Supplementary Figure S1: Normal cells have consistently higher resistance to PIKfyve and p38MAPK inhibition than cancer cells. Supplementary Figure S2: JNK and p44/p42 MAP kinases protein and phosphorylation levels do not correlate with sensitivity to PIKfyve inhibition. Supplementary Figure S3: Manipulation of p38MAPK protein levels in resistant and sensitive cells changes their sensitivity to PIKfyve inhibition. Supplementary Figure S4: Synergistic cytotoxic effects of combined PIKfyve and p38MAPK inhibition. Supplementary Figure S5: Total cell count and SEMs in support of the data presented in Fig.3. Supplementary Figure S6: Inhibition of PIKfyve and/or p38MAPK kinases does not trigger apoptosis or activate the p53 stress response. Supplementary Figure S7: Combined PIKfyve and p38MAPK inhibition blocks autophagy in all cells. Supplementary Figure S8: WX8 is a specific inhibitor of PIKfyve.
ARTICLE ABSTRACT
In nutrient-poor conditions, autophagy buffers metabolic stress and counteracts the effects of chemotherapy and radiation on cancer cells, which depend on autophagy for survival. However, clinical trials targeting autophagy have failed to produce successful anticancer treatments using currently available inhibitors. Recent studies have shown that PIKfyve kinase inhibitors disrupt lysosome function in autophagy and can selectively kill certain cancer cells. Analysis of biochemical changes caused by PIKfyve inhibition revealed that resistant cells contain significantly higher levels of cellular p38MAPK protein and phosphorylation. Expression of the lysosomal protein, lysosomal-associated membrane protein 2, carrying phosphomimetic mutations of the p38MAPK phosphorylation sites prevented all effects caused by PIKfyve inhibition–induced lysosome dysfunction. Thus, the activation of p38MAPK in response to PIKfyve inhibition revealed a novel compensatory role in maintaining lysosome function in autophagy. The functional cooperation between the cellular PIKfyve and p38MAPK pathways in regulating lysosome homeostasis was especially important in cancer cells. Combined inhibition of PIKfyve and p38MAPK activities synergistically blocked autophagy-mediated protein degradation, prevented cathepsin maturation, and markedly reduced the viability of multiple cancer cell types without affecting the viability of normal cells. Furthermore, combined PIKfyve and p38MAPK inhibitors synergistically reduced tumor growth in mice bearing xenografts of human colorectal adenocarcinoma, suggesting a novel way to target cancer cells by prolonged inhibition of autophagy using lower drug concentrations.
This study demonstrates that PIKfyve and p38MAPK cooperate to regulate lysosome homeostasis and their combined inhibition synergistically blocks autophagy to reduce cancer cell viability in vitro and in vivo.
