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  • Flach, Johanna  (2)
  • Hofmann, Franziska  (2)
  • Palme, Iris  (2)
  • 1
    Online Resource
    Online Resource
    Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations
    Ferrata Storti Foundation (Haematologica) ; 2020
    In:  Haematologica Vol. 106, No. 11 ( 2020-09-14), p. 2906-2917
    Details
    In: Haematologica, Ferrata Storti Foundation (Haematologica), Vol. 106, No. 11 ( 2020-09-14), p. 2906-2917
    Abstract: Somatic mutations in genes coding for splicing factors, e.g., SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with myelodysplastic syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease, therefore, making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation- associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with the low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association between R-loops and ATR sensitivity and the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.
    Type of Medium: Online Resource
    ISSN: 1592-8721 , 0390-6078
    URL: Article
    DOI: 10.3324/haematol.2020.254193
    Language: Unknown
    Publisher: Ferrata Storti Foundation (Haematologica)
    Publication Date: 2020
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  • 2
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    Online Resource
    R-Loops-Induced ATR Signaling As a Potential Therapeutic Target in Myelodysplastic Syndrome
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4218-4218
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 4218-4218
    Abstract: Introduction: Somatic mutations in genes coding for splicing factors (e.g. SF3B1, U2AF1 and SRSF2) are found in about 50% of patients with Myelodysplastic Syndrome (MDS). These mutations have been shown to frequently occur early in the mutational hierarchy of the disease making them particularly attractive therapeutic targets. Recent research has revealed an association of splicing factor mutations (sfm) with elevated levels of DNA:RNA intermediates (R-loops), which induce replication stress and downstream activation of the ataxia telangiectasia and Rad3-related protein (ATR) pathway. The aim of this work was to investigate R-loops-associated ATR signaling as a novel therapeutic concept in primary CD34+ MDS patient cells carrying sfm, and to identify possible novel options for combination therapy. Methods: Using quantitative immunofluorescence microscopy we assessed levels of R-loops in primary CD34+ bone marrow cells isolated from MDS patients (n=23) with and without sfm. In addition, we evaluated the direct association of R-loops with induction of replication stress and activation of associated signaling by analyzing replication fork progression rates and phosphorylation of ATR target proteins. Furthermore, we determined the in vitro sensitivity of mutant (n=12) and non-mutant CD34+ (n=10) cells of MDS patients towards ATR inhibitors (VE-821 and AZD6738) alone and in combination with splicing modulator Pladienolide B and investigated the impact on DNA damage accumulation and apoptosis. We also performed these experiments in cord blood derived CD34+ cells overexpressing SRSF2P95H without other MDS-associated cellular alterations. Results: We found significantly elevated levels of R-loops in CD34+ cells from MDS patients carrying sfm compared to non-splicing factor mutated (non-sfm) MDS cells and healthy controls (mean MFI= 177 (sfm) vs. 78 (non-sfm) vs. 91 (healthy), p 〈 0.0001). These were primarily found in the stem and progenitor as well as myeloid cell compartments, while lymphoid cells showed normal levels. Induced R-loops caused delayed replication fork dynamics as determined by fiber assay (mean fork speed= 0.282 kb/min (sfm) vs. 0.401 kb/min (non-sfm), p ≤0.0001), which in association with elevated levels of single-strand DNA marker replication protein A (RPA) strongly suggests the presence of replication stress in splicing factor mutated CD34+ cells. When exposed to ATR inhibitors in vitro, splicing factor mutant cells showed a significantly elevated sensitivity towards these drugs (normalized mean IC50= 0.89 µM (sfm) vs. 2.84 µM (non-sfm), p ≤0.001) associated with heightened levels of DNA damage and apoptosis, both of which were further increased by the addition of splicing modulator Pladienolide B. We also confirmed a direct correlation of R-loops associated ATR signaling with the presence of mutant SRSF2P95H by lentiviral overexpression in cord blood CD34+ cells. Conclusion: Overall, our results identify ATR as a promising novel therapeutic target in MDS with splicing factor mutations and provide a preclinical rationale for combination therapy with splicing modulator drugs. Disclosures Nolte: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-126431
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
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