Targeting Non-Apoptotic Pathways with the Cell Permeable TAT-Conjugated NOTCH1 RAM Fragment for Leukemia and Lymphoma
Ryota Uchimura1, Shinpei Nishimura2, Mikako Ozaki1
1Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan.
Abstract:
Targeting nonapoptotic cell death offers a promising strategy for overcoming apoptosis resistance in cancer. In this study, we developed Tat-Ram13, a 25-mer peptide that fuses the NOTCH1 intracellular domain fragment RAM13 with a cell-penetrating HIV-1 TAT, for the treatment of T-cell acute lymphoblastic leukemia with aberrant NOTCH1 mutation. Tat-Ram13 significantly downregulated NOTCH1-target genes in T-ALL cell lines. Furthermore, the peptide had potent cytotoxic effects on various human leukemia and lymphoma cell lines. However, it did not affect normal lymphocytes and monocytes, some subsets of leukemia cells, or adherent tumor cells. This cell-selective cytotoxic activity was closely correlated with the peptide uptake via macropinocytosis in leukemia cells. In leukemia cells, Tat-Ram13 triggered rapid cell death. This cell death involved mitochondrial membrane depolarization and extracellular release of lactate dehydrogenase and high-mobility group box-1 protein without activation of caspase-3 or cleavage of PARP-1. These results suggest that Tat-Ram13 cell death is nonapoptotic and mediated by rapid plasma membrane rupture. Moreover, alanine scanning analysis identified four critical hydrophobic amino acids in the RAM13 domain essential for its cytotoxicity. Consequently, these results suggest that Tat-Ram13 is a tumor-selective, nonapoptotic cell death-inducing agent for treating refractory leukemia and lymphomas with apoptosis resistance.
Insights
Tat-Ram13 peptide induces nonapoptotic cell death in leukemia and lymphoma cells, offering a new strategy against apoptosis-resistant cancers. This targeted agent selectively kills cancer cells without harming normal lymphocytes.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis resistance is a major challenge in cancer therapy, particularly in leukemia and lymphomas.
- Targeting nonapoptotic cell death pathways presents a promising alternative strategy.
- NOTCH1 signaling plays a role in T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis.
Purpose of the Study:
- To develop and evaluate Tat-Ram13, a novel peptide for targeting nonapoptotic cell death in refractory leukemia and lymphomas.
- To investigate the mechanism of action and cell selectivity of Tat-Ram13.
Main Methods:
- Development of Tat-Ram13, a peptide combining RAM13 (NOTCH1 intracellular domain fragment) and HIV-1 TAT.
- Assessment of Tat-Ram13's effects on NOTCH1-target genes and cell viability in various cancer cell lines and normal lymphocytes.
- Investigation of the cell death mechanism, including macropinocytosis, mitochondrial membrane potential, and caspase/PARP-1 activation.
- Alanine scanning mutagenesis to identify critical residues for cytotoxicity.
Main Results:
- Tat-Ram13 significantly downregulated NOTCH1-target genes in T-ALL cell lines.
- The peptide exhibited potent, selective cytotoxicity against human leukemia and lymphoma cell lines, sparing normal lymphocytes.
- Cell death was mediated by macropinocytosis, mitochondrial depolarization, and plasma membrane rupture, independent of caspase-3 or PARP-1 activation.
- Four critical hydrophobic amino acids in the RAM13 domain were identified as essential for Tat-Ram13's cytotoxicity.
Conclusions:
- Tat-Ram13 is a novel peptide that induces selective nonapoptotic cell death in leukemia and lymphoma cells.
- It represents a potential therapeutic agent for apoptosis-resistant hematological malignancies.
- The findings highlight the therapeutic potential of targeting nonapoptotic cell death pathways.
