Mechanistic analysis of an extracellular signal–Regulated kinase 2–Interacting compound that inhibits mutant BRAF-expressing melanoma cells by inducing oxidative stress

Article abstract of DOI:10.1124/jpet.120.000266

Constitutively active extracellular signal–regulated kinase (ERK) 1/2 signaling promotes cancer cell proliferation and survival. We previously described a class of compounds containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-1–mediated transcription and growth of cancer cells containing active ERK1/2 due to mutations in Ras G-proteins or BRAF, Proto-oncogene B-RAF (Rapidly Acclerated Fibrosarcoma) kinase. The current study identified chemical features required for biologic activity and global effects on gene and protein levels in A375 melanoma cells containing mutant BRAF (V600E). Saturation transfer difference-NMR and mass spectrometry analyses revealed interactions between a lead compound (SF-3-030) and ERK2, including the formation of a covalent adduct on cysteine 252 that is located near the docking site for ERK/FXF (DEF) motif for substrate recruitment. Cells treated with SF-3-030 showed rapid changes in immediate early gene levels, including DEF motif–containing ERK1/2 substrates in the Fos family. Analysis of transcriptome and proteome changes showed that the SF-3-030 effects overlapped with ATP-competitive or catalytic site inhibitors of MAPK/ERK Kinase 1/2 (MEK1/2) or ERK1/2. Like other ERK1/2 pathway inhibitors, SF-3-030 induced reactive oxygen species (ROS) and genes associated with oxidative stress, including nuclear factor erythroid 2–related factor 2 (NRF2). Whereas the addition of the ROS inhibitor N-acetyl cysteine reversed SF-3-030–induced ROS and inhibition of A375 cell proliferation, the addition of NRF2 inhibitors has little effect on cell proliferation. These studies provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a ROS-dependent mechanism.

Full text of DOI:10.1124/jpet.120.000266

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Title Page:
Mechanistic analysis of an ERK2-interacting compound that inhibits
mutant-BRAF expressing melanoma cells by inducing oxidative stress
Ramon Martinez III1, Weiliang Huang1, Ramin Samadani, Bryan Mackowiak, Garrick Centola,
Lijia Chen, Ivie L. Conlon, Kellie Hom, Maureen A. Kane, Steven Fletcher, and Paul Shapiro*
Department of Pharmaceutical Sciences, University of Maryland-Baltimore School of Pharmacy
20 N. Penn St., Baltimore, Maryland 21201 USA
1Authors contributed equally
*Correspondence
1

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a. running title: Mechanisms of a novel inhibitor of active ERK1/2 signaling.
b.
Paul Shapiro
20 Penn St., Room 561, Baltimore, MD 21201
Telephone: 410-706-8522
FAX: 410-706-5017
Email: pshapiro@rx.umaryland.edu
c. Number of:
Text pages: 28
Tables: 3
Figures: 6
References: 88
Number of words:
Abstract: 249
Introduction: 738
Discussion: 1470
d. Nonstandard abbreviations: ERK1/2, extracellular signal-regulated kinase-1/2; IEG,
immediate early genes; AP-1, activator protein-1; NRF2, nuclear factor erythroid 2-related factor
2; MAP3K8, mitogen-activated protein kinase kinase kinase-8; ROS, reactive oxygen species.
2

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Abstract
Constitutively active extracellular signal-regulated kinase (ERK1/2) signaling promotes cancer
cell proliferation and survival. We previously described a class of compounds containing a 1,1-
dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that targeted ERK2 substrate
docking sites and selectively inhibited ERK1/2-dependent functions, including activator protein-
1 (AP-1)-mediated transcription, and growth of cancer cells containing active ERK1/2 due to
mutations in Ras G-proteins or BRAF kinase. The current study identified chemical features
required for biological activity and global effects on gene and protein levels in A375 melanoma
cells containing mutant BRAF (V600E). STD-NMR and mass spectrometry analyses revealed
interactions between a lead compound (SF-3-030) and ERK2 including the formation of a
covalent adduct on cysteine 252 located near the docking site for ERK, FXF (DEF) motif for
substrate recruitment. Cells treated with SF-3-030 showed rapid changes in immediate early
gene (IEG) levels including DEF motif containing ERK1/2 substrates in the Fos family. Analysis
of transcriptome and proteome changes showed that the SF-3-030 effects overlapped with ATP-
competitive or catalytic site inhibitors of MEK1/2 or ERK1/2. Like other ERK1/2 pathway
inhibitors, SF-3-030 induced reactive oxygen species (ROS) and genes associated with oxidative
stress, including nuclear factor erythroid 2-related factor 2 (NRF2). Whereas the addition of the
ROS inhibitor N-acetyl cysteine reversed SF-3-030 induced ROS and inhibition of A375 cell
proliferation, the addition of NRF2 inhibitors have little effect on cell proliferation. These studies
provide mechanistic information on a novel chemical scaffold that selectively regulates ERK1/2-
targeted transcription factors and inhibits the proliferation of A375 melanoma cells through a
ROS-dependent mechanism.
3

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Significance Statement
Constitutive activation of the extracellular signal-regulated kinase (ERK1/2) pathway drives the
proliferation and survival of many cancer cell types. Given the diversity of cellular functions
regulated by ERK1/2, the current studies have examined the mechanism of a novel chemical
scaffold that targets ERK2 near a substrate binding site and inhibits select ERK functions. Using
transcriptomic and proteomic analyses, we provide a mechanistic basis for how this class of
compounds inhibits melanoma cells containing mutated BRAF and active ERK1/2.
4

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Introduction
De-regulated protein kinase activity that promotes cell proliferation and survival is a
hallmark of many cancers. Thus, targeted inhibition of protein kinases with small molecules have
become an important therapeutic strategy to treat cancer (Wu et al., 2016). The extracellular
signal-regulated kinase-1 and 2 (ERK1/2) proteins are members of the mitogen activated protein
kinase (MAPK) family and major regulators of intracellular signaling events that provide cells
with a proliferation and survival advantage. Constitutive activation of ERK1/2 occurs through
mutations or elevated expression of upstream activators, including receptor tyrosine kinases
(RTKs), Ras G-proteins, and Raf kinases (Mendelsohn and Baselga, 2006; Bennasroune et al.,
2004; Reuter et al., 2000; Shapiro, 2002; Arora and Scholar, 2005). Activating mutations in
MEK1, a direct activator of the ERK1/2 proteins, may contribute to de-regulated ERK1/2
activity (Emery et al., 2009). In addition, activating mutations in a conserved glutamate in a
region of ERK2 associated with protein-protein interactions are associated with the progression
of cutaneous T-cell lymphomas (Mahalingam et al., 2008; da Silva Almeida et al., 2015; Arvind
et al., 2005). A number of small molecular weight anti-cancer drugs targeting the ATP binding or
catalytic sites on RTKs, BRAF, and MEK1/2 are being used in the clinic to block ERK1/2
signaling (Flaherty and McArthur, 2010; Zhong and Bowen, 2011; Wu et al., 2015). Despite
promising initial responses, resistance to these kinase inhibitors is often observed and subsequent
therapeutic options are limited. Drug resistance occurs through several mechanisms, including
acquired mutations in the kinase catalytic site targeted by the compounds, activation of
alternative mechanisms to turn on proteins downstream of the targeted kinase, or increases in
compensatory signaling pathways that provide a survival advantage independent of the kinase
being targeted (Poulikakos and Solit, 2011; Poulikakos and Rosen, 2011; Fedorenko et al., 2015;
5

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Tuma, 2011). Recent efforts aimed at overcoming inherent or acquired resistance to existing
kinase inhibitors are directly targeting ERK1/2 proteins (Morris et al., 2013; Maik-Rachline and
Seger, 2016; Germann et al., 2017) and highlight the need to identify alternative approaches to
inhibit constitutively active kinase signaling pathways that promote drug resistance and drive the
survival of cancer cells.
Using computational modeling and biological testing, we have previously identified small
molecules that are designed to selectively inhibit ERK1/2 signaling by disrupting specific
substrate interactions (Samadani et al., 2015; Hancock et al., 2005; Chen et al., 2006; Boston et
al., 2011). This approach targeted two defined substrate docking sites on ERK2; the D-
recruitment site (DRS) or F-recruitment site (FRS) that interact with substrates containing D-
domains or DEF motifs, respectively (Burkhard et al., 2011; Lee et al., 2011; Sammons et al.,
2019). Previous studies suggest that most ERK1/2 substrates contain D-domains, although some
substrates, such as the ternary complex factor Elk-1, contain both a D-domain and a DEF motif
(von Kriegsheim et al., 2009). Several transcription factors that promote cancer cell growth,
including c-Myc and Fos family proteins contain the DEF motif, which is characterized by a
phenylalanine-any amino acid-phenylalanine (FXF) sequence that is 6-10 amino acids away
from the serine or threonine phosphorylation site. Hydrophobic interactions between the FXF
motif and the recruitment site on ERK1/2 facilitate kinase-substrate interactions necessary for
efficient phosphoryl transfer. To selectively target DEF motif containing transcription factors
that promote tumor growth, we previously described the identification of a class of compounds
containing a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate scaffold that inhibited
DEF motif containing substrates of the activator protein-1 (AP-1) family (Samadani et al., 2015).
This class of compounds selectively inhibited the proliferation of melanoma cells harboring
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BRAF mutations and constitutively active ERK1/2. These compounds were also effective at
inhibiting melanoma cell lines that are inherently resistant to clinically relevant inhibitors of
mutated BRAF due to elevated mitogen-activated protein kinase kinase kinase-8 (MAP3K8)
(Samadani et al., 2015).
To define the structure activity relationship for the 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-
benzenesulfonate-based compounds, the current studies synthesized several analogues and
identified a single double bond in the sulfur heterocycle that was essential for the compound’s
biological activity. The findings also demonstrate that this class of compounds forms a covalent
interaction with ERK2 near the FRS and have differential effects on the expression levels of
ERK1/2-regulated transcription factors containing DEF motifs. The most active compound, SF-
3-030, was examined for global effects on gene and protein levels to evaluate ERK1/2-dependent
and independent mechanisms of action. The findings support a mechanism by which SF-3-030
induced an oxidative stress response that mediated the inhibition of melanoma cells containing
mutated and constitutively active BRAF.
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Materials and Methods
Compound synthesis: All reactions were performed in oven-dried glassware under an inert (N2)
atmosphere. Anhydrous solvents were used as supplied without further purification. Chemicals
were purchased from Sigma-Aldrich (St. Louis, MO), Alfa Aesar (Ward Hill, MA), or Oakwood
Chemicals (West Columbia, SC). Reactions were monitored by thin-layer chromatography
(TLC), visualizing with KMnO4 stain and/or UV as appropriate. Silica gel flash column
chromatography was performed with 60Å, 230-400 mesh silica gel, and crude reaction mixtures
1
were first adsorbed onto silica gel from CH2Cl2 at room temperature (RT). H and 13C NMR
spectra were recorded on a Varian 400 MHz NMR spectrometer (Bruker amaZon X) at 25 °C.
Chemical shifts are reported in parts per million (ppm) and are referenced to residual non-
deuterated solvent peak (CHCl3: δH 7.26, δC 77.2; DMSO: δH 2.50, δC 39.5). Prior to biological
testing, final compounds were confirmed to be >95% pure by HPLC chromatography. Detailed
methods of the chemical synthesis are provided in the supplemental data.
Chemicals and antibodies: The MEK1/2 inhibitor AZD6244 (Cat. No. BV-2234-5), 6-(4-
bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide),
was purchased from Axxora (Farmingdale, NY). The ERK1/2 inhibitor, SCH772984 (Cat. No.
S7101), ((R)-1-(2-oxo-2-(4-(4-(pyrimidin-2-yl)phenyl)piperazin-1-yl)ethyl)-N-(3-(pyridin-4-yl)-
1H-indazol-5-yl)pyrrolidine-3-carboxamide) was purchased from Selleckchem (Houston, TX).
Antioxidants N-acetyl L-cysteine (NAC) (Cat. No. A7250), sodium pyruvate (Cat. No. P2256),
and mannitol (Cat. No. M4125) were purchased from Sigma Aldrich (St. Louis, MO). NRF2
inhibitors ML385 (Cat. No. SML 1833) and Brusatol (Cat. No. SML 1868) were purchased from
Sigma Aldrich. Antibodies against total c-Fos (Cat. No. 2250), FosB/B2 (Cat. No. 2251), Fra1
(Cat. No. 5281), c-Jun (Cat. No. 9165), c-Myc (Cat. No. 5605), Elk-1 (Cat. No. 9182), ß-actin
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(Cat. No. 4970) and cleaved PARP (Cat. No. 9541) were purchased from Cell Signaling
Technology (Beverly, MA). The phospho-specific ERK1/2 (pThr183/pTyr185)(Cat. No. M9692)
and α-tubulin (Cat. No. T6074) antibodies were from Sigma Aldrich. Phosphorylation-specific
antibodies against histone H3 (pSer10)(Cat. No. 9701), Elk-1 (pS383)(Cat. No. 9186), MEK1/2
(pSer217/pSer221)(Cat. No. 9121) were purchased from Cell Signaling Technology. Antibodies
against ERK2 (Cat. No. sc-154), MEK 1/2 (Cat. No. sc-81504), HMOX-1 (Cat. No. sc-136960)
and NRF2 (Cat. No. sc-722) were from Santa Cruz Biotechnology. A second NRF2 antibody
(Cat. No. ab 137550), and the NQO1 antibody (Cat. No. ab34173) were purchased from Abcam
(Cambridge, MA). The FoxD3 (Cat. No. 631701) antibody was purchased from BioLegend (San
Diego CA). The OSGIN-1(Cat. No. 15248-1-AP), GCLM (Cat. No. 14241-1-AP), SRXN1 (Cat.
No. 14273-1-AP), GCLC (Cat. No. 12601-1-AP), AKR1B10 (Cat. No. 18252-1-AP), G6PD
(Cat. No. 66373-1-Ig), and TXNRD1 antibodies (Cat. No. 11117-1-AP) were purchased from
ProteinTech (Rosemont, IL). The xCT antibody (Cat. No. NB300-317SS) was purchased from
Novus Biologicals (Centennial, CO).
Cell lines: Cell lines used included HeLa cervical carcinoma cells, Jurkat T-cell leukemia cells,
and NRas mutated promyelocytic leukemia HL-60 cells. Melanoma cell lines tested included
A375 and SK-Mel-28 (homozygous BRAF V600E mutation), SK-MEL-5 (heterozygous BRAF
V600E mutation), SK-Mel-2 (BRAF wild type, NRas Q61R mutation), and RPMI-7951 cells
(heterozygous BRaf mutation), which are inherently resistant to BRAF inhibitors due to elevated
MAP3K8 (Johannessen et al., 2010). Cells were purchased from American Type Culture
Collection (ATCC; Manassas, VA) and grown in DMEM or EMEM plus 10% FBS. All media
were supplemented with penicillin and streptomycin. The cell lines have been authenticated by
evaluating short tandem repeat DNA profiles and matching with the ATCC database. Cell lines
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are routinely tested for mycoplasma contamination using the MycoAlert detection kit (Lonza;
Walkersville, MD).
Cell viability/apoptosis assays: Cells were seeded at 5,000 cells/well in 96-well plates, cultured
overnight, and treated for 48 hours with the indicated dose of compounds. IC50 values were
determined using 9 data points and 3 fold dilutions of 0.001 – 10 M for AZD6244 and
SCH772984 or 0.1 – 100 M for SF-3-030. Cell viability was measured according to
manufacturer’s instructions using the fluorescent CellTiter Blue Assay (Promega; Madison,
WI) or the CellTiter-Glo® Luminescent Cell Viability Assay (Promega; Madison, WI). To
determine the minimal exposure time needed to induce an apoptotic response, cells were exposed
to test compounds for various times (0-24 hours), the cells were washed, and then fresh medium
was added without test compound for a total of 24 hours. Relative apoptosis was measured by
immunoblotting for the cleaved form of poly (ADP-ribose) polymerase (PARP).
Reactive oxygen species measurements: Reactive oxygen species (ROS) were measured using
the cell-permeable fluorogenic reagent CellROX Deep Red according to manufacturer’s
instructions (Thermo Fisher Scientific; Waltham, MA). Briefly, A375 melanoma cells were
seeded at 104 cells per well in a 96-well plate. After 24 hours, the cells were co-incubated with
SF-3-030 ± various ROS inhibitors, as well as 5 µM of CellROX Deep Red reagent for 1 hour at
37°C. Subsequently, cells were washed three times with DMEM containing no Phenol Red. Live
cell fluorescence was imaged using a Nikon Eclipse Ti confocal microscope, using a 10X
objective magnification. Total fluorescent intensity was obtained in triplicate wells from 4
frames of view per well (~500-1000 cells/frame) with a biological duplicate performed for each
treatment condition. The mean ± standard deviations were determined using NIS-Elements
analysis software.
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RNAseq analysis: A375 cells were treated for 1 hour with 10 M AZD6244 or 25 M SF-3-030
and total RNA was isolated (RNAeasy, Qiagen; Hilden, Germany) from treated and untreated
cells in duplicate samples. Differential expression analysis was done by GENEWIZ (South
Plainfield, NJ.) using Illumina Genome Analyzer and HiSeq instruments in the FastQ format as
previously described (Anders et al., 2013). Data were analyzed using Ingenuity Pathway
Analysis software. Changes in expression were shown for genes with false discovery rates of <
0.05.
Immunoassays: Immunoblot analysis of protein expression, phosphorylation, and cell
proliferation were done as previously described (Samadani et al., 2015). Cells were washed with
cold phosphate buffered saline (PBS, pH 7.2, Invitrogen; Carlsbad, CA) and protein lysates were
collected with 2X SDS-PAGE sample buffer (4% SDS, 5.7M -mercaptoethanol, 0.2M Tris pH
6.8, 20% glycerol, 5mM EDTA) Proteins were separated by SDS-PAGE, analyzed by
immunoblotting, and detected using enhanced chemiluminesence (PierceTM ECL, Thermo Fisher
Scientific). Immunoblots were quantified via densitometry using the Azure c300
Chemiluminsecent Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA) for
image capture and data quantified on the Image StudioTM Lite Quantification software (v5.2, Li-
COR Biotechnology, Lincoln, NE, USA). Immunoassays were also performed using Wes™
Simple Western (ProteinSimple, San Jose, CA, USA). Electropherograms were quantified using
Compass 225 for SW software (v3.1.7, ProteinSimple, San Jose, CA, USA) applying a Gaussian
peak fit distribution for determining area under the curve.
Covalent modification analysis by high resolution liquid chromatography-tandem mass
spectrometry: His-tagged ERK2 was purified from E. coli BL21 (DE3) cells transformed with
wild type erk2 construct using the previously described method (Burkhard et al., 2011). For
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covalent modification analysis, in vitro kinase reactions containing 100 µg of purified ERK2,
1mM ATP, 1X NEBuffer™ for Protein Kinases (New England Biolabs, Ipswich, MA), and 50
µM SF-3-030 were incubated for 2 hours at 25°C. After the reactions, ERK2 protein was
desalted, reduced, alkylated and trypsinolyzed on filter as described previously (Wisniewski et
al., 2009; Erde et al., 2014). Tryptic peptides were separated on a nanoACQUITY UPLC
analytical column (BEH130 C18, 1.7 μm, 75 μm x 200 mm, Waters Corporation; Milford, MA)
over a 165-minute linear acetonitrile gradient (3 – 40%) with 0.1 % formic acid on a Waters
nano-ACQUITY UPLC system (Waters Corporation; Milford , MA) and analyzed on a coupled
Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific; San
Jose, CA) as described (Williamson et al., 2016). Full scans were acquired at a resolution of
120,000, and precursors were selected for fragmentation by higher-energy collisional
dissociation (normalized collision energy at 32 %) for a maximum 3-second cycle. Tandem mass
spectra were searched against the ERK2 protein sequence using a Sequest HT algorithm (Eng et
al., 2008) and a MS Amanda algorithm (Dorfer et al., 2014) with a maximum precursor mass
error tolerance of 10 ppm. Possible substitution (SN2’ and SN2, +115.9932), Michael addition
(+324.0126) and carbamidomethylation of cysteine were treated as dynamic modifications.
Resulting hits were validated at a maximum false discovery rate of 0.01 using a semi-supervised
machine learning algorithm Percolator (Kall et al., 2007). The probabilities of modification sites
were computed using a ptmRS algorithm (Taus et al., 2011).
Saturation Transfer Difference-Nuclear Magnetic Resonance (STD-NMR) analysis: STD-
NMR analysis of ligand binding to ERK2 as was done previously described for p38 MAP
kinases (Shah et al., 2017). A 1 mM stock solution of SF-3-030 was made in 85% D2O:15% d6-
DMSO (v/v). STD-NMR samples contained 150 mM NaCl, 50 mM phosphate (pH 7), 200 μM
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SF-3-030, and 5 μM ERK2 protein in D2O. Spectra of both compound and ligand bound protein
were recorded on an Agilent DD2 500-MHz spectrometer equipped with a 5-mm inverse proton–
fluorine–carbon–nitrogen probe head at 25°C. Further detailed methods of the NMR protocol
used are provided in the supplemental data.
Differential protein expression by high-resolution liquid chromatography tandem mass
spectrometry: A375 cells grown on 10 cm plates were treated for 4 and 12 hours with 0.1%
DMSO vehicle, 25 M SF-3-030 or 10 M SCH772984. Following one wash in cold PBS, the
cells were collected by scraping twice with cold PBS, centrifuged at 3000 rpm for 2 min. and the
cell pellets were stored at -80°C. Cells were lysed in 4 % sodium deoxycholate, reduced,
alkylated and trypsinolyzed on filter as described (Wisniewski et al., 2009). Tryptic peptides
were separated on a nanoACQUITY UPLC analytical column (CSH130 C18, 1.7 μm, 75 μm x
200 mm, Waters Corporation; Milford, MA) over a 180 min linear acetonitrile gradient (3 –
43%) with 0.1 % formic acid on a Waters nano-ACQUITY UPLC system (Waters Corporation;
Milford, MA), and analyzed on a coupled Thermo Scientific Orbitrap Fusion Tribrid mass
spectrometer (Thermo Scientific; San Jose, CA) as described (Williamson et al., 2016). Full
scans were acquired at a resolution of 120,000 and precursors were selected for fragmentation by
higher-energy collisional dissociation (normalized collision energy at 30%) for a maximum 3-
second cycle. Tandem mass spectra were searched against a UniProt human reference proteome
using a SEQUEST HT algorithm (Eng et al., 2008) with a maximum precursor mass error
tolerance of 10 ppm. Resulting hits were validated at a maximum false discovery rate of 0.01
using a semi-supervised machine learning algorithm Percolator (Kall et al., 2007). Abundance
ratios were measured by comparing the MS1 peak volumes of peptide ions, whose identities
were confirmed by MS2 sequencing as described above. Label-free quantifications were
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performed using an aligned AMRT (Accurate Mass and Retention Time) cluster quantification
algorithm (Qi et al., 2012). Pathway and gene ontology analysis were performed with Qiagen
Ingenuity and Panther GO databases, as described (Kramer et al., 2014; Mi et al., 2017). Proteins
showing at least a doubling in expression as compared to untreated cells, with an FDR adjusted
ANOVA p-value <0.05, were considered significantly changed and used for further analysis.
FDR corrected Fisher's exact p-values of < 0.05 using a previously described procedure for
multiple testing were used in the gene ontology analyses to identify biological processes,
molecular functions, and cellular components associated with observed protein changes
(Hochberg, 1995). Ingenuity pathway database was used to predict canonical pathways and
upstream regulators according to the proteins that were significantly different using an absolute
activation z-score of > 2 for at least one condition with a Fisher's exact test p-value < 0.05.
Lactate dehydrogenase (LDH)/cytotoxicity assay: Cells were seeded 5,000 cells/well in 96-
well plates, cultured overnight, and treated for 48 hours with the indicated dose of compound SF-
3-030. Cytotoxicity via LDH release was measured according to manufacturer’s instructions
using the CyQUANTTM LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific: Waltham, MA).
Statistical analysis: Graphical statistical analysis was performed using a one-tailed ANOVA
with a 95% confidence interval using GraphPad-Prism V.5.01. Three biological replicates were
chosen for data subjected to statistical analysis. The data represent descriptive statistics and were
used to summarize key findings. Results were considered statistically significant if the p-value is
less than 0.05.
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Results
The double bond in the sulfur heterocycle is required for biological activity. Our previous
studies described a novel class of small molecules containing a 1,1-dioxido-2,5-dihydrothiophen-
3-yl 4-benzenesulfonate scaffold that selectively inhibited BRAF mutated melanoma cells
containing constitutively active ERK1/2 signaling (Samadani et al., 2015). A limited structure-
activity relationship study was performed to determine key chemical features required for the
compound’s biological activity. Modifications of the parent compound, SF-3-026, focused on the
benzene ring, the linker region connecting the two ring structures, and removal of the double
bond in the sulfur heterocycle (Fig. 1). The analogues were tested at a single high dose for
effects on viability of cancer cell lines containing BRAF mutations (A375, RPMI-7951, and SK-
Mel-28), NRas mutation (HL60), or no known mutations in ERK1/2 pathway proteins (HeLa and
Jurkat) (Fig. 1). RPMI-7951 cells are inherently resistant to BRAF targeted inhibitors due to
upregulated expression of MAP3K8 (Johannessen et al., 2010). In agreement with our previous
studies (Samadani et al., 2015), SF-3-026, SF-3-027, SF-3-029, and SF-3-030 showed selective
inhibition of cancer cell lines with activating mutations in the ERK1/2 pathway (Fig. 1). The
inhibitory effects were lost in compounds where the double bond in the sulfur heterocycle was
removed (Fig. 1). Select modifications to the benzene group or linker in the absence of the
double bond in the sulfur heterocycle had no effect on cell viability indicating the 1,1-dioxido-
2,5-dihydrothiophen-3-yl moiety was essential for the compound’s biological activity (Fig. 1).
Previous studies showed that catalytic site inhibitors of MEK1/2 and ERK1/2 are selective
for cells with activating BRAF or Ras mutations (Yeh et al., 2007; Morris et al., 2013).
Similarly, we determined the IC50 values for SF-3-030 and ATP dependent/catalytic site
inhibitors of MEK1/2 and ERK1/2 in four cancer cell lines and show that all compounds favored
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inhibition of cells containing activating BRAF and Ras mutations (Supplemental Table 1). The
reported doubling times for A375, HeLa, and Jurkat cells are ~25-30 hours compared to ~50
hours for the RPMI-7951 cells indicating that each compound’s potency is independent of cell
growth rates. These findings provide additional support that SF-3-030 inhibitory effects on cell
growth are correlated with ERK1/2 activity.
The effects of SF-2-110 (inactive control), SF-3-026, and SF-3-030 (Supplemental Fig. 1A)
on cell viability were further evaluated in dose – response assays using four melanoma cell lines
containing either BRAF or NRas mutations. As expected, SF-2-110 did not affect the viability of
any of the cell lines up to 50 M (Supplemental Figs. 1B-E). In contrast, SF-3-026 and SF-3-030
inhibited all cell lines with IC50 values in the 5-10 M range with SF-3-030 showing the highest
potency against SK-MEL-28 and SK-MEL-2 cells (Supplemental Figs. 1B-E). These findings
indicate that the sensitivity of cancer cells in vitro to the lead compounds is independent of the
BRAF or NRas mutational status.
SF-3-030 directly interacts with ERK2 through non-covalent and covalent mechanisms. Given
the higher inhibitory potency of SF-3-030, we evaluated whether this compound directly
interacted with ERK2. Using Saturation Transfer Difference-Nuclear Magnetic Resonance
(STD-NMR), it was confirmed that SF-3-030 interactions with ERK2 occur primarily through a
reversible interaction of the naphthalene group and the sulfur heterocycle (Supplement Fig. 2).
However, the requirement for the double bond in the sulfur heterocycle of SF-3-030 for
biological activity suggested that the compound’s mechanism of action involves the formation of
covalent adducts with cysteine residues. Two possible SF-3-030 adducts could result from a
Michael addition or a substitution reaction that would increase the molecular weight of a cysteine
by 324 or 116 Da, respectively (Fig. 2A). To test this, ERK2 was incubated with SF-3-030 and
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covalent adducts were analyzed by high-resolution liquid chromatography-tandem mass
spectrometry. Of the seven cysteine residues in ERK2, cysteine 252 (C252) was the predominant
residue modified by a 116 Da mass, suggesting a substitution reaction and decomposition of SF-
3-030 (Fig. 2A/B & Supplemental Figure 3). C252 is located near the DEF-motif recruitment
site, which is consistent with its proposed mechanism of targeting DEF motif containing
substrates (Samadani et al., 2015). Other major MAP kinases such as p38α and JNK1 do not
have a cysteine located at this site. We have also previously determined that SF-3-030 had no
effect on p38 MAP kinase signaling (Samadani et al., 2015), suggesting that SF-3-030 is not
acting as a random alkylating agent or affecting similar MAP kinases. These data suggest that the
initial binding interactions of SF-3-030 with ERK2 involve non-covalent interactions that
position the compound to form a covalent bond between C252 and the sulfur heterocycle group.
SF-3-030 selectively regulates ERK-dependent immediate early gene expression. We next
examined relative protein levels of ERK1/2-regulated immediate early genes (IEG). Previously,
we showed that a 1 hour treatment with SF-3-030 caused differential changes in AP-1 protein
levels characterized by inhibition of Fra-1, FosB, and the alternative splice variant FosB2 but no
effect on c-Fos levels (Samadani et al., 2015). The relative levels of these proteins along with
other AP-1 members were examined in A375 cells treated for 0-24 hours with SF-3-030 or the
ERK1/2 catalytic site inhibitor SCH772984 as a positive control. Similar to our previous studies
(Samadani et al., 2015), a 1 hour treatment with SF-3-030 or SCH772984 had little effect on c-
Fos levels (Fig. 3A). However, after 2 hours treatment, c-Fos increased only with SF-3-030 and
persisted for up to 24 hours (Fig. 3A). Another major component of the AP-1 complex, c-Jun,
increased with either SF-3-030 or SCH772984 treatments (Fig. 3A). It should be noted that the
increased levels of c-Fos and c-Jun do not correlate with increased AP-1 activity, which we’ve
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shown to be inhibited in these and other cell lines treated with SF-3-030 (Samadani et al., 2015;
Defnet et al., 2019b).
Other Fos family members, including FosB/B2 and Fra-1 decreased in cells treated with
SCH772984 (Fig. 3A). Cells treated with SF-3-030 also showed decrease Fra-1 levels whereas
FosB/B2 levels transiently decreased followed by an increased after 4 hours as observed with c-
Fos (Fig. 3A). The levels of c-Myc, a potential target for treating melanoma (Polsky and Cordon-
Cardo, 2003; Korkut et al., 2015), showed a rapid inhibition by SF-3-030 when compared to
SCH772984 (Fig. 3A). Interestingly, c-Myc levels increased back to basal levels after 24 hour
treatment with SF-3-030 (Fig. 3A). However, this dose of SF-3-030 is lethal to ~90% of A375
cells after 48 hours exposure (Supplement Fig. 1B). A transient increase in the ERK1/2-mediated
phosphorylation of Elk-1, a ternary complex factor involved in regulating c-fos and other IEG
(Shaw and Saxton, 2003), was observed in SF-3-030 treated cells and correlated with ERK1/2
phosphorylation (Fig. 3B). However, there was no evidence that MEK1/2, the upstream activator
of ERK1/2, was activated indicating SF-3-030 was acting at the level of ERK1/2 (Supplemental
Fig. 4).
The dose-dependent effect of the active compounds SF-3-026 and SF-3-030 on IEG levels
were examined after a 4 hour exposure. The inactive control, SF-2-110, had no effect on c-Fos,
c-Jun, Fra-1, FosB/B2, or c-Myc levels (Fig. 3C). In contrast, SF-3-026 and SF-3-030 caused a
dose-dependent decrease in Fra-1, FosB/B2, and c-Myc protein levels but an increase in c-Jun
and c-Fos (Fig. 3C). The increased potency of SF-3-030 in affecting IEG protein levels
compared to the parent compound SF-3-026 is consistent with our previous studies (Samadani et
al., 2015). It is also noted that SF-3-030 at a dose near its IC50 (~5 M) for inhibition of cell
proliferation was as potent at inhibiting Fra-1 and c-Myc protein levels as the 5 M doses of
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AZD6244 or SCH77294 (Fig. 3C). Quantitative analysis of c-Fos, Fra-1, and c-Myc protein
levels after exposure with 25µM SF-2-110 or SF-3-030 is shown in Figure 3D. Together, these
findings demonstrate that the lead compounds can differentially affect ERK1/2-regulated
transcription factors.
It was next determined whether the changes in IEG expression observed after 1-2 hours with
SF-3-030 (Fig. 3A) coincided with the exposure time needed to induce an apoptotic response. In
these experiments, A375 cells were exposed to SF-3-030 for various times, washed to remove
excess compound, and then incubated with serum-supplemented growth medium for a total of 24
hrs. Apoptosis was evident after 2 hours exposure to SF-3-030 as measure by the appearance of
cleaved PARP (Fig. 3E). This exposure time also coincided with the loss of histone H3
phosphorylation at Ser10, which is a marker of mitosis and is observed during the transcription
of selective IEGs in response to ERK1/2 pathway activation (Sassone-Corsi et al., 1999). Loss of
histone H3 phosphorylation after 2 hours exposure to SF-3-030 suggests that mitotic progression
is inhibited and that phosphorylation of S10 is dispensable for regulating the transcription of
IEG, such as c-Fos and c-Jun as previously reported (Drobic et al., 2010).
SF-3-030 regulates ERK1/2-dependent and independent transcription. RNAseq analysis
was used to measure early changes in gene expression patterns and identify on- and off-target
effects of SF-3-030. Total RNA was isolated from A375 cells treated for 1 hour with an IC90
dose (25µM) of SF-3-030 or the MEK inhibitor AZD6244 (10 µM) as a positive control for
ERK1/2 pathway inhibition. The IC90 dose for SF-3-030 was used as it was determined to have
no general cytotoxicity in LDH assays (data not shown; (Defnet et al., 2019a)). Approximately
16,000 transcripts were identified in each condition and statistically significant changes (≥1.5
fold) in transcript expression between treated and untreated cells were reported. We identified 38
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or 68 transcripts in cells treated with SF-3-030 or AZD6244, respectively, which showed
significant decreases with 12 genes common to both treatments (Supplemental Table 2). ERK1/2
pathway targets involved in cell growth and survival that decreased with both treatments
included c-Myc, CTGF, SOX11, and GATA3. AZD6244 also inhibited transcription of c-Fos,
Egr-1, and IER3 genes, which are elevated in BRAF V600E mutated melanoma cells and may
promote ERK-mediated growth of these tumor types (Pratilas et al., 2009). The inhibition of c-
Myc by SF-3-030, AZD6244, and SCH772984 at the RNA and protein levels (Fig. 3) supports a
shared mechanism of targeting ERK-mediated signaling by these compounds.
The data also revealed that AZD6244 inhibited many negative regulators of the ERK1/2
pathway, including Sprouty (SPRY) and dual specificity phosphatases (DUSP) transcripts
(Supplemental Table 2). SPRY1 was the only negative regulator of ERK1/2 signaling that was
inhibited by both AZD6244 and SF-3-030 (Supplemental Table 2). Additional growth-related
genes that decreased only with AZD6244 treatment included regulators of epidermal growth
factor (EGF) receptor signaling including c-Fos, TGFA, and HBEGF (Supplemental Table 2). In
contrast, only SF-3-030 inhibited a unique set of growth and angiogenesis promoting genes
including CYR61 (also called IGF-binding protein 10), which may promote tumor progression
during hypoxia (Kunz et al., 2003), and BCL6, which has been associated with poor overall
survival of melanoma patients (Alonso et al., 2004).
There were 14 or 24 transcripts that increased >1.5 fold following treatment with
SF-3-030
or AZD6244, respectively, relative to untreated cells, and only one gene, CHMP4C, was
common to both treatments (Supplemental Table 2). CHMP4C is involved in sorting and
delivery of endosomal vesicles to the lysosome and in cell cycle checkpoint control during
cytokinesis (Carlton et al., 2012). Ingenuity Pathway Analysis (IPA) indicated that several genes
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upregulated in response to
treatment, including heme oxygenase-1 (HMOX1), c-Fos,
SF-3-030
c-Jun, and DnaJ homolog subfamily B member 1 (DNAJB1) (Supplemental Table 2), were
consistent with activation of a nuclear factor erythroid 2-related factor 2 (NRF2) mediated
oxidative stress response. However, given the exposure to
was only 1 hour, it is likely
SF-3-030
that other transcription factors besides NRF2 are regulating these genes at this time point.
Other genes upregulated by included MAP3K14 (also called NF-kappa-beta-
SF-3-030
inducing kinase, NIK), which activates NF-κB and the early growth reponse-1 (Egr-1)
transcription factor, which may have tumor suppressor functions through regulation of p53 (Shin
et al., 2006). Transcripts that increased only in response to AZD6244 treatment included SOX2
and FOXD3, which are associated with survival of melanoma-initiating cells, metastasis and
drug resistance (Santini et al., 2014; Abel et al., 2013). The activation status of the top 50
upstream regulators predicted to be affected following treatment with
or AZD6244
SF-3-030
was determined using IPA software. Using Z-scores of >2 or <-2 to indicate activation or
inactivation, respectively, and p values of <0.03, AZD6244 predictably inhibited many
regulators of ERK1/2 and other MAP kinase signaling pathways (Supplemental Table 3). In
contrast,
was predicted to affect only 6 regulators, including activation of EGR1 as
SF-3-030
indicated previously (Supplement Table 3). Given some overlap with AZD6244-regulated genes,
these data suggest that treatment affects fewer cellular signaling events than AZD6244
SF-3-030
and that early MAP kinase signaling events largely remain intact.
SF-3-030 enhances markers of mitochondrial dysfunction and oxidative stress. To gain further
insight into the mechanism of SF-3-030 inhibition of cell proliferation, we next used high-
resolution liquid chromatography-tandem mass spectrometry to determine the changes in levels
of soluble proteins in A375 cells treated for 4 or 12 hours with SCH772984 or SF-3-030. After 4
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hours, 22 (SF-3-030) or 16 (SCH772984) proteins were identified that showed significant
(p<0.05) changes compared to untreated cells (Supplemental Table 4). A summary of proteins
that showed statistically significant increases or decreases (≥1.5 fold) with SF-3-030 or
SCH772984, as compared to untreated controls, is shown in table 1A/B. At the 4 hour time point,
only one protein, adenosylhomocysteinase (AHCY), which regulates the generation of the S-
adenosylmethionine methyl donor, was inhibited with SF-3-030 or SCH772984 treatment.
Proteins that increased after exposure to SF-3-030 included the zinc finger protein 774 (ZNF774)
that has recently been shown to have tumor suppressor functions in hepatocellular carcinoma
(Guan et al., 2020), transcription factor 4 (TCF4), which regulates cell differentiation, and
HMOX1 (Table 1A). SF-3-030 decreased cytochrome c-type heme lyase (HCCS) levels
suggesting defects in mitochondrial electron transport function (Table 1A). SCH772984
increased levels of the transmembrane and TPR repeat-containing protein 1 (TMTC1), which
indicated disruption of calcium homeostasis in the endoplasmic reticulum (Sunryd et al., 2014),
and decreased the tetratricopeptide repeat protein (TTC29) whose function is currently unknown
(Table 1B).
After 12 hours, 44 and 48 proteins showed significant changes (p<0.05) with SF-3-030 and
SCH772984 treatment, respectively, as compared to untreated cells (Supplemental Table 5).
Proteins that increased or decreased ≥1.5 fold with SF-3-030 or SCH772984 treatments relative
to untreated controls are shown in Tables 2A or B, respectively. Common to SF-3-030 or
SCH772984 treatments was increased levels of TCF4 and cytochrome c oxidase assembly
protein (SCO2) and decreased levels of a protein involved in ribosome biogenesis (RSL24D1).
SCO2 has been associated with p53-mediated apoptosis signaling pathways through the
generation of ROS (Madan et al., 2013).
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Unique to SF-3-030 was a significant increase in the oxidative stress-induced growth
inhibitor 1 (OSGIN1) protein (Table 2A), which is regulated by oxidized lipids and NRF2 during
oxidative stress to promote cytochrome c release from mitochondria (Li et al., 2007). MEMO1, a
protein involved in the sustained production of ROS (MacDonald et al., 2014), was also
increased in cells exposed to SF-3-030 (Tables 1A and 2A). As was observed after 4 hours,
HCCS levels were also decreased after 12 hour exposure to SF-3-030 (Tables 1A and 2A)
further supporting compromised mitochondria function. Together, these findings indicate SF-3-
030 induced signaling events that reduce mitochondrial function and increase ROS. After 12
hours exposure to SCH772984 a significant induction of the Forkhead box protein D3 (FOXD3)
was observed (Table 2B) and is consistent with previous studies that show upregulation of this
protein promotes resistance to Raf and MEK inhibitors (Abel et al., 2013). Immunoblot analysis
confirmed the induction of FOXD3 expression in A375 cells treated with SCH772984 but not in
SF-3-030 treated cells (Supplemental Fig. 5).
Based on the observed changes in protein levels, IPA pathway analysis suggested a potential
role for SF-3-030 induction of NRF2-mediated oxidative stress response and heme degradation
in agreement with mitochondrial dysfunction. These findings are consistent with previous studies
that implicate the ERK1/2 pathway in regulating oxidative phosphorylation in melanoma cells
and the induction of ROS following treatment with inhibitors of BRAF and MEK1/2 (Haq et al.,
2013; Cesi et al., 2017; Yuan et al., 2020). Immunoblot analysis confirmed that SF-3-030
treatment rapidly induced NRF2 levels that were sustained for at least 24 hours and that NRF2
induction could be inhibited by co-treatment with the ROS inhibitor N-acetylcysteine (NAC)
(Fig. 4A-C).
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SF-3-030 inhibition of A375 cell proliferation is dependent on ROS induction but independent
of NRF2. The transcriptome and proteome data provided evidence for the generation of ROS in
cells treated with
. We confirmed the increased ROS production in A375 cells after a 1
SF-3-030
hour treatment with
, which was partially inhibited by co-treatment with several ROS
SF-3-030
inhibitors (Fig. 5A). NAC was the only ROS inhibitor that restored A375 cell proliferation in the
presence of (Fig. 5B). Similarly, only NAC reversed induction of c-Fos (Fig.
SF-3-030
SF-3-030
5C). While these data indicate that NAC may mitigate ROS production by
to restore
SF-3-030
A375 cell proliferation, there was also the possibility that NAC directly forms adducts with
SF-
, which would reduce the compound’s effective concentration and biological activity.
3-030
However, using thin layer chromatography to separate compounds, we found no evidence that
NAC directly interacted with following a 1 or 24 hour incubation in both PBS and
SF-3-030
methanol solvent systems (data not shown).
Induction of NRF2 has been implicated in protecting against oxidative stress induced by anti-
cancer drugs and may contribute to drug resistance (Telkoparan-Akillilar et al., 2019). To assess
whether NRF2 induction protected cells from
inhibition, we co-treated A375 cells with
SF-3-030
the NRF2 inhibitors, ML-385 or brusatol (Singh et al., 2016; Olayanju et al., 2015; Ren et al.,
2011). and sulforaphane, as a positive control, induced NRF2 and target genes such as
SF-3-030
HMOX-1, OSGIN-1, NAD(P)H dehydrogenase [quinone] 1 (NQO1), glutamate-cysteine ligase
modifier subunit (GCLM), and sulfiredoxin 1 (SRXN1) (Fig. 6A/B and Supplemental Fig. 6A).
In contrast, protein levels for other NRF2-regulated genes, including aldo-keto reductase family
1 member B10 (AKR1B10), glutamate-cysteine ligase catalytic subunit (GCLC), glucose-6-
phosphate dehydrogenase (G6PD), cystine/glutamate transporter/solute carrier family 7 member
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11 (xCT/SLC7A11), and thioredoxin reductase 1 (TXNRD1) were not induced by
(Supplemental Fig. 7).
SF-3-030
As compared to sulforaphane,
induced a more robust and sustained induction of
SF-3-030
HMOX-1 indicating qualitative differences in NRF2 activators. Brusatol was a potent inhibitor
of or sulforaphane induction of NRF2 and its target genes. Although ML-385 inhibited
SF-3-030
HMOX-1 induced by sulforaphane after 24 hours (supplemental Fig. 6A/B), it was not as
effective as brusatol at inhibiting NRF2 signaling induced by (Fig. 6A). ML-385 had
SF-3-030
no effect on cell proliferation up to 50 µM, however brusatol inhibited A375 cells in a dose
dependent manner (Fig. 6 C/D). Co-treatment with brusatol or ML-385 had little effect on
SF-3-
or sulforaphane inhibition of A375 cell proliferation, indicating NRF2 induction was
030
dispensable for the cell response to these compounds (Fig. 6E/F, supplement Fig. 6C/D). Dose-
dependent inhibition of A375 cells by sulforaphane or brusatol alone supports the high
sensitivity these cells have to fluctuations in ROS (Meierjohann, 2014). Together, these data
indicate that
-mediated inhibition of A375 melanoma cell proliferation is through a
SF-3-030
mechanism that is ROS-dependent, and NRF2-independent.
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Discussion
The first objective of the current studies was to elucidate the structure activity relationship of
a 1,1-dioxido-2,5-dihydrothiophen-3-yl 4-benzenesulfonate chemical scaffold that we previously
identified to selectively inhibit cancer cells containing activated ERK1/2 signaling (Samadani et
al., 2015). The dependence on a double bond in the sulfur heterocycle for biological activity
indicated the compound’s mechanism of action involved the formation of covalent adducts with
cysteine residues on ERK1/2 and perhaps other proteins through Michael addition chemistry.
SF-3-030 was confirmed via STD-NMR and mass spectrometry-based analyses to interact with
ERK2 via non-covalent interactions and the formation of a covalent adduct on C252 near the
FRS. While the formation of covalent adducts raises the concern about specificity and off-target
effects, we have found no evidence that SF-3-030 leads to changes in other MAP kinase
signaling pathways or randomly interacts with cysteine using the ROS inhibitor NAC (data not
shown). This indicates that the chemical structure of SF-3-030 confers target selectivity and
prevents random interactions with sulfhydryl groups.
There is the potential that SF-3-030 forms a covalent adduct with KEAP1 the negative
regulator of NRF2. KEAP1 has up to 21 cysteine residues that may be modified by chemical
stressors to relieve KEAP1 inhibition and allow NRF2 activation (Dayalan Naidu and Dinkova-
Kostova, 2020). Sulforaphane, which modifies KEAP1 on C151 to activate NRF2, showed
qualitative differences in the expression of NRF2-regulated genes as compared to SF-3-030 (Fig.
6 and supplemental Fig. 6). It will be interesting to evaluate whether SF-3-030 also modifies
KEAP1 cysteine residues and how they impact NRF2 functions. As indicated in the current
studies, SF-3-030 induction of NRF2 did not appear to affect A375 cell proliferative responses to
SF-3-030.
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Despite past concerns about safety and off-target toxicity, advances in structure function
relationships and medicinal chemistry have generated renewed interest in the development of
irreversible binding drugs for treating disease (Singh et al., 2011). There are several small
molecule anti-cancer drugs in the clinic that form covalent adducts on receptor and non-receptor
tyrosine kinases through Michael addition (reviewed in (Zhao and Bourne, 2018)). Afatinib,
osimertinib, and neratinib form irreversible adducts on cysteine residues in the catalytic sites of
EGF receptors for the treatment of non-small cell lung cancers and breast cancers while ibrutinib
targets Bruton’s tyrosine kinase (BTK) for the treatment of lymphoma/leukemia. Additionally,
there is renewed interest in targeting ERK1/2 with small molecules via allosteric inhibition of
substrate interactions sites, such as the D-recruitment site (DRS) (Sammons et al., 2019). Recent
studies have identified a small molecule inhibitor that covalently binds to a conserved cysteine
(C159) in the DRS of ERK2 (Kaoud et al., 2019). This compound selectively blocked the
activation of ERK1/2, did not modify other members of the MAP kinase family, and inhibited
the proliferation of melanoma cells with BRAF (V600E) mutations that were resistant to BRAF
inhibitors (Kaoud et al., 2019). Future studies will be aimed at identifying if cysteine residues on
other proteins may be modified by SF-3-030 to establish their role in cell signaling and growth
inhibition.
The second objective of the studies was to evaluate global effects of SF-3-030 on gene and
protein changes to gain insight into this chemical scaffold’s mechanism of action. The
transcriptome and proteome data show that the overall number of genes and proteins affected by
the lead compound, SF-3-030, are less than or similar to the number of proteins targeted by
known ERK1/2 or MEK1/2 ATP competitive or catalytic site inhibitors. Similar to the ATP-
competitive ERK1/2 pathway inhibitors, SF-3-030 appears to be more selective for cancer cells
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containing constitutively activating ERK1/2 pathway mutations. While rapidly dividing cells are
typically more sensitive to anti-cancer drugs, SF-3-030 effects on a select number of cells with
activating ERK1/2 pathway mutations were shown to be independent of cell doubling times.
Although further studies with additional cell lines will be needed to verify this observation, these
data indicate that off-target effects on non-transformed cells may be limited.
Transcriptome and proteome analyses indicated that SF-3-030 induced rapid changes in the
levels of ERK1/2 regulated transcription factors consistent with an oxidative stress response.
Transcription factors such as c-Fos and c-Jun are elevated in response to oxidative or metabolic
stress (Webster et al., 1994) and ROS-mediated induction of ERK1/2 can enhance Elk-1
mediated transcription and c-Fos expression (Muller et al., 1997). It was intriguing that SF-3-030
caused a robust induction of c-Fos but inhibited c-Myc protein levels (Fig. 3) and that these
changes occurred at the level of transcription (Supplemental Table 2). Other examples where
proteins levels increase due to SF-3-030 induction of transcription include c-Jun and HMOX1. In
contrast, SF-3-030 also inhibited Fra-1 levels but there was no evidence this occurred through
effects on transcription (Fig. 3 and Supplemental Table 2). It is possible that the 1 hour time
point used to evaluate transcriptome changes by RNAseq did not capture changes in Fra-1
transcription. The expression of Fra-1 and other Fos family proteins are regulated by c-Fos and
subsequent AP-1 complex activity (Milde-Langosch, 2005). However, c-Fos induction in the
current studies does not correlate with increased AP-1 activity, which we previously showed to
be inhibited by SF-3-030 (Samadani et al., 2015; Defnet et al., 2019a). While apoptosis can be
induced by c-Fos induction (Preston et al., 1996), further studies will be needed to clarify the
requirement for c-Fos expression in SF-3-030 mediated cell death in the absence of AP-1
activity.
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ERK1/2 activation increases the expression of c-Myc (Kerkhoff et al., 1998), which can
protect melanoma cells against oxidative stress (Benassi et al., 2006). In contrast, SF-3-030
mediated downregulation of c-Myc and its potential role in responding to elevated ROS by
regulating the synthesis of antioxidants such as glutathione (Gao et al., 2009). Following
exogenous hydrogen peroxide-induced oxidative stress, ERK1/2 phosphorylation of c-Myc at
serine 62 facilitates c-Myc recruitment to the glutamate-cysteine ligase catalytic subunit (GCLC)
promoter and the expression of GCLC, the rate limiting enzyme involved in glutathione
synthesis (Benassi et al., 2006). However, in the current studies, the dramatic inhibition of c-Myc
protein levels by SF-3-030 after 8 hours (Fig. 3A) is not observed with GCLC (Supplement Fig.
7). Nonetheless, targeted inhibition of c-Myc with ERK1/2 pathway inhibitors is a suggested
approach to decrease the proliferation of melanoma cells and overcome drug resistance
(Ciuffreda et al., 2009; Korkut et al., 2015).
The role of reactive oxygen species (ROS) in promoting or inhibiting cancer cells is subject
to debate and there is evidence to support that either increased or decreased ROS production can
sensitize cancer cells to growth inhibition and cell death (Trachootham et al., 2009; Galadari et
al., 2017). While the use of anti-oxidant strategies may protect against some cancers
(Trachootham et al., 2009), there are limited clinical data that supports the beneficial effects of
using anti-oxidant supplements to reduce the risk of developing cancer (Goodman et al., 2011).
However, the mechanisms used by many anti-cancer drugs to kill cells involves increased
oxidative stress (Trachootham et al., 2009). Melanocytes and melanoma cells are particularly
sensitive to ROS and the ROS dose will likely determine whether cells are able to adapt or will
die (Meierjohann, 2014), Additional evidence supports the use of oxidative stress to inhibit
melanoma cell metastasis (Piskounova et al., 2015). While ROS activation may not be sufficient
30