CREB1 and GSK3 Isoforms as Potential Adaptive Signaling Nodes in PI3K/Akt/mTOR Inhibitor Treated Philadelphia...

Philadelphia chromosome-positive (Ph+) B-cell acute lymphoblastic leukemia (ALL), driven by the BCR-ABL1 fusion, remains highly aggressive, with poor prognosis despite tyrosine kinase inhibitor (TKI) therapy. Aberrant PI3K/Akt/mTOR pathway activation contributes to resistance, warranting identification of predictive kinase biomarkers. To identify and validate critical phospho-kinase markers within the PI3K/Akt/mTOR axis as candidate adaptive signaling nodes and exploratory prognostic markers in Ph+ B-ALL. Network pharmacology using STRING and Cytoscape mapped the protein-protein interactions, identifying high-centrality nodes. Functional enrichment analyses (GO/KEGG) were then performed via ShinyGO. Experimentally, phospho-kinase arrays profiled 39 kinase phosphorylation sites in Ph+ B-ALL cells (SUPB-15) treated with PI3K/Akt/mTOR inhibitors (Rapamycin, GDC-0941, GSK690693, Perifosine). Gene expression was validated by RT-qPCR in 100 Ph+ B-ALL patients and protein validation employed Western blot in 50 patient samples with 15 and five healthy controls, respectively. Network analysis identified CREB1 and GSK3 isoforms as central hub regulators. Phospho-kinase profiling revealed p-CREB (Ser133) phosphorylation upregulation following Perifosine, GDC-0941, and Rapamycin treatment, with no statistically significant change after GSK690693. p-GSK3α/β (Ser21/9) phosphorylation showed a statistically significant increase after Perifosine, with non-significant trends after GDC-0941, and a significant reduction following Rapamycin. Hierarchical clustering then divided proteins into four distinct clusters positioning CREB1 in cluster 3 and GSK3α/β in cluster 2 as candidate adaptive response markers based on their phosphorylation profiles. RT-qPCR demonstrated marked downregulation of GSK3α (approximately 71%), GSK3β (63%), and CREB1 (65%) transcripts in patients versus controls, with even greater suppression seen in cell lines. Conversely, Western blot revealed a significant 2.49 fold elevated p-CREB (Ser133) (β = 1.317, p = 0.000347) and a phospho-GSK3α/β (Ser21/9) level that did not differ significantly between patients and controls (β = 0.467, p = 0.466), revealing mRNA-protein discordance indicative of compensatory post-transcriptional and post-translational regulation. These findings represent treatment-associated and disease-state associations between altered phosphorylation states and the Ph+ B-ALL context; functional validation remains an essential next step. mRNA-protein discordance with transcriptional downregulation (63-71%) contrasting with elevated p-CREB levels (2.49-fold, p = 0.000347) suggests post-translational compensatory mechanisms under therapeutic pressure. CREB1 and GSK3 isoforms emerge as central network nodes that might potentially mediate adaptive resistance, although functional validation is required. It should be noted that the evidence presented is associative rather than causal and a direct link between the observed phosphorylation changes and clinical drug resistance has not been established in this study. Sustained CREB phosphorylation despite PI3K/Akt/mTOR inhibition implicates alternative kinase pathways (MAPK/ERK, CaMK), supporting rational combination therapy strategies. These findings position p-CREB and p-GSK-3α/β as candidate biomarkers, highlighting the value of phospho-proteomics over transcriptomics alone in capturing pathway activation. Multi-omics integration may prove essential for overcoming adaptive resistance in ALL patients.