Simultaneous activation and late-stage inhibition of autophagy induces autophagy-dependent cell death in leukemia

Resistance to apoptosis is a major challenge in cancer therapy, especially in relapsed or refractory leukemia. Autophagy, a conserved lysosomal degradation pathway, plays a complex dual role: it can promote tumor survival under stress, yet its excessive activation can trigger autophagy-dependent cell death (ACD). This paradox has inspired a novel therapeutic strategy that simultaneously activates and inhibits specific stages of autophagy to push cells toward death. However, the molecular mechanisms underlying this approach remain poorly understood, particularly in non-solid tumors like leukemia. This study investigated the effects of simultaneous autophagy activation and stage-specific inhibition on cell death in human Jurkat E6.1 leukemic T cells. Cells were treated with rapamycin (RAPA) to induce autophagy, combined with either chloroquine (CQ) to block late-stage degradation or 3-methyladenine (3-MA) to inhibit early-stage autophagosome formation. Cell viability was measured by MTS assay, while autophagy flux was evaluated using a combination of MDC staining, mCherry-eGFP-LC3 fluorescence imaging and Western blot analysis for LC3. To delineate the mode of cell death, we accessed apoptosis via caspase-3 cleavage and employed pharmacological inhibitors of apoptosis (z-VAD-FMK) and necrosis (Necrostatin-1). Simultaneous autophagy induction with RAPA and late-stage inhibition with CQ (RAPA + CQ) triggered massive autophagosome accumulation and significantly enhanced cell death. In contrast, early-stage inhibition with 3-MA (RAPA + 3-MA) prevented autophagic vesicle formation and abrogated the cytotoxic effect. Importantly, this cell death occurred independently of both apoptosis and necroptosis, suggesting a direct result of excessive and unprocessed autophagosomes, consistent with ACD. In conclusion, our findings demonstrate that trapping cells in a state of high autophagic flux through simultaneous induction and late stage inhibition, is a potent strategy to trigger non-apoptotic cell death in leukemia. This approach represents a promising therapeutic strategy to overcome apoptosis-resistant leukemia.