Targeting Non-Apoptotic\nPathways with the Cell Permeable\nTAT-Conjugated NOTCH1 RAM Fragment for Leukemia and Lymphoma Cells

Targeting nonapoptotic cell death offers a promising\nstrategy for\novercoming apoptosis resistance in cancer. In this study, we developed\nTat-Ram13, a 25-mer peptide that fuses the NOTCH1 intracellular domain\nfragment RAM13 with a cell-penetrating HIV-1 TAT, for the treatment\nof T-cell acute lymphoblastic leukemia with aberrant NOTCH1 mutation.\nTat-Ram13 significantly downregulated NOTCH1-target genes in T-ALL\ncell lines. Furthermore, the peptide had potent cytotoxic effects\non various human leukemia and lymphoma cell lines. However, it did\nnot affect normal lymphocytes and monocytes, some subsets of leukemia\ncells, or adherent tumor cells. This cell-selective cytotoxic activity\nwas closely correlated with the peptide uptake via macropinocytosis\nin leukemia cells. In leukemia cells, Tat-Ram13 triggered rapid cell\ndeath. This cell death involved mitochondrial membrane depolarization\nand extracellular release of lactate dehydrogenase and high-mobility\ngroup box-1 protein without activation of caspase-3 or cleavage of\nPARP-1. These results suggest that Tat-Ram13 cell death is nonapoptotic\nand mediated by rapid plasma membrane rupture. Moreover, alanine scanning\nanalysis identified four critical hydrophobic amino acids in the RAM13\ndomain essential for its cytotoxicity. Consequently, these results\nsuggest that Tat-Ram13 is a tumor-selective, nonapoptotic cell death-inducing\nagent for treating refractory leukemia and lymphomas with apoptosis\nresistance.