MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance.

Chemoresistance constitutes a major challenge in the treatment of triple-negative breast cancer (TNBC). Mixed-Lineage Kinase 4 (MLK4) is frequently amplified or overexpressed in TNBC where it facilitates the aggressive growth and migratory potential of breast cancer cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. Here, we demonstrate that MLK4 promotes TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. We observed that MLK4 knock-down or inhibition sensitized TNBC cell lines to chemotherapeutic agents in vitro. Similarly, MLK4-deficient cells displayed enhanced sensitivity towards doxorubicin treatment in vivo. MLK4 silencing induced persistent DNA damage accumulation and apoptosis in TNBC cells upon treatment with chemotherapeutics. Using phosphoproteomic profiling and reporter assays, we demonstrated that loss of MLK4 reduced phosphorylation of key DNA damage response factors, including ATM and CHK2, and compromised DNA repair via non-homologous end-joining pathway. Moreover, our mRNA-seq analysis revealed that MLK4 is required for DNA damage-induced expression of several NF-кB-associated cytokines, which facilitate TNBC cells survival. Lastly, we found that high MLK4 expression is associated with worse overall survival of TNBC patients receiving anthracycline-based neoadjuvant chemotherapy. Collectively, these results identify a novel function of MLK4 in the regulation of DNA damage response signaling and indicate that inhibition of this kinase could be an effective strategy to overcome TNBC chemoresistance.

Inhibition of the ʟ-glutamine transporter ASCT2 sensitizes plasma cell myeloma cells to proteasome inhibitors.

Proteasome inhibitors (PIs), used in the treatment of plasma cell myeloma (PCM), interfere with the degradation of misfolded proteins leading to activation of unfolded protein response (UPR) and cell death. However, despite initial strong antimyeloma effects, PCM cells eventually develop acquired resistance to PIs. The pleiotropic role of ʟ-glutamine (Gln) in cellular functions makes inhibition of Gln metabolism a potentially good candidate for combination therapy. Here, we show that PCM cells, both sensitive and resistant to PIs, express membrane Gln transporter (ASCT2), require extracellular Gln for survival, and are sensitive to ASCT2 inhibitors (ASCT2i). ASCT2i synergistically potentiate the cytotoxic activity of PIs by inducing apoptosis and modulating autophagy. Combination of ASCT2 inhibitor V9302 and proteasome inhibitor carfilzomib upregulates the intracellular levels of ROS and oxidative stress markers and triggers catastrophic UPR as shown by upregulated spliced Xbp1 mRNA, ATF3 and CHOP levels. Moreover, analysis of RNA sequencing revealed that the PI in combination with ASCT2i reduced the levels of Gln metabolism regulators such as MYC and NRAS. Analysis of PCM patients' data revealed that upregulated ASCT2 and other Gln metabolism regulators are associated with advanced disease stage and with PIs resistance. Altogether, we identified a potent therapeutic approach that may prevent acquired resistance to PIs and may contribute to the improvement of treatment of patients suffering from PCM.

Upregulation of MLK4 promotes migratory and invasive potential of breast cancer cells.

Metastasis to distant organs is a major cause for solid cancer mortality, and the acquisition of migratory and invasive phenotype is a key factor in initiation of malignancy. In this study we investigated the contribution of Mixed-Lineage Kinase 4 (MLK4) to aggressive phenotype of breast cancer cells. Our TCGA cancer genomic data analysis revealed that amplification or mRNA upregulation of MLK4 occurred in 23% of invasive breast carcinoma cases. To find the association between MLK4 expression and the specific subtype of breast cancer, we performed a transcriptomic analysis of multiple datasets, which showed that MLK4 is highly expressed in triple-negative breast cancer compared to other molecular subtypes. Depletion of MLK4 in cell lines with high MLK4 expression impaired proliferation and anchorage-dependent colony formation. Moreover, silencing of MLK4 expression significantly reduced the migratory potential and invasiveness of breast cancer cells as well as the number of spheroids formed in 3D cultures. These in vitro findings translate into slower rate of tumor growth in mice upon MLK4 knock-down. Furthermore, we established that MLK4 activates NF-κB signaling and promotes a mesenchymal phenotype in breast cancer cells. Immunohistochemical staining of samples obtained from breast cancer patients revealed a strong positive correlation between high expression of MLK4 and metastatic potential of tumors, which was predominantly observed in TNBC subgroup. Taken together, our results show that high expression of MLK4 promotes migratory and invasive phenotype of breast cancer and may represent a novel target for anticancer treatment.