Targeting Cbl-b by a small molecular inhibitor potentiates T cell receptor signaling and suggests rational combination strategies.

Casitas B-lineage lymphoma-b (Cbl-b), an E3 ubiquitin ligase, is a novel rising cancer immunotherapy target negatively regulating T cell response, yet the effects of its pharmacological inhibition on T cell activation remain unexplored. Here, we present a systematic pharmacological analysis of Cbl-b inhibition in T cells using a selective small-molecule Cbl-b inhibitor (Cbl-bi) which targets Cbl-b directly. Cbl-bi potentiates T-cell receptor (TCR) signaling in both Jurkat and primary murine T cells, as evidenced by increased phosphorylation of phospholipase C gamma 1 (PLCγ1) and extracellular signal-regulated kinase (Erk) and elevated interleukin-2 production following anti-CD3/CD28 stimulation. Transcriptomic profiling of Cbl-b-inhibited T cells revealed a pronounced activation and inflammatory signature, with enrichment of nuclear factor kB (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. Pharmacological interrogation of downstream signaling demonstrated that MAPK pathway activity is essential for the enhanced activation conferred by Cbl-b inhibition. Moreover, co-inhibition of autophagy or hematopoietic progenitor kinase 1 (HPK1) synergistically amplified the pro-activation effect of Cbl-b inhibition. Collectively, these findings provide mechanistic insight into Cbl-b's immunosuppressive role and identify rational combination strategies targeting Cbl-b, autophagy, or HPK1 to robustly potentiate T cell function for immunotherapy of cancer.