@phdthesis{Mestermann2020,
  author    = {Mestermann, Katrin},
  title     = {Pharmacological control of CAR T-cells by dasatinib},
  doi       = {10.25972/OPUS-18056},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180562},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Cellular therapies using chimeric antigen receptor (CAR) modified T cells to eradicate tumor cells have been a major breakthrough in the treatment of hematologic malignancies. However, there are no measures to control CAR T cell activity after infusion, which is mostly required in cases of CAR T cell overreaction, e.g. cytokine release syndrome, or in the case of T cell failure, e.g. caused by exhaustion. In our study, we identified the tyrosine kinase inhibitor (TKI) dasatinib (© Sprycel) as a suitable agent to steer CAR T cells in vitro and in vivo. We show that single treatment of CD4+ and CD8+ CAR T cells with dasatinib conferred either partial or complete inhibition, depending on the applied concentration. The blockade was immediate and encompassed spe-cific lysis, cytokine secretion and proliferation following antigen encounter. The mechanism relied on reduced phosphorylation of key kinases in the CAR signaling cascade, which led to abrogation of nuclear factor of activated T-cells (NFAT) signaling. Importantly, inhibition was fully reversible by dasatinib withdrawal. In vivo, dasatinib blocked CAR T cell function without impairing the engraftment of CAR T cells or their subsequent anti tumor function once dasatinib administration was discontinued. We therefore introduce dasatinib as a new tool to efficiently block CAR T cells in vitro and in vivo, with data suggesting that dasatinib can be used in a clinical setting to mitigate toxicity after adaptive transfer of CAR modified T cells and other forms of T cell based immunotherapy. Additionally we show that intermittent inhibition of CAR T cells by dasatinib im-proves the efficacy of CAR T cell therapy. By pausing T cells for short periods of time in vi-vo, upregulation of programmed death protein 1 (PD-1) and subsequent induction of exhaus-tion was prevented, which increased the expansion of T cells and the rate of tumor eradica-tion. Our data therefore suggest that dasatinib can additionally be used to overcome T cell exhaustion that is induced by massive tumor burden and upregulation of inhibitory receptors.},
  subject      = {Immuntherapie},
  language  = {en}
}
@phdthesis{Schmitt2010,
  author    = {Schmitt, Kathrin},
  title     = {Identification and Characterization of GAS2L3 as a Novel Mitotic Regulator in Human Cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-52704},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Precise control of mitotic progression is vital for the maintenance of genomic integrity. Since the loss of genomic integrity is known to promote tumorigenesis, the identification of knew G2/M regulatory genes attracts great attention. LINC, a human multiprotein complex, is a transcriptional activator of a set of G2/M specific genes. By depleting LIN9 in MEFs, a core subunit of LINC, Gas2l3 was identified as a novel LINC target gene. The so far uncharacterized Gas2l3 gene encodes for a member of the family of growth arrest specific 2 (GAS2) proteins, which share a highly conserved putative actin binding CH and a putative microtubule binding GAS2 domain. In the present study GAS2L3 was identified as a LINC target gene also in human cells. Gene expression analysis revealed that GAS2L3 transcription, in contrast to all other GAS2 family members, is highly regulated during the cell cycle with highest expression in G2/M. The GAS2L3 protein showed a specific localization pattern during the M phase: In metaphase, GAS2L3 localized to the mitotic spindle, relocated to the spindle midzone microtubules in late anaphase and concentrated at the midbody in telophase where it persisted until the end of cytokinesis. Overexpression of a set of different GAS2L3 deletion mutants demonstrated that the localization to the mitotic microtubule network is dependent on the C-terminus, whereas the midbody localization is dependent on full length GAS2L3 protein. Additionally, exclusive overexpression of the CH domain induced the formation of actin stress fibers, suggesting that the CH domain is an actin binding domain. In contrast, the GAS2 domain was neither needed nor sufficient for microtubule binding, indicating that there must be an additional so far unknown microtubule binding domain in the C-terminus. Interestingly, immunoblot analysis also identified the C-terminus as the domain responsible for GAS2L3 protein instability, partially dependent on proteasomal degradation. Consistent with its specific localization pattern, GAS2L3 depletion by RNAi demonstrated its responsibility for proper mitosis and cytokinesis. GAS2L3 depletion in HeLa cells resulted in the accumulation of multinucleated cells, an indicator for chromosome mis-segregation during mitosis. Also the amount of cells in cytokinesis was enriched, indicating failures in completing the last step of cytokinesis, the abscission. Strikingly, treatment with microtubule poisons that lead to the activation of the spindle assembly checkpoint (SAC) indicated that the SAC was weakened in GAS2L3 depleted cells. Although the exact molecular mechanism is still unknown, fist experiments support the hypothesis that GAS2L3 might be a regulator of the SAC master kinase BUBR1. In conclusion, this study provides first evidence for GAS2L3 as a novel regulator of mitosis and cytokinesis and it might therefore be an important guardian against tumorigenesis.},
  subject      = {Mensch},
  language  = {en}
}