Project description:HeLa/16E6-16E7 cells after expression of wild-type E2 compared to an E2 DNA-binding mutant Keywords: Wild-type E2 Infection vs. Mutant E2 Infection
Project description:To get insights into the function of RPAP3, we characterized its partners by performing a proteomic analysis in human cells. We fused the Cterm part of RPAP3 domain to GFP and stably expressed it in HeLa cells using site-specific integration with the Flp-In system. Following differential labeling of GFP-RPAP3-Cter and control cells with isotopically labelled amino-acids (SILAC), whole cell extracts were immuno-precipitated (IP) with anti-GFP antibodies and immunoprecipitates were subjected to quantitative mass-spectrometry analysis. Similar experiments were then performed on two mutated form of RPAP3 that can no longer with the two essential AAA+ ATPases RUVBL1/RUVBL2, important partners of Wild type RPAP3 Cterm. 3 batchs of SILAC analysis K0R0 control Hela H9 vs K4R6 x-FLAG- GFP- RPAP3 Cterm Wild type K0R0 control Hela H9 vs K4R6 x-FLAG- GFP- RPAP3 Cterm mutant 1 R623A-M626A K0R0 control Hela H9 vs K4R6 x-FLAG- GFP- RPAP3 Cterm mutant 2 F630A-S632A
Project description:<p>Although multi-agent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die due to chemo-refractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape and pattern of clonal evolution at relapse in pediatric ALL cases. These analyses showed that ALL relapses originate from a common ancestral precursor clone of the diagnosis and relapsed populations and frequently harbor mutations implicated in chemotherapy resistance. RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Notably, while some cases showed emergence of RAS mutant clones at relapse, in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations. Mechanistically, functional dissection of mouse and human wild type Kras and mutant Kras (Kras G12D) isogenic leukemia cells demonstrated induction of methotrexate resistance, but also improved response to vincristine, in mutant Kras- expressing lymphoblasts. These results identify chemotherapy driven selection as a central mechanism of leukemia clonal evolution and pave the road for the development of tailored personalized therapies for the treatment of relapsed ALL. </p>
