RNA sequencting of CLL cells treated by BTK degrader

BTK plays a critical role in B cell malignancies survival. BTK inhibitor was successfully used as first line treatment for CLL in clinical. The emerging unmet needs is new segments are needed for ibrutinib R/R patients. The purpose of this study is to investigate genomic changes and signaling pathway differences after CLL cells were treated with BTK inhibitor (ibrutinib) or degrader (NRX0492). Overall design: 4 IGHV unmutated primary CLL samples were selected for drug treatment. CLL cells were purified (negtive selection) using CD3+ MACS Cell Separation Columns following manufacturer's protocol (Miltenyi Biotec). Cells were treated with DMSO, 1 µM ibrutinib, 2 nM hook, or 2 nM NRX-0492 for 18 hours .After treatment, cells were seperated into two groups, treatment verification group and RNA extraction group, for stimulation. For treatment verification group, cells were stimulated by 20 µg/mL F(ab')2 anti-human IgM (Jackson ImmunoResearch Laboratories) at 37°C for 15 min and were detected by flow to make sure drug and stimulation work well. For RNA extration group, cells were harvest for RNA extration after 6 hours anti-IgM stimulation. Purified RNA was sequenced, and data was analyzed by bioinfomatics scientist.