MAP Kinase Interacting Kinase-mediated translation in Diffuse Large B-Cell Lymphoma

Abstract

The Ras MAPK pathway was the first pathway to be identified as oncogenic. Since its discovery, many attempts have been made to target this pathway and have systematically failed despite a variety of targeting strategies. I therefore chose to target oncogenesis via a set of kinases downstream of the Mitogen Activated Protein Kinases (MAPKs), called Mitogen Activated Protein Kinase Interacting Kinases, or MNKs, using both large-scale and directed biochemistry to better understand their effect. Augmentation of protein translation has been observed in a number of human malignancies; specifically, augmentation of cap-dependent protein translation in diffuse large B-cell lymphoma (DLBCL) was previously demonstrated in the Gartenhaus lab. This cancer-associated phenomenon results in the dysregulation of many proto-oncogenes and tumor suppressors, leading to cellular abnormalities. MNK kinases phosphorylate eIF4E1 at serine 209, resulting in oncogene-favorable mRNA translation. Several cancers, and not their normal counterparts, have been shown to require MNK signaling. We demonstrate that MNK signaling is required and amplified in primary DLBCL using immunohistochemistry and RT-qPCR. We show that MNK signaling may be targetable using p38 MAPK inhibitors in DLBCL, based on both viability and signaling. Interestingly, p38 inhibition also resulted in growth arrest from a cell cycle block rather than apoptosis, as shown by flow cytometry. For further analysis, we generated custom cell lines expressing both MNK and eIF4E1 genetic mutations and found, using immunoprecipitation and cap-pull-down experiments, that MNKs impact eIF4E1 cap binding in DLBCL and regulate alternative eIF4F complexes, functioning as a molecular switch for translation complexes. Following the observation of altered eIF4F complexes, we found that eIF4E family members may work in part through 5' mRNA motifs. Through bioinformatics analysis of the transcriptome and translatome data from micro-arrays in DLBCL, potential motifs were discovered. Interestingly we also found that eIF4E1 up-regulates NF-?B in select DLBCL. Our results disagree with the canonical belief that eIF4E proteins work exclusively through binding of the 5' mRNA-cap, and further show that MNKs drive mRNA translation in DLBCL and normal B-cells, with the potential to impact all cell types. Our work provides novel insight into the mechanistic basis of malignant mRNA translation and presents several previously unknown protein associations that function through heretofore-unrelated signaling pathways, offering a potential for future targeting in a clinical setting.