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. 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.

RAS家族属于小GTP酶（small GTPases）家族，是人类癌症中最常发生突变的基因家族之一。在胰腺导管腺癌（pancreatic ductal adenocarcinoma, PDAC）中，约95%的病例携带激活性KRAS突变，突变位点主要集中于12、13或61号密码子，其中G12D是最常见的突变类型（占比40%）。与之相反，KRASQ61L突变虽为组成型激活，但在PDAC患者肿瘤中几乎不存在。这提示KRASQ61L可能参与独特的等位基因特异性信号通路，从而限制其驱动肿瘤发生的能力。阐明限制该突变发生的机制，将有助于我们理解驱动肿瘤发生的关键KRAS效应分子与信号通路。为探究这些机制，我们在同基因永生化胰腺细胞系中采用了严格调控的多西环素诱导型KRAS表达系统，可直接比较KRASQ61L与常见PDAC突变型KRASG12D的功能差异。结合TurboID邻近标记技术与RNA测序（RNA sequencing），我们绘制了早期效应分子互作与转录应答图谱，发现KRASQ61L可更强程度地过度激活ERK/MAPK通路，导致ERK1/2的核转位增加。最后，胰腺细胞对KRASG12D的过表达具有较高耐受性，但KRASQ61L过表达会导致细胞增殖受损与凋亡增加。上述研究结果为长期存在的致癌信号通路"Goldilocks"模型提供了实验依据——即ERK/MAPK通路过度激活反而不利于肿瘤发生。本研究为PDAC中KRASQ61L相对缺失的现象提供了机制层面的解释，同时加深了我们对KRAS等位基因特异性脆弱性的理解，可为未来靶向KRAS驱动型胰腺癌的治疗策略提供参考。本RNA测序实验旨在探究不同KRAS突变在人胰腺导管上皮环境中对转录程序的影响。我们采用了经工程改造的HPNE细胞（human pancreatic nestin-expressing cells），该细胞可表达多西环素诱导型野生型KRAS（KRASWT），或三种致癌性KRAS突变体之一：KRASG12D、KRASG12R或KRASQ61L。将每一株同基因细胞系（HPNE-KRASWT、HPNE-KRASG12D、HPNE-KRASG12R、HPNE-KRASQ61L）用多西环素处理48小时，以诱导转基因表达。