Cell death in NF-κB-dependent tumour cell lines as a result of NF-κB trapping by linker-modified hairpin decoy oligonucleotide

AbstractThe transcription factor NF-κB is frequently activated in cancer, and is therefore a valuable target for cancer therapy. Decoy oligodeoxynucleotides (ODNs) inhibit NF-κB by preventing its binding to the promoter region of target genes. Few studies have used NF-κB-targeting with ODNs in cancer. Using a hairpin NF-κB-decoy ODN we found that it induced growth inhibition and cell death in NF-κB-dependent tumour cell lines. The ODN colocalized with the p50 subunit of NF-κB in cells and directly interacted with it in nuclear extracts. In TNFα-treated cells the ODN and the p50 subunit were found in the cytoplasm suggesting that the complex did not translocate to the nucleus. Transcriptional activity of NF-κB was efficiently inhibited by the ODN, whereas a scrambled ODN was without effect on transcription. Thus, ODN-mediated inhibition of NF-κB can efficiently promote cell death in cancer cells providing a potentially powerful approach to tumour growth inhibition

A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes.</p> <p>Results</p> <p>New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1.</p> <p>Conclusions</p> <p>Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate protein/DNA interactions. The finding of a STAT3-specific hpdODN establishes the first rational basis for designing STAT3 DBD-specific inhibitors.</p

Inhibition of a new AXL isoform (AXL3) induces apoptosis of mantle cell lymphoma cells

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma having a poor overall survival that is in need for the development of new therapeutics. In this study, we report the identification and expression of a new isoform splice variant of the tyrosine kinase receptor AXL in MCL cells. This new AXL isoform, called AXL3, lacks the ligand-binding domain of the commonly described AXL splice variants and is constitutively activated in MCL cells. Interestingly, functional characterization of AXL3, using CRISPRi, revealed that only the knockdown of this isoform leads to apoptosis of MCL cells. Importantly, pharmacological inhibition of AXL activity resulted in a significant decrease in the activation of well-known pro-proliferative and survival pathways activated in MCL cells (i.e.b-catenin, AKT, and NF-kB). Therapeutically, pre-clinical studies using a xenograft mouse model of MCL indicated that bemcentinib is more effective than ibrutinib in reducing the tumour burden and to increase the overall survival. Our study highlights the importance of a previously unidentified AXL splice variant in cancer and the potential of bemcentinib as a targeted therapy for MCL

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

<p>Abstract</p> <p>Background</p> <p>The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear.</p> <p>Results</p> <p>The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death.</p> <p>Conclusions</p> <p>The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.</p

Cytogenetic complexity in chronic lymphocytic leukemia: definitions, associations and clinical impact

Recent evidence suggests that complex karyotype (CK) defined by the presence of 653 chromosomal aberrations (structural and/or numerical) identified by chromosome banding analysis (CBA) may be relevant for treatment decision-making in chronic lymphocytic leukemia (CLL). However, many challenges towards routine clinical application of CBA remain. In a retrospective study of 5290 patients with available CBA data, we explored both clinicobiological associations and the clinical impact of CK in CLL. We found that patients with 655 abnormalities, defined as high-CK, exhibit uniformly dismal clinical outcome, independently of clinical stage, TP53 aberrations (deletion of chromosome 17p and or TP53 mutations, TP53abs) and the expression of somatically hypermutated (M-CLL) or unmutated (U-CLL) immunoglobulin heavy variable genes (IGHV). Thus, they contrasted CK cases with 3 or 4 aberrations (low-CK and intermediate-CK, respectively) who followed aggressive disease courses only in the presence of TP53abs. At the other end of the spectrum, patients with CK and +12,+19 displayed an exceptionally indolent profile. Building upon CK, TP53abs and IGHV gene somatic hypermutation status, we propose a novel hierarchical model where patients with high-CK exhibit the worst prognosis, while M-CLL lacking CK or TP53abs as well as CK with +12,+19 show the longest overall survival. In conclusion, CK should not be axiomatically considered unfavorable in CLL, representing a heterogeneous group with variable clinical behavior. High-CK with 655 chromosomal aberrations emerges as prognostically adverse, independently of other biomarkers. Prospective clinical validation is warranted before finally incorporating high-CK in risk stratification of CLL

Pre-therapeutic assessment of chronic lymphocytic leukemia: which genetic assessment and why?

A l’ère des traitements ciblés, la prise en charge de la leucémie lymphoïde chronique (LLC) a évolué. Le bilan génétique préthérapeutique est indispensable aujourd’hui pour le choix de la stratégie thérapeutique adaptée à chaque patient. La LLC est une hémopathie dont l’évolution clinique est hétérogène, un tiers des patients n’étant jamais traité. D’autre part, les rechutes sont fréquentes et les patients reçoivent souvent plusieurs lignes de traitement. Les marqueurs génétiques sont de deux types: (1) prédictifs de la réponse au traitement, qui orientent le choix thérapeutique et/ ou (2) pronostiques, qui aident è stratifier les patients pour un meilleur suivi. La décision d’entreprendre un traitement reste basée sur le stade clinique. Le bilan génétique va permettre d’orienter le traitement vers la chimiothérapie ou les traitements ciblés. En effet, il est basé sur le statut mutationnel de TP53 et des IGHV (gène de la partie variable des chaînes lourdes des immunoglobulines). Le statut muté de TP53 contre-indique la chimiothérapie basée sur les analogues de purines comme la fludarabine. Le statut IGHV muté est prédictif d’une très bonne réponse à cette dernière. D’autres marqueurs sont pronostiques et vont aider à stratifier les patients en fonction du risque d’évolution. Les recommandations actuelles, en dehors des essais cliniques, sont d’évaluer le statut mutationnel de TP53–déletion 17p (del[17p]) et mutations–avant toute ligne de traitement et le statut mutationnel des IGHV ainsi que la présence de la délétion 11q, 13q, la trisomie 12 par hybridation in situ en fluorescence (FISH), un caryotype conventionnel étant recommandé

Dérégulations transcriptionnelles et lymphoproliférations B associées au virus d'Epstein-Barr

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude d'un marqueur diagnostique et pronostique dans les syndromes lymphoprolifératifs B CD5 positif (CLLU1)

Les syndromes lymphoprolifératifs (SLP) B CD5 positif regroupent la leucémie lymphoïde chronique (LLC) et le lymphome du manteau (MCL) bien identifiés. Mais il existe des cas de SLP B CD5 positif inclassables très peu étudiés à ce jour. Nous les avons défini par exclusion, non LLC (score de Matutes < 4) et non MCL (ne surexprimant pas la cycline D1). Récemment, un nouveau marqueur pronostique de LLC, CLLU1, a été découvert. Nous avons évalué l expression de CLLU1 dans ces 3 types de SLP et répertorié les données des autres marqueurs pronostiques de LLC. Pour la LLC, contrairement au MCL, on détecte une expression de CLLU1, corrélée aux autres marqueurs pronostiques péjoratifs classiques. Nous confirmons l intérêt diagnostique et pronostique de CLLU1 dans notre cohorte. Pour les SLP inclassables, l expression de CLLU1 est hétérogène. L étude comparative des caractéristiques cliniques, cytologiques et des marqueurs pronostiques de LLC a permis de différencier cette entité de la LLC.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Place de la protéine LNK dans les MPN (myeloproliferative neoplasm)

PARIS-BIUP (751062107) / SudocSudocFranceF