The heterodimerization (HD) domain of NOTCH1, responsible for association of NOTCH1 extracellular and transmembrane regions after furin-mediated cleavage of NOTCH1 precursor, is one of the hotspots for gain-of-function NOTCH1 mutations in T-cell acute lymphoblastic leukemia (T-ALL) (Weng et al. 2004). NOTCH1 HD domain mutants are responsive to ligand binding, but the activation (through cleavage of S2 and S3 sites and release of the intracellular domain NICD1) also happens spontaneously, in the absence of DLL and JAG ligands (Malecki et al. 2006). The following NOTCH1 HD domain mutants were directly functionally studied by Malecki et al.: NOTCH1 V1576E, NOTCH1 F1592S, NOTCH1 L1593P, NOTCH1 L1596H, NOTCH1 R1598P, NOTCH1 I1616N, NOTCH1 I1616T, NOTCH1 V1676D, NOTCH1 L1678P, NOTCH1 I1680N, NOTCH1 A1701P and NOTCH1 I1718T; other frequent NOTCH1 HD domain mutants (NOTCH1 L1574P, NOTCH1 L1574Q and NOTCH1 L1600P) are assumed to behave in a similar way.