Integrated transcriptomic and proteomic profiling of RXRA-overexpressing chronic myeloid leukemia cells identifies protein kinase D as a candidate target for imatinib sensitization

Achoimre

Background Retinoid-X-receptor alpha ( RXRA ) is downregulated in chronic myeloid leukemia (CML). We previously demonstrated that overexpression of RXRA sensitizes CML cells to imatinib, but the mechanisms underlying this effect remain undefined. Objective To characterize the molecular programs associated with RXRA-mediated imatinib sensitization through integrated transcriptomic–proteomic profiling, and to identify and examine pharmacologically actionable downstream effectors in CML cell lines and primary patient-derived CD34 + cells. Methods RXRA -overexpressing ( RXRA -OE) and empty vector (EV) LAMA84 cells were profiled by RNA sequencing and mass spectrometry-based proteomics. Four concordantly downregulated candidates ( PRKD1 , UCHL1 , FSCN1 and NTRK3 ), prioritized by magnitude of concordant downregulation, biological relevance and inhibitor availability, were confirmed by qRT-PCR and immunoblotting. Pharmacological inhibition of each candidate combined with imatinib was assessed in CML cell lines, primary CML CD34 + cells and healthy-donor CD34 + controls. Results RXRA overexpression produced 7,053 differentially expressed transcripts and 1,668 differentially abundant proteins (technical triplicates of one sample per group), with 711 concordant gene–protein pairs (Pearson r = 0.64). RXRA -overexpressing cells scored higher for genes downregulated in hematopoietic stem cells and for the granulocyte–monocyte progenitor signature, with enriched myeloid gene sets and reduced expression of receptor tyrosine kinase and growth-factor signaling genes. Enzymes of glycolysis, the pentose phosphate pathway and sterol biosynthesis were downregulated, while lipid catabolic genes were upregulated. Among the four candidates, the serine/threonine kinase PRKD1 and the deubiquitinase UCHL1 emerged as candidate imatinib-sensitizing targets: the pan-PKD inhibitor CRT0066101 and the UCHL1 inhibitor LDN-57444 each synergized with imatinib and reduced clonogenic potential. PRKD1 protein was detected in all four CML cell lines tested. Combined PKD inhibition and imatinib reduced the viability and colony-forming capacity of primary CML CD34 + cells. In healthy-donor CD34 + cells the live-cell fraction did not differ between treatments (n = 5), and colony formation was assessed in two donors; these comparisons were limited by sample size and selectivity for CML cells was not established. Conclusion Integrated multi-omics associates RXRA overexpression with myeloid lineage-associated and metabolic expression signatures in imatinib-sensitized CML cells, and identifies PRKD1 as a candidate effector whose pharmacological targeting with a pan-PKD inhibitor enhances imatinib efficacy in vitro .