Transcriptional modulation of differentiation-associated markers during ATRA and ATO treatment in acute promyelocytic leukaemia cells

Background
Acute promyelocytic leukaemia (APL), a cytogenetically and molecularly distinct subtype of acute myeloid leukaemia, is characterised by the t(15;17) translocation that generates the oncogenic PML-RARα fusion protein, disrupting normal myeloid differentiation. Differentiation therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has transformed APL treatment, yet the early transcriptional dynamics of key regulatory genes remain underexplored. PU.1, a master transcription factor that orchestrates myeloid lineage specification, is frequently suppressed in APL, contributing to the differentiation arrest. In contrast, Annexin A1, a calcium-dependent phospholipid-binding protein, plays multifaceted roles in apoptosis, inflammatory resolution, and has emerging associations with granulocytic maturation. Together, these markers represent valuable indicators of early differentiation and therapeutic response in APL models.

Aims
To evaluate early transcriptional changes in PU.1 and Annexin A1 expression following ATRA and ATO treatment in NB4 APL cells, monotherapy and in combination, and to assess their suitability as early transcriptional biomarkers of therapeutic response.

Methods
NB4 cells were treated with ATRA (1μM), ATO (0.5μM), or their combination for 2 and 6 hours. Quantitative PCR (qPCR) was performed to assess relative mRNA expression levels of PU.1 and Annexin A1, using β-actin as an endogenous control. Fold changes were calculated using the ΔΔCq method, normalising against untreated controls. PCR products were run on 2% agarose gel stained with ethidium bromide/SYBR Safe to assess amplification specificity. Experiments were performed in triplicate.

Results
PU.1 transcription was significantly upregulated at 2 hours post-treatment with ATRA (4.1-fold) and ATO (2.5-fold), with the ATRA + ATO combination showing a synergistic but transient rise (1.9-fold). At 6 hours, ATRA-induced PU.1 expression further increased (5.2-fold), whereas expression declined in the combination group, suggesting a short-lived transcriptional peak. Annexin A1 showed limited transcriptional induction (<1.5-fold across conditions), consistent with post-transcriptional regulation observed in protein-level analyses. These dynamics indicate a time-dependent, treatment-specific modulation of PU.1, supporting its potential as a sensitive early marker of therapeutic response in APL.

Conclusion
This qPCR-based study identifies PU.1 as a robust and rapidly inducible transcriptional marker in response to ATRA and ATO, supporting its potential as an early indicator of therapeutic differentiation in APL cells. The lack of significant Annexin A1 mRNA upregulation despite prior protein-level changes suggests post-transcriptional control mechanisms. These findings provide further molecular insight into ATRA/ATO-induced differentiation in APL and warrant investigation in patient-derived samples to assess their translational utility in clinical diagnostics or response monitoring.