RNA sequencing of SW1990 KRAS-G12D or KRAS-G12D/K128R knockin cell lines

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not yet fully understood. K128 ubiquitination is the most prevalent modification of the GTPase core domain in both NRAS and KRAS. The ubiquitination at K128 was decreased in cancer samples compared to normal tissue. We found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAP), NF1 and RASA1, which increases RAS binding to GAP proteins and promotes GAP-mediated GTP hydrolysis. K128 ubiquitination is transiently induced by growth factors or cytokines, which restricts the extent of wild-type RAS activation in a GAP-dependent manner. In contrast, the loss of K128 ubiquitination in the KRAS-G12D mutant promotes tumor growth by suppressing RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Dysregulation of K128 ubiquitination unleashes both wild-type and mutant RAS signaling and triggers a senescence-associated secretory phenotype, driving RAS-driven tumorigenesis.