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Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.SignificanceLoss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs.

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3-10. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.