LKB1 biology: assessing the therapeutic relevancy of LKB1 inhibitors

Abstract

Liver Kinase B1 (LKB1), encoded by Serine-Threonine Kinase 11 (STK11), is a master kinase that regulates cell migration, polarity, proliferation, and metabolism through downstream adenosine monophosphate-activated protein kinase (AMPK) and AMPK-related kinase signalling. Since genetic screens identified STK11 mutations in Peutz-Jeghers Syndrome, STK11 mutants have been implicated in tumourigenesis labelling it as a tumour suppressor. In support of this, several compounds reduce tumour burden through upregulating LKB1 signalling, and LKB1-AMPK agonists are cytotoxic to tumour cells. However, in certain contexts, its role in cancer is paradoxical as LKB1 promotes tumour cell survival by mediating resistance against metabolic and oxidative stressors. LKB1 deficiency has also enhanced the selectivity and cytotoxicity of several cancer therapies. Taken together, there is a need to develop LKB1-specific pharmacological compounds, but prior to developing LKB1 inhibitors, further work is needed to understand LKB1 activity and regulation. However, investigating LKB1 activity is strenuous as cell/tissue type, mutations to the LKB1 signalling pathway, STE-20-related kinase adaptor protein (STRAD) binding, Mouse protein 25-STRAD binding, splicing variants, nucleocytoplasmic shuttling, post-translational modifications, and kinase conformation impact the functional status of LKB1. For these reasons, guidelines to standardize experimental strategies to study LKB1 activity, associate proteins, spliced isoforms, post-translational modifications, and regulation are of upmost importance to the development of LKB1-specific therapies. Therefore, to assess the therapeutic relevancy of LKB1 inhibitors, this review summarizes the importance of LKB1 in cell physiology, highlights contributors to LKB1 activation, and outlines the benefits and risks associated with targeting LKB1.

Keywords: Adenosine Monophosphate-Activated Protein Kinase (AMPK); Adenosine Monophosphate-Activated Protein Kinase-related Kinases (ARKs); Autophosphorylation; Drug Discovery; LKB1 Inhibitors; Liver Kinase B1 (LKB1); Mouse Protein 25 (MO25); Post-translational Modifications; STE-20-related Kinase Adaptor Protein (STRAD); Tumour Suppressor.

Fig. 1

The effect of STRAD and MO25 on LKB1 nucleocytoplasmic shuttling and function Catalytically active LKB1 resides in the cytoplasm but is stored in the nucleus where it has little activity. The nuclear localization signal within LKB1 binds to importinɑ/β, which allows LKB1 to cross nuclear import receptors. STRADɑ/β binds to the kinase domain increasing the cytoplasmic retention of LKB1. STRADβ prevents binding to importin ɑ/β in the cytoplasm whereas STRADɑ binds to nuclear export receptors, such as exportin7 and CRM1, which allows LKB1 to cross nuclear export receptors. LKB1 activity is greatest when mouse protein 25 (MO25) binds STRAD and STRAD is bound to LKB1. LKB1 SL26 mutants have little enzymatic activity because they are unable to bind STRAD leading to the nuclear retention of LKB1 SL26

Fig. 2

Pathways targeted by the LKB1-STRAD-MO25 complex LKB1-STRAD-MO25 phosphorylate AMPK and AMPK-related kinases (SIKs, BRSKs, MARKs, SNRK, and NUAKs). Both Ca2+/calmodulin-dependent kinase kinase-β (CAMKK2) and LKB1-STRAD-MO25 phosphorylate AMPKɑ on T172. Once phosphorylated AMPKɑ forms an active heterotrimeric complex with AMPKβ and AMPKγ. The active AMPK complex regulates cell polarity, migration, and activates catabolic processes. The AMPK complex is also activated by increasing the AMP:ATP ratio. The LKB1-MARK pathway activates Hippo kinases to impede proliferation and angiogenesis, inhibits Snail to block migration, and regulates polarity and microtubule dynamics by phosphorylating Tau or microtubule-associated proteins (MAPs). The LKB1-BRSK pathway regulates neuronal development and polarity through Tau phosphorylation. The LKB1-SIK pathway antagonizes cyclic AMP response element-binding protein (CREB) and Tax transcription while promoting dendritic cell avoidance. The LKB1-NUAK pathway regulates cell adhesion/detachment and axon branching. NUAK also antagonizes NF-κB activity promoting apoptosis while inhibiting angiogenesis and proliferation. The LKB1-SNRK pathway antagonizes proliferation by inhibiting β-catenin activity and promotes skeletal muscle contraction, glucose transport, and cardiac development

Fig. 3

Regulating the functional status of LKB1 LKB1 activity is regulated through protein-protein interactions, lipid binding, and post-translational modifications. Heat-shock protein (HSP) 90 and cell division cycle 37 (CDC37) increase LKB1 activity (measured by AMPK activation) via stabilizing LKB1 and blocking its proteasome-dependent degradation. However, LKB1 activity is disrupted by 14-3-3 binding to phosphorylated T336, Fyn binding to PIPPSP motifs and phosphorylating tyrosine residues (Y), and Nur77 binding, which all promote LKB1 nuclear retention. Several post-translational modifications including ubiquitination, phosphorylation, farnesylation, acetylation, sumolation, S-Nitrosylation, and 4-hydroxy-trans-2-nonenal (HNE) adducts may enhance or antagonize LKB1 activity. Ubiquitination of K41, K44, K48, K62, and K63 are mediated by Skp2-SCF, FBXO22, and RNF146. There are several sites of autophosphorylation on LKB1 including T185, T336, T363, and T402. Upstream kinases including proviral integration site for MuLV (PIM), protein kinase B (Akt), cyclin-dependent kinases (CDKs), v-Raf murine sarcoma viral oncogene homolog B1 (B-RAF), Rsk, extracellular signal-regulated kinase (ERK), Aurora A, and Fyn inhibit LKB1 through Y261, Y365, S299, S325, and S334 phosphorylation whereas protein kinase A (PKA), protein kinase C (PKC), Rsk, and ataxia telangiectasia mutated (ATM)-dependent phosphorylation at T363, S307, S399, and S428 enhance LKB1 activity. Farnesylation at C430 increases LKB1 activity, promotes LKB1-membrane association, and is key for LKB1 regulating migration and polarity. Acetylation of K44, K48, K64, K96, K97, K296, K311, K416, K423, and K431 is regulated through a balance of activity by N-ɑ-acetyltransferase 20 (NAA20) acetylases and sirtuins (SIRTs) deacetylases. SIRT activity has been associated with HECT and RLD domain containing E3 ubiquitin ligase 2 (HERC2) ubiquitinating LKB1 prior to its degradation via proteasomes. Alternatively, SIRTs may enhance LKB1 activity by increasing its affinity for STRAD. Sumolaytion of K96, K178, and K235 is mediated by SUMO1 and SUMO2. SUMO1 enhances LKB1-dependent AMPK phosphorylation whereas SUMO2 disrupts STRAD binding, which increases LKB1 nuclear retention. S-Nitrosylation of C430 dampens LKB1 activity by promoting its degradation via proteasomes. Another antagonist of LKB1 activity is the formation of HNE adducts on K97