A previous study identified 65 TP53 variants in AML patients from TCGA and BeatAML cohorts, with R248Q being the most frequent. This mutation is a known hotspot, functionally dominant-negative, and predicted to be pathogenic. Although structural analyses show no major changes, evidence indicates it impairs p53 function. Synergism assays identified Kasumi1 as the optimal model.

This study aimed to assess whether APR-246 reactivates mutated p53 in Kasumi1 (R248Q) cells and to evaluate its effects combined with clinically relevant chemotherapeutics.

Kasumi1 cells were cultured in RPMI with 20% FBS and treated with APR-246 (0, 2.5, 5, 10 µM) for 72 hours. For combination assays, cells received: (i) 0 or 6.25 µM APR-246; (ii) 0.5 µM cytarabine alone or with APR-246; (iii) 0.06 µM daunorubicin alone or combined; (iv) 1.25 µM venetoclax alone or combined with APR-246. After treatment, cells were centrifuged, washed with PBS, and pellets used for protein extraction with lysis buffer (Tris-HCl, Triton X-100, PMSF, Na₃VO₄, NaF, Na₄P₂O₇, EDTA). Samples were incubated on ice, centrifuged, run on SDS-PAGE, transferred to nitrocellulose, blocked with 5% skim milk, incubated with primary and HRP-conjugated secondary antibodies, and detected by ECL™ according to manufacturer instructions.

Upon increasing concentrations of APR-246, we assessed the expression of key proteins, including p53 (drug target), BAX (pro-apoptotic marker), p21 (a p53- responsive cell cycle regulator), γH2AX (marker of DNA damage), PARP1 (apoptosis marker), SQSTM1/p62 and LC3B (autophagy markers), and α-tubulin (loading control). APR-246 treatment resulted in a dose-dependent reduction in p53, p62, and LC3B-I/II levels, accompanied by upregulation of BAX, p21, and cleaved PARP1, indicating induction of apoptosis and autophagic flux. Notably, combination treatments further increased γH2AX levels and PARP1 cleavage compared to single-agent controls, suggesting a synergistic enhancement of DNA damage and apoptotic signaling.

These findings corroborate literature reports that APR-246 restores mutated p53 functionality. The observed p53 decrease may result from reactivation, leading to negative feedback-mediated degradation pathways. Enhanced apoptotic and cell cycle arrest markers, coupled with reduced autophagy proteins, confirm pathway restoration. Synergistic effects in combination assays emphasize APR-246’s potential as an adjuvant to standard chemotherapy in TP53-mutated AML. APR-246 exhibits functional activity in Kasumi1 cells harboring the TP53 R248Q mutation by promoting apoptosis, reducing autophagy, and increasing DNA damage. Its combination with drugs in clinical settings potentiates these effects, underscoring synergistic therapeutic potential. Future studies will include in silico docking, molecular dynamics simulations, and in vitro complementary molecular analyses such as apoptosis assays and PCR-based gene expression.