CRISPR-Cas9 screening of KSHV-transformed cells identifies XPO1 as a vulnerable target of cancer cells by inducing p62 SQSTM1-mediated p53 activation in PML bodies

Mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export promotes uncontrolled cell proliferation and growth transformation of cancer cells. We performed a genome-wide CRISPR-Cas9 screening in matched primary and KSHV-transformed cells, and identified genes that were pro-growth and growth-suppressive of both types of cells, of which exportin XPO1 was a critical factor for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by siRNA-mediated knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells, as well as cell lines of other types of cancer including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest and p53 activation, which depended on the formation of PML nuclear bodies. Furthermore, XPO1 induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Our findings highlight a novel mechanism of p53 activation and identify XPO1 as a vulnerable target of cancer cells. Overall design: To identify key genes responsible for KSHV-related survival, a CRISPR pooled libraries containing sgRNAs that specifically target all known cellular genes were transduced into Cas9-expressing KSHV-transformed primary rat mesenchymal embryonic stem cells (KMM) and control primary rat mesenchymal stem cells (MM). Genomic DNA from surviving MM and KMM cells at day 1, 4, 11 and 21 post-transduction were collected and sequenced, and the results were analyzed for the gain or loss of sgRNAs.