p53 dosage impedes Kras G12D - and Kras Q61R -mediated tumorigenesis

25 Mice engineered with G12D versus Q61R mutant of Kras exhibit differences in the number 26 and grade of tumors. Namely, the incidence or grade of oral or forestomach squamous epithelial 27 lesions was more prevalent in the Kras G12D background while hematolymphopoietic disease was 28 more prevalent in the Kras Q61R background. Loss of the Trp53 gene encoding p53 enhances the 29 ability of oncogenic Kras to initiate tumorigenesis in carcinogen and genetic models of lung 30 cancer, while an extra copy of Trp53 ( Super p53 ) was recently shown to suppress Kras-induced 31 tumorigenesis in a genetic model of this disease. Given this, we evaluated whether such an extra 32 copy of Trp53 would alter tumorigenesis upon global activation of a modified Kras allele 33 engineered with either a G12D or Q61R mutation. We report that an increase in p53 dosage 34 generally reduced tumor number or grade across a number of organs in a manner largely 35 independent of the type of Kras mutation, which was sufficient to extend lifespan in the less 36 aggressive background of a Kras G12D initiating mutation.


Introduction
The RAS superfamily of small GTPases is comprised of the genes KRAS, NRAS, and HRAS in humans (Kras, Nras, and Hras in mice) [1].Activated growth factor receptors are known to lead to the switching of RAS from an inactive GDP-bound to an active GTP-bound state.Once activated, RAS recruits proteins with RAS-binding domains or Ras-associating domains, thereby activating these proteins to propagate signaling.Hydrolysis of GTP returns RAS to the inactive GDP-bound state, terminating the signaling [2].However, in a fifth or more of human cancers, point mutations at one of the three hotspot sites render RAS constitutively active, which is well established to initiate tumorigenesis [1][2][3][4].Between the three RAS genes, three hotspots, with six possible substitutions there are more than 50 potential oncogenic mutations, yet cancers tend to have a particular bias, or a 'RAS mutation tropism', towards an often unique subset of these mutations [5].Case in point, the most common RAS mutations in lung cancer are G12C/V/D in KRAS [6], but Q61R/K in NRAS in melanoma [7].Expression of different RAS mutants in mice can also affect the nature of tumors arising.
For example, expressing a Kras G12D , but not an Nras G12D allele in the skin of mice leads to melanoma [8].Three oncogenic Kras mutations were more commonly detected in tumors arising in mice infected with an sgRNA library designed to generate all 12 oncogenic mutations at codons G12 and G13 [9].We recently created Cre-inducible (lox-STOP-lox or LSL) Kras alleles encoded by common (com) codons and either of the two biochemically distinct mutations G12D or Q61R.
Globally activating these two via Cre expressed from the ubiquitous Rosa26 locus led to differences in the severity and in some cases even type of tumors arising in a manner specific to mutation type.Namely, the incidence or grade of oral or forestomach squamous epithelial lesions was more prevalent in the Kras G12D background while hematolymphopoietic disease was more prevalent in the Kras Q61R background [10].
The patterns of tumors induced by these two alleles is ostensibly a product of how a normal cell responds to different activation levels or specific mutant oncoproteins.In this regard, oncogenic RAS can both induce proliferation or senescence, the latter of which is mediated in part by the tumor suppressor p53 [11].Recently, "super p53" mice encoding an extra copy of Trp53 [12] were shown to suppress spontaneous Kras-driven lung and lymphoma tumorigenesis arising in an LA1-Kras G12D background, but not radiation-induced lymphomas [13].This prompted us to ask whether an increase in p53 dosage would alter the severity or type of tumors arising between different oncogenic mutations.We thus generated mice with the two experimental genotypes of Kras LSL-comG12D/+ ;Super p53 and Kras LSL-comQ61R/+ ;Super p53 and the two control genotypes of Kras LSL-comG12D/+ and Kras LSL-comQ61R/+ in the Rosa26 CreERT2/+ background, which allows ubiquitous Cre expression in a broad range of tissues [14].The Kras LSL alleles were activated by tamoxifen treatment and the mice were analyzed for the presence of hematolymphopoietic, oral and forestomach, and lung tumors, as well as tumors in other organs.We report here that an increase in p53 dosage generally suppressed tumor number or type across several organs, largely independent of the type of Kras mutation, which was sufficient to extend lifespan in the less aggressive Kras G12D background.This implies that regardless of whether a tissue is preferentially induced by the level or type of mutant of Kras, an extra copy of Trp53 may suppress initiation and/or progression of the resulting tumors.

Results
The experimental design to explore the influence of p53 dosage on

Fig 1. The effect of p53 dosage on Kras-driven tumorigenesis. (A)
Experimental design to study the effect of the Super p53 allele on tumorigenesis upon globally activating Kras LSL-comG12D versus Kras LSL-comQ61R alleles by tamoxifen in a Rosa26 CreER2/+ background.(B) Kaplan-Meier survival curve of Rosa26 CreER2/+ mice with the indicated genotypes (0 days refers to the last tamoxifen injection).
Dotted lines: 50% survival.Pairwise comparison for statistical analysis is provided in S2 Fig.
Examples of H&E-stained slides of the indicated tissues are provided in S3 Fig.

An increase in p53 dosage provides an overall survival benefit in mice in which Kras G12D is globally activated
The time of moribundity, which was previously shown to precede death due to cancer [10], was plotted for all four cohorts using the Kaplan-Meier method [15].We previously reported the median survival of Kras comG12D/+ and Kras comQ61R/+ mice after tamoxifen injections was 14 and 51 days, respectively, in the Rosa26 CreERT2/+ background [10].With increased p53 dosage, the median survival was unchanged in the Kras comQ61R/+ cohort but increased by 23 days (51 to 74 days) in the Kras comG12D/+ mice, which equates to 45% increase in survival (Fig allele.

An increase in p53 dosage alters the severity of tumorigenesis across multiple organs
To examine the effect of an increase in p53 dosage on tumorigenesis, the lung, thymus, stomach, pancreas, spleen, oral epithelia, liver, and kidney were removed from five to eight mice from each of the four cohorts and paraffin embedded and H&E stained (Fig 1A).Two slides from each of these eight organs from all animals in the study were analyzed for the incidence and grading of tumors by a board-certified veterinary pathologist (S3 Fig) .In terms of the number of mice with tumors in these organs, we find, not surprisingly, that organs previously shown to be resistant to Kras-induced tumorigenesis [10], namely the kidney, liver, and pancreas, remained to be determined.Thus, the additional copy of Trp53 generally reduced the incidence of tumors detected in a wide spectrum of organs in a manner largely independent of mutation type.

Oral and forestomach squamous epithelial lesions
We previously reported that oral and forestomach squamous epithelial lesions were more prevalent and aggressive in a Kras LSL-comG12D/+ compared to a Kras LSL-comQ61R/+ genotype, suggesting that these two tissues were particularly sensitive to the ability of the G12D mutant of Kras to induce tumorigenesis [10].To explore the impact of an extra allele of Trp53 on the tumorigenesis in these two tissues, the aforementioned H&E-stained slides were analyzed and graded as either mild, moderate, or marked atypical squamous hyperplasia (ASH) or as squamous papilloma.In

Hematolymphopoietic lesions
We previously reported that hematolymphopoietic disease was more severe either through increased Kras expression or in the Kras comQ61R/+ mutant background, suggesting that this tissue was particularly sensitive to the level of active Kras to induce tumorigenesis [10].Given this, we compared the number of mice with myeloproliferative disease as well as the number and grade of mice with lymphomas.Specifically, the aforementioned H&E slides of all tissues from each of the four genotypes were analyzed for the presence of hematolymphopoietic lesions.As previously reported, the myeloproliferative disease was only detected in mice with Q61R mutation [10] and the percentage of animals with myeloproliferative disease decreased from

Lung lesions
Upon comparing the incidence and grading of the lesions in the lungs of all four cohorts, we found that with increased p53 dosage, the severity of the peripheral lesions was reduced in both Kras comG12D/+ and Kras comQ61R/+ mice.Specifically, with extra copy of Trp53, percentage of animals with peripheral hyperplasia decreased from 100% to 87% in the Kras comG12D/+ background, while peripheral hyperplasia were actually absent in the Kras comQ61R/+ background (Fig 4A).However, there was no difference in other types of lung tumors (bronchiolar hyperplasia and adenomas), other than an increase in bronchiolar hyperplasia in the Kras comG12D/+ background (S5A,B Fig).
Thus, the addition of an extra copy of Trp53 appeared to have the greatest effect on peripheral lung hyperplasia.

Discussion
We report that increasing p53 dosage extended lifespan in Rosa26 CreERT2/+ ;Kras LSL-comG12D/+ mice by one statistical comparison, and this was associated with a decrease in the incidence and/or grade of lesions in the four organs pathologically analyzed, namely the oral cavity, forestomach, hematolymphopoietic compartment, and lung (except for lung bronchiolar hyperplasia).An additional copy of Trp53 did not, however, extend lifespan in the Rosa26 CreERT2/+ ;Kras LSL-comQ61R/+  mice.Nevertheless, the number of these mice without lesions in the oral cavity, forestomach, and hematolymphopoietic compartment increased, and peripheral hyperplasia in the lung were entirely absent.While the similar lifespan between Kras LSL-comQ61R/+ and Kras LSL-comQ61R/+ ;Super p53 mice may reflect a mutation-specific effect, we favor a model whereby the median survival of 2 weeks is simply too short for an extra copy of Trp53 to alter lifespan.We also note that the effect of an extra copy of Trp53 did not always suppress tumorigenesis.For example, the percentage of animals with lung tumors in the Kras comQ61R/+ genotype or hematolymphopoietic cancers in the We note the following caveats to this study.First, due to the breeding strategy, the number of littermates with the control genotypes Rosa26 CreERT2/+ ;Kras LSL-comG12D/+ or Rosa26 CreERT2/+ ;Kras LSL-comQ61R/+ was extremely limited, and thus we chose to make comparisons to the control genotypes reported in our previous study as all experimental steps were performed in the same manner.Thus, some trends may be the product of the natural variation in tumorigenesis rather than a bona fide phenotypic difference.However, the reduction in the number and/or grade of tumors was a reoccurring theme in the Super p53 background, supporting this as a real phenomenon.Second, we have not measured the level of p53 protein in the Trp53 +/+ versus Super p53 backgrounds to confirm an increase in the latter is linked to the observed phenotypes.However, by the very nature of providing an extra copy of this gene, p53 protein was previously demonstrated to increase with genotoxic insult in mouse embryonic fibroblasts with subsequent decrease in cell cyle arrest [12].Third, we appreciate that the Kras LSL- comG12D/+ and Kras LSL-comQ61R/+ alleles have altered the normal structure of the Kras gene, and hence limit the degree that these two alleles can be compared to other modified Kras alleles.
Nevertheless, by using two alleles constructed in the identical fashion, meaningful comparisons could be made.In summary, with these exceptions and caveats, the extra copy of Trp53 appears to be generally suppressive regardless of the type of oncogenic Kras mutation used to initiate tumorigenesis, although the effect was clearly greater when tumorigenesis is less aggressive.
While admittedly very speculative, we suggest that given these findings, perhaps then naturally occurring variations in the level of p53 protein in the human population may influence the likelihood that a spontaneously arising KRAS mutation initiates tumorigenesis, which may find value in predicting susceptibility to Kras-driven cancers or in development of therapeutic strategies against KRAS-induced cancers.

Mouse strains
Kras LSL-comG12D/+ and Kras LSL-comQ61R/+ mice were previously described and were from a pure 129 background [10].Super p53 mice were kindly provided by David Kirsch (University of Toronto) and were from C57BL/6 background.Rosa26 CreERT2/CreERT2 mice were obtained from The Jackson Laboratory (strain #008463).All animals derived in subsequent crosses were selected by genotyping using the genotyping methods previously described [10,12].All animal experiments were approved by Duke IACUC.
of control animals in the study to a similar number in the experimental cohort to make statistical power evenly distributed across cohorts.Thus, tumorigenesis comparisons were made against 5 to 6 randomly chosen mice from the control cohorts with random distribution of males and females in Kras LSL-comQ61R/+ and Kras LSL-comG12D/+ genotypes in the Rosa26 CreERT2/+ background [10].
Tumorigenesis studies were performed in the same manner as reported previously (Fig 1A) [10].
Namely, tamoxifen (Sigma-Aldrich, T5648-5G, CAS# 10540-29-1) was dissolved in corn oil (Sigma-Aldrich, C8267) and filter sterilized.At six to eight weeks of age, experimental cohorts of 7 to 9 mice with random distribution of males and females in Kras LSL-comG12D/+ ;Super p53 and Kras LSL- comQ61R/+ ;Super p53 genotypes were injected intraperitoneally with 250 μg/g body weight of tamoxifen four times every 48 hrs.Tamoxifen injections were performed under anestesia by isoflurane to prevent suffering.After recombination of the LSL cassette with tamoxifen, mice were observed daily during injections, 1 week after last injection, and weekly thereafter.Mice were humanely euthanized with carbon dioxide inhalation followed by removal of vital organs within 2 hrs of detection of moribundity humane endpoint.To prevent suffering and pain, we defined moribundity humane endpoint as any of the visible signs of sudden behaviroal change, poor/hunched posture, lost hair coat condition, sudden activity level change, painful facial expression, signs of pain that was not anticipated by the study plan, weight loss of exceeding 15% compared to an age-matched reference, and cardiopulmonary disorders.Selected tissues, including lung, thymus, stomach, pancreas, spleen, oral epithelia, liver, and kidney, were removed at necropsy followed by fixation in 10% formalin (VWR, 89370-094) for 24-48 hrs, then were stored in 70% ethanol (VWR, 89125-166) until routine processing. 2 animals from the Kras LSL-comQ61R/+ ;Super p53 genotype were found dead within 24 hrs of the first tamoxifen injection, thus, were not included in either of the reported survival or tumorigenesis studies as we could not perform necropsy in a timely manner.Tissues were sliced with one-to-twomillimeter thickness.Tissue slices were embedded in paraffin with the flat sides down, sectioned at a depth of 5 μm, and stained by the H&E method by IDEXX Laboratories.All H&E slides were evaluated by a board-certified veterinary pathologist with experience in murine pathology in a blinded fashion.

Statistical analysis
Statistical analyses were performed using GraphPad Prism software version 9. A p-value of less than 0.05 was considered statistically significant.
1B).Although pairwise comparisons did not show statistically significant differences between experimental and control cohorts using the Log rank test (S2A,B Fig), there was a statistically significant increase (p value <0.05) in the median survival of the Kras comG12D/+ mice with an extra allele of Trp53 using the Gehan-Breslow-Wilcoxon test (S2B Fig), which weighs early time points higher [16].Thus, an extra copy of p53 appears to extend lifespan in the context of the less aggressive Kras comG12D/+ tumor-free (Fig 1C).However, with two exceptions, there was a reduction in mice with oral, forestomach, lung, and hematolymphopoietic lesions in the both the Kras comG12D/+ and Kras comQ61R/+ mice in the Super p53 background (Fig 1C).The two exceptions were the number of animals with hematolymphopoietic and lung lesions was the same in Kras comG12D/+ and Kras comQ61R/+ mice, respectively, with versus without an extra copy of Trp53 (Fig 1C).Whether this reflects underlying biology or the limited number of mice with an extra copy of p53 remains both the Kras comG12D/+ and Kras comQ61R/+ background, increased p53 dosage reduced the severity of the oral squamous epithelial lesions (Fig2A).Namely, the percentage of animals with oral squamous epithelial lesions decreased with increased p53 dosage in the Kras comG12D/+ and Kras comQ61R/+ background by 20% and 40%, respectively.Furthermore, the percentage of mice with the Kras comG12D/+ alleles that had the most severely graded oral lesions of squamous papilloma decreased from 100% to 80% with an extra copy of Trp53, while there was a clear shift from more severe lesions of marked and moderate ASH to less severe lesions of mild ASH or no lesions in the Kras comQ61R/+ background with increased p53 dosage (Fig2A).The effect was similar in forestomach squamous epithelial lesions, although less pronounced (Fig2B).Specifically, the percentage of animals with forestomach squamous epithelial lesions decreased with increased p53 dosage in Kras comG12D/+ and Kras comQ61R/+ background by 25% and 40%, respectively.Furthermore, in both Kras comG12D/+ and Kras comQ61R/+ background, increasing p53 dosage led to a shift from more severe to less severe squamous epithelial lesion types in the forestomach, which was more noticeable in the Kras comQ61R/+ genotype (Fig2B).The observation that the number and grade of oral and forestomach squamous epithelial lesions decreased in the super p53 background supports the contention that an additional copy of Trp53 suppresses both tumor initiation and progression independent of the nature of the initiating Kras mutation.

Fig 2 .
Fig 2. The effect of p53 dosage on Kras-driven oral and forestomach tumorigenesis.Percent of

Fig 3 .
Fig 3.The effect of p53 dosage on Kras-driven hematolymphopoietic tumorigenesis.Percent of
5.1 (GraphPad Software).Pairwise comparisons of the survival plots of each cohort with and without an extra allele of Trp53 were performed with Log-rank (Mantel-Cox) and Gehan-Breslaw-Wilcoxon test[17] (S2B-C Fig).For comparisons of number of lesions per animal, one-way ANOVA with Bonferroni's multiple-comparisons test with a single pooled variance and a 95% CI were used (S5A-B Fig).