Gruffaz, M. | bioRxiv Science

bioRxiv2019

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

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.

cancer biology↗

bioRxiv2018

Repurposing cytarabine for treating primary effusion lymphoma by targeting KSHV latent and lytic replications

Oncogenic Kaposis sarcoma-associated herpesvirus (KSHV) is etiologically linked to primary effusion lymphoma (PEL), an aggressive and non-treatable malignancy commonly found in AIDS patients. In this study, we performed a high throughput screening of 3,731 characterized compounds, and identified cytarabine approved by FDA for treating numerous types of cancer as a potent inhibitor of KSHV-induced PEL. We showed the high efficacy of cytarabine in the growth inhibition of various PEL cells by inducing cell cycle arrest and apoptosis. Cytarabine inhibited host DNA and RNA syntheses and therefore induced cellular cytotoxicity. Furthermore, cytarabine inhibited viral DNA and RNA syntheses and induced the the rapid degradation of KSHV major latent protein LANA, leading to the suppression of KSHV latent replication. Importantly, cytarabine effectively inhibited active KSHV replication and virion production in PEL cells. Finally, cytarabine treatments not only effectively inhibited the initiation and progression of PEL tumors, but also induced regression of grown PEL tumors in a xenograft mouse model. Together, our study has identified cytarabine as novel therapeutic agent for treating PEL as well as eliminating KSHV persistent infection.\n\nImportancePrimary effusion lymphoma is an aggressive malignancy caused by Kaposis sarcoma-associated herpesvirus. The outcome of primary effusion lymphoma is dismal without specific treatment. Through a high throughput screening of characterized compounds, we identified a FDA-approved compound cytarabine as a potent inhibitor of primary effusion lymphoma. We showed that cytarabine induced regression of PEL tumors in a xenograft mouse model. Cytarabine inhibited host and viral DNA and RNA syntheses, resulting in the induction of cytotoxicity. Of interest, cytarabine induced the degradation of KSHV major latent protein LANA, hence suppressing KSHV latent replication, which is required for PEL survival. Furthermore, cytarabine inhibited KSHV lytic replication program, preventing virion production. Our findings identified cytarabine as novel therapeutic agent for treating PEL as well as for eliminating KSHV persistent infection. Since cytarabine is already approved by the FDA, it might be an ideal candidate for repurposing for PEL therapy and for further evaluation in advanced clinical trials.

microbiology↗