60 research outputs found
Identification et caractĂ©risation de polymorphismes gĂ©nĂ©tiques impliquĂ©s dans la rĂ©ponse Ă lâimatinib dans la leucĂ©mie myĂ©loĂŻde chronique
Chronic myeloid leukemia (CML) is a rare myeloproliferative syndrome treated by tyrosine kinase inhibitors, such as imatinib. Despite its efficacy, resistance to treatment is a persistent clinical issue. Notably, genetic variants causing alterations in apoptosis may explain heterogeneity of imatinib sensitivity between patients. First, the goal was to look for candidate variants. For that purpose, a panel of 45 apoptotic genes was assessed by next-generation sequencing on 24 CML patients, 12 sensitive and 12 resistant to imatinib treatment. Using informatics tools, 473 polymorphisms were detected. As the number of sequenced samples was limited, novel statistical methods had to be developed to interpret the results. The variant frequency in resistant and sensitive patients was compared to variant frequency in the general population and visualized using descriptive statistics. This strategy allowed to obtain a restricted list of 95 polymorphisms that might be involved in resistance to the treatment. Then, genes were ranked according to variant allele enrichment. At the end, three candidate genes were chosen and sequenced for 103 CML patients. This methodology, automated using a computer algorithm, permitted to highlight a nonsynonymous variant in the BCL RAMBO gene, significantly found more often in resistant patients. Second, the objective was to characterize the role of this variant in response to imatinib using model cell lines modified by CRISPR-Cas9 technology. BCL-RAMBO knock-out cells were obtained and allowed to demonstrate the major role of BCL-RAMBO protein in apoptosis inhibition. Additionally, cell lines carrying the variant were created using a new CRISPR-Cas9 mediated technique: the whole exon carrying the nucleotide of interest was replaced with a variant exon. The amino acid change induced by the identified polymorphism was associated with a loss of sensitivity to imatinib treatment in these cell lines as suggested after patient sequencing. These data indicate that BCL-RAMBO, anti apoptotic factor in a CML cell line, could become a novel therapeutic target to overcome drug inefficacy for a subset of resistant patients.La leucĂ©mie myĂ©loĂŻde chronique (LMC) est un syndrome myĂ©loprolifĂ©ratif rare traitĂ© par des inhibiteurs de tyrosine kinase, tel que lâimatinib. MalgrĂ© son efficacitĂ©, la rĂ©sistance au traitement est un problĂšme rĂ©current. Des variants gĂ©nĂ©tiques responsables dâune altĂ©ration de la mort cellulaire programmĂ©e (apoptose) pourraient notamment expliquer lâhĂ©tĂ©rogĂ©nĂ©itĂ© de la rĂ©ponse au traitement entre les patients. Dans un premier temps, lâobjectif Ă©tait de rechercher des variants candidats. Pour cela un panel de 45 gĂšnes impliquĂ©s dans lâapoptose a Ă©tĂ© Ă©tudiĂ© par sĂ©quençage nouvelle gĂ©nĂ©ration chez 24 patients atteints de LMC, 12 rĂ©pondeurs et 12 rĂ©sistants au traitement par imatinib. A lâaide dâoutils informatiques, 473 polymorphismes ont Ă©tĂ© dĂ©tectĂ©s. Le nombre de patients Ă©tudiĂ©s Ă©tant limitĂ©, de nouvelles mĂ©thodes statistiques ont dĂ» ĂȘtre dĂ©veloppĂ©es pour analyser les rĂ©sultats obtenus. Tout dâabord, les frĂ©quences de survenue des variants chez les patients rĂ©sistants et rĂ©pondeurs ont Ă©tĂ© comparĂ©es aux frĂ©quences observĂ©es dans la population gĂ©nĂ©rale et visualisĂ©es par une approche de statistiques descriptives. Cette stratĂ©gie a permis de rĂ©duire la liste Ă 95 polymorphismes pouvant ĂȘtre impliquĂ©s dans la rĂ©sistance au traitement. Par la suite, les gĂšnes ont Ă©tĂ© classĂ©s selon leur enrichissement en allĂšles variants. Au final, trois gĂšnes candidats ont Ă©tĂ© choisis et sĂ©quencĂ©s chez 103 patients. Cette mĂ©thodologie, automatisĂ©e grĂące Ă un algorithme informatique, a permis de mettre en Ă©vidence, un variant non synonyme dans le gĂšne BCL RAMBO, retrouvĂ© plus frĂ©quemment chez les patients rĂ©sistants de maniĂšre significative. Dans un second temps, lâobjectif Ă©tait de caractĂ©riser le rĂŽle de ce variant dans la rĂ©ponse Ă lâimatinib Ă lâaide de lignĂ©es cellulaires modifiĂ©es par la technologie CRISPR-Cas9. Des cellules nâexprimant plus la protĂ©ine ont Ă©tĂ© obtenues et ont permis de mettre en Ă©vidence le rĂŽle majeur de la protĂ©ine BCL RAMBO dans lâinhibition de lâapoptose. Des lignĂ©es cellulaires portant le variant candidat ont Ă©galement Ă©tĂ© crĂ©Ă©es Ă lâaide dâune nouvelle technique utilisant CRISPR-Cas9 : lâexon entier contenant le nuclĂ©otide dâintĂ©rĂȘt a Ă©tĂ© remplacĂ© par un exon modifiĂ©. La modification dâun acide aminĂ© induite par le variant a Ă©tĂ© associĂ© Ă une perte de la sensibilitĂ© au traitement par imatinib dans ces lignĂ©es, comme suggĂ©rĂ© aprĂšs sĂ©quençage des patients. Ces donnĂ©es indiquent que BCL-RAMBO, facteur anti-apoptotique dans une lignĂ©e modĂšle de LMC, pourrait devenir une nouvelle cible thĂ©rapeutique afin de surmonter la rĂ©sistance Ă lâimatini
Identification and characterisation of genetic polymorphisms associated to imatinib sensitivity in chronic myeloid leukemia
La leucĂ©mie myĂ©loĂŻde chronique (LMC) est un syndrome myĂ©loprolifĂ©ratif rare traitĂ© par des inhibiteurs de tyrosine kinase, tel que lâimatinib. MalgrĂ© son efficacitĂ©, la rĂ©sistance au traitement est un problĂšme rĂ©current. Des variants gĂ©nĂ©tiques responsables dâune altĂ©ration de la mort cellulaire programmĂ©e (apoptose) pourraient notamment expliquer lâhĂ©tĂ©rogĂ©nĂ©itĂ© de la rĂ©ponse au traitement entre les patients. Dans un premier temps, lâobjectif Ă©tait de rechercher des variants candidats. Pour cela un panel de 45 gĂšnes impliquĂ©s dans lâapoptose a Ă©tĂ© Ă©tudiĂ© par sĂ©quençage nouvelle gĂ©nĂ©ration chez 24 patients atteints de LMC, 12 rĂ©pondeurs et 12 rĂ©sistants au traitement par imatinib. A lâaide dâoutils informatiques, 473 polymorphismes ont Ă©tĂ© dĂ©tectĂ©s. Le nombre de patients Ă©tudiĂ©s Ă©tant limitĂ©, de nouvelles mĂ©thodes statistiques ont dĂ» ĂȘtre dĂ©veloppĂ©es pour analyser les rĂ©sultats obtenus. Tout dâabord, les frĂ©quences de survenue des variants chez les patients rĂ©sistants et rĂ©pondeurs ont Ă©tĂ© comparĂ©es aux frĂ©quences observĂ©es dans la population gĂ©nĂ©rale et visualisĂ©es par une approche de statistiques descriptives. Cette stratĂ©gie a permis de rĂ©duire la liste Ă 95 polymorphismes pouvant ĂȘtre impliquĂ©s dans la rĂ©sistance au traitement. Par la suite, les gĂšnes ont Ă©tĂ© classĂ©s selon leur enrichissement en allĂšles variants. Au final, trois gĂšnes candidats ont Ă©tĂ© choisis et sĂ©quencĂ©s chez 103 patients. Cette mĂ©thodologie, automatisĂ©e grĂące Ă un algorithme informatique, a permis de mettre en Ă©vidence, un variant non synonyme dans le gĂšne BCL RAMBO, retrouvĂ© plus frĂ©quemment chez les patients rĂ©sistants de maniĂšre significative. Dans un second temps, lâobjectif Ă©tait de caractĂ©riser le rĂŽle de ce variant dans la rĂ©ponse Ă lâimatinib Ă lâaide de lignĂ©es cellulaires modifiĂ©es par la technologie CRISPR-Cas9. Des cellules nâexprimant plus la protĂ©ine ont Ă©tĂ© obtenues et ont permis de mettre en Ă©vidence le rĂŽle majeur de la protĂ©ine BCL RAMBO dans lâinhibition de lâapoptose. Des lignĂ©es cellulaires portant le variant candidat ont Ă©galement Ă©tĂ© crĂ©Ă©es Ă lâaide dâune nouvelle technique utilisant CRISPR-Cas9 : lâexon entier contenant le nuclĂ©otide dâintĂ©rĂȘt a Ă©tĂ© remplacĂ© par un exon modifiĂ©. La modification dâun acide aminĂ© induite par le variant a Ă©tĂ© associĂ© Ă une perte de la sensibilitĂ© au traitement par imatinib dans ces lignĂ©es, comme suggĂ©rĂ© aprĂšs sĂ©quençage des patients. Ces donnĂ©es indiquent que BCL-RAMBO, facteur anti-apoptotique dans une lignĂ©e modĂšle de LMC, pourrait devenir une nouvelle cible thĂ©rapeutique afin de surmonter la rĂ©sistance Ă lâimatinibChronic myeloid leukemia (CML) is a rare myeloproliferative syndrome treated by tyrosine kinase inhibitors, such as imatinib. Despite its efficacy, resistance to treatment is a persistent clinical issue. Notably, genetic variants causing alterations in apoptosis may explain heterogeneity of imatinib sensitivity between patients. First, the goal was to look for candidate variants. For that purpose, a panel of 45 apoptotic genes was assessed by next-generation sequencing on 24 CML patients, 12 sensitive and 12 resistant to imatinib treatment. Using informatics tools, 473 polymorphisms were detected. As the number of sequenced samples was limited, novel statistical methods had to be developed to interpret the results. The variant frequency in resistant and sensitive patients was compared to variant frequency in the general population and visualized using descriptive statistics. This strategy allowed to obtain a restricted list of 95 polymorphisms that might be involved in resistance to the treatment. Then, genes were ranked according to variant allele enrichment. At the end, three candidate genes were chosen and sequenced for 103 CML patients. This methodology, automated using a computer algorithm, permitted to highlight a nonsynonymous variant in the BCL RAMBO gene, significantly found more often in resistant patients. Second, the objective was to characterize the role of this variant in response to imatinib using model cell lines modified by CRISPR-Cas9 technology. BCL-RAMBO knock-out cells were obtained and allowed to demonstrate the major role of BCL-RAMBO protein in apoptosis inhibition. Additionally, cell lines carrying the variant were created using a new CRISPR-Cas9 mediated technique: the whole exon carrying the nucleotide of interest was replaced with a variant exon. The amino acid change induced by the identified polymorphism was associated with a loss of sensitivity to imatinib treatment in these cell lines as suggested after patient sequencing. These data indicate that BCL-RAMBO, anti apoptotic factor in a CML cell line, could become a novel therapeutic target to overcome drug inefficacy for a subset of resistant patients
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