Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

Abstract

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

Combined elevation of TRIB2 and MAP3K1 indicates poor prognosis and chemoresistance to temozolomide in glioblastoma

“…Abnormally elevated TRIB2 was previously identified as the clinically relevant factor in various subtypes of human cancers . As described previously, TRIB2 promotes tumorigenesis and induces therapeutic resistance by activating Wnt‐mediated downstream activation of Hippo signaling and the YAP pathway through transcriptional control of gene expression and TRIB2‐mediated posttranslational regulation of protein stability in liver cancer cells .…”

“…Abnormally elevated TRIB2 was previously identified as the clinically relevant factor in various subtypes of human cancers. [5][6][7][8] As MAP3K1 is a 196-kDa serine/threonine kinase that is activated by various stimuli and cell stresses, including growth factors, cytokines, and microtubule disruption. 35 Our present study showed elevated MAP3K1 expression in glioma, which is conversely associated with a poor prognosis and therapeutic resistance in glioma.…”

“…5 Recent studies have indicated that TRIB2 functions as a crucial oncogene that regulates a wide range of cellular processes, including tumorigenesis, proliferation, invasion, and therapeutic resistance, in various subtypes of cancer, such as small cell lung cancer, liver cancers, colorectal cancer, and acute myeloid leukemia. [5][6][7][8] Foulkes et al 9 demonstrated that TRIB2 expression is significantly increased in acute myeloid leukemia and promotes tumor proliferation by inactivating CCAATenhancer-binding protein α (C/EBPα). Moreover, Wnt/TCF signaling-dependent activation of TRIB2 stimulates the proliferation of liver cancer cells through a stabilization effect on yes-associated protein (YAP).…”

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

“…That some of the active compound-cysteine interactions led to protein degradation without requiring a separate E3 ligase-directing ligand (e.g., EV-3-mediated degradation of BIRC2/3) underscores the potential for covalent modification by small molecules to directly affect protein stability in cells (Foulkes et al, 2018;Jones, 2018;Schreiber, 2019;Yang et al, 2019). We do not yet understand how EV-3 promotes BIRC2 and BIRC3 degradation, but this could involve disruption of protein-protein interactions proximal to the compound-modified cysteines in these proteins.…”

An evolutionary-conserved redox regulatory mechanism in human Ser/Thr protein kinases

“…When compared to WT Aurora A, incorporation of the C290A mutation had no effect on protein thermostability (T m ~40 °C, fig. S4B) measured by DSF , Foulkes et al, 2018 or ∆T m values induced by ATP or MLN8237 binding ( fig. S4C).…”