Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.

Loss of cell-cycle control is a hallmark of cancer. Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) are serine-threonine kinases important for the G1–S-phase transition of the cell cycle (1). Activation of CDK4/6 by binding to cyclin D results in phosphorylation of the tumor suppressor protein, Rb. In its unphosphorylated state, Rb binds and inhibits the transcription factor E2F, thereby maintaining the cells in the G1 phase of the cell cycle (1). However, upon phosphorylation by CDK4/6, Rb dissociates from E2F, allowing the latter to activate the transcription of genes responsible for S-phase transition (1).

The CDK4/6 inhibitor, palbociclib, has revolutionized the treatment of estrogen receptor–positive (ER+) breast cancer and is currently being tested for efficacy in a myriad of malignancies including colon cancer, glioblastoma, ovarian cancer, and pancreatic cancer (2). In melanoma, palbociclib has been combined with inhibitors of MAPK signaling in preclinical studies and, more recently in clinical trials (NCT04720768; refs. 3, 4). Importantly, palbociclib is being tested in melanoma subtypes that have few effective treatment options (5, 6). Furthermore, early-phase clinical trials are also underway investigating the efficacy of palbociclib as a single agent in melanomas with copy number variations of CDK4 or cyclin D1 (7). There are no targeted therapies that improve survival for patients with NRAS-mutant, NF1-mutant, or triple wild-type metastatic melanoma (5, 6, 8). Hence, CDK4/6 inhibitors may represent a promising addition to the therapeutic armamentarium for the management of BRAF wild-type melanomas.

Downstream of the MAPK pathway are the MAP Kinase-interacting serine/threonine-protein kinases 1 and 2 (MNK1 and MNK2; ref. 8). These proteins are the exclusive kinases for eukaryotic translation initiation factor 4E (eIF4E) on serine 209, the 5′cap binding protein within the eIF4F complex, which regulates mRNA translation (8). Studies in human and murine melanomas have shown that the phosphorylation of eIF4E on serine 209 supports tumor progression and decreases overall survival (9, 10). Of note, increased phosphorylation of eIF4E by MNK1/2 has been observed in response to chemotherapeutics and targeted inhibitors (11, 12). The latter is perhaps not entirely surprising, as feedback loops, whereby the inhibition of one pathway is compensated for by the upregulation of other signaling pathways, are a common mechanism of acquired therapy resistance (13). The activity of eIF4E is also regulated by the PI3K/AKT/mTOR pathway (8). Activation of the PI3K-AKT/mTOR pathway results in the phosphorylation of eIF4E-binding proteins, 4EBP1/2 (8). Phosphorylation of 4EBP1/2, by mTOR, releases eIF4E and results in activated translation (8). Studies have demonstrated that inhibition of the PI3K/AKT/mTOR pathway in conjunction with palbociclib results in substantially decreased tumor growth (14–17). Furthermore, new studies have also implicated a role for CDK4 in regulating the availability of eIF4E through its phosphorylation of 4EBP1/2 (18). Herein, we present data that eIF4E phosphorylation, which is induced downstream of activated MAPK-MNK1/2 signaling, is induced in melanoma and breast cancer cell lines in response to palbociclib treatment. We thus set forth to test the hypothesis that the antitumor effects of palbociclib are limited by its ability to promote the downstream compensatory phosphorylation of eIF4E. Indeed, our data support that the combination of MNK1/2 inhibitors and palbociclib cooperate as a potentially important new therapeutic approach to the management of melanoma and other therapy-resistant cancers.

Cell lines and reagents

BLM, NRAS-mutant human melanoma cells, were a generous gift from Ghanem Ghanem (Institut Jules Bordet, Bruxelles). MEWO, NF1-mutant human melanoma cells, were a generous gift from Ian Watson (McGill University). BLM, MEWO, MCF7, T47D, CHL-1, and HEK293T cell lines were cultured in DMEM (Wisent bioproducts #319–005-CL) containing 10% FBS and 100 IU/mL penicillin and 100 IU/mL streptomycin at 37°C and 5% CO2. HEK293T and palbociclib-resistant MCF7 cells were a generous gift from Dr. Sidong Huang. T47D-parental and matched palbociclib-resistant cell lines were a generous gift from Dr. David Cescon and were cultured in RPMI supplemented with 10% FBS (19). All experiments were initiated within 5 passages of thawing a master stock of cells. Cell lines were routinely tested for Mycoplasma using the e-Myco VALID Mycoplasma PCR Detection Kit. Identity of cell lines was verified by short tandem repeat profiling. Palbociclib (#S1116) was purchased from Selleck Chemicals and dissolved according to the manufacturer's instructions to a stock concentration of 10 mmol/L. During experiments, cells were exposed to palbociclib at concentrations ranging from 25 nmol/L to 500 nmol/L. For in vivo experiments, palbociclib monhohydrochloride was purchased from MedChemExpress (#HY-50767A). SEL201 (20) was provided by RYVU therapeutics. eFT508 was purchased from Selleckchem (#S8275). Unless specified otherwise, SEL201 and eFT508 drug treatments were performed at 2.5 μmol/L and 0.5 μmol/L respectively.

Colony formation assay

Cells were seeded in 6-well plates at densities of 2,000 to 5,000 cells per well and allowed to adhere overnight. Media was changed the next day and drug was added. At experimental endpoint, the cells were stained with 0.5% crystal violet in 70% ethanol for 1 hour. The plates were scanned and clonogenic outgrowth was quantified manually using ImageJ, or were dissolved using 1 mL of 10% glacial acetic acid per well for 1 hour on a shaker. Subsequently, absorbance was measured at 590 nm and relative differences were graphed. Clonogenic assays on CHL-1 were performed at The Peter MacCallum Cancer Centre, Victoria, Australia.

Immunoblotting

500,000 to 1,000,000 cells were treated for 48 hours. Lysates were prepared using RIPA containing 50 mmol/L Tris HCl pH8.0, 150 mmol/L NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 5 mmol/L EDTA, protease (Roche, #11697498001), and phosphatase inhibitors (Roche #4906845001). Cells were lysed using 50 μL of complete RIPA per 1 million cells and sonicated before centrifuging at max speed for 15 minutes. Protein concentration was measured by Bradford assay. 50 μg of protein lysate were PAGE-separated (40% Acrylamide/Bis Solution, 37.5:1; Bio-Rad, #1610148) and transferred onto PVDF membranes (Roche #0301004001), blocked for 1 hour in 5% nonfat milk, and incubated with primary antibody overnight at 4°C. The following day, the membranes were washed and incubated with secondary antibody for 1 hour. The membranes were developed using Amersham ECL Western Blotting Reagent (#RPN2106) or Immobilon Western Chemiluminescent HRP Substrate (#WBKLS0500). Antibody details can be found in Supplementary Table S1.

Quantitative PCR

RNA was prepared using E.Z.N.A. Total RNA Kit (Omega Bio-tek). cDNA was prepared from 1 μg of total RNA using iScript cDNA Synthesis Kit (Bio-Rad). Gene expression was quantified using the Applied Biosystems 7500 Fast Real-Time PCR System using GoTaq Green Master Mix (Promega). Primer details can be found in Supplementary Table S2.

Lentivirus production and transduction

Lentiviral plasmids were co-transfected with the packaging plasmids Pax2 and MD2G into HEK293T cells using calcium phosphate precipitation. Viral supernatant was harvested 72 hours post transfection, spun down at 500 × g for 5 minutes, and filtered through a 0.45-μm filter. 500 μL of viral supernatant were used to transduce 100,000 cells in the presence of 8 μg/mL polybrene for 24 hours. The following day, media was changed, and transduced cells were selected using 2 μg/mL of puromycin.

Cell-cycle analysis

20,000 to 50,000 cells were treated with inhibitors for 3 days and then washed twice with ice-cold PBS containing 1% FBS. Cells were stained with 50 μg/mL propidium iodide solution in hypotonic buffer (0.1% Triton X-100 and 0.1% sodium citrate) for at least 20 minutes in the dark. At least 10,000 cycling cells were analyzed using BD LSR Fortessa II. Data was analyzed using FlowJo VX (RRID:SCR_008520).

dCas9 cell line generation

BLM cells were transduced with vectors encoding dCas9 (Addgene, #46911) Infected cells were single-cell sorted and clones were expanded and selected for proliferation rates that matched the parental cell lines. Single-guide RNA (sgRNA)’s were cloned in to pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene, #60955). sgRNA sequences can be found in Supplementary Table S3.

Fluorescence Ubiquitination Cell-Cycle Indicator cell line generation

Cells were virally transduced with Fluorescence Ubiquitination Cell-Cycle Indicator (FUCCI) vector expressing mKO-CDT1 and clover-Geminin. Populations of cells expressing medium to high levels of mKO and clover were sorted into culture and used for further experiments.

Senescence assay

One hundred cells were seeded in 12-well plates and subsequently treated with indicated concentrations of SEL201, palbociclib, or their combination for 7 days. Senescence staining was performed using Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, #9860) according to the manufacturer's protocol. After the incubation period, images were taken and approximately equal numbers of total cells were quantified across treatments. Total and β-gal positive cells were manually counted using ImageJ software (RRID:SCR_003070).

In vivo experiments

Animal experiments were conducted according to the regulations established by the Canadian Council of Animal Care, and protocols approved by the McGill University Animal Care and Use Committee (2015–7672). Tyr::CreER/BRafCA/+/Ptenlox/lox mice were treated topically with 4-hydroxytamoxifen (4-HT) for 3 consecutive days. Fifteen days post 4-HT exposure, mice were randomized into 4 cohorts and treated independently with vehicle, palbociclib (120 mg/kg), eFT508 (1 mg/kg), or the combination of both drugs by oral gavage. Endpoint was determined when the tumors ulcerated or reached a volume of approximately 1,500 mm3. BLM cells were injected into the right flank of immunodeficient NOD/SCID mice. When tumor volumes reached around 80 to 100 mm3, the mice were randomized into 4 cohorts and treated independently with vehicle, palbociclib (120 mg/kg), eFT508 (1 mg/kg), or the combination of both drugs. Endpoint was determined when the tumor volume reached approximately 2,000 mm3. All in vivo drug treatments were administered on a 5-day-on and 2-day-off schedule.

Mass spectrometry–based proteomics

BLM cells were treated with either monotherapy or the combination of the two for 48 hours. At end point the cells were washed with PBS, harvested by scraping, flash frozen in liquid nitrogen and stored at −80°C before processing. Detailed methods for sample preparation, acquisition and subsequent data analysis can be found in the supplemental methods.

Immunohistochemistry

Briefly, tumor sections were stained for phosphorylated eIF4E (phospho-eIF4E), and counterstained with 20% Harris-modified hematoxylin (Thermo Fisher Scientific). Slides were scanned and phospho-eIF4E levels were assessed by calculating the area of positive and negative cells using the pixel classification feature on QuPath software (threshold score range: negative > 0.9 < positive). Antibody details can be found in Supplementary Table S1.

Statistical analysis

Unless otherwise specified, all experiments were performed in a minimum of 3 biological replicates. All in vitro and in vivo data are represented as mean ± SD. Student t test or one-Way ANOVA (Tukey post hoc test) were applied for statistical tests presented, using GraphPad Prism Version 9.0.0 (RRID:SCR_002798). The specific statistical analysis for each figure is listed in Supplementary Table S4. P values < 0.05 were considered significant. Log-rank test was applied to Kaplan–Meier analyses in in vivo experiments. P values are specified in the figure itself, the figure legend, or in Supplementary Table S4.

Data availability

Proteomics data from our mass spectrometry experiments have been deposited to the PRIDE database (RRID:SCR_012052) with the dataset accession PXD033390. All supporting data have been included in Supplementary Tables in the manuscript or are available upon request from the corresponding authors.

Combined inhibition of CDK4/6 and MNK1/2 decreases clonogenic outgrowth of melanoma and breast cancer cell lines

Several anticancer therapies have been shown to trigger the activation of the MNK1/2–eIF4E axis, a well-known pro-survival pathway (11, 12). We found that the CDK4/6 inhibitor, palbociclib, induced an increase in the expression of phospho-eIF4E, compared with vehicle-control treated cells in the NRAS- and NF1-mutated melanoma cell lines BLM and MEWO, respectively (Fig. 1A, and B). The phosphorylation of eIF4E enhances the translation of mRNAs which encode pro-survival proteins (10, 20, 21). Thus, we hypothesized that the therapeutic efficacy of palbociclib is limited by its ability to promote the phosphorylation of eIF4E. To test this, we blocked the activity of MNK1/2, the only known kinases for serine 209 on eIF4E, predicting that this may sensitize cancer cells to the antitumor effects of palbociclib. We co-treated BLM cells with palbociclib and the MNK1/2 inhibitor SEL201 and showed significantly decreased colony formation versus either single agent (Fig. 1C). This effect is recapitulated in BLM cells exposed to a combination of palbociclib and eFT508 (22), a MNK1/2 inhibitor currently in clinical testing (NCT03616834, NCT04261218, NCT04622007). Similarly, in NF1-mutant MEWO cells combined pharmacologic inhibition of CDK4/6 and MNK1/2 resulted in significantly decreased clonogenic outgrowth (Fig. 1D). We observed suppression of phospho-Rb and phospho-eIF4E attributed to palbociclib and MNK1/2 inhibitor treatment, respectively (Fig. 1E and F). We next assessed whether MEWO cells in which we stably silenced MNK1 and MNK2 using shRNA (MEWO shMKNK1/2) were sensitized to palbociclib. We observed that MEWO cells with MNK1/2 knocked down have significantly impaired clonogenic outgrowth when treated with palbociclib compared with their scramble counterparts (Fig. 1G). Similarly, we used the dCas9 Clustered Regularly Interspersed Short Palindromic Repeats interference system to genetically repress the transcription of MKNK1 and MKNK2, to best recapitulate the pharmacologic inhibition of MNK1/2 in BLM cells. BLM cells that are deficient in MNK1/2 are more sensitive to palbociclib compared with their scramble counterparts (Supplementary Fig. S1A). Repression of MKNK1 expression was measured by immunoblotting, and expression of MKNK2 was measured only by quantitative PCR, as currently available MNK2 antibodies are not specific (Fig. 1H and I; Supplementary Fig. S1B and S1C).

Figure 1.

Cotargeting MNK1/2 and CDK4/6 decreases clonogenic outgrowth of melanoma cancer cell lines. A and B, The phosphorylation of eIF4E is induced by palbociclib treatment in BLM and MEWO melanoma cells at 24 hours. Numbers above the p-eIF4E panels indicate the relative densitometry values for the expression of p-eIF4E/eIF4E calculated from the values of 3 biological replicates per cell line. C, Clonogenic assay demonstrating the effects of palbociclib in combination with 2.5 μmol/L SEL201 or 0.5 μmol/L eFT508 in BLM melanoma cells across 3 (P+S) or 4 (P+E) biological replicates. (one-way ANOVA P50 vs. P50+S, P = 0.0009; P100 vs. P100+S, P = 0.0011) (one-way ANOVA P50 vs. P50+E, P = 0.0638; P100 vs. P100+E, P = 0.002). D, Clonogenic assay demonstrating the effect of palbociclib in combination with 2.5 μmol/L SEL201 in MEWO melanoma cells across 3 biological replicates. (one-way ANOVA P100 vs. P100+S, P = 0.0058). E and F, Phosphorylation of Rb and eIF4E is repressed upon palbociclib and SEL201 exposure at 48 hours in BLM and MEWO, respectively. G, Representative clonogenic assay demonstrating that MEWO cells deficient in MKNK1 and MKNK2 are sensitive to palbociclib across 2 biological replicates (one-way ANOVA shCTL P50 vs. shMNK1/2 P50, P = 0.0037; shCTL P100 vs. shMNK1/2 P100, P = 0.0101). H, Immunoblot demonstrating MNK1 knockdown in MEWO-modified cells. I, qPCR data demonstrating MKNK2 knockdown in MEWO-modified cells.

Figure 1.

Cotargeting MNK1/2 and CDK4/6 decreases clonogenic outgrowth of melanoma cancer cell lines. A and B, The phosphorylation of eIF4E is induced by palbociclib treatment in BLM and MEWO melanoma cells at 24 hours. Numbers above the p-eIF4E panels indicate the relative densitometry values for the expression of p-eIF4E/eIF4E calculated from the values of 3 biological replicates per cell line. C, Clonogenic assay demonstrating the effects of palbociclib in combination with 2.5 μmol/L SEL201 or 0.5 μmol/L eFT508 in BLM melanoma cells across 3 (P+S) or 4 (P+E) biological replicates. (one-way ANOVA P50 vs. P50+S, P = 0.0009; P100 vs. P100+S, P = 0.0011) (one-way ANOVA P50 vs. P50+E, P = 0.0638; P100 vs. P100+E, P = 0.002). D, Clonogenic assay demonstrating the effect of palbociclib in combination with 2.5 μmol/L SEL201 in MEWO melanoma cells across 3 biological replicates. (one-way ANOVA P100 vs. P100+S, P = 0.0058). E and F, Phosphorylation of Rb and eIF4E is repressed upon palbociclib and SEL201 exposure at 48 hours in BLM and MEWO, respectively. G, Representative clonogenic assay demonstrating that MEWO cells deficient in MKNK1 and MKNK2 are sensitive to palbociclib across 2 biological replicates (one-way ANOVA shCTL P50 vs. shMNK1/2 P50, P = 0.0037; shCTL P100 vs. shMNK1/2 P100, P = 0.0101). H, Immunoblot demonstrating MNK1 knockdown in MEWO-modified cells. I, qPCR data demonstrating MKNK2 knockdown in MEWO-modified cells.

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CDK4/6 inhibitors are clinically indicated for the treatment of ER+ metastatic breast cancer, but the majority of patients will eventually develop resistance to CDK4/6i (23). Thus we wanted to determine whether our results extended to ER+ breast cancer. Consistent with our data in melanoma, palbociclib treatment of two ER+ breast cancer cell lines, MCF7 and T47D, resulted in increased phosphorylation of eIF4E (Fig. 2A, and B). Moreover, the combination of palbociclib and SEL201 significantly decreased the clonogenic outgrowth of MCF7 cells compared with either single agent (Fig. 2C). Similar results were obtained in T47D, wherein the combination significantly decreased clonogenicity compared with either of the single agents alone (Fig. 2D). Through immunoblotting, we observed suppression of phospho-Rb and phospho-eIF4E attributed to palbociclib and MNK1/2 inhibitor treatment, respectively (Fig. 2E, and F).

Figure 2.

Combined inhibition of MNK1/2 and CDK4/6 represses clonogenic outgrowth in breast cancer cell lines. A and B, The phosphorylation of eIF4E is induced by palbociclib treatment in MCF7 and T47D breast cancer cells at 24 hours. Numbers above the p-eIF4E panels indicate the relative densitometry values for the expression of p-eIF4E/eIF4E calculated from the values of three biological replicates per cell line. C, Clonogenic assay demonstrating the effects of palbociclib in combination with 2.5 μmol/L SEL201 in MCF7 across three biological replicates (one-way ANOVA P25 vs. P25+S, P = < 0.0001; P50 vs. P50+S, P = < 0.0001). D, Clonogenic assay demonstrating the effects of palbociclib in combination with SEL201 in T47D across three biological replicates (one-way ANOVA P25 vs. P25+S, P = 0.0483; P50 vs. P50+S, P = 0.0379). E and F, Phosphorylation of Rb and phosphorylation of eIF4E is repressed upon palbociclib and SEL201 exposure, respectively, at 48 hours in MCF7 and T47D.

Figure 2.

Combined inhibition of MNK1/2 and CDK4/6 represses clonogenic outgrowth in breast cancer cell lines. A and B, The phosphorylation of eIF4E is induced by palbociclib treatment in MCF7 and T47D breast cancer cells at 24 hours. Numbers above the p-eIF4E panels indicate the relative densitometry values for the expression of p-eIF4E/eIF4E calculated from the values of three biological replicates per cell line. C, Clonogenic assay demonstrating the effects of palbociclib in combination with 2.5 μmol/L SEL201 in MCF7 across three biological replicates (one-way ANOVA P25 vs. P25+S, P = < 0.0001; P50 vs. P50+S, P = < 0.0001). D, Clonogenic assay demonstrating the effects of palbociclib in combination with SEL201 in T47D across three biological replicates (one-way ANOVA P25 vs. P25+S, P = 0.0483; P50 vs. P50+S, P = 0.0379). E and F, Phosphorylation of Rb and phosphorylation of eIF4E is repressed upon palbociclib and SEL201 exposure, respectively, at 48 hours in MCF7 and T47D.

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Combined inhibition of CDK4/6 and MNK1/2 results in decreased expression of proteins involved in DNA replication, cell cycle, and mitosis

We next sought to determine the mechanism by which SEL201 and palbociclib cooperate to inhibit clonogenic outgrowth of cancer cells. MNK1/2 inhibitors are well known to repress the synthesis of a subset of proteins with roles in cell-cycle regulation and cell survival (20, 24). Therefore, we used mass spectrometry–based quantitative proteomics to identify differentially expressed proteins upon combined SEL201 and palbociclib. Hierarchical clustering of differentially expressed proteins revealed a unique protein expression signature in cells treated with the combination of palbociclib and SEL201, compared with single agents alone (Fig. 3A). Moreover, we observed a large cluster (cluster 9) of proteins that have uniquely decreased expression in the combination therapy compared with SEL201 or palbociclib alone (Fig. 3A; Supplementary Table S5). Pathway analysis of cluster 9 revealed multiple enriched pathways involved in cell-cycle dynamics including hallmark E2F targets (M5925), cell cycle (R-HSA-1640170), and chromosome segregation (GO:0007059) in cells treated with the combination therapy (Fig. 3B; Supplementary Table S5). Upon closer inspection, we observed that the cells treated with the combination had a repressed E2F protein expression signature (Supplementary Fig. S2A). In depth analysis identified that the combination repressed the protein expression of critical cell-cycle regulators that are frequently associated with poor prognosis in multiple malignancies including TOP2a (25), AURKB (26), KIF4A (27), RRM2 (28; Fig. 3C), TPX2 (29), and INCENP (30). Network analysis of cluster 9 further demonstrated that proteins frequently associated with AURKB were near collectively repressed in the combination (Supplementary Fig. S2B; red solid circles), resulting in deficiencies in resolution of sister chromatid cohesion (R-HSA-2500257), separation of sister chromatids (R-HSA-2467813), and chromosome, centromeric region (GO:0000775). Pathway analysis of other clusters represented in the heatmap of differentially expressed proteins identified other potentially relevant clusters, which appeared to be driven by the single agent treatments (Supplementary Fig. S2C–S2L).

Figure 3.

Combined inhibition of MNK1/2 and CDK4/6 results in repression of critical mitotic regulators. A, Heatmap showing differential protein expression between BLM cells treated with vehicle, SEL201, palbociclib, or the combination therapy at 48 hours. B, Pathway analysis of proteins in cluster 9 shows repression of key mitotic proteins in the combination. C, Combined inhibition of MNK1/2 and CDK4/6 further decreases the expression of E2F target proteins associated with mitosis. D–F, Immunoblot validating that the combination of SEL201 plus palbociclib further represses the expression of critical mitotic enhancers in BLM, MCF7, and T47D at 48 hours. Numbers below the AURKB panels indicate the relative densitometry values for the expression of AURKB/loading control calculated from the values of 2 biological replicates per cell line. G, Immunoblot demonstrating that palbociclib represses the expression of critical mitotic enhancers in MNK1/2-deficient BLM-dCas9 cells at 48 hours.

Figure 3.

Combined inhibition of MNK1/2 and CDK4/6 results in repression of critical mitotic regulators. A, Heatmap showing differential protein expression between BLM cells treated with vehicle, SEL201, palbociclib, or the combination therapy at 48 hours. B, Pathway analysis of proteins in cluster 9 shows repression of key mitotic proteins in the combination. C, Combined inhibition of MNK1/2 and CDK4/6 further decreases the expression of E2F target proteins associated with mitosis. D–F, Immunoblot validating that the combination of SEL201 plus palbociclib further represses the expression of critical mitotic enhancers in BLM, MCF7, and T47D at 48 hours. Numbers below the AURKB panels indicate the relative densitometry values for the expression of AURKB/loading control calculated from the values of 2 biological replicates per cell line. G, Immunoblot demonstrating that palbociclib represses the expression of critical mitotic enhancers in MNK1/2-deficient BLM-dCas9 cells at 48 hours.

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Next, we validated the identified changes in proteins that are required for mitosis by immunoblot. Consistently, in BLM, MCF7 and T47D, expression of proteins critical for chromosome segregation, such as AURKB, survivin, and TPX2, were further reduced in cells treated with the combination, compared with single agents (Fig. 3D to F). Moreover, qPCR analysis of BLM and MCF7 cells treated with the combination demonstrated that the repressed expression of these proteins was also detected at the mRNA level (Supplementary Fig. S2M and S2N). To further validate these effects, we treated the MNK1/2 deficient BLM cells with palbociclib. MNK1/2 deficient BLM cells treated with palbociclib for 48 hours showed a further decrease in AURKB, survivin, and TPX2 expression compared with scramble control and MNK1/2 knockdown alone (Fig. 3G). These results suggest that dual inhibition of MNK1/2 and CDK4/6 repress the expression of key mitotic proteins that are commonly associated with poor prognosis.

Combined inhibition of CDK4/6 and MNK1/2 results in G1 cell-cycle arrest, suppression of cyclin A expression, and increased cellular senescence

Palbociclib and SEL201 are both able to repress the expression of critical cell-cycle regulators (2, 21, 24, 31). Moreover, based on the pathway analysis from cluster 9 in our quantitative proteomics, we examined the impact of combined palbociclib and SEL201 treatment on progression through the cell cycle. The FUCCI system employs two fluorescent proteins (mKusabira-Orange, and Clover), each fused to different regulators of the cell cycle, (cdt1, and geminin) respectively (32). During G1, cells fluoresce orange due to the proteasomal degradation of clover-geminin, while mKO-cdt1 expression is sustained. In subsequent phases of the cell cycle, mKO-cdt1 is degraded while clover-geminin expression increases, resulting in cells that fluoresce green in G2 (32). When we treated FUCCI stable-BLM cells with palbociclib and SEL201 we observed that the cells treated with the combination therapy had an increased accumulation in the G1 phase of the cell cycle, compared with either single agent alone (Fig. 4A).

Figure 4.

Inhibition of MNK1/2 and CDK4/6 results in G1 cell-cycle arrest, suppression of cyclin A expression, and increased cellular senesence. A, The combination of palbociclib and SEL201 induces G1 cell-cycle arrest as measured by the FUCCI-tagged BLM melanoma cells (bar graph represents the average ± SD of 2 biological replicates). B, The combination of palbociclib and SEL201 induces G1 cell-cycle arrest as measured by Propidium Iodide-stained BLM melanoma cells (bar graph represents the average ± SD of 2 biological replicates). C, Cell-cycle analysis demonstrated that the combination induces G1 cell-cycle arrest in MCF7 cells (bar graph represents the average ± SD of 2 biological replicates). D, Immunoblot demonstrating increased expression of endogenous cell-cycle inhibitor p27 and decreased expression in cyclin A in BLM-FUCCI cells treated with the combination at 48 hours. Numbers below p27 and cyclin A blots indicate average relative densitometry values of target protein/loading control across 3 biological replicates. E and F, Immunoblot demonstrating increased expression of endogenous cell-cycle inhibitor p27 and decreased expression of cyclin A in BLM, MCF7 cells treated with the combination at 48 hours. Numbers below the p27 and cyclin A panels indicate the relative densitometry values for the expression of target protein/loading control across 3 biological replicates per cell line. G, BLM cells treated with palbociclib and SEL201 have significantly more senescent cells compared with either monotherapy across 2 biological replicates. Scale bar, 150 μm; (one-way ANOVA; palbociclib vs. palbociclib+SEL201, P = 0.0047; SEL201 vs. palbociclib+SEL201, P = 0.003). H, BLM cells treated with palbociclib and SEL201 express higher levels of senescence markers at 48 hours. I, MCF7 cells treated with palbociclib and SEL201 have significantly more senescent cells compared with either monotherapy across 3 biological replicates. Scale bar, 75 μm; (one-way ANOVA; palbociclib vs. palbociclib+SEL201, P = 0.0017; SEL201 vs. palbociclib+SEL201, P = <0.0001).

Figure 4.

Inhibition of MNK1/2 and CDK4/6 results in G1 cell-cycle arrest, suppression of cyclin A expression, and increased cellular senesence. A, The combination of palbociclib and SEL201 induces G1 cell-cycle arrest as measured by the FUCCI-tagged BLM melanoma cells (bar graph represents the average ± SD of 2 biological replicates). B, The combination of palbociclib and SEL201 induces G1 cell-cycle arrest as measured by Propidium Iodide-stained BLM melanoma cells (bar graph represents the average ± SD of 2 biological replicates). C, Cell-cycle analysis demonstrated that the combination induces G1 cell-cycle arrest in MCF7 cells (bar graph represents the average ± SD of 2 biological replicates). D, Immunoblot demonstrating increased expression of endogenous cell-cycle inhibitor p27 and decreased expression in cyclin A in BLM-FUCCI cells treated with the combination at 48 hours. Numbers below p27 and cyclin A blots indicate average relative densitometry values of target protein/loading control across 3 biological replicates. E and F, Immunoblot demonstrating increased expression of endogenous cell-cycle inhibitor p27 and decreased expression of cyclin A in BLM, MCF7 cells treated with the combination at 48 hours. Numbers below the p27 and cyclin A panels indicate the relative densitometry values for the expression of target protein/loading control across 3 biological replicates per cell line. G, BLM cells treated with palbociclib and SEL201 have significantly more senescent cells compared with either monotherapy across 2 biological replicates. Scale bar, 150 μm; (one-way ANOVA; palbociclib vs. palbociclib+SEL201, P = 0.0047; SEL201 vs. palbociclib+SEL201, P = 0.003). H, BLM cells treated with palbociclib and SEL201 express higher levels of senescence markers at 48 hours. I, MCF7 cells treated with palbociclib and SEL201 have significantly more senescent cells compared with either monotherapy across 3 biological replicates. Scale bar, 75 μm; (one-way ANOVA; palbociclib vs. palbociclib+SEL201, P = 0.0017; SEL201 vs. palbociclib+SEL201, P = <0.0001).

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It is possible that anticancer agents that deregulate cell-cycle progression can induce unwanted fluorescence kinetics in the FUCCI system, which are not consistent with actual changes in the phases of the cell cycle (33). Using traditional flow cytometry and propidium iodide staining, BLM cells showed increased accumulation of cells in the G1 phase of the cell cycle with combined SEL201 and palbociclib treatment compared with either single agent (Fig. 4B). Similarly, in MCF7, the combined treatment with palbociclib and SEL201 increased the proportion of cells in G1, compared with either single agent (Fig. 4C). Cyclin A is an E2F target gene whose overexpression has been associated with poor patient outcomes (34). Although palbociclib represses cyclin A expression, palbociclib-resistant cells fail to maintain repressed cyclin A levels (16, 35). Through immunoblotting, we observed that the expression of cyclin A is further repressed in BLM, and MCF7 cells treated with combined SEL201 and palbociclib, compared with either of the single agents (Fig. 4DF).

In our proteomics analysis, we observed that BLM cells treated with the combination of palbociclib and SEL201 had elevated levels of p27 compared with either monotherapy. Recent studies have shed light on the dual roles p27 plays in cell-cycle control. Cells devoid of p21 or p27, paradoxically, cannot form cyclin D1-CDK4 complexes (36). Not only does p27 facilitate the interaction between cyclin D1 and CDK4, but, p21 or p27 also enhance CDK4 activity by enabling CDK-activating kinase (CAK)-mediated phosphorylation of the T-loop in CDK4 (37, 38). However, despite these distinct activating mechanisms, increased levels of p21 and p27 strongly inhibit cyclin D1-CDK4 complex formation (37, 39). We observed that the treatment of BLM and BLM-FUCCI cells with combined palbociclib and SEL201 led to increased p27 levels, compared with either single agent alone (Fig. 4D, E). Similarly, MCF7 cells treated with SEL201 have an increased expression of p27 (Fig. 4F). Cellular accumulation of the cell-cycle inhibitor p27 is an established marker of senescence (40). We observed that our cells treated with the combination were enlarged and flattened; a phenotype characteristic of cells undergoing senescence (Fig. 4G; ref. 41). Palbociclib has been demonstrated to induce senescence (3, 4, 42), and in our BLM model this effect was mild (Fig. 4G). However, the combination of palbociclib with SEL201 resulted in a significant increase in senescence compared with either single agent as measured by β–galactosidase activity (Fig. 4G). An initial pathway analysis of cluster 2 from our proteomics data (Fig. 3A) revealed an enrichment of proteins related to Senescence and autophagy in cancer (WP615; Supplementary Fig. S2D; Supplementary Table S5). The latter prompted a more focused analysis of the proteomics data, revealing an augmented expression of proteins frequently implicated in senescence, including proteins among the senescence-associated secretory phenotype (Supplementary Table S6). In support of the proteomics data, immunoblotting of BLM cells treated for 48 hours with the combination of palbociclib and SEL201 revealed an increased expression of pro-senescence markers ATG7, ISG15, Fibronectin, and PDCD4 (Fig. 4H; Supplementary Table S6). Similarly, MCF7 cells treated with the combination resulted in significantly increased β–galactosidase activity compared with either monotherapy at 7 days (Fig. 4I). Overall, these data indicate that the combined inhibition of MNK1/2 and CDK4/6 results in an accumulation of cells in G1 during short-term exposure, and prolonged exposure results in cells becoming senescent.

Inhibition of MNK1/2 overcomes resistance to palbociclib

Multiple mechanisms lead to the acquisition of resistance to palbociclib, including loss of RB, loss of PRMT5 regulation, increased cyclin E expression, or activating mutations in the PI3K pathway (14, 23, 31). Several studies have also demonstrated that aberrant mRNA translation may be a mode of acquired resistance to therapies, including palbociclib (16, 43). To understand whether inhibitors of MNK1/2 hold promise in CDK4/6 inhibitor resistant disease, we tested previously described palbociclib-resistant CHL-1 (CHL-1-PalboR) melanoma cells (31). CHL-1 cells harbor no mutations in BRAF, NF1, and NRAS (triple-WT; ref. 31). We observed that the combined inhibition of MNK1/2 and CDK4/6 significantly repressed clonogenic outgrowth of CHL-1 parental melanoma cells (Fig. 5A). Moreover, CHL-1-PalboR cells treated with MNK1/2 inhibition were resensitized to palbociclib (Fig. 5A).

Figure 5.

Pharmacologic inhibition of MNK1/2 overcomes palbociclib resistance in models of melanoma and breast cancer. A, Clonogenic outgrowth of CHL-1 palbociclib-resistant melanoma cells is repressed upon exposure to 2.5 μmol/L SEL201 across 3 biological replicates (CHL-1 Parental: one-way ANOVA P30 vs. P30+S, P = <0.0001; P100 vs. P100+S, P = 0.0037; CHL-1 PalboR: one-way ANOVA P30 vs. P30+S, P = <0.0001; P100 vs. P100+S P = <0.0001; P300 vs. P300+S P = <0.0001). B, Clonogenic outgrowth of MCF7 palbociclib-resistant breast cancer cells is repressed upon exposure to 2.5 μmol/L SEL201 across 3 biological replicates (MCF7 Parental: one-way ANOVA P25 vs. P25+S P = 0.001; P100 vs. P100+S P = 0.1864; MCF7 PalboR: one-way ANOVA P25 vs. P25+S P = <0.0001; P100 vs. P100+S P = < 0.0001). C, MCF7 palbociclib-resistant breast cancer cells have a higher basal level of phospho-eIF4E compared with their parental counterpart at 48 hours. D, Palbociclib-resistant MCF7 cells express higher levels of MKNK2 compared with MCF7 parental cells across 2 biological replicates (unpaired t test P = 0.046). E, T47D palbociclib-resistant breast cancer cells are resensitized to palbociclib upon exposure to 2.5 μmol/L SEL201 exposure across 3 biological replicates (T47D Parental: one-way ANOVA P25 vs. P25+S P = < 0.0001; P100 vs. P100+S P = 0.0016; T47D PalboR: one-way ANOVA P25 vs. P25+S P = < 0.0001; P100 vs. P100+S P = < 0.0001; P250 vs. P250+S P = < 0.0001; P500 vs. P500+S P = < 0.0001). F, T47D palbociclib-resistant breast cancer cells have a higher basal level of phospho-eIF4E compared with their parental counterpart. G, Palbociclib-resistant T47D cells express higher levels of MKNK2 compared with T47D parental cells across 4 biological replicates (unpaired t test P = 0.0036).

Figure 5.

Pharmacologic inhibition of MNK1/2 overcomes palbociclib resistance in models of melanoma and breast cancer. A, Clonogenic outgrowth of CHL-1 palbociclib-resistant melanoma cells is repressed upon exposure to 2.5 μmol/L SEL201 across 3 biological replicates (CHL-1 Parental: one-way ANOVA P30 vs. P30+S, P = <0.0001; P100 vs. P100+S, P = 0.0037; CHL-1 PalboR: one-way ANOVA P30 vs. P30+S, P = <0.0001; P100 vs. P100+S P = <0.0001; P300 vs. P300+S P = <0.0001). B, Clonogenic outgrowth of MCF7 palbociclib-resistant breast cancer cells is repressed upon exposure to 2.5 μmol/L SEL201 across 3 biological replicates (MCF7 Parental: one-way ANOVA P25 vs. P25+S P = 0.001; P100 vs. P100+S P = 0.1864; MCF7 PalboR: one-way ANOVA P25 vs. P25+S P = <0.0001; P100 vs. P100+S P = < 0.0001). C, MCF7 palbociclib-resistant breast cancer cells have a higher basal level of phospho-eIF4E compared with their parental counterpart at 48 hours. D, Palbociclib-resistant MCF7 cells express higher levels of MKNK2 compared with MCF7 parental cells across 2 biological replicates (unpaired t test P = 0.046). E, T47D palbociclib-resistant breast cancer cells are resensitized to palbociclib upon exposure to 2.5 μmol/L SEL201 exposure across 3 biological replicates (T47D Parental: one-way ANOVA P25 vs. P25+S P = < 0.0001; P100 vs. P100+S P = 0.0016; T47D PalboR: one-way ANOVA P25 vs. P25+S P = < 0.0001; P100 vs. P100+S P = < 0.0001; P250 vs. P250+S P = < 0.0001; P500 vs. P500+S P = < 0.0001). F, T47D palbociclib-resistant breast cancer cells have a higher basal level of phospho-eIF4E compared with their parental counterpart. G, Palbociclib-resistant T47D cells express higher levels of MKNK2 compared with T47D parental cells across 4 biological replicates (unpaired t test P = 0.0036).

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As therapeutic resistance to CDK4/6 inhibition is an emerging clinical problem in patients with breast cancer, we next tested the response of palbociclib-resistant breast cancer cells to MNK1/2 inhibition. For this, we treated a previously described MCF7 model of acquired resistance to palbociclib (MCF7-PalboR) (43) with SEL201. Similar to our melanoma model of palbociclib-resistance, treatment of MCF7-PalboR cells with SEL201 resensitized them to palbociclib (Fig. 5B). In agreement with the results obtained in CHL-1 and MCF7, SEL201 also resensitized T47D palbociclib-resistant breast cancer cells (T47D-PalboR) to palbociclib (Fig. 5D). Importantly, we observed that MCF7-PalboR and T47D-PalboR cells expressed higher levels of phospho-eIF4E compared with their parental counterparts (Fig. 5C and F). qPCR analysis of these cells revealed that the palbociclib-resistant MCF7 and T47D cells express higher levels of MKNK2 (Fig. 5D and G). These data support our hypothesis that activation of the MNK1/2–eIF4E axis may be a novel mechanism associated with resistance to palbociclib. In concordance with these data, Pancholi and colleagues recently demonstrated that palbociclib-resistant MCF7 cells express higher levels of MKNK2 and increased ERK/MAPK signaling (44). Overall, these data suggest that palbociclib-resistant cells may have a higher reliance on the MNK1/2–eIF4E axis for survival, and that MNK1/2 inhibition may be a therapeutic vulnerability in palbociclib-resistant cancer cells.

Targeting MNK1/2 and CDK4/6 delays tumor progression and increases overall survival in vivo in murine models of melanoma

Loss of PTEN has been demonstrated to promote resistance to MAPK therapy (45). Moreover, there are conflicting studies as to whether PTEN loss alters sensitivity to CDK4/6 inhibitors. Recent work has demonstrated that PTEN-loss promotes resistance to palbociclib (46), while others have shown that PTEN-loss confers sensitivity to palbociclib (47). With this in mind, we next sought to test the in vivo efficacy of the MNK1/2 inhibitor eFT508, which is currently being tested in clinical trials, in combination with palbociclib using the well described Tyr::CreER/BRafCA/+/Ptenlox/lox conditional melanoma model. This melanoma model allows for 4-HT inducible, melanocyte-targeted BRAFV600E expression, and simultaneous PTEN inactivation. 12 to 15 days post 4-HT treatment, hyperpigmented lesions were observed and treatments were initiated (10). We observed a significant inhibition of tumor outgrowth in the combination treatment arm compared with the palbociclib monotherapy arm at day 54, when tumor volume was assessed at approximately 1,000 mm3 (Fig. 6A). Maintaining this tumor volume threshold, we observed that the mice in the combination arm had a median overall survival of 75 days compared with mice in the palbociclib-single agent arm, which achieved a median overall survival of 70.5 days. In addition, assessing survival when tumor volumes reached 1,500 mm3, we observed a significant overall survival advantage in the mice treated with the combination therapy, compared with either monotherapy (Fig. 6B). Median overall survival was as follows for each cohort: combination (83 days) > palbociclib (74 days) > eFT508 (67.5 days) > Vehicle (54 days). No overt toxicity was observed in the combination compared with single agents, as measured by mouse body weight (Supplementary Fig. S3E). eFT508 administration in mice demonstrated on target-engagement, as shown by the repression of phospho-eIF4E expression in the tumors (Fig. 6C and D). Furthermore, we observed that murine tumors treated with the combination exhibited decreased expression of AURKB and survivin (Fig. 6D), recapitulating our in vitro immunoblotting results in Fig. 3. Similar results, tumor growth delay and increased overall survival with no overt toxicity, were obtained in a BLM xenograft mouse model (Fig. 6E and F; Supplementary Fig. S3J). In toto, our data support the use of MNK1/2 inhibitors to augment the therapeutic benefit of palbociclib in melanoma.

Figure 6.

Cotargeting MNK1/2 and CDK4/6 in vivo improves overall survival in murine models of melanoma. A, Tumor growth curves of Tyr::CreER/BRafCA/+/Ptenlox/lox mice treated with vehicle, single-agent palbociclib, eFT508, or a combination of the two. B, Kaplan–Meier demonstrates significant survival advantage of Tyr::CreER/BRafCA/+/Ptenlox/lox murine melanomas treated with the combination compared with vehicle or either monotherapy (log-rank test). C, Phosphorylation of eIF4E is repressed in vivo in response to eFT508 treatment. Scale bar, 2 mm; (one-way ANOVA; veh vs. P+E, P = 0.0140; P vs. P+E, P = 0.0171). D, Representative immunoblots of Tyr::CreER/BRafCA/+/Ptenlox/lox tumors demonstrating repressed expression of AURKB and survivin in the combination compared with palbociclib. E, Tumor growth curves of BLM xenografts in NOD/SCID mice treated with vehicle, single-agent palbociclib, eFT508, or a combination of the two. F, Kaplan–Meier demonstrates significant survival advantage of NOD/SCID mice engrafted with BLM melanoma cells and treated with the combination compared with vehicle or either monotherapy. (log-rank test). G, Model depicting the impact of MNK1/2-eIF4E inhibition in combination with palbociclib in melanoma and breast cancer cells. Our data suggest that the efficacy of palbociclib is limited by its ability to promote the phosphorylation of eIF4E. Blocking the increase in palbociclib-mediated phosphorylation of eIF4E results in augmented antitumor activity in therapy-naïve or palbociclib-resistant cells.

Figure 6.

Cotargeting MNK1/2 and CDK4/6 in vivo improves overall survival in murine models of melanoma. A, Tumor growth curves of Tyr::CreER/BRafCA/+/Ptenlox/lox mice treated with vehicle, single-agent palbociclib, eFT508, or a combination of the two. B, Kaplan–Meier demonstrates significant survival advantage of Tyr::CreER/BRafCA/+/Ptenlox/lox murine melanomas treated with the combination compared with vehicle or either monotherapy (log-rank test). C, Phosphorylation of eIF4E is repressed in vivo in response to eFT508 treatment. Scale bar, 2 mm; (one-way ANOVA; veh vs. P+E, P = 0.0140; P vs. P+E, P = 0.0171). D, Representative immunoblots of Tyr::CreER/BRafCA/+/Ptenlox/lox tumors demonstrating repressed expression of AURKB and survivin in the combination compared with palbociclib. E, Tumor growth curves of BLM xenografts in NOD/SCID mice treated with vehicle, single-agent palbociclib, eFT508, or a combination of the two. F, Kaplan–Meier demonstrates significant survival advantage of NOD/SCID mice engrafted with BLM melanoma cells and treated with the combination compared with vehicle or either monotherapy. (log-rank test). G, Model depicting the impact of MNK1/2-eIF4E inhibition in combination with palbociclib in melanoma and breast cancer cells. Our data suggest that the efficacy of palbociclib is limited by its ability to promote the phosphorylation of eIF4E. Blocking the increase in palbociclib-mediated phosphorylation of eIF4E results in augmented antitumor activity in therapy-naïve or palbociclib-resistant cells.

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Our data show that simultaneous inhibition of MNK1/2 and CDK4/6 work together to repress clonogenic outgrowth, increase G1 cell-cycle arrest, senescence and prolong the survival of melanoma bearing mice. Pathway analysis of the proteome of cells treated with the combination showed an overrepresentation of markers associated with cell-cycle and chromosome segregation. Notably, we observed that the combination further repressed the expression of numerous mitotic regulators, including TOP2a, AURKB, KIF4A, RRM2, TPX2, and INCENP, compared with either single agent alone. This is of particular importance as increased expressions of these markers have been associated with poor prognosis in multiple cancers (25–30).

The combination of palbociclib with hormone therapy has significantly improved clinical outcome of ER+ breast cancer (23). Furthermore, MNK1/2 inhibitors are actively being tested for efficacy in advanced breast cancer (NCT04261218). Similar to our data in melanoma, MCF7 breast cancer cells treated with the combination of MNK1/2 and CDK4/6 inhibitors have decreased clonogenic outgrowth compared with single agents. Melanoma and breast cancer cells exposed to the combination demonstrate increased G1 accumulation, and reduced expression of cyclin A. In addition, we observed increased expression of p27 and senescence in cell treated with the combination. Similar to BLM, we observe that breast cancer cells treated with the combination have reduced levels of critical mitotic regulators, such as TPX2, AURKB, and survivin. Strikingly, we observed no evidence of G2–M cell-cycle arrest in melanoma or breast cancer cells treated with the combination. Interestingly, repression of AURKB activity has also been demonstrated to induce a G1 arrest in non–small cell lung cancer cells (48). It would be very interesting to test whether the repression of AURKB through combined inhibition of CDK4/6 and MNK1/2 directly impacts accumulation of cells in G1–S-phase of the cell cycle.

Of clinical relevance, our data demonstrate that breast cancer cells that acquire resistance to palbociclib have increased levels of phosphorylated-eIF4E. Although we are continuing to explore the mechanism via which the phosphorylation of eIF4E increases, our data and that of others (44), suggest it may be due to increased expression of MKNK2 (Fig. 6G). Pharmacologic inhibition of MNK1/2 in palbociclib-resistant cells results in re-sensitization to palbociclib. Given that CDK4/6 inhibitors are being clinically investigated as single agents in melanoma (NCT02465060, NCT02857270, and NCT02791334), we tested whether palbociclib-resistant melanoma cells were sensitive to MNK1/2 inhibitors. Similar to breast cancer, CHL1 palbociclib-resistant melanoma cells are resensitized to palbociclib upon MNK1/2 inhibition.

In therapy naïve cells, increased levels of phospho-eIF4E in response to chemotherapeutic and targeted agents have been previously reported. One study demonstrated that treatment of pancreatic ductal adenocarcinoma cells with gemcitabine results in increased SRSF1-mediated MNK2b splicing that results in increased phospho-eIF4E (13). Furthermore, blocking this increase in phospho-eIF4E results in increased apoptosis in gemcitabine treated cells (13). Other studies have also implicated a role for increased phospho-eIF4E in resistance to stress and DNA damaging agents through the selective translation of cyclin D1, HuR, and Mcl-1 mRNA's (49). Our future work will be aimed at identifying the subset of mRNAs, which are most efficiently translated in response to CDK4/6 inhibitors.

Overall, our data demonstrate the MNK1/2–eIF4E axis to be an exploitable salvage pathway in treatment naïve and palbociclib-resistant models (Fig. 6G). Palbociclib has recently demonstrated modest antitumor activity in acral lentiginous melanoma (50). In addition, we have previously shown that the oncogenicity of KIT-mutant acral melanomas are highly reliant on the MNK1/2–eIF4E axis, and, by blocking MNK1/2 we are able to repress the oncogenicity of these melanomas (20). Albeit the single-efficacy of MNK1/2 inhibitors are mild, we would predict that the addition of MNK1/2 inhibitors would potently augment the efficacy of palbociclib, and perhaps that of other CDK4/6 inhibitors, in melanoma subtypes with limited treatment options. Furthermore, although speculative, one might envision the use of phospho-eIF4E as a potential biomarker to predict the onset of resistance to palbociclib.

A.A.N. Rose reports grants from Canada First Research Excellence Fund, CIHR, Canadian Cancer Society, Conquer Cancer Foundation of ASCO, and Jewish General Hospital Foundation; grants from TransMedTech Institute during the conduct of the study; personal fees from Novartis; and personal fees from Pfizer outside the submitted work. K.E. Sheppard reports grants from National Health and Medical Research Council Australia during the conduct of the study. D.W. Cescon reports grants and personal fees from AstraZeneca, Gilead, GlaxoSmithKline, Merck, Pfizer, and Roche; personal fees from Exact Sciences and Eisai; grants and nonfinancial support from Inivata; personal fees from Novartis; and personal fees from Inflex outside the submitted work; in addition, D.W. Cescon has a patent for (US62/675,228) for methods of treating cancers characterized by a high expression level of spindle and kinetochore associated complex subunit 3 (ska3) gene, issued. F.A. Mallette reports grants from Canadian Institutes of Health Research during the conduct of the study. R.P. Zahedi reports he is CEO of MRM Proteomics Inc. W.H. Miller reports grants from Merck, CIHR, TFRI, and SWCRF; grants from CCSRI during the conduct of the study; personal fees from Merck, Bristol-Myers Squibb, Roche, Novartis, Amgen, GSK, Mylan, EMD Serono, and Sanofi; other support from BMS, Novartis, GSK, Roche, AstraZeneca, MethylGene, MedImmune, Bayer, Amgen, Merck, Incyte, Pfizer, Ocellaris Pharma, and Astellas; and other support from Alkermes outside the submitted work. No disclosures were reported by the other authors.

S.A. Prabhu: Conceptualization, resources, data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. O. Moussa: Conceptualization, resources, formal analysis, investigation, methodology, writing–original draft, writing–review and editing. C. Gonçalves: Validation, investigation, writing–review and editing. J.H. LaPierre: Software, validation, investigation, writing–review and editing. H. Chou: Investigation. F. Huang: Validation, investigation, writing–review and editing. V.R. Richard: Investigation, writing–review and editing. P.Y.M. Ferruzo: Validation. E.M. Guettler: Investigation. I. Soria-Bretones: Software, investigation, writing–review and editing. L. Kirby: Validation, investigation, writing–review and editing. N. Gagnon: Investigation. J. Su: Investigation. J. Silvester: Investigation. S.S. Krisna: Investigation. A.A.N. Rose: Investigation. K.E. Sheppard: Resources, writing–review and editing. D.W. Cescon: Resources, investigation, methodology, writing–review and editing. F.A. Mallette: Resources, investigation, writing–review and editing. R.P. Zahedi: Resources, methodology, writing–review and editing. C.H. Borchers: Software, investigation, writing–review and editing. S.V. del Rincon: Conceptualization, resources, supervision, funding acquisition, investigation, methodology, writing–original draft, project administration, writing–review and editing. W.H. Miller Jr.: Conceptualization, resources, supervision, funding acquisition, investigation, methodology, writing–original draft, project administration, writing–review and editing.

This research was funded by the Canadian Institutes of Health Research (CIHR; grant PJT-162260 to SVDR and grant PJT-156269 to W.H. Miller Jr. and S.V. del Rincon). S.A. Prabhu and S.S. Krisna hold FRQS studentships. O. Moussa holds an FRQS scholarship and was awarded the Andy-Lena Chabot award by the Cancer Research Society. F. Huang was sponsored by a McGill Faculty of Medicine graduate studentship and received a McGill Integrated Cancer Research Training Program graduate studentship. We are grateful to Genome Canada for financial support through the Genomics Technology Platform (GTP: 264PRO). We are also grateful for financial support from the Terry Fox Research Institute. C.H. Borchers is grateful for support from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada) and the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, Quebec, Canada). We thank Christian Young for valuable expertise in designing and executing flow cytometry experiments. We thank Dr. Josie Ursini-Siegel for providing the NOD-SCID mice and Dr. Luc Furic for providing reagents. Special thanks to Dr. Henry Yu for invaluable discussions.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

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