Predictive biomarkers for response to trametinib in non-small cell lung cancer

2.1Cell lines and cell culture

The NSCLC cell lines A427 (HTB-53), CALU-3 (HTB-55), CALU-6 (HTB-56), NCI-H1299 (CRL-5803), NCI-H1693 (CRL-5887), NCI-H1838 (CRL-5899), NCI-H2087 (CRL-5922), NCI-H2170 (CRL-5928), NCI-H292 (CRL-1848), NCI-H358 (CRL-5807) and NCI-H460 (HTB-177) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). COR-L105 (Cat # 92031918) was purchased from MilliporeSigma (St. Louis, MO, USA). All these cell lines were authenticated. All cell lines were cultured in RPMI-1640 (ATCC Cat # 30-2001) with the exception of A427 and CALU-3 which were cultured in EMEM (ATCC Cat # 30-2003). All cells were cultured at 37°C in a humid atmosphere of 5% CO2 in medium supplemented with 10% fetal bovine serum (FBS; Thomas Scientific Cat # FB5001, Swedesboro, NJ, USA) and a penicillin-streptomycin solution of 100 units/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Cat # 15140163, Waltham, MA, USA).

2.2Reagents and antibodies

Trametinib (Mekinist; GSK1120212) was obtained from Selleck Chemicals (Cat # S2673; Houston, TX, USA) and prepared per their instructions. Antibodies for ERK (p44/p42 MAPK; ERK1/2; Cat # 4695), Phospho-ERK (Thr202/Tyr204; Cat # 4376), Cyclin D1 (Cat # 2922 & 55506), p27KIP1 (Cat # 3688), GAPDH (HRP Conjugate; Cat # 3683), Phospho-AKT (Ser473; Cat # 4060), AKT (pan; Cat # 4685), Phospho-4E-BP1 (Thr70; Cat # 9455), 4E-BP1 (Cat # 9644), LC3-A/B (Cat # 4108), SQSTM1/p62 (Cat # 5114), Beclin-1 (Cat # 3495), PARP (Cat # 9532), Vimentin (Cat # 5741), E-Cadherin (Cat # 14472), Bcl-2 (Cat # 4223), Phospho-p-Rb (Ser807/811; Cat # 8516), Rb (Cat # 9309), Caspase-3 (Cat # 9662), Caspase-7 (Cat # 9494), and α-Tubulin (Cat # 2125) were from Cell Signaling Technologies (CST; Danvers, MA, USA). Antibody for Ki-67 (SP6; Cat # 275 R) used for immunocytochemistry and western blot was from Cell Marque (Rocklin, CA, USA). Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Bio-Rad, Cat # 1706515) and Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Bio-Rad, Cat # 1706516) were from Bio-Rad Laboratories (Hercules, CA, USA). All antibodies were used according to the manufacturers’ instructions.

2.3Colony formation assay

The assay was performed as previously shown by Franken et al. [11]. Cells were seeded as single cell suspensions of 150, 200 or 300 cells per well according to the plating efficiency of the cell lines in 6 well plates with media containing 10% FBS and incubated overnight. Cells were then treated with trametinib (0, 0.15, 0.46, 1.4, 4.1, 12.3, 37, 111, 333 and 1000 nM) in media containing 10% FBS and incubated for 72 h. Following trametinib treatment, drug media was removed and replaced with media containing 10% FBS. Cells were incubated over several days to allow colony maturation. Once colonies in the untreated control samples proliferated to at least 50 cells per colony, media were removed and cells were fixed overnight at room temperature with 2 mL of fixative solution (1-part glacial acetic acid to 7-parts methanol). The next day, fixative was removed, and wells were gently rinsed with 2 mL tap water. Cells were then stained for 1 h at room temperature using 2 mL of staining solution (methanol containing 0.5% Coomassie blue). Excess stain was decanted, wells were rinsed with 2 mL of tap water and allowed to dry overnight. Stained colonies were visually marked and counted using a bright field microscope.

2.4Preparation of whole cell extracts for western blotting

One million cells per well were seeded into 6-well plates and incubated overnight in media supplemented with 10% FBS. The next day, media were replaced with media containing 0.5% FBS and incubated an additional 24 h (“FBS starvation” condition). Cells were then treated with trametinib (0, 0.016, 0.4, 10 and 250 nM) in media containing 0.5% FBS and incubated for 24 or 48 h. For total protein extraction, media were collected from each condition to retrieve floating cells. Then, adherent cells were washed with Hanks buffered saline solution (HBSS). Wash solutions were collected to retrieve detached cells and combined with the previously collected media. Attached cells were harvested with Accutase (Life Tech., Cat # A1110501) and combined with the previously collected media and wash solutions. Each pool was then centrifuged (200 g, 5 min) at room temperature to pellet cells. Cells were washed with ice-cold PBS and centrifuged again. Cell pellets were resuspended for lysing using 250 μL of 1X lysis buffer (Bio-Rad; Cat # 10024042) containing additional protease (CST; Cat # 5871 S) and phosphatase (CST; Cat # 5870 S) inhibitor cocktails and incubated for 10 min at 4°C on ice. Cell lysates were clarified with centrifugation (10,000 g) at 4°C for 10 min. Cell lysate concentrations were quantified using the Bio-Rad DC Protein Assay (Bio-Rad, Cat # 5000116).

2.5Western blotting

For western blots, an equal amount of protein (6 μg) was loaded into sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; Mini-Protean TGX Precast Gel; Bio-Rad) with 7.5% (Cat # 4561024), 10% (Cat # 4561035), 12% (Cat # 4561045), or 4-15% (Cat # 4561085). Proteins were separated at 200 V for 30 minutes in 1X Tris/Glycine/SDS buffer (Bio-Rad, Cat # 1610772) and transferred with transfer buffer (Trans-Blot Turbo Transfer Kit; Bio-Rad, Cat # 1704273) to polyvinylidene fluoride (PVDF) membranes using a Trans-Blot Turbo (Bio-Rad, Cat # 1704155). Membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in tris-buffered saline with Tween 20 (TBST; 15 mM Tris-HCl, pH 7.5, 200 mM NaCl and 0.1% Tween 20). Membranes were incubated with primary antibodies overnight at 4°C and the next day with rocking agitation for 1 h at room temperature; all primary antibodies were diluted in TBST following manufacturers’ specifications. Membranes were washed three times for 5 min each in TBST and incubated with goat anti-rabbit or -mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibodies diluted 1 : 5,000 in TBST with 5% non-fat dry milk for 1 h at room temperature. Membranes were washed three times for 10 min each in TBST and target proteins were identified using Clarity Western ECL Substrate (Bio-Rad, Cat # 1705061) and autoradiography film (GenHunter, Cat # B581, Nashville, TN, USA).

2.6Immunocytochemistry

Immunocytochemistry was performed using the BioGenex kit. For the assay, 10,000 cells per chamber were seeded with 10% FBS-containing media in an 8-chambered slide (Thermo Fisher Scientific, Cat # 154941). After overnight incubation, chamber media were replaced with 0.5% FBS-containing media. Cells were incubated an additional 24 h for growth and then treated with trametinib (0, 0.4, 10 and 250 nM) for 24 and 48 h. Cells were then fixed with 10% neutral buffered formalin solution for 10 min, washed twice with PBS and stored in 70% solution. For antigen retrieval, chambers slides were rinsed in PBS for 10 min, placed in PBS-Tween 20 (0.05%) for 10 min, incubated in 10 mM sodium citrate pH 6.0 containing 0.05% Tween 20 for 25 min at 90°C, cooled at room temperature for 20 min, washed in PBS-Tween 20 (0.05%) for 10 min, blocked with 3% hydrogen peroxide (Cat # HK111) and washed with Super Sensitivetrademark wash buffer (Cat # HK583-5K). Next, chamber slides were blocked with Power Blocktrademark solution (Cat # HK0830189) for 10 min and cells were incubated with Ki-67 antibody at a 1 : 200 dilution in enhanced antibody diluent at 4°C overnight. The following day, chambers were washed three times with Super Sensitivetrademark wash buffer, cells were incubated with Super Enhancertrademark (Cat # HK518-50K) signal detection for 20 min and again washed three times with wash buffer. Cells were then incubated with pre-diluted poly-HRP reagent (Cat # HK519) (mix of anti-rabbit/-mouse HRP provided in kit) for 30 min in the dark. Chambers were washed again three times with wash buffer and incubated with diaminobenzidine (DAB) chromogen substrate (Cat # HK128) for 7 min in the dark. Chamber slides were rinsed gently under running water for 1 min and dipped in counterstain Mayer’s Hematoxylin (Cat # HK100) for 1 min. Cells were imaged using a bright field microscope (Carl Zeiss, Jena, Germany) with a 6D Canon camera. Ki-67 expression was quantified with ImageJ software, counting 100 cells per treatment, per cell line. Data are represented as the average intensity obtained from two independently repeated experiments.

2.7Apoptosis, necrosis and healthy cells assessment

Apoptosis and necrosis events were detected using the PromoKine l Kit (Cat # PK-CA707-30018, PromoCell GmBH, Heidelberg, Germany). For the assay, after 48 h of trametinib treatment in adherent culture, cells were harvested similarly as described above in “Preparation of Whole Cell Extracts”, except PBS was supplemented with 2.5 mM CaCl2. Each sample (containing 200,000 cells) was resuspended in 26.5 μL of 1X staining solution and incubated at room temperature for 15 min in the dark. Cells were then washed in 1X binding buffer and centrifuged to pellet. Cell pellets were resuspended in 50 μL of 1X binding buffer. And 10 μL of each sample was loaded into a microfluidic chamber slide and observed under 20X magnification using a confocal microscope (Leica Microsystems, IL, USA). Three images were taken and a total of 100 cells per treatment, per cell line were analyzed using ImageJ software. Ethidium homodimer III-positive cells were scored as ‘necrotic cells’ while annexin V-stained cells were scored as ‘apoptotic cells,’ and cells stained by both were scored as ‘late-apoptotic cells.’

3.1NSCLC cell lines have varied sensitivity to trametinib

Anti-cancer activity of trametinib was assessed using a CFA [11] in a panel of twelve EGFR WT and BRAF WT NSCLC cell lines with varied RAS gene alterations (Table 1). As they are BRAF WT, these NSCLC patient models would not be approved for trametinib therapy per the FDA-approved sequence-based companion diagnostic test [6]. We stratified cell lines into three categories: highly, moderately and minimally sensitive to trametinib. We used trametinib IC50 values of < 10 nM for highly sensitive (CALU-6, CALU-3, H1299, H2087, COR-L105 and H292), 10-100 nM for moderately sensitive (A427, H1693 and H358) and > 100 nM for minimally sensitive cell lines (H1838, H460 and H2170; Fig. 1; Table 1).

Fig. 1

Anti-proliferative activity of trametinib on NSCLC cell lines. A colony forming assay (CFA) was used to measure the anti-proliferative activity of trametinib on the full panel of 12 NSCLC cell lines. Cells were plated at their own optimal plating efficiency. Each cell line was treated with trametinib concentrations ranging from 0.15 nM to 1μM for 72 h and allowed to grow for 7 days to form colonies. Colonies were counted and averaged for each concentration, then compared to vehicle control. Data are represented with mean and standard deviation of representative experiment. Data are representative of two or three independent experiments.

3.2ERK is not a good predictive marker for trametinib response

Since MAP Kinase (ERK) is an effector substrate of MEK, we assessed ERK phosphorylation status (T202/204) as a predictive biomarker for trametinib. Phospho-ERK (p-ERK) was assessed at various trametinib doses and exposure times. First, we studied short-term (0.5 h) responses in 4 representative cell lines: CALU-6 (highly sensitive), A427 (moderately sensitive) and H1838 and H2170 (minimally sensitive). Regardless of sensitivities in the CFA reference assay, all four cell lines showed strong downregulation of p-ERK with 10 and 250 nM trametinib at 0.5 h (Supplementary Figure 1). Furthermore, at extended treatments of 24 and 48 h, all 12 cell lines showed universal strong downregulation of p-ERK with 10 and/or 250 nM trametinib, with no total ERK change (Fig. 2). Therefore, trametinib is an effective inhibitor of proximal MAP kinase activity irrespective of ultimate response category and p-ERK is not suited to predict longer-term fate in NSCLC. Since this proximal biomarker did not correlate to overall trametinib response, we assessed intermediate and distal biomarkers for predictive potential.

Fig. 2

Inhibitory effect of trametinib on ERK phosphorylation. Western blots were used to measure the effect of trametinib on the levels of phosphorylated ERK (pERK) and total ERK on the full panel of 12 NSCLC cell lines. Cell lines were treated with vehicle control, 0.016, 0.4, 10 or 250 nM trametinib for 24 or 48 h. Western blot images are representative of two independent experiments.

3.3Trametinib causes cell cycle and growth arrest

To further elucidate downstream pathways of trametinib response, we investigated changes in cell cycle markers. Trametinib can induce p27KIP1 expression in cancer [12–14]. We confirmed a clear dose-dependent increase in p27KIP1 expression at 24 and 48 h almost universally in our cell lines, irrespective of overall trametinib sensitivities (Fig. 3). p27KIP1 expression was strongly induced with 10 nM in most cell lines while in H1693, H292 and H1838 the increase was less pronounced. This indicates that p27KIP1 expression changes are not predictive of overall trametinib response.

Fig. 3

Induction of G1/S arrest by trametinib. The full panel of 12 cell lines were treated with 0, 0.016, 0.4, 10 or 250 nM trametinib for 24 or 48 h. Expression levels of the G1/S phase proteins cyclin D1 and p27 were determined by western blotting. Six ug of protein was loaded in each lane for each sample. Equal loading of protein was determined by GAPDH. Western blot images are representative of two independent experiments.

The RAS/RAF/MEK pathway influences cyclin D1 expression [13], therefore, we examined cyclin D1 levels following trametinib treatment. In most cell lines, we observed a strong decrease in cyclin D1 expression with 10 and 250 nM trametinib at 24 and 48 h, irrespective of overall trametinib sensitivities (Fig. 3). Interestingly, whereas cyclin D1 was undetectable in H292 using antibody clone E3P5 S (Cat # 55506), it showed a similar down-regulation of cyclin D1 with a different antibody (Cat # 2922) (Supplementary Figure 1). Other studies have shown that the functional determinant for cyclin D1 downregulation is the phosphorylation status of retinoblastoma protein (pRb; S807/811) [13]. Therefore, we analyzed this with the four representative cell lines described above. In all four cell lines, phospho-pRb (p-pRb) was downregulated with 250 nM trametinib at 24 and 48 h (Supplementary Figure 1). Together, these data indicate that regardless of overall sensitivities, trametinib induces cell cycle arrest at G1/S phase in NSCLC cells. Specifically, trametinib-induced p27KIP1 accumulation is functional and inhibits cyclin D1-Cdk4/6 activity resulting in G1/S phase arrest. Since all NSCLC cell lines with varied RAS mutation status showed similar p-ERK and cyclin D1 responses, it is unlikely that G1/S arrest is the primary cause of the varied trametinib responses.

3.4Trametinib induces apoptotic and/or non-apoptotic cell death in NSCLC cell lines

To further elucidate trametinib-induced cellular outcomes, we investigated types of cell death. First, we examined PARP cleavage to examine apoptosis induction. In CALU-6 and H1299 (highly sensitive) and H358 (moderately sensitive) cells, we observed strong dose-dependent increases of cleaved PARP (c-PARP) after 24 and 48 h trametinib treatment, but in COR-L105 (highly sensitive) the increase occurred only at 48 h (Fig. 4). Furthermore, in CALU-6 we observed caspase-3/7 cleavage (Supplementary Figure 1). In contrast, c-PARP induction was not observed in other trametinib-sensitive cell lines including CALU-3 and H2087 (highly sensitive) and A427 and H1693 (moderately sensitive). In A427, we observed moderate c-PARP induction with 0.4 nM trametinib followed by an overall decrease at higher doses. Minimally sensitive cell lines H1838 and H2170 showed no c-PARP with increasing trametinib dosing (Fig. 4).

Fig. 4

Growth inhibitory and apoptotic effects of Trametinibs. The expression of the proliferation marker Ki67 and the apoptosis marker cleaved PARP were determined by western blotting in the full panel of 12 cell lines. Cells were treated with 0, 0.016, 0.4, 10 or 250 nM trametinib for 24 h or 48 h. Six ug of protein was loaded in each lane for each sample. Equal loading of protein was determined by GAPDH. Western blot images are representative of two independent experiments.

Since trametinib-induced PARP cleavage was not observed in 5 of our 9 sensitive cell lines, we investigated the possibility of the role of anti-apoptotic protein Bcl-2 and induction of necrotic cell death. We observed strong-to-moderate basal Bcl-2 expression in A427, H460, H1299, CALU-3, CORL-105 and H2170 cells, while in H2087 and H1838 we observed minimal expression (Fig. 5). Below detectable Bcl-2 expression was observed in CALU-6, H358, H1693 and H292. Bcl-2 expression levels do not indicate potential apoptotic response to trametinib in NSCLC cell lines, though it is possible that other pro-/anti-apoptotic factors play a role.

Fig. 5

Epithelial-to-mesenchymal transition (EMT) and anti-apoptotic status of NSCLC cell lines. The basal expression of EMT markers vimentin and E-cadherin and the anti-apoptotic Bcl2 protein was analyzed by western blotting of the full panel of 12 NSCLC cell lines. Equal loading of protein was determined by GAPDH. For nine cell lines, high exposure time blots are shown as needed to confirm the expression of respective proteins. Western blot images are representative of two independent experiments.

Additionally, we measured cell death using the PromoKine Apoptotic/Necrotic/Healthy Cells Detection Kit in the same four representative cell lines. After 72 h of trametinib treatment we observed a marked increase in cell death in the highly sensitive CALU-6 (+90%) and moderately sensitive A427 (+55%) cell lines but not in our minimally sensitive cell lines H1838 (0%) and H2170 (∼15%) (Supplementary Figure 2). Taken together, these data indicate that trametinib causes a strong increase in apoptotic and marginal necrotic death in sensitive lines.

3.5Trametinib induces autophagy in trametinib-resistant NSCLC cell lines

Most MTAs are known to induce cytostatic effects rather than cytotoxicity. We observed strong cytotoxic effects in our most sensitive cell line CALU-6 (Fig. 4 and Supplementary Figures 1 and 2). Since the apoptotic response in other cell lines was less pronounced, we investigated the possibility of trametinib-induced autophagy in NSCLC cell lines.

We observed no increase in LC3-II:LC3-I ratio in highly (CALU-6), moderately (A427) or minimally sensitive (H2170) cells (Fig. 6). Minimally sensitive H1838 cells showed a dose-dependent increase in LC3-II:LC3-I ratio. H1838 had significantly higher levels of LC3-II over LC3-I in our untreated control. Furthermore, in H1838, p62 levels were downregulated with 250 nM trametinib at 24 h indicating a state of autophagic flux, yet at 48 h p62 increased, indicating autophagy inhibition. We did not observe dose- or time-dependent changes in autophagy marker Beclin-1 expression in H1838, H2170 or A427 cells with trametinib. In CALU-6, Beclin-1 was slightly downregulated with 250 nM trametinib at both 24 and 48 h (Fig. 6). This result is consistent with apoptosis marker c-PARP response in CALU-6 (Fig. 4).

Fig. 6

Autophagy as a protective mechanism against trametinib. Autophagy flux (LC3 and p62) and initiation (Beclin 1) was determined by performing immunoblotting of one highly sensitive (CALU-6), one moderately sensitive (A427) and two minimally sensitive (H1838 & H2170) cell lines. Cells were treated with 0, 0.016, 0.4, 10 or 250 nM trametinib for 24 or 48 h. Six ug of protein was loaded in each lane for each sample. Equal loading of protein was determined by GAPDH. Western blot images are representative of two independent experiments.

3.6Epithelial-to-mesenchymal transition (EMT) does not predict trametinib sensitivity

EMT plays a role in resistance to MTAs in cancer including MEK inhibitors in KRAS mutated lung cancers [10]. To determine if EMT status is predictive of trametinib response, we examined the expression of vimentin and E-cadherin. In highly sensitive CALU-6, CALU-3, H1299 and moderately sensitive A427, we detected strong vimentin expression, indicative of mesenchymal characteristics (Fig. 5). In the highly (H2087, CALU-3 and H292), moderately (H1693 and H358), and minimally sensitive (H1838 and H2170) cell lines we observed strong E-cadherin expression (Fig. 5), indicating an epithelial characteristic. Interestingly, CALU-3 strongly expressed both EMT markers indicating the possibility of a heterogenous cell population. Neither marker was detected in H460 cells, potentially due to detection limits (Fig. 5). Cell lines with epithelial characteristic have varied responses from highly to minimally sensitive to trametinib. These data indicate EMT status cannot predict trametinib response.

3.7Ki67 does not predict trametinib sensitivity

Static Ki67 abundance is a well-known cell proliferation marker in cancer and clinical trials have used Ki67 expression as a biomarker for trametinib response [15]. We examined Ki67 expression following trametinib treatment using western blot and ICC. In trametinib-sensitive CALU-6 and H1299 cells at 24 and 48 h treatment, we observed a strong dose-dependent decrease in Ki67 expression, as well as a clear protein degradation pattern by western blot (Fig. 4). Highly sensitive H2087, moderately sensitive A427 and minimally sensitive H460 showed downregulation of Ki67 expression at higher trametinib doses at 24 and 48 h without protein degradation pattern (Fig. 4). Meanwhile, highly sensitive H292 and moderately sensitive H1693 and H358 showed effective downregulation of Ki67 from 0.4nM onwards at 48 h. Interestingly, in highly sensitive CALU-3 and COR-L105, and minimally sensitive H1838 and H2170, we observed similar decreases in Ki67 with 250 nM trametinib dose only at 48 h (Fig. 4).

We performed ICC staining in the representative cell lines after trametinib treatments to confirm western blot data. In CALU-6 and A427 there were very few surviving cells with a fraction of them Ki67 positive with 250 nM trametinib at 48 h. H1838 showed a marginal decrease in total surviving cells with 250 nM at 48 h. H2170 showed little change upon treatment. This ICC Ki67 expression data was consistent with our western blot data (Supplementary Figure 3). These data indicate that Ki67 response is not predictive of overall trametinib response.

3.8Trametinib activates AKT and therapy-induced senescence

AKT is a downstream effector of both EGFR and RAS signaling through the PI3K/AKT pathway and involved in promoting cell survival and senescence. MEK inhibitors are known to induce compensatory AKT activity as a cell survival mechanism [16, 17]. To better elucidate potential cell survival mechanisms or therapy induced senescence following trametinib treatment, we examined the expression of key markers of the PI3K/AKT pathway.

Highly sensitive CALU-3 and H2087, moderately sensitive H1693 and H358, as well as minimally sensitive H1838, H460 and H2170 showed dose-dependent increases in AKT phosphorylation (p-AKT) (Fig. 7). Slight differences were evident with the highly sensitive CALU-6 and moderately sensitive A427 which showed a sudden and abrupt increase in p-AKT expression at 10 nM (at 24 and 48 h) and 250 nM (at 48 h) trametinib, respectively. We did not see strong increases in p-AKT in highly sensitive H1299, COR-L105 or H292 (Fig. 7). This suggests that p-AKT modulation following trametinib treatment is complex and an unsuitable standalone predictive biomarker.

Fig. 7

Effects of trametinib on PI3K/AKT/mTOR pathway. The expression of the cell survival signaling protein phosphorylated AKT and growth regulation (phosphorylated 4EBP1), were determined by western blotting in the full panel of 12 cell lines using varied concentrations of trametinib at 24 or 48 h. Six μg of protein was loaded in each lane for each sample. Equal loading of protein was determined by GAPDH. Western blot images are representative of two independent experiments.

Being downstream of PI3K/AKT, we also studied the mTOR pathway effector protein 4E-BP1. In highly sensitive CALU-6, CALU-3, COR-L105 and moderately sensitive H358 we observed a marked decrease in phospho-4E-BP1 (Ser70) (p-4E-BP1) with increasing trametinib dosing at 24 and 48 h (Fig. 4). In moderately sensitive A427 and minimally sensitive H1838, we observed an increase in p-4E-BP1 expression with increasing trametinib at 24 and 48 h. Interestingly, in highly sensitive H1299, H292, H2087, moderately sensitive H1693 and minimally sensitive H460, we observed a dose dependent decrease of p-4E-BP1 at 24 h and an increase at 48 h, indicating senescence was induced (Fig. 7). Minimally sensitive H2170 expressed consistently high levels of p-4E-BP1 at 48 h with and without trametinib. Downregulation of p-ERK is known to cause G1/S-phase cell cycle arrest of cancer cells; in response to this, a compensatory AKT/mTOR pathway activation, which induces protein synthesis, would suggest a senescent characteristic [18]. Based on that, our parallel analysis of p-4E-BP1 responses can predict a cytostatic response following trametinib treatment. To measure trametinib-induced senescence, we performed β-galactosidase assay staining. In moderately sensitive H1693 and minimally sensitive H460 cells we observed a marked increase in β-galactosidase staining with 250 nM trametinib at 72 h. Surprisingly, in highly sensitive H1299 cells, which showed only marginal increase in p-4E-BP1 expression at 48 h, were distinctly positive for β-galactosidase (Supplementary Figure 4). Many p-4E-BP1 expressing cell lines like H2087, H292, A427 and H1838 did not show this senescence characteristic by β-galactosidase staining (Supplementary Figure 4). However, it is possible that therapy-induced senescent cells may not show β-galactosidase staining based on reports describing an increase in lysosomal content through many cell divisions cause β-galactosidase staining in senescent cells [19, 20]. It is also possible that our cell lines, based on unique cellular contexts, may not all commit to senescence. Altogether, these data indicate that trametinib causes primarily a cytostatic effect in NSCLC cell lines.