Author response: Genetically manipulating endogenous Kras levels and oncogenic mutations in vivo influences tissue patterning of murine tumorigenesis
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Despite multiple possible oncogenic mutations in the proto-oncogene KRAS, unique subsets of these mutations are detected in different cancer types. As KRAS mutations occur early, if not being the initiating event, these mutational biases are ostensibly a product of how normal cells respond to the encoded oncoprotein. Oncogenic mutations can impact not only the level of active oncoprotein, but also engagement with proteins. To attempt to separate these two effects, we generated four novel Cre-inducible (LSL) Kras alleles in mice with the biochemically distinct G12D or Q61R mutations and encoded by native (nat) rare or common (com) codons to produce low or high protein levels. While there were similarities, each allele also induced a distinct transcriptional response shortly after activation in vivo. At one end of the spectrum, activating the KrasLSL-natG12D allele induced transcriptional hallmarks suggestive of an expansion of multipotent cells, while at the other end, activating the KrasLSL-comQ61R allele led to hallmarks of hyperproliferation and oncogenic stress. Evidence suggests that these changes may be a product of signaling differences due to increased protein expression as well as the specific mutation. To determine the impact of these distinct responses on RAS mutational patterning in vivo, all four alleles were globally activated, revealing that hematolymphopoietic lesions were permissive to the level of active oncoprotein, squamous tumors were permissive to the G12D mutant, while carcinomas were permissive to both these features. We suggest that different KRAS mutations impart unique signaling properties that are preferentially capable of inducing tumor initiation in a distinct cell-specific manner. Editor's evaluation This article addresses the long-standing question of why specific mutations in RAS are associated with tumors arising in distinct tissues; whether this reflects biological expression or functionality. The authors utilize a clever mouse genetic approach to modulate both which Kras mutation is expressed and how much of that Kras mutant is expressed, expressing the same Kras mutation at either a low level (due to rare codon utilization) or at a higher level (through common codon utilization). This study convincingly supports the conclusion that different KRAS mutations impart unique signaling properties that are preferentially capable of inducing tumor initiation in a distinct cell-specific manner. https://doi.org/10.7554/eLife.75715.sa0 Decision letter eLife's review process Introduction Why specific driver mutations track with different cancers is unknown, yet speaks to the very origins of cancer, with implications for early detection and prevention. This is particularly well illustrated with the RAS family of small GTPases, comprised of KRAS, NRAS, and HRAS. Focusing on KRAS, the most commonly mutated of the three (Prior et al., 2020), single-point mutations at one of three hotpot positions (G12, G13, and Q61) result in six possible substitutions that inhibit the intrinsic or extrinsic GTPase activity of the protein. These mutations render KRAS constitutively GTP-bound and active, which is well known to be oncogenic (Simanshu et al., 2017). Although this amounts to 18 possible oncogenic mutations, specific subsets tend to be found in different cancer types. For example, the most common oncogenic KRAS mutation is G12C in non-small cell lung cancer but Q61H in plasma cell myeloma (Prior et al., 2020). Mice similarly exhibit a bias of specific oncogenic Kras mutations towards different cancer types as different mutant Kras alleles have different tumorigenic potential when activated in different tissues (Li et al., 2018; Poulin et al., 2019; Winters et al., 2017; Wong et al., 2020; Zafra et al., 2020). Different oncogenic mutations also affect the ability of Nras to induce tumorigenesis in mice, as do the same mutations in different RAS isoforms (Burd et al., 2014; Haigis et al., 2008; Kong et al., 2016; Murphy et al., 2022). While this tissue 'tropism' of cancers towards specific RAS mutations has been appreciated for decades (Bos, 1989), the underlying mechanism is unclear. Variation in the ability of specific KRAS mutants to be tumorigenic (or not) in different tissues ostensibly results from differences in oncogenic signaling between mutants and the response of normal cells thereof. Generally speaking, oncogenic signaling is a product of the amplitude of the signal (quantitative signaling) and/or the pathways engaged (qualitative signaling). In terms of quantitative signaling, different mutations can exhibit different degrees of activation (GTP-loading) and/or different sensitivities to positive (Ras GTP exchange factors [RasGEFs]) or negative (RAS GTPase activating proteins [RasGAPs]) regulators (Gebregiworgis et al., 2021; Munoz-Maldonado et al., 2019; Simanshu et al., 2017). Various methods to manipulate quantitative Kras signaling, such as through modulating recombination rates (Singh et al., 2021), homozygous expression of the mutant allele (Wang et al., 2011), changing codon usage to increase protein expression (Pershing et al., 2015), additional pharmacological activation of the mitogen-activated protein kinase (MAPK) pathway (Cicchini et al., 2017), and so forth (Li et al., 2018), all affect tumorigenesis. On the other hand, perhaps one of the best examples of qualitative differences in KRAS signaling is the G12R mutant. Unlike the more canonical G12D mutation, the G12R mutant exhibits reduced the RAS effector PI3Kα binding and PI3K/AKT signaling (Hobbs et al., 2020). Activating an inducible KrasLSL-G12R allele in the pancreas led to fewer early premalignant lesions compared to the much more tumorigenic KrasLSL-G12D allele (Zafra et al., 2020). Despite an appreciation that different KRAS mutations can manifest in quantitative or qualitative signaling differences, how each contributes to the mutational patterns of this oncogene in cancer is unclear. KRAS mutations are often initiating, being sufficient to induce tumorigenesis in mice and truncal in many human cancers. Thus, the bias of specific cancers towards distinct KRAS mutations could arise from potential tissue-specific differences of normal cells in the mutagenic process or repair capacity and/or in their responses to the nature of the signaling imparted by specific KRAS mutations (Li et al., 2018). In regard to the latter, determining the immediate response of normal cells to different KRAS mutations in vivo may help elucidate the mutational patterning of this oncogene. Identifying an oncogenic mutation arising in the KRAS gene from the cell-of-origin prior to becoming a tumor is challenging in humans. Mice, on the other hand, provide an ideal model system to experimentally explore this phenomenon as the point of tumor initiation can be precisely defined using inducible oncogenic Kras alleles. To therefore explore why different cancer types have a bias towards specific KRAS mutations, we created four novel inducible murine Kras alleles with different oncogenic mutations that were expressed at either low or high levels. In this way, we hypothesize that tissues in which tumor initiation is driven by the level of oncogenic signaling would preferentially develop tumors at higher protein levels while those driven by a specific mutant would preferentially develop tumors with just one of the two mutants. We chose two completely different oncogenic mutations for these alleles, G12D and Q61R. G12D places a negatively-charged headgroup into the catalytic cleft of RAS and blocks extrinsic (RasGAP-mediated) GTPase activity (Parker et al., 2018). On the other hand, Q61R replaces the catalytic amino acid with one that has a positively-charged headgroup, disrupting the position of the active site water molecule necessary for intrinsic GTP hydrolysis (Buhrman et al., 2010). Q61 is also essential for extrinsic GTP hydrolysis, as it stabilizes the transition state via hydrogen bonds to the γ-phosphate and nucleophilic water while providing another hydrogen bond to the RasGAP arginine finger (Grigorenko et al., 2007; Kötting et al., 2008; Rabara et al., 2019; Scheffzek et al., 1997). Comparing these two mutants directly reveals Q61R to have significantly lower GTP exchange and hydrolysis rates than G12D in Nras (Burd et al., 2014) and reduced RasGAP-mediated GTP hydrolysis rates in Kras (Rabara et al., 2019), akin to other substitutions at these two positions (Gebregiworgis et al., 2021; Smith et al., 2013). In those few cases in which the tumorigenic potential of the G12D and Q61R mutants of the same Ras isoform has been directly compared in mice, tissue-specific expression of the G12D mutant was less oncogenic than Q61R in both Nras-induced melanoma (Burd et al., 2014) and Kras-induced myeloproliferative neoplasm (Kong et al., 2016). To alter the expression of these two mutants, the first three coding exons of Kras were fused and encoded by either their native rare codons, which is known to retard Kras protein expression, or common codons to increase Kras protein expression (Ali et al., 2017; Fu et al., 2018; Lampson et al., 2013; Pershing et al., 2015). We chose the novel approach of altering mammalian codon usage to modulate protein expression in vivo (Li and Counter, 2021; Pershing et al., 2015; Sasine et al., 2018) as no additional elements are required to change protein levels, providing a simple, reproducible, and uniform way of modulating Kras levels in mice and derived cell lines. These four alleles were activated and immediately thereafter the in vivo transcriptome was determined in the lungs. This revealed that while these four alleles certainly shared similarities, each also induced unique transcriptional responses. Increased expression shifted the transcriptional hallmarks from those indicative of an expansion of multipotent cells to that of hyperproliferation and oncogenic stress. Changing the mutation type shifted the hallmark of estrogen response in the G12D mutant to that of the p53 pathway and DNA repair in the Q61R mutant. All four alleles were then globally activated, revealing that hematolymphopoietic lesions were permissive to the level of active oncoprotein, squamous tumors were preferentially permissive to the G12D mutant, while carcinomas tended to be permissive to both these changes. These findings support tissue-specific responses to the degree and type of signaling imparted by different oncogenic mutations molds the tissue tropism of cancers towards specific oncogenic KRAS mutations. Results A panel of inducible oncogenic Kras alleles designed to separate the effects of mutation type from the activity level of the oncoprotein To explore how specific cancers are driven by distinct KRAS mutations, we reasoned that effects unique to a mutation could be separated from those imparted by activation level by simply comparing two different oncogenic mutants expressed at high or low levels. To this end, we created the four novel Cre-inducible oncogenic Kras alleles KrasLSL-natG12D, KrasLSL-natQ61R, KrasLSL-comG12D, and KrasLSL-comQ61R (Figure 1A). Each began with an LSL transcriptional/translational repressor sequence (STOP) flanked by loxP sites (Jackson et al., 2001) engineered into the intron of Kras following the first non-coding exon to provide temporal and spatial control of gene expression. This was followed by a fusion of the first three coding exons encoded by either their native (nat) rare codons, which are known to retard protein expression, or 93 of these rare codons converted to their common (com) counterparts to increase protein expression (Figure 1—figure supplement 1 and Figure 1—source data 6). Each version contained either a G12D or Q61R mutation. As noted above, these two mutants alter RAS activity in a biochemically different manner (Munoz-Maldonado et al., 2019; Simanshu et al., 2017), with Q61R reported to yield higher levels of active (GTP-bound) Ras (Burd et al., 2014; Kong et al., 2016; Pershing et al., 2015). This was followed by the next intron containing an FRT-NEO-FRT cassette for ES selection, which was excised via Flp-mediated recombination in the resultant animals, after which the Flp transgene was outbred. Finally, the remainder of the gene was left intact so as to generate the two Kras4a and Kras4b isoforms, as both contribute to tumorigenesis (To et al., 2008), potentially through unique protein interactions (Amendola et al., 2019). Figure 1 with 6 supplements see all Download asset Open asset Conditional KrasLSL alleles with different oncogenic mutations and codon usage. (A) Schematic of generating and activating KrasLSL alleles with the first three coding exons fused and encoded by native (nat) versus common (com) codons with either a G12D or Q61R mutation. (B) PCR genotyping of two independently derived mouse embryonic fibroblast (MEF) cultures (two biological replicates) with the indicated Kras alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) and the unrecombined (KrasLSL*, 389 bp) and recombined (LoxP recombined, 616 bp) KrasLSL allelic products. Gel images were cropped and color inverted for optimal visualization. Full-length gel images are provided in Figure 1—source data 2. (C) Expression levels, determined by immunoblot with an anti-Kras antibody, RAS activity levels, determined by RBD pull-down (RBD-PD) of lysates collected from MEF cells derived from mice with the indicated Kras alleles in the presence of Cre recombinase (CRE). MEF cultures derived from KrasLSL-natG12D/+ mice in the absence of Cre recombinase were used as negative control. MEF cultures were either serum starved overnight (starved) or serum starved overnight followed by serum stimulation for 5 min (stimulated). 20% of the elute from RBD-PD and 30 μg total protein from the total cell lysates were loaded. Tubulin serves as loading control. One of two biological replicates; see Figure 1—figure supplement 3 for the second biological replicate. Full-length gel images are provided in Figure 1—source data 3. Figure 1—source data 1 Full-length gel images of RBD pull downs. Full-length gel images from RAF1-RBD pull downs (top) and whole-cell lysates (bottom) from HEK-HTs ectopically expressing engineered Kras constructs shown in Figure 1—figure supplement 2. https://cdn.elifesciences.org/articles/75715/elife-75715-fig1-data1-v1.png Download elife-75715-fig1-data1-v1.png Figure 1—source data 2 Full-length gel image of genotyping of mouse embryonic fibroblast (MEF) cultures derived from the KrasLSL alleles in Figure 1B. PCR genotyping of two independently derived MEF cultures with the indicated KrasLSL alleles in the absence and presence of Cre recombinase (CRE) to detect the unaltered wild-type Kras allele product (WT, 488 bp) as well as the unrecombined (KrasLSL, 389 bp) and recombined (LoxP recombined, 616 bp) Kras allelic products. Gel images were color inverted for better visualization. Red box depicts region shown in Figure 1B. https://cdn.elifesciences.org/articles/75715/elife-75715-fig1-data2-v1.zip Download elife-75715-fig1-data2-v1.zip Figure 1—source data 3 Full-length gel images of RBD pull-downs in MEFs. Full-length gel images from RBD pull-downs (left) and whole-cell lysates (right) from MEFs derived from KrasLSL alleles with serum starvation, or serum starvation followed by serum stimulation shown in Figure 1C, and same conditions with a second clone of MEF cultures with serial dilutions of 500 μg lysate (left) and 200 μg lysate (right) used for RBD-PD as shown in Figure 1—figure supplement 3. Red box depicts regions shown in Figure 1C and Figure 1—figure supplement 3. https://cdn.elifesciences.org/articles/75715/elife-75715-fig1-data3-v1.png Download elife-75715-fig1-data3-v1.png Figure 1—source data 4 Full-length gel images of Kras expression and activity using RBD pull-downs from lung tissue. Full-length gel images from RAF1-RBD pull-downs (RBD-PD, top) and whole-cell lysates (bottom) of lungs from mice with KrasLSL alleles seven days after tamoxifen injection as shown in Figure 1—figure supplement 4A. Immunoblots of two separate pull downs from two biological replicates are shown as in Figure 1—figure supplement 4B. Red box depicts the regions shown in Figure 1—figure supplement 4B. https://cdn.elifesciences.org/articles/75715/elife-75715-fig1-data4-v1.png Download elife-75715-fig1-data4-v1.png Figure 1—source data 5 Ct values from the qRT-PCR analysis in Figure 1—figure supplement 5. https://cdn.elifesciences.org/articles/75715/elife-75715-fig1-data5-v1.xlsx Download elife-75715-fig1-data5-v1.xlsx Figure 1—source data 6 Sequence of coding exons 1 to 3 of the four KrasLSL alleles. Sequence alignment of the codons in the coding exons 1 to 3 of the indicated KrasLSL alleles in comparison to the murine wild-type sequence (Kras). Top: amino acid sequence. Red nucleotides: optimized codons. Green highlight: G12D mutation. Blue highlight: Q61R mutation. https://cdn.elifesciences.org/articles/75715/elife-75715-fig1-data6-v1.xlsx Download elife-75715-fig1-data6-v1.xlsx To confirm that the expression and activity of these four versions of oncogenic Kras was reflected in their design, we ectopically expressed the corresponding FLAG-tagged murine Kras cDNAs in HEK-HT cells that critically depend upon oncogenic RAS for tumorigenesis (Hahn et al., 1999). The total amount of ectopic Kras protein was then determined by immunoblot with an anti-FLAG antibody, and the level of GTP-bound and active Kras was determined by pull-down with a Ras binding domain (RBD) peptide followed by immunoblot or ELISA. Based on common codons increasing protein expression and Q61R leading to higher levels of GTP-bound RAS, the four constructs display the expected stepwise increase in RBD-bound Kras in the ascending order of KrasnatG12D<KrasnatQ61R <KrascomG12D<KrascomQ61R in both assays (Figure 1—figure supplement 2, Figure 1—source data 1). The effect of codon usage and mutation type on Kras activity in derived murine cells To evaluate the endogenous functionality of this allelic set, we derived and characterized two separate mouse embryonic fibroblast (MEF) cultures from each of the genotypes KrasLSL-natG12D/+, KrasLSL-natQ61R/+, KrasLSL-comG12D/+, and KrasLSL-comQ61R/+, and validated that all four alleles could be recombined upon the expression of Cre recombinase (Figure 1B, Figure 1—source data 2). We then confirmed that this allelic set yields an increase in endogenous Kras activity consistent with codon usage and mutation type. These pairs of MEF cultures expressing Cre were either serum starved or stimulated to assess basal or stimulated Kras activity, respectively. RBD pull-down followed by immunoblot demonstrated the stepwise increase in Kras expression and activity levels in the ascending order of KrasnatG12D<KrasnatQ61R <KrascomG12D<KrascomQ61R in both conditions (Figure 1C, Figure 1—figure supplement 3, Figure 1—source data 3). Thus, endogenous expression of these four alleles revealed that the Q61R mutant is more active than the G12D mutant, and changing rare codons to common increases the expression and activity of endogenous Kras oncoprotein. The effect of codon usage and mutation type on Kras activity in vivo To validate these findings in vivo, we crossed the KrasLSL-natG12D/+, KrasLSL-natQ61R/+, KrasLSL-comG12D/+, and KrasLSL-comQ61R/+ genotypes into a Rosa26CreERT2/+ background, which expresses a tamoxifen-inducible Cre from the endogenous Rosa26 promoter that is active in a broad spectrum of tissues (Ventura et al., 2007). Two adult mice from each of the four derived cohorts, as well as the control strain (Rosa26CreERT2/+), were injected with tamoxifen, and seven days later humanely euthanized, their lungs removed, and duplicate samples of derived protein lysates subjected to RBD pull-down followed by immunoblot (Figure 1—figure supplement 4A). As in the case of the MEF cultures, we observed a stepwise increase in Kras expression and activity consistent with the codon usage and oncogenic mutation in this allelic set (Figure 1—figure supplement 4B C, Figure 1—source data 4). To assess whether this was reflected in the expression of downstream target genes, we generated the same four cohorts of mice, but in this case isolated RNA from the lungs of euthanized mice seven days after tamoxifen injection and determined the level of mRNA encoded by five known Ras target genes by qRT-PCR (Figure 1—figure supplement 5A). This revealed the expected increase in three such transcripts when Kras was encoded with common codons, which was further increased in the Q61R-mutant background (Figure 1—figure supplement 5B C, Figure 1—source data 5). To further validate this effect at the protein level, we performed reverse-phase protein array (RPPA) analysis of lung tissue upon activation of the two extreme alleles, KrasLSL-natG12D versus KrasLSL-comQ61R in the Rosa26CreERT2/+ background, with Rosa26CreERT2/+ serving as a control. Three mice from each genotypes were injected with tamoxifen and seven days later euthanized, their lungs removed, embedded, and derived protein lysates subjected to RPPA analysis. When normalized to the control tissue, there was a clear increase in the level of phosphorylated proteins of the MAPK pathway and downstream transcription factors upon activating the KrasLSL-comQ61R allele compared to activating the KrasLSL-natG12D allele (Figure 1—figure supplement 6, Supplementary file 1). Thus, the increase in Kras activity imparted by the Q61R mutant or by common codons observed in cultured cells is recapitulated in lung tissue. The effect of codon usage and mutation type on Kras biological activity in vivo We next assessed whether these alleles were proportionally tumorigenic in a side-by-side comparison in the same organ. Each of these four alleles were crossed into a CC10CreER/+ (and Rosa26CAG-fGFP/+) background, and the resultant mice were injected with tamoxifen to specifically induce Cre-mediated recombination in the lung, after which every month thereafter for 6 months, five mice from each of the four cohorts were euthanized (Figure 2—figure supplement 1A). The lungs from all mice were visually analyzed for the presence of surface pulmonary tumors (Figure 2—figure supplement 1B), and in addition, two H&E-stained sections from pairs of mice were assayed for the presence and type of pulmonary tumors by a veterinarian pathologist to the (Figure This revealed a stepwise increase in and tumor of pulmonary lesions in with the increased activity of the in the ascending order of KrasnatG12D<KrasnatQ61R <KrascomG12D<KrascomQ61R (Figure These tumorigenic were the result of activating the inducible Kras alleles in the lungs as five control type mice in the same background that were injected with tamoxifen to develop tumors after months, the of the study (Figure 2—figure supplement 1C, Finally, we in a few that the alleles preferentially lesions were more with the Q61R-mutant alleles (Figure 2—figure supplement We that this allelic set exhibits an increase in tumorigenic potential consistent with the activity of the encoded but potentially may also display of effects on tumorigenesis. Figure 2 with 1 supplement see all Download asset Open asset effect upon activating each oncogenic Kras (A) of H&E-stained lung sections and (B) the tumor from analysis of two lung sections from five mice with the indicated KrasLSL alleles in a background at each of the indicated The response of lung tissue to Kras alleles with different codon usage or mutations in vivo As the different tumor types and that upon activation of each of these four inducible oncogenic Kras alleles are a product of tumor these effects in how cells immediately respond to these different We compared the immediate transcriptional as a of the changes upon activation of each in the of three adult mice from each of the four inducible oncogenic Kras alleles in Rosa26CreERT2/+ background were injected with tamoxifen, and seven days later the were euthanized, their lungs removed, and RNA was isolated for transcriptome (Figure supplement 1A). The resultant a increase in the of genes expressed with the level of active Kras assessed by the RBD pull-down in lung we and genes were expressed change than 2, and less than upon activation of the KrasLSL-natG12D, KrasLSL-natQ61R, KrasLSL-comG12D, and KrasLSL-comQ61R alleles, compared to the wild-type Kras allele (Figure Figure supplements Figure data 1). analysis of these four revealed that the transcriptome upon activating both alleles encoded by native rare codons from both alleles encoded with common the G12D mutant was most distinct from the Q61R mutant in the background (Figure supplement when two mutants were the of expressed genes specific to each allele increased from the G12D to the Q61R mutant when Kras was encoded with common codons more than it when encoded with native rare codons (Figure supplement 6, Figure data 1). Comparing the of the two Kras alleles encoded with native rare versus common codons revealed that the two alleles KRAS hallmarks while the two alleles induced signaling to oncogenic such as an increase in signaling, and hallmarks and a in and hallmarks (Figure Figure supplement 1C, Figure data 2). We this as expression level the degree of oncogenic signaling in this allelic Comparing the G12D versus Q61R revealed both alleles induced the hallmark while the Q61R-mutant alleles induced DNA and and that there is a response activated by the Q61R-mutant alleles (Figure Figure supplement 1C, Figure data 2). Figure 3 with supplements see all Download asset Open asset transcriptome induced by each oncogenic Kras (A) of the change versus of the genes expression in each allele compared to the wild-type Kras allele in the are provided in Figure supplements and patterns of the indicated hallmarks by Kras codon usage (B) and mutation type hallmarks with a are is to RAS activity for better visualization. All hallmarks upon activating KrasLSL alleles with an are provided in Figure supplement analysis of the genes in the hallmarks in analysis of the lungs of versus mice seven days after tamoxifen Figure data 1 expressed genes in each allele from Figure supplements Download Figure data 2 of the hallmarks in Figure supplement Download Figure data 3 Ct values from the qRT-PCR analysis in Figure supplement Download To validate the transcriptional responses detected by we four to six genes in a of Two adult mice from each of the four cohorts and control mice were injected with tamoxifen as above, and seven days later the were euthanized, their lungs removed, RNA and the level of transcripts determined by We expression patterns to the transcriptome in the hallmarks of via and which increase with Kras activity
Source: eLife
Institutions: Duke Medical Center, George Mason University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4294904243