Background Acute myeloid leukemia (AML) presents significant therapeutic challenges, particularly due to the resistance to chemotherapy agents like daunorubicin (DNR). One of the proteins implicated in this resistance is protein disulfide isomerase A3 (PDIA3/ERP57), which plays a crucial role in tumor progression and drug resistance. However, the specific mechanisms by which PDIA3 influences chemosensitivity and autophagy in AML are not well understood. In this study, we explored the role of PDIA3 in contributing to DNR resistance and assessed the potential of silibinin, a natural inhibitor of PDIA3, to improve chemosensitivity in AML.

Methods We conducted an analysis of PDIA3 expression in 92 samples from patients with acute myeloid leukemia (AML), which included individuals who were newly diagnosed, in complete remission, or experiencing relapse/refractory disease, alongside 44 samples from healthy donors by qRT-PCR. Our functional studies involved modulating PDIA3 through shRNA knockdown in THP1 and U937 cell lines, as well as overexpressing it in the Kasumil-1 cell line. We assessed autophagy by employing transmission electron microscopy (TEM), monitoring the conversion of LC3-I to LC3-II, and evaluating p62 degradation. Additionally, we examined apoptosis through the cleavage of caspase-3 and PARP, and assessed drug sensitivity using CCK-8 assays. To explore resistance mechanisms, RNA sequencing were utilized to screen 160 genes associated with resistance, while co-immunoprecipitation was performed to confirm the interaction between PDIA3 and mTOR. The activity of the AKT/mTOR pathway was analyzed via Western blotting. We measured autophagy flux using chloroquine (HCQ) and ATG5 siRNA. In vivo experiments involved treating U937 xenografts in nude mice with silibinin at a dosage of 200 mg/kg/day, with or without the addition of daunorubicin (DNR) at 2 mg/kg. Furthermore, we conducted a phase II clinical trial that randomized 76 newly diagnosed AML patients to receive standard daunorubicin and cytarabine (DA) treatment, either with or without silibinin.

Results PDIA3 mRNA was found to be significantly overexpressed in patients with acute myeloid leukemia (AML) compared to healthy donors, showing a 3.2-fold increase (P<0.001). This elevated level of PDIA3 was associated with high-risk cytogenetics (P=0.003) and a higher incidence of relapse or refractory disease (P=0.002). In contrast, patients with low PDIA3 levels demonstrated higher complete remission rates, achieving 82.1% compared to 41.4% in those with high PDIA3 levels (P<0.01). Furthermore, knocking down PDIA3 resulted in a significant reduction in the IC50 of daunorubicin (DNR) by 58% in THP1 cells and 62% in U937 cells (P<0.001), while overexpressing PDIA3 led to a 2.3-fold increase in IC50 in Kasumil-1 cells. RNA sequencing revealed 830 genes dependent on PDIA3, with notable enrichment in the autophagy pathway (NES=−2.1, FDR=0.003). The knockdown of PDIA3 also enhanced autophagic flux, indicated by a 4.2-fold increase in the ratio of LC3-II to LC3-I (P<0.01), and promoted apoptosis. Additionally, silibinin at a concentration of 50 μM was shown to suppress PDIA3 expression by 65% (P<0.001) and reduce DNR IC50 by 40–60%. Mechanistically, silibinin inhibited the phosphorylation of mTOR at Ser2448 and restored autophagy. In xenograft models, the combination of silibinin and DNR resulted in a synergistic reduction of tumor volume by 68% (P<0.001) and weight by 72%. Clinically, the combination of daunorubicin and silibinin improved complete remission rates to 69.2% compared to 42.0% (P=0.008) and increased rates of uMRD to 68.4% versus 28.9% (P<0.001), while also shortening the duration of hematologic recovery from anemia (18±3 days compared to 25±4 days, P=0.02) without increasing toxicity.

Conclusion PDIA3 plays a significant role in promoting resistance to daunorubicin (DNR) in acute myeloid leukemia (AML) by inhibiting autophagy through the activation of the AKT/mTOR signaling pathway. Silibinin, identified as an inhibitor of PDIA3, has been shown to restore autophagy and enhance chemosensitivity in both in vitro and in vivo models. This research highlights PDIA3 as a promising therapeutic target and positions silibinin as a potentially effective chemosensitizer for the treatment of AML.

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2025
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