Small molecule modulators of TOX protein re-invigorate T cell activity

The TOX protein (thymocyte selection-associated high mobility group box) is a critical transcription factor implicated in both T acute lymphoblastic leukemia (T-ALL) and CD8⁺ T cell exhaustion. Gene perturbation studies suggest that inhibiting TOX may have therapeutic implications for both leukemia and T cell exhaustion. However, due to its complex molecular mechanisms and intrinsically disordered structure, TOX has not been effectively targeted by small molecules to date. In this study, we used small molecule microarray (SMM) screening and biochemical assays to identify a series of TOX protein-protein interaction (PPI) inhibitors. We identified KI-TOX-A3 as a TOX protein binder and potent TOX PPI inhibitor. In T-ALL, KI-TOX-A3 revealed selective cytotoxicity and proteosome-dependent TOX degradation. In CD8⁺ T cells, KI-TOX-A3 potently reversed T cell exhaustion by decreasing surface inhibitory receptors, increasing expression of effector cytokines, and enhancing cancer cell killing activity. We also demonstrate the utility of KI-TOX-A3 to probe potential epigenetic regulatory mechanisms of TOX via KAT7 acetylation in T cells.