Antiapoptotic proteins Bcl2 and BclX do not protect chronic myeloid leukemia cells from imatinib-mediated growth arrest

Imatinib (Glivec, Gleevec, STI571), a Bcr-Abl kinase inhibitor, is the most used drug in chronic myeloid leukemia. Imatinib induces apoptosis in a number of CML-derived cell lines, including K562. However, in order to achieve hematological remissions it is required chronic treatment with the drug, a fact inconsistent with a cytotoxic mechanism of imatinib in vivo. In this work we have analysed the effects of imatinib on the proliferation and apoptosis of K562-derived cell lines with constitutive expression of the anti-apoptotic genes Bcl2 and BclX. We found that imatinib-mediated apoptosis was completely abrogated in both Bcl2- and BclX-cell lines. However, imatinib inhibited proliferation, although growth rate was higher than in parental K562. We conclude that, besides its apoptotic effect, imatinib acts through an apoptosis-independent mechanism to arrest cell growth.The work was supported by grant PM98-0109 and SAF2002-04193 from Spanish Ministry of Science and Technology to J.L.Peer Reviewe

MYC in chronic myeloid leukemia: Induction of aberrant DNA synthesis and association with poor response to imatinib

El pdf es la versión post-print.-- et al.Untreated chronic myeloid leukemia (CML) progresses from chronic phase to blastic crisis (BC). Increased genomic instability, deregulated proliferation, and loss of differentiation appear associated to BC, but the molecular alterations underlying the progression of CML are poorly characterized. MYC oncogene is frequently deregulated in human cancer, often associated with tumor progression. Genomic instability and induction of aberrant DNA replication are described as effects of MYC. In this report, we studied MYC activities in CML cell lines with conditional MYC expression with and without exposure to imatinib, the front-line drug in CML therapy. In cells with conditional MYC expression, MYC did not rescue the proliferation arrest mediated by imatinib but provoked aberrant DNA synthesis and accumulation of cells with 4C content. We studied MYC mRNA expression in 66 CML patients at different phases of the disease, and we found that MYC expression was higher in CML patients at diagnosis than control bone marrows or in patients responding to imatinib. Further, high MYC levels at diagnosis correlated with a poor response to imatinib. MYC expression did not directly correlate with BCR-ABL levels in patients treated with imatinib. Overall our study suggests that, as in other tumor models, MYC-induced aberrant DNA synthesis in CML cells is consistent with MYC overexpression in untreated CML patients and nonresponding patients and supports a role for MYC in CML progression, possibly through promotion of genomic instability. ©2011 AACR.This work was supported by a grant from the Ministerio de Ciencia e Innovacion of Spain (SAF08-01581) and by the RTICC (Red Temática de Investigación Cooperativa en Cancer, RD06/0020/0017) to J. León and a grant from Instituto de Salud Carlos III (FIS08/0829) to M.D. Delgado.Peer Reviewe

High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Myc (c-Myc) counteracts p27 effects, and low p27 usually correlates with high Myc expression in human cancer. However there is no information on the co-expression of both genes in chronic lymphocytic leukemia (CLL). We found a lack of correlation between RNA and protein levels of p27 and Myc in CLL cells, so we determined the protein levels by immunoblot in 107 cases of CLL. We observed a high p27 protein expression in CLL compared to normal B cells. Ectopic p27 expression in a CLL-derived cell line resulted in cell death resistance. Surprisingly, Myc expression was very low or undetectable in most CLL cases analyzed, with a clear correlation between high p27 and low Myc protein levels. This was associated with low Skp2 expression, which is consistent with the Skp2 role in p27 degradation and with SKP2 being a Myc target gene. High Myc expression did not correlate with leukemia progression, despite that cell cycle-related Myc target genes were upregulated. However, biochemical analysis showed that the high p27 levels inhibited cyclin-Cdk complexes even in Myc expressing CLL cells. Our data suggest that the combination of high p27 and low Myc is a marker of CLL cells which is mediated by Skp2.The work has been funded by grants SAF11-23796 from MINECO and RD12/0036/0033 (to JL), and RD12/0036/0004 (to DC) and RD12/0036/0060 (to MAP) from Instituto Carlos III. These funding was co-sponsored by the European Union FEDER program. JC was recipient of a Fellowship from Fundación Marqués de Valdecilla and from the University of Cantabria.Peer Reviewe

Treatment‐free remission after dasatinib in patients with chronic myeloid leukaemia in chronic phase with deep molecular response: Final 5‐year analysis of DASFREE

Patients with chronic myeloid leukaemia in chronic phase (CML-CP) who have a sustained deep molecular response (DMR) are eligible to discontinue treatment and attempt treatment-free remission (TFR). In the DASFREE study (ClinicalTrials.gov; NCT01850004), the 2-year TFR rate after dasatinib discontinuation was 46%; here we present the 5-year update. Patients with a stable DMR after ≥2 years of dasatinib therapy discontinued treatment and were followed for 5 years. At a minimum follow-up of 60 months, in 84 patients discontinuing dasatinib, the 5-year TFR rate was 44% (n = 37). No relapses occurred after month 39 and all evaluable patients who relapsed and restarted dasatinib (n = 46) regained a major molecular response in a median of 1.9 months. The most common adverse event during the off-treatment period was arthralgia (18%, 15/84); a total of 15 withdrawal events were reported in nine patients (11%). At the 5-year final follow-up, almost half of the patients who discontinued dasatinib after a sustained DMR maintained TFR. All evaluable patients who experienced a relapse quickly regained a DMR after restarting dasatinib, demonstrating that dasatinib discontinuation is a viable and potentially long-term option in patients with CML-CP. The safety profile is consistent with the previous report

High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

Myc (c-Myc) counteracts p27 effects, and low p27 usually correlates with high Myc expression in human cancer. However there is no information on the co-expression of both genes in chronic lymphocytic leukemia (CLL). We found a lack of correlation between RNA and protein levels of p27 and Myc in CLL cells, so we determined the protein levels by immunoblot in 107 cases of CLL. We observed a high p27 protein expression in CLL compared to normal B cells. Ectopic p27 expression in a CLL-derived cell line resulted in cell death resistance. Surprisingly, Myc expression was very low or undetectable in most CLL cases analyzed, with a clear correlation between high p27 and low Myc protein levels. This was associated with low Skp2 expression, which is consistent with the Skp2 role in p27 degradation and with SKP2 being a Myc target gene. High Myc expression did not correlate with leukemia progression, despite that cell cycle-related Myc target genes were upregulated. However, biochemical analysis showed that the high p27 levels inhibited cyclin-Cdk complexes even in Myc expressing CLL cells. Our data suggest that the combination of high p27 and low Myc is a marker of CLL cells which is mediated by Skp2

Validation of mutated <i>CEBPA</i> bZIP as a distinct prognosis entity in acute myeloid leukemia: a study by the Spanish PETHEMA registry

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Networking for advanced molecular diagnosis in acute myeloid leukemia patients is possible: the PETHEMA NGS-AML project

Next-generation sequencing (NGS) has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia (AML). However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. In order to overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for NGS panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse AML. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized NGS analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group. This cooperative nationwide strategy provides advanced molecular diagnostic for AML patients of the PETHEMA group (clinicaltrials gov. Identifier: NCT03311815)

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Myc/p27 balance in chronic lymphocytic leukemia

Trabajo presentado al "22th Biennial Congress of the European Association for Cancer Research" celebrado en Barcelona del 7 al 10 de julio de 2012.-- et al.[Background]: Chronic Iymphocytic leukemia (CLL) is the most common leukemia in the western world. CLL is characterized by the accumulation of CD5+ 8 Iymphocytes. However, clinical outcome of CLL may be different. Some patients have an indolent leukemia with long survival while others experience an aggressive disease. Contrary to other human malignancies, the cyclin-dependent kinase inhibitor p27kip1 (p27) has been described to be overexpressed in CLL cells. On the other hand, Myc is an oncogenic transcription factor overexpressed in many human tumours. In recent years, work in several cellular models have demonstrated that Myc, through different mechanisms, antagonize p27 express ion and its anti-proliferatice activity. Thus we set out to study the interaction functional between p27 and Myc in 8-CLL and whether changes in p27/Myc ratio correlated with the clinic features of the disease. [Material and Methods]: Protein and mRNA levels of Myc and p27 in CLL patients and Mec1 cells were analyzed by Western 810t and real time RTPCR. We studied p27 and Myc localization by immunofluorescence. Annexin assay was performed to study the fludarabine resistance. SPSS and GraphPad Prism were used for statistical studies. [Results]: We studied expression levels of p27 and Myc in more than 100 CLL patients using peripheral blood, tonsil and CD19+ Iymphocytes as control. p27 and Myc levels were inversely correlated. Thus, the ratio between p27 (overexpressed) and Myc (downregulated) appears inverted in CLL with respect to the other tumours so far known. Moreover, low p27 and high Myc express ion correlated with the expression of Skp2, a subunit of ubiquitin protein ligase complex SCF which is the main responsible for p27 degradation. Skp2 is a Myc target gene and promotes the degradation of p27, suggesting a pathway Myo-Skp2-p27 in CLL. To investigate why p27 is expressed in CLL we overexpressed in a CLL-derived cellline (Mec1) and observed that high levels of p27 provide resistance to fludarabine treatment. In fact, p27 expression was strongly correlated with Bcl2 levels, an antiapoptotic protein, in CLL samples. [Conclusions]: Our data provide new insights into the p27/Myc balance in CLL cells. The high percentage of p27 overexpressed patients suggests an important function in CLL. causing low levels of Myc and increasing resistan ce to apoptosis, likely through collaboration with 8c12. These resu lts suggest a functional explanation so as why p27 is overexpressed in CLL.Peer Reviewe

c-myc expression in cell lines derived from chronic myeloid leukemia

Letter to the Editor.-- Open AccessWe analyzed proliferation and c-myc expression in three chronic myeloid leukemia (CML)-derived cell lines treated with interferon-α, hydroxyurea, busulfan and imatinib. We found that c-Myc levels did not universally correlate with CML cell proliferation and that c-Myc down-regulation correlated to imatinib activity but not to imatinib-induced apoptosis.This study was supported by grants SAF2002-04193 from Spanish Ministry of Science and Technology to J.L., and FIS01-1129 from Spanish Ministry of Health to M.D.D.Peer Reviewe