FUNCTIONAL REACTIVATION OF MUTANT P53 R248Q BY EPRENETAPOPT TRIGGERS CELL CYCLE ARREST AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA
- 1Universidade de São Paulo, São Paulo, SP, Brazil
Abstract
Background: TP53-mutated acute myeloid leukemia (AML) carries a dismal prognosis. Eprenetapopt (APR-246), a p53 reactivator, is under investigation as a targeted therapeutic strategy. Among mutations identified in our cohort, R248Q was the most frequent, a dominant-negative hotspot mutation considered pathogenic; structural analyses revealed no significant alterations, though data indicate impaired p53 function. Aim: This study aimed to evaluate the ability of APR-246 to reactivate the functional conformation of mutant p53 (R248Q) in Kasumi-1 cells. Materials and Methods: Kasumi-1 cells were cultured in RPMI with 20% FBS and treated with eprenetapopt (0, 2.5, 5, 10 µM) for 48–72h. Protein extracts were analyzed by Western blotting. Gene expression was assessed by RT-qPCR. Cell cycle and apoptosis were evaluated by flow cytometry using Annexin V/propidium iodide staining. Results: Increasing eprenetapopt doses reduced p53, p62, and LC3B-I/II levels, with concomitant upregulation of BAX, p21, and cleaved PARP1, indicating apoptosis and enhanced autophagic flux. RT-qPCR showed strong transcriptional reactivation of p53 targets after 10 µM eprenetapopt: CDKN1A mRNA increased ∼90-fold (p<0.001), correlating with cell cycle arrest, while BH3-only mediators BBC3/PUMA (∼5.5-fold) and PMAIP1/NOXA (∼6.3-fold) were upregulated (p<0.001). Conversely, BAX and MDM2 transcripts showed slight downregulation (p<0.001, p<0.05), suggesting cytotoxicity is driven mainly by the p21/PUMA/NOXA axis. Cell cycle analysis showed dose-dependent changes: untreated cells displayed normal distribution across G0/G1 and S phases with minimal Sub-G1 fragmentation, whereas increasing concentrations progressively reduced proliferative populations and induced G2/M arrest at intermediate doses (p<0.01, 5 µM), followed by a marked increase in Sub-G1 at the highest dose (p<0.001), reflecting genomic collapse. Apoptosis assays confirmed these findings: baseline apoptosis was below 15%, while treatment induced a dose-dependent shift toward Annexin V-positive populations, reaching ∼70% and ∼80% apoptosis at 5 and 10 µM, respectively (p<0.001 vs. control). Discussion: These findings indicate that eprenetapopt restores transcriptional and functional activity of mutant p53, driving G2/M arrest and robust apoptosis in Kasumi-1 cells. Marked CDKN1A induction confirms recovery of p53 transactivation capacity, consistent with the observed G2/M retention, while PUMA/NOXA upregulation indicates transcriptional reactivation of the intrinsic apoptotic program. Notably, BAX protein increased despite reduced mRNA, suggesting post-transcriptional regulation, possibly via PUMA/NOXA-mediated displacement of BAX from anti-apoptotic complexes (BCL-2/BCL-XL), reinforcing the central role of the p21/PUMA/NOXA axis over BAX transcriptional induction. Decreased MDM2 mRNA further suggests partial restoration of the p53-MDM2 feedback loop. Concurrent reduction of p62 and LC3B-I/II alongside decreased total p53 suggests autophagy acts as an auxiliary pathway favoring degradation of destabilized mutant p53 and amplifying apoptotic signaling. Conclusion: Collectively, these findings demonstrate that eprenetapopt effectively reactivates mutant p53 transcriptional function, translating into significant cytotoxicity in TP53-mutated AML cells, supporting its therapeutic potential in this poor-prognosis subgroup.
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BibTeX
@article{bertoline2026functional,
title = {{FUNCTIONAL REACTIVATION OF MUTANT P53 R248Q BY EPRENETAPOPT TRIGGERS CELL CYCLE ARREST AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA}},
author = {Letícia Machado Favery BERTOLINE and Maria Fernanda Lopes CARVALHO and Natália Sudan PARDUCCI and Keli LIMA and João Agostinho MACHADO NETO},
journal = {Hematology, Transfusion and Cell Therapy},
year = {2026},
month = oct,
doi = {10.1016/j.htct.2026.107597},
url = {https://doi.org/10.1016/j.htct.2026.107597},
note = {CC BY},
}
RIS
TY - JOUR
TI - FUNCTIONAL REACTIVATION OF MUTANT P53 R248Q BY EPRENETAPOPT TRIGGERS CELL CYCLE ARREST AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA
AU - Letícia Machado Favery BERTOLINE
AU - Maria Fernanda Lopes CARVALHO
AU - Natália Sudan PARDUCCI
AU - Keli LIMA
AU - João Agostinho MACHADO NETO
PY - 2026
DA - 2026/10/23
JO - Hematology, Transfusion and Cell Therapy
DO - 10.1016/j.htct.2026.107597
UR - https://doi.org/10.1016/j.htct.2026.107597
AB - Background: TP53-mutated acute myeloid leukemia (AML) carries a dismal prognosis. Eprenetapopt (APR-246), a p53 reactivator, is under investigation as a targeted therapeutic strategy. Among mutations identified in our cohort, R248Q was the most frequent, a dominant-negative hotspot mutation considered pathogenic; structural analyses revealed no significant alterations, though data indicate impaired p53 function. Aim: This study aimed to evaluate the ability of APR-246 to reactivate the functional conformation of mutant p53 (R248Q) in Kasumi-1 cells. Materials and Methods: Kasumi-1 cells were cultured in RPMI with 20% FBS and treated with eprenetapopt (0, 2.5, 5, 10 µM) for 48–72h. Protein extracts were analyzed by Western blotting. Gene expression was assessed by RT-qPCR. Cell cycle and apoptosis were evaluated by flow cytometry using Annexin V/propidium iodide staining. Results: Increasing eprenetapopt doses reduced p53, p62, and LC3B-I/II levels, with concomitant upregulation of BAX, p21, and cleaved PARP1, indicating apoptosis and enhanced autophagic flux. RT-qPCR showed strong transcriptional reactivation of p53 targets after 10 µM eprenetapopt: CDKN1A mRNA increased ∼90-fold (p<0.001), correlating with cell cycle arrest, while BH3-only mediators BBC3/PUMA (∼5.5-fold) and PMAIP1/NOXA (∼6.3-fold) were upregulated (p<0.001). Conversely, BAX and MDM2 transcripts showed slight downregulation (p<0.001, p<0.05), suggesting cytotoxicity is driven mainly by the p21/PUMA/NOXA axis. Cell cycle analysis showed dose-dependent changes: untreated cells displayed normal distribution across G0/G1 and S phases with minimal Sub-G1 fragmentation, whereas increasing concentrations progressively reduced proliferative populations and induced G2/M arrest at intermediate doses (p<0.01, 5 µM), followed by a marked increase in Sub-G1 at the highest dose (p<0.001), reflecting genomic collapse. Apoptosis assays confirmed these findings: baseline apoptosis was below 15%, while treatment induced a dose-dependent shift toward Annexin V-positive populations, reaching ∼70% and ∼80% apoptosis at 5 and 10 µM, respectively (p<0.001 vs. control). Discussion: These findings indicate that eprenetapopt restores transcriptional and functional activity of mutant p53, driving G2/M arrest and robust apoptosis in Kasumi-1 cells. Marked CDKN1A induction confirms recovery of p53 transactivation capacity, consistent with the observed G2/M retention, while PUMA/NOXA upregulation indicates transcriptional reactivation of the intrinsic apoptotic program. Notably, BAX protein increased despite reduced mRNA, suggesting post-transcriptional regulation, possibly via PUMA/NOXA-mediated displacement of BAX from anti-apoptotic complexes (BCL-2/BCL-XL), reinforcing the central role of the p21/PUMA/NOXA axis over BAX transcriptional induction. Decreased MDM2 mRNA further suggests partial restoration of the p53-MDM2 feedback loop. Concurrent reduction of p62 and LC3B-I/II alongside decreased total p53 suggests autophagy acts as an auxiliary pathway favoring degradation of destabilized mutant p53 and amplifying apoptotic signaling. Conclusion: Collectively, these findings demonstrate that eprenetapopt effectively reactivates mutant p53 transcriptional function, translating into significant cytotoxicity in TP53-mutated AML cells, supporting its therapeutic potential in this poor-prognosis subgroup.
SN - 2531-1379
SN - 2531-1387
ER -