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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.

List of differentially expressed proteins annotated by cluster with pathway analysis in BLM cells

Supplementary Figure 1

The spindle assembly checkpoint maintains genomic integrity. A key component is tyrosine threonine kinase (TTK, also known as Mps1). TTK antagonism is hypothesized to cause genomic instability and cell death. Interrogating The Cancer Genome Atlas revealed high TTK expression in lung adenocarcinomas and squamous cell cancers versus the normal lung (P < 0.001). This correlated with an unfavorable prognosis in examined lung adenocarcinoma cases (P = 0.007). TTK expression profiles in lung tumors were independently assessed by RNA in situ hybridization. CFI-402257 is a highly selective TTK inhibitor. Its potent antineoplastic effects are reported here against a panel of well-characterized murine and human lung cancer cell lines. Significant antitumorigenic activity followed independent treatments of athymic mice bearing human lung cancer xenografts (6.5 mg/kg, P < 0.05; 8.5 mg/kg, P < 0.01) and immunocompetent mice with syngeneic lung cancers (P < 0.001). CFI-402257 antineoplastic mechanisms were explored. CFI-402257 triggered aneuploidy and apoptotic death of lung cancer cells without changing centrosome number. Reverse phase protein arrays (RPPA) of vehicle versus CFI-402257–treated lung cancers were examined using more than 300 critical growth-regulatory proteins. RPPA bioinformatic analyses discovered CFI-402257 enhanced MAPK signaling, implicating MAPK antagonism in augmenting TTK inhibitory effects. This was independently confirmed using genetic and pharmacologic repression of MAPK that promoted CFI-402257 anticancer actions. TTK antagonism exerted marked antineoplastic effects against lung cancers and MAPK inhibition cooperated. Future work should determine whether CFI-402257 treatment alone or with a MAPK inhibitor is active in the lung cancer clinic.

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.

Figure S1: The structure of CFI-402257. Figure S2: TTK mRNA expression profiles in lung cancer cases with different smoking patterns, as shown in panel A. In this left panel N means no smoking history while Y indicates the presence of a smoking history. That was displayed in the right panel as never, former or current smokers. Panel B displays the anti-proliferative effects of CFI-402257 relative to vehicle treatments in lung adenocarcinoma cell lines. The area under the curve was calculated based on the proportion of control values and log10 dosage values to generate dose-response curves. Error bars are standard deviations. Figure S3: Immunoblot analysis of ED1 (A) and A549 (B) lung cancer cells after independent transfection with TTK-targeting (siRNA1 and siRNA2) or control siRNAs. Figure S4: Representative photomicrographs of 393P (A) or H1299 (B) lung cancer cell line-derived tumors after 21 days of CFI-402257 or vehicle treatments. (C) Syngeneic 393P tumor-bearing mice were treated with vehicle, 8.5 mg/kg CFI-402257 orally for 5 days, after which tumors were harvested for RPPA analyses (n = 5 mice per group). Comparison of the 8.5mg/kg CFI-402257-treated group to vehicle-treated groups. The proteins displayed above the red line are significantly different (P < 0.05). Figure S5: (A) The association between survival and expression of MAP2K1/MAP2K2 and TTK in combination using TCGA. Green: MAP2K low/TTK low, purple: MAP2K low/TTK high, orange: MAP2K high/TTK low and red: MAP2K high/TTK high. (B) The combination index (CI) for each dosage pair was calculated. The dash line indicates CI=1.0. Figure S6: TTK mutations in lung adenocarcinomas (A) and squamous cell carcinomas (B) in TCGA database.

Supplementary Figure 2

Supplementary Figure 3

Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) are standard first-line treatments for metastatic ER