Therapeutic targeting of the NOTCH1 and neddylation pathways in T cell acute lymphoblastic leukemia

Gamma Secretase Inhibitors (GSIs) effectively block the activation of oncogenic protein Notch homolog-1 (NOTCH1), a frequent event in T-cell Acute Lymphoblastic Leukemia (T-ALL). However, their clinical application is hampered by severe gastrointestinal toxicity due to the inhibition of NOTCH1 signaling and subsequent increase in goblet cell differentiation in the gut. Genome-wide CRISPR loss-of-function (LOF) screen in the colon cancer cell line LS174T identified the neddylation pathway as a main regulator of goblet cell differentiation upon NOTCH1 inhibition. Consistently, genetic deletion or pharmacologic inhibition of the neddylation pathway with the small molecule inhibitor MLN4924 rescued GSI-induced differentiation and cell death in LS174T cells. Mechanistically, neddylation inhibition by MLN4924 increases the protein stability of Hairy and enhancer of split-1 (HES1), a known NOTCH1 target and a regulator of absorptive and secretory cell fate decisions. Combined treatment with GSI and MLN4924 in murine Notch1-induced model of T-ALL showed leukemia regression and improved overall survival without any associated gut toxicity. Overall, these results substantiate the potential of targeting NOTCH1 and neddylation pathway in the treatment of NOTCH1-induced T-ALL. To elucidate the mechanisms mediating LS174T (adenocarcinoma cell line) differentiation upon NOTCH inhibition, we performed gene expression profiling in LS174T cells treated with vehicle (DMSO) or GSI (CompE) (250 nm) for 48 hours We extracted RNA from triplicate cultures of LS174T cells treated for 48 h with vehicle (DMSO) or CompE (250 nM) using RNeasy Plus Mini kit (Qiagen) according to the manufacturer’s protocol. RNA of each sample was amplified and labeled using the 3’ IVT Express Kit (Affymetrix) and hybridized on Affymetrix Human U133 Plus 2.0 arrays according to the manufacturer’s protocol as previously described (Real PJ et al., 2009). We normalized inter array intensity differences using the Dchip 36 package and selected for analysis the 10,896 probes with the least variation among experimental replicas.