Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.

Arrestin-3 interaction with maternal embryonic leucine-zipper kinase.

Maternal embryonic leucine-zipper kinase (MELK) overexpression impacts survival and proliferation of multiple cancer types, most notably glioblastomas and breast cancer. This makes MELK an attractive molecular target for cancer therapy. Yet the molecular mechanisms underlying the involvement of MELK in tumorigenic processes are unknown. MELK participates in numerous protein-protein interactions that affect cell cycle, proliferation, apoptosis, and embryonic development. Here we used both in vitro and in-cell assays to identify a direct interaction between MELK and arrestin-3. A part of this interaction involves the MELK kinase domain, and we further show that the interaction between the MELK kinase domain and arrestin-3 decreases the number of cells in S-phase, as compared to cells expressing the MELK kinase domain alone. Thus, we describe a new mechanism of regulation of MELK function, which may contribute to the control of cell fate.

Computational insights into the binding of IN17 inhibitors to MELK.

The protein kinase MELK is an important kinase in cell signaling and has shown to be a promising anti-cancer target. Recent work has resulted in a novel small molecule scaffold targeting MELK, IN17. However, there has been little structural information or physical understanding of MELK-IN17 interactions. Using Tinker-OpenMM on GPUs, we have performed free energy simulations on MELK binding with IN17 and 11 derivatives. This series of studies provides structural insights into how substitution on IN17 leads to differences in complex structure and binding thermodynamics. In addition, this study serves as an assessment of the current capabilities of the AMOEBA forcefield, accelerated by GPU computing, to serve as a molecular-dynamics-based free energy simulation platform for lead optimization.

MELK: a potential novel therapeutic target for TNBC and other aggressive malignancies.

INTRODUCTION: There is an unmet need in triple-negative breast cancer (TNBC) patients for targeted therapies. Maternal embryonic leucine zipper kinase (MELK) is a promising target for inhibition based on the abundance of correlative and functional data supporting its role in various cancer types. Areas covered: This review endeavors to outline the role of MELK in cancer. Studies covering a range of biological functions including proliferation, apoptosis, cancer stem cell phenotypes, epithelial-to-mesenchymal transition, metastasis, and therapy resistance are discussed here in order to understand the potential of MELK as a clinically significant target for TNBC patients. Expert opinion: Targeting MELK may offer a novel therapeutic opportunity in TNBC and other cancers. Despite the abundance of correlative data, there is still much we do not know. There are a lack of potent, specific inhibitors against MELK, as well as an insufficient understanding of MELK's downstream substrates. Addressing these issues is the first step toward identifying a patient population that could benefit from MELK inhibition in combination with other therapies.

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth.

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes.

Advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) have permitted phosphoproteomic analysis on a grand scale, but ongoing challenges specifically associated with confident phosphate localization continue to motivate the development of new fragmentation techniques. In the present study, ultraviolet photodissociation (UVPD) at 193 nm is evaluated for the characterization of phosphopeptides in both positive and negative ion modes. Compared to the more standard higher energy collisional dissociation (HCD), UVPD provided more extensive fragmentation with improved phosphate retention on product ions. Negative mode UVPD showed particular merit for detecting and sequencing highly acidic phosphopeptides from alpha and beta casein, but was not as robust for larger scale analysis because of lower ionization efficiencies in the negative mode. HeLa and HCC70 cell lysates were analyzed by both UVPD and HCD. While HCD identified more phosphopeptides and proteins compared to UVPD, the unique matches from UVPD analysis could be combined with the HCD data set to improve the overall depth of coverage compared to either method alone.

From in Silico Discovery to intra-Cellular Activity: Targeting JNK-Protein Interactions with Small Molecules.

The JNK-JIP1 interaction represents an attractive target for the selective inhibition of JNK-mediated signaling. We report a virtual screening (VS) workflow, based on a combination of three-dimensional shape and electrostatic similarity to discover novel scaffolds for the development of non-ATP competitive inhibitors of JNK targeting the JNK-JIP interaction. Of 352 (0.13%) compounds selected from the NCI diversity set more than 22% registered as hits in a biochemical kinase assay. Several compounds discovered to inhibit JNK activity under standard kinase assay conditions also impeded JNK activity in HEK293 cells. These studies led to the discovery that the lignan (-)-zuonin A inhibits JNK-protein interactions with a selectivity of 100-fold over ERK2 and p38 MAPKα. These results demonstrate the utility of a virtual screening protocol to identify novel scaffolds for highly selective, cell-permeable inhibitors of JNK-protein interactions.

Manipulating JNK signaling with (--)-zuonin A.

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (-)-zuonin A. Here we report the asymmetric synthesis of (-)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (-)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (-)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (-)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (-)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (-)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (-)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein-protein interactions within MAPK cascades.

Development of JNK2-selective peptide inhibitors that inhibit breast cancer cell migration.

Despite their lack of selectivity toward c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP(10)-Δ-TAT(i)) or to a poly arginine sequence (JIP(10)-Δ-R(9)) enabled the potent inhibition of JNK2 (IC(50) ≈ 90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the overexpression of GFP-JNK2α. Both JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2α. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP(10)-Δ-TAT(i) containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP(10)-Δ-TAT(i) and JIP(10)-Δ-R(9) inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined and found to inhibit p38MAPKα in an in vitro cell-free assay with little propensity to inhibit JNK isoforms.