“Oh, Dear We Are in Tribble”: An Overview of the Oncogenic Functions of Tribbles 1

Simple Summary Pseudokinases have evolved from conventional kinases and differ from them in their inability to phosphorylate their substrates. The Tribbles pseudokinases have evolved from the CAMK family of kinases and include three members: Tribbles 1, 2, and 3. Tribbles 1, which evolved later in metazoan lineage, appears to have a regulatory function. It interacts with a variety of substrates through different domains embedded in its molecular structure. Each interaction regulates cellular processes involved in cell division, survival, and metabolism. These processes also extrapolate to cancer development and other diseases. Based on its involvement in disorders and therapy resistance, it can be considered a potential candidate for drug development. The improved knowledge about its function can be utilized to design small-molecule inhibitors against TRIB1. Abstract Pseudokinases are catalytically inactive proteins in the human genome that lack the ability to transfer phosphate from ATP to their substrates. The Tribbles family of pseudokinases contains three members: Tribbles 1, 2, and 3. Tribbles 1 has recently gained importance because of its involvement in various diseases, including cancer. It acts as a scaffolding protein that brings about the degradation of its substrate proteins, such as C/EBPα/β, MLXIPL, and RAR/RXRα, among others, via the ubiquitin proteasome system. It also serves as an adapter protein, which sequesters different protein molecules and activates their downstream signaling, leading to processes, such as cell survival, cell proliferation, and lipid metabolism. It has been implicated in cancers such as AML, prostate cancer, breast cancer, CRC, HCC, and glioma, where it activates oncogenic signaling pathways such as PI3K-AKT and MAPK and inhibits the anti-tumor function of p53. TRIB1 also causes treatment resistance in cancers such as NSCLC, breast cancer, glioma, and promyelocytic leukemia. All these effects make TRIB1 a potential drug target. However, the lack of a catalytic domain renders TRIB1 “undruggable”, but knowledge about its structure, conformational changes during substrate binding, and substrate binding sites provides an opportunity to design small-molecule inhibitors against specific TRIB1 interactions.


Introduction
The human genome encodes a total of 581 protein kinases, of which 50 are catalytically inactive or weak and termed pseudokinases because they lack at least one residue in their conserved catalytic site [1].Some pseudokinases retaining weak kinase activity include ErbB3/HER3, in which a couple of amino acids in its catalytic domain are replaced by other amino acids (Glu740> His and Asp815> Asn), but it is still able to bind ATP and catalyze phosphoryl transfer with lower efficiency [2].Pseudokinases are known to perform a variety of physiological functions, such as allosteric regulation of other enzymes (e.g., VRK3 [3]), scaffolding of signaling components (e.g., Tribbles family [4]), and acting as molecular switches (e.g., MLKL [5]).Many pseudokinases are implicated in several diseases, such as cancer, metabolic disorders, and autoimmune diseases, among others [6].One such pseudokinase is mammalian tribbles homolog 1 (TRIB1), a member of the Tribbles family of pseudokinases, which has emerged to play a role in several diseases, including cancer, and is the focus of this review.

Structure and Function of TRIB1
TRIB1 is encoded by the TRIB1 gene on chromosome 8 (8q24.13locus) in the human genome.It is also known as C8FW, TRB1, GIG2, and SKIP1.TRIB1 was first identified in 1997 by Wilkin and colleagues (human clone C8FW), and in spite of a ~30% sequence identity with the protein kinase family, they did not observe an ATP binding site in their isolated clone [7].The half-life of TRIB1 mRNA is less than 1 h [8], and protein turnaround is about 2 h [9].The TRIB1 protein has two isoforms, isoforms 1 and 2. The isoform 1 encoded by the longer transcript is the full-length protein (41 kDa), whereas the translation of isoform 2 is initiated at a downstream start codon, and the encoded protein contains a shorter N-terminus (23.48 kDa) [10].TRIB1 is highly expressed in the bone marrow, liver, thyroid gland, urinary bladder, adipose tissue, gall bladder, and parathyroid gland [11].TRIB1 protein is found in both the cytoplasmic [12] and nuclear [13] compartments of the cell.Several transcription factor binding sites are predicted to be present in the TRIB1 gene promoter 5 ′ UTR region, including E2F, C/EBPβ, and NF-1 [4].Similarly, many miRNAs also putatively bind to the 3 ′ UTR of TRIB1 mRNA and regulate its gene expression and function [14].ATF3 regulates TRIB1 gene expression in response to proteasome inhibition in hepatocyte cell lines [15].

Evolution and Structure of TRIB1
TRIB1 belongs to the Tribbles family of pseudokinases, comprised of TRIB1, TRIB2, TRIB3, and a distantly related member, STK40.The name Tribbles comes from their homology with the domain structure of the Drosophila tribbles protein [16].In 2000, TRIB1 was first described to have a function during Drosophila gastrulation in the modulation of String/Cdc25 proteins in mesoderm cells [16][17][18].TRIB1 orthologs are mainly encountered in later metazoan lineages (the vertebrates), as opposed to TRIB2, which is also observed in the oldest metazoans [4].This is suggestive of the fact that TRIB1 evolved later in evolution, possibly to accommodate complex cell signaling processes in higher organisms.
The structure of TRIB1 is composed of three domains: The N-terminal domain (AA 1-90), the central pseudokinase domain (AA 91-330), and the C-terminal domain (AA 331-373) [19,20].The N-terminal domain of TRIB1 contains a putative nuclear localization signal spanning residues 33-51, which is shown to be required for nuclear localization [9].The N-terminal domain is also rich in proline (P), glutamate (E), serine (S), and threonine (T) residues (AA 53-88), which are characteristic of PEST domains; however, it is also shown to be unassociated with TRIB1 instability [15].The pseudokinase domains of TRIB1, 2, and 3 share sequence similarity with the canonical kinase domain of CAMK (calcium/calmodulindependent protein kinase) [21].Furthermore, at the amino acid level, TRIB1 shares 71% sequence similarity with TRIB2 and 54% sequence similarity with TRIB3 in its pseudokinase domain region.The structural features of the pseudokinase domain that distinguish it from a canonical kinase include a bent αC helix and divergent glycine-rich loop, both of which are required for ATP binding in a bona fide protein kinase.The Asp-Phe-Gly (DFG) motif is replaced by the Ser-Leu-Glu (SLE) sequence that occludes the nucleotide binding pocket.All of these structural/sequence changes represent a deformed N-terminal lobe and prevent the binding of ATP to TRIB1, thus lacking or inhibiting any phosphoryl transfer, which in turn classifies it as a pseudokinase [22].The pseudokinase domain also allows for binding of interacting partners and substrates, with C/EBPα/β (AA 168-293 [23]) being its most characterized substrate so far.The C-terminal domain of TRIB1 houses two important protein-protein interaction motifs: ILLHPWF (AA 332-339 [12]) that binds MEK1/2, and DQIVPE (AA 355-360 [24]) for binding COP1 E3 ubiquitin ligase.It has been shown by Murphy et al. that there is an intramolecular interaction between the C-terminal tail of TRIB1 and its pseudokinase domain, which in turn increases the stability of the TRIB1 protein [22].This interaction is also observed in the CAMK group of protein kinases, where the C-terminal regulatory region makes multiple inhibitory interactions with its catalytic core to play an autoinhibitory role [25].Both TRIB2 and TRIB3 also contain a MEK1/2 binding site, I(L/D)LHPW(F/L) (AA 30(2/9)-3(07/15)), and a COP1 binding site (D/A)Q(L/V)VPD (AA 3(26/33)-3(31/37)) on their C-termini [26].Overall, TRIB2 (343 amino acids) and TRIB3 (358 amino acids) are smaller proteins than TRIB1, with a shorter N-terminal domain as revealed by amino acid alignment.

Functions of TRIB1
TRIB1 has been recognized as an adapter protein that provides a scaffold for the degradation of its substrates (summarized in Table 1).The most characterized substrate of TRIB1 is C/EBPα, a lineage-specific transcription factor that undergoes degradation via COP1 E3 ubiquitin ligase [22].Binding of C/EBPα to the TRIB1 pseudokinase domain initiates a conformational change in the SLE motif to release the COP1 binding motif on the C-terminal domain, in turn relieving the autoinhibition.This substrate-bound conformation of TRIB1 is known as the "SLE-in" conformation and corresponds to the active protein kinase conformation [23].COP1 then binds to the C-terminus through its WD40 domain [24] and causes ubiquitination of C/EBPα at its lysine residues, ultimately causing its degradation through the ubiquitin proteasome system [27].After the degradation of C/EBPα, the C-terminal domain folds itself back onto the pseudokinase domain, and the autoinhibitory conformation is restored.This conformation is known as "SLE-out" and is similar to an inactive protein kinase [28].The tribbles protein also controls the protein levels of slbo, the Drosophila homolog of the C/EBP family of transcription factors, via degradation and inhibits border cell migration during oogenesis [29].Furthermore, because the C/EBP family of transcription factors determines the switch between granulopoiesis and monopoiesis [30], degradation of C/EBPα by TRIB1 regulates myeloid cell differentiation [31].TRIB1 has also been shown to negatively regulate retinoic acid receptor (RAR) signaling by forming a complex with it and its binding partner RXR (retinoid X receptor) [32].
The other primary function of TRIB1 is the sequestration of signaling molecules and subsequent activation of the downstream pathways (summarized in Table 1).As mentioned above, the pseudokinase and the C-terminal domains of TRIB1 contain sites that bind to Akt and MEK1/2, respectively.Drosophila tribbles have been shown to interact with Akt1 through its kinase-like domain and inhibit its activation [33].Another report shows that TRIB1 binds to Akt, possibly at its pseudokinase domain, and causes its activation [34].TRIB3 has also been shown to interact with Akt1 through its pseudokinase domain [35].The mediation of Akt activation and its downstream signaling by TRIB1 suggests that TRIB1 is a modulator of the PI3K-Akt pathway [36].Alternatively, TRIB1 also interacts with MEK1/2 through its C-terminal domain and regulates the activation of its downstream protein, ERK [37].TRIB1 also interacts with MKK4 through its pseudokinase domain and modulates vascular smooth muscle cell proliferation and chemotaxis by activating the downstream JNK pathway [38].TRIB1 also plays a role during M1 and M2 macrophage polarization of murine bone marrow-derived macrophages (BMDMs) by modulating the JAK/STAT pathway [39].TRIB1 has been shown to be important for the differentiation of tissue-resident M2-like macrophages (F4/80 + MR + ), which are involved in the maintenance of adipose tissues [31].

TRIB1 and Lipid Metabolism
TRIB1 is known to play an important role in lipid metabolism.It was first identified to be associated with triglycerides, LDL, and HDL cholesterol in a genome-wide association study (GWAS) [50].TRIB1 knockout mice exhibit increased triglyceride and plasma cholesterol levels, which are accompanied by downregulation of lipogenic genes such as acetyl-CoA carboxylase and fatty acid synthase, among others [51].Liver-specific deletion of TRIB1 has revealed that elevated levels of C/EBPα increase the transcription of fatty acid synthesis genes and result in pathologic phenotypes, suggesting a role of TRIB1 in keeping the transcription of lipogenic genes in check via degradation of C/EBPα [52,53].However, on the contrary, a recent report suggests that adipocyte specific knockdown of TRIB1 results in reduced plasma triglyceride and cholesterol levels along with increased adiponectin secretion, which are associated with improved metabolic health in humans [54].TRIB1 also interacts with the hepatic lipogenic master regulator MLXIPL (MLX interacting protein like), also known as ChREBP, and causes its degradation, leading to transcriptional inhibition of genes involved in liponeogenesis [40].The physical interaction of TRIB1 with HNF4A has also been reported, which plays a role in liver and intestinal gene expression as well as lipid metabolism [41].TRIB1 also acts as a scaffold for the SAP18 (Sin3A-associated protein) and mSin3A interactions, which modulate the expression of MTTP (microsomal TG transfer protein) in the mouse liver, ultimately impacting lipid metabolism [42].TRIB1 also has the capability to suppress liver regeneration by modulating redox homeostasis in hepatocytes via blockade of Nrf2 nuclear translocation [43].TRIB1 genetic loci are also associated with liver function in terms of certain enzymes' secretion, such as alanine transaminase (ALT), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT), which are commonly used as markers of certain liver ailments, suggesting it to be a genetic trait that might be inherited among humans [55].

TRIB1 and Innate Immunity
TRIB1 regulates NF-кB through various mechanisms, namely, regulation of p100 expression and inhibition of IKKα via phosphorylation by Akt on T23, among others.Regulation of NF-кB by TRIB1 is supported by TRIB1 knockdown studies, which resulted in downregulation of NF-кB-transcribed genes such as CCND1 (cyclin D1) and IL-8 (interleukin 8), suggesting a role of TRIB1 in cell cycle transition and inflammation [36].Furthermore, the IL-8 promoter contains AP-1 and C/EBPα binding sites, both of which have been shown to be regulated by TRIB1 [19].TRIB1 is also shown to interact with the RelA subunit of NF-кB in white adipocytes and acts as its transcriptional co-activator to regulate client cytokine gene expression [44].Interaction of TRIB1 with FoxP3, the master regulator of regulatory T cells (Tregs), is reported in human adherent cell lines and Tregs [45,56].TRIB1 also increases IL-2 production in activated T cells through NFAT (nuclear factor of activated T cells) [57].TRIB1-mediated downregulation of C/EBPβ results in altered TLR-mediated signaling in macrophages, especially in the context of C/EBPβ-induced gene expression [58].Knockdown of TRIB1 in macrophages inhibits their migration and increases production of TNFα [59].TRIB1 is identified as a potential biomarker for chronic antibody-mediated allograft rejection due to its increased mRNA levels present in peripheral blood mononuclear cells of patients with deteriorating renal graft function [60].Taken together, these reports suggest that TRIB1 may have a function in innate immunity as well.The functional overview of TRIB1 is outlined in Figure 1. and regulate TCR (T cell receptor) signaling.The pseudokinase domain is the longest domain and acts as a scaffold and sequester a variety of proteins.It is known to bind NF-кB, Foxp3, HDAC1, HNF4A, Akt, and MKK4 and influence processes such as inflammation, cell cycle regulation, p53 deacetylation, lipid metabolism, Treg regulation, cell survival, and proliferation.The pseudokinase domain also binds substrates for proteasomal degradation such as C/EBPα/β, MLXIPL (ChREBP), FERMT2, RAR/RXRα, and CD72.Degradation of these substrates affects processes such as myeloid cell differentiation, lipid metabolism, adipocyte maintenance, transcriptional downregulation, and autoimmunity.The pseudokinase domain also contains an SLE sequence as a substitute for the DFG motif that is responsible for binding ATP in a canonical kinase.The C-terminal domain binds MEK1/2 at ILLHPW sequence and activates downstream MAPK signaling to induce cell proliferation.COP1 E3 ligase also binds at the DQIVPE sequence in the C-terminal domain to result in degradation of TRIB1 substrates through the ubiquitin proteasome system.CUL4A/B is another ubiquitin ligase that is shown to bind TRIB1.Nrf2 also binds to TRIB1 through the C-terminal domain, and this interaction has been implicated in liver regeneration.

Role of TRIB1 in Disease
Based on its various functions described above, TRIB1 has been implicated in numerous diseases and malignancies.Its close proximity to MYC and its involvement in fundamental pathways such as MAPK signaling, NF-кB signaling, and the PI3K-Akt pathway have led to the identification of TRIB1 as an oncogene that plays a pivotal role in tumor progression and maintenance.Furthermore, its involvement in lipid metabolism and innate immunity causes other disease modalities such as atherosclerosis and inflammation.

Acute Myeloid Leukemia (AML)
It was observed in an AML patient that TRIB1 was overexpressed along with MYC, both of which reside on the same chromosomal region (8q24) 2.25 Mb apart [61].AML is characterized by the accumulation of hematopoietic progenitor cells in the bloodstream due to genetic alterations affecting their differentiation [62].As mentioned earlier, C/EBPα is the lineage-specific transcription factor that is targeted for degradation by TRIB1 [22,30].It was later discovered that overexpressed TRIB1 drives Hoxa9-induced leukemogenesis by decreasing C/EBPα protein levels, thereby modulating the enhancer programs at the Erg and Spns2 loci [63].Hoxa9 and Meis1 are normally expressed in early progenitor cells and decrease in expression upon their terminal differentiation [64].In the context of AML, Hoxa9 and Meis1 are overexpressed in primary bone marrow cells and cause leukemogenesis [65][66][67].Hoxa9 is a regulator of the primitive hemopoietic compartment, and Meis1 is a transcription factor critical for leukemia stem cell regulation [68].Specific interaction between TRIB1 and the coactivation of Hoxa9/Meis1 is reported in AML [65].TRIB1-mediated activation of the MAPK pathway also plays a role in AML by enhancing the self-renewal of malignant bone marrow cells [12].

Prostate Cancer
TRIB1 and cMYC are observed to be co-amplified in prostate cancer as well.TRIB1 mRNA levels are also similarly elevated in the prostate of PTEN null mice, exhibiting increased proliferation potential [69].TRIB1 overexpression causes increased sphere formation in prostate cancer cell lines and increased tumor formation in a xenograft mouse model [70].TRIB1 influences the prostate cancer tumor microenvironment by causing CD136+ macrophage infiltration, promoting M2 macrophage differentiation, and affecting cytokine secretion by inhibiting IкB-ζ (NF-кB inhibitor zeta) [71].

Breast Cancer
TRIB1 and cMYC co-amplification is further observed in breast cancer patients [46] and associated with reduced overall and disease-free survival [46].TRIB1 regulates both the G1/S [36] and G2/M transition [46] in breast cancer cells.A recent study utilizing the qPLEX-RIME technique [72] has revealed that TRIB1 interacts with the β-catenin co-factor FERMT2 (FERM Domain Containing Kindlin 2) and ER co-factor ZBTB7A (Zinc Finger and BTB Domain Containing 7A) in breast cancer cells, giving further insights into the TRIB1 interactome and its involvement in transcriptional regulation through β-catenin and ER [46].TRIB1 reduces DR5 protein levels in breast cancer cells through its elevated NF-кB signaling, thus decreasing TRAIL-induced apoptosis [36].Heightened expression of TRIB1 in tumor associated macrophages (TAMs) influences the breast cancer tumor microenvironment by regulating oncogenic cytokine expression [73].

Colorectal Cancer (CRC)
TRIB1 has been shown to be amplified in both CRC cell lines and patients [74].TRIB1 and MYC are co-amplified in CRC cells.TRIB1 protein expression significantly correlates with the expression of proteins such as p-ERK, p-MEK, Akt, PTEN, cleaved caspase 3, and MYC in CRC cells [75].TRIB1 overexpression correlates with poor overall survival in CRC patients.TRIB1 promotes migration and invasion of CRC cells through the activation of FAK/Src and ERK pathways, resulting in an upregulation of MMP-2 expression [76].

Other Cancers
The TRIB1 gene is also associated with pancreatic cancer in a Chinese Han population [77].TRIB1 is observed to be highly expressed in bone marrow mononuclear cells of multiple myeloma (MM) patients with progressive disease compared to those in remission.Such patients have an increased percentage of M2 macrophages, suggesting that TRIB1 plays a role in M2 macrophage polarization in MM [78].TRIB1 plays a role in hepatocellular carcinoma (HCC) as well, as evidenced by its upregulation in HCC cell lines and tumor tissues.TRIB1 promotes cell proliferation, migration, invasion, and epithelial-tomesenchymal transition (EMT) in HCC cell lines.TRIB1 upregulation is accompanied by p53 downregulation and increased β-catenin signaling [79], as observed in breast cancer cells.A previous report suggests that TRIB1 can downregulate p53's transcriptional activity by forming a complex with HDAC1, leading to HDAC1 catalyzed p53 deacetylation, thereby reducing its DNA binding activity [47].Moreover, it has been shown in glioma and primary GBM cells that TRIB1 reduces p53 levels through a similar mechanism [34,80].Increased TRIB1 mRNA levels have been correlated with poor overall survival of glioma patients [34].This mechanism suggests that TRIB1 could play a central role in tumor initiation and maintenance by modulating the tumor suppressor activities of p53.

TRIB1 and Other Diseases
In addition to cancer, TRIB1 has been implicated in metabolic and lipid disorders, according to several GWAS.TRIB1 variants are directly associated with a reduced risk of myocardial infarction in humans [51].Similarly, a previous review has discussed that TRIB1 is linked to the etiology of Crohn's disease, non-alcoholic fatty liver disease, dyslipidemia, and cardiovascular disease [81].Increased TRIB1 gene expression is encountered in the coronary arteries of patients with ischemic heart disease [38].Genetic deletion of TRIB1 promotes atherosclerosis in mice, which is accompanied by pronounced hyperlipidemia and hepatic and systemic inflammation [82].However, lack of TRIB1 in myeloid cells decreases early atheroma formation and reduces atherosclerosis burden in mouse models [83].A recent meta-analysis reveals that the TRIB1 SNP (Rs2954029) A allele present in the 8q24 locus is positively associated with the risk of coronary artery disease (CAD) combined with stroke [84].Several studies have also explored the association between TRIB1 and non-alcoholic liver disease and hepatic steatosis in terms of SNPs (rs6982502, rs2954021, rs17321515, rs2954029) in different populations [40, [85][86][87].Transcriptomics studies in similar mouse models reveal genes enriched in VLDL assembly, TG biosynthesis, gluconeogenesis, and glycogen synthesis pathways are upregulated after TRIB1 overexpression [40, [85][86][87].In order to exploit TRIB1's ability to modulate lipid metabolism, Nagiec et al. identified a small molecule BRD0418, a benzofuran compound that increases TRIB1 expression in HepG2 cells, leading to reduced VLDL production, cholesterol biosynthesis, and increased LDL uptake [88].The cytokine oncostatin M, which is shown to upregulate LDLR gene transcription in HepG2 cells [89], also acts by increasing TRIB1 gene expression [88].Another tricyclic glycal compound, BRD8518, was later identified to have higher potency than BRD0418 in inducing TRIB1 and LDLR gene expression.This compound affects the MAPK pathway but is not suitable for in vivo testing because of its ADME profile [90].TRIB1 is also suggested to influence obesity by regulating brown adipose tissue mitochondrial function via respiratory chain complex III [91].Of note, TRIB1 is shown to negatively regulate B cells in systemic lupus erythematosus via its interaction with CD72, an effector of autoimmunity [48].

Role of TRIB1 in Cancer Therapy Resistance
Therapy resistance has always been considered the biggest roadblock in cancer treatment and relapse.Based on the above discussion, it is clear that TRIB1 plays an important role in the malignant transformation of various tumors and modulates several mechanisms for tumor sustenance.Several reports have shown that its involvement in various pathways, such as PI3K-AKT, MAPK, p53-HDAC1, and NF-кB, among others, suggests TRIB1 plays a role in cancer therapy resistance.TRIB1 was identified as an exclusive downstream effector of mutant PIK3CA in genetically modified lung epithelial cells [92].A later report showed that downregulation of p53 by the TRIB1-HDAC1 interaction induces resistance to cisplatin chemotherapy and cancer stem cell enrichment in non-small cell lung carcinoma (NSCLC) [93].Similarly, the TRIB1 gene was identified as part of a gene signature induced in breast cancer cell lines in response to long-term paclitaxel treatment, causing treatment resistance [94].TRIB1 overexpression causes resistance to ATRA (all-trans retinoic acid) treatment during acute promyelocytic leukemia in exclusively sensitive myeloid cells expressing the PML/RARA fusion protein by preventing their differentiation through the downregulation of C/EBPα [95].We and others have shown that TRIB1 mRNA and protein levels are upregulated by radiation and temozolomide treatment in glioma cells, causing a decrease in treatment-induced cell death [34,80].In primary GBM cells, TRIB1 causes upregulation of the ERK and Akt pathways and modulation of p53 function through COP1 and HDAC1, further contributing to therapy resistance [34].TRIB1 negatively regulates the anti-tumor cytokine IL-15 in TAMs in breast tumors, causing a decrease in T-cells that promote anti-tumor responses [73].TRIB1 has been identified as a negative regulator of T-cell receptor (TCR) signaling via its interaction with MALT1, a TCR activator, leading to disruption of MALT1 signaling complexes responsible for optimal T cell activation and function [49].Deletion of TRIB1 from T cells has been reported to improve PD-L1 immune checkpoint blockade, suggesting a role for TRIB1 in immunotherapy resistance [96].A recent report has similarly shown that in a CRC mouse model, T cell recruitment to the tumor was improved after TRIB3 loss, thereby sensitizing the tumor to immune checkpoint blockade therapy [97].The roles of TRIB1 in cancer and therapy resistance are further discussed in Table 2.

Potential Strategies to Target TRIB1
In light of the above discussion, it is clear that TRIB1 is involved in the tumor maintenance and therapy resistance of various cancers.Therefore, inhibiting TRIB1 can be considered a potential strategy for targeted cancer therapy that would alter the function/activity of important proteins such as Akt and MAPK in normal cells.In general, TRIB1 and many other pseudokinases have been considered "undruggable" for a very long time because, unlike conventional kinases, they do not possess an ATP binding domain, which has so far been the most druggable site in these proteins.On the contrary, the pseudokinases that possess weak catalytic activity (such as TRIB2, the JAK family, and HER3) have been targeted by small molecules against their catalytic site and demonstrated inhibition of their pseudokinase activities.There are a variety of FDA-approved JAK inhibitors for different diseases that act not only by targeting the ATP binding domain but also through allosteric inhibition [103].Ruxolitinib was the first FDA-approved small-molecule inhibitor of JAK1/2 for the treatment of BCR-ABL1-negative myeloproliferative neoplasms and acts through competitive inhibition of the ATP-binding catalytic site on the kinase domain [104].
Given the variety of functions performed by TRIB1, several approaches can be utilized to disrupt its efficient operation.The most prominent function of TRIB1 is to act as a scaffold for the sequestration of signaling molecules, for which it adapts "SLE-in" (on) and "SLE-out" (off) conformations that allow for the substrates/effectors to bind.Smallmolecule compounds locking TRIB1 in the SLE-in conformation would allow for blocking any substrate from binding, and locking in the SLE-out conformation would maintain TRIB1 in its auto-inhibitory conformation.Jamieson et al. have tested several compounds for their potential to stabilize TRIB1 in either of these conformations and reported moderate success [23].Foulkes et al. reported that pre-existing EGFR inhibitors can target the lowaffinity ATP binding site on TRIB2 and cause either its stabilization or destabilization [105].Mutant K-Ras has been an "undruggable" target for years in lung cancer treatment, but the design of a covalent inhibitor that locks this protein in its "inactive conformation" has provided great promise in targeting this protein [106].The recent resolution of the TRIB1 crystal structure and deeper understanding of the mechanisms of TRIB1 action have revealed new substrates and sites that can be used to design small-molecule inhibitors that are not only effective but are also blood-brain-barrier permeable.
Another potential approach for targeting TRIB1 could be the induction of its degradation through PROTACs (proteolysis targeting chimeras) and HyT (hydrophobic tagging) to reduce its levels inside the cell.PROTACs are bifunctional molecules that cause the degradation of a protein(s) of interest by bringing them within close proximity of an E3 ligase [107].HyT, on the other hand, tags protein(s) of interest with hydrophobic fragments so that they are recognized as misfolded and damaged proteins and are therefore cleared through protein quality control mechanisms [108].The pseudokinase Her3 has been targeted using both approaches.PROTAC selectively degrades HER3 but does not abrogate its downstream signaling [109], whereas HyT induces partial HER3 degradation and blocks HER3-dependent signaling in cell line models [110].PROTAC 23 developed against IRAK3 pseudokinase exhibits degradation of around 98% IRAK3 protein in THP1 cells [111].MLKL is another pseudokinase that has been successfully targeted using PRO-TAC 36 in the TSZ model of necroptosis, demonstrating an inhibition of cell death [112].Other alternative approaches worth exploration include allosteric inhibition, antibodies, proteasome inhibitors, and irreversible inhibitors, among others.

Conclusions
TRIB1 plays a variety of roles in both health and disease.Although it may not phosphorylate any downstream substrates like a canonical kinase, it certainly acts as an intermediate for the sequestration of proteins to carry out important physiological functions such as differentiation, lipid metabolism, and innate immune function, among others.As was identified by Sanchez-Vega et al., there are at least ten cell signaling pathways that are found to be altered in different cancers [113].Of these pathways, TRIB1 has been shown to play a role in the RTK/RAS, PI3K, Myc, p53, and cell cycle pathways in different tumor types, suggesting the importance of this pseudokinase in oncogenic signaling.TRIB1 and pseudokinases in general have only recently gained limelight in the cancer field for providing a new insight into tumorigenic processes.The implication of TRIB1 in malignant transformation also provides an opportunity for drug development for effective therapies.However, the lack of a catalytic domain on this protein may pose some hurdles, but its alternating "active" and "inactive" conformations hold potential as an attractive vulnerability to design effective drugs that would block its scaffolding function and disrupt downstream oncogenic signaling.PROTACs are an up-and-coming approach that have shown success in hitting undruggable targets and therefore warrants further exploration with regards to TRIB1.In conclusion, targeting TRIB1 presents an opportunity for cancer treatment as well as other metabolic disorders in which TRIB1 has been shown to play a role.

Figure 1 .
Figure 1.Overview of TRIB1 structure and functions.TRIB1 has three domains: An N-terminal domain, a pseudokinase domain, and a C-terminal domain.All three domains bind to distinct substrates

Table 1 .
The interacting partners of TRIB1.

Table 2 .
TRIB1 in cancer and treatment resistance.