Fig 2: Cbl-b-Notch1 axis regulation. Adenosine induces immunosuppression in CD8+ T-cells in the tumor microenvironment by activating the adenosine A2A receptor (A2AR), thus causing downregulation of Notch1 and suppression of effector functions. A2AR controls Notch1 levels by regulating the ubiquitin ligase Casitas B-lineage lymphoma b (Cbl-b), which mediates the ubiquitination and degradation of Notch1. A2AR promotes Cbl-b via Suppressor of T-cell receptor signaling 1 (STS-1) Tyr-phosphatase, possibly through Tyr-dephosphorylation of Cbl-b. A2AR, together with other receptors, including T-cell receptor (TCR) and co-stimulation receptor CD28, may control Notch1 levels in T-cells and, in turn, effector functions by modulating Cbl-b. This model places Cbl-b at the core of a regulatory axis, which, by integrating positive and negative signals from different receptors, determines the fate of Notch1 and effector functions. Pharmacological inhibition of Cbl-b blocks this axis and reactivates Notch1 and effector functions.

Fig 3: Cbl-b ubiquitinates and degrades Notch1. (A, B) Notch1 or Cbl-b co-immunoprecipitation in protein lysates from 293T cells were transfected with various Myc-tagged Notch1 and/or HA-tagged Cbl-b constructs, as indicated. (C) Ubiquitinated Notch1 protein levels detected upon immunoprecipitation of Notch1 in 293T cells co-transfected with a His-tagged NICD construct and various HA-tagged Cbl-b constructs, as indicated. (D) Notch1 activation in 293T cells measured using the Hes-luciferase reporter gene system. (E) Protein levels of Notch1 and IFN-gamma production in primary activated CD8+ T-cells isolated from C57BL6 (WT) or Cbl-b -/- (Cbl-b KO). Densitometry (O.D.) results for N1TM normalized by ß-actin are shown below the panel. The graphs show averages ± standard deviation from three independent experiments. **p < 0.01, ***p < 0.001 two tailed T-test with equal variance. Negative control refers to samples immunoprecipitated using rabbit IgG instead of anti-Notch1/HA-tag/Cbl-b antibody. None, non-transfected cells. EV, empty vector.

Fig 4: Yangxinshi inhibits the p-samd2, a-sma, and Cbl-b genes expression. (a) Western Blot analysis of p-samd2, a-sma, and Cbl-b genes' expression treated with low glucose (5.5 mM) or high glucose (50 mM) and with or without Yangxinshi (each group has three samples, and each experiment was repeated three times). (b) Quantitative results of Western blot in.

Fig 5: Cbl-b inhibitors enhance anti-PD1/PDL1 efficacy in tumor-derived organoids. Organoids were obtained from syngeneic TNBC, (A) C0321, (B) M-WNT, and colon cancer, (C) MC38, models and treated with vehicle (control, DMSO) or 5µM of NTX-512 or NTX-477, alone or in combination with 1µg/ml anti-PD1 or 1µg/ml anti-PDL1. The plots show cancer cell death in organoids. Scale bars length is indicated above each bar (µm). The graphs show averages ± standard deviation from =10 organoids from independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. NS, non-significant.