Activation Of Ribosomal S6 Kinase Research Articles

EGFR inhibition with erlotinib induces membrane accumulation of aquaporin-2 via PDK1 and RSK activation

In the canonical aquaporin-2 (AQP2) signaling pathway, vasopressin (VP) binds to its V2 receptor, which in turn activates adenylate cyclase and protein kinase A (PKA), resulting in AQP2 S256 phosphorylation and accumulation of AQP2 in the membrane. The epidermal growth factor receptor (EGFR) inhibitor erlotinib also increases AQP2 S256 phosphorylation and membrane accumulation, yet it does not increase cAMP or PKA activity. We hypothesize that the ribosomal s6 kinase (RSK), a downstream effector in the EGFR-MAPK/ERK pathway, is the terminal kinase phosphorylating AQP2 in this novel PKA-independent pathway activated by erlotinib, and that the phosphoinositide dependent kinase-1 (PDK1), a well-established activator of RSK, is indispensable for erlotinib-induced AQP2 activation. Using our LLC-AQP2 cell model, we show that erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation are abolished by the specific RSK inhibitor BI-D1870. RSK knockdown with siRNA also blocked AQP2 S256 phosphorylation and membrane accumulation, as did RSK knockout with CRISPR/cas9 editing. Next, rat kidney slices were incubated in Hank’s buffer with or without BI-D1870 and treated with erlotinib. BI-D1870 inhibited AQP2 membrane accumulation in collecting duct principal cells, supporting the relevance of this pathway in kidney cells in situ. An in-vitro kinase assay using purified proteins demonstrated that RSK directly phosphorylates AQP2 at the S256 residue. Additionally, erlotinib consistently increased phosphorylation of RSK T359, a residue associated with RSK’s active state. In canonical RSK activation, a phosphorylation cascade results in PDK1 binding to and activating RSK. We found that PDK1 inhibition with an experimental compound PS-222 also blocked erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation. This implicates PDK1 along with RSK in the novel erlotinib-induced pathway of AQP2 traffcking. Our data show that RSK and PDK1 are terminal effectors in the novel, PKA-independent pathway of AQP2 activation induced by erlotinib. Further elaboration of this new non-canonical pathway promises to uncover potential pharmacological targets for the treatment of water balance disorders. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4’s transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Antitumor activity of extracellular signal-regulated kinases 1/2 inhibitor BVD-523 (ulixertinib) on thyroid cancer cells.

This study aimed to investigate BVD-523 (ulixertinib), an adenosine triphosphate (ATP)-dependent extracellular signal-regulated kinases 1/2 inhibitor, for its antitumor potential in thyroid cancer. Ten thyroid cancer cell lines known to carry mitogen-activated protein kinase (MAPK)-activated mutations, including v-Raf murine sarcoma viral oncogene homolog B (BRAF) and rat sarcoma virus (RAS) mutations, were examined. Cells were exposed to a 10-fold concentration gradient ranging from 0 to 3000 nM for 5 days. The half-inhibitory concentration was determined using the Cell Counting Kit-8 assay. Following BVD-523 treatment, cell cycle analysis was conducted using flow cytometry. In addition, the impact of BVD-523 on extracellular signal-regulated kinase (ERK)- dependent ribosomal S6 kinase (RSK) activation and the expression of cell cycle markers were assessed through western blot analysis. BVD-523 significantly inhibited thyroid cancer cell proliferation and induced G1/S cell cycle arrest dose-dependently. Notably, cell lines carrying MAPK mutations, especially those with the BRAF V600E mutation, exhibited heightened sensitivity to BVD-523's antitumor effects. Furthermore, BVD-523 suppressed cyclin D1 and phosphorylated retinoblastoma protein expression, and it robustly increased p27 levels in an RSK-independent manner. This study reveals the potent antitumor activity of BVD-523 against thyroid cancer cells bearing MAPK-activating mutations, offering promise for treating aggressive forms of thyroid cancer.

The Role of p90 Ribosomal S6 Kinase (RSK) in Tyrosine Kinase Inhibitor (TKI)-Induced Cardiotoxicity.

Targeted therapy, such as tyrosine kinase inhibitors (TKIs), has been approved to manage various cancer types. However, TKI-induced cardiotoxicity is a limiting factor for their use. This issue has raised the need for investigating potential cardioprotective techniques to be combined with TKIs. Ribosomal S6-kinases (RSKs) are a downstream effector of the mitogen-activated-protein-kinase (MAPK) pathway; specific RSK isoforms, such as RSK1 and RSK2, have been expressed in cancer cells, in which they increase tumour proliferation. Selective targeting of those isoforms would result in tumour suppression. Moreover, activation of RSKs expressed in the heart has resulted in cardiac hypertrophy and arrhythmia; thus, inhibiting RSKs would result in cardio-protection. This review article presents an overview of the usefulness of RSK inhibitors that can be novel agents to be assessed in future research for their effect in reducing cancer proliferation, as well as protecting the heart from cardiotoxicity induced by TKIs.

Kaposi's Sarcoma-Associated Herpesvirus Immediate Early Proteins Trigger FOXQ1 Expression in Oral Epithelial Cells, Engaging in a Novel Lytic Cycle-Sustaining Positive Feedback Loop.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that can replicate in oral epithelial cells to promote viral transmission via saliva. To identify novel regulators of KSHV oral infection, we performed a transcriptome analysis of KSHV-infected primary human gingival epithelial (HGEP) cells, which identified the gene coding for the host transcription factor FOXQ1 as the top induced host gene. FOXQ1 is nearly undetectable in uninfected HGEP and telomerase-immortalized gingival keratinocytes (TIGK) cells but is highly expressed within hours of KSHV infection. We found that while the FOXQ1 promoter lacks activating histone acetylation marks in uninfected oral epithelial cells, these marks accumulate in the FOXQ1 promoter in infected cells, revealing a rapid epigenetic reprogramming event. To evaluate FOXQ1 function, we depleted FOXQ1 in KSHV-infected TIGK cells, which resulted in reduced accumulation of KSHV lytic proteins and viral DNA over the course of 4 days of infection, uncovering a novel lytic cycle-sustaining role of FOXQ1. A screen of KSHV lytic proteins demonstrated that the immediate early proteins ORF45 and replication and transcription activator (RTA) were both sufficient for FOXQ1 induction in oral epithelial cells, indicating active involvement of incoming and rapidly expressed factors in altering host gene expression. ORF45 is known to sustain extracellular signal-regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) pathway activity to promote lytic infection. We found that an ORF45 mutant lacking RSK activation function failed to induce FOXQ1 in TIGK cells, revealing that ORF45 uses a shared mechanism to rapidly induce both host and viral genes to sustain lytic infection in oral epithelial cells. IMPORTANCE The oral cavity is a primary site of initial contact and entry for many viruses. Viral replication in the oral epithelium promotes viral shedding in saliva, allowing interpersonal transmission, as well as spread to other cell types, where chronic infection can be established. Understanding the regulation of KSHV infection in the oral epithelium would allow for the design of universal strategies to target the first stage of viral infection, thereby halting systemic viral pathogenesis. Overall, we uncover a novel positive feedback loop in which immediate early KSHV factors drive rapid host reprogramming of oral epithelial cells to sustain the lytic cycle in the oral cavity.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Defective synaptic plasticity in a model of Coffin-Lowry syndrome is rescued by simultaneously targeting PKA and MAPK pathways.

Empirical and computational methods were combined to examine whether individual or dual-drug treatments can restore the deficit in long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse observed in a cellular model of Coffin-Lowry syndrome (CLS). The model was produced by pharmacological inhibition of p90 ribosomal S6 kinase (RSK) activity. In this model, coapplication of an activator of the mitogen-activated protein kinase (MAPK) isoform ERK and an activator of protein kinase A (PKA) resulted in enhanced phosphorylation of RSK and enhanced LTF to a greater extent than either drug alone and also greater than their additive effects, which is termed synergism. The extent of synergism appeared to depend on another MAPK isoform, p38 MAPK. Inhibition of p38 MAPK facilitated serotonin (5-HT)-induced RSK phosphorylation, indicating that p38 MAPK inhibits activation of RSK. Inhibition of p38 MAPK combined with activation of PKA synergistically activated both ERK and RSK. Our results suggest that cellular models of disorders that affect synaptic plasticity and learning, such as CLS, may constitute a useful strategy to identify candidate drug combinations, and that combining computational models with empirical tests of model predictions can help explain synergism of drug combinations.

Conformations of p90 Ribosomal S6 Kinase (RSK) Activation

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie downstream of the Ras‐MAPK pathway and regulate cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N‐terminal kinase (NTK), and a C‐terminal kinase (CTK) in the same polypeptide chain. The NTK is a member of the AGC kinase superfamily, and the CTK is a member of the CAMK family. A flexible linker bridges the NTK and CTK, and embedded within the linker is the highly conserved tail of AGC kinases (AGC tail) which contains the conserved turn and hydrophobic motifs (HF). RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal‐regulated kinase (ERK2) and 3‐phosphoinositide‐dependent protein kinase 1 (PDK1). ERK2 phosphorylates the CTK and the turn motif. The phosphorylated CTK is then able to phosphorylate the linker at the HF, this enables PDK1 to bind the linker and then phosphorylate and activate the NTK2. The precise structural details of RSK kinase activation are not fully understood and a more rigorous biochemical characterization could provide valuable insight into how kinase complexes assemble and how the linker facilitates multiple binding and phosphorylation events.To gain a better understanding of the FL RSK2‐ERK2 signaling complex we have used cryoEM and hydrogen deuterium exchange mass spectrometry (HDX) to gain insight into the overall shape and solvent accessibility of the overall complex. We have determined a low‐resolution density map of the RSK2‐ERK2 complex, revealing a compact shape. Additionally, all three kinase domains are relatively well protected from solvent, with the exception of the activation loops of the CTK, and the aC helix of the NTK. While the tails of RSK2 and linker (AGC tail) are highly solvent exposed. We hypothesize that the linker region needs such solvent accessibility in order to enable the various interactions and phosphorylation events necessary for RSK activation. We have also utilized peptide array binding experiments to determine the residues within the linker that are necessary to enable ERK2 phosphorylation of the turn motif, CTK phosphorylation of the HF, and PDK1 binding and subsequent phosphorylation of the NTK activation loop. ERK2 appears to bind to the linker and NTK in a multivalent fashion via the C‐lobe (300‐320) and the N‐lobe. The CTK seems to primarily interact with HF in the linker. And PDK1 binds to both the HF and a conserved FDX1‐2Y/F motif in the linker.

P70S6 kinase regulates oligodendrocyte differentiation and is active in remyelinating lesions.

The p70 ribosomal S6 kinases (p70 ribosomal S6 kinase 1 and p70 ribosomal S6 kinase 2) are downstream targets of the mechanistic target of rapamycin signalling pathway. p70 ribosomal S6 kinase 1 specifically has demonstrated functions in regulating cell size in Drosophila and in insulin-sensitive cell populations in mammals. Prior studies demonstrated that the mechanistic target of the rapamycin pathway promotes oligodendrocyte differentiation and developmental myelination; however, how the immediate downstream targets of mechanistic target of rapamycin regulate these processes has not been elucidated. Here, we tested the hypothesis that p70 ribosomal S6 kinase 1 regulates oligodendrocyte differentiation during developmental myelination and remyelination processes in the CNS. We demonstrate that p70 ribosomal S6 kinase activity peaks in oligodendrocyte lineage cells at the time when they transition to myelinating oligodendrocytes during developmental myelination in the mouse spinal cord. We further show p70 ribosomal S6 kinase activity in differentiating oligodendrocytes in acute demyelinating lesions induced by lysophosphatidylcholine injection or by experimental autoimmune encephalomyelitis in mice. In demyelinated lesions, the expression of the p70 ribosomal S6 kinase target, phosphorylated S6 ribosomal protein, was transient and highest in maturing oligodendrocytes. Interestingly, we also identified p70 ribosomal S6 kinase activity in oligodendrocyte lineage cells in active multiple sclerosis lesions. Consistent with its predicted function in promoting oligodendrocyte differentiation, we demonstrate that specifically inhibiting p70 ribosomal S6 kinase 1 in cultured oligodendrocyte precursor cells significantly impairs cell lineage progression and expression of myelin basic protein. Finally, we used zebrafish to show in vivo that inhibiting p70 ribosomal S6 kinase 1 function in oligodendroglial cells reduces their differentiation and the number of myelin internodes produced. These data reveal an essential function of p70 ribosomal S6 kinase 1 in promoting oligodendrocyte differentiation during development and remyelination across multiple species.

Activation of Downstream mTORC1 Target Ribosomal Protein S6 Kinase (S6K) Can Be Found in a Subgroup of Dutch Patients with Granulomatous Pulmonary Disease.

Mechanistic target of rapamycin complex 1 (mTORC1) has been linked to different diseases. The mTORC1 signaling pathway is suggested to play a role in the granuloma formation of sarcoidosis. Recent studies demonstrated conflicting data on mTORC1 activation in patients with sarcoidosis by measuring activation of its downstream target S6 kinase (S6K) with either 33% or 100% of patients. Therefore, the aim of our study was to reevaluate the percentage of S6K activation in sarcoidosis patients in a Dutch cohort. To investigate whether this activation is specific for sarcoid granulomas, we also included Dutch patients with other granulomatous diseases of the lung. The activation of the S6K signaling pathway was evaluated by immunohistochemical staining of its downstream effector phospho-S6 in tissue sections. Active S6K signaling was detected in 32 (43%) of the sarcoidosis patients. Twelve (31%) of the patients with another granulomatous disorder also showed activated S6K signaling, demonstrating that the mTORC1 pathway may be activated in a range for different granulomatous diseases (p = 0.628). Activation of S6K can only be found in a subgroup of patients with sarcoidosis, as well as in patients with other granulomatous pulmonary diseases, such as hypersensitivity pneumonitis or vasculitis. No association between different clinical phenotypes and S6K activation can be found in sarcoidosis.

Bisdemethoxycurcumin Promotes Apoptosis and Inhibits the Epithelial-Mesenchymal Transition through the Inhibition of the G-Protein-Coupled Receptor 161/Mammalian Target of Rapamycin Signaling Pathway in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is one of the leading causes of cancer death in the world and lacks an effective targeted therapy. G-protein-coupled receptor 161 (GPR161) has been demonstrated to perform the functional regulations on TNBC progression and might be a potential new target for TNBC therapy. This study showed the effects of bisdemethoxycurcumin (BDMC) on GPR161 regulation, indicating that BDMC effectively inhibited GPR161 expression and downregulated GPR161-driven signaling. BDMC showed the potent inhibitory effects on TNBC proliferation through suppressing GPR161-mediated mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase (p70S6K) activation. Besides, in this study, we discover the mechanism of GPR161-driven TNBC metastasis, linking to GPR161-mediated twist-related protein 1 (Twist1)/matrix metallopeptidase 9 (MMP9) contributing to the epithelial-mesenchymal transition (EMT). BDMC effectively repressed GPR161-mediated TNBC metastasis via inhibiting Twist1/MMP9-induced EMT. The three-dimensional invasion assay also showed that BDMC significantly inhibited TNBC invasion. The combination treatment of BDMC and rapamycin enhanced the inhibition of TNBC proliferation and metastasis through increasing the blockage of mTOR activation. Furthermore, this study also observed that BDMC activated the caspase 3/9 signaling pathway to induce TNBC apoptosis. Therefore, BDMC could be applicable to anticancer therapy, especially targeting on the GPR161-driven cancer type.

Abstract 14369: Smooth Muscle Specific Deletion of RSK2 Suppresses Vasoconstriction of Resistance Arteries

Introduction: Intracellular Ca 2+ is a vital second messenger that plays a fundamental role in regulating intraluminal pressure- and agonist-induced vasoconstriction. Our laboratory previously demonstrated that intraluminal pressure and G-protein coupled receptor agonist stimulation activate p90 ribosomal S6 kinase 2 (RSK2). RSK2, in turn, phosphorylates the regulatory light chain of myosin (RLC 20 ) to induce constriction of small, resistance-sized arteries. More recently, we discovered that resistance arteries from global KO RSK2 mice exhibited a more dilated luminal diameter, reduced myogenic tone and RLC 20 phosphorylation, and decreased blood pressure. However, it is unclear whether the observed effects are a result of RSK2 global deletion in the mouse which may cause developmental abnormalities affecting the cardiovascular system. Hypothesis: We hypothesize that smooth muscle-specific RSK2 signaling in mouse resistance arteries is a significant regulator of myogenic tone and arterial stiffness. Methods: Inducible smooth muscle-specific KO mice for RSK2 (RSK2 SMC-/- ) were generated to study the role of RSK2 signaling in the regulation of vascular tone. Pressure myography studies assessed vascular reactivity in 4 th - and 5 th -order mesenteric arteries (MAs) isolated from RSK2 SMC-/- and RSK2 fl/fl mice. Results: The downregulation of normal RSK2 expression in resistance arteries was confirmed by Western blot. Our data shows that the intraluminal pressure-induced constriction of MAs is significantly attenuated in RSK2 SMC-/- mice (n=7 each group). Additionally, RSK2 SMC-/- resistance arteries experienced a delay in development of myogenic tone at 40 mmHg indicating that RSK2 signaling is sensitive to small changes in intraluminal pressure. Furthermore, MAs from RSK2 SMC-/- mice exhibited decreased vascular stiffness (indicated by enhanced strain, and rightward shift of stress-strain relationship) when compared to MAs from RSK2 fl/fl mice. Conclusions: This study provides novel evidence that smooth muscle RSK2 signaling contributes to pressure-induced vasoconstriction, and vascular stiffness. Inhibiting smooth muscle RSK2 activity may represent a novel strategy for rescuing vascular function in hypertensive patients.

Activation of RSK2 upregulates SOX8 to promote methotrexate resistance in gestational trophoblastic neoplasia

Resistance to chemotherapy is frequently driven by aberrantly activated kinases in cancer. Herein, we characterized the global phosphoproteomic alterations associated with methotrexate (MTX) resistance in gestational trophoblastic neoplastic (GTN) cells. A total of 1111 phosphosites on 713 proteins were significantly changed, with highly elevated Ribosomal S6 Kinase 2 (RSK2) phosphorylation (pS227) observed in MTX-resistant GTN cells. Activation of RSK2 promoted cell proliferation and survival after MTX treatment in GTN cell models. Interestingly, RSK2 might play an important role in the regulation of reactive oxygen species (ROS) homeostasis, as manipulation of RSK2 activation affected ROS accumulation and SOX8 expression in GTN cells. In addition, overexpression of SOX8 partly rescued cell proliferation and survival in RSK2-depleted MTX-resistant GTN cells, suggesting that SOX8 might serve as a downstream effector of RSK2 to promote MTX resistance in GTN cells. Highly activated RSK2/SOX8 signaling was observed in MTX-resistant GTN specimens. Further, the RSK2 inhibitor BIX02565 effectively reduced SOX8 expression, induced ROS accumulation, and enhanced MTX-induced cytotoxicity in vitro and in vivo. Collectively, our findings suggested that RSK2 activation could promote MTX resistance via upregulating SOX8 and attenuating MTX-induced ROS in GTN cells, which may help to develop experimental therapeutics to treat MTX-resistant GTN.Resistance to chemotherapy is frequently driven by aberrantly activated kinases. RSK2 was identified as a potential regulator of methotrexate resistance in gestational trophoblastic neoplastic cells. RSK2 activation promotes methotrexate resistance via upregulation of SOX8 and attenuation of reactive oxygen species. RSK2 inhibitors might therefore be useful to treat methotrexate-resistant gestational trophoblastic neoplasia.

Structural Studies and Dynamics of p90 Ribosomal S6 Kinase (RSK)

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) lie downstream of the Ras-MAPK pathway and regulates cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N-terminal kinase (NTK), and a C-terminal kinase (CTK) in the same polypeptide chain. RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal-regulated kinase (ERK) and 3-phosphoinositide-dependent protein kinase 1 (PDK1). The precise structural details of RSK kinase activation are not fully understood and a more rigorous structural characterization could provide valuable insight into how kinase complexes assemble and how kinases activate other kinases. RSK activation begins with ERK binding to and activating the CTK. A major goal of this project is to determine the structure of the full length RSK-ERK complex primarily utilizing cryoEM to gain insight into the orientations of three kinase domains relative to one another and to identify specific interaction networks that exist within the complex. We have determined a low resolution density map of the RSK-ERK complex using negative stain EM and can identify each kinase domain.

EPHA2 Interacts with DNA-PKcs in Cell Nucleus and Controls Ionizing Radiation Responses in Non-Small Cell Lung Cancer Cells

Simple SummaryDespite the introduction of targeted therapies against genomic drivers in non-small cell lung cancer (NSCLC), e.g., mutated epidermal growth factor receptor (EGFR) and EML4-ALK fusion or immune checkpoint blockade, many patients are receiving radiation therapy (RT). Thus, RT is important for the treatment of advanced NSCLC, where it is used alone or combined with targeted agents or chemotherapy. Unfortunately, a large fraction of the NSCLC cases show resistance to ionizing radiation (IR), calling for further understanding of the underlying mechanisms and novel ways to IR sensitization. Here, we demonstrate a novel function of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) pathway that controls cellular IR response. Moreover, we show that EphA2 can be targeted for IR sensitization of resistant NSCLC cells. Thus, we suggest that further understanding of this mechanism may allow for new RT sensitization approaches to treat LC.Ephrin (EFN)/Erythropoietin-producing human hepatocellular receptors (Eph) signaling has earlier been reported to regulate non-small cell lung cancer (NSCLC) cell survival and cell death as well as invasion and migration. Here, the role of Ephrin type-A receptor 2 (EphA2) on the DNA damage response (DDR) signaling and ionizing radiation (IR) cellular effect was studied in NSCLC cells. Silencing of EphA2 resulted in IR sensitization, with increased activation of caspase-3, PARP-1 cleavage and reduced clonogenic survival. Profiling of EphA2 expression in a NSCLC cell line panel showed a correlation to an IR refractory phenotype. EphA2 was found to be transiently and rapidly phosphorylated at Ser897 in response to IR, which was paralleled with the activation of ribosomal protein S6 kinase (RSK). Using cell fractionation, a transient increase in both total and pSer897 EphA2 in the nuclear fraction in response to IR was revealed. By immunoprecipitation and LC-MS/MS analysis of EphA2 complexes, nuclear localized EphA2 was found in a complex with DNA-PKcs. Such complex formation rapidly increased after IR but returned back to basal level within an hour. Targeting EphA2 with siRNA or by treatment with EFNA1 ligand partly reduced phosphorylation of DNA-PKcs at S2056 at early time points after IR. Thus, we report that EphA2 interacts with DNA-PKcs in the cell nucleus suggesting a novel mechanism involving the EphA2 receptor in DDR signaling and IR responsiveness.

Abstract PS11-33: A first-in-human Phase 1/1b multicenter, open-label dose escalation study to assess safety and tolerability of PMD-026, a first-in-class oral RSK inhibitor, in metastatic breast cancer patients

Abstract Background: Metastatic breast cancer (mBC) remains an aggressive disease with limited durable treatment options; the worst prognosis among the breast cancer subtypes is typically seen in metastatic triple-negative breast cancer (mTNBC). Given that unmet need, we sought to identify an actionable molecular target to combat mTNBC. Promising preclinical activity identified p90 ribosomal s6 kinase 2 (RSK2) as a key kinase in mTNBC. PMD-026 is a potent, oral, small molecular RSK inhibitor with high selectivity for the RSK2 isoform. RSK is a major convergence point in the important MAPK and PDK-1 signaling pathways, which drive TNBC cell survival, proliferation, and drug resistance. Methods: The primary aim of this single-arm, open-label, first-in-human, phase 1/1b study (NCT04115306) is to evaluate the safety of single agent PMD-026 in patients with mBC. Secondary endpoints are clinical activity, pharmacokinetics (PK) and correlative biomarker expression on tumor specimens. Patients are dosed orally once or twice daily in 21-day cycles with measures to adapt the dosing schedule based on the PK data, as needed. In dose escalation, patients must have mBC with evaluable or measurable disease by RECIST v1.1. In dose expansion, patients must have mTNBC with measurable disease by RECIST v1.1. Patients must have progressed on or after standard of care therapy. Tumor tissue is required to retrospectively correlate RSK2 activity with clinical outcomes via immunohistochemistry using a CAP/CLIA certified companion diagnostic (CDx). Results: Twelve mBC patients (ER+ mBC n=5, mTNBC n=7) who have failed standard chemotherapy as well as targeted therapies such as CDK4/6 inhibitors and immunotherapies have been enrolled to date. Patients have been treated in escalating cohorts of 25, 50, 100, 200, 400 (200 q12) or 600 mg (300 q12) of PMD-026 administered orally daily. At 400 mg the dose schedule was changed from daily to q12 hrs based on PK results to optimize drug exposure over a 24-hr timeframe in patients. The PK of PMD-026 showed linear exposure and a high volume of distribution. The AUC was ~9100 hr*ng/ml on Day 1 when PMD-026 was dosed at 200 mg qd demonstrating high exposure. In addition, when dosed at 200 mg q12 hrs, PMD-026 serum levels approached the preclinically established desired level of 1 µM over 24 hrs. In the 200 mg q12 hrs cohort, adverse events consisted of G2 GERD (n=1) and G2 neutropenia (n=1). Initial signs of activity were observed as CT-identified necrosis in a neck node metastasis (n=1) and transient decrease in CA 27-29 (n=1). While the 200 mg q12 hrs dose was generally well-tolerated, there were 2 dose limiting toxicities at 300 mg q12 hrs including syncope (n = 1) and vomiting with dehydration leading to reversible acute kidney injury (n = 1). To further understand the patient population, RSK2 activation was assessed in tumor samples from all patients. RSK2 was activated in all of the tumors and the H-Score ranged from 110 to 198 using the CDx platform. Conclusions: Preliminary evidence indicates that PMD-026 is well-tolerated at dose levels up to 200 mg q12 with initial signs of activity; pharmacokinetics showed good linear exposure. Updated safety, clinical activity, pharmacokinetic and biomarker analyses will be presented; target accrual for Phase 1b Expansion is approximately 20 mTNBC patients. (clinicaltrials.gov NCT04115306). Citation Format: Murali Beeram, Judy S. Wang, Pavani Chalasani, Lida Mina, Amita Patnaik, Mary Rose Pambid, Aarthi Jayanthan, My-my Huynh, Gerrit Los, Sandra E. Dunn, F. Andrew Dorr. A first-in-human Phase 1/1b multicenter, open-label dose escalation study to assess safety and tolerability of PMD-026, a first-in-class oral RSK inhibitor, in metastatic breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-33.

Simultaneous Targeting of RSK and AKT Efficiently Inhibits YB-1-Mediated Repair of Ionizing Radiation-Induced DNA Double-Strand Breaks in Breast Cancer Cells

Y-box binding protein 1 (YB-1) overexpression is associated with chemotherapy- and radiation therapy resistance. Ionizing radiation (IR), receptor tyrosine kinase ligands, and mutation in KRAS gene stimulate activation of YB-1. YB-1 accelerates the repair of IR-induced DNA double-strand breaks (DSBs). Ribosomal S6 kinase (RSK) is the main kinase inducing YB-1 phosphorylation. We investigated the impact of RSK targeting on DSB repair and radiosensitivity. The triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, and Hs 578T, in addition to non-TNBC cell lines MCF7, HBL-100, and SKBR3, were used. MCF-10A cells were included as normal breast epithelial cells. The RSK inhibitor LJI308 was used to investigate the role of RSK activity in S102 phosphorylation of YB-1 and YB-1-associated signaling pathways. The activation status of the underlying pathways was investigated by Western blotting after treatment with pharmacologic inhibitors or transfection with siRNA. The impact of LJI308 on DSB repair and postirradiation cell survival was tested by the γH2AX foci and the standard clonogenic assays, respectively. LJI308 inhibited the phosphorylation of RSK (T359/S363) and YB-1 (S102) after irradiation, treatment with EGF, and in cells expressing a KRAS mutation. LJI308 treatment slightly inhibited DSB repair only in some of the cell lines tested. This was shown to be due to PI3K-dependent stimulation of AKT or constitutive AKT activity mainly in cancer cells but not in normal breast epithelial MCF-10A cells. Simultaneous targeting of AKT and RSK strongly blocked DSB repair in all cancer cell lines, independent of TNBC status or KRAS mutation, with a minor effect in MCF-10A cells. Cotargeting of RSK- and AKT-induced radiation sensitivity in TNBC MDA-MB-231 and non-TNBC MCF7 cells but not in MCF-10A cells. Simultaneous targeting of RSK and AKT might be an efficient approach to block the repair of DSBs after irradiation and to induce radiosensitization of breast cancer cells.

Synthesis and Biological Evaluation of 4'-Substituted Kaempfer-3-ols.

The synthesis of two series of five kaempfer-3-ols was described. The first set all have a C-3 hydroxyl group and the second has a carboxymethoxy ether at the C-3 position. Both series have variable substitution at the C-4' position (i.e., OH, Cl, F, H, OMe). Both kaempferols and carboxymethoxy ethers were evaluated for their ability to inhibit ribosomal s6 kinase (RSK) activity and cancer cell proliferation.

Role of p90 ribosomal S6 kinase in long-term synaptic facilitation and enhanced neuronal excitability

Multiple kinases converge on the transcription factor cAMP response element-binding protein (CREB) to enhance the expression of proteins essential for long-term synaptic plasticity and memory. The p90 ribosomal S6 kinase (RSK) is one of these kinases, although its role is poorly understood. The present study exploited the technical advantages of the Aplysia sensorimotor culture system to examine the role of RSK in long-term synaptic facilitation (LTF) and long-term enhancement of neuronal excitability (LTEE), two correlates of long-term memory (LTM). Inhibition of RSK expression or RSK activity both significantly reduced CREB1 phosphorylation, LTF, and LTEE, suggesting RSK is required for learning-related synaptic plasticity and enhancement in neuronal excitability. In addition, knock down of RSK by RNAi in Aplysia sensory neurons impairs LTF, suggesting that this may be a useful single-cell system to study aspects of defective synaptic plasticity in Coffin-Lowry Syndrome (CLS), a cognitive disorder that is caused by mutations in rsk2 and associated with deficits in learning and memory. We found that the impairments in LTF and LTEE can be rescued by a computationally designed spaced training protocol, which was previously demonstrated to augment normal LTF and LTM.

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition. We have identified inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without potent cytotoxicity. The structure-activity data from this study will be used as a platform to develop novel RSK2 inhibitors.