Fig 2: MNK inhibitors increase CD8+ T cells in tumors but induce a T cell exhaustive phenotype.Mice with established syngeneic pancreatic (KPC-344) and thyroid (TBP-3868) tumors were randomized and treated with DMSO (vehicle control), the MNK inhibitor CGP57380 (25 mg/kg), or the MNK inhibitor eFT508 (1 mg/kg) daily. (A and C) The collected tumors were analyzed for p-eIF4E expression by immunofluorescence staining and CD8+ T cells by IHC staining. Scale bar: 100 µm. The absolute number of stained CD8+ T cells per 10× section was quantified by ImageJ and averaged from 5 individual sections. (B and D) The collected tumors were digested and analyzed by flow cytometry for the number of CD8+ T cells. (E and F) Tumor-infiltrating CD8+ T cells were analyzed by flow cytometry for the expression of CD69, granzyme B (GzmB), perforin-1 (Prf1), and PD-1. Data points in B, D, E, and F represent individual tumors. (G and H) Tumor size was monitored and measured using calipers. Tumor volumes (V) were calculated using the formula V = (W2 × L)/2, where W is tumor width and L is tumor length. Arrows indicate start of treatment. Data are shown as the mean ± SEM, and analysis was done using 1-way ANOVA followed by Dunnett’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: MNK inhibitors potentiate an immunosuppressive phenotype in BM-derived monocytes (BMDMs).(A) BMDMs were cultured in L929-conditioned media for 5 days before treating for 24 hours with IL-4 (25 ng/mL) to induce alternatively activated (M2) polarization. M2-polarized BMDMs were cocultured with CFSE-stained splenic CD8+ T cells for 72 hours in the presence of DMSO, CGP57380 (10 µM), or eFT508 (1 µM). T cell proliferation, as determined by the percentages of dividing T cells and T cell numbers, was analyzed by flow cytometry. (B) The CD8+ T cells were also analyzed by flow cytometry for the expression of granzyme B (GzmB), perforin-1 (Prf1), and TNF-a. MFI was calculated using FlowJo. (C) M2-polarized BMDMs were treated with DMSO, CGP57380 (CGP, 10 µM), or eFT508 (eFT, 1 µM) for 48 hours. Expression of phosphorylated eIF4ES209 (p-eIF4E) was analyzed by Western blotting with HSP90 as loading control. Expression levels of select classically activated (M1) and M2 genes were evaluated by qPCR with GAPDH used as a house-keeping gene and averaged from 3 independent experiments to generate heatmaps. Data and data points in A are shown as the mean ± SEM and biological replicates respectively. Data and data points in B are shown as the mean ± SD and technical replicates, respectively, and data are representative of 3 independent experiments. Analysis was done using 1-way ANOVA followed by Dunnett’s multiple comparison test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig 4: LARP1 recruits mTORC1 to LARP1-interacting mRNPs in a manner dependent of mTORC1 activity.(A) Endogenous LARP1 co-IPs endogenous mTORC1 in HEK293T cells. LARP1 antibody 087A, but not 088A, co-IPs mTORC1. The LARP1 antibody, 087A or 088A recognizes amino acids 275–325 or 575–625 of LARP1, respectively. (B) The effect of RNAse A on the interaction of LARP1 with mTORC1 and other mRNA binding proteins (PABP1 and eIF4E). Endogenous LARP1 was IPed from the lysates treated with RNAse A. (C) The effect of RNase A on the interaction of LARP1 with the components of the initiation complex. Endogenous eIF4G1 was IPed from the lysates treated with RNAse A. (D) LARP1 interacts with mTORC1 in a manner dependent on mTORC1 activity. (E) LARP1 co-IPs mTORC1 in a growth factor/amino acid stimulation-dependent manner.DOI: http://dx.doi.org/10.7554/eLife.25237.007

Fig 5: KSHV induces the hypoxia response to upregulate the HIF2a/eIF4E2 translation-initiation complex in normoxia(A) HIFs and eIF4E2 protein levels in normoxic siControl and siHIF1ß iSLK.KSHV219 cells. These cells were silenced as in Figure 1.(B) Expression of HIFs and eIF4E2 in siControl and siHIF1ß KSHV-negative SLK cells.(C) Fold change of eIF4E2 and HIF2a mRNA and protein level relative to siControl at 24 h post-reactivation in cells from (A) (n = 3; mean ± SD; *p < 0.001, two-way ANOVA with Tukey’s post-test).(D) HIFs expression in siControl and sieIF4E2 iSLK.KSHV219 cells cultured in normoxia.(E) HIFs protein levels in KSHV-negative SLK cells that were silenced with siControl or sieIF4E2.(F) Translation efficiency of HIF2a in siControl and sieIF4E2 iSLK.KSHV219 cells 48 h post-reactivation. HIF2a mRNA levels in all fractions were measured using qRT-PCR (n = 3), and each fraction CT value was normalized to the 80S fraction CT.(G) HIF2a pull-down levels after eIF4E2 and eIF4E1 RIPs in reactivated cells 24 h post-DOX (n = 3; mean ± SD; ***p < 0.0001, two-way ANOVA with Tukey’s post-test).(H) HIF1a, HIF1ß, and eIF4E2 protein levels in siControl and siHIF2a iSLK.KSHV219 cells cultured in normoxia.(I) Feedforward activation mechanism for eIF4E2 translation initiation in normoxia. KSHV-upregulated HIFs drive eIF4E2 expression, and the eIF4E2-containing complex allows HIF2a translation, further enhancing viral mRNA translation initiation by eIF4E2.