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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A model for RAS mutation patterns in cancers: finding the sweet spot.

The three RAS genes - HRAS, NRAS and KRAS - are collectively mutated in one-third of human cancers, where they act as prototypic oncogenes. Interestingly, there are rather distinct patterns to RAS mutations; the isoform mutated as well as the position and type of substitution vary between different cancers. As RAS genes are among the earliest, if not the first, genes mutated in a variety of cancers, understanding how these mutation patterns arise could inform on not only how cancer begins but also the factors influencing this event, which has implications for cancer prevention. To this end, we suggest that there is a narrow window or 'sweet spot' by which oncogenic RAS signalling can promote tumour initiation in normal cells. As a consequence, RAS mutation patterns in each normal cell are a product of the specific RAS isoform mutated, as well as the position of the mutation and type of substitution to achieve an ideal level of signalling.

Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability.

In human cancers, oncogenic mutations commonly occur in the RAS genes KRAS, NRAS, or HRAS, but there are no clinical RAS inhibitors. Mutations are more prevalent in KRAS, possibly suggesting a unique oncogenic activity mediated by KRAS-specific interaction partners, which might be targeted. Here, we determine the specific protein interactomes of each RAS isoform by BirA proximity-dependent biotin identification. The combined interactomes are screened by CRISPR-Cas9 loss-of-function assays for proteins required for oncogenic KRAS-dependent, NRAS-dependent, or HRAS-dependent proliferation and censored for druggable proteins. Using this strategy, we identify phosphatidylinositol phosphate kinase PIP5K1A as a KRAS-specific interactor and show that PIP5K1A binds to a unique region in KRAS. Furthermore, PIP5K1A depletion specifically reduces oncogenic KRAS signaling and proliferation, and sensitizes pancreatic cancer cell lines to a MAPK inhibitor. These results suggest PIP5K1A as a potential target in KRAS signaling for the treatment of KRAS-mutant cancers.

Copper Chelation as Targeted Therapy in a Mouse Model of Oncogenic BRAF-Driven Papillary Thyroid Cancer.

Purpose: Sixty percent of papillary thyroid cancers (PTC) have an oncogenic (V600E) BRAF mutation. Inhibitors of BRAF and its substrates MEK1/2 are showing clinical promise in BRAFV600E PTC. PTC progression can be decades long, which is challenging in terms of toxicity and cost. We previously found that MEK1/2 require copper (Cu) for kinase activity and can be inhibited with the well-tolerated and economical Cu chelator tetrathiomolybdate (TM). We therefore tested TM for antineoplastic activity in BRAFV600E -positive PTC.Experimental Design: The efficacy of TM alone and in combination with current standard-of-care lenvatinib and sorafenib or BRAF and MEK1/2 inhibitors vemurafenib and trametinib was examined in BRAFV600E-positive human PTC cell lines and a genetically engineered mouse PTC model.Results: TM inhibited MEK1/2 kinase activity and transformed growth of PTC cells. TM was as or more potent than lenvatinib and sorafenib and enhanced the antineoplastic activity of sorafenib and vemurafenib. Activated ERK2, a substrate of MEK1/2, overcame this effect, consistent with TM deriving its antineoplastic activity by inhibiting MEK1/2. Oral TM reduced tumor burden and vemurafenib in a BrafV600E -positive mouse model of PTC. This effect was ascribed to a reduction of Cu in the tumors. TM reduced P-Erk1/2 in mouse PTC tumors, whereas genetic reduction of Cu in developing tumors trended towards a survival advantage. Finally, TM as a maintenance therapy after cessation of vemurafenib reduced tumor volume in the aforementioned PTC mouse model.Conclusions: TM inhibits BRAFV600E -driven PTC through inhibition of MEK1/2, supporting clinical evaluation of chronic TM therapy for this disease. Clin Cancer Res; 24(17); 4271-81. ©2018 AACR.

Wnt signaling suppresses MAPK-driven proliferation of intestinal stem cells.

Intestinal homeostasis depends on a slowly proliferating stem cell compartment in crypt cells, followed by rapid proliferation of committed progenitor cells in the transit amplifying (TA) compartment. The balance between proliferation and differentiation in intestinal stem cells (ISCs) is regulated by Wnt/ß-catenin signaling, although the mechanism remains unclear. We previously targeted PORCN, an enzyme essential for all Wnt secretion, and demonstrated that stromal production of Wnts was required for intestinal homeostasis. Here, a PORCN inhibitor was used to acutely suppress Wnt signaling. Unexpectedly, the treatment induced an initial burst of proliferation in the stem cell compartment of the small intestine, due to conversion of ISCs into TA cells with a loss of intrinsic ISC self-renewal. This process involved MAPK pathway activation, as the proliferating cells in the base of the intestinal crypt contained phosphorylated ERK1/2, and a MEK inhibitor attenuated the proliferation of ISCs and their differentiation into TA cells. These findings suggest a role for Wnt signaling in suppressing the MAPK pathway at the crypt base to maintain a pool of ISCs. The interaction between Wnt and MAPK pathways in vivo has potential therapeutic applications in cancer and regenerative medicine.

Wild-type Kras expands and exhausts hematopoietic stem cells.

Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Krasex3op) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that Kras protein levels were significantly increased in bone marrow (BM) HSCs in Krasex3op/ex3op mice, demonstrating that the translation of Kras in HSCs is normally constrained by rare codons. Krasex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells, and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in Erk1/2 phosphorylation and cyclin-dependent kinase 4 and 6 (Cdk4/6) activation. Furthermore, both the enhanced colony-forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced expansion of HSCs, progenitor cells, and B cells in a hematopoietic cell-autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence, which may speak to the importance of codon usage in mammalian biology.

Copper Chelation Inhibits BRAFV600E-Driven Melanomagenesis and Counters Resistance to BRAFV600E and MEK1/2 Inhibitors.

MEK1/2 and BRAFV600E inhibitors are used to treat BRAFV600E-positive melanoma, with other cancers under evaluation. Genetic perturbation of copper import or pharmacologic reduction of copper with the clinical copper chelator TTM inhibits MEK1/2 kinase activity and reduces BRAFV600E-driven tumorigenesis. In this study, we report that TTM inhibited transformed growth of melanoma cell lines resistant to BRAF or MEK1/2 inhibitors and enhanced the antineoplastic activity of these inhibitors. TTM also provided a survival advantage in a genetically engineered mouse model of melanoma, and when accounting for putative overdosing, trended toward an increase in the survival benefit afforded by BRAF inhibition. This effect was phenocopied by genetically inhibiting copper import in tumors, which was linked to a reduction in MAPK signaling. Thus, TTM reduces copper levels and MAPK signaling, thereby inhibiting BRAFV600E-driven melanoma tumor growth. These observations inform and support clinical evaluation of TTM in melanoma. Cancer Res; 77(22); 6240-52. ©2017 AACR.

A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution.

Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS/PIK3CA double-mutant colorectal cancers (CRCs) with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.

Codon bias imposes a targetable limitation on KRAS-driven therapeutic resistance.

KRAS mutations drive resistance to targeted therapies, including EGFR inhibitors in colorectal cancer (CRC). Through genetic screens, we unexpectedly find that mutant HRAS, which is rarely found in CRC, is a stronger driver of resistance than mutant KRAS. This difference is ascribed to common codon bias in HRAS, which leads to much higher protein expression, and implies that the inherent poor expression of KRAS due to rare codons must be surmounted during drug resistance. In agreement, we demonstrate that primary resistance to cetuximab is dependent upon both KRAS mutational status and protein expression level, and acquired resistance is often associated with KRASQ61 mutations that function even when protein expression is low. Finally, cancer cells upregulate translation to facilitate KRASG12-driven acquired resistance, resulting in hypersensitivity to translational inhibitors. These findings demonstrate that codon bias plays a critical role in KRAS-driven resistance and provide a rationale for targeting translation to overcome resistance.