotoxic stress, possibly by modulating the DDR pathway. Since genotoxic agents are fundamental parts of antineoplastic treatment, further investigation of the mechanisms these lncRNAs impact would provide novel and interesting avenues for overcoming treatment resistance.

3.
PLoS One ; 12(3): e0174124, 2017.
Article in English | MEDLINE | ID: mdl-28346506

ABSTRACT

Pre-B cell childhood acute lymphoblastic leukemia (pre-B cALL) is a heterogeneous disease involving many subtypes typically stratified using a combination of cytogenetic and molecular-based assays. These methods, although widely used, rely on the presence of known chromosomal translocations, which is a limiting factor. There is therefore a need for robust, sensitive, and specific molecular biomarkers unaffected by such limitations that would allow better risk stratification and consequently better clinical outcome. In this study we performed a transcriptome analysis of 56 pre-B cALL patients to identify expression signatures in different subtypes. In both protein-coding and long non-coding RNAs (lncRNA), we identified subtype-specific gene signatures distinguishing pre-B cALL subtypes, particularly in t(12;21) and hyperdiploid cases. The genes up-regulated in pre-B cALL subtypes were enriched in bivalent chromatin marks in their promoters. LncRNAs is a new and under-studied class of transcripts. The subtype-specific nature of lncRNAs suggests they may be suitable clinical biomarkers to guide risk stratification and targeted therapies in pre-B cALL patients.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Leukemic , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA, Long Noncoding/genetics , Child , Child, Preschool , Cohort Studies , Female , Humans , Male , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/metabolism , Precursor Cells, B-Lymphoid/pathology
4.
Oncotarget ; 8(5): 7477-7488, 2017 Jan 31.
Article in English | MEDLINE | ID: mdl-27980230

ABSTRACT

Childhood acute lymphoblastic leukemia (cALL) is the most common pediatric cancer and, despite an 85% cure rate, still represents a major cause of disease-related death in children. Recent studies have implicated long non-coding RNAs (lncRNAs) in cALL etiology, progression, and treatment response. However, barring some exceptions little is known about the functional impact of lncRNAs on cancer biology, which limits their potential as potential therapeutic targets. We wanted to investigate the functional role of lncRNAs identified as specifically overexpressed in pre-B cALL by whole-transcriptome sequencing. Here we report five lncRNAs specifically upregulated in pre-B cALL that had significant impacts on cancer hallmark traits such as cell proliferation, migration, apoptosis, and treatment response. In particular, silencing of the RP11-137H2.4 lncRNA effectively restored normal glucocorticoid (GC) response in a GC-resistant pre-B cALL cell line and specifically modulated expression of members of both the NRAS/BRAF/NF-κB MAPK cascade and cell cycle pathways. Since GC form the cornerstone of cALL chemotherapy and resistance in cALL confers a dismal prognosis, characterizing RP11-137H2.4'sexact role and function in this process will be critical to the development of new therapeutic approaches to overcome GC resistance in children treated for cALL.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Prednisolone/pharmacology , RNA, Long Noncoding/genetics , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Child , Child, Preschool , Female , Gene Expression Regulation, Leukemic , Humans , MAP Kinase Signaling System/drug effects , Male , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA Interference , RNA, Long Noncoding/metabolism , Time Factors , Transfection
5.
J Pathol ; 237(1): 14-24, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25965880

ABSTRACT

A tyrosine kinase network composed of the TAM receptor AXL and the cytoplasmic kinases LYN and SYK is involved in nilotinib-resistance of chronic myeloid leukaemia (CML) cells. Here, we show that the E3-ubiquitin ligase CBL down-regulation occurring during prolonged drug treatment plays a critical role in this process. Depletion of CBL in K562 cells increases AXL and LYN protein levels, promoting cell resistance to nilotinib. Conversely, forced expression of CBL in nilotinib-resistant K562 cells (K562-rn) dramatically reduces AXL and LYN expression and resensitizes K562-rn cells to nilotinib. A similar mechanism was found to operate in primary CML CD34(+) cells. Mechanistically, the E3-ligase CBL counteracts AXL/SYK signalling, promoting LYN transcription by controlling AXL protein stability. Surprisingly, the role of AXL in resistance was independent of its ligand GAS6 binding and its TK activity, in accordance with a scaffold activity for this receptor being involved in this cellular process. Collectively, our results demonstrate a pivotal role for CBL in the control of a tyrosine kinase network mediating resistance to nilotinib treatment in CML cells.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Proto-Oncogene Proteins/metabolism , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , src-Family Kinases/metabolism , Enzyme Stability , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Ligands , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-cbl/genetics , RNA Interference , Receptor Protein-Tyrosine Kinases/genetics , Syk Kinase , Time Factors , Transfection , src-Family Kinases/genetics , Axl Receptor Tyrosine Kinase
6.
Leuk Res Treatment ; 2012: 861301, 2012.
Article in English | MEDLINE | ID: mdl-23213550

ABSTRACT

We used K562 cells sensitive or generated resistant to imatinib or nilotinib to investigate their response to mycophenolic acid (MPA). MPA induced DNA damage leading to cell death with a minor contribution of apoptosis, as revealed by annexin V labeling (up to 25%). In contrast, cell cycle arrest and positive staining for senescence-associated ß-galactosidase activity were detected for a large cell population (80%). MPA-induced cell death was potentialized by the inhibition of autophagy and this is associated to the upregulation of apoptosis. In contrast, senescence was neither decreased nor abrogated in autophagy deficient K562 cells. Primary CD34 cells from CML patients sensitive or resistant to imatinib or nilotinib respond to MPA although apoptosis is mainly detected. These results show that MPA is an interesting tool to overcome resistance in vitro and in vivo mainly in the evolved phase of the disease.

7.
Blood ; 118(8): 2211-21, 2011 Aug 25.
Article in English | MEDLINE | ID: mdl-21730355

ABSTRACT

In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.


Subject(s)
Antineoplastic Agents/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , src-Family Kinases/metabolism , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Neoplasm , Base Sequence , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/physiology , Female , Fusion Proteins, bcr-abl/metabolism , Gene Expression , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , K562 Cells , Male , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proteomics , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Syk Kinase , src-Family Kinases/genetics , Axl Receptor Tyrosine Kinase
8.
Trends Biochem Sci ; 33(4): 171-80, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18353649

ABSTRACT

The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.


Subject(s)
Gene Expression Regulation, Enzymologic , I-kappa B Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Enzyme Activation , Gene Expression Regulation, Neoplastic , Humans , I-kappa B Kinase/physiology , Models, Biological , Models, Genetic , NF-kappa B/metabolism , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/physiology , Signal Transduction , Ubiquitin/metabolism
9.
J Biol Chem ; 282(43): 31131-46, 2007 Oct 26.
Article in English | MEDLINE | ID: mdl-17823124

ABSTRACT

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKepsilon-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKepsilon-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKepsilon upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKepsilon-mediated Lys(63)-linked polyubiquitination, a mechanism that does not require TBK1-IKKepsilon kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKepsilon complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys(63)-linked polyubiquitination of their scaffold protein, TANK.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , I-kappa B Kinase/immunology , Lipopolysaccharides/immunology , Lysine/metabolism , Protein Serine-Threonine Kinases/immunology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Enzyme Activation/immunology , Genes, Reporter , HeLa Cells , Humans , I-kappa B Kinase/metabolism , Interferon Regulatory Factors/immunology , Interferon Regulatory Factors/metabolism , Kidney/cytology , Lipopolysaccharides/metabolism , Luciferases/metabolism , Lysine/chemistry , Macrophages/immunology , Macrophages/metabolism , Mice , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Transfection , Two-Hybrid System Techniques , Ubiquitination
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