Relaxin-2 Preconditioning Promotes Dexamethasone Responsiveness in Molecularly Distinct Glucocorticoid-Resistant T-ALL Models

Introduction: Glucocorticoids remain essential components of acute lymphoblastic leukemia (ALL) therapy; however, glucocorticoid resistance is a major cause of treatment failure and relapse, particularly in T-cell ALL (T-ALL). A major mechanism of glucocorticoid resistance is reduced glucocorticoid receptor (GR) expression, leading to impaired GR signaling. Interestingly, relaxin-2 (RLN-2) has been shown to increase GR expression at both mRNA and protein levels in THP-1 monocytic leukemia cells. However, the role of RLN-2 in glucocorticoid responsiveness in ALL remains unknown. This study evaluated whether RLN-2 preconditioning influences dexamethasone (DEX) responsiveness in resistant T-ALL cells.

Methods: Two molecularly distinct glucocorticoid-resistant T-ALL models were used, CCRF-CEM-R and LOUCY-R, generated by long-term dexamethasone exposure. Resistance was confirmed by reduced responsiveness to DEX, lack of apoptosis induction, and decreased GR protein levels. Cells were preincubated with RLN-2 (0.1 nM) for 9 days, followed by DEX (1 µM) treatment for 3 days. AT001 and RU486 (1 µM) were used in selected experiments to assess pathway specificity. Treatment response was assessed using an Alamar Blue metabolic assay; flow cytometric analysis of viability, apoptosis, and cell-cycle distribution; and immunofluorescence analyses of GR subcellular localization.

Results: DEX treatment alone showed limited biological activity in resistant T-ALL cells, confirming glucocorticoid resistance. RLN-2 preconditioning altered cellular response to subsequent DEX exposure, resulting in reduced metabolic activity and cell viability, accompanied by treatment-associated changes in apoptosis and cell-cycle distribution compared with DEX-treated controls. These effects were associated with enhanced GR nuclear translocation, suggesting restoration of GR signaling as a potential mechanism underlying altered DEX responsiveness. Similar trends were observed across both models despite their molecular heterogeneity.

Conclusions: RLN-2 preconditioning modulates dexamethasone responsiveness in glucocorticoid-resistant T-ALL models. The observed effects suggest a potential role for RLN-2 in restoring GR-dependent signaling. These findings support further investigation of RLN-2 as a potential strategy for improving glucocorticoid responsiveness in resistant ALL.