Novel Potent and Selective Inhibitors of p90 Ribosomal S6 Kinase Reveal the Heterogeneity of RSK Function in MAPK- Driven Cancers
Ida Aronchik1, Brent A. Appleton1, Stephen E. Basham1, Kenneth Crawford1, Mercedita Del Rosario1, Laura V. Doyle1, William F. Estacio1, Jiong Lan2, Mika K. Lindvall1, Catherine A. Luu1, Elizabeth Ornelas1, Eleni Venetsanakos1, Cynthia M. Shafer1, and Anne B. Jefferson1
Abstract
The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its
substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer.
Implications: Two novel potent and selective RSK inhibitors will now allow a full assessment of the potential of RSK as a therapeutic target for oncology. Mol Cancer Res; 12(5); 803–12. ©2014 AACR.
Introduction
The p90 ribosomal S6 kinase (RSK) comprises a family of four closely related proteins that are widely expressed in cancer cell lines and tissues and are activated in response to a number of growth factors and hormones. RSK substrate phosphorylation has been functionally linked to cell survival, proliferation, and more recently to cell motility (1–4). RSK kinases have a unique structure containing two nonidentical kinase domains, N-terminal and C-terminal, separated by a linker region. The currently favored model for RSK activa- tion entails ERK (extracellular signal–regulated kinase) phos- phorylation of Thr573/577 (residue numbering for RSK1/ RSK2 amino acid sequence) in C-terminal kinase domain of RSK, which ultimately leads to a phosphorylation-based docking site for PDK1 in the hydrophobic motif of RSK on Ser380/386. PDK1 phosphorylation of residue Ser
Authors’ Affiliations: 1Novartis Institutes for BioMedical Research, Emeryville, California; and 2Shanghai Haiyan Pharmaceutical Technology Co., Ltd., Shanghai
Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).
Corresponding Author: Brent A. Appleton, Novartis Institutes for BioMed- ical Research, 4560 Horton Street, Emeryville, CA, 94608. Phone: 510-923- 8228; Fax: 510-655-9910; E-mail: [email protected]
doi: 10.1158/1541-7786.MCR-13-0595
©2014 American Association for Cancer Research.
221/227 then activates the N-terminal kinase domain of RSK, which in turn phosphorylates numerous nuclear and cytoplasmic proteins that account for the diverse cellular roles of RSK. Because the essential first step of RSK activa- tion is its phosphorylation by ERK, RSK is positioned to be a critical effector of mitogen-activated protein kinase (MAPK) signaling that accompanies mutational activation of KRAS or BRAF in cancer. Indeed, substrates described for RSK have been used to predict proliferative and antiapoptotic func- tions as well as contributions to invasion and motility, possibly associated with transition from an epithelial-to- mesenchymal phenotype (1, 2, 4, 5).
One of physiologic substrates of RSK with a particularly diverse set of downstream effects is Y-box–binding protein 1 (YB1). YB1 regulates transcription and translation by bind- ing to its recognition motifs in both DNA and RNA to regulate key cell processes such as proliferation, motility, and stemness characteristics (6–11). Two kinases, RSK and AKT, have been studied as regulators of YB1 function through phosphorylation of YB1 on Serine 102 (12, 13). Previous studies have described somewhat opposing roles for phospho-YB1 as a modulator of both transcription and translation (8, 11, 14). Thus, we hypothesize that YB1 has the potential to be strongly phosphorylated by RSK in the context of its activation downstream of mutated KRAS or BRAF, and thereby to serve as both a mediator of functional effects of RSK and a marker for its activity.
803
Exploration of RSK and its substrate phosphorylation role in cancer has been previously hindered by the lack of optimal loss-of-function tools. Published inhibitors of RSK have helped gain some understanding of the role of RSK in cellular signaling. However, these molecules have limited use in dissecting RSK biology and in the assessment of RSK as a potential cancer target due to poor selectivity, or because they only target a subset of RSK family isoforms. For instance, the irreversible pyrrolopyrimidine RSK inhibitor (FMK) does not inhibit RSK3 because this isoform lacks a properly positioned active site cysteine needed for covalent binding (15). Another widely used RSK inhibitor, BI-D1870, potently inhibits all RSK isoforms in biochemical assays by binding to its N-terminal kinase domain, yet also has numerous off-target activities (16, 17).
In this study, we introduce two potent and highly selective inhibitors of the four RSK isoforms that overcome these issues. We then use these inhibitors to assess the potential of RSK as a therapeutic target in oncology by exploring the correlation between YB1 phosphorylation and RSK inhib- itor–induced functional effects across a panel of MAPK- activated cell lines.
Materials and Methods
Materials
All cell lines used in this study were obtained from the American Type Culture Collection (ATCC), were authen- ticated by the SNP6 profile, and were used for experiments within 30 passages of authentication. Cell propagation was in accordance with the ATCC recommended conditions. Cell media, FBS, PBS, and trypsin were supplied by CellGro (Mediatech, Inc.) and Cambrex/BioWhittaker. Soft agar and TransIT-LT1 transfection reagent were acquired from VWR International. Growth factor–reduced Matrigel was pur- chased from Collaborative Biomedical Products. Primary antibodies for Western blotting were acquired from Cell Signaling Technology. The secondary anti-mouse and anti- rabbit antibodies conjugated with horseradish peroxidase were obtained from GE Healthcare UK Ltd. Secondary antibodies conjugated to fluorescent probes as well as rho- damine–phalloidin for immunofluorescence were purchased from Molecular Probes/Invitrogen.
Kinase inhibitors
LJH685 and LJI308 were synthesized using methodology described in Supplementary Methods. All kinase inhibitors were synthesized in house and dissolved in dimethyl sulf- oxide (DMSO) 99.9% high-performance liquid chroma- tography grade (Sigma-Aldrich). The final dilution was performed in the treatment medium. Unless otherwise specified, cells were treated for 4 hours with inhibitors at the following concentrations, which were selected to achieve
complete inhibition based on testing a range of doses of each: 10 mmol/L LJH685 or LJI308, 6 mmol/L FMK-PA, 1.0 mmol/L PD0325901, 50 mmol/L I-9, 5 mmol/L MK2206, 3 mmol/L KU-0063794, and 250 nmol/L RAD001 or equiv- alent volume of DMSO for negative control samples.
Inhibition of RSK1, RSK2, and RSK3 activity
Enzymatic activity of RSK isoforms 1, 2, and 3 (PV4049, PV4051, and PV3846) was assessed using recombinant full- length RSK protein purchased from Invitrogen (Life Tech- nologies). RSK1 (1 nmol/L), RSK2 (0.1 nmol/L), or RSK3 (1 nmol/L) was allowed to phosphorylate 200 nmol/L peptide substrate (biotin-AGAGRSRHSSYPAGT-OH) in the presence of ATP at concentration equal to the Km for
ATP for each enzyme (RSK1, 5 mmol/L; RSK2, 20 mmol/L; and RSK3, 10 mmol/L) and appropriate dilutions of RSK inhibitors. Additional details are provided in Supplementary
Methods.
KinomeScan
Kinase selectivity profiling was carried out by KinomeScan (Ambit/DiscoveRx). Data are reported as 100 minus the percentage of control, in which larger numbers indicate more complete binding.
pYB1 quantification
A quantitative electrochemiluminescence (ECL) assay was developed to measure cellular levels of YB1 protein phos- phorylated at Ser102. This assay was built using ECL reagents from MesoScale Discovery (MSD). Specifically, cell lysate generated using radioimmunoprecipitation assay
lysis buffer (Sigma; R0278) was added to unblocked 96-well high-bind ECL plates and incubated overnight at 4◦C on a plate shaker. The following day, plates were washed with 1 wash buffer followed by 2-hour room temperature incuba- tion with 10% bovine serum albumin diluted in 1 tris wash
buffer (50 nm Tris pH 7.5, 0.15 mol/L NaCl, and 0.02% Tween-20). Plates were then washed with 1 wash buffer followed by 2-hour room temperature incubation with a phospho-specific Ser102 YB1 antibody (Cell Signaling
Technologies; C34A2; 1.3 mg/mL). Plates were washed
again with 1 wash buffer and then incubated for 2 hours
at room temperature with a sulfo-tag goat anti-rabbit sig- naling antibody (MSD; 0.5 mg/mL). Plates underwent a final wash before addition of 1.5 MSD read buffer and detection
of the phospho-YB1 signal in a MSD Sector 6000 plate reader. We routinely ran this assay using a mouse BAF cell line engineered to express activated fibroblast growth factor receptor (FGFR), but it is readily applicable to other cell lines with sufficient baseline phosphorylation of YB1. Although total YB1 levels were not affected by inhibitor treatment, we routinely normalized the phospho-YB1 signal to total YB1 signal for EC50 calculation. Total YB1 was detected through the same protocol described for phospho-YB1, but used a
total YB1 antibody (Santa Cruz Biotechnology; SC-101198 (59-Q); 1 mg/mL) with a sulfo-tag goat anti-mouse antibody (MSD; 0.5 mg/mL) for detection.
Assessment of cell growth
Cell growth in anchorage-dependent and anchorage- independent assays was quantified using CellTiter-Glo or Alamar Blue reagents according to supplier protocols. Details of the assays are provided in Supplementary Methods.
Cell-cycle analysis and apoptosis detection
MDA-MB-231 and H358 cells were assessed for cell-cycle distribution by propidium iodide staining (details in Sup- plementary Methods). For caspase-3 activation analysis, the cells were plated on minimal attachment plates (Nano-
culture plates; Scivax USA, Inc.) and treated with indicated doses of LJH685 in presence of 1 mmol/L NucView 488 probe (Biotium Inc.). The presence of cleaved fluorescent
substrate indicative of active caspase-3 was detected at 3- hour intervals as signal emitted at 488 nm using IncuCyte fluorescent microscopy Essen Bioscience.
Results
LJH685 and LJI308 are potent and specific RSK inhibitors
To address the functional role of RSK, a novel series of inhibitors of the ATP-binding domain of the N-terminal RSK kinase domain were developed. Two closely related difluoro- phenyl pyridine representatives, LJH685 and LJI308, are shown in Fig. 1. LJH685 and LJI308 both inhibit RSK1, 2, and 3 biochemical activities with IC50 of 0.004 to 0.013
mmol/L (Fig. 1A).
As a common shortcoming of many RSK inhibitors is their
poor selectivity profile, we used KinomeScan kinase–bind- ing screening assays to assess the selectivity profiles of
LJH685 and LJI308 binding to 96 kinases. LJH685 and LJI308 bound nearly 100% of RSK2 at 10 mmol/L con-
Figure 1. LJH685 and LJI308 are potent and specific RSK inhibitors. A, chemical structure of LJH685 and LJI308. IC50 for RSK inhibition and the
percentage of target binding at 10 mmol/L inhibitor are shown below. NT,
not tested. B, visualization of KinomeScan kinase competition–binding data for LJH685 versus BI-D1870-AE binding to 96 kinases at 10 mmol/L concentration. Larger circles indicate higher percentage binding.
Figure 2. Crystal structure of LJH685 bound to RSK2 confirms binding to the ATP site with pyridine nitrogen as the hinge binder. The inhibitor demonstrates nonplanar conformation in which the three aromatic rings retain a propeller-shaped arrangement. The coordinates and structure factors for RSK2 in complex with LJH685 have been deposited to the rcsb (http://www.rcsb.org) with the PDB ID code 4NUS.
centration, but bound none of the other kinases to the same extent, suggesting good selectivity for RSK. Furthermore, when LJI308 was tested in an expanded panel of 442 kinases, it bound the targeted N-termini of remaining RSK isoforms, RSK1, RSK3, and RSK4 to similar extent (Supplementary Table S1). Overall, only MEK4, S6K1, and HIPK1-3 bound either compound to the same degree. To determine whether the binding of LJI308 to MEK4, S6K1, and HIP kinases translates into inhibition of enzyme activity, we tested LJI308 in biochemical activity assays for these kinases.
LJI308 inhibited S6K1 with an IC50 of 0.8 mmol/L, repre- senting a 200-fold lower inhibition than that of RSK2. LJI308 inhibited MEK4 less than 50% at 10 mmol/L and HIP kinase 1 less than 50% at 1 mmol/L. This suggests that
none of these kinases are inhibited with similar potency as
the RSK isoforms, and that LJH685 and LJI308 are excep- tionally selective inhibitors of RSK1, 2, 3, and 4.
BI-D1870-AE was tested against the same panel of 96 kinases and resulted in RSK2 binding to 99.9%, whereas it bound type II receptor for bone morphogenetic protein kinase, Polo-like kinases (PLK1 and PLK3), and several other kinases, including aurora kinase B and phosphatidylinositol- 4,5-bisphosphate 3-kinasecatalyticsubunitgammatoasimilar extentastheon-targeteffecton RSK2 (Fig. 1B; Supplementary Table S1). By this comparison, LJH685 and LJI308 have significantly improved specificity over BI-D1870-AE.
To confirm the binding site for LJH685 in RSK and to
better understand its selectivity, we solved the crystal struc- ture of LJH685 (Fig. 2; PDB ID code 4NUS). LJH685 bound RSK2 in a nonplanar conformation in which the three aromatic rings form a propeller-shaped arrangement. Furthermore, the difluorophenyl ring is binding to the gatekeeper area and is twisted 49 degrees from the plane of the pyridine ring. In most kinases, the area around the gatekeeper prefers substituents in the same plane as the hinge-binding moiety. To accommodate the propeller
arrangement, Phe212 of the DFG motif flips out in a conformation reminiscent of the classical type II, DFG-out orientation. However, Asp211 of the DFG blocks access to the hydrophobic pocket and interacts with the difluorophe- nyl ring and the catalytic Lys100. Thus, we propose that the nonplanar shape of LJH685 and its conformational prop- erties distinct from the currently available and commonly used inhibitor dihydropteridinone (BI-D1870) contribute to its significantly improved selectivity profile (unpublished data, M.K. Lindvall, B.A. Appleton, C.M. Shafer). Further- more, given the biochemical profile of structurally related molecules LJH685 and LJI308 and their mode of targeting RSK N-terminal kinase via interaction with the ATP-bind- ing site, these compounds are nearly identical with respect to RSK inhibition and are used interchangeably in further experiments.
LJH685 potently and selectively inhibits RSK in cells
We tested the ability of these compounds to inhibit RSK in a cellular context. The description of YB1 as a direct substrate for RSK made it an excellent candidate cellular marker for RSK activity. Experiments using EGF-stimulated COS7 cells as a model system for active MAPK signaling confirmed that in that context, YB1 is directly phosphorylated on Ser102 by
RSK (Supplementary Fig. S1). We, therefore, used pSer102- YB1 as a cellular marker for RSK and asked whether its phosphorylation in MDA-MB-231 cancer cells that bear activating mutations within the MAPK signaling pathway could be modulated by RSK inhibitors. As shown in Fig. 3A, LJH685 efficiently reduced phosphorylation of YB1 at sub- micromolar concentrations and caused nearly complete inhi- bition at higher concentrations. In comparison, the C-ter- minal RSK inhibitor, FMK-PA, was similarly potent, but never achieved complete inhibition of YB1 phosphorylation even at high concentrations, possibly because it does not inhibit RSK3. To further quantitate RSK inhibition in cells, we developed an antibody-binding ECL-based assay that quantifies cellular levels of YB1 phosphorylated at Ser102.
LJH685 and LJI308 blocked YB1 Ser102 phosphorylation with similar potency (EC50 0.2–0.3 mmol/L) and a reproducible maximum inhibition of >90% at 20 mmol/L
concentration, consistent with the qualitative results by
Western blotting (Figs. 3 and 4). Because a number of other proteins have also been described as phosphorylated by RSK, we confirmed that phosphorylation of a second of these, the antiapoptotic protein BAD at Ser112, is also inhibited by LJH685 at concentrations that correlate well with inhibition of pYB-1 (Supplementary Fig. S2).
Figure 3. LJH685 modulates YB1 phosphorylation but affects phosphorylation of S6RP only in combination with mTOR inhibitors. MDA-MB-231 (A and B) and H358 (B) cells were treated with the indicated kinase inhibitors for 4 hours either alone or in combinations. Cell lysates were analyzed by Western blot analysis with indicated antibodies.
Figure 4. Inhibition of RSK in cells correlates with decreased cell number and broad phosphorylation effects only in an anchorage-independent setting. A, growth of MDA-MB-231 and H358 cells was assessed in anchorage-dependent setting using CellTiter-Glo assay (blue diamonds) and in anchorage- independent setting using Alamar blue staining (red squares) with increasing concentrations of LJH685 in each growth environment. The percentage of control cell number (y-axis) was compared with the percentage of control cellular Ser102 YB1 phosphorylation (red triangles) measured using quantitative ECL assay. EC50 for each assay is shown below the curve. B, MDA-MB-231 cells were grown in uncoated plastic or ultra-low bind plates and treated with the indicated kinase inhibitors for 3 hours. Cell lysates were analyzed by Western blot analysis with indicated antibodies.
We next compared LJH685 and LJI308 with additional inhibitors of the MAPK signaling pathway and the parallel phosphoinositide 3-kinase (PI3K) signaling pathway for their ability to inhibit phosphorylation of YB1. In these experiments, we used MDA-MB-231 and NCI-H358 (H358) cells that have KRAS mutations that activate MAPK signaling and represent established models for late-stage invasive triple-negative breast cancer and non–small cell lung carcinoma, respectively. The cells were treated with
10 mmol/L LJH685, LJI308, or 6 mmol/L FMK-PA for 4
hours. In addition, inhibitors of MEK (MAP–ERK kinase;
PD0325901), ERK (I-9), AKT (MK2206), and two mTOR
inhibitors targeting catalytic and allosteric domains of the molecule, respectively (KU-0063794 and RAD001), were compared with LJH685 and LJI308 in this study system (15, 18–21). The doses were chosen based on previous data to give full target modulation by each compound (data not shown). As with LJH685 and LJI308, MAPK pathway inhibitors cause substantial inhibition of YB1 phosphoryla- tion on Ser102 in both cell lines (Fig. 3B, left), consistent with the role of these kinases in activating RSK (published and data not shown). Strikingly, although previous publications impli- cate AKT in YB1 activation (12, 13), AKT inhibition did not affect YB1 phosphorylation, confirming the specificity of phospho-YB1 modulation by RSK in these cells (also see Supplementary Fig. S2). Surprisingly, in H358 cells inhibi- tion of mTOR with either KU-0063794 or RAD001 caused partial inhibition of YB1 phosphorylation. This suggests that in this cell line, mTOR can influence YB1 phosphorylation, either directly or via cross-talk with RSK (Fig. 3B, left).
Ribosomal S6 protein (S6RP), which is directly involved in modulation of protein synthesis (22), represents another protein sometimes described as a RSK substrate but well
established as phosphorylated by mTOR. Recent studies point to potential RSK involvement in direct S6RP enzy- matic activation as well as indirect activation via phosphor- ylation of raptor, a member of mTOR complex 1 (mTORC1; ref. 23). Therefore, to examine this signaling convergence node, we assessed the levels of S6RP phosphor- ylated on Serine 235/236. As shown in Fig. 3B left, the levels of S6RP phosphorylation were not convincingly modulated by LJH685or LJI308, whereas both allosteric and catalytic mTOR inhibitors gave strong inhibition of S6RP phosphor- ylation (Fig. 3B, left). Interestingly, in both cell lines the combination of the AKT inhibitor or either of the mTOR inhibitors with LJH685 caused an additive inhibition of S6RP phosphorylation (Fig. 3B, right). These data suggest that RSK is a minor contributor and mTOR kinase a major contributor to phosphorylation of S6RP, but that YB1 is predominantly phosphorylated by RSK.
LJH685 RSK inhibition correlates with antiproliferative effects in MAPK pathway–dependent cancer cell lines only in anchorage-independent growth setting
We next examined the functional effects of LJH685 and, based on recent findings that implicate RSK in anchorage- independent growth modulation and cell transformation (24), assessed the effect of RSK inhibition in both attached and anchorage-independent cell culture settings. MDA- MB-231 and H358 cells were plated on conventional plastic tissue-culture plates as well as in soft agar and treated with increasing doses of LJH685. Cell proliferation was assessed using CellTiter-Glo reagent and Alamar blue stain for the two growth settings, respectively. YB1 phosphorylation was measured using the ECL assay in a parallel set of cells plated on plastic and identically treated with inhibitors as for the
CellTiter-Glo determination. As Fig. 4A demonstrates, the growth of both cell lines in soft agar was efficiently inhibited using LJH685, with EC50 values of 0.73 and 0.79 mmol/L in
MDA-MB-231 and H358, respectively. Surprisingly, the
growth of either cell line in an attached setting was only affected by LJH685 at concentrations significantly above the
doses at which RSK is fully inhibited, with an EC50 in this setting of 48 and 66 mmol/L in these cell lines, respectively. The inhibition of YB1 phosphorylation tracked closely
with the cell growth inhibition in soft agar setting, with EC50 of 0.39 and 0.57 mmol/L in each cell line, demon- strating that RSK inhibition was fully achieved, irrespective
of the differential growth response. In a control experiment, we confirmed that the concentration at which LJH685 inhibits pYB1 in the anchorage-independent setting corre- lates well with the concentration at which it inhibits pYB1 on plastic (Supplementary Fig. S2). The correlation of LJH685 EC50 values for pYB1 and soft agar growth inhibition suggests that anchorage-independent growth in these cell lines is dependent on RSK kinase activity. In contrast, the inability of LJH685 to inhibit growth of the same cell lines on plastic indicates that in the attached growth setting, these cells do not depend on RSK function for proliferation.
To further examine the signaling events underlying the differential growth effects of RSK inhibition, we used ultra- low bind cell culture plates, which maintain anchorage- independent cell growth without a scaffold or matrix and serve as a basic three-dimensional (3D) surrogate system. This system possesses the advantage over soft agar in that it allows for sufficient cell recovery and protein extraction for analysis, and cells in this setting were similarly sensitive to RSK inhibitor (data not shown). MDA-MB-231 cells plated in the anchorage-independent setting or on conventional plastic plates were treated with LJH685, mTOR inhibitor, or vehicle control DMSO for 4 hours, and the total cell lysates were analyzed for presence of phosphorylated and total S6RP and YB1 proteins using Western blot analysis. As shown in Fig. 4B, YB1 phosphorylation was efficiently inhibited by LJH685 and was unaffected by RAD001 in either growth setting, confirming that in this cell line YB1 remains under the control of RSK and is not affected by mTOR activity in either attached or anchorage-independent growth settings. In contrast, LJH685 efficiently reduced phosphorylation of S6RP in the anchorage-independent setting. This suggests that S6RP becomes an RSK-dependent substrate in the anchorage-independent setting and may be a critical medi- ator of the growth inhibition in soft agar.
To explore the possibility that there may be functional effects of RSK inhibition in additional cell lines, we assessed the effects of LJH685 across a panel of 21 cell lines derived from five lineages representing models of lung, colon, pan- creatic, breast, and melanoma cancers. These cell lines were selected on the basis of MAPK cascade activating mutations, as well as the functional sensitivity of these cell lines to MEK inhibitors (data not shown). RAF and KRAS mutation status as well as cell line lineage are shown for each cell line (Table 1, column 1–3). As assessed by quantitative ECL p- YB1 assay described above, LJH685 inhibits YB1phosphor-
ylation across the cell lines with IC50 ranging from 0.28 mmol/L to over the limit of assay detection, with 15 cell lines exhibiting IC50 below 3.4 mmol/L (Table 1, column 4).
For the cell lines with the most potent inhibition of YB1
phosphorylation, the highest concentrations of LJH685 resulted in >80% maximum inhibition of YB1, establishing that YB1 phosphorylation in those cells was predominantly
controlled by RSK. As with the MDA-MB-231 and H358 cell lines, LJH685 did not potently inhibit the growth of any of the tested cell lines in the attached setting (column 6). Surprisingly, only two cell lines in addition to MDA-MB- 231 and H358, WM1799 and NCI-H2122, demonstrated a close correlation between phospho-YB1 inhibition and growth inhibition in soft agar (columns 4 and 5), suggesting that only these cell lines are also dependent on RSK for growth in this setting. Thus, our data demonstrate that the effect of RSK inhibition in cell lines with aberrantly activated MAPK signaling is highly growth-setting specific and cannot be stratified by either lineage or specific mutation status. In addition, although we confirm YB1 as a direct substrate for RSK in an expanded cell line set, we are unable to demon- strate a robust and consistent functional dependence on YB1 phosphorylation.
RSK involvement in cell-cycle regulation and apoptosis induction in MAPK-driven cancer cell lines is anchorage- setting specific
Although we demonstrate that RSK inhibition by LJH685 results in reduced numbers of cells for several cell lines in soft agar, it was not yet clear what aspect of cell growth in this setting is dependent on RSK enzymatic activity. Thus, we conducted an additional series of cell growth assays in an attempt to deconvolute the cell dependency on RSK in the soft agar anchorage-independent growth assay.
First, to understand whether RSK signaling is necessary only to support growth in the absence of attachment, or whether the lower cell density in soft agar contributes to the differences in RSK inhibition responses, we used several other surrogate assays for anchorage-independent growth. H358 cells proved resistant to growing in a Matrigel matrix, but MDA-MB-231 cells could be grown in this matrix and
exhibited 66% growth inhibition at 10 mmol/L LJH685
with a concomitant morphology change in this setting
(Supplementary Fig. S3). This finding suggested that these cells remain dependent on RSK in this alternate anchorage- independent assay. We next tested MDA-MB-231 and H358 cell lines for RSK dependency while growing on Scivax NanoCulture assay plates. Scivax plates enable a low level of cell attachment due to a nanoscale pattern printed on the bottom of assay plates that promotes 3D cell growth. MDA-MB-231 and H358 cells cultured for 10 days on Scivax plates grew as loosely adherent clusters of cells (Fig. 5A; Supplementary Table S2). LJI308 was equally inhibitory to H358 growth in this setting as in soft agar, whereas MDA-MB-231 was less potently inhibited by LJI308 in this minimal attachment setting.
Third, we tested the effects of LJI308 on H358 and MDA- MB-231 cells in colony formation assays. This assay format
Mutation status
pYB1
Soft agar
Attached
Cancer type/cell line RAF KRAS EC50/(Max I) EC50 EC50
Lung
Calu6 Q61K 9.95 (58) >100 >100
NCI-H2122 G12C 0.63 (87) 61, >33 26
NCI-H358 G12C 0.57 (91) 0.79 66
NCI-H727 G12V 0.64 (81) 30 >100
NCI-H2087 wt (N-Ras Q61K) 0.28 (82) 1.1 64
Colon
SW620 G12V 3.23 (74) >100 >100
SW480
HT29
V600E G12V
Q61L 2.22 (61)
>20 (43) >100
>100 >100
>100
Pancreatic
Capan-2 G12V 0.9 (78) >100 >100
MiaPaCa2 G12C 3.35 (64) 62, >33 >100
SW1990
Panc02.03 G12D
G12A/G12D 1.6 (69)
>20 (31) >100
>100 56
>100
Breast
MDA-MB-231 G464V G13D 0.39 (93) 0.73 48
Melanoma
A375 V600E 0.38 (96) >100 52
G361 V600E 0.49 (88) 38 70
Colo205 V600E >20 (48) >100 >100
Malme3M V600E 0.73 (87) 60 16.3
WM1799 V600E 1.55 (61) 3 92
WM983B
WM266-4 V600E
V600D/V600E >20 (37)
0.37 (88) 11.2
22, >33 89
>100
tests the role of cell density in growth characteristics of cells by establishing sparse but attached growth conditions. As demonstrated in Fig. 5B, H358 colony numbers were reduced more than 2-fold by a concentration of LJI308 that inhibits colony formation of these cells in soft agar. This result suggests that in addition to dependency on RSK for anchorage-independent growth, H358 cells are also dependent on RSK for growth on plastic when seeded at a low starting density. An alternative explanation is that RSK dependency in H358 cells is only observed after longer periods of incubation with LJH685 because the anchorage- independent and colony formation assays are performed with 10 days of compound incubation compared with 3 days of compound incubation in standard cell proliferation assays in the attached setting. MDA-MB-231 cells, on the other hand, seem to be less potently inhibited by LJI308 for colony formation on plastic than for growth in soft agar just as they were somewhat less potently inhibited in the Scivax assay above, suggesting that these cells may rely on RSK activity primarily to survive anchorage independence. The difference in cell line sensitivity in colony formation and Scivax assays further underscores the cell line–specific nature of RSK dependence within the subset of cell lines showing sensitivity to LJH685 and LJI308.
RSK has been previously described as mediating acti- vation of several substrates such as Elk1, c-Fos, and p27, which either directly regulate cell-cycle kinases or drive expression of cell-cycle regulator proteins (25–27). Thus, we examined the effect of RSK inhibition on cell-cycle progression and apoptosis induction in either attached or unattached MDA-MB-231 and H358 cells. For both cell lines, there was some decrease in the percentage of cells in S- and G2-phases asa result of plating with no attachment on low-bind plates, but neither cell line showed strong effects of RSK inhibition on distribution within the phases of cell cycle (Supplementary Fig. S4). However, as shown in Fig. 5C in both MDA-MB-231 and H358 cell lines, RSK inhibition in the anchorage-independent growth conditions led to a doubling of cell numbers in the sub-G1 phase of the cell cycle suggesting that this con- centration of LJH685 could be inducing apoptosis. To confirm this, we tracked caspase-3 activity by noncyto- toxic NucView488 fluorescent probe in both cell lines while growing in the Scivax minimal attachment plates in the presence of increasing doses of LJH685. As shown
in Fig. 5D, caspase-3 increased with time in both MDA- MB-231 and in H358 cells treated with 10 mmol/L RSK inhibitor. These results confirm that in some cell lines
Figure 5. RSK inhibition affects MDA-MB-231 and H358 colony formation, growth with minimum attachment, and viability. A and B, MDA-MB-231 or H358 cell lines were grown with the indicated concentrations of LJI308 as colonies on minimal attachment Scivax plates (A) or as colonies on conventional tissue- culture plates (B). Cell-cycle distribution and viability were determined after treatment with 10 mmol/L LJH685 on attached or anchorage-independent
conditions for 5 days (C) or after treatment with increasing LJH685 doses during growth in Scivax plates and assessed for caspase-3 activation (D). ω, P 0.005
at 10 mmol/L LJH685 dose compared with DMSO-treated control. Data depicted are for period after stabilization of specific caspase-3 signal.
such as MDA-MB-231 and H358, RSK contributes to survival in the anchorage-independent setting despite other differences in the nature of dependency on RSK.
Discussion
Aberrant activation of the MAPK and PI3K signaling pathways is a hallmark of many cancer types. Convergent signaling nodes between these pathways could present new targeting points to achieve an effective therapeutic outcome. RSK represents such a point of convergence by incorporating sequential activation steps from both ERK and PDK1 and by phosphorylating numerous downstream substrates associat- ed with cell proliferation, motility, and cell polarity (1, 2, 4, 5). However, our understanding of this critical signaling node has been previously hindered by the lack of potent and specific tools that enable a deeper understanding of the role
of RSK in cancer signaling networks. In this study, we introduce two N-terminal RSK kinase inhibitors, LJH685 and LJI308, that are highly selective for all RSK isoforms. The selectivity of these inhibitors can possibly be explained by the unusual propeller conformation of the compound that can be accepted by the N-terminal kinase domain of RSK. The potency, selectivity, and effectiveness of LJH685 and LJI308 at inhibiting RSK activity in cells makes these molecules excellent new tools to better understand the role of RSK in MAPK pathway signaling and function.
We used LJH685 and LJI308 in functional cell viability assays in a panel of cell lines that bear activating mutations in the MAPK signaling cascade and are sensitive to MEK inhibitors. Surprisingly, LJH685 and LJI308 only inhibited the growth of a narrow subgroup of these cells, and only when these cells were grown in an anchorage-independent
setting or at low cell densities in colony formation assays. Notably, although phosphorylation of YB1 at Ser102 was well inhibited by LJH685 and LJI308, this did not closely correlate with widespread functional consequences. These results were surprising given recent studies that have linked YB1 to important physiologic roles in cancer progression, including the expression of FGFR, proliferating cell nuclear antigen, MAP Kinase interacting serine/threonine kinase, and matrix metalloproteinase 2, among others (11, 12, 14, 28, 29). Many of these studies investigate the importance of YB1 protein without specifically testing the role of Ser102 phosphorylation. Although previous publications have indi- cated that YB1 is critical for these widespread roles, our results with LJH685 and LJI308 suggest that the importance of phosphorylation at Ser102 may be more nuanced.
Another intriguing possibility is that additional RSK substrates or the cell growth context may influence the functional consequence of inhibiting phosphorylation of YB1. For instance, we find that in the subgroup of sensitive cell lines, phosphorylation of S6RP, a component of the 40S ribosome, which plays an integral role in protein translation, is mediated by RSK preferentially in an anchorage-indepen- dent setting and correlates with the loss of cell viability under these growth conditions. Furthermore, we find that RSK and mTOR both contribute to phosphorylation of S6RP even in cells grown under attached conditions, pointing to conver- gence of signaling inputs. This result is consistent with the recent finding by Carriere and colleagues (23) that RSK is a key activator of mTORC1 complex protein RAPTOR. Although we find YB1 phosphorylation to be predominantly controlled by RSK under both anchorage-dependent and anchorage-independent growth conditions, RSK substrates such as S6RP may be redundantly phosphorylated by other kinases in an anchorage-dependent context but exclusively regulated by RSK in the anchorage-independent setting.
Interestingly, even within the subgroup of cell lines sensitive to LJH685, we detect differences in their depen-
dency on RSK for survival, attesting to the high variability of RSK involvement in cellular survival mechanisms. Our study suggests that a role for RSK signaling in anchorage- independent survival persists in certain cancer contexts, but cannot be generalized to apply wherever there is mutational activation of the MAPK signaling cascade. The effects of signaling pathway cross-talk and incomplete functional dependence on a single protein phosphoryla- tion may mask the subtle roles of the substrate phosphor- ylation of RSK.
Disclosure of Potential Conflicts of Interest
J. Lan is a research investigator III with Novartis. No potential conflicts of interest were disclosed by the other authors.
Authors’ Contributions
Conception and design: I. Aronchik, B.A. Appleton, K. Crawford, J. Lan,
M.K. Lindvall, E. Venetsanakos, C.M. Shafer, A.B. Jefferson
Development of methodology: S.E. Basham, K. Crawford, L.V. Doyle, C.A. Luu Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): I. Aronchik, B.A. Appleton, S.E. Basham, M. Del Rosario, L.V. Doyle,
W.F. Estacio, E. Ornelas, A.B. Jefferson Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- tational analysis): I. Aronchik, B.A. Appleton, S.E. Basham, W.F. Estacio,
M.K. Lindvall, A.B. Jefferson
Writing, review, and/or revision of the manuscript: I. Aronchik, B.A. Appleton,
S.E. Basham, K. Crawford, W.F. Estacio, C.A. Luu, E. Venetsanakos, C.M. Shafer,
A.B. Jefferson Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Del Rosario, C.A. Luu
Study supervision: S.E. Basham
Grant Support
The Berkeley Center for Structural Biology is supported in part by the NIH, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, and Office of Basic Energy Sciences of the U.S. Department of Energy under contract no. DE-AC02-05CH112311.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received November 14, 2013; revised January 27, 2014; accepted February 5,
2014; published OnlineFirst February 20, 2014.
References
1. Doehn U, Hauge C, Frank SR, Jensen CJ, Duda K, Nielsen JV, et al. RSK is a principal effector of the RAS–ERK pathway for eliciting a coordinate promotile/invasive gene program and phenotype in epithelial cells. Mol Cell 2009;35:511–22.
2. Zhang Z, Liu R, Townsend PA, Proud CG. p90(RSK)s mediate the activation of ribosomal RNA synthesis by the hypertrophic agonist phenylephrine in adult cardiomyocytes. J Mol Cell Cardiol 2013; 59:139–47.
3. Saha M, Carriere A, Cheerathodi M, Zhang X, Lavoie G, Rush J, et al. RSK phosphorylates SOS1 creating 14-3-3-docking sites and nega- tively regulating MAPK activation. Biochem J 2012;447:159–66.
4. Li P, Goto H, Kasahara K, Matsuyama M, Wang Z, Yatabe Y, et al. P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation. Mol Biol Cell 2012;23:1582–92.
5. Lee S, Shuman JD, Guszczynski T, Sakchaisri K, Sebastian T, Cope- land TD, et al. RSK-mediated phosphorylation in the C/EBP{beta} leucine zipper regulates DNA binding, dimerization, and growth arrest activity. Mol Cell Biol 2010;30:2621–35.
6. Eliseeva IA, Kim ER, Guryanov SG, Ovchinnikov LP, Lyabin DN. Y-box- binding protein 1 (YB-1) and its functions. Biochemistry 2011;76: 1402–33.
7. Evdokimova V, Tognon C, Ng T, Ruzanov P, Melnyk N, Fink D, et al. Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial–mesenchymal transition. Cancer Cell 2009;15:402–15.
8. Evdokimova V, Ovchinnikov LP, Sorensen PH. Y-box binding protein 1: providing a new angle on translational regulation. Cell Cycle 2006; 5:1143–7.
9. Lyabin DN, Eliseeva IA, Skabkina OV, Ovchinnikov LP. Interplay between Y-box-binding protein 1 (YB-1) and poly(A) binding protein (PABP) in specific regulation of YB-1 mRNA translation. RNA Biol 2011;8:883–92.
10. Mouneimne G, Brugge JS. YB-1 translational control of epithelial– mesenchyme transition. Cancer Cell 2009;15:357–9.
11. Stratford AL, Fry CJ, Desilets C, Davies AH, Cho YY, Li Y, et al. Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells. Breast Cancer Res 2008;10: R99.
12. Astanehe A, Finkbeiner MR, Krzywinski M, Fotovati A, Dhillon J, Berquin IM, et al. MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibi- tion. Oncogene 2012;31:4434–46.
13. Basaki Y, Hosoi F, Oda Y, Fotovati A, Maruyama Y, Oie S, et al. Akt- dependent nuclear localization of Y-box-binding protein 1 in acquisi- tion of malignant characteristics by human ovarian cancer cells. Oncogene 2007;26:2736–46.
14. Stratford AL, Habibi G, Astanehe A, Jiang H, Hu K, Park E, et al. Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy. Br Cancer Res 2007;9:R61.
15. Cohen MS, Hadjivassiliou H, Taunton J. A clickable inhibitor reveals context-dependent autoactivation of p90 RSK. Nat Chem Biol 2007;3:156–60.
16. Sapkota GP, Cummings L, Newell FS, Armstrong C, Bain J, Frodin M, et al. BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo. Biochem J 2007;401:29–38.
17. Frodin M. A RSK kinase inhibitor reporting its selectivity in vivo. Nat Chem Biol 2007;3:138–9.
18. Nyfeler B, Bergman P, Triantafellow E, Wilson CJ, Zhu Y, Radetich B, et al. Relieving autophagy and 4EBP1 from rapamycin resistance. Mol Cell Biol 2011;31:2867–76.
19. Barrett SD, Bridges AJ, Dudley DT, Saltiel AR, Fergus JH, Flamme CM, et al. The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901. Bioorganic Med Chem Lett 2008;18:6501–4.
20. Yap TA, Yan L, Patnaik A, Fearen I, Olmos D, Papadopoulos K, et al. First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors. J Clin Oncol 2011;29:4688–95.
21. Martinez-Botella G, Hale MR, Maltais F, Tang Q, Straub J, Vertex Pharmaceuticals Inc. Pyrrole compounds as inhibitors of erk protein kinase, synthesis thereof and intermediates thereto. United States patent WO 2005113541 A1 2005 May 13.
22. Roux PP, Shahbazian D, Vu H, Holz MK, Cohen MS, Taunton J, et al. RAS/ERK signaling promotes site-specific ribosomal protein S6 phos- phorylation via RSK and stimulates cap-dependent translation. J Biol Chem 2007;282:14056–64.
23. Carriere A, Cargnello M, Julien LA, Gao H, Bonneil E, Thibault P, et al. Oncogenic MAPK signaling stimulates mTORC1 activity by promoting RSK-mediated raptor phosphorylation. Curr Biol 2008; 18:1269–77.
24. Xian W, Pappas L, Pandya D, Selfors LM, Derksen PW, de Bruin M, et al. Fibroblast growth factor receptor 1-transformed mammary epithelial cells are dependent on RSK activity for growth and survival. Cancer Res 2009;69:2244–51.
25. Oh YT, Liu X, Yue P, Kang S, Chen J, Taunton J, et al. ERK/ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expres- sion through co-activation of CHOP and Elk1. J Biol Chem 2010;285:41310–9.
26. De Cesare D, Jacquot S, Hanauer A, Sassone-Corsi P. Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene. Proc Natl Acad Sci U S A 1998;95:12202–7.
27. Fujita N, Sato S, Tsuruo T. Phosphorylation of p27Kip1 at threonine 198 by p90 ribosomal protein S6 kinases promotes its binding to 14-3- 3 and cytoplasmic localization. J Biol Chem 2003;278:49254–60.
28. Matsumoto K, Abiko S, Ariga H. Transcription regulatory complex including YB-1 controls expression of mouse matrix metalloprotei- nase-2 gene in NIH3T3 cells. Biol Pharm Bull 2005;28:1500–4.
29. Gu C, Oyama T, Osaki T, Kohno K, Yasumoto K. Expression of Y box- binding protein-1 correlates with DNA topoisomerase IIalpha and proliferating cell nuclear antigen expression in lung cancer. Anticancer Res 2001;21:2357–62.