The Exploration of PI3K

Abstract

Introduction BCR-ABL1-negative classic myeloproliferative neoplasms (MPN) include primary myelofibrosis, polycythemia vera, and essential thrombocythemia. Calreticulin (CALR) 52 bp deletion and 5 bp insertion were discovered to be involved in MPN pathogenesis. Calreticulin, a Ca2+-binding chaperone, is implicated in the regulation of Ca2+, protein folding, and avariety of signaling pathways. It was reported that mutant CALR results increased JAK/STAT and other signaling pathways activation. However, these data contrast with another study‘s observation that CALR mutant cells are not dependent on JAK/STAT signaling. Therefore, there is still a need to explore molecular mechanisms activated in CALR mutant cells. Aim The aim of our study was to characterize the effects of specific JAK/STAT, PI3K/Akt/mTOR, and Hedgehog signaling inhibitors in CALR Del52 mutated cells. Methods The UT-7 cell line was employed in our study. CRISPR/Cas9 system was chosen for CALR 52 bp deletion initiation in cells. After selection of potential DNA targets in gDNA, corresponding tabs were cloned into a vector (pSpCas9(BB)-2A-Puro (PX459) V2.0) that is optimized for Cas9and RNA-guided expression in eukaryotic cells. For plasmid and HDR template electrotransfer into UT-7 cells, the following parameters were applied: 1 HV pulse of 1600 V/cm with 500 µspulse duration (the electroporation system BTX T820 was used). The transfected cells weres elected in puromycin (1 μg/ml). Further, we explored the potent effect of targeting JAK/STAT, PI3K/Akt/mTOR, and Hedgehog pathways with specific agents in CALR Del52mutant and CALR WT cells. We evaluated the effect of CYT387 (JAK1/2 inhibitor), RAD001(mTOR inhibitor), and HPI-1 (Gli1/2 inhibitor) agents. The effect of signaling pathways [...].