Long non-coding RNA DARS-AS1 promotes tumor progression by suppressing PACT-mediated PKR activation

Cancer cells evolve various mechanisms to overcome cellular stresses and maintain progression. Protein kinase R (PKR) and its protein activator (PACT) are the initial responders in monitoring diverse stress signals and lead to cell proliferation inhibition and cell apoptosis in consequence. However, the regulation of PACT-PKR pathway in cancer cells is largely unknown. Herein, we identify that the long non-coding RNA (lncRNA) aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) is directly involved in the inhibition of the PACT-PKR pathway and promotes cancer cell proliferation. Using large-scale CRISPRi functional screening of 971 cancer-associated lncRNAs, we find that DARS-AS1 is associated with significantly enhanced cancer cell proliferation. Accordingly, knocking down DARS-AS1 inhibits cell proliferation of multiple cancer cell lines and promotes cancer cell apoptosis in vitro and significantly reduces tumor growth in vivo. Mechanistically, DARS-AS1 directly binds to the activator domain of PACT and prevents PACT-PKR interaction, thereby decreasing PKR activation, eIF2a phosphorylation and inhibiting apoptotic cell death. Clinically, DARS-AS1 is broadly expressed across multiple cancers and the increased expression of this lncRNA indicates poor prognosis. This study elucidates the lncRNA DARS-AS1 directed cancer-specific modulation of the PACT-PKR pathway and provides a novel target for cancer prognosis and therapeutic treatment. Overall design: The pool of sgRNA oligos was synthesized at CustomArray, Inc. (Bothewell, WA) and cloned into a pgRNA-humanized plasmid (Addgene #44248). To produce virus particles, 12 µg pooled pgRNA-humanized plasmids, 7.2 µg psPAX2, and 4.8 µg pMD2.G were co-transfected into 5×106 HEK293T cells in a 10 cm dish using DNAfect transfection reagent (CWBIO, Beijing, China), by following the manufacturer's instructions. Virus particles were harvest at 48 hr and 72 hr after transfection and filtered through 0.45 µm filter membrane. For screening, SW620 cells expressing dCas9/KRAB fusion proteins were generated by viral transduction. Modified SW620 cells were infected with the viral library in four independent infection experiments at MOI 0.1-0.3 and selected with 2 µg/mL puromycin (Sigma, St. Louis, MO) over 2 days. After, cells were in vitro cultured for 18 days at minimum library coverage of 500 cells/sgRNA for screening.