PO-186 Generation of the GLI1, GLI2 and GLI3 knock-out ovarian cancer cell lines using the CRISPR/Cas9

Abstract

Material and methods Immunohistochemical scoring for total-STAT3, pY705 and pS727 was done in 75 paired core-biopsy and post-NACT TNBC samples. Bioluminescence resonance energy transfer based sensor (PhosphoBRET) was developed by N-terminal fusion of STAT3 with Nanoluc (donor) and TurboFP635 (acceptor) and validated on multiple cancer cell lines (MCF7, A549, HT1080, PC3) or drug inhibitors (Niclosamide, Stattic). We also engineered 3'UTR STAT3shRNA cells overexpressing Nanoluc-STAT3 variants i.e. Wild type [wt], Y705F, S727A, K685R and performed biochemical as well as phenotypic assays to study biological role of STAT3 pathway. Results and discussions Over 90% TNBC cases showed positive staining for pS727 as compared to pY705 STAT3 indicating hyperactivation of non-canonical pathway. 3'UTR STAT3 shRNA cells over-expressing S727A PTM mutant, showed decreased pY705 expression along with abrogation of K685 acetylation. The level of downstream STAT3 targets like Myc, cyclin D1 etc. remained intact while some of the novel target genes (Her2, E-Cadherin and ERa) showed elevated expression in S727A and K685R mutants. Additionally, S727A or K685R mutant also exhibited 2-fold decrease in overall cell proliferation and survival potential. The designed STAT3-PhosphoBRET sensor demonstrated upto 3-fold gain in BRET ratio (p<0.01) using IL6 or EGF ligand. Further, PhosphoBRET sensor expressing either Y705F or S727A or K685R showed increased BRET ratio (p<0.001) implicating higher STAT3 dimerization and activation. Additionally, the PhosphoBRET platform was also extended to test various STAT3 inhibitors, of which Niclosamide was identified as a potent dual blocker (pS727 and pY705) of STAT3 activation for the first time. Conclusion Predominant expression of pS727 STAT3 in TNBC cases along with in vitro data implicate crucial role of non canonical (pS727 and acK685) pathway in controlling STAT3 dimerization and downstream function independent of pY705 activation. Hence, future drug design strategy should aim both arms of STAT3 pathway to completely abrogate its oncogenic function.