CRISPR-Cas9 screening of KSHV-transformed cells identifies XPO1 as a vulnerable target of cancer cells

Abstract

The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, of which the cancer vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a CRISPR-Cas9 screening in a unique model of matched primary and oncogenic KSHV-transformed cells, and identified genes that were pro-growth and growth-suppressive of both cells, of which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by siRNA knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells, and cell lines of other cancers including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation but the mechanism of p53 activation differed among different types of cancer cells. p53 activation depended on the formation of PML nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken together, we have identified novel pro-growth and growth-suppressive genes of primary and cancer cells, and demonstrated XPO1 as a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML-and p62-dependent mechanism of p53 activation in some types of cancer cells.\n\nImportanceUsing a model of oncogenic virus KSHV driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations, and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both pro-growth and growth-suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated exportin XPO1 as a critical factor for the survival of transformed cells. Using a XPO1 inhibitor KPT-8602 and by siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells, as well as gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53 but the mechanism of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate XPO1 as a vulnerable target of cancer cells, and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML-and p62-mediated mechanism of p53 activation in some types of cancer cells.