The kinase Mnk2 is a substrate of the MAPK pathway and phosphorylates the translation initiation factor eIF4E. In humans, MKNK2, the gene encoding for Mnk2, is alternatively spliced yielding two splicing isoforms with differing last exons: Mnk2a, which contains a MAPK-binding domain, and Mnk2b, which lacks it. We found that the Mnk2a isoform is downregulated in breast, lung, and colon tumors and is tumor suppressive. Mnk2a directly interacts with, phosphorylates, activates, and translocates p38α-MAPK into the nucleus, leading to activation of its target genes, increasing cell death and suppression of Ras-induced transformation. Alternatively, Mnk2b is pro-oncogenic and does not activate p38-MAPK, while still enhancing eIF4E phosphorylation. We further show that Mnk2a colocalization with p38α-MAPK in the nucleus is both required and sufficient for its tumor-suppressive activity. Thus, Mnk2a downregulation by alternative splicing is a tumor suppressor mechanism that is lost in some breast, lung, and colon tumors.

Differential exposure of tumor cells to blood-borne and angiocrine factors results in diverse metabolic microenvironments conducive for non-genetic tumor cell diversification. Here, we harnessed a methodology for retrospective sorting of fully functional, stroma-free cancer cells solely on the basis of their relative distance from blood vessels (BVs) to unveil the whole spectrum of genes, metabolites, and biological traits impacted by BV proximity. In both grafted mouse tumors and natural human glioblastoma (GBM), mTOR activity was confined to few cell layers from the nearest perfused vessel. Cancer cells within this perivascular tier are distinguished by intense anabolic metabolism and defy the Warburg principle through exercising extensive oxidative phosphorylation. Functional traits acquired by perivascular cancer cells, namely, enhanced tumorigenicity, superior migratory or invasive capabilities, and, unexpectedly, exceptional chemo-and radioresistance, are all mTOR dependent. Taken together, the study revealed a previously unappreciated graded metabolic zonation directly impacting the acquisition of multiple aggressive tumor traits.

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.

The gene encoding the kinase Mnk2 (MKNK2) is alternatively spliced to produce two isoforms—Mnk2a and Mnk2b. We previously showed that Mnk2a is downregulated in several types of cancer and acts as a tumor suppressor by activation of the p38–MAPK stress pathway, inducing apoptosis. Moreover, Mnk2a overexpression suppressed Ras-induced transformation in culture and in vivo. In contrast, the Mnk2b isoform acts as a pro-oncogenic factor. In this study, we designed modified-RNA antisense oligonucleotides and screened for those that specifically induce a strong switch in alternative splicing of the MKNK2 gene (splice switching oligonucleotides or SSOs), elevating the tumor suppressive isoform Mnk2a at the expense of the pro-oncogenic isoform Mnk2b. Induction of Mnk2a by SSOs in glioblastoma cells activated the p38–MAPK pathway, inhibited the oncogenic properties of the cells, re-sensitized the cells to chemotherapy and inhibited glioblastoma development in vivo. Moreover, inhibition of p38–MAPK partially rescued glioblastoma cells suggesting that most of the anti-oncogenic activity of the SSO is mediated by activation of this pathway. These results suggest that manipulation of MKNK2 alternative splicing by SSOs is a novel approach to inhibit glioblastoma tumorigenesis.

Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA-peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.