c-Cbl targets PD-1 in immune cells for proteasomal degradation and modulates colorectal tumor growth

Abstract

Casitas B lymphoma (c-Cbl) is an E3 ubiquitin ligase and a negative regulator of colorectal cancer (CRC). Despite its high expression in immune cells, the effect of c-Cbl on the tumor microenvironment remains poorly understood. Here we demonstrate that c-Cbl alters the tumor microenvironment and suppresses Programmed cell death-1 (PD-1) protein, an immune checkpoint receptor. Using syngeneic CRC xenografts, we observed significantly higher growth of xenografts and infiltrating immune cells in c-Cbl+/− compared to c-Cbl+/+ mice. Tumor-associated CD8+ T-lymphocytes and macrophages of c-Cbl+/− mice showed 2–3-fold higher levels of PD-1. Functionally, macrophages from c-Cbl+/− mice showed a 4–5-fold reduction in tumor phagocytosis, which was restored with an anti-PD-1 neutralizing antibody suggesting regulation of PD-1 by c-Cbl. Further mechanistic probing revealed that C-terminus of c-Cbl interacted with the cytoplasmic tail of PD-1. c-Cbl destabilized PD-1 through ubiquitination- proteasomal degradation depending on c-Cbl’s RING finger function. This data demonstrates c-Cbl as an E3 ligase of PD-1 and a regulator of tumor microenvironment, both of which were unrecognized components of its tumor suppressive activity. Advancing immune checkpoint and c-Cbl biology, our study prompts for probing of PD-1 regulation by c-Cbl in conditions driven by immune checkpoint abnormalities such as cancers and autoimmune diseases.

Targeting the ubiquitination/deubiquitination process to regulate immune checkpoint pathways

“…The C-terminus of c-Cbl interacts with the cytoplasmic tail of PD-1, leading to PD-1 ubiquitination and proteolysis. 42 Moreover, both c-Cbl and Cbl-b downregulate PD-L1 expression after the inhibition of PI3K/Akt, Jak/Stat, and MAPK-Erk signaling. 43 These findings suggest that c-Cbl/Cbl-b is negatively associated with PD-1/PD-L1 expression and that targeting c-Cbl/Cbl-b may sensitize cancer patients to ICB treatments.…”

Targeting ubiquitin signaling for cancer immunotherapy

“…Another E3 ligase, c-Cbl, has been shown to interact with the cytoplasmic tail of PD1 and mediate ubiquitin-dependent PD1 degradation in the tumor microenvironment. 99 Ubiquitination and deubiquitination also regulate the stability of PD1 ligand 1 (PD-L1) in tumor cells, which contributes to the regulation of T cell function and antitumor immunity. 100 , 101 …”

“…Another E3 ligase, c-Cbl, has been shown to interact with the cytoplasmic tail of PD1 and mediate ubiquitin-dependent PD1 degradation in the tumor microenvironment. 99 Ubiquitination and deubiquitination also regulate the stability of PD1 ligand 1 (PD-L1) in tumor cells, which contributes to the regulation of T cell function and antitumor immunity. 100,101 Another family of T cell costimulatory/coinhibitory receptors includes members of the TNFR superfamily, many of which are induced along with T cell activation and play a particularly important role in regulating effector and memory T cell responses.…”

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy

“…The other Cbl E3 ligase, c-Cbl, also affects PD-1, but contrary to Cbl-b, as a negative regulator. c-Cbl binds to the cytosolic tail of PD-1 and, acting as an E3 ligase, ubiquitinates PD-1 for its proteasomal degradation [ 211 ] ( Figure 3 ). Consequently, genetic reduction of c-Cbl ( c-Cbl +/− ) elevates PD-1 expression in CD8 + T cells and macrophages [ 211 ].…”