MAPK15 mediates BCR-ABL1-induced autophagy and regulates oncogene-dependent cell proliferation and tumor formation

<p>A reciprocal translocation of the <i>ABL1</i> gene to the <i>BCR</i> gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel “druggable” link between BCR-ABL1 and human CML.</p

ERK8 kinase domain model.

<p>(A), Multiple sequence alignment between ERK8 and the selected templates FUS3 and ERK2. Numbering is referred to human ERK8 cDNA sequence as defined in Uniprot accession number Q8TD08. Consensus code: “yellow” indicates positions which have a single, fully conserved residue; “green” indicates conservation between groups of strongly similar properties; “blue” indicates conservation between groups of weakly similar properties. Gatekeeper residue is in bold and indicated by a full black circle. The TEY activation motif is in red (activation loop spans from the DFG motif to the APE motif, residues 155–187). The region in square brackets has been substituted (starting from the position indicated with the dashed red line) with the alignment highlighted in the bottom square that includes p38α. (B), Model of the ERK8 kinase domain (residues 12–345 of the full-length 1–544 protein) obtained by means of homology modeling protocol. Conserved kinase domain features are indicated, β-sheets colored in yellow, α-helices colored in red, loops colored in green, TEY activation motif colored in blue. (C), Superimposition of the same ERK8 model (grey) with the ERK2 template (purple). (D), Evolution of ERK8 structure with the MD refinement. Superimposition of the ERK8 model (grey), used as MD input, with the representative final structure (the refined ERK8 structure) (cyan) obtained after the simulation. (E), Superimposition of the refined ERK8 model (cyan) with the ERK2 template (purple).</p

Flowchart of the <i>in silico</i> protocol.

<p>Computational steps applied to select all the hit compounds to be tested <i>in vitro</i>. In each set the percentage of success rate refers to the ratio between the number of active molecules and the number of tested molecules in the following experimental screening: purified GST-ERK8 protein (50 ng/sample) was used in kinase assays. Candidate compounds were dissolved in dimethyl sulfoxide (DMSO) and tested at fixed concentration of 50 µM (an equal volume of DMSO was added to control samples). Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry. Molecules were classified as active when the residual kinase activity was less than 50% in comparison to control samples.</p

Pharmacophore models.

<p>(A), Left panel, Structure-based pharmacophore generated from the Mg⁺⁺ loaded ERK8/ADP complex (coordinates were taken from the refined ERK8 structure) by using the Ligandscout software. Right panel, Structure-based pharmacophore generated by the GRID-based pharmacophore modeling approach, starting from the ligand-bound refined structure of ERK8. Features code: HYD = hydrophobic; HBA = H-bond acceptor; HBD = H-bond donor; AROM = aromatic ring; grey spheres are excluded volumes. (B), The two ligand-based pharmacophores generated with the training set of 18 different inhibitors active towards ERK8 (from Bain J, et al., 2007). Features code same as above.</p

A resistant ERK8_F92I mutant confirms the predicted ATP pocket-binding mode.

<p>(A), Representative structures from MD simulation of the complex between ITT57 and both ERK8_WT (left panel) and ERK8_F92I mutant (right panel). The residue at position 92 is labeled and showed as sticks. The ITT57 ligand is showed as sticks. Protein residues and ligand atoms are colored by atom type. (B), GST tagged ERK8_WT and ERK8_F92I proteins (200 ng/sample) were used in kinase assays in presence of the indicated concentrations of ITT53, ITT57 and Ro-318220 molecules. Using the paper-spotted kinase assay technique, we quantified and normalized the activities of the WT and of the mutant protein. MBP phosphorylation levels were evaluated by β-counting protocol of triplicates and results expressed as percentage of residual kinase activity compared with control samples. Significance (p-value) was obtained by one-way ANOVA test. Asterisks were attributed for the following significance values: p<0.05 (*), p<0.01 (**), p<0.001 (***).</p

<i>In vitro</i> characterization.

<p>(A), Dose/response curves for ITT53 and ITT57 on GST-ERK8_Bac. Results are reported as residual MBP phosphorylation levels compared with the control (DMSO). The average results of two independent experiments done in triplicate ± SD are plotted with the curve-fitting PRISM software (GraphPad). The concentration of drug that inhibited activity by 50% (IC₅₀) is shown. (B), ITT53, ITT57 and Ro-318220 ATP competition assay on GST-ERK8_Bac. Inhibition values are reported as percentage of residual MBP phosphorylation levels (i.e., residual kinase activity) compared with the control (DMSO). Results for the two indicated concentrations of ITT53, ITT57 and Ro-318220 (top, middle, bottom panel, respectively) at four different ATP doses were plotted. The average results of two independent experiments done in triplicate ± SD are plotted.</p

Gatekeeper mutants.

<p>(A), Multiple sequence alignment of gatekeeper region among different members of the MAPK and CDK families of kinases. The position corresponding to the gatekeeper residue is highlighted. (B), Superimposition of the refined ERK8 structure (cyan) and CDK2 (magenta) X-ray structure. (C), Western Blot control of GST-fusion proteins from <i>E. coli</i>. Each lane was loaded with 100 ng of purified protein. ERK8_KD sample (lane 6) is a point mutant on the conserved lysine (Lys, K) in position 42 to arginine (Arg, R). (D), Representative kinase assay blot of gatekeeper mutants (200 ng/sample of purified protein) (upper panel). Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry. Coomassie staining verified that equal amounts of substrate were loaded (lower panel). Quantification of kinase activity in comparison to WT, as scored by MBP phosphorylation, from three independent experiments is reported in the lower panel.</p

Effect of selected molecular scaffolds on bacterial and eukaryotic GST-ERK8.

<p>(A), Molecular structure of selected compounds. (B), Binding mode of each compound as obtained after the molecular docking step. The ITT molecules are showed as sticks and colored by atom type. ERK8 protein structure is represented by secondary structure cyan elements. (C), Samples of GST-ERK8 from <i>E. coli</i> with the indicated concentration of inhibitors were subjected to kinase assay. Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry (upper panel). Coomassie staining verified that equal amounts of substrate were loaded (lower panel). (D), The average results of three independent experiments done in duplicate ± SD are plotted. (E), Samples of GST-ERK8_Bac with the indicated concentration of inhibitors were subjected to kinase assay. Reactions were resolved by SDS-PAGE and ³²P incorporation on MBP was estimated by densitometry (upper panel). Coomassie staining verified that equal amounts of substrate were loaded (lower panel). (F), The average results of three independent experiments done in duplicate ± SD are plotted.</p

Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4‑<i>d</i>]pyrimidines for Targeting Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo­[3,4-<i>d</i>]­pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo­[3,4-<i>d</i>]­pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. <i>In vitro</i> HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds

Aptamer Functionalization of Nanosystems for Glioblastoma Targeting through the Blood–Brain Barrier

Polymeric nanoparticles (PNPs) may efficiently deliver in vivo therapeutics to tumors when conjugated to specific targeting agents. Gint4.T aptamer specifically recognizes platelet-derived growth factor receptor β and can cross the blood–brain barrier (BBB). We synthesized Gint4.T-conjugated PNPs able of high uptake into U87MG glioblastoma (GBM) cells and with astonishing EC₅₀ value (38 pM) when loaded with a PI3K-mTOR inhibitor. We also demonstrated in vivo BBB passage and tumor accumulation in a GBM orthotopic model