Pancreatic cells are resistant to KRASQ61L expression due to hyperactivation of ERK/MAPK signaling and induction of apoptosis

The RAS family of small GTPases is among the most frequently mutated gene families in human cancer. In pancreatic ductal adenocarcinoma (PDAC), ~95% of cases harbor an activating KRAS mutation, primarily at codon 12, 13, or 61, with G12D the most common overall (40%). In contrast, the KRASQ61L mutation, though constitutively active, is virtually absent in PDAC patient tumors. This suggests that KRASQ61L may engage in distinct, allele-specific signaling that limits its ability to drive tumorigenesis. Determining the mechanisms that limit the occurrence of this mutation will aid in our understanding of the critical KRAS effectors and pathways that drive tumorigenesis. To investigate these mechanisms, we utilized a tightly controlled doxycycline-inducible KRAS expression system in an isogenic, immortalized pancreatic cell line, enabling direct comparison of KRASQ61L to the common PDAC mutant KRASG12D. Using TurboID proximity labeling alongside RNA sequencing, we mapped early effector interactions and transcriptional responses, revealing that KRASQ61L induces greater hyperactivation of the ERK/MAPK pathway, resulting in increased nuclear translocation of ERK1/2. Finally, pancreatic cells are highly tolerant to overexpression of KRASG12D, but KRASQ61L overexpression leads to impaired proliferation and increased apoptosis. These findings provide experimental support for the long-standing "Goldilocks" model of oncogenic signaling, where too much ERK/MAPK pathway activation is detrimental to tumorigenesis. Our work offers a mechanistic explanation for the relative absence of KRASQ61L in PDAC and contributes to our understanding of KRAS allele-specific vulnerabilities, which can inform future therapeutic strategies targeting KRAS-driven pancreatic cancer. Overall design: This RNA sequencing experiment was performed to investigate how different KRAS mutations impact transcriptional programs in a human pancreatic ductal epithelial context. We used HPNE (human pancreatic nestin-expressing) cells engineered to express doxycycline-inducible versions of wild-type KRAS (KRASWT), or one of three oncogenic KRAS mutants: KRASG12D, KRASG12R, or KRASQ61L. Each isogenic cell line (HPNE-KRASWT, HPNE-KRASG12D, HPNE-KRASG12R, HPNE-KRASQ61L) was treated with doxycycline for 48 hours to induce transgene expression.