MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate.

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α-MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α-MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.

Requirement for epithelial p38α in KRAS-driven lung tumor progression.

Malignant transformation entails important changes in the control of cell proliferation through the rewiring of selected signaling pathways. Cancer cells then become very dependent on the proper function of those pathways, and their inhibition offers therapeutic opportunities. Here we identify the stress kinase p38α as a nononcogenic signaling molecule that enables the progression of KrasG12V-driven lung cancer. We demonstrate in vivo that, despite acting as a tumor suppressor in healthy alveolar progenitor cells, p38α contributes to the proliferation and malignization of lung cancer epithelial cells. We show that high expression levels of p38α correlate with poor survival in lung adenocarcinoma patients, and that genetic or chemical inhibition of p38α halts tumor growth in lung cancer mouse models. Moreover, we reveal a lung cancer epithelial cell-autonomous function for p38α promoting the expression of TIMP-1, which in turn stimulates cell proliferation in an autocrine manner. Altogether, our results suggest that epithelial p38α promotes KrasG12V-driven lung cancer progression via maintenance of cellular self-growth stimulatory signals.

DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF.

MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.

Optimal linker length for small molecule PROTACs that selectively target p38α and p38ß for degradation.

We report the design of hetero-bifunctional small molecules that selectively target p38α and p38ß for degradation. These proteolysis targeted chimeras (PROTACs) are based on an ATP competitive inhibitor of p38α and p38ß, which is linked to thalidomide analogues to recruit the Cereblon E3 ubiquitin ligase complex. Compound synthesis was facilitated by the use of a copper catalyzed "click" reaction. We show that optimization of the linker length and composition is crucial for the degradation-inducing activity of these PROTACs. We provide evidence that these chemical compounds can induce degradation of p38α and p38ß but no other related kinases at nanomolar concentrations in several mammalian cell lines. Accordingly, the PROTACs inhibit stress and cytokine-induced p38α signaling. Our compounds contribute to understanding the development of PROTACs, and provide a useful tool to investigate functions of the p38 MAPK pathway and its involvement in diseases.

Regulation of tumor angiogenesis and mesenchymal-endothelial transition by p38α through TGF-ß and JNK signaling.

The formation of new blood vessels is essential for normal development, tissue repair and tumor growth. Here we show that inhibition of the kinase p38α enhances angiogenesis in human and mouse colon tumors. Mesenchymal cells can contribute to tumor angiogenesis by regulating proliferation and migration of endothelial cells. We show that p38α negatively regulates an angiogenic program in mesenchymal stem/stromal cells (MSCs), multipotent progenitors found in perivascular locations. This program includes the acquisition of an endothelial phenotype by MSCs mediated by both TGF-ß and JNK, and negatively regulated by p38α. Abrogation of p38α in mesenchymal cells increases tumorigenesis, which correlates with enhanced angiogenesis. Using genetic models, we show that p38α regulates the acquisition of an endothelial-like phenotype by mesenchymal cells in colon tumors and damage tissue. Taken together, our results indicate that p38α in mesenchymal cells restrains a TGF-ß-induced angiogenesis program including their ability to transdifferentiate into endothelial cells.