Genetically manipulating endogenous Kras levels and oncogenic mutations in vivo influences tissue patterning of murine tumorigenesis.

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.

Treatment with the nitric oxide synthase inhibitor L-NAME provides a survival advantage in a mouse model of Kras mutation-positive, non-small cell lung cancer.

Oncogenic mutations in the gene KRAS are commonly detected in non-small cell lung cancer (NSCLC). This disease is inherently difficult to treat, and combinations involving platinum-based drugs remain the therapeutic mainstay. In terms of novel, pharmacologically actionable targets, nitric oxide synthases (NOS) have been implicated in the etiology of KRAS-driven cancers, including lung cancer, and small molecular weight NOS inhibitors have been developed for the treatment of other diseases. Thus, we evaluated the anti-neoplastic activity of the oral NOS inhibitor L-NAME in a randomized preclinical trial using a genetically engineered mouse model of Kras and p53 mutation-positive NSCLC. We report here that L-NAME decreased lung tumor growth in vivo, as assessed by sequential radiological imaging, and provided a survival advantage, perhaps the most difficult clinical parameter to improve upon. Moreover, L-NAME enhanced the therapeutic benefit afforded by carboplatin chemotherapy, provided it was administered as maintenance therapy after carboplatin. Collectively, these results support the clinical evaluation of L-NAME for the treatment of KRAS mutation-positive NSCLC.

Rare codons capacitate Kras-driven de novo tumorigenesis.

The KRAS gene is commonly mutated in human cancers, rendering the encoded small GTPase constitutively active and oncogenic. This gene has the unusual feature of being enriched for rare codons, which limit protein expression. Here, to determine the effect of the rare codon bias of the KRAS gene on de novo tumorigenesis, we introduced synonymous mutations that converted rare codons into common codons in exon 3 of the Kras gene in mice. Compared with control animals, mice with at least 1 copy of this Kras(ex3op) allele had fewer tumors following carcinogen exposure, and this allele was mutated less often, with weaker oncogenic mutations in these tumors. This reduction in tumorigenesis was attributable to higher expression of the Kras(ex3op) allele, which induced growth arrest when oncogenic and exhibited tumor-suppressive activity when not mutated. Together, our data indicate that the inherent rare codon bias of KRAS plays an integral role in tumorigenesis.

Rare codons regulate KRas oncogenesis.

Oncogenic mutations in the small Ras GTPases KRas, HRas, and NRas render the proteins constitutively GTP bound and active, a state that promotes cancer. Ras proteins share ~85% amino acid identity, are activated by and signal through the same proteins, and can exhibit functional redundancy. Nevertheless, manipulating expression or activation of each isoform yields different cellular responses and tumorigenic phenotypes, even when different ras genes are expressed from the same locus. We now report a novel regulatory mechanism hardwired into the very sequence of RAS genes that underlies how such similar proteins impact tumorigenesis differently. Specifically, despite their high sequence similarity, KRAS is poorly translated compared to HRAS due to enrichment in genomically underrepresented or rare codons. Converting rare to common codons increases KRas expression and tumorigenicity to mirror that of HRas. Furthermore, in a genome-wide survey, similar gene pairs with opposing codon bias were identified that not only manifest dichotomous protein expression but also are enriched in key signaling protein classes and pathways. Thus, synonymous nucleotide differences affecting codon usage account for differences between HRas and KRas expression and function and may represent a broader regulation strategy in cell signaling.

RALA and RALBP1 regulate mitochondrial fission at mitosis.

Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission. Equal distribution of mitochondria to daughter cells during mitosis requires fission. Mitotic mitochondrial fission depends on both the relocalization of the large GTPase DRP1 to the outer mitochondrial membrane and phosphorylation of Ser 616 on DRP1 by the mitotic kinase cyclin B-CDK1 (ref. 2). We now report that these processes are mediated by the small Ras-like GTPase RALA and its effector RALBP1 (also known as RLIP76, RLIP1 or RIP1; refs 3, 4). Specifically, the mitotic kinase Aurora A phosphorylates Ser 194 of RALA, relocalizing it to the mitochondria, where it concentrates RALBP1 and DRP1. Furthermore, RALBP1 is associated with cyclin B-CDK1 kinase activity that leads to phosphorylation of DRP1 on Ser 616. Disrupting either RALA or RALBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B-CDK1 converge on RALA and RALBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.