Kinase LKB1 Research Articles

Role of SIK1 in tumors: Emerging players and therapeutic potentials (Review).

Salt‑induced kinase 1 (SIK1) is a serine/threonine protein kinase that is a member of the AMP‑activated protein kinase family. SIK is catalytically activated through its phosphorylation by the upstream kinase LKB1. SIK1 has been reported to be associated with numerous types of cancer. The present review summarizes the structure, regulatory factors and inhibitors of SIK1, and also describes how SIK1 is a signal regulatory factor that fulfills connecting roles in various signal regulatory pathways. Furthermore, the anti‑inflammatory effects of SIK1 during the early stage of tumor occurrence and its different regulatory effects following tumor occurrence, are summarized, and through collating the tumor signal regulatory mechanisms in which SIK1 participates, it has been demonstrated that SIK1 acts as a necessary node in cancer signal transduction. In conclusion, SIK1 is discussed independent of the SIKs family, its research results and recent progress in oncology are summarized in detail with a focus on SIK1, and its potential as a therapeutic target is highlighted, underscoring the need for SIK1‑targeted regulatory strategies in future cancer therapy.

Impact of protein and small molecule interactions on kinase conformations.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Impact of protein and small molecule interactions on kinase conformations

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

Abstract 4450: MYC upregulates MCT4 expression and promotes metabolic reprogramming and enhanced lactate utilization in LKB1-deficient NSCLC

Abstract STK11, which encodes the serine/threonine kinase LKB1, is the second most commonly altered tumor suppressor in non-small cell lung cancer (NSCLC). LKB1 loss is frequently co-mutated with KRAS and associated with primary resistance to immunotherapy, which is partially due to MCT4-dependent lactate secretion. However, the mechanisms driving MCT4 elevation and metabolic reprogramming remains unclear. We hypothesized that increased MYC expression, which is associated with LKB1 loss, plays a pivotal role in metabolic alterations, promoting lactate incorporation and maintaining redox homeostasis. To explore the impact of lactate on tumor cells with LKB1 loss, we performed metabolic profiling on KRAS mutant (K) murine lung cancer cells with or without LKB1 knockout (KL) treated with either glucose or lactate as the carbon source. PLS analysis indicated that there was no notable difference between K and KL cells after glucose treatment. However, we observed significant changes in metabolites between K and KL cells when treated with lactate. Differentially altered metabolites included TCA-related components such as acetyl-CoA and glutamate, indicating enhanced lactate utilization in KL tumor cells. We also noticed that KL cells showed increased oxidative phosphorylation (OXPHOS) with the addition of lactate, with increased GSH/GSSH and NADPH/NADP+ ratios, suggesting that, as compared to LKB1-intact cells, LKB1-deficient cells had an enhanced ability to utilize OXPHOS and maintain redox homeostasis with lower levels of ROS generation when utilizing lactate as an energy source. However, in KL cells, MYC knockout (KO) or MCT4 KO partially abolished the lactate-induced OXPHOS and increased ROS. Moreover, [U-13C]lactate tracing revealed that isotopologues were significantly enriched in TCA components in KL cells, and this was reversed by either MYC KO or MCT4 KO, suggesting that MYC and MCT4 were critical for lactate reutilization in KL tumor cells. We observed that MYC could directly bind to the promoter region and transcriptionally activate MCT4 expression, and restoring MCT4 in MYC KO cells enhanced lactate incorporation and decreased ROS levels. Finally, we injected KL murine tumor cells with or without MYC/MCT4 KO into mice and infused [U-13C]lactate to detect the isotopologues distribution in vivo. KL tumors showed significantly enhanced lactate incorporation as compared to K tumors, while MYC KO or MCT4 KO reversed these phenotypes. Collectively, our data indicates that in LKB1-deficient tumors, upregulation of MYC promotes MCT4 expression and increases lactate incorporation and utilization, and that can be inhibited by targeting MYC or MCT4. These findings provide insight into the mechanisms driving the metabolic reprogramming and aggressive phenotype of KRAS-mutant LKB1-deficient tumors and identify a novel therapeutic strategy for this recalcitrant subgroup. Citation Format: Yu Qian, David Molkentine, Yifan Kong, Qian Huang, Chendong Yang, Alissa Poteete, Alvaro P. Guimaraes, Peixin Jiang, Ferdinandos Skoulidis, Alexandre Reuben, John D. Minna, Ralph J. DeBerardinis, John V. Heymach. MYC upregulates MCT4 expression and promotes metabolic reprogramming and enhanced lactate utilization in LKB1-deficient NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4450.

LKB1 regulates macrophage metabolism and functional polarization in immunomodulation

Abstract Macrophages play critical roles in maintaining tissue homeostasis and modulating immune responses. In response to microenvironmental cytokines, macrophages coordinate cellular signaling networks and diverse transcriptional programs to dictate their phenotypical and functional heterogeneity. For instance, LPS/IFNγ and IL-4 induce classically (M1) and alternatively (M2) activated macrophages, respectively. Remodeling cellular metabolism has been highlighted a key process underlying macrophage functional polarization. However, the precise mechanisms coordinating macrophage metabolism and polarization remain elusive. We report here that the kinase LKB1, a well-known negative regulator of mTOR signaling pathway, serves as a metabolic checkpoint that connects cellular metabolic reprogramming to functional polarization in macrophages. We found that genetic depletion of LKB1 in macrophages polarized their differentiation towards M2-like macrophages, characterized by enhanced expression of M2-associated markers, independently of mTOR and STAT6 signaling pathways. In contrast, loss of LKB1 had no substantial impact on M1-like macrophages. Moreover, LKB1-defienct macrophages orchestrated reprogramming of mitochondrial metabolism to facilitate M2 polarization and tumor immune evasion. Collectively, our studies establish a previously unappreciated role for LKB1 in connecting macrophage metabolism and functional polarization in modulating tumor immunity. This work was partially supported by the Herman B Wells Center and Riley Children’s Foundation.

Functions of LKB1 in neural crest development: The story unfolds.

Neural crest cells (NCCs) are highly motile, multipotent, embryonic cells that delaminate from the dorsal edges of the neural tube. NCCs follow stereotypical long-range migratory pathways to reach target organs during development, where they give rise to multiple derivatives. The identification of reservoirs of neural crest stem cells that persist to adulthood has recently aroused renewed interest in the biology of NCCs. In this context, several recent studies have demonstrated the essential role of the metabolic kinase LKB1 in NCC establishment. This review surveys how LKB1 governs the formation and maintenance of several neural crest derivatives, including facial bones, melanocytes, Schwann cells, and the enteric nervous system. We also detail the underlying molecular mechanisms that involve downstream effectors of LKB1, in particular the contribution of the AMPK-mTOR signaling pathway to both polarity and metabolic processes. Collectively, these recent discoveries open promising perspectives for new therapeutic applications for the treatment of neural crest disorders.

Phosphorylation of LKB1 by PDK1 Inhibits Cell Proliferation and Organ Growth by Decreased Activation of AMPK.

The master kinase LKB1 is a key regulator of se veral cellular processes, including cell proliferation, cell polarity and cellular metabolism. It phosphorylates and activates several downstream kinases, including AMP-dependent kinase, AMPK. Activation of AMPK by low energy supply and phosphorylation of LKB1 results in an inhibition of mTOR, thus decreasing energy-consuming processes, in particular translation and, thus, cell growth. LKB1 itself is a constitutively active kinase, which is regulated by posttranslational modifications and direct binding to phospholipids of the plasma membrane. Here, we report that LKB1 binds to Phosphoinositide-dependent kinase (PDK1) by a conserved binding motif. Furthermore, a PDK1-consensus motif is located within the kinase domain of LKB1 and LKB1 gets phosphorylated by PDK1 in vitro. In Drosophila, knockin of phosphorylation-deficient LKB1 results in normal survival of the flies, but an increased activation of LKB1, whereas a phospho-mimetic LKB1 variant displays decreased AMPK activation. As a functional consequence, cell growth as well as organism size is decreased in phosphorylation-deficient LKB1. Molecular dynamics simulations of PDK1-mediated LKB1 phosphorylation revealed changes in the ATP binding pocket, suggesting a conformational change upon phosphorylation, which in turn can alter LKB1's kinase activity. Thus, phosphorylation of LKB1 by PDK1 results in an inhibition of LKB1, decreased activation of AMPK and enhanced cell growth.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

Abstract 863: LKB1 inhibits NFkB to suppress cancer stem cells in triple negative breast cancer

Abstract Breast cancer is the most common malignancy diagnosed in US women and is the second leading global cause of death for women. Triple Negative Breast Cancer (TNBC) is a more aggressive subtype of breast cancer that is categorized by negative expression of estrogen receptors, progesterone receptors, and amplification of HER2/neu. Because of its receptor phenotype, traditional hormone therapy fails to produce significant short- or long-term benefits for patients. Additionally, chemotherapeutic and surgical interventions often result in relapse and development of a more aggressive disease with higher rates of growth, metastasis, and drug resistance. TNBC also tends to affect women younger than 40 as well as black women, making it a disease of disparities. The Liver Kinase B1 (LKB1/STK11) signaling pathway has been well characterized to have a role in defining cellular responses to energy homeostasis, metabolism, as well as tumor suppressive capabilities. It functions as a master regulator through a set of 14 known kinase targets to maintain its role in cell-type specific regulation. Peutz Jeghers Syndrome, a genetic disorder with a predisposition to developing malignancies, and many other solid tumor cancers are associated with a loss of expression, somatic mutation, or functional deficiencies of LKB1 activity. While studies have been done to understand the role of LKB1 signaling, its role and mechanism in the regulation of TNBC remains unknown. We previously demonstrated LKB1 expression to be associated with increased survival rates for patients diagnosed with TNBC. Therefore, we hypothesize that LKB1 activity in TNBC cell lines will inhibit cancer function. To test this, TNBC cell lines were transfected to overexpress LKB1 with vector transfections generated for controls. Initial molecular results demonstrate a reversal of EMT gene expression by qPCR in cells that overexpressed LKB1. Flow cytometry analysis of LKB1 overexpressing cells show a decrease in the GD2+ cancer stem cells (CSCs). Additionally, functional assays show a reduction growth and proliferation in 3D culture systems. Interestingly, there was no effect on proliferation in standard 2D culture conditions. GD2 was previously identified to be regulated by NFkB signaling. Because LKB1 overexpression resulted in the suppression of CSCs, we hypothesized that LKB1 expression suppresses NFkB signaling. To test this, we ran a western blot panel for NFkB antibodies. Interestingly, LKB1 OE cells had lower levels of IKKα, IKKß, and p65 compared to pcDNA controls. This suggests that LKB1 functions to inhibit NFkB signaling, resulting in the suppression of the CSC phenotype in TNBC. To summarize, LKB1 expression inhibits NFkB signaling, which leads to decreased CSCs. This results in reversal of EMT genes and diminished capabilities to form spheres. Future studies will be dedicated to identifying which of the 14 downstream kinases LKB1 activates to exert its functions. Citation Format: Khoa Nguyen, Madlin Alzoubi, Thomas Cheng, Katherine Hebert, Steven Elliott, Hassan Yousefi, Suresh Alahari, Matthew Burow, Bridgette Collins-Burow. LKB1 inhibits NFkB to suppress cancer stem cells in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 863.

Abstract 1036: Targeting bone morphogenetic protein 6 as a therapeutic target for LKB1-inactivated lung cancer

Abstract Lung cancer is among the most commonly diagnosed cancers and the leading cause of cancer-related death globally, with approximately 85% of all lung cancers having non-small cell lung cancer (NSCLC). The protein kinase LKB1 is the 3rd most frequently mutated gene in lung adenocarcinoma, and serves as a master regulator of cell metabolism and growth. LKB1-inactivating mutations have a significant impact on NSCLC development. Although KRAS mutation is most frequently co-mutated with the tumor suppressor p53, the strongest cooperation was seen with homozygous inactivation of LKB1. Loss of LKB1 on a KRAS-mutant (KL) background leads to increased tumor burden, shortened survival time and increased metastasis compared to loss of TP53 (KP). Furthermore, inactivation or loss of LKB1 correlated with poor prognosis, where tumors are typically aggressive and chemoresistant. To discover therapeutic vulnerabilities for this aggressive genetic subtype of lung adenocarcinoma, we evaluated the contribution of multiple single- and double-genetic alterations (KRAS G12D (K), LKB1 knockdown (L), and p53 knockdown (P)) to 3D invasion phenotypes using immortalized human bronchial epithelial cells (HBEC3-KT) and conducted RNA transcriptome profiling of the invading HBEC3-KT 3D spheroids. We found that bone morphogenetic protein 6 (BMP-6) is upregulated in invasive KL spheroids only compared to K and KP, suggesting that the loss of both KL drives increased BMP6 expression. Consistent with this finding, the iron regulator hepcidin, which is regulated by BMP6 is also upregulated in KL spheroids. Both BMP-6 and hepcidin expression can be repressed by restoring exogenous LKB1 in KL spheroids suggesting that LKB1 loss is at least in part responsible for the overexpression of the BMP6/hepcidin pathway. In addition, inhibition of BMP-6 signaling via BMP type I receptor ALK2 using small molecule inhibitor LDN214117 suppressed of cell invasion in 3D culture and decreased tumor growth in a KL syngeneic mouse model. Collectively, our findings show that upregulation of hepcidin via BMP6 signaling and followed by iron sequestration in tumor cells is part of the metastatic invasion strategy of LKB1 inactivated NSCLC. Therefore, current clinical strategies targeting BMP6 signaling could improve outcomes for LKB1-mutant lung cancer patients. Citation Format: Junghui Koo, Chang-Soo Seong, Bhakti Dwivedi, Carol Tucker-Burden, Adam I. Marcus, Melissa Gilbert-Ross. Targeting bone morphogenetic protein 6 as a therapeutic target for LKB1-inactivated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1036.

Caspase cleavage and nuclear retention of the energy sensor AMPK-α1 during apoptosis.

SummaryAMP-activated protein kinase (AMPK) coordinates energy homeostasis during metabolic and energy stress. We report that the catalytic subunit isoform AMPK-α1 (but not α2) is cleaved by caspase-3 at an early stage during induction of apoptosis. AMPK-α1 cleavage occurs following Asp529, generating an ∼58-kDa N-terminal fragment (cl-AMPK-α1) and leading to the precise excision of the nuclear export sequence (NES) from the C-terminal end. This cleavage does not affect (1) the stability of pre-formed heterotrimeric complexes, (2) the ability of cl-AMPK-α1 to become phosphorylated and activated by the upstream kinases LKB1 or CaMKK2, or (3) allosteric activation by AMP or A-769662. Importantly, cl-AMPK-α1 is only detectable in the nucleus, consistent with removal of the NES, and ectopic expression of cleavage-resistant D529A-mutant AMPK-α1 promotes cell death induced by cytotoxic agents. Thus, we have elucidated a non-canonical mechanism of AMPK activation within the nucleus, which protects cells against death induced by DNA damage.

Methods to assess small molecule allosteric modulators of the STRAD pseudokinase.

With the increased appreciation of the biological relevance of pseudokinase (PSK) allostery, the broadening of small molecule strategies to target PSK function is of particular importance. We and others have pursued the development of small molecule allosteric modulators of the STRAD pseudokinase by targeting its ATP binding pocket. The purpose of this effort is to modulate the function of the LKB1 tumor suppressor kinase, which exists in a trimer with the STRAD PSK and the adaptor protein MO25. Here we provide detailed guidance regarding the different methods we have used for medium throughput screening to identify STRAD ligands and measure their impact on LKB1 kinase activity. Our experience supports preferential use of direct measurements of LKB1 kinase activity, and demonstrates the limitations of indirect assessment methods in the development trans-acting allosteric modulators.

A Novel Pyrazolo[3,4-d]pyrimidine Induces Heme Oxygenase-1 and Exerts Anti-Inflammatory and Neuroprotective Effects.

The anti-oxidant enzyme heme oxygenase-1 (HO-1) is known to exert anti-inflammatory effects. From a library of pyrazolo[3,4-d]pyrimidines, we identified a novel compound KKC080096 that upregulated HO-1 at the mRNA and protein levels in microglial BV-2 cells. KKC080096 exhibited anti-inflammatory effects via suppressing nitric oxide, interleukin-1β (IL-1β), and iNOS production in lipopolysaccharide (LPS)-challenged cells. It inhibited the phosphorylation of IKK and MAP kinases (p38, JNK, ERK), which trigger inflammatory signaling, and whose activities are inhibited by HO-1. Further, KKC080096 upregulated anti-inflammatory marker (Arg1, YM1, CD206, IL-10, transforming growth factor-β [TGF-β]) expression. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, KKC080096 lowered microglial activation, protected the nigral dopaminergic neurons, and nigral damage-associated motor deficits. Next, we elucidated the mechanisms by which KKC080096 upregulated HO-1. KKC080096 induced the phosphorylation of AMPK and its known upstream kinases LKB1 and CaMKKbeta, and pharmacological inhibition of AMPK activity reduced the effects of KKC080096 on HO-1 expression and LPS-induced NO generation, suggesting that KKC080096-induced HO-1 upregulation involves LKB1/AMPK and CaMKKbeta/AMPK pathway activation. Further, KKC080096 caused an increase in cellular Nrf2 level, bound to Keap1 (Nrf2 inhibitor protein) with high affinity, and blocked Keap1-Nrf2 interaction. This Nrf2 activation resulted in concurrent induction of HO-1 and other Nrf2-targeted antioxidant enzymes in BV-2 and in dopaminergic CATH.a cells. These results indicate that KKC080096 is a potential therapeutic for oxidative stress- and inflammation-related neurodegenerative disorders such as Parkinson’s disease.

Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in the liver and secreted as biliary glycoprotein 1 (BGP1) via bile canaliculi (BCs). CEACAM1-LF is a 72 amino acid cytoplasmic domain mRNA splice isoform with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ceacam1−/− or Ser503Ala transgenic mice have been shown to develop insulin resistance and nonalcoholic fatty liver disease; however, the role of the human equivalent residue, Ser508, in lipid dysregulation is unknown. Human HepG2 hepatocytes that express CEACAM1 and form BC in vitro were compared with CEACAM1−/− cells and CEACAM1−/− cells expressing Ser508Ala null or Ser508Asp phosphorylation mimic mutations or to phosphorylation null mutations in the tyrosine ITIMs known to be phosphorylated by the tyrosine kinase Src. CEACAM1−/− cells and the Ser508Asp and Tyr520Phe mutants strongly retained lipids, while Ser508Ala and Tyr493Phe mutants had low lipid levels compared with wild-type cells, indicating that the ITIM mutants phenocopied the Ser508 mutants. We found that the fatty acid transporter CD36 was upregulated in the S508A mutant, coexpressed in BCs with CEACAM1, co-IPed with CEACAM1 and Src, and when downregulated via RNAi, an increase in lipid droplet content was observed. Nuclear translocation of CD36 associated kinase LKB1 was increased sevenfold in the S508A mutant versus CEACAM1−/− cells and correlated with increased activation of CD36-associated kinase AMPK in CEACAM1−/− cells. Thus, while CEACAM1−/− HepG2 cells upregulate lipid storage similar to Ceacam1−/− in murine liver, the null mutation Ser508Ala led to decreased lipid storage, emphasizing evolutionary changes between the CEACAM1 genes in mouse and humans.

NUAK Kinases: Brain-Ovary Axis.

Liver kinase B (LKB1) and adenosine monophosphate (AMP)-activated protein kinase (AMPK) are two major kinases that regulate cellular metabolism by acting as adenosine triphosphate (ATP) sensors. During starvation conditions, LKB1 and AMPK activate different downstream pathways to increase ATP production, while decreasing ATP consumption, which abrogates cellular proliferation and cell death. Initially, LKB1 was considered to be a tumor suppressor due to its loss of expression in various tumor types. Additional studies revealed amplifications in LKB1 and AMPK kinases in several cancers, suggesting a role in tumor progression. The AMPK-related proteins were described almost 20 years ago as a group of key kinases involved in the regulation of cellular metabolism. As LKB1-downstream targets, AMPK-related proteins were also initially considered to function as tumor suppressors. However, further research demonstrated that AMPK-related kinases play a major role not only in cellular physiology but also in tumor development. Furthermore, aside from their role as regulators of metabolism, additional functions have been described for these proteins, including roles in the cell cycle, cell migration, and cell death. In this review, we aim to highlight the major role of AMPK-related proteins beyond their functions in cellular metabolism, focusing on cancer progression based on their role in cell migration, invasion, and cell survival. Additionally, we describe two main AMPK-related kinases, Novel (nua) kinase family 1 (NUAK1) and 2 (NUAK2), which have been understudied, but play a major role in cellular physiology and tumor development.

Nuclear UHRF1 is a gate-keeper of cellular AMPK activity and function.

The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis. Although much has been learned on how low energy status and glucose starvation activate AMPK, how AMPK activity is properly controlled in vivo is still poorly understood. Here we report that UHRF1, an epigenetic regulator highly expressed in proliferating and cancer cells, interacts with AMPK and serves to suppress AMPK activity under both basal and stressed conditions. As a nuclear protein, UHRF1 promotes AMPK nuclear retention and strongly suppresses nuclear AMPK activity toward substrates H2B and EZH2. Importantly, we demonstrate that UHRF1 also robustly inhibits AMPK activity in the cytoplasm compartment, most likely as a consequence of AMPK nucleocytoplasmic shuttling. Mechanistically, we found that UHRF1 has no obvious effect on AMPK activation by upstream kinases LKB1 and CAMKK2 but inhibits AMPK activity by acting as a bridging factor targeting phosphatase PP2A to dephosphorylate AMPK. Hepatic overexpression of UHRF1 showed profound effects on glucose and lipid metabolism in wild-type mice but not in those with the liver-specific knockout of AMPKα1/α2, whereas knockdown of UHRF1 in adipose tissue led to AMPK activation and reduced sizes of adipocytes and lipogenic activity, highlighting the physiological significance of this regulation in glucose and lipid metabolism. Thus, our study identifies UHRF1 as a novel AMPK gate-keeper with critical roles in cellular metabolism.

Epigenetic control of metastasis

Cancer Metastasis is when cancer becomes deadly. Unsuccessful searches for driver mutations of metastasis have led some to explore alternative epigenetic causes. Pierce et al. found that the protein kinase LKB1, better known for influencing cancer through regulation of metabolism, had a tumor-suppressive role in one mouse lung cancer model. This tumor develops largely because of epigenomic effects on chromatin accessibility. Loss of LKB1 activates distinct waves of epigenetic remodeling in primary and metastatic tumor cells. Metastasis was mediated by activation of the transcription factor SOX17. The authors were then able to detect a small population of cells in primary tumors with the epigenetic characteristics of later metastatic cells, revealing a promising therapeutic target. Nat. Cell Biol. 23 , 915 (2021).

Abstract 79: Crosstalk of PIM and LKB1 kinases in driving growth of prostate cancer cells

Abstract The oncogenic PIM kinases and the tumor-suppressive LKB1 kinase have been linked to regulation of cell growth and proliferation. Here we have investigated their potential interactions as well as their relative impacts on tumorigenic growth of prostate cancer cells. The cellular functions of PIM and LKB1 kinases were evaluated using either pan-PIM inhibitors or CRISPR/Cas9-based genomic editing, with which all three PIM family members and/or LKB1 were knocked out from PC3 prostate cancer cells. In addition to cell-based 2D proliferation assays, we examined tumor growth using the chick embryo chorioallantoic membrane (CAM) xenograft model. In this report, we show that inhibition of PIM expression or activity results in increased phosphorylation of AMPK at Thr172 in an LKB1-dependent fashion. With in vitro kinase assays, we demonstrate that LKB1 is a novel direct substrate for PIM kinases and that Ser334 is one of the PIM target sites in LKB1. Accordingly, wild-type LKB1, but not the phosphodeficient S334A mutant can restore PIM inhibitor-induced AMPK phosphorylation in LKB1 knock-out cells. Whereas loss of LKB1 exaggerates formation of PC3-based tumors in the CAM xenograft model, co-deletion of PIM kinases attenuates it. The impairment of cell proliferation and tumor growth in cells lacking both PIM and LKB1 kinases not only underscores the potential crosstalk in signaling between these kinases, but also suggest that PIM inhibitors could be used to restrain growth of LKB1-deficient tumors. Citation Format: Kwan Long Mung, William Eccleshall, Niina M. Santio, Adolfo Rivero-Müller, Cecilia Sahlgren, Päivi J. Koskinen. Crosstalk of PIM and LKB1 kinases in driving growth of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 79.

Abstract 2937: Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth

Abstract The goal of this study is to define the role of NEDD9 in formation of progression of non-small cell lung cancer (NSCLC). NSCLC has a low survival rate, with metastasis contributing to the vast majority of deaths. NEDD9 expression has been shown to be repressed by LKB1, and upregulated in LKB1-mutated NSCLC; in these tumors, elevated NEDD9 promotes metastasis, serving as a scaffolding intermediate in the integrin and receptor tyrosine kinase signaling cascades, promoting pro-invasive signaling. In this study, we for the first time assessed the consequences for cancer formation after introduction of a constitutive null allele of Nedd9 (KPN mice) in the Krastm4Tyj/J/Trp53tm1Brn/J (KP mice) Adeno-cre inducible murine model of NSCLC, in the context of intact Lkb1. Unexpectedly, KPN mice presented with significantly accelerated NSCLC tumorigenesis and enhanced tumor invasion. Orthotopic re-implantation of KP and KPN tumor into syngeneic mice with wt and null Nedd9 genotypes demonstrated that the effect of Nedd9 loss on promoting tumor growth is cell-autonomous. Proteomic analysis of KPN versus KP tumors revealed that more aggressive phenotypes occurred in spite of depressed activity of the pro-proliferative, pro-invasive Nedd9 partner proteins Src and Fak. Treatment of mice with the Src inhibitor dasatinib significantly reduced, but did not eliminate, tumor growth in KP and KPN mice. As an alternative hypothesis, we considered that Ras-dependent NSCLC tumors require active autophagy, a process dependent on activation of the kinases Lkb1 and Ampk; given Lkb1 represses Nedd9, we investigated whether Nedd9 reciprocally regulates Lkb1. We found that KPN tumors specifically and significantly upregulated active Lkb1 based on a mechanism of post-transcriptional activation, causing increased expression of the Lkb1 substrate Ampk and downstream effectors of autophagy, including elevated appearance of LC3-positive autophagosomes in KPN NSCLC tumor tissue. NEDD9 depletion in human NSCLC cell lines similarly elevated LKB1 activation. In vivo treatment of KPN and KP mice with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors, emphasizing the importance of autophagy for the growth differential. In sum, these data for the first time identify and define a previously unknown role for the pro-metastatic protein NEDD9 in control of autophagy, based on a role as a negative regulator of the Lkb1-Ampk signaling axis in NSCLC. Citation Format: Alexander Y. Deneka, Meghan Kopp, Anna S. Nikonova, Anna Gaponova, Anna Kiseleva, Douglas Flieder, Ilya Serebriiskii, Harvey Hensley, Erica Golemis. Nedd9 controls autophagy to limit non-small cell lung cancer (NSCLC) growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2937.