A genome‐wide CRISPR screen identifies FBXO42 involvement in resistance toward MEK inhibition in NRAS‐mutant melanoma

Abstract NRAS mutations are the most common alterations among RAS isoforms in cutaneous melanoma, with patients harboring these aggressive tumors having a poor prognosis and low survival rate. The main line of treatment for these patients is MAPK pathway‐targeted therapies, such as MEK inhibitors, but, unfortunately, the response to these inhibitors is variable due to tumor resistance. Identifying genetic modifiers involved in resistance toward MEK‐targeted therapy may assist in the development of new therapeutic strategies, enhancing treatment response and patient survival. Our whole‐genome CRISPR‐Cas9 knockout screen identified the target Kelch domain‐containing F‐Box protein 42 (FBXO42) as a factor involved in NRAS‐mutant melanoma‐acquired resistance to the MEK1/2 inhibitor trametinib. We further show that FBXO42, an E3 ubiquitin ligase, is involved in the TAK1 signaling pathway, possibly prompting an increase in active P38. In addition, we demonstrate that combining trametinib with the TAK1 inhibitor, takinib, is a far more efficient treatment than trametinib alone in NRAS‐mutant melanoma cells. Our findings thus show a new pathway involved in NRAS‐mutant melanoma resistance and provide new opportunities for novel therapeutic options.

Despite decades of research on RAS, it is still regarded as being "undruggable" (Stephen, Esposito, Bagni, & McCormick, 2014). Therefore, MAPK pathway inhibitors, such as MEK inhibitors (MEKi), are the typical therapeutic approach when it comes to NRAS-mutant melanoma (Munoz-Couselo, Adelantado, Ortiz, Garcia, & Perez-Garcia, 2017;Santarpia, Lippman, & El-Naggar, 2012). Trametinib is an FDAapproved, allosteric inhibitor of MEK1/2 used to treat NRAS-mutant melanoma, both as a monotherapy and in combination with other anti-cancer drugs (Johnson & Puzanov, 2015). However, these current therapies used to treat patients with NRAS-mutant melanoma are not very efficient, owing to the aggressive nature of tumor cells and complex changes in molecular signaling (Johnson & Puzanov, 2015). Thus, identifying genetic modifiers involved in such resistance mechanisms is of great importance. We here report on a novel mechanism of drug resistance in NRAS-mutant melanoma.
Our whole-genome CRISPR-Cas9 knockout (KO) screen in NRAS Q61R melanoma cells revealed the Kelch domain-containing F-Box protein 42 (FBXO42), an E3 ubiquitin ligase (Sun et al., 2009), involvement in resistance toward trametinib treatment. We further show that FBXO42 is involved in the TAK1 signaling pathway, leading to increased P38 activation. Thus, based on these observations, we demonstrate that combining trametinib with takinib, a TAK1 inhibitor is a far more efficient treatment than monotreatment with trametinib in NRAS-mutant melanoma cells.
Put together, our findings reveal a novel mechanism of tumor resistance to MEK inhibition in NRAS-mutant melanoma, and potentially offering a new therapeutic strategy. MM130405, MM130926, and MM130227 cell lines were cultured in RPMI-1640. All melanoma cell lines were supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin and grown at 37°C in 5% CO 2 for 5-15 passages. All cells have been authenticated by sequencing and were tested routinely for mycoplasma using Mycoplasma EZ-PCR test kit (#20-700-20, Biological Industries, Kibbutz Beit Ha'emek).

| CRISPR-Cas9 mediated genome-wide screen
SK-MEL-147 cells were lentivirally transduced with two GeCKO libraries (A and B) at a MOI of 0.3 aiming to ensure that most cells receive only one viral construct (Shalem et al., 2014). Briefly, 5 × 10 6 cells were plated in 10 cm 2 dishes. 48 hr after infection, cells were selected with puromycin (1 µg/ml) for 14 days. Cells were split into two pools: One arm was subjected to 100 nM MEKi trametinib treatment (GSK1120210 Selleckchem), whereas the other arm was left untreated. Colonies formed in the drug-treated arm were individually picked and expanded. For identification of sgRNAs in the individual clones, genomic DNA was isolated and sgRNAs were recovered by PCR amplification. Amplified DNA fragments were cloned into the TOPO TA-cloning vector (450071, Invitrogen), followed by identification of the sgRNA by Sanger sequencing.

| CRISPR cloning
Cloning of sgRNAs into the LentiCRISPRv2 vector was performed as described (http://www.genome-engin eering.org/crisp r/). Briefly, the LentiCRISPRv2 plasmid was digested with BsmBI and gel-purified. DNA oligonucleotides (Invitrogen) were annealed and ligated into the digested vector. Target sgRNA oligonucleotide sequences are listed in Figure S8.

Significance
As NRAS-mutant melanoma tumors are mostly resistant to MEK inhibitors, investigating signaling pathways that lead to resistance has taken a center stage. Our data show that the TAK1 pathway is involved in resistance toward MEK inhibition in NRAS-mutant melanoma. These findings have clinical implications as they may lead to the development of combined inhibitor therapy toward MEK and TAK1, which could be an effective treatment for melanoma patients harboring an NRAS mutation.

| Colony formation assay
Colony formation assays were performed by seeding 500 K cells in 6-well plates. The medium was refreshed twice per week for 2 weeks, and then, the plates were fixed in 4% formaldehyde solution, stained with crystal violet (0.01% in dH2O), and photographed.

| Cell viability assays
Melanoma cell lines were seeded into 96-well plates (3,000 cells per well). On the next day, trametinib (GSK1120210 Selleckchem)/ selumetinib (AZD6244, AstraZeneca) was added to the plate's wells at increasing concentrations, from 1 pM to 100 μM, in three replicates, with DMSO as a negative control. After an additional 72 hr, cell proliferation was assessed using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega). Analysis was performed using graph-Pad Prism. The combined effect of TAK1 and MEK inhibition on cell proliferation was tested by adding to the plate wells an increasing concentration of the MEK inhibitor trametinib, from 1 pM to 100 μM, and a constant concentration of 5 μM takinib (HY-103490, BioTAG). Cells were evaluated for viability after 72 hr as described above.

| RNA sequencing analysis
RNA capture was performed with TruSeq Library Prep Kit v2 (Illumina) and sequenced on a HiSeq4000. RNA counts were quantified from single-end reads using STAR aligner (Dobin et al., 2013).

| Immunohistochemistry
Tissue sections (4 μm thick) were deparaffinized in xylene, rehydrated using graded concentrations of ethanol, and rinsed in distilled water. Heat-induced epitope retrieval was performed in 10 mM citrate buffer at pH 6.0 for 10 min at 95°C. Sections were allowed to cool for one hour and then rinsed in distilled water. Endogenous peroxidase activity was blocked for 30 min. with hydrogen peroxide. For nonspecific binding, sections were blocked with 20% normal horse serum and 0.1% triton. Following blocking treatment, primary antibody (Rabbit anti-human FBXO42 obtained from Abcam [ab81638]) was diluted 1:25 and incubated overnight at 4°C. Detection was accomplished using a biotinylated secondary goat anti-rabbit antibody, followed by application of streptavidin-peroxidase conjugate solution and exposure to 3-3′-diamino-benzidine (Sigma). Slides then were counterstained with hematoxylin (Sigma), dehydrated, and mounted with permanent media. Stained sections were examined and photographed on a bright-field scanner (Pannoramic SCAN II slide scanner) equipped with Carl Zeiss objectives (10×; 20×; 40×; 60×).

| Pooled stable expression
To produce lentiviruses, the following FBXO42 constructs were generated: FBXO42 was tagged with Flag at the N-terminus (pCDH1-FBXO42), FBXO42Δfbox was tagged with Flag at the Cterminus, and FBXO42Δkelch was tagged with Flag at the N-terminus. Deletion mutations were a kind gift from Yongfeng Shang (Peking University Health Science Center). Plasmids were co-transfected into HEK293T cells seeded at 2.5 × 10 6 per T75 flask with psPAX2 and pMD2.G helper plasmids using TurboFect as described by the manufacturer. Virus-containing media were harvested 60 hr after transfection, filtered, aliquoted, and stored at −80°C. The lentiviruses for FBXO42 and its mutants were used to infect SK-MEL-147, MM130926, and MM130227 as previously described (Arafeh et al., 2015).

| RAS activation assay
Two 15-cm plates with SK-MEL-147 melanoma cells were treated with 100 nM trametinib or DMSO as control, for 24 hr. Ras-GTP levels were detected using a Ras activation kit (Millipore), following the manufacturer's instructions (Merck). RAS-GTP activation was quantified by using Image Lab software (Bio-Rad).

| Identification of FBXO42-interacting proteins
Immunoprecipitation of FBXO42 in SK-MEL-147 cells was performed as previously described.
Immunoprecipitations were washed five times with lysis buffer and then resuspended with sample buffer before denaturation and separation by SDS-PAGE on 10% mini gels.
The proteins in the gel were visualized with an Imperial™ protein stain (Thermo Scientific), then reduced with 3 mM DTT (60°C for 30 min), modified with 10 Mm iodoacetamide in 100 mM ammonium bicarbonate (in the dark, room temperature for 30 min), and digested in 10% acetonitrile and 10 mM ammonium bicarbonate with either modified trypsin or chymotrypsin (Promega) at a 1:10 enzyme-tosubstrate ratio, overnight at 37°C. Alternatively, the proteins in a mixture in 8 M urea and 100 mM ammonium bicarbonate were reduced and modified as described and digested in 2 M urea, 25 mM ammonium bicarbonate with modified trypsin or chymotrypsin (Promega) at a 1:50 enzyme-to-substrate ratio. The resultant peptides were carbamidomethyl on Cys was accepted as a static modification. The minimal peptide length was set to six amino acids, and a maximum of two miscleavages was allowed. Peptide-and protein-level false discovery rates (FDRs) were filtered to 1% using the target-decoy strategy. Semi-quantitation was done by calculating the peak area of each peptide based on its extracted ion currents (XICs), and the area of the protein was determined by averaging the three most intense peptides from each protein.

| A function-based genomic screen in NRASmutant melanoma cells identifies FBXO42 loss driving trametinib resistance
In order to identify genes essential to maintain sensitivity toward the MEKi trametinib in NRAS-mutant melanoma, we conducted a wholegenome CRISPR-Cas9 knockout screen ( Figure 1a).  Figure   S1c). We checked the resistance effect of these cell lines toward an additional potent and highly selective MEK1/2 inhibitor, selumetinib, (Kim & Patel, 2014) and received similar results (Figure 2c,d).
These data confirm that KO of FBXO42 in NRAS-mutant melanoma cells leads to resistance toward trametinib treatment.

| Identification of novel FBXO42 binding partners
FBXO42 is an integral component of the SCF ubiquitin ligase complex. FBXO42 specifically associates with Skp1, Cul1, and Rbx1, the constituents of the SCF complex, an association which is dependent on the F-box domain (Cardozo & Pagano, 2004;Petroski & Deshaies, 2005). The FBXO42 Kelch domain is responsible for the binding of an interactor protein regulated by SCF ubiquitination (Yan et al., 2015). In an effort to better understand the mechanistic role of FBXO42 in NRAS-mutant melanoma, we per- Interestingly, this analysis leads to the identification of a novel interaction with MAP3K7 also known as TAK1 and its regulators, TAB1, TAB2, and ITCH (Roh, Song, & Seki, 2014) (Figure 5c and Table S1). TAK1 activation is triggered by diverse stimuli, including pro-inflammatory cytokines such as IL-1 and TNF. TAK1 culminates in downstream activation of NF-κB, P38, JNK, and ERK (Adhikari, Xu, & Chen, 2007;Ajibade et al., 2012). The binding to TAB1 and TAB2 proteins forms a complex required for TAK1 autophosphorylation-induced activation. Furthermore, TAK1 is negatively regulated by the E3 ubiquitin ligase, ITCH (Roh et al., 2014;Shibuya et al., 1996).
We identified the indicated TAK1 binding partners only in WT FBXO42 and FBXO42ΔFbox samples but not in the FBXO42Δkelch sample, emphasizing that they might be potential interactors.
These results suggest that FBXO42 has a role in the TAK1 signaling pathway.

| FBXO42 KO leads to TAK1 signaling activation
It has been shown that TAK1 phosphorylates and activated members of the mitogen-activated protein kinase kinase (MKK) family, which, in turn, phosphorylate and activate JNK and P38 kinases (Adhikari et al., 2007;Ajibade et al., 2012). Indeed, an increase in pP38 expression, together with a decrease in p-JNK, was identified in FBXO42 KO samples compared to the control (Figure 5d,e and S4). This is consistent with the ability of P38 MAPK to negatively regulate JNK (Gupta et al., 2015).
To further assess the involvement of the TAK1 signaling pathway in trametinib resistance in NRAS mutant FBXO42 KO melanoma cells, we combined trametinib with takinib, the potent and selective TAK1 inhibitor (Totzke et al., 2017), and tested their inhibition of FBXO42 KO cell growth. We found that they achieved greater efficacy than treatment with trametinib or takinib monotreatment (Figure 5f,g and Figure S5-7).

| D ISCUSS I ON
In recent years, the genetic landscape of melanoma has been extensively studied (Cancer Genome Atlas Network, 2015;Hodis et al., 2012;Krauthammer et al., 2012). On the basis of exome and genome sequencing studies, BRAF and NRAS were identified as the most commonly mutated genes in cutaneous melanoma patients (Krauthammer et al., 2012). As NRAS and BRAF activate the MAPK pathway, this led to the development of highly selective kinase inhibitors that target this pathway (Tsao, Chin, Garraway, & Fisher, 2012). However, acquired tumor resistance toward these targeted therapies is a significant therapeutic obstacle (Neel & Bivona, 2017 Mass spectrometry analysis further showed that FBXO42 is involved in the TAK1 signaling pathway. TAK1 belongs to the MAP3K7 F I G U R E 5 FBXO42 KO activates the MAPK pathway and TAK1 signaling leading to trametinib resistance. (a) SK-MEL-147 RAS-GTP levels were assessed by RAS pull-down assay, and treated samples were incubated with 100 nM trametinib for 24 hr. RAS-GTP/RAS ratios are representative of three independent experiments. Trametinib-treated and non-treated samples were compared to their sgCtrl. n = 3, *p < .05, **p < .01 one-way ANOVA followed by Tukey's test. (b) SK-MEL-147 cells were treated with 100 nM trametinib for 24 hr. Cell lysates were analyzed by immunoblot. pERK/ERK ratios were calculated from two independent experiments. Untreated samples were blotted with non-sensitive ECL; trametinib-treated samples were blotted with sensitive ECL. Trametinib-treated and non-treated samples were compared to their sgCtrl. n = 2, *p < .05, **p < .01, ***p < .001, one-way ANOVA followed by Tukey's test. (c) Cells stably expressing FLAG-FBXO42 were immunoprecipitated with anti-Flag. Bound proteins were eluted and analyzed by mass spectrometry. The identified proteins are listed. FLAG-FBXO42ΔKelch and FLAG-FBXO42ΔF-box were used as control. (d,e) SK-MEL-147 and MZ-MEL-2 cell lines were treated with 100 nM trametinib for 24 hr. Cell lysates were analyzed by immunoblot with the indicated antibodies. pP38/P38 ratios were generated using Image Lab (Bio-Rad). (f,g) Dose-response curves generated using SK-MEL-147 and MZ-MEL-2 cell lines, representing the delta between cells treated with trametinib (0.001-10 μM) and cells treated with the combination of trametinib (0.001-10 μM) and takinib (2.5 or 5 μM, respectively). Error bars, SD  (Sakurai et al., 2002). It is well established that P38 signaling plays a central role in the regulation of cellular responses to stress, as well as the induction and progression of inflammation-related diseases, inflammation-induced cancer, and various cancers (Grossi, Peserico, Tezil, & Simone, 2014;Yin et al., 2016). Our findings add support to the attempt to try and inhibit this signaling pathway.
Moreover, we demonstrate that combining the MEK inhibitor trametinib with the TAK1 inhibitor, takinib achieves far greater efficacy than treatment with trametinib alone in NRAS-mutant melanoma cells.
Put together, our findings reveal a novel mechanism leading to tumor resistance toward MEK inhibition in NRAS-mutant melanoma ( Figure 6). Namely, that FBXO42 has a role in trametinib resistance via the TAK1 signaling pathway, thus providing new opportunities for novel therapeutic options.