ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) is characterized by ectopic expression of transcription factors such as TAL1, TLX1/3 and NKX2-1. NKX2-1 is overexpressed in 5% of T-ALL patients, driving transcriptional induction and metabolic addiction to the serine/glycine synthesis pathway in cancer cells. However, a more complete picture of the transcriptional targets of NKX2-1 and its role in T-ALL pathogenesis is missing. We generated a CRISPR-Cas9 NKX2-1 knockout (KO) model in the NKX2-1 expressing RPMI-8402 (RPMI) T-ALL cell line. KO of NKX2-1 in RPMI cells induced apoptosis while not affecting cell cycle progression. RNA-seq on RPMI WT versus NKX2-1 KO cells showed significant downregulation of gene sets related to T-cell differentiation upon NKX2-1 KO. Additionally, we observed that NKX2-1 promoted a more mature phenotype in RPMI cells, by mildly decreasing CD34 and increasing CD2 expression. Furthermore, gene sets related to epigenetics and DNA damage were enriched in the RNA-seq data. Interestingly, DNA damage machinery related proteins, such as RPA1, appeared among the binding partners of NKX2-1 in IP-MS, and this interaction was confirmed in PDX cells. In agreement with this, we found significantly lower levels of DNA damage marks gH2AX and H3K27me3 in the presence of NKX2-1. NKX2-1 expression was associated with decreased sensitivity to topoisomerase inhibitor etoposide, and to HDAC inhibitors vorinostat and romidepsin. Furthermore, these agents resulted in lower induction of gH2AX and H3K27me3 in NKX2-1 expressing cells. Finally, NKX2-1 positive T-ALL patients showed overexpression of DNA repair genes in public RNA-seq data compared to NXK2-1 negative T-ALL, further supporting a critical role of NKX2-1 in protecting cells against DNA damage. Mechanistically, we identified RUNX1 as NKX2-1 binding co-factor through motif analysis of NKX2-1 ChIP-seq peaks and in our NKX2-1 IP-MS experiment. NKX2-1/RUNX1 binding was confirmed in RPMI and in PDX cells. Interestingly, NKX2-1 and RUNX1 IP-MS experiments revealed that most NKX2-1 binding partners also bound to RUNX1, and that presence of NKX2-1 altered the co-factors of RUNX1. Moreover, iRegulon performed on T-ALL patients revealed that genes that are differentially expressed in NKX2-1+ T-ALL samples were regulated by RUNX1. Finally, we observed that RUNX1 inhibitor, Ro5-3335, provoked a significantly higher cell death in the presence of NKX2-1. Summing up, in addition to induction of metabolic serine/glycine synthesis addiction, our data indicate that NKX2-1 is essential to protect T-ALL cells against DNA damage. Furthermore, we observed that NKX2-1 is a co-factor of RUNX1, regulating its binding partners and activity.
