Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression.

BACKGROUND: Because obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (-)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PURPOSE: This study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. METHODS: Preadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. RESULTS: Here, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. CONCLUSIONS: These findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

Empagliflozin mitigates cardiac hypertrophy through cardiac RSK/NHE-1 inhibition.

BACKGROUND: SGLT2i reduce cardiac hypertrophy, but underlying mechanisms remain unknown. Here we explore a role for serine/threonine kinases (STK) and sodium hydrogen exchanger 1(NHE1) activities in SGLT2i effects on cardiac hypertrophy. METHODS: Isolated hearts from db/db mice were perfused with 1 µM EMPA, and STK phosphorylation sites were examined using unbiased multiplex analysis to detect the most affected STKs by EMPA. Subsequently, hypertrophy was induced in H9c2 cells with 50 µM phenylephrine (PE), and the role of the most affected STK (p90 ribosomal S6 kinase (RSK)) and NHE1 activity in hypertrophy and the protection by EMPA was evaluated. RESULTS: In db/db mice hearts, EMPA most markedly reduced STK phosphorylation sites regulated by RSKL1, a member of the RSK family, and by Aurora A and B kinases. GO and KEGG analysis suggested that EMPA inhibits hypertrophy, cell cycle, cell senescence and FOXO pathways, illustrating inhibition of growth pathways. EMPA prevented PE-induced hypertrophy as evaluated by BNP and cell surface area in H9c2 cells. EMPA blocked PE-induced activation of NHE1. The specific NHE1 inhibitor Cariporide also prevented PE-induced hypertrophy without added effect of EMPA. EMPA blocked PE-induced RSK phosphorylation. The RSK inhibitor BIX02565 also suppressed PE-induced hypertrophy without added effect of EMPA. Cariporide mimicked EMPA's effects on PE-treated RSK phosphorylation. BIX02565 decreased PE-induced NHE1 activity, with no further decrease by EMPA. CONCLUSIONS: RSK inhibition by EMPA appears as a novel direct cardiac target of SGLT2i. Direct cardiac effects of EMPA exert their anti-hypertrophic effect through NHE-inhibition and subsequent RSK pathway inhibition.

p90RSK pathway inhibition synergizes with cisplatin in TMEM16A overexpressing head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) constitutes one of the most common types of human cancers and often metastasizes to lymph nodes. Platinum-based chemotherapeutic drugs are commonly used for treatment of a wide range of cancers, including HNSCC. Its mode of action relies on its ability to impede DNA repair mechanisms, inducing apoptosis in cancer cells. However, due to acquired resistance and toxic side-effects, researchers have been focusing on developing novel combinational therapeutic strategies to overcome cisplatin resistance. In the current study, we identified p90RSK, an ERK1/2 downstream target, as a key mediator and a targetable signaling node against cisplatin resistance. Our results strongly support the role of p90RSK in cisplatin resistance and identify the combination of p90RSK inhibitor, BI-D1870, with cisplatin as a novel therapeutic strategy to overcome cisplatin resistance. In addition, we have identified TMEM16A expression as a potential upstream regulator of p90RSK through the ERK pathway and a biomarker of response to p90RSK targeted therapy in the context of cisplatin resistance.

Identification of RSK substrates using an analog-sensitive kinase approach.

The p90 ribosomal S6 kinases (RSK) family of serine/threonine kinases comprises four isoforms (RSK1-4) that lie downstream of the ERK1/2 mitogen-activated protein kinase pathway. RSKs are implicated in fine tuning of cellular processes such as translation, transcription, proliferation, and motility. Previous work showed that pathogens such as Cardioviruses could hijack any of the four RSK isoforms to inhibit PKR activation or to disrupt cellular nucleocytoplasmic trafficking. In contrast, some reports suggest nonredundant functions for distinct RSK isoforms, whereas Coffin-Lowry syndrome has only been associated with mutations in the gene encoding RSK2. In this work, we used the analog-sensitive kinase strategy to ask whether the cellular substrates of distinct RSK isoforms differ. We compared the substrates of two of the most distant RSK isoforms: RSK1 and RSK4. We identified a series of potential substrates for both RSKs in cells and validated RanBP3, PDCD4, IRS2, and ZC3H11A as substrates of both RSK1 and RSK4, and SORBS2 as an RSK1 substrate. In addition, using mutagenesis and inhibitors, we confirmed analog-sensitive kinase data showing that endogenous RSKs phosphorylate TRIM33 at S1119. Our data thus identify a series of potential RSK substrates and suggest that the substrates of RSK1 and RSK4 largely overlap and that the specificity of the various RSK isoforms likely depends on their cell- or tissue-specific expression pattern.

P90 ribosomal S6 kinases: A bona fide target for novel targeted anticancer therapies?

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases. They are downstream effectors of the Ras/ERK/MAPK signaling cascade. ERK1/2 activation directly results in the phosphorylation of RSKs, which further, through interaction with a variety of different downstream substrates, activate various signaling events. In this context, they have been shown to mediate diverse cellular processes like cell survival, growth, proliferation, EMT, invasion, and metastasis. Interestingly, increased expression of RSKs has also been demonstrated in various cancers, such as breast, prostate, and lung cancer. This review aims to present the most recent advances in the field of RSK signaling that have occurred, such as biological insights, function, and mechanisms associated with carcinogenesis. We additionally present and discuss the recent advances but also the limitations in the development of pharmacological inhibitors of RSKs, in the context of the use of these kinases as putative, more efficient targets for novel anticancer therapeutic approaches.

A compound directed against S6K1 hampers fat mass expansion and mitigates diet-induced hepatosteatosis.

The ribosomal protein S6 kinase 1 (S6K1) is a relevant effector downstream of the mammalian target of rapamycin complex 1 (mTORC1), best known for its role in the control of lipid homeostasis. Consistent with this, mice lacking the S6k1 gene have a defect in their ability to induce the commitment of fat precursor cells to the adipogenic lineage, which contributes to a significant reduction of fat mass. Here, we assess the therapeutic blockage of S6K1 in diet-induced obese mice challenged with LY2584702 tosylate, a specific oral S6K1 inhibitor initially developed for the treatment of solid tumors. We show that diminished S6K1 activity hampers fat mass expansion and ameliorates dyslipidemia and hepatic steatosis, while modifying transcriptome-wide gene expression programs relevant for adipose and liver function. Accordingly, decreased mTORC1 signaling in fat (but increased in the liver) segregated with defective epithelial-mesenchymal transition and the impaired expression of Cd36 (coding for a fatty acid translocase) and Lgals1 (Galectin 1) in both tissues. All these factors combined align with reduced adipocyte size and improved lipidomic signatures in the liver, while hepatic steatosis and hypertriglyceridemia were improved in treatments lasting either 3 months or 6 weeks.

Design, synthesis and anti-breast cancer evaluation of biaryl pyridine analogues as potent RSK inhibitors.

In order to discover and develop the new RSK kinase inhibitor, 50 pyridyl biaryl derivatives were designed and synthesized with LJH685 as the lead compound and their anti-tumor ability was tested. The results showed that the ability of 7d compound to inhibit the phosphorylation of YB-1 was comparable to that of LJH685. Among them, after preliminary screening, compound 7d showed good activity in inhibiting cell proliferation. Therefore, we took 7d as an example and performed molecular docking analysis on it. Judging from the overlapping combination diagram with LJH685, the results have verified that compound 7d has a similar skeleton to LJH685 and has a similar docking effect with RSK. Therefore, compound 7d is in line with the RSK inhibitor we designed and could be developed to a promising anti-tumor drug in the future.

ZCL-082, a boron-containing compound, induces apoptosis of non-Hodgkin's lymphoma via targeting p90 ribosomal S6 kinase 1/NF-κB signaling pathway.

INTRODUCTION: Despite the rapid progress in the diagnosis and treatment, the prognosis of some types of non-Hodgkin's lymphoma (NHL), especially those with double-hit or double-expressor genotypes, remains poor. Novel targets and compounds are needed to improve the prognosis of NHL. METHODS: We investigated the effect of ZCL-082, a novel boron-containing compound with anti-proliferating activity against ovarian cancer cells, on NHL cells and human peripheral blood mononuclear cells by CCK-8 assay, Annexin V/PI double staining assay, RH123/PI double staining, Western blot, and immunohistochemistry. NF-κB pathway activity was analyzed using luciferase reporter gene assay and RT-PCR. The location of p65 was detected by immunofluorescence and nuclear/cytoplasmic fractionation assay. Immunoprecipitation and chromatin immunoprecipitation assays were used to detect the binding between p65 and p300. CETSA and molecular docking assay were carried out to test the interaction between ZCL-082 and p90 ribosomal S6 kinase 1 (RSK1). Kinase reaction was conducted to examine the inhibition of RSK1 kinase activity by ZCL-082. RESULTS: We found that ZCL-082 can induce the apoptosis of various NHL cell lines in vitro and in vivo. ZCL-082 significantly inhibits TNFα- or LPS-induced NF-κB activation without disturbing TNFα-induced IκBα degradation or the nuclear translocation and DNA-binding ability of p65. However, ZCL-082 markedly suppresses the phosphorylation of p65 on Ser536 and the interaction between p65 and p300. The overexpression of the phosphomimetic mutant of p65 at Ser536 partially abrogates ZCL-082-induced cell death. We further found that ZCL-082 directly binds to and inhibits the activity of RSK1. RSK1 can phosphorylate RelA/p65 on Ser536 and its overexpression is associated with the poor prognosis of lymphoma. The overexpression of RSK1 partially rescues ZCL-082-induced cell death. Molecular docking studies show that ZCL-082 fits well with the N-terminal kinase domain of RSK1. Furthermore, the combination of ZCL-082 and BCL-2 inhibitor ABT-199 has a synergistic apoptosis-inducing effect against double-hit lymphoma cell line OCI-Ly10. DISCUSSION: We found that ZCL-082 is a highly promising anti-lymphoma compound that targets RSK1 and interferes with the RSK1/NF-κB signaling pathway. The combination of ZCL-082 with BCL-2 inhibitor may represent a novel strategy to improve the outcome of double-hit or double-expressor lymphoma.

A novel ribosomal protein S6 kinase 2 inhibitor attenuates the malignant phenotype of cutaneous malignant melanoma cells by inducing cell cycle arrest and apoptosis.

Malignant melanoma (MM) is a highly life-threatening tumor causing the majority of the cutaneous cancer-related deaths. Previously, ribosomal protein S6 kinase 2 (RSK2), the downstream effector of the MAPK pathway, represents a therapeutic target in melanoma. AE007 is discovered as a targeted RSK2 inhibitor, and subsequent results showed that AE007 inhibits RSK2 by directly binding to its protein kinase domain. AE007 causes cell cycle arrest and cellular apoptosis, thereby dramatically inhibiting proliferation, migration, and invasion of melanoma cells. Nevertheless, melanocytes and keratinocytes are not affected by this compound. In addition, suppression of RSK2 abrogates the inhibitory effect of AE007 on melanoma cell proliferation. AE007 treatment significantly inhibits the expression of Cyclin D1, Cyclin B1, CDK2, and Bcl-2, while raises the cleavage of PARP. Moreover, RNA sequencing results show that AE007 treatment can affect the genes expression profile, including the expression of cell cycle and DNA replication genes. In conclusion, AE007 is a promising melanoma therapeutic agent by targeting RSK2.

RSK Inhibition Induces Apoptosis by Downregulating Protein Synthesis in a Variety of Acute Myeloid Leukemia Cell Lines.

Fms-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1/2 (IDH1/2) mutations drive malignancy in acute myeloid leukemia (AML), which accounts for approximately 40% of AML cases. Treatment with FLT3 or IDH1/2 inhibitors is used for such patients; however, it is not considered for most patients with AML who lack mutations on the respective genes. In this study, p90 ribosomal S6 kinase (RSK) was found to serve as a new therapeutic target in various AMLs with or without FLT3 mutations. BI-D1870, a potent inhibitor of RSK, significantly suppressed the proliferation of AML cell lines, among which three encoded wild-type FLT3 and three contained FLT3 driver mutations, compared with chronic myeloid leukemia K562 cells or other adherent cancer cells. BI-D1870 inhibited protein synthesis by dephosphorylating the p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 in all AML cells except KG-1a cells. Meanwhile, the expression of microtubule-associated protein light chain 3B-I and -II increased in KG-1a cells treated with BI-D1870. BI-D1870 induced caspase-dependent apoptosis in all AML cells, including KG-1a cells. We next investigated the synergistic effect of BI-D1870 with cytarabine, a traditional anticancer drug used in AML. Synergistic effects of BI-D1870 and cytarabine were not observed in any of the cell lines. The findings suggested that BI-D1870 alone exerts an adequate antiproliferative effect on AML with or without FLT3 mutations and serves as a novel AML therapeutic agent.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 µM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

Identifying requirements for RSK2 specific inhibitors.

Identifying isoform-specific inhibitors for closely related kinase family members remains a substantial challenge. The necessity for achieving this specificity is exemplified by the RSK family, downstream effectors of ERK1/2, which have divergent physiological effects. The natural product, SL0101, a flavonoid glycoside, binds specifically to RSK1/2 through a binding pocket generated by an extensive conformational rearrangement within the RSK N-terminal kinase domain (NTKD). In modelling experiments a single amino acid that is divergent in RSK3/4 most likely prevents the required conformational rearrangement necessary for SL0101 binding. Kinetic analysis of RSK2 association with SL0101 and its derivatives identified that regions outside of the NTKD contribute to stable inhibitor binding. An analogue with an n-propyl-carbamate at the 4" position on the rhamnose moiety was identified that forms a highly stable inhibitor complex with RSK2 but not with RSK1. These results identify a SL0101 modification that will aid the identification of RSK2 specific inhibitors.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors.

The RSK2 kinase is the downstream effector of the Ras/Raf/MEK/ERK pathway, that is often aberrantly activated in acute myeloid leukemia (AML). Recently, we reported a structure-activity study for BI-D1870, the pan-RSK inhibitor, and identified pteridinones that inhibited cellular RSK2 activity that did not result in concomitant cytotoxicity. In the current study, we developed a series of pyrrolopyrimidines and purines to replace the pteridinone ring of BI-D1870, with a range of N-substituents that extend to the substrate binding site to probe complementary interactions, while retaining the 2,6-difluorophenol-4-amino group to maintain interactions with the hinge domain and the DFG motif. Several compounds inhibited cellular RSK2 activity, and we identified compounds that uncoupled cellular RSK2 inhibition from potent cytotoxicity in the MOLM-13 AML cell line. These N-substituted probes have revealed an opportunity to further examine substituents that extend from the ATP- to the substrate-binding site may confer improved RSK potency and selectivity.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. METHODS: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. RESULTS: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. CONCLUSIONS: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Design, Synthesis and Biological Evaluation of Arylpyridin-2-yl Guanidine Derivatives and Cyclic Mimetics as Novel MSK1 Inhibitors. An Application in an Asthma Model.

Mitogen- and Stress-Activated Kinase 1 (MSK1) is a nuclear kinase, taking part in the activation pathway of the pro-inflammatory transcription factor NF-kB and is demonstrating a therapeutic target potential in inflammatory diseases such as asthma, psoriasis and atherosclerosis. To date, few MSK1 inhibitors were reported. In order to identify new MSK1 inhibitors, a screening of a library of low molecular weight compounds was performed, and the results highlighted the 6-phenylpyridin-2-yl guanidine (compound 1a, IC50~18 µM) as a starting hit for structure-activity relationship study. Derivatives, homologues and rigid mimetics of 1a were designed, and all synthesized compounds were evaluated for their inhibitory activity towards MSK1. Among them, the non-cytotoxic 2-aminobenzimidazole 49d was the most potent at inhibiting significantly: (i) MSK1 activity, (ii) the release of IL-6 in inflammatory conditions in vitro (IC50~2 µM) and (iii) the inflammatory cell recruitment to the airways in a mouse model of asthma.

Garcinia cambogia suppresses adipogenesis in 3T3-L1 cells by inhibiting p90RSK and Stat3 activation during mitotic clonal expansion.

Obesity is associated with an increase in adipose tissue, which is mediated by hyperplasia and hypertrophy. Therefore, inhibiting cell proliferation during mitotic clonal expansion (MCE) is one of the major strategies for preventing obesity. The antagonistic effects of Garcinia cambogia (G. cambogia) on obesity have been studied in animal experimental models. However, the effects of G. cambogia extract on MCE, and the underlying molecular mechanisms, are poorly understood. In this study, 3T3-L1 cells were used to investigate whether G. cambogia extract affected cell proliferation during MCE and to identify target molecules for any anti-adipogenic activity. G. cambogia extract suppressed isobutylmethylxanthine and dexamethasone-and-insulin (MDI)-induced adipogenesis at an early stage by attenuating MCE. In G. cambogia extract-treated preadipocytes, MDI-induced cell proliferation and cell cycle progression were inhibited by G0 /G1 arrest due to an increase in p21 and p27 expression, and inhibition of cyclin-dependent kinase 2, cyclin E1 expression, and retinoblastoma (Rb) phosphorylation. In addition, the MDI-induced phosphorylation and subsequent translocation into the nucleus of p90 ribosomal S6 kinase (p90RSK) and signal transducer and activator of transcription (Stat) 3 were suppressed. Specific inhibitors of p90RSK (FMK) and Stat3 (stattic) regulated cell proliferation and adipogenesis. In conclusion, this study demonstrated that G. cambogia extract inhibited MCE by regulating p90RSK, Stat3, and cell cycle proteins, leading to G0 /G1 arrest. These findings provide new insight into the mechanism by which G. cambogia suppresses adipocyte differentiation and show that p90RSK is critical for adipogenesis as a new molecular target.

Evaluation of imidazo[2,1-b]thiazole-based anticancer agents in one decade (2011-2020): Current status and future prospects.

Several reports have highlighted imidazo[2,1-b]thiazole derivatives as potential antiproliferative agents. They act through kinase inhibition, tubulin inhibition, and other molecular mechanisms of action. In the current article, we reviewed the imidazo[2,1-b]thiazole-based compounds that were reported as anticancer agents. Their biological characteristics as well as structure-activity relationship (SAR) have been reviewed and evaluated. Our main focus was on the reports published in the literature from 2011 to 2020.

Discovery of a novel dual-target inhibitor against RSK1 and MSK2 to suppress growth of human colon cancer.

Colon cancer is the most aggressive tumor in both men and women globally. As many the chemotherapeutic regimens have adverse side effects and contribute to the resistance and recurrence, therefore, finding novel therapeutic targets and developing effective agents are urgent. Based on the TCGA and GTEx database analysis, RSK1 and MSK2 were found abnormal expressed in colon cancer. RSK1 and MSK2 were overexpressed in colon cancer tissues confirmed by western blot and IHC. After knocking down RSK1 or MSK2, cell proliferation and anchorage-independent cell growth were markedly inhibited. Using a computer docking model, we identified a novel dual-target inhibitor, APIO-EE-07, that could block both RSK1 and MSK2 kinase activity in a dose-dependent manner. APIO-EE-07 inhibited cell growth and induced apoptosis and also increased expression of Bax as well as cleaved caspase-3 and -PARP in colon cancer cells by downregulating RSK1 and MSK2 downstream targets, including CREB and ATF1. Furthermore, APIO-EE-07 decreased tumor volume and weight in human patient-derived xenografts tumors implanted in SCID mice. In summary, our results demonstrate that RSK1 and MSK2 are the potential targets for the treatment of colon cancer. APIO-EE-07, a novel dual-target inhibitor of RSK1 and MSK2, can suppress the growth of colon cancer by attenuating RSK1 and MSK2 signaling.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.