Nemo‐like kinase modulates glucocorticoid‐induced erythroid progenitor differentiation by regulating stability of the glucocorticoid receptor

Glucocorticoid receptor (GR) is a critical regulator of erythroid progenitor proliferation, while Nemo-like kinase (NLK) is reported to be hyperactivated in Diamond-Blackfan anemia (DBA), suggesting a possible cross talk. Here, we demonstrate that NLK directly interacts with multiple domains of GR and promotes its ubiquitin-mediated proteasomal degradation. Co-immunoprecipitation assays confirmed endogenous NLK-GR interaction in K562 cells, enhanced by proteasomal inhibition. NLK overexpression downregulated levels of GR in a kinase-dependent manner across HEK293T, K562, and MEL cells, an effect reversed by MG132 or a ubiquitination-defective mutant. NLK directly phosphorylated GR at Ser226, as shown by in vitro kinase assays and site-specific immunoblotting. Conversely, NLK depletion reduced basal GR phosphorylation while increasing total GR. We identified OTS167 as a direct NLK inhibitor through cellular thermal shift and kinase assays. OTS167 suppressed NLK autophosphorylation and decreased Ser226 phosphorylation of GR, stabilizing GR protein. Functionally, among all the inhibitors tested, OTS167 maximally inhibited proliferation of K562 and MEL cells by 40-90%. OTS167 also induced erythroid differentiation in K562 and MEL by increasing CD71/TER119 expression and benzidine-positive cells by 60-80%, while NLK overexpression inhibited hemin-induced benzidine staining by 25%. In primary human CD34⁺ cells, NLK and GR exhibited inverse temporal expression during erythropoiesis. OTS167 or dexamethasone expanded CD71⁺ and CFU-E populations and enhanced proliferation (Ki67⁺) across BFU-E, CFU-E, and proerythroblast stages. Conversely, dexamethasone upregulated NLK, suppressing GR and suggesting a feedback loop. Thus, NLK-mediated GR downregulation constrains erythropoiesis, and its inhibition by OTS167 promotes erythroid expansion, revealing a targetable pathway in erythroid disorders.