Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence

Paula Carpintero-Fernández,Michela Borghesan,Olga Eleftheriadou,Tom P Mitchell,Belen Pan-Castillo,Alejandro Yuste,Maria Mayan,Ana O’Loghlen,Muge Ogrunc,Thomas D Nightingale,Juan Antonio Fafián-Labora

doi:10.1038/s41419-022-04569-3

Abstract

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

Highlights

A key characteristic of cancer cells is the deregulation of cyclindependent kinases (CDKs) leading to uncontrolled cell proliferation
We ability of sgF9, sgRNA targeting PROZ (sgPROZ) and sgRNA targeting RB1 (sgRB) to prevent the proliferation arrest screened a panel of 22 cancer cell lines for their response to induced by Palbo was confirmed by performing proliferation different CDK4/6 inhibitors and we show that F9 loss-of-function curves at different days after treatment (Fig. 2B, C)
A better understanding of the mechanisms regulating senescence induced by CDK4/6 inhibitors is needed in order to increase therapy efficacy and patient stratification in cancer

Summary

INTRODUCTION

A key characteristic of cancer cells is the deregulation of cyclindependent kinases (CDKs) leading to uncontrolled cell proliferation. The molecular mechanisms by which CDK4/ 6 inhibitors induce senescence in cancer cells and the genes involved in conferring drug resistance or lack of response to these inhibitors are unknown, preventing patient stratification prior to Palbociclib treatment [18]. In this study, using a human genome-wide CRISPR/Cas library we identified genes whose loss-of-function prevent the proliferative arrest induced by Palbociclib in MCF7 breast cancer cells. We ability of sgF9, sgPROZ and sgRB to prevent the proliferation arrest screened a panel of 22 cancer cell lines for their response to induced by Palbo was confirmed by performing proliferation different CDK4/6 inhibitors and we show that F9 loss-of-function curves at different days after treatment (Fig. 2B, C). We could observe an increase in the mRNA expression levels of F9

RESULTS

Findings

DISCUSSION