The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.

• MeSH
• ATM protein genetika   metabolismus   MeSH
• biotest MeSH
• chemorezistence genetika   MeSH
• chronická lymfatická leukemie farmakoterapie   genetika   metabolismus   patologie   MeSH
• doxorubicin farmakologie   MeSH
• epigeneze genetická MeSH
• lidé MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• mutace * MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• poškození DNA MeSH
• protinádorové látky farmakologie   MeSH
• regulace genové exprese u leukemie * MeSH
• RNA nádorová genetika   MeSH
• senzitivita a specificita MeSH
• vidarabin analogy a deriváty   farmakologie   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• doxorubicin MeSH
• fludarabine MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• protinádorové látky MeSH
• RNA nádorová MeSH
• vidarabin MeSH

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

• MeSH
• apoptóza MeSH
• ATM protein metabolismus   MeSH
• chronická lymfatická leukemie genetika   MeSH
• delece genu MeSH
• doxorubicin farmakologie   MeSH
• fosfoproteiny genetika   MeSH
• genom lidský MeSH
• histony metabolismus   MeSH
• imidazoly farmakologie   MeSH
• kohortové studie MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U2 genetika   MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• nádorový supresorový protein p53 genetika   MeSH
• piperaziny farmakologie   MeSH
• poškození DNA MeSH
• prognóza MeSH
• průtoková cytometrie MeSH
• receptor Notch1 genetika   MeSH
• regulace genové exprese u leukemie * MeSH
• sestřihové faktory MeSH
• vidarabin analogy a deriváty   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• doxorubicin MeSH
• fludarabine MeSH Prohlížeč
• fosfoproteiny MeSH
• H2AX protein, human MeSH Prohlížeč
• histony MeSH
• imidazoly MeSH
• malý jaderný ribonukleoprotein U2 MeSH
• nádorový supresorový protein p53 MeSH
• NOTCH1 protein, human MeSH Prohlížeč
• nutlin 3 MeSH Prohlížeč
• piperaziny MeSH
• receptor Notch1 MeSH
• sestřihové faktory MeSH
• SF3B1 protein, human MeSH Prohlížeč
• TP53 protein, human MeSH Prohlížeč
• vidarabin MeSH

In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.

• MeSH
• analýza přežití MeSH
• B-lymfocyty účinky léků   metabolismus   patologie   MeSH
• buněčné klony MeSH
• chronická lymfatická leukemie farmakoterapie   genetika   mortalita   patologie   MeSH
• dospělí MeSH
• klonální evoluce účinky léků   genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• protinádorové látky aplikace a dávkování   škodlivé účinky   MeSH
• recidiva MeSH
• regulace genové exprese u leukemie * MeSH
• retrospektivní studie MeSH
• senioři MeSH
• signální transdukce MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH
• protinádorové látky MeSH
• TP53 protein, human MeSH Prohlížeč

ATM abnormalities are frequent in chronic lymphocytic leukemia and represent an important prognostic factor. Sole 11q deletion does not result in ATM inactivation by contrast to biallelic defects involving mutations. Therefore, the analysis of ATM mutations and their functional impact is crucial. In this study, we analyzed ATM mutations in predominantly high-risk patients using: i) resequencing microarray and direct sequencing; ii) Western blot for total ATM level; iii) functional test based on p21 gene induction after parallel treatment of leukemic cells with fludarabine and doxorubicin. ATM dysfunction leads to impaired p21 induction after doxorubicin exposure. We detected ATM mutation in 16% (22 of 140) of patients, and all mutated samples manifested demonstrable ATM defect (impaired p21 upregulation after doxorubicin and/or null protein level). Loss of ATM function in mutated samples was also evidenced through defective p53 pathway activation after ionizing radiation exposure. ATM mutation frequency was 34% in patients with 11q deletion, 4% in the TP53-defected group, and 8% in wild-type patients. Our functional test, convenient for routine use, showed high sensitivity (80%) and specificity (97%) for ATM mutations prediction. Only cells with ATM mutation, but not those with sole 11q deletion, were resistant to doxorubicin. As far as fludarabine is concerned, this difference was not observed. Interestingly, patients from both these groups experienced nearly identical time to first treatment. In conclusion, ATM mutations either alone or in combination with 11q deletion uniformly led to demonstrable ATM dysfunction in patients with chronic lymphocytic leukemia and mutation presence can be predicted by the functional test using doxorubicin.

• MeSH
• ATM protein antagonisté a inhibitory   genetika   fyziologie   MeSH
• chromozomální delece MeSH
• chronická lymfatická leukemie diagnóza   genetika   patologie   MeSH
• dospělí MeSH
• doxorubicin farmakologie   MeSH
• kohortové studie MeSH
• leukocyty mononukleární účinky léků   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidské chromozomy, pár 11 genetika   MeSH
• mutace genetika   MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• doxorubicin MeSH