CD40LG/CD28-Mediated Rho GTPase Signaling Drives Survival and Chemoresistance in Non-ETP T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by unfavorable clinical outcomes. Despite significant progress in deciphering the genetic and epigenetic landscapes of T-ALL, the underlying molecular mechanisms, particularly in non-early T-cell precursor (non-ETP) T-ALL, remain incompletely understood. In this study, functional assays were performed using three well-characterized non-ETP T-ALL cell lines. In vivo therapeutic efficacy was evaluated using non-ETP T-ALL xenograft models. Transcriptomic profiling was performed by RNA sequencing (RNA-seq) followed by bioinformatic analysis. Publicly available clinical datasets from T-ALL patients were mined to analyze survival outcomes. We found that activation of CD40 ligand (CD40LG) or CD28 accelerates cell-cycle progression and enhances the migratory capacity of non-ETP T-ALL cells, with CD40LG uniquely upregulating CXCR4 to mediate bone marrow tropism. Further RNA-seq and functional validation identified Rho GTPase signaling, specifically RhoA/Rac1/Rac2, as a pivotal downstream effector of CD40LG/CD28, leading to therapeutic resistance to PI3K inhibition. Pharmacological blocking RhoA or Rac1 using small-molecule compounds not only induces remarkable cytotoxicity but also sensitizes resistant cells to PI3K inhibitors, both in vitro and in vivo. Clinically, elevated expression of