S857 THE NUCLEOTIDE KINASE NADK CONSTITUTES A METABOLIC VULNERABILITY OF NOTCH1‐DRIVEN T‐ALL

Abstract

Background:Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer with a cumulative risk of ∼1 in 2,000 children by the age of 15 years and an increasing incidence over the last 30 years.Activating mutations in the NOTCH1gene are found in more than 60% of cases of T‐cell Acute Lymphoblastic leukaemia (T‐ALL) and are being targeted therapeutically with compounds such as γ‐secretase inhibitors (GSIs) and Notch inhibiting antibodies (mAbs).Aims:To identify novel therapeutic vulnerabilities of Notch1 driven T‐ALL.Methods:A CRISPR‐Cas9 drop‐out screen was used to identify genetic vulnerabilities of NOTCH1‐driven T‐ALL. Amongst these was the nucleotide kinase NADK, a novel therapeutic vulnerability of T‐ALL that we chose to investigate in downstream studies. These included in‐vitro and in‐vivo validation of NADK as a therapeutic target using CRISPR/gRNA and a compound inhibitor, and investigation of its function and relationship NOTCH1 activation using diverse approaches.Results:A genome wide CRISPR‐Cas9 screen was performed in T‐ALL cell lines driven by NOTCH1 overexpression (Jurkat, CEM‐CCRF), or mutation (PEER) and in a line not dependent on NOTCH1 (Loucy) (Figure 1A). We identified 69 NOTCH‐specific essential genes, including well‐known players in NOTCH signaling such as NOTCH1, RBPJ, BCL11B, GATA3 and CTPB1. We also identified a separate group of genes associated to cellular pathways involved in the reduction of reactive oxygen species (ROS). One of these was NADK, the gene for nicotinamide adenine dinucleotide kinase, which we investigate further. NADK drives the conversion of NAD+to NADP+, which in its reduced form (NADPH) is used in the reduction of ROS levels through the production of reduced Glutathione. Exposure of Loucy (NOTCH1‐WT) to H2O2 showed that it was not able to reduce intracellular ROS levels as efficiently as NOTCH1‐driven lines, suggesting increased NADK activity in the latter. In keeping with this, intracellular NADP+/NADPH levels that were significantly higher in NOTCH1‐driven cell lines than in Loucy (Figure 1C). In addition, pharmacological or genetic inhibition of NADK led to a significant increase in ROS levels in the NOTCH1‐driven Jurkat cells, while Loucy cells were not affected (Figure 1B). It was previously reported that, in both human and mouse T‐ALL cells, the intracellular domain of Notch1 (NiCD) suppresses production of ROS levels. This was confirmed by demonstrating that the intracellular levels of NADP+/NADPH increased by more than two‐fold when the human NiCD was overexpressed in Loucy (Figure 1F). In‐vivo experiments demonstrated that genetic or pharmacological inhibition of NADK reduced leukaemia proliferation and significantly prolonged the survival of mice xenografted with Jurkat or PEER, but not Loucy (Figure 1D/1E). We hypothesize that NOTCH1 drives ROS detoxification through NADK activation, a function required to support its proliferative effect. This frames NADK as a therapeutic vulnerability in NOTCH1‐driven T‐ALL (Figure 1G).Summary/Conclusion:Our findings propose that the intracellular domain of NOTCH1 activates NADK to enhance the production NADP+/NADPH, a function required to support it proliferative effects. This frames NADK as a putative novel therapeutic vulnerability of NOTCH1‐driven T‐ALL and potentially other NOTCH1‐driven cancers.image