Ras
isoforms H-, N-, and K-ras are each mutated in specific
cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane,
Ras proteins are laterally segregated into
isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in
cancer drug development. We therefore conducted a chemical screen with commercial and in-house
natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a
statin and farnesyl-
transferase inhibitor, we surprisingly identified five
protein synthesis inhibitors as positive regulators. Using commonly employed
cycloheximide as a representative compound, we show that
protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data,
cycloheximide treatment activated both Erk and Akt
kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly,
cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with
cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of
cycloheximide to promote
tumor cell growth was also reflected in its ability to increase
breast cancer cell
tumors grown in ovo. These results illustrate the possibility of identifying Ras-
isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from
protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain
tumor cell types.