Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth.

Ras is mutated in up to 30% of cancers, including 90% of pancreatic ductal adenocarcinomas, causing it to be constitutively GTP-bound, and leading to activation of downstream effectors that promote a tumorigenic phenotype. As targeting Ras directly is difficult, there is a significant effort to understand the downstream biological processes that underlie its protumorigenic activity. Here, we show that expression of oncogenic Ras or direct activation of the MAPK pathway leads to increased mitochondrial fragmentation and that blocking this phenotype, through knockdown of the mitochondrial fission-mediating GTPase Drp1, inhibits tumor growth. This fission is driven by Erk2-mediated phosphorylation of Drp1 on Serine 616, and both this phosphorylation and mitochondrial fragmentation are increased in human pancreatic cancer. Finally, this phosphorylation is required for Ras-associated mitochondrial fission, and its inhibition is sufficient to block xenograft growth. Collectively, these data suggest mitochondrial fission may be a target for treating MAPK-driven malignancies.

Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo.

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.

Copper is required for oncogenic BRAF signalling and tumorigenesis.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

Tumour maintenance is mediated by eNOS.

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.

p53 dosage can impede KrasG12D- and KrasQ61R-mediated tumorigenesis.

Mice engineered with a G12D versus Q61R mutation in Kras exhibited differences in tumorigenesis. Namely, the incidence or grade of oral or forestomach squamous epithelial lesions was more prevalent in the KrasG12D background while hematolymphopoietic disease was more prevalent in the KrasQ61R background. Loss of the Trp53 gene encoding the tumor suppressor p53 enhances the ability of oncogenic Kras to initiate tumorigenesis in carcinogen and genetic models of lung cancer. Conversley, an extra copy of Trp53 (Super p53) was recently shown to suppress Kras-induced tumorigenesis in a genetic model of this disease. Given this, we evaluated whether an extra copy of Trp53 would alter tumorigenesis upon global activation of a modified Kras allele engineered with either a G12D or Q61R mutation. We report that an increase in p53 dosage significantly reduced the incidence or grade of oral and forestomach squamous tumors induced by either G12D and Q61R-mutant Kras. The incidence of myeloproliferative disease was also significantly reduced with increased p53 dosage in the KrasQ61R background. Both the percentage of mice with lung tumors and total number of adenomas per animal were unchanged. However, the incidence and grade of peripheral atypical alveolar hyperplasia was significantly decreased in both backgrounds with increased p53 dosage. Finally, the number of foci of bronchioloalveolar hyperplasia per animal significantly increased with increased p53 dosage in the KrasG12D background. These results suggest that an extra copy of p53 can impede oncogenic Kras driven tumorigenesis in some tissues.

Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance.

While tumors become addicted to oncogenes like Ras, the microenvironment in which tumor cells reside changes during tumorigenesis; the cells are surrounded initially by normal tissue and later by tumor tissue. Hence, we asked if Ras exerts its oncogenic effects through the same set of effectors during different stages of tumorigenesis. We now show in human cells that the Ras effector pathways MAPK, RalGEF, and PI3K are required to initiate tumor growth. Conversely, activation of the PI3K/AKT pathway replaced Ras once tumors formed, although other effectors were still activated independently of Ras, presumably by factors provided upon the establishment of a tumor microenvironment. Thus, as tumorigenesis progresses the addiction of cancers to their initiating oncogene is reduced to, at least in the case of Ras, the PI3K/AKT pathway.

Activation of RalA is critical for Ras-induced tumorigenesis of human cells.

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

An ultra-sensitive method to detect mutations in human RAS templates.

The RAS family of small GTPases is mutated in roughly a fifth of human cancers. Hotspot point mutations at codons G12, G13, and Q61 account for 95% of all these mutations, which are well established to render the encoded proteins oncogenic. In humans, this family comprises three genes: HRAS, NRAS, and KRAS. Accumulating evidence argues that oncogenic RAS point mutations may be initiating, as they are often truncal in human tumours and capable of inducing tumorigenesis in mice. As such, there is great interest in detecting oncogenic mutation in the RAS genes to understand the origins of cancer, as well as for early detection purposes. To this end, we previously adapted the microbial ultra-sensitive Maximum Depth Sequencing (MDS) assay for the murine Kras gene, which was capable of detecting oncogenic mutations in the tissues of mice days after carcinogen exposure, essentially capturing the very first step in tumour initiation. Given this, we report here the adaption and details of this assay to detect mutations in a human KRAS sequence at an analytic sensitivity of one mutation in a million independently barcoded templates. This humanized version of MDS can thus be exploited to detect oncogenic mutations in KRAS at an incredible sensitivity and modified for the same purpose for the other RAS genes.

Non-canonical genomic driver mutations of urethane carcinogenesis.

The carcinogen urethane induces pulmonary tumors in mice initiated by an incredibly specific Q61L/R oncogenic mutation in the proto-oncogene Kras. Previous Whole-Exome Sequencing of urethane-induced tumors revealed a bias towards A➙T/G and G➙A substitutions. Subsequent ultra-sensitive Maximum-Depth Sequencing of Kras shortly after urethane exposure suggest a further refinement to CA➙CT/G substitutions. As C182AA➙C182T/GA substitutions in Kras result in Q61L/R mutations, the extreme bias of urethane towards these genomic driver mutations can be ascribed to the specificity of the carcinogen for CA➙CT/G substitutions. However, we previously found that changing rare codons to common in the Kras gene to increase protein expression shifted mutations in urethane-induced tumors away from Kras, or when detected in Kras, to G12D mutations that are usually rarely detected in such tumors. Moreover, the loss of p53 partially reversed this effect, generating tumors with either Q61L/R or G12D oncogenic Kras mutations, or no Kras mutations, presumably due to other genomic driver mutations. Determining the origin of these G12D and other unknown non-canonical genomic driver mutations would provide critical insight into the extreme bias of carcinogens for specific genomic driver mutations. We thus compared the types of Single Nucleotide Variations detected by previously performed Maximum-Depth Sequencing immediately after urethane exposure to the mutation signatures derived from Whole Exome Sequencing of urethane-induced tumors. This identified two types of non-canonical mutations. First, a V637E oncogenic mutation in the proto-oncogene Braf that conforms to the mutation signature of urethane, suggesting that the mutational bias of the carcinogen may account for this non-canonical mutation, similar to that for canonical Q61L/R mutations in Kras. Second, G12D and Q61H mutations in Kras that did not fit this mutation signature, and instead shared similarity with Single Nucleotide Variations detected by Maximum-Depth Sequencing from normal cells, suggesting that perhaps these mutations were pre-existing. We thus posit that when canonical Kras mutations are selected against that the carcinogen may instead promote the expansion of pre-existing genomic driver mutations, although admittedly we cannot rule out other mechanisms. Interrogating the mutation signatures of human lung cancers similarly identified KRAS genomic driver mutations that failed to match the mutation signature of the tumor. Thus, we also speculate that the selection for non-canonical genomic driver mutations during urethane carcinogenesis may reflect the process by which discordance between genomic driver mutations and mutational signatures arises in human cancers.

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.

Distinct responses to rare codons in select Drosophila tissues.

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in an evolutionarily young testis-specific gene, RpL10Aa. Optimizing RpL10Aa codons disrupts female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.

cPLA2 regulates the expression of type I interferons and intracellular immunity to Chlamydia trachomatis.

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.

Signaling levels mold the RAS mutation tropism of urethane.

RAS genes are commonly mutated in human cancer. Despite many possible mutations, individual cancer types often have a 'tropism' towards a specific subset of RAS mutations. As driver mutations, these patterns ostensibly originate from normal cells. High oncogenic RAS activity causes oncogenic stress and different oncogenic mutations can impart different levels of activity, suggesting a relationship between oncoprotein activity and RAS mutation tropism. Here, we show that changing rare codons to common in the murine Kras gene to increase protein expression shifts tumors induced by the carcinogen urethane from arising from canonical Q61 to biochemically less active G12Kras driver mutations, despite the carcinogen still being biased towards generating Q61 mutations. Conversely, inactivating the tumor suppressor p53 to blunt oncogenic stress partially reversed this effect, restoring Q61 mutations. One interpretation of these findings is that the RAS mutation tropism of urethane arises from selection in normal cells for specific mutations that impart a narrow window of signaling that promotes proliferation without causing oncogenic stress.

Using BioID to Characterize the RAS Interactome.

Identifying the proteins that associate with RAS oncoproteins has great potential, not only to elucidate how these mutant proteins are regulated and signal but also to identify potential therapeutic targets. Here we describe a detailed protocol to employ proximity labeling by the BioID methodology, which has the advantage of capturing weak or transient interactions, to identify in an unbiased manner those proteins within the immediate vicinity of oncogenic RAS proteins.

Oncogenic KRAS is dependent upon an EFR3A-PI4KA signaling axis for potent tumorigenic activity.

The HRAS, NRAS, and KRAS genes are collectively mutated in a fifth of all human cancers. These mutations render RAS GTP-bound and active, constitutively binding effector proteins to promote signaling conducive to tumorigenic growth. To further elucidate how RAS oncoproteins signal, we mined RAS interactomes for potential vulnerabilities. Here we identify EFR3A, an adapter protein for the phosphatidylinositol kinase PI4KA, to preferentially bind oncogenic KRAS. Disrupting EFR3A or PI4KA reduces phosphatidylinositol-4-phosphate, phosphatidylserine, and KRAS levels at the plasma membrane, as well as oncogenic signaling and tumorigenesis, phenotypes rescued by tethering PI4KA to the plasma membrane. Finally, we show that a selective PI4KA inhibitor augments the antineoplastic activity of the KRASG12C inhibitor sotorasib, suggesting a clinical path to exploit this pathway. In sum, we have discovered a distinct KRAS signaling axis with actionable therapeutic potential for the treatment of KRAS-mutant cancers.

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

Capturing the primordial Kras mutation initiating urethane carcinogenesis.

The environmental carcinogen urethane exhibits a profound specificity for pulmonary tumors driven by an oncogenic Q61L/R mutation in the gene Kras. Similarly, the frequency, isoform, position, and substitution of oncogenic RAS mutations are often unique to human cancers. To elucidate the principles underlying this RAS mutation tropism of urethane, we adapted an error-corrected, high-throughput sequencing approach to detect mutations in murine Ras genes at great sensitivity. This analysis not only captured the initiating Kras mutation days after urethane exposure, but revealed that the sequence specificity of urethane mutagenesis, coupled with transcription and isoform locus, to be major influences on the extreme tropism of this carcinogen.

Expression of transgenes enriched in rare codons is enhanced by the MAPK pathway.

The ability to translate three nucleotide sequences, or codons, into amino acids to form proteins is conserved across all organisms. All but two amino acids have multiple codons, and the frequency that such synonymous codons occur in genomes ranges from rare to common. Transcripts enriched in rare codons are typically associated with poor translation, but in certain settings can be robustly expressed, suggestive of codon-dependent regulation. Given this, we screened a gain-of-function library for human genes that increase the expression of a GFPrare reporter encoded by rare codons. This screen identified multiple components of the mitogen activated protein kinase (MAPK) pathway enhancing GFPrare expression. This effect was reversed with inhibitors of this pathway and confirmed to be both codon-dependent and occur with ectopic transcripts naturally coded with rare codons. Finally, this effect was associated, at least in part, with enhanced translation. We thus identify a potential regulatory module that takes advantage of the redundancy in the genetic code to modulate protein expression.

ELR+ CXC chemokines and oncogenic Ras-mediated tumorigenesis.

The small GTPase Ras is mutated to remain in the active oncogenic state in one-third of human cancers, thereby promoting tumorigenesis. It has recently come to light that one consequence of oncogenic Ras signaling is secretion of cytokines vascular endothelial growth factor (VEGF), interleukin 6 (IL6), hCXCL1 (Gro-alpha) and hCXCL8 (IL8). As the latter two belong to the ELR+ Cys-X-Cys (CXC) chemokine family, we investigated whether the entire family of ELR+ CXC chemokines plays a role in oncogenic Ras-mediated tumorigenesis. We now demonstrate that oncogenic Ras induced the expression and secretion of the ELR+ CXC chemokine family in different tumorigenic human cells and that these chemokines are elevated in tumor specimens. Moreover, genetic ablation of the common receptor for these chemokines, mCXCR2, reduced oncogenic Ras-driven tumorigenesis in mice. Taken together, we suggest that oncogenic Ras induces the secretion of the ELR+ CXC chemokine family to promote tumorigenesis. This chemokine signature may identify the presence of Ras activation in cancer and perhaps even serve as targets for oncogenic Ras-driven tumor cells.