MNK1 Inhibitor CGP57380 Overcomes the Activation of eIF4E Induced By mTORC1 Inhibitor: The Mechanism of Synergic Killing on T-ALL Cells

Background: In spite of the impressive outcomes achieved in acute lymphoblastic leukemia (ALL), current therapeutic strategies are never adequate for patients with higher risk ALL, including T-ALL. Meanwhile, toxicities and high rates of relapse of chemotherapy pose an enormous challenge to us. Therefore, novel and effective therapeutic approaches are needed for the patients suffering from this disease.

Aberrantly activation of mTOR pathway in T-ALL promotes tumor progression, thus targeting mTOR is thought as an effective therapeutic strategy for the treatment of T-ALL. But to our disappointment, mTOR C1 inhibitors rapamycin and its analogs (rapalogs) showed a limited efficacy in ALL, suggesting the existence of resistance pathways.

In this study, we found that the activation of eukaryotic translation initiation factor 4E (eIF4E) is highly associate with resistant to mTORC1 inhibitor RAD001 (everolimus ) in T-ALL cells, and the combination of MNK1 inhibitor CGP57380, which blocks the MNK1-mediated phosphorylation of eIF4E, with RAD001 showed a great synergic killing effect on T-ALL cells, suggesting that this combination therapy represents a promising therapeutic approach for T-ALL.

Methos: T-ALL cell lines Jurkat, CEM, Molt-4 were treated by MNK1 inhibitor GCP57380 and RAD001 (mTOR C1 inhibitor) alone or together in our study. The cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after drug exposure. Cell apoptosis was determined using annexin V and DAPI staining and analyzed by flow cytometry or fluorescence microscopy. The relevant changes of proteins occured secondary to drug exposure were examined by Western blotting analysis. CalcuSyn software based on Chou and Talalay analysis was used in calculating Combination Index (CI) value in the combination regimen.

Result: After 24 h or 48 h of treatment, the proliferation of the T-ALL cells was significantly inhibited by GCP57380 in a time- and dose-dependent manner, with IC50 of about 8 µM, as indicated by MTT assay. Then we found a limited efficacy of RAD001 in treating with T-ALL cells, with the largest inhibition ratio of about 40%. Meanwhile, the RAD001 induced growth inhibition and apoptosis remarkably enhanced by the addition of GCP57380, measured through Annexin V/PI staining by cytometry, and PARP cleavage measured by Western blotting. Conjoint analysis showed a significant synergistic effect in the combination group (Cl < 1).

Importantly, mechanism’s studies showed RAD001 induced up-regulation of p-eIF4E in a time- and dose- dependent manner in T-ALL cells. It is reported that eIF4E plays a crucial role in eukaryotic translation and related to chemoresistance. Thus the up-regulation of eIF4E induced by RAD001 could have an association with the drug-resistance and provided the basis to the combination of GCP57380 with RAD001. As we expected, the addition of GCP57380 greatly decreased the level of p-eIF4E comparing with using RAD001 alone, indicating a Mnk1-dependent eIF4E activation in T-ALL cells treated by RAD001. Furthermore, we found the levels of p-4EBP1 suffering its biggest drop in the combination group. As we know, 4EBP-1, a downstream molecular of mTOR pathway, may block the function of eIF4E through bonding to it. This finding suggested that 4EBP-1 might be a molecular cross-point between the mTORC1 and MNK1 pathways, making it a further target for T-ALL therapy.

Conclusion: Taken together, these data shows that mTOR C1 inhibitor RAD001 could induce a MNK1-mediated eIF4E activation and the addition of MNK1 inhibitor CGP57380 may synergetically enhance RAD001 induced anti-T-ALL efficacy. We also find the 4EBP1 may be a cross-talking between the mTOR C1 and MNK1 pathways. These data provide a justification for early phase clinical trials of MNK1 inhibitor GCP57380 in combination with RAD001 in patients with high risk T-cell acute lymphoblastic leukemia.