Ubiquitination in T cell activation and checkpoint inhibition

preface

The advent of T-cell-based immunotherapies has dramatically changed the treatment paradigm
for cancer patients.
Despite success, currently approved immunotherapy regimens still have limitations
.
Therefore, a deeper understanding of the molecular mechanisms of T cell activation and inhibition is needed to reasonably expand the targets and possibilities
of improved immunotherapy.

Protein ubiquitination downstream of immune signaling pathways is essential
for positive and negative regulation of almost all immune responses, especially T cell activation.
A large number of studies have shown that ubiquitination exerts a variety of cellular and molecular roles by controlling the function, localization and stability of proteins, and the regulation of ubiquitin-dependent pathways can significantly change the activation of T cells and enhance anti-tumor response
.
Therefore, technologies that selectively use ubiquitin-related enzymes for cancer treatment are being actively developed, and targeted ubiquitination offers great possibilities
for advancing T cell immunotherapy.

Protein ubiquitination

Ubiquitination is a post-translational modification, ubiquitin is a small protein consisting of 76 amino acids, usually covalently linked
by a chain of ubiquitin to lysine residues within the substrate protein.
Ubiquitination affects protein function in a variety of ways, such as by affecting protein stability, turnover, cell localization, and inducing conformational changes
that affect interactions with other proteins.

This process is initiated by the E1 ubiquitin activator, which is activated using ATP, followed by the transfer of ubiquitin monomers to the E2 ubiquitin-binding enzyme
.
Ubiquitin is eventually transferred from E2 to substrate under the action of E3 ubiquitin ligase
.

Ubiquitination is multifaceted, and substrate proteins can be labeled
with only one ubiquitin (monoubiquitinated), multiple monoubiquitins (multiple monoubiquitinated), or multiple ubiquitin chains (polyubiquitinated).
Since ubiquitin itself has seven lysine (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) and one N-terminal methionine residue (M1), ubiquitination is possible, so several structurally distinct homogeneous/isotypic, mixed/heterotypical, and branched polyubiquitin chains
can be formed.

In addition to being the main regulator of proteins that degrade proteins through proteosomes, ubiquitination is actually a multifunctional regulator of almost all cell signaling cascades
.
The fate of ubiquitinated proteins depends on the amount of ubiquitin added and the ubiquitin amino acids that form ubiquitin junctions
.
In this way, and due to the multiple possibilities of different chains in the topology, ubiquitin constitutes a highly versatile signal
.

Monoubiquitination and polyubiquitination are involved in endocytosis of membrane receptors, as well as protein degradation, localization, and protein-protein interactions, and these types of chains are also involved
in TGF-β signaling.
Among the homotypic polyubiquitin chains, the Lys48 and Lys63 linked chains are the most abundant, driving proteasome degradation and regulating a variety of cell signaling events
, respectively.
The Lys11 linker regulates the cell cycle and mitochondrial autophagy, and the M1 linkage is essential for the intrinsic response to inflammation, specifically to regulate NF-κB signaling
.

Cellular processes regulated by the ubiquitin linkage chains of Lys6-, Lys27-, Lys29-, and Lys33 remain less clear
.
The Lys27 linkage chain appears to be important
for regulating innate immunity, cell cycle, and mitochondrial function.
The Lys6 strand appears to be involved in DNA repair and mitochondrial autophagy, while the Lys29 strand is associated
with Wnt signaling as well as protein toxic stress and the cell cycle.
The Lys33 chain plays a key role
in Golgi post-transport and T cell receptor (TCR) regulation.

In addition, it is very important that the assembly of ubiquitin on the substrate can be reversed by the action of deubiquitinase (DUB), which hydrolyzes and removes ubiquitin from the substrate, making ubiquitination a transient modification, and DUB is essential
for maintaining physiological ubiquitin balance.
This imbalance of balance is associated with the pathogenesis of human diseases, particularly cancer, infection, neurodegeneration, and immune disorders, and as a result, DUB has begun to become an attractive drug target for inhibitor-based therapies
.

The ubiquitin pathway in TCR/CD3 activation

E3 ligase

The E3 ubiquitin ligase Casitas B lymphoma (Cbl-B) is the most characteristic E3 ligase and a major player in
T cell activation.
When T cells are activated, it is recruited into TCR at immune synapses, exerting multiple inhibitory mechanisms
downstream of TCR.

Cbl-b interacts
with key TCR signalsome molecules such as LCK, SLP76, ZAP70, Vav1, PKCθ, and PI3K through its multiple protein interaction domains.
Cbl-b works synergistically with the E3 ligase Itch to mediate the polyubiquitination of the Lys33-linked TCR-ζ, which does not target the TCR receptor for degradation or endocytosis, but rather prevents its phosphorylation and further binding
to downstream ZAP70 kinases.
Through these interactions, as well as ubiquitination of some signaling body components, Cbl-b strongly inhibits T cell activation
.
Cbl-b knockout mice have overactive T cells whose activation does not require CD28, and autoimmune damage
has never been described in Cbl-b knockout mice.
Therefore, Cbl-b has multiple checkpoint inhibition and minimal autoimmune toxicity, making it a strong candidate
for future targeted cancer immunotherapy.

The gene associated with lymphocyte impotence (GRAIL) is a transmembrane E3 ligase that localizes endosomes and also negatively regulates T cell activation
.
TCR-mediated T cell activation promotes GRAIL expression, which in turn hinders T cell activation
.
Mechanically, GRAIL stabilizes and activates Rho-guanine dissociation inhibitors (RhoGDI) through non-degradable ubiquitin chains, leading to cytoskeletal rearrangement and defects in
IL-2 secretion.

NRDP1 is another E3 ligase
that impairs TCR signaling.
After T cell activation, NRDP1 adds the Lys33 polyubiquitin nondegradable chain to the ZAP70 kinase
.
Lys33-linked ubiquitin chains help recruit Sts1 and Sts2 phosphatases, ultimately dephosphorylating and inactivating
ZAP70.

In addition to acting on proximal signaling in vitro, E3 ligase controls key transduction events
leading to nuclear translocation of transcription factors for T cell activation by further acting downstream on inhibiting T cell activation.
For example, after TCR/CD28 ligation, the E3 ligase Pellino1 (PELI1) blocks the NF-κB pathway by labeling c-Rel with a Lys48 polyubiquitin chain, resulting in its proteasome degradation
.

DUBs

Deubiquitinase A20 and CYLD, which play an important role in regulating NF-κB signaling in native immune cells, can also significantly alter T cell function
.
The conditional deletion of A20 in peripheral T cells results in CD8+ T cells having enhanced NF-κB signaling that can be activated more efficiently even at low antigen doses to produce higher levels of IL-2 and IFN-γ
.

CYLD can impair T cell activation by NF-κB by deubiquitinating
the Lys63 ubiquitin chain of TAK1 kinase downstream of the CBM complex.
CYLD-deficient mice developed colitis with increased
T cell frequency and activation.

Using a similar mechanism of action, deubiquitinase USP18 inhibits TCR
by deubiquitinase USP18 on TAK1.
Gene deletion of USP18 leads to overactivation of NF-κB and NFAT and overproduction
of IL-2 in T cells.
In contrast, desubiquiterase USP9X and USP12 actively modulate the NF-κB pathway by removing inhibitory ubiquitin chains from BCL10, thereby blocking the assembly
of CBM signaling complexes.
Thus, T cells lacking USP9X or USP12 exhibit lower levels of NF-κB activation upon TCR activation, as well as decreased
proliferation and cytokine production.

TCR internalization

The amplitude and duration of TCR signaling are determined
by the balance of internalization, recirculation, and degradation of cell membrane receptors.
Upon antigen stimulation, the TCR/CD3 complex is ubiquitinated, which is essential
for the internalization of surface receptors.
Cbl-b has been reported to promote the internalization of TCR alone or in combination with the E3 ligase c-Cbl, which also interferes with activation and aggregation, thereby disrupting the stability of immune synapses and further weakening TCR signaling
.

Similarly, the E3 ligase GRAIL can downregulate the expression
of surface TCR/CD3 complexes by proteasome polyubiquitination and degradation of CD3ζ molecules.
Recent studies have shown that surface expression of CAR receptors for CAR-T therapy is also affected
by ubiquitin-mediated endocytosis and degradation.
The poor persistence of CAR-T cells injected into the patient is the main limitation of
this tumor immunotherapy.
A recent study was able to improve the stability of CAR by mutating all lysine residues in the cytoplasmic domain, which effectively bypasses the ubiquitination-dependent lysosomal degradation of CARs, and as a result, these CAR-T cells improve the persistence of CAR and signaling from endosomes, while enhancing effector function to generate a durable anti-tumor immune response
in mouse models.

The ubiquitin pathway downstream of the co-stimulatory receptor

CD28

The E3 ligase Cbl-b is a major inhibitor of CD28-mediated signaling, and mechanically, the E3 ligase Cbl-b inhibits the CD28-dependent PI3K pathway
by binding to and ubiquitinating the p85β sub-of PI3K.

E3 ligase not only hinders CD28 signaling, but also promotes CD28 signaling
.
For example, TCR/CD28 activates NFAT involving the E3 ligase TRAF6
.
TRAF6 is recruited to immune synapses to interact with the scaffold protein LAT and enhance LAT phosphorylation and TCR signaling
by Lys63 ubiquitination.

To fully activate T cells, CD28 coreceptors must overcome Cbl-b inhibition
.
CD28 signaling blocks the Cbl-b inhibition pathway by triggering post-translational modifications of Cbl-b, ultimately leading to its proteasome degradation
.
Among them, the E3 ligase NEDD4 plays an important role, and NEDD4 has been reported to bind and ubiquitinated Cbl-b for proteasome degradation
under CD28 co-stimulation.

Other co-stimulatory receptors

Similar to CD28, the co-stimulatory receptors 4-1BB, CD40L, OX40, and GITR of members of the tumor necrosis factor receptor (TNFR) family synergistically promote T cell activation, especially proliferation and cytokine production
.
As TNFRs, they lack intrinsic enzyme function and rely on several E3 ligases, including TRAF (TRAF1/2/3), cIAP1/2, and LUBAC, to guide a large number of Lys63, Lys48, and linear ubiquitination events
in the signal transduction cascade leading to NF-κB activation.
Deubiquiterase A20, CYLD, and OTULIN can clear these ubiquitination events to prevent NF-κB activation
.

Inhibitory receptor downstream ubiquitin pathway

CTLA-4

So far, there have been no reports of direct ubiquitination of CTLA-4 by E3, and no records
of ubiquitination sites in CTLA-4.
However, there was a significant overall decrease in ubiquitin modification in CTLA-4-deficient T cells, which strongly demonstrates the importance
of ubiquitination events in the CTLA-4 pathway.
In addition, experimental evidence suggests that the primary CTLA-4 inhibitory function is mediated by key T cell inhibitory E3 ligases, which also regulate T cell activation, namely Cbl-b, ITCH, and GRAIL
.

In addition, Cbl-b, ITCH, and GRAIL are fundamental drivers of T cell impotence, and these three E3 ligases also play an important immunosuppressive role
by regulating the development and function of Treg.
Cbl-b and IITCH are involved in TGF-β mediated Foxp3 regulation, and the lack of both E3 ligases impairs TGF-β-induced development of Foxp3+Tregs (iTreg), resulting in low expression of iTreg and functional defects
.

PD-1

Unlike CTLA-4, some reports highlight the critical role
of ubiquitination in the PD-1/PD-L1 system.
For PD-1 receptors on T cells, Cbl-b is also significantly involved as a key mediator of the PD-1 inhibition pathway, and T cells lacking Cbl-b are resistant to PD-1 inhibition
.
Without Cbl-b, PD-1 is inefficient in inhibiting IFN-γ production or inducing cell death following T cell activation
.

Another Cbl E3 ligase c-Cbl also affects PD-1, but in contrast to Cbl-b, it is a negative regulator.

c-Cbl binds to the cytoplasmic tail of PD-1 and performs proteasome degradation
as E3 ligase, ubiquitinated PD-1.
Thus, c-Cbl reduction improves PD-1 expression
in CD8+ T cells and macrophages.

Similarly, the E3 ligase FBXO38 controls the T cell antitumor response
by mediating PD-1 degradation.
Mechanistically, FBXO38 is degraded
by labeling PD-1 by adding Lys48 polyubiquitin chains at Lys233.
Mouse models of melanoma and colorectal cancer lacking FBXO38 had a higher tumor burden, which was associated with
high PD-1 expression in tumor-invasive T cells.

PD-L1

The tumor level of PD-L1 is an important determinant of
tumor immunity.
Several studies have determined that cancer cells use EGFR signaling to stabilize PD-L1 expression to evade T cell immunity
.
E3 ligase recognizes and ubiquitin-labeled PD-L1 for proteolysis, relying mainly on glycogen synthase kinase 3 (GSK3α/β).

GSK3β phosphorylates the C-terminal domain of PD-L1 to recruit E3 ligase β-TrCP for PD-L1 ubiquitin-dependent degradation
.
EGFR signaling counteracts the GSK3β/β-TrCP/PD-L1 degradation pathway
by inducing massive glycosylation of PD-L1 near the GSK3β phosphorylation site.

Another study further showed that GSK3α/ARIH1 is another kinase/E3 ligase pair
that induces PD-L1 proteolysis.
The E3 ligase ARIH1 recognizes GSK3α-dependent phosphorylation of PD-L1 at S279/S283, followed by ubiquitination and degradation
of PD-L1 Lys48.
RECENTLY, EGFR HAS ALSO BEEN FOUND TO INCREASE PD-L1 LEVELS BY ADDITIONALLY INTERFERING WITH MEMBRANE BINDING TO THE MARCH8 E3 LIGASE, WHICH ALSO TARGETS PD-L1 FOR DEGRADATION
.
Finally, c-Cbl overexpression, alone or in combination with Cbl-b, can also reduce STAT3/AKT/ERK phosphorylation, thereby downregulating PD-L1 expression and increasing anti-tumor response
.

Cullin3 SPOP and TRIM21E3 ligase further disrupt the stability of PD-L1 by ubiquitination-dependent degradation, and their action is again dependent on kinases, specifically the cyclin-dependent kinases CDK4 and CDK5
.
However, CDK4 assists PD-L1 degradation by stabilizing Cullin3 SPOP, while CDK5 negatively interferes with the effect of
TRIM21 on PD-L1.

In addition, PD-L1 is also stable by several other deubiquitinase enzymes, namely OTUB1, USP9X, and USP7
.
These studies have potential therapeutic implications because they demonstrate that inhibiting or clearing any of these DUBs can re-energize the anti-tumor response in mice and make cancer cells sensitive
to T cell killing.

Development of oncology drugs for ubiquitin-dependent pathways

Until now, the targeting of ubiquitin-dependent pathways has been largely based on small molecule inhibitors that can block the catalytic domain of ubiquitin-associated enzymes or prevent them from binding to substrates or other regulatory proteins through direct binding
or allostericity.
Currently, more than 200 compounds have been developed, many of which are undergoing preclinical and clinical trials
for cancer treatment.

Most ubiquitin-based small molecule compounds are designed to target the essential ubiquitin pathway
in cancer cells.
With the advent of T-cell-based tumor immunotherapy, targeted inhibition of selective E3 and DUB has been studied, among which the key intracellular checkpoint T cell inhibitor Cbl-b has great potential
.
Studies have successfully downregulated Cbl-b levels in T cells using RNA interference, which has produced excellent in vitro and preclinical results in different adoptive T cell metastases and is well tolerated by cancer patients and is currently in clinical trials (NCT02166255 and NCT03087591).

In addition, preclinical trials have been conducted against small molecules of IAPs, MDM2, and USP7 and demonstrated at least partial enhancement of in vivo anti-tumor immunity
when used alone or in combination.

Ubiquitin-dependent pathways that regulate PD-L1 degradation in cancer cells are also being tested
.
For example, a competitive palmitoylation inhibitor (CPP-S1) to prevent palmitoylation of PD-L1, which in turn promotes PD-L1 ubiquitin-mediated degradation
.
The EGFR signaling pathway that controls the ubiquitination and degradation of PD-L1 is also effectively blocked in vivo in different ways, including EGFR small molecule inhibitors, copper chelators and small molecules targeting CSN5, all of which significantly enhance the anti-tumor response
of T cells.

Proximity-based therapeutics, including PROTAC or molecular glue techniques, are also rapidly evolving to target proteasome degradation of several specific tumor proteins, and more than a dozen clinical trials are currently validating the therapeutic potential
of these ubiquitination-based technologies.

brief summary

The data suggest that modulating several ubiquitin-dependent pathways, particularly the ubiquitin-dependent enzymes involved, can unleash intense, long-lasting, and targeted anti-tumor responses
.
Tremendous progress has also been made in the field of ubiquitin, with the development of a variety of new technologies and methods that can specifically target almost all ubiquitin-dependent enzymes or use them to target substrate proteins of interest, including tumor proteins
.
Looking ahead, it is believed that regulation of these important ubiquitin-dependent pathways will soon become a viable alternative to
improving targeted tumor immunotherapy.

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