Gewählte Publikation:

Scheipl, S.
“Novel Therapeutic Targets for Chordoma”
Doktoratsstudium der Medizinischen Wissenschaft; Humanmedizin; [ Dissertation ] Graz Medical University; 2016. pp. [OPEN ACCESS]
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Autor*innen der Med Uni Graz:

Scheipl Susanne

Betreuer*innen:

Leithner Andreas

Liegl-Atzwanger Bernadette

Rinner Beate

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Abstract:

Chordomas are rare malignant bone tumours with a poor prognosis. The mainstay of treatment is surgery. Even though there have been advances in irradiation therapy, therapeutic options are limited, particularly in advanced and metastasising disease. In view of this unmet need, the author conducted three studies in order to identify potential therapeutic targets for chordoma. These approaches are summarised in this thesis. The first study assesses whether the insulin-growth factor-1 (IGF-1) receptor (IGF-1R) could be a potential target for specific inhibition in chordomas. For that reason we studied the immunohistochemical (IHC) expression of IGF-1R and its ligands, IGF-1 and IGF-2, in a series of 50 clinical chordoma samples (34 primary tumours, 16 recurrences). Thirty-eight chordomas (76%) expressed IGF-1R, 46 (92%) stained positive for IGF-1 and 25 (50%) for IGF-2. Staining was strong and diffuse in 18 (36%; IGF-1R), 32 (64%; IGF-1) and 8 (16%; IGF-2) chordomas, respectively. In a second study, we investigated histone deacetylase (HDAC) inhibitors as potential epigenetic therapeutics. We performed IHC on 50 chordoma samples using antibodies against HDACs 1-6. We then tested three pan-HDAC inhibitors, vorinostat (SAHA), panobinostat (LBH-589) and belinostat (PXD101), in our chordoma cell line, MUG-Chor1, by Western blots, cell cycle analysis and various apoptosis assays. IHC was negative for HDAC1, positive for HDAC2 in the majority of specimens (n=36; 72%) and positive for HDACs 3-6 in all available specimens (n=43; 86%). he HDAC inhibitors SAHA and LBH-589 induced a significant increase of G2/M phase cells and cleaved caspase-3 (p=0.0003 and p=0.0014 after 72h, respectively), while PXD101 did not. For SAHA and LBH-589, a peak in caspase 3/7 activity and PARP cleavage confirmed apoptosis. At the University College London (UCL) Cancer Institute we chose a more comprehensive approach and undertook a phenotypic compound screen against 1,097 compounds on three well-characterised chordoma cell lines. A total of 154 compounds were selected from the single concentration screen (1 µM) and further profiled in a 10-point dose-response (EC50) format. EC50 profiling was conducted in chordoma cell lines as well as in normal dermal fibroblasts. The latter were included as we aimed to identify compounds which selectively targeted chordoma cells but not fibroblasts. Twenty-seven of these 154 compounds displayed chordoma selective growth-inhibitory effects. Twenty-one of these 27 compounds (78%) targeted the epidermal growth factor receptor/erythroblastic leukaemia viral oncogene homolog (EGFR/ERBB) family. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were responsive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as our most promising compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). These compounds induced apoptosis in responsive cell lines and suppressed p-EGFR and its downstream-markers in a dose-dependent manner. Analyses of chemical trends and substituent patterns suggested that EGFR inhibitors with small aniline substituents in the 4-position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib significantly reduced tumour growth in two xenograft mouse models. One of the resistant cell lines (U-CH2) was shown to express high levels of p-MET, a recognised bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected. Our findings were consistent with the (p-) EGFR expression reported in the majority of clinical samples. Based on the promising preclinical data on EGFR inhibition, Chordoma Foundation and associated researchers and clinicians are currently planning a prospective, randomised clinical trial on advanced chordoma.

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