BMP4 induces a p21-dependent cell state shift in glioblastoma linking mesenchymal transition to senescence
Bone morphogenetic protein 4 (BMP4) has emerged as a potential glioblastoma therapy due to its anti-proliferative effect via SOX2 downregulation and differentiation promotion. However  BMP4 responses vary across and within tumors. Our previous data indicate that BMP4 induces a mesenchymal-like cell state  which is associated with therapy-resistance and tumor recurrence  similar to senescence in cancer. In this study  we investigated BMP4?s potential to induce senescence in primary glioblastoma cells  including proneural- and mesenchymal-like clones derived from the same tumor. BMP4 treatment induced senescence-associated genes and phenotypic changes  such as cell enlargement  senescence-associated-?-gal expression  lamin B1 downregulation  and elevated p21 levels. Importantly  the most robust senescence induction was observed in the mesenchymal-like clone  compared to its proneural counterpart. Notably  mesenchymal-like cells displayed elevated basal levels of p21 and other senescence-associated markers  suggesting a convergence of mesenchymal and senescent traits. p21 knockout abolished BMP4-induced senescence  maintaining proliferation and cell size despite SOX2 downregulation. Additionally  senolytic treatment effectively eliminated senescent cells through apoptosis  thereby favoring survival of cells retaining normal p21 levels.  Our findings demonstrate BMP4?s ability to induce p21-dependent senescence in glioblastoma  particularly in therapy-resistant mesenchymal-like cells. These insights provide potential therapeutic strategies targeting senescence pathways in this challenging disease. Overall design: The human glioblastoma cell line U3065MG and two clonal derivates (clone-271 and clone-475) were used in the study. Cells were treated with recombinant BMP4  protein (Thermo Fisher Scientific) at 10 ng/ml between 7-33 days. For knockout of the p21 gene, the p21 Waf1/Cip1 CRISPR/Cas9 KO plasmids (sc-400013) and the p21-specific homology-directed repair (HDR) plasmid containing puromycin resistance gene and RFP gene (sc-400013-HDR) were transfected into cells according to the manufacturer's recommendations (Santa Cruz). For siRNA experiments, cells were seeded one day prior to siRNA transfection using Lipofectamine RNAiMAX (Thermo Fisher Scientific), overnight. siRNA used were Smad4 Silencer Select Pre-designed siRNA (1 nM, #4390824, ID s8403) and negative control siRNA (#4390843) (Thermo Fisher Scientific). After 2 days, BMP4 was added to the cultures. Cells were re-transfected on day 8 and used for analyses on day 12 and 14. For senolytic experiments, navitoclax (ABT-263, Selleck Chemicals) was used at a 1000 nM concentration.