P072 | REDOX VULNERABILITY IN PLZF::RARA-POSITIVE ACUTE PROMYELOCYTIC LEUKEMIA REVEALS THERAPEUTIC POTENTIAL OF HIGH-DOSE ASCORBATE AND AZACITIDINE

The PLZF::RARA (ZBTB16-RARA) fusion defines a rare, incurable subtype of acute promyelocytic leukemia (APL), characterized by therapy resistance and transcriptional repression of genes required for myeloid differentiation. Since the antioxidant system plays a key role in therapy sensitivity, we evaluated the expression of two key regulators, FOXO3a and G6PD, in primary blasts from patients with PLZF::RARA+ APL, PML::RARA+ APL, AML, normal bone marrow (NBM), and in a ZnSO₄-inducible PLZF::RARA-expressing cell line (U937-B412), compared with a control line (U937-MT). In PLZF::RARA+ patient samples, G6PD expression (n = 5; 0.7 ± 0.5), was lower than in NBM (n = 5; 1.6 ± 1.2) but comparable to APL’s (n = 10; 0.8 ± 0.5) and AML (n = 7; 0.7 ± 0.5). In B412 cells, PLZF::RARA induction significantly reduced G6PD expression at 6h at both the mRNA (0.65 ± 0.38 vs. 1.5 ± 0.2 in MT; p = 0.04) and protein levels although not statistically significant (MT: 2±1.2 vs B412: 1.3±0.1). mRNA expression of FOXO3a, key regulator of oxidative stress responses, was downregulated in PLZF::RARA APL (n = 4; 0.3 ± 0.1), compare with NBM (n = 6; 1.9 ± 2.5) and similar to PML::RARA APL and AML blast cells. In B412 cells, PLZF::RARA suppressed FOXO3a at both protein (0.35 ± 0.07 vs. 1.55 ± 0.07 in MT; p = 0.003) and mRNA levels (0.9 ± 0.2 vs. 1.6 ± 0.4; p = 0.04). Consequently, catalase enzymatic activity was reduced in B412 cells by approximately 34.5% compared to the MT control (0.091 vs. 0.139, respectively), while SOD1 protein expression (by Western blot) was also decreased. Both are FOXO3a targets, indicating impaired antioxidant defenses. This suggests that PLZF::RARA+ cells are sensitive to oxidative stress. High-dose ascorbate, a known pro-oxidant, generates H₂O₂ and ROS, which can overwhelm these compromised systems. Treatment of B412 cells with azacitidine (100 ng/mL), an epigenetic therapy agent, and ascorbate (1 mM) significantly increased apoptosis, as shown by mitochondrial membrane depolarization (ΔΨm: Ctrl 3%, AZA 28.6%, ASC 41.6%, AZA+ASC 62.7%) and cleavage of caspase-3 and PARP1. These data indicate that PLZF::RARA represses key antioxidant regulators, creating a redox vulnerability that can be exploited by pro-oxidant therapy. The combination of azacitidine and high-dose ascorbate shows enhanced efficacy and may offer a novel therapeutic approach in PLZF::RARA-positive APL.