Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses

Abstract

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

Keywords: Melanoma; Oncology.

Figure 1. Phospho-eIF4E deficiency decreases melanoma outgrowth and metastasis.

(A) Schematic diagram of the Tyr::CreER/BRaf^CA/+/Pten^lox/lox/Eif4e^WT/WT (eIF4E^WT) and the Tyr::CreER/BRaf^CA/+/Pten^lox/lox/Eif4e^KI/KI (eIF4E^KI) murine melanoma model. (B) Tumor growth curve (top) and representative pictures (bottom) of eIF4E^WT and eIF4E^KI mice (n = 20 per group) after topical administration of 4-HT. Two-way ANOVA (comparing eIF4E^WT, eIF4E^HET, and eIF4E^KI as presented in Supplemental Figure 1A). (C) Primary tumor weight (day 50) of eIF4E^WT (n = 14) and eIF4E^KI (n = 12) mice. (D) Percentages (left) and representative images (right) of Ki67-positive melanoma cells in eIF4E^WT and eIF4E^KI primary melanoma sections (day 50; n = 8 per genotype; scale bars: 50 μm). (E) Kaplan-Meier curves showing overall survival of eIF4E^WT and eIF4E^KI mice (n = 21 per group). Log-rank test. (F) Inguinal lymph node (iLN) sizes measured by ultrasound (top left), representative iLN pictures (day 48; bottom left; scale bars: 2 mm), and representative images of H&E-stained iLNs (day 48; right; scale bars: 50 μm) of eIF4E^WT and eIF4E^KI mice (n = 10 per group). (G) Cervical lymph nodes (cLNs) were resected from eIF4E^WT and eIF4E^KI mice (n = 10 per group) with size-matched primary tumors (500–800 mm³). Number of metastasis-positive cLNs per mouse (top) and representative images of H&E-stained cLNs (bottom; scale bars: 20 μm) are presented. Mann-Whitney test. (C, D, and F) Two-sided unpaired t test. All values are represented as mean ± SEM.

Figure 2. Phospho-eIF4E–deficient murine and human melanomas are more differentiated.

(A) Representative eIF4E^WT (n = 16) and eIF4E^KI (n = 15) primary tumor histology with H&E staining. (B) Representative IHC images showing the expression of S100 (left) and the percentage of pigmented area in the S100-positive region (right) in the eIF4E^WT and eIF4E^KI tumors. (C) Representative Western blot of the indicated proteins in 4 eIF4E^WT and 4 eIF4E^KI melanomas (top left; see all blots in Supplemental Figure 12) with quantification of MITF level normalized to β-actin (bottom left and right). (D and E) Representative IHC images with scores showing the expression of Melan-A (D) and GP100 (E) in eIF4E^WT and eIF4E^KI melanomas. (F) Top: IHC images showing the expression of phospho-eIF4E and Melan-A in 2 representative tumors from a total of 29 patients with melanoma. One patient sample with high phospho-eIF4E expression (score > 2.5/5) and one patient sample with low phospho-eIF4E expression (score = 0.5–1/5) are shown. Bottom: IHC scoring of Melan-A in phospho-eIF4E^hi and phospho-eIF4E^lo patient-derived melanomas. (G) Western blot analysis of the indicated proteins in human HBL melanoma cells stably expressing shCtrl and shMKNK1/2 (representative of n = 3). (A–E) Melanomas were resected on day 50. (A, D, and F) Scale bars: 2 mm. (B and E) Scale bars: 100 μm. (B–F) Number of biological replicates is indicated in each graph. (B and C) Two-sided unpaired t test. (D–F) Mann-Whitney test. All values are represented as mean ± SEM.

Figure 3. Phospho-eIF4E–deficient melanoma cells resist phenotype switching via inhibiting NGFR mRNA translation.

(A) Western blot analysis of the indicated proteins in murine melanoma cell lines MDMel-WT⁷³, MDMel-KI⁶¹, D4M.3a, and B16-F10 (representative of n = 3). (B) Percent invasion of MDMel-KI⁶¹ cells relative to MDMel-WT⁷³ cells (top) and representative images (bottom; original magnification, ×10; n = 3). Two-way ANOVA with Tukey’s test (comparing all groups shown in Supplemental Figure 3B). (C) Representative immunofluorescence images showing the expression of NGFR in eIF4E^WT and eIF4E^KI tumors (day 50; top; n = 8 per group; scale bars: 30 μm) and the percentage of samples with high, medium, and low expression of NGFR from each group (bottom). (D) Top: Western blot analysis of NGFR expression in the murine melanoma cell lines MDMel-WT⁸⁸, MDMel-WT⁷³, MDMel-KI⁶¹, and MDMel-KI⁵⁸ (representative of n = 3). Bottom: Fold change of Ngfr mRNA in MDMel-KI⁶¹ cells relative to MDMel-WT⁷³ cells, normalized to m36B4 (Rplp0) as a reference gene (n = 4). Two-way ANOVA with Tukey’s test (comparing all groups shown in Supplemental Figure 3F). (E) Polysome profiles of MDMel-WT and MDMel-KI cells (representative of n = 3). (F) Percentage of transcripts in each polysomal fraction quantified by quantitative real-time PCR (top) and representative image showing rRNAs and PCR-amplified cDNA fragments of the indicated targets (bottom; n = 3). Multiple unpaired 2-tailed t test. (G) Left: Percent invasion of MDMel-WT⁷³ cells with Ngfr knockdown relative to the control group (top), and representative images (bottom; original magnification, ×10; see also Supplemental Figure 3G). One-way ANOVA. Right: Western blot confirming knockdown of NGFR (n = 3). (H) Left: Western blot analysis of the indicated proteins in DMSO- or SEL201-treated MDMel-WT cells. Right: Percent invasion of SEL201-treated cells relative to control (top), and representative images (bottom; original magnification, ×10; representative of n = 3). Two-sided unpaired t test. All values are represented as mean ± SD.

Figure 4. Phospho-eIF4E–deficient melanomas have an altered secretome.

(A) Schematic of the experimental design for the membrane-based cytokine arrays. (B) Representative images of the cytokine arrays showing the expression of secreted factors present in the conditioned medium derived from eIF4E^WT and eIF4E^KI primary melanoma cultures (n = 2 mice per genotype). (C) Concentration of CCL5 and IL-6 in the conditioned medium derived from the eIF4E^WT and eIF4E^KI primary melanoma cultures (n = 6 mice per genotype). (D) Fold change of the indicated mRNAs in MDMel-KI cells relative to MDMel-WT cells, normalized to m36B4 (Rplp0) as a reference gene (n = 3 for Igfbp6, n = 4 for Angptl4, n = 5 for the rest). Bottom: Zymography to assess MMP-9 activity in the conditioned medium of MDMel-WT and MDMel-KI cells (representative of n = 3). (E) Concentration of CCL5 in the conditioned medium of MDMel-WT and MDMel-KI cells (n = 3). (F) Percentage of transcripts in each polysomal fraction quantified by quantitative real-time PCR (n = 3). Multiple unpaired 2-tailed t test. (G) Representative image showing PCR-amplified cDNA fragments of the indicated targets (n = 3). The loading control (m36B4) is the same as for the data in Figure 3F, as the samples were run in parallel. (H) Fold change of indicated mRNAs in siNgfr-2–transfected (see Supplemental Table 4) MDMel-WT cells relative to the control group, normalized to m36B4 (Rplp0) (n = 3). (I) Correlation of the expression of indicated genes with the expression of NGFR (HTSeq [https://xenabrowser.net/datapages/?dataset=TCGA-SKCM.htseq_fpkm.tsv&host=https%3A%2F%2Fgdc.xenahubs.net&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443], fragments per kilobase of transcript per million mapped reads [FPKM]) in Genomic Data Commons (GDC) TCGA Melanoma data set (SKCM cohort, n = 477). Spearman rank-order. (C–E and H) Two-sided unpaired t test. (C) Data are represented as mean ± SEM. (D–H) Values represent the mean ± SD.

Figure 5. Phospho-eIF4E deficiency impairs melanoma immunosuppression.

(A) Immune cell populations infiltrated into the melanomas from BRaf^CA/+/Pten^lox/lox eIF4E^WT and eIF4E^KI mice (day 50). (B) Representative eIF4E^WT and eIF4E^KI tumor samples (day 50) with IHC staining for granzyme B (left; scale bars: 100 μm) and corresponding scores (right). (C) Schematic of ex vivo experimental designs. (D) Percentage IFN-γ–producing CD8⁺ cells, stimulated and cultured in the conditioned medium from eIF4E^WT or eIF4E^KI primary melanoma cultures (WT-CM, KI-CM) or regular medium for 72 hours. (E) MDSC inhibition of IFN-γ production in CD8⁺ T cells, cultured in WT-CM or KI-CM, relative to corresponding MDSC-free control group. (F–H) MDSC migration toward WT-CM, KI-CM, or regular medium (F); medium containing recombinant murine CCL5 (G); and WT-CM or KI-CM upon maraviroc (MVC; 100 nM) treatment (H). (I) Percent division of CD8⁺ T cells isolated from the iLNs of eIF4E^WT and eIF4E^KI tumor-bearing mice, cultured alone or with B16-F10 melanoma cells. One data point was excluded (Grubbs’ test). (J) Percent viability of B16-F10 cells cocultured with T cells from eIF4E^WT and eIF4E^KI tumor-bearing animals. See also Supplemental Figure 6I. (K) Percent viability of B16-F10 cells, silenced or not for Melan-A (siMlana-1; see Supplemental Table 4), cocultured with T cells from eIF4E^WT and eIF4E^KI tumor-bearing animals, relative to corresponding control groups. Number of biological replicates (mice) is indicated in each graph. For ex vivo assays (C–K), all tumor-bearing mice were sacrificed between days 35 and 38. (A and B) Two-sided unpaired t test. (D, F, and G) One-way repeated-measures (RM) ANOVA with Tukey’s test. (E and H–K) Two-way RM ANOVA with Šidák correction. All values are represented as mean ± SEM.

Figure 6. Tumor cell–intrinsic and –extrinsic phospho-eIF4E facilitates melanoma outgrowth and metastasis.

(A, D, and G) Schematic of the experimental design. (B and C) Growth curve (B) and tumor weight (C, left; day 22) with representative pictures (C, right; scale bars: 5 mm) of MDMel-WT– and MDMel-KI–derived melanomas. (E and F) Growth curve (E) and tumor weight (F, left; day 21) with representative pictures (F, right; scale bars: 5 mm) of D4M.3a-derived melanomas in BLK6-WT and BLK6-KI mice. (H and I) Number of metastatic nodules (H, left) with representative pictures of India Ink–inflated lungs (H, right), and percentage of tumor area (I, left) with representative images of H&E-stained lung sections (I, right; scale bars: 4 mm), from BLK6-WT mice and BLK6-KI mice, after tail vein injection of LWT1 cells (day 21). (B and E) Two-way ANOVA. (C, F, H, and I) Two-sided unpaired t test. All values are represented as mean ± SEM.

Figure 7. The MNK1/2-eIF4E axis regulates PD-L1 expression on DCs.

(A–C) PD-L1 expression on DCs in primary tumors (left) and draining iLNs (right) from BRaf^CA/+/Pten^lox/lox eIF4E^WT and eIF4E^KI mice (A), vehicle- or SEL201-treated BRaf^CA/+/Pten^lox/lox eIF4E^WT mice (B), and vehicle- or SEL201-treated BLK6-WT mice bearing YUMMER1.7-derived melanomas (C). (D) PD-L1 expression on DCs isolated from BLK6-WT mice and cultured ex vivo with or without SEL201 for 18 hours. (E and F) T cells were isolated from spleens of OT-1 mice and cocultured with OVA peptide–pulsed WT-DCs and KI-DCs for 72 hours. (E) Percentage CD25⁺CD44⁺ cells (left) and IFN-γ⁺CD44⁺ cells (right) out of CD8⁺ T cells. (F) IFN-γ expression on all CD8⁺ T cells (left) and IFN-γ⁺CD8⁺ T cells (right). See Supplemental Figure 8A for detailed experimental design. Number of biological replicates is indicated in each graph. Two-sided unpaired t test. All values are represented as mean ± SEM.

Figure 8. MNK1/2 inhibitor sensitizes melanoma to anti–PD-1 immunotherapy.

(A) Growth curve (left) and representative pictures (right, week 5) of melanomas in eIF4E^WT mice given control (Vehicle+IgG), monotherapies (Vehicle+αPD-1, SEL201+IgG), or combination therapy (SEL201+αPD-1). (B) Kaplan-Meier curves showing overall survival of mice in each group. (C) Number of metastasis-positive cLNs per mouse with representative images of H&E-stained cLNs (scale bars: 100 μm) is presented for each group. (D) Percentage of CD103⁺ DCs out of non-B non-T cells in the iLNs from animals in each indicated group. (E) Relative plasma cytokine and chemokine levels in the control and combination therapy groups, detected by the MAGPIX multiplexing system (Thermo Fisher Scientific). (F and G) Melanoma growth (F, left), representative pictures (F, right; day 21), and Kaplan-Meier curves showing overall survival (G) of BLK6-WT mice bearing YUMMER1.7-derived melanoma, given the indicated treatments. (C–E) Mice were sacrificed after indicated treatment for 5 weeks. (F and G) See individual tumor growth in Supplemental Figure 11A. Number of biological replicates (mice) is indicated in each graph. (A and F) Two-way RM ANOVA with Tukey’s test. (C and D) Two-way ANOVA with Tukey’s test. (B and G) Log-rank test. All values represent the mean ± SEM.

Figure 9. Combination of MNK1/2 inhibitor and anti–PD-1 immunotherapy increases specific intratumoral T cell subsets.

(A and B) Melanoma growth (A, left), representative pictures (A, right; day 18), and Kaplan-Meier curves showing overall survival (B) of BLK6-WT mice bearing B16-pdl1–derived melanoma, given the indicated treatments. (C–E) Abundance of indicated immune cell populations in B16-pdl1–derived melanomas or draining iLNs of each treatment group. Mice were sacrificed at endpoint, indicated in B, with 1 mouse (Vehicle+αPD-1) removed from panel B because of a non–tumor-related death (see Supplemental Figure 11B). TIL, tumor-infiltrating lymphocyte. (A and B) See individual tumor growth in Supplemental Figure 11B. Number of biological replicates (mice) is indicated in each graph. (A) Two-way RM ANOVA with Tukey’s test. (B) Log-rank test. (C–E) Two-way ANOVA with Tukey’s test. All values represent the mean ± SEM.