KRAS primes the road for WNT-driven tumour initiation

The RAS Dialogue Blog posts are written by RAS experts sharing the latest research, updates, and scientific RAS news. The content is curated by the RAS Initiative.

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Activating mutations in the KRAS oncogene occur in 30-40% of colorectal tumours^1,2, yet KRAS activation alone is insufficient to drive neoplastic transformation in intestinal epithelial cells^3–5. Instead, the most common initiating event is the inactivation of APC, a central regulator of WNT signalling^6–9. The high frequency of APC loss in hereditary intestinal polyposis syndromes and early-stage sporadic lesions, together with the observation that KRAS and TP53 mutations typically arise in more advanced disease, underpins a classical view that colorectal cancer (CRC) develops in a sequential series of discrete steps⁸. In this model, APC-mutant cells arise first and acquire malignant potential only after accumulating additional oncogenic mutations with each step progressively conferring increased fitness in cancer initiating clones. 

However, evidence recently published suggests an alternative temporal ordering in intestinal tumorigenesis. Previously we identified, in histologically normal human colon, both small and large patches composed of dozens to hundreds of intestinal glands (termed crypts) harbouring the same activating KRAS mutations that are found in human CRCs ^10,11. These large patches arise from increased crypt fission rates driven by KRAS activation, consistent with previous mouse modelling studies reporting similar phenomena¹². In follow-up studies, we also detected oncogenic mutations in additional cancer-associated genes, including PTEN, SMAD4, and ARID1A¹³. Investigating the possible role that these mutations play in colorectal cancer initiation, and how they might interact with canonical initiating events such as APC loss, motivated us to devise mouse modelling approaches to test if the order of acquisition of mutations impacted tumorigenesis.

Our recent publication in Nature¹⁴ tried to address this question using two complementary experimental protocols. Under one condition, we generated mouse cohorts carrying defined cancer driver mutations (including Kras^G12D) in the adult intestinal epithelium, that alone are insufficient to generate tumours, followed by exposure to the potent alkylating mutagen N-ethyl-N-nitrosourea (ENU), which induces random point mutations that are also individually inefficient in generating tumours. This was termed a “priming” protocol. In the second approach, the order of events was reversed: mice were first treated with ENU and the known defined driver mutations were then subsequently induced at different time points. This strategy was termed the “rescue” protocol, for reasons that will become clear later. To identify ENU-induced mutations, the resulting tumours were then sequenced.

ENU treatment of mice primed by Kras^G12D activation or by deletion of one allele of Apc (Apc^het) resulted in a striking three orders of magnitude increase in intestinal tumour burden compared to mice without priming, together with a markedly shorter median survival (Figure 1a). Other priming events, including Fbxw7^nulland Trp53^null, also displayed marked increases in tumour burden. While all cohorts developed tumours in the small intestine, only Apc^het, Kras^G12D, Trp53^het/Notch1-ICD^het, and Trp53^null/Notch1-ICD^het cohorts displayed colonic tumours, providing an early indication that tissue context influences tumour outcomes including regional differences along the intestinal tract.

DNA sequencing showed that most tumours were initiated by ENU-induced mutations in either Apc or Ctnnb1. However, the relative frequencies of these initiating events were determined by the priming background. For example, approximately 90% of the tumours arising in ENU-treated Apc^het-primed mice contained at least one Apc mutation, compared with tumours from Kras^G12D-primed mice that showed a high prevalence of Ctnnb1 mutations (~85%).  We further observed selection for specific Ctnnb1 mutations that promote its   stabilisation^15–19. For example, p.G34E predominated in tumours from Kras^G12D- and Trp53^null-primed mice, while p.S37F and pD32G were more frequent in Fbxw7^null-primed cohorts. Notably, p.S33P was exclusive to the Kras^G12D cohort. Together, these findings suggested that even among priming events that favour transformation through β-catenin stabilisation, distinct mutational outcomes are selectively favoured. After confirming that the most efficient priming events did not alter overall the number of mutations, the best explanation for our observations is that priming creates a permissive environment, albeit to different degrees, for ENU-induced initiating mutations.

Our findings raise a key question: what happens to cells carrying ENU-induced initiating mutations in the absence of priming? Are such cells retained, able to divide and clonally expand within the epithelium and yet remain incapable of tumour initiation, or are they lost and if so, at what rate? Reasoning that retained initiating events could be identified by rescuing their tumour-promoting capacity post hoc, we applied the “rescue” protocol, reversing the order of treatment by inducing pro-oncogenic fields 10–30 days after ENU mutagenesis. Compared with the corresponding priming experiments, a rapid and substantial decline in tumour multiplicity across all cohorts was observed, including a 97% reduction in Kras^G12D-rescued mice. Importantly, rescued tumours were still driven by Apc and Ctnnb1 mutations. A quantitative assessment of selection bias revealed negative selection against both Apc and Ctnnb1 mutations in the absence of priming, with a stronger negative bias observed for most Ctnnb1 mutations.

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Our experimental approach in mice points to a central role for priming in shaping tumour initiation. A logical next step was to search for evidence of analogous events impacting selection of APC mutations in human CRC. We analysed publicly available genomic data from colorectal tumours and filtered it to include cancers with two APC-truncating mutations²⁰. Then APC domain retention was examined in CRCs with co-occurring APC and KRAS mutations to assess whether KRAS-activating mutations influence the selection of APC truncations. This revealed that the C-terminal APC truncation in CRCs with no KRAS mutations commonly retained one 20 amino-acid(aa) repeat located in the mutation cluster region (MCR), while those with KRAS-activating mutations tended to retain two repeats (Figure 2). The 20aa repeats bind to β-catenin and regulate its stability. Retention of more repeats increases β-catenin degradation, reducing WNT-signalling transcriptional output^15,21–23. Interestingly, this pattern was observed across all KRAS mutational hotspots (G12, G13, Q61, K117, and A146). 

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Motivated by these results, we next analysed polyps (believed to represent earlier CRC precursors) that harboured one or two APC mutations²⁴. In this cohort, we observed a more modest trend toward increased retention of 20aa repeats in KRAS-mutant polyps compared to that observed in CRCs with KRAS-activating mutations. However, analysis of the variant allele frequency (VAF) values, an indicator of the relative proportion of the polyp containing a given mutation, for KRAS and APC revealed a striking pattern: as the KRAS/APC VAF ratio increased, 20aa repeat retention progressively resembled that observed in CRCs. This suggests that polyps with higher relative KRAS VAFs (consistent with earlier acquisition of KRAS mutations) may be at increased risk of progression, consistent with priming dictating mutational order and that primed polyps are at higher risk of progression.

Our experimental data indicate that priming creates a permissive environment that facilitates transformation by ENU-induced, WNT pathway–activating mutations in Apc and Ctnnb1 that would otherwise be rapidly eliminated by strong negative selection. Kras^G12D emerges as the most efficient priming event in models of sporadic CRC. While elucidating the molecular mechanisms underpinning this negative selection and its reversal by priming was beyond the scope of this study, the shared regulation of protein stability by β-TrCP and GSK3β provides a plausible point of contact between the WNT and RAS pathways and an important avenue for future investigation^25–29.

Together, our convergent mouse and human data strongly support a model in which KRAS-mutant crypt patches in histologically normal colon epithelium actively shape colorectal cancer initiation by favouring the fixation of WNT-activating mutations, thereby providing an alternative to the canonical sequence of events in CRC progression. A key implication of this priming model is that APC loss is highly adaptable, capable of achieving effective oncogenic complementation through multiple mutational solutions that readily accommodate pre-existing mutations arising during ageing. Consequently, prevention strategies aimed at limiting the availability of primed tissue for malignant transformation would need to be population-based, applied early in life, and preferentially target the most efficient priming events: KRAS-activating mutations.

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References

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