Abstract Colonic stem cells (CSCs) reside within spatially organized niches exposed to a gradient of growth factor signals, including EGF secreted by stromal cells at the crypt base. Despite this gradient, CSCs exhibit paradoxical signaling patterns that deviate from canonical ligand-receptor models, maintaining high Akt activity and suppressed ERK activation. Using patient-derived colonic organoids (PDCOs), we find that differentiated cells display pulsatile ERK activity, whereas stem cell compartments are ERK-silent through an Akt-directed inhibitory phosphorylation of Raf-1 at serine 259. Raf-1S259 phosphorylation thereby insulates the stem cell compartment from excessive MAPK activation despite abundant EGF stimulation. Disruption of this Raf-1S259 insulation, either through transient ERK activation or Akt inhibition, leads to rapid stem cell differentiation. Conversely, oncogenic mutations such as KRASG12V or non-phosphorylatable Raf-1S259A abolish this regulatory control, producing constitutively high ERK activation along with sustained Akt signaling. This dual activation drives loss of epithelial patterning, expansion of undifferentiated cells, and morphological dysplasia reminiscent of early colorectal cancer. Quantitative live-cell imaging reveals distinct ERK dynamics underlying cell fates: healthy differentiation is characterized by pulsatile ERK activity in non-stem cells, whereas sustained ERK activation marks transformed CSCs. Remarkably, transient reintroduction of ERK pulsatility or Akt suppression rescues differentiation even under oncogenic conditions. Together, these findings suggest that the temporal dynamics of ERK activity, maintained by an Akt-Raf-1 insulation circuit, are critical for preserving epithelial organization and could be leveraged to reverse early transformation in colorectal cancers. Citation Format: Lauren Riede, Alex Borowiec, Woody March-Steinman, Jayati Chakrabarti, Sohini Mallick, Curtis Thorne, Andrew Paek, Kelvin Pond. Loss of ERK pulsatility drives transformation of colonic organoids through collapse of Akt-mediated signaling insulation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr LB281.
Riede et al. (Fri,) studied this question.