Molecular dynamics of PU.1 and annexin A1 in acute promyelocytic leukaemia cells following ATRA/ATO therapy

Background:
Acute promyelocytic leukaemia (APL), a distinct subtype of acute myeloid leukaemia, is characterised by the t(15;17) translocation and resultant PML-RARα fusion oncoprotein, which blocks myeloid differentiation. The combined use of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) represents the current gold-standard therapy for APL. However, the early molecular responses and mechanisms underlying their synergistic differentiation effect remain under-characterised. In particular, the roles of PU.1, a master myeloid transcription factor, and Annexin A1, a phospholipid-binding protein involved in apoptosis and granulocytic maturation, may serve as early markers of treatment response.

Aims:
To investigate early transcriptional and protein-level expression dynamics of PU.1 and Annexin A1 in NB4 APL cells treated with ATRA, ATO, or their combination, and to evaluate their potential utility as early biomarkers of differentiation in APL.

Methods:
NB4 cells were treated with 1μM ATRA, 0.5μM ATO, or their combination. mRNA and protein expression levels of PU.1 and Annexin A1 were assessed at 6- and 24-hours post-treatment using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. β-actin was used as the internal loading control. Protein levels were semi-quantitatively assessed using densitometry and visual comparison. Experiments were performed in triplicate.

Results:
Baseline mRNA expression of PU.1 and Annexin A1 was detected in untreated NB4 cells. At 6-hours, PU.1 protein expression was upregulated following both ATRA- and ATO-monotherapy and synergistic ATRA + ATO treatment. Annexin A1 was upregulated with monotherapy but suppressed with combination therapy, indicating an early antagonistic interaction. At 24-hours, PU.1 remained upregulated with ATRA monotherapy, was unchanged with ATO, and was lost with combination treatment. Annexin A1 expression was progressively downregulated across all treatments, with complete loss of expression under 24-hour ATRA + ATO. Notably, mRNA levels for both genes remained relatively stable, suggesting a post-transcriptional regulatory mechanism. The inverse temporal expression patterns of PU.1 and Annexin A1, particularly under synergistic treatment, are consistent with established models of myeloid differentiation and may indicate a time-sensitive feedback or stress-response axis relevant to therapeutic efficacy.

Conclusion
This study reveals early, transient modulation of PU.1 and Annexin A1 expression during ATRA/ATO therapy in APL cells. PU.1 upregulation represents an early differentiation signal, whereas Annexin A1 suppression may reflect apoptotic or anti‑inflammatory shifts during combined treatment. The loss of both proteins at 24-hour implies feedback inhibition or treatment‑induced cellular stress. Their divergent expression dynamics highlight potential utility as early biomarkers of treatment response and warrant validation in patient-derived samples.