Various cell lines including B-ALL (Nalm6, Reh), T-ALL (Jurkat, CCRF-CEM, CEM/C1), CML (HL-60), and THP-1cells were evaluated; the majority responded poorly to erastin, except for Reh cells. Recent evidence suggests that autophagy might facilitate ferroptosis by degrading anti-ferroptosis regulators, revealing the interaction between autophagy and ferroptosis (Liu et al., 2020; Zhou et al., 2020). was higher in Jurkat and CCRF-CEM cells than in Reh cells. The sensitivity could be modified by the autophagy activator rapamycin (Rapa) and the autophagy inhibitor chloroquine (CQ). Rapa sensitized ALL cells to erastin-induced ferroptosis. In ALL xenograft mice, the combination treatment of Rapa and erastin resulted in longer survival time than those observed with erastin or Rapa treatment alone. VDAC3 was regulated by autophagy post-transcriptionally, mainly via NCRW0005-F05 the ubiquitin-proteasome system (UPS). FBXW7 was verified as a specific E3 ligase of VDAC3. knockdown attenuated VDAC3 degradation by suppressing its ubiquitination, thereby increasing the sensitivity of ALL cells to erastin. Conclusion: Autophagy regulated erastin-induced ferroptosis via the FBXW7-VDAC3 axis. Rapa sensitized ALL cells to erastin-induced ferroptosis both and negative. Antibodies and Chemicals The following antibodies were used at the indicated dilution for immunoblotting (IB) and immunoprecipitation (IP) analyses: VDAC3 (1:800 for IB, 3?g for IP; Proteintech, Wuhan, China), FBXW7 (1:800 for IB; 3?g for IP, Proteintech), Ubiquitin (1:800 for IB, Proteintech), and -Actin (1:8,000 for IB, Proteintech). Horseradish peroxidase (HRP) labeled secondary antibody conjugates were purchased from Molecular Probes (Abbkine, Beijing, China). Erastin (APExBIO, Beijing, China), ferrostatin-1 (Fer-1), rapamycin (Rapa), MK-2206, chloroquine (CQ), 3-MA, bafilomycin A1 (BafA1), MG132, and imidazole ketone erastin (IKE) were obtained from Selleck Chemicals (Houston, TX). Cell Viability and Cell Mortality Assays Cell viability was detected by the MTS assay (Promega, Madison, WI). Briefly, cells were equally seeded into 96-well plates with various concentrations of erastin (0.01, 0.1, 1, 10, and 100?mol/l). DMSO (Sigma-Aldrich, St. Louis, MO) was used as a negative control. Cell viability was measured using the MTS kit at 24?h. Absorbance was tested at 490?nm using a microplate analyzer (BioTek, Winooski, VT). Cell mortality was detected by a trypan blue assay (Beyotime Biotechnology, Shanghai, China). In brief, cells were stained with trypan blue and counted under an inverted microscope. The blue-stained cells were considered dead, and five random fields per insert were counted. Cell mortality was calculated according to the following formula: cell mortality (%) = number of dead cells/total number of cells 100. RNA Isolation and Quantitative Real-Time PCR Total RNA was extracted using RNAiso Plus Reagent (TaKaRa, Dalian, China) and dissolved in 10?l of RNase-free water. The purity and concentration were determined using the NanoPhotometer 50 (Implen, Munich, Germany). Next, 1?g of total RNA was used for reverse transcription to synthesize cDNA using the PrimeScript RT Reagent Kit (TaKaRa). For quantitative real-time polymerase chain reaction (qRT-PCR), the amplification system consisted of 2?l of cDNA, 1?l of forward and reverse primers, 10?l SYBR Premix Ex TaqII (2), 0.4?l of ROX Reference Dye (50), and ddH2O for a total NCRW0005-F05 volume of 20?l. The amplification conditions were as follows: initial denaturation at 95C for 30?s, followed by 40 cycles of 95C for 5?s and 60C for 30?s. Relative expression levels were quantified using the 2 2?Ct method. The primer sequences were as follows (synthesized by GenScript, Nanjing, China): 0.05, ** 0.01, *** 0.001. To verify whether autophagy could regulate ferroptosis in ALL cells, the cell mortality of Jurkat, CCRF-CEM, and Reh cells were evaluated after treatment with erastin and Rapa or CQ. As shown in Figure 1C, the mortality rates of Jurkat and CCRF-CEM cells were significantly higher for the combination of Rapa with erastin than for Rapa or erastin alone ( 0.001 for both). Jurkat and CCRF-CEM cells were NCRW0005-F05 further treated with the ferroptosis inhibitor ferrostatin-1 (Fer-1). Fer-1 attenuated the combined effect of Rapa and erastin in cell death, indicating that Rapa participated in the ferroptosis. In addition, Rapa promoted erastin-induced ferroptosis in other ALL cell lines (Nalm6 and Sup-B15 cells, in Supplementary Figure S1). These results indicated that Rapa promotes erastin-induced ferroptosis in ALL cells. Moreover, Jurkat, CCRF-CEM, Nalm6, and Sup-B15 cells were treated with another autophagy activator, MK-2206, and similar results were obtained (Figure 1D). These data indicated that autophagy activation could APO-1 promote the erastin-induced ferroptosis in ALL cells. In contrast, autophagy inhibitors such as CQ, BafA1, and 3-MA reduced Reh cell mortality induced by erastin ( 0.001 for all, Figure 1E). These findings demonstrated that activation of autophagy promoted the ferroptosis of ALL cells induced by erastin, and blockage of autophagy protected.