In silico and in vitro screening of small molecule Inhibitors against SYT-SSX1 fusion protein in synovial sarcoma.

Synovial sarcoma (SS) is characterized by a tumour specific chromosomal translocation t(X;18) (p11;q11) which results in the formation of SYT-SSX1 fusion protein. This fusion protein represents a clear therapeutic target and molecules specifically targeting SYT-SSX1 fusion protein are currently not available. In this study, SYT-SSX1 fusion protein sequence was retrieved from Uniprot and 3D structure was generated using I-TASSER modeling program. A structure based computational screening approach has been employed using Glide docking software to identify potential SYT-SSX1 small molecule inhibitors that bind to the junction region of the fusion protein. The obtained inhibitors were further filtered based on the docking score and ADME/T properties. Ten best fit compounds were chosen for in vitro studies. The anti-proliferative activities of these 10 compounds were screened in Yamato, ASKA (carries SYT-SSX1 fusion protein) and other sarcoma cell lines such as A673, 143B to understand the specificity of inhibition of the chosen compounds. The in vitro activity was compared against HEK293 cell lines. The compound 5-fluoro-3-(1-phenyl-1H-tetraazol-5-yl)-1H-indole (FPTI) was found to be selectively cytotoxic in synovial sarcoma cell lines (Yamato and ASKA) and this compound also showed insignificant anti proliferative activity on other cell lines. Further, target gene expression study confirmed that FPTI treatment down-regulated SYT-SSX1 and modulated its downstream target genes. Cell cycle analysis revealed the involvement of an apoptotic mechanism of cell death. Further experimental validations may elucidate the therapeutic potentials of FPTI against SYT-SSX1 fusion protein.

NOTCH1 and FBXW7 mutations favor better outcome in pediatric South Indian T-cell acute lymphoblastic leukemia.

The NOTCH1 signaling pathway is essential for hematopoiesis and a critical regulatory step for T-cell proliferation and maturation. The E3 ubiquitin ligase FBXW7 controls NOTCH1 protein stability. Mutations in NOTCH1/FBXW7 activate NOTCH signaling and are of prognostic significance in patients with T-cell acute lymphoblastic leukemia (T-ALL). In this study we analyzed NOTCH1 and FBXW7 mutations in 50 South Indian T-ALL patients treated by a modified ALL BFM 95 regimen. The hot spot exons (HD-N, HD-C, TAD, and PEST) of NOTCH1 and exons 9 of the 10 of FBXW7 were polymerase chain reaction amplified and sequenced. In total, 20 of the 50 (40%) T-ALL patients revealed heterozygous mutations in the NOTCH1 domains, and a predominance of missense mutations in HD-N (70%) and PEST (15%) domains. FBXW7 mutations were detected in 5 of the 50 (10%) T-ALL patients. T-ALL patients with NOTCH1/FBXW7 mutations expressed higher protein level of NOTCH1 compared with patients without NOTCH1/FBXW7 mutations. Six of the mutations detected in NOTCH1 were not reported previously. When tested in a Dual Luciferase Renilla reporter assay some of these conferred increased NOTCH activity, suggesting that these are activating mutations. Importantly, 13 of the 20 (65%) NOTCH1/FBXW7-mutated T-ALL patients showed a good prednisone response (P=0.01) and a better clinical outcome compared with NOTCH1/FBXW7 nonmutated patients (P=0.03). These data suggest that NOTCH1/FBXW7 mutations are present in T-ALL patients from Southern India and may be useful biomarkers to predict prognosis in T-ALL.