NKX2-1 regulates cell survival, maturation, and DNA-damage responses as a cofactor of RUNX1 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by ectopic expression of transcription factors, including NKX2-1, which is overexpressed in 5% of patients. NKX2-1 is associated with a cortical immunophenotype and drives metabolic addiction to the serine/glycine synthesis pathway in T-ALL. However, a complete picture of the role of NKX2-1 in T-ALL pathogenesis is missing. We characterized a CRISPR-Cas9 NKX2-1 knockout model of RPMI-8402, the only known NKX2-1-expressing T-ALL cell line, and validated obtained results in patient samples. NKX2-1 knockout caused a less mature immunophenotype and promoted cell cycle progression, in line with direct transcriptional repression of CDK6 by NKX2-1 that we observed. Furthermore, NKX2-1 protected T-ALL cells from apoptosis and DNA damage. The NKX2-1 protein directly bound DNA repair factors, such as RPA1 and RPA2, and presence of NKX2-1 resulted in differential expression of gene sets related to DNA damage repair in RPMI-8402 cells and patient samples. Furthermore, NKX2-1 positive cells showed less induction of DNA damage and apoptosis upon treatment with etoposide, a DNA damaging chemotherapy agent that is clinically used to treat T-ALL. Mechanistically, our data supported that RUNX1 is an important co-factor for NKX2-1 transcriptional regulation in T-ALL cells, and that NKX2-1 modulated the composition of RUNX1 protein complexes. Notably, NKX2-1 expressing cells showed higher sensitivity towards RUNX1 inhibition, suggesting a cooperative role in regulating T-ALL cell survival. This work reveals a critical role of NKX2-1 in enhancing T-ALL cell survival through DNA damage protection, and identifies RUNX1 as an important cofactor in T-ALL pathogenesis.