The RAS genes are mutated in a fifth or more of human cancers (Prior et al., 2020), which is well established to be tumorigenic (Pylayeva-Gupta et al., 2011). There are three individual RAS genes in humans, KRAS, NRAS, and HRAS, three primary sites mutated in human cancers, G₁₂, G₁₃, and Q₆₁, and six possible amino acid substitutions at each site arising from a point mutation. As such, there are 54 possible oncogenic point mutations, not including rare non-canonical mutations (Hobbs et al., 2016). Mapping these mutations to human cancers reveals a distinct pattern or ‘tropism’ in which individual cancer types are characterized by specific RAS mutations (Li et al., 2018). The same can be said for other oncogenes. For example, the most frequent EGFR point mutation in glioblastoma is G₅₉₈V/A, but L₈₆₁Q in lung adenocarcinoma; the most frequent IDH1 mutation in low-grade glioma is R₁₃₂H, but R₁₃₂C in melanoma, and so on (Chang et al., 2016). While these mutational biases are well described, the mechanism responsible is not.

One hint to the mechanism underlying RAS mutation tropism is that oncogenic RAS mutations are thought to occur early. Focusing on lung cancer, in humans oncogenic KRAS mutations have been detected in premalignant lesions (Kanda et al., 2012) as well as in multiple regions within the same tumor (Zhang et al., 2014a), indicative of an early origin (Wistuba and Gazdar, 2006). In mice, oncogenic Kras mutations are capable of initiating tumors in carcinogen (McCreery and Balmain, 2017) and genetically engineered (Kwon and Berns, 2013) lung cancer models. As early driver mutations, it follows that RAS mutation tropism is a reflection of the normal cells in which the mutation first occurred. By its very classification, oncogenic RAS can induce proliferation (Pylayeva-Gupta et al., 2011). However, high oncogenic signaling through the MAPK effector pathway of RAS can paradoxically induce an oncogenic stress response in normal cells mediated by the tumor suppressors p16 and p53, which leads to the growth arrest termed senescence (Muñoz-Espín and Serrano, 2014). Indeed, hyperactivation of MAPK signaling via the combination of Kras^G12V, Braf^D631A, and loss of the remaining wildtype Braf allele activated p53 and impeded lung tumorigenesis, an effect rescued by pharmacological inhibition of the MAPK pathway (Nieto et al., 2017).

Accumulating evidence suggests that different oncogenic mutations can be biochemically distinct (Muñoz-Maldonado et al., 2019; Smith et al., 2013). Relevant to this study, a G₁₂D mutation sterically inhibits the catalytic cleft (Parker et al., 2018) while Q₆₁R replaces the catalytic amino acid (Buhrman et al., 2010). A direct comparison of G₁₂D and Q₆₁R mutations in Nras revealed that the former has lower GTP loading and tumorigenic potential in the skin (Burd et al., 2014) and hematopoietic system (Kong et al., 2016). Indeed, a panel of G_12/13 Kras mutants introduced by Cas9-mediated gene editing in the lung revealed widely different tumorigenic potentials between different mutants (Winters et al., 2017). Taken together, we hypothesize that the level of oncogenic signaling may dictate the type of mutation conducive to initiate tumorigenesis in a normal cell, and hence play a role in RAS mutation tropism.

One challenge to testing this hypothesis is trying to backtrack to catch a single, ostensibly random mutagenic event in one gene from one normal cell, decades before manifesting as cancer in humans. However, in mice the moment of tumor initiation can be precisely defined as the point of carcinogen exposure, with the added benefit that carcinogens mimic the spontaneous nature of human cancers. Carcinogenesis is also an ideal model of RAS mutation tropism. Case in point, the carcinogen urethane found in fermented foods and alcoholic products (Gowd et al., 2018) primarily induces pulmonary tumors with a very specific Kras^Q61L or Kras^Q61R driver mutation, depending on the strain (Dwyer-Nield et al., 2010). These oncogenic mutations are also a strong match to the mutation signature of this carcinogen. Urethane-induced mutations conform to the A>T/G consensus sequence derived from comprehensive whole-exome sequencing urethane-induced tumors (Westcott et al., 2015), and the even more restricted CAN>CT/GN sequence determined shortly after urethane exposure by different ultra-sensitive sequencing approaches (Li et al., 2020; Valentine et al., 2020). Such mutations at position CA₁₈₂A give rise to the Q₆₁L/R oncogenic mutations characteristic of this carcinogen. We thus capitalized on this extreme bias of urethane for Kras^Q61L/R-mutant pulmonary tumors to genetically elucidate the effect of oncogenic signaling levels on RAS mutation tropism.

To explore the effect of oncogenic signaling levels on the selection of initiating oncogenic mutations, we genetically enhanced Kras translation to increase oncogenic activity or inactivated p53 to inhibit the cellular response to oncogenic stress in mice exposed to urethane. In regards to the first genetic change, we compared the native Kras^nat allele that is naturally enriched in rare codons and correspondingly poorly translated (Lampson et al., 2013) to the Kras^ex3op allele, in which 27 rare codons in exon 3 (which is not the site of oncogenic mutations) were converted to common, leading to roughly twice as much Kras protein in the lungs or derived cells of mice (Pershing et al., 2015). In regards to the second genetic change, we evaluated retaining or conditionally inactivating the Trp53 gene specifically in the lung, which has been shown to suppress oncogenic stress due to high mutant Kras expression in this tissue in vivo (Feldser et al., 2010; Junttila et al., 2010). Using this approach, we show here that the canonical Q₆₁L/R mutations are selected against in the more highly expressed Kras^ex3op allele in urethane-induced tumors, even though the carcinogen favors this mutation. Instead, biochemically less active G₁₂ mutations are detected, which, upon the loss of p53, partially shifts back to Q₆₁L/R mutations. p53 loss also promoted the expansion of the normally rare G₁₂ mutants in the Kras^nat allele, which was accompanied by an imbalance between the mutant and wildtype transcripts suggestive of higher expression. Moreover, tumors characterized by Q₆₁ mutations or the less active G₁₂ mutants when coupled with allelic imbalance exhibited similar transcript levels of three genes known to be activated by oncogenic RAS, suggesting a similar degree of oncogenic signaling. Taken together, these data support a narrow window of signaling conducive to initiate tumorigenesis in a normal cell. Namely, that imparted by the more active Q₆₁ mutations in Kras^nat allele or less active G₁₂ mutations in the higher expressed Kras^ex3op allele. p53 loss reprograms this mutation tropism, allowing more active mutations Q₆₁L/R in the Kras^ex3op allele as well as promoting or permitting allelic imbalance of less active G₁₂ mutations in the Kras^nat allele to now induce tumorigenesis. Selection for an oncogenic mutation imparting an optimal level of signaling in normal cells thus appears to influence the RAS mutation tropism of urethane.

Here, we show that p53 loss reprograms the RAS mutation tropism of urethane. On one hand, loss of this tumor suppressor shifts the canonical Q₆₁ oncogenic mutations that are normally detected in the unperturbed Kras^nat allele of urethane-induced tumors towards the usually rare G_12/13 mutations, but in conjunction with an increase in the mutant:wildtype ratio. On the other hand, the loss of p53 shifts the prevalence of G_12/13 mutations in the codon-optimized and more highly translated Kras^ex3op allele towards Q₆₁ mutations. The one common theme in both these shifts is the potential for higher oncogenic signaling, which argues that the degree of oncogenic signaling dictates the type of mutation conducive to initiate tumorigenesis in normal cells (Figure 5). In agreement, the expression of three known RAS downstream target genes was similar between Kras^ex3op with Q₆₁ mutations and Kras^nat with G_12/13 mutations when coupled with a high mutant:wildtype ratio, suggesting that expression differences render Q₆₁ and G_12/13 mutations fungible. While there is no question that the level of oncogenic signaling can influence tumorigenesis, what is new here is that the sensitivity of normal cells to oncogenic signaling may underlie the type of oncogenic mutation selected, which is relevant to the RAS mutation tropism of human cancers. We also note that neither the loss of p53 nor the change in codon usage in Kras altered the mutation signature of urethane, arguing that a selection for an optimal oncogenic signal supersedes the mutational bias of this carcinogen. Such a finding speaks to the somewhat complexing discordance often observed between the mutation signature of tumors and the corresponding driver mutation in human cancers (Dietlein et al., 2020; Temko et al., 2018), even in cancers in which the mutational signature can be ascribed to a specific mutagenic process (Buisson et al., 2019).

Figure 5

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There are three presumptions to the above model (Figure 5). First, Q₆₁ oncogenic mutations in Kras activate the MAPK pathway more potently than G_12/13 mutations. In agreement, a Q₆₁R mutation in Nras and/or Kras has a much (1000 times) slower intrinsic hydrolysis rate, higher GTP-loading, and more robust activation of the MAPK pathway, and when activated in the skin or hematopoietic system in vivo, is more tumorigenic than the corresponding G₁₂D mutation (Burd et al., 2014; Kong et al., 2016; Pershing et al., 2015). We also find that the expression of three downstream Ras target genes is higher in tumors with a Q₆₁ mutation compared to G_12/13 mutation in the absence of an allelic mRNA imbalance. One caveat, however, is that neither the level of the oncoproteins nor their signaling was directly measured at the protein level as the small amount of tumor material available was devoted to DNA/RNA analysis. Second, increasing Kras level increases signaling. In agreement, the Kras^ex3op allele has been documented to express more protein in the lung (Pershing et al., 2015) and increase ERK phosphorylation in hematopoietic stem cells (Sasine et al., 2018). Altering rare codons to common in ectopic or endogenous human KRAS also leads to higher translation, KRAS protein expression, and/or MAPK activation (Ali et al., 2017; Fu et al., 2018; Lampson et al., 2013; Pershing et al., 2015; Peterson et al., 2020). It remains possible, however, that rare codons are differentially translated (Quax et al., 2015) or higher Kras protein expression reduces MAPK signaling through negative feedback (Shin et al., 2009) specifically in the cell of origin of these pulmonary tumors. We also note the caveats that only three downstream target genes were assayed, and at the transcriptional rather than protein level, and further, that these three genes reside in just one effector pathway of RAS. Third, high oncogenic signaling is a negative selection, and this selection is p53-dependent. It is formally possible that the effect of p53 loss on the mutational bias of urethane is linked to cell fate or other factors instead of suppressing oncogene stress, but this would have to occur very quickly since urethane exposure shortly followed tamoxifen injection. We also note that oncogenic stress and p53 induction could not be measured in individual normal cells immediately after acquiring different oncogenic mutations by urethane mutagenesis, but both these points are well supported in multiple model systems (Muñoz-Espín and Serrano, 2014). As such, it is reasonable to propose that the level of oncogenic signaling is a driving force in the selection of the type of oncogenic Kras mutations initiating tumorigenesis (Figure 5). Capturing signaling in individual normal cells by single-cell transcriptomics immediately after induction of different oncogenic Kras mutations may provide a platform to interrogate this model. Further, application of CRISPR-Cas9-based barcoding tools that allow simultaneous lineage tracing and transcriptome analysis of individual cells within a tumor (Bowling et al., 2020; Quinn et al., 2021) could provide a clearer picture of the signaling dynamics throughout the course of tumor evolution.

We note that urethane has a bias towards CAN’CT/GN mutations that are absent in both strand orientations at codons encoding G₁₂ and G₁₃, and the mutagenic signature of urethane was the same in the Kras^nat and Kras^ex3op alleles. This suggests that G_12/13 oncogenic mutations are extremely rare, with urethane either inducing these mutations directly at a low frequency or inducing a cooperating mutation or non-mutational event that promotes a rare, pre-existing Kras-mutant cell to expand. In either scenario, the very fact that tumors arise with G_12/13 oncogenic mutations argues for potent selection of an extremely rare oncogenic mutation over the far more common mutations favored by the carcinogen. Furthermore, if these mutations are indeed pre-existing, such a finding elevates the importance of promotional events (secondary mutations, inflammation, etc.) in the process of tumor initiation (Balmain, 2020). Perhaps related, it is interesting that the loss of p53 did not shift all the oncogenic mutations in Kras^ex3op from G₁₂ to Q₆₁, which is unexpected given the rarity of G₁₂ mutations induced by urethane. One interpretation is that oncogenic G₁₂ mutations may be superior to Q₆₁ mutations in some manner, which is interesting insofar as G₁₂ mutations are more common in KRAS than Q₆₁ mutations in human cancers (Prior et al., 2020).

We also note an imbalance in the mRNA ratio of the mutant:wildtype Kras alleles in some urethane-induced tumors. Amplification of oncogenic Kras alleles has been previously documented in various mouse cancer backgrounds (Chung et al., 2017; Mueller et al., 2018; Westcott et al., 2015). What is new, however, is that this tracked with the type of oncogenic mutation, and even more interesting, in opposite directions. Namely, we observed a bias towards a high mutant:wildtype mRNA ratio in G_12/13-mutant Kras alleles in the absence of p53. Few tumors show amplification of the G_12/13-mutant allele based on genomic copy number analysis (Figure 4—figure supplement 2), pointing towards transcriptional or post-transcriptional mechanisms. One interpretation of these findings is that loss of p53 allows expansion of tumors with G_12/13-mutant Kras alleles that are more highly expressed, an observation borne out by signaling analysis. Increasing MAPK signaling by paradoxical activation with a BRAF inhibitor expands the number of cell types that are tumorigenic upon activation of the LSL-Kras^G12D allele in the lung (Cicchini et al., 2017), hence it is quite possible that a similar phenomenon is at play here as well. Such higher expression could be a product of natural variation of gene expression, a mutation caused by urethane, or p53 loss itself.

We also observed a bias towards a low mutant:wildtype mRNA ratio in Q₆₁-mutant Kras alleles. Such a finding is consistent with this mutation inducing high oncogenic signaling, and hence a selection against increased expression. Interestingly, this ratio was low irrespective of the p53 status. Perhaps there is an upper limit to oncogenic signaling, even in the absence of p53, that is tumor-promoting. In support, we note that G_12/13-mutant tumors with a high mutant:wildtype mRNA ratio had the same Ras signaling output as Q₆₁-mutant tumors, at least as assessed by the expression level of the three tested genes downstream of Ras. Indeed, RAS-mutant human tumors, which often have disruption of p53 and other tumor suppressors, typically do not have BRAF or EGFR mutations and vice versa outside of drug resistance settings (Cancer Genome Atlas Research Network et al., 2018). Moreover, co-expressing oncogenic RAS with oncogenic BRAF or EGFR can be growth suppressive (Cisowski et al., 2016; Unni et al., 2018).

In conclusion, multiple independent lines of investigation support a model whereby the sensitivity of a normal cell to a narrow range of oncogenic signaling dictates the nature of the mutation conducive to tumor initiation. Too low, a mutation fails to induce proliferation, as supported by the finding that less active G₁₂ mutants are rarely recovered in the Kras^nat allele unless accompanied by an allelic imbalance permitted or fostered by the absence of p53. Too high, a mutation induces arrest, as supported by the finding that more active Q₆₁ mutants are not recovered from the more highly expressed Kras^ex3op allele unless oncogene-induced senescence is suppressed by the loss of p53 (Figure 5). Moreover, this selection pressure exceeds the mutational bias of the mutagen, as supported by the observations that G₁₂ mutations fail to match the urethane mutational signature, yet nevertheless arise at a high frequency in urethane-induced tumors in a Kras^ex3op background. Such findings speak to the extreme mutational bias of urethane-induced tumors, and perhaps more broadly, provide mechanistic insight into the RAS mutation tropism observed in human cancers.

All raw sequencing data has been deposited to NCBI Sequence Read Archive (SRA) under accession number PRJNA663179.

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Author details

1. Siqi Li

   Pharmacology and Cancer Biology, Duke University, Durham, United States

   Present address

   Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

2. Christopher M Counter

   Pharmacology and Cancer Biology, Duke University, Durham, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Visualization, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   count004@mc.duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0748-3079

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