Integration of transcriptomics, proteomics and loss-of-function screening reveals WEE1 as a target for combination with dasatinib against proneural glioblastoma.

Glioblastoma is characterized by a pronounced resistance to therapy with dismal prognosis. Transcriptomics classify glioblastoma into proneural (PN), mesenchymal (MES) and classical (CL) subtypes that show differential resistance to targeted therapies. The aim of this study was to provide a viable approach for identifying combination therapies in glioblastoma subtypes. Proteomics and phosphoproteomics were performed on dasatinib inhibited glioblastoma stem cells (GSCs) and complemented by an shRNA loss-of-function screen to identify genes whose knockdown sensitizes GSCs to dasatinib. Proteomics and screen data were computationally integrated with transcriptomic data using the SamNet 2.0 algorithm for network flow learning to reveal potential combination therapies in PN GSCs. In vitro viability assays and tumor spheroid models were used to verify the synergy of identified therapy. Further in vitro and TCGA RNA-Seq data analyses were utilized to provide a mechanistic explanation of these effects. Integration of data revealed the cell cycle protein WEE1 as a potential combination therapy target for PN GSCs. Validation experiments showed a robust synergistic effect through combination of dasatinib and the WEE1 inhibitor, MK-1775, in PN GSCs. Combined inhibition using dasatinib and MK-1775 propagated DNA damage in PN GCSs, with GCSs showing a differential subtype-driven pattern of expression of cell cycle genes in TCGA RNA-Seq data. The integration of proteomics, loss-of-function screens and transcriptomics confirmed WEE1 as a target for combination with dasatinib against PN GSCs. Utilizing this integrative approach could be of interest for studying resistance mechanisms and revealing combination therapy targets in further tumor entities.

Identification of epigenetic modifiers essential for growth and survival of AML1/ETO-positive leukemia.

Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.

In Vivo Genome-Wide Pooled RNAi Screens in Cancer Cells to Identify Determinants of Chemotherapy/Drug Response.

Large-scale RNAi screens (i.e., genome-wide arrays and pools) can reveal the essential biological functions of previously uncharacterized genes. Due to the nature of the selection process involved in screens, RNAi screens are also very useful for identifying genes involved in drug responses. The information gained from these screens could be used to predict a cancer patient's response to a specific drug (i.e., precision medicine) or identify anti-cancer drug resistance genes, which could be targeted to improve treatment outcomes. In this capacity, screens have been most often performed in vitro. However, there is limitation to performing these screens in vitro: genes which are required in only an in vivo setting (e.g., rely on the tumor microenvironment for function) will not be identified. As such, it can be desirable to perform RNAi screens in vivo. Here we outline the additional technical details that should be considered for performing genome-wide RNAi drug screens of cancer cells under in vivo conditions (i.e., tumor xenografts).

Functional Genomic Screening During Somatic Cell Reprogramming Identifies DKK3 as a Roadblock of Organ Regeneration.

Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf-3 (DKK3) is identified as novel factor for organ regeneration using combined transcription-factor-induced reprogramming and RNA-interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three-germ-layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3-null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical-Wnt-signaling in Dkk3-null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog-signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical-Wnt-, and Hedgehog-signaling.

An in vivo genome-wide shRNA screen identifies BCL6 as a targetable biomarker of paclitaxel resistance in breast cancer.

Paclitaxel is a common breast cancer drug; however, some tumors are resistant. The identification of biomarkers for paclitaxel resistance or sensitivity would enable the development of strategies to improve treatment efficacy. A genome-wide in vivo shRNA screen was performed on paclitaxel-treated mice with MDA-MB-231 tumors to identify genes associated with paclitaxel sensitivity or resistance. Gene expression of the top screen hits was associated with tumor response (resistance or sensitivity) among patients who received neoadjuvant chemotherapy containing paclitaxel. We focused our validation on screen hit B-cell lymphoma 6 (BCL6), which is a therapeutic target in cancer but for which no effects on drug response have been reported. Knockdown of BCL6 resulted in increased tumor regression in mice treated with paclitaxel. Similarly, inhibiting BCL6 using a small molecule inhibitor enhanced paclitaxel treatment efficacy both in vitro and in vivo in breast cancer models. Mechanism studies revealed that reduced BCL6 enhances the efficacy of paclitaxel by inducing sustained G1/S arrest, concurrent with increased apoptosis and expression of target gene cyclin-dependent kinase inhibitor 1A. In summary, the genome-wide shRNA knockdown screen has identified BCL6 as a potential targetable resistance biomarker of paclitaxel response in breast cancer.

A Small-Scale shRNA Screen in Primary Mouse Macrophages Identifies a Role for the Rab GTPase Rab1b in Controlling Salmonella Typhi Growth.

Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium's adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled S. Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.

USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells.

Altering ubiquitination by disruption of deubiquitinating enzymes (DUBs) affects hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. Here, we systematically inactivate DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNA (shRNA) screens. We find that multiple DUBs may be individually required for hematopoiesis and identify ubiquitin-specific protease 15 (USP15) as essential for HSC maintenance in vitro and in transplantations and Usp15 knockout (KO) mice in vivo. USP15 is highly expressed in human hematopoietic tissues and leukemias. USP15 depletion in murine progenitors and leukemia cells impairs in vitro expansion and increases genotoxic stress. In leukemia cells, USP15 interacts with and stabilizes FUS (fused in sarcoma), a known DNA repair factor, directly linking USP15 to the DNA damage response (DDR). Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSCs and leukemia cells.

DECIPHER pooled shRNA library screen identifies PP2A and FGFR signaling as potential therapeutic targets for diffuse intrinsic pontine gliomas.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive pediatric brain tumors that are characterized by a recurrent mutation (K27M) within the histone H3 encoding genes H3F3A and HIST1H3A/B/C. These mutations have been shown to induce a global reduction in the repressive histone modification H3K27me3, which together with widespread changes in DNA methylation patterns results in an extensive transcriptional reprogramming hampering the identification of single therapeutic targets based on a molecular rationale. METHODS: We applied a large-scale gene knockdown approach using a pooled short hairpin (sh)RNA library in combination with next-generation sequencing in order to identify DIPG-specific vulnerabilities. The therapeutic potential of specific inhibitors of candidate targets was validated in a secondary drug screen. RESULTS: We identified fibroblast growth factor receptor (FGFR) signaling and the serine/threonine protein phosphatase 2A (PP2A) as top depleted hits in patient-derived DIPG cell cultures and validated their lethal potential by FGF ligand depletion and genetic knockdown of the PP2A structural subunit PPP2R1A. Further, pharmacological inhibition of FGFR and PP2A signaling through ponatinib and LB-100 treatment, respectively, exhibited strong tumor-specific anti-proliferative and apoptotic activity in cultured DIPG cells. CONCLUSIONS: Our findings suggest FGFR and PP2A signaling as potential new therapeutic targets for the treatment of DIPGs.

Functional genomics screen with pooled shRNA library and gene expression profiling with extracts of Azadirachta indica identify potential pathways for therapeutic targets in head and neck squamous cell carcinoma.

Tumor suppression by the extracts of Azadirachta indica (neem) works via anti-proliferation, cell cycle arrest, and apoptosis, demonstrated previously using cancer cell lines and live animal models. However, very little is known about the molecular targets and pathways that neem extracts and their associated compounds act through. Here, we address this using a genome-wide functional pooled shRNA screen on head and neck squamous cell carcinoma cell lines treated with crude neem leaf extracts, known for their anti-tumorigenic activity. We analyzed differences in global clonal sizes of the shRNA-infected cells cultured under no treatment and treatment with neem leaf extract conditions, assayed using next-generation sequencing. We found 225 genes affected the cancer cell growth in the shRNA-infected cells treated with neem extract. Pathway enrichment analyses of whole-genome gene expression data from cells temporally treated with neem extract revealed important roles played by the TGF-ß pathway and HSF-1-related gene network. Our results indicate that neem extract affects various important molecular signaling pathways in head and neck cancer cells, some of which may be therapeutic targets for this devastating tumor.

HIV-1 Fusion with CD4+ T cells Is Promoted by Proteins Involved in Endocytosis and Intracellular Membrane Trafficking.

The HIV-1 entry pathway into permissive cells has been a subject of debate. Accumulating evidence, including our previous single virus tracking results, suggests that HIV-1 can enter different cell types via endocytosis and CD4/coreceptor-dependent fusion with endosomes. However, recent studies that employed indirect techniques to infer the sites of HIV-1 entry into CD4+ T cells have concluded that endocytosis does not contribute to infection. To assess whether HIV-1 enters these cells via endocytosis, we probed the role of intracellular trafficking in HIV-1 entry/fusion by a targeted shRNA screen in a CD4+ T cell line. We performed a screen utilizing a direct virus-cell fusion assay as readout and identified several host proteins involved in endosomal trafficking/maturation, including Rab5A and sorting nexins, as factors regulating HIV-1 fusion and infection. Knockdown of these proteins inhibited HIV-1 fusion irrespective of coreceptor tropism, without altering the CD4 or coreceptor expression, or compromising the virus' ability to mediate fusion of two adjacent cells initiated by virus-plasma membrane fusion. Ectopic expression of Rab5A in non-permissive cells harboring Rab5A shRNAs partially restored the HIV-cell fusion. Together, these results implicate endocytic machinery in productive HIV-1 entry into CD4+ T cells.

TRPS1 Is a Lineage-Specific Transcriptional Dependency in Breast Cancer.

Perturbed epigenomic programs play key roles in tumorigenesis, and chromatin modulators are candidate therapeutic targets in various human cancer types. To define singular and shared dependencies on DNA and histone modifiers and transcription factors in poorly differentiated adult and pediatric cancers, we conducted a targeted shRNA screen across 59 cell lines of 6 cancer types. Here, we describe the TRPS1 transcription factor as a strong breast cancer-specific hit, owing largely to lineage-restricted expression. Knockdown of TRPS1 resulted in perturbed mitosis, apoptosis, and reduced tumor growth. Integrated analysis of TRPS1 transcriptional targets, chromatin binding, and protein interactions revealed that TRPS1 is associated with the NuRD repressor complex. These findings uncover a transcriptional network that is essential for breast cancer cell survival and propagation.

RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes.

The potent proinflammatory cytokine interleukin (IL)-1 triggers gene expression through the NF-κB signaling pathway. Here, we investigated the cofactor requirements of strongly regulated IL-1 target genes whose expression is impaired in p65 NF-κB-deficient murine embryonic fibroblasts. By two independent small-hairpin (sh)RNA screens, we examined 170 genes annotated to encode nuclear cofactors for their role in Cxcl2 mRNA expression and identified 22 factors that modulated basal or IL-1-inducible Cxcl2 levels. The functions of 16 of these factors were validated for Cxcl2 and further analyzed for their role in regulation of 10 additional IL-1 target genes by RT-qPCR. These data reveal that each inducible gene has its own (quantitative) requirement of cofactors to maintain basal levels and to respond to IL-1. Twelve factors (Epc1, H2afz, Kdm2b, Kdm6a, Mbd3, Mta2, Phf21a, Ruvbl1, Sin3b, Suv420h1, Taf1, and Ube3a) have not been previously implicated in inflammatory cytokine functions. Bioinformatics analysis indicates that they are components of complex nuclear protein networks that regulate chromatin functions and gene transcription. Collectively, these data suggest that downstream from the essential NF-κB signal each cytokine-inducible target gene has further subtle requirements for individual sets of nuclear cofactors that shape its transcriptional activation profile.

Functional Genome-wide Screen Identifies Pathways Restricting Central Nervous System Axonal Regeneration.

Axonal regrowth is crucial for recovery from CNS injury but is severely restricted in adult mammals. We used a genome-wide loss-of-function screen for factors limiting axonal regeneration from cerebral cortical neurons in vitro. Knockdown of 16,007 individual genes identified 580 significant phenotypes. These molecules share no significant overlap with those suggested by previous expression profiles. There is enrichment for genes in pathways related to transport, receptor binding, and cytokine signaling, including Socs4 and Ship2. Among transport-regulating proteins, Rab GTPases are prominent. In vivo assessment with C. elegans validates a cell-autonomous restriction of regeneration by Rab27. Mice lacking Rab27b show enhanced retinal ganglion cell axon regeneration after optic nerve crush and greater motor function and raphespinal sprouting after spinal cord trauma. Thus, a comprehensive functional screen reveals multiple pathways restricting axonal regeneration and neurological recovery after injury.

Pooled Generation of Lentiviral Tetracycline-Regulated microRNA Embedded Short Hairpin RNA Libraries.

Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.

Identification of essential transcription factors for adequate DNA damage response after benzo(a)pyrene and aflatoxin B1 exposure by combining transcriptomics with functional genomics.

DNA damage mediates widespread changes in transcription through activation or repression of transcription factors (TFs). However, the consequences of regulating specific TFs for the outcome of the DNA repair process remain incompletely understood. Here, we combined transcriptomics and TF binding prediction with functional genomics to identify TFs essential for adequate DNA repair in HepG2 liver cells after a non-cytotoxic dose of carcinogens benzo(a)pyrene (BaP) (2µM) and aflatoxin B1 (AFB1) (5µM). BaP and AFB1 induced a largely common transcriptional response, mediated by similar TFs. A lentiviral shRNA screen knocking down the top31 identified TFs, was performed to determine their effect on DNA repair by assessing phosphorylation of H2AX (γ-H2AX). In addition to the top candidate p53, we identified several other interesting TFs that modulated γ-H2AX after BaP and AFB1 treatment. Validation studies confirmed the role of p53 in reducing γ-H2AX formation and DNA breaks measured by COMET assay after BaP and AFB1 exposure. Expression of the cell cycle inhibitor p21 was profoundly impaired upon p53 knock-down. In addition, the expression of 2 genes involved in nucleotide exchange repair, DDB2 and XPC was significantly reduced in p53 knock-down cells. Although p63 knock-down affected DNA damage upon BaP treatment this was not associated with altered expression of DDB2 or XPC. Finally, knock-down of ARNT reduced γ-H2AX in response to BaP, which was associated with reduced CYP1A1 expression. Importantly, our results suggest a new role for ARNT and its dimerization partner AHR in the occurrence of H2AX phosphorylation after AFB1 treatment. These data show that modulation of TF activity impacts on the repair of BaP- and AFB1-induced DNA damage. Our study also demonstrates the potential of combining functional genomics with genome-wide expression analysis to identify yet unknown causal relationships, thereby aiding in the interpretation of complex biological systems.

Synergistic interactions with PI3K inhibition that induce apoptosis.

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition.

The mTOR Complex Controls HIV Latency.

A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

High-Throughput Screening of Tyrosine Kinase Inhibitor Resistant Genes in CML.

Genome-wide RNA interference (RNAi) screening in mammalian cells has proven to be a powerful tool for identifying new genes and molecular pathways relevant to many cellular processes and diseases. For example, screening for genes that, when inactivated, lead to resistance to cancer therapeutic drugs can reveal new mechanisms for how resistance develops and identify potential targetable strategies to overcome drug resistance. Here, we describe a detailed procedure for performing a high-throughput RNAi screen using a genome-wide human short hairpin RNA (shRNA) library for identifying tyrosine kinase inhibitor (TKI)-resistance genes in a human CML cell line model.

HDAC Inhibitors.

Lysine acetylation in proteins is one of the most abundant posttranslational modifications in eukaryotic cells. The dynamic homeostasis of lysine acetylation and deacetylation is dictated by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Important substrates for HATs and HDACs are histones, where lysine acetylation generally leads to an open and transcriptionally active chromatin conformation. Histone deacetylation forces the compaction of the chromatin with subsequent inhibition of transcription and reduced gene expression. Unbalanced HAT and HDAC activity, and therefore aberrant histone acetylation, has been shown to be involved in tumorigenesis and progression of malignancy in different types of cancer. Therefore, the development of HDAC inhibitors (HDIs) as therapeutic agents against cancer is of great interest. However, treatment with HDIs can also affect the acetylation status of many other non-histone proteins which play a role in different pathways including angiogenesis, cell cycle progression, autophagy and apoptosis. These effects have led HDIs to become anticancer agents, which can initiate apoptosis in tumor cells. Hematological malignancies in particular are responsive to HDIs, and four HDIs have already been approved as anticancer agents. There is a strong interest in finding adequate biomarkers to predict the response to HDI treatment. This chapter provides information on how to assess HDAC activity in vitro and determine the potency of HDIs on different HDACs. It also gives information on how to analyze cellular markers following HDI treatment and to analyze tissue biopsies from HDI-treated patients. Finally, a protocol is provided on how to detect HDI sensitivity determinants in human cells, based on a pRetroSuper shRNA screen upon HDI treatment.

Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma.

Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS-RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers.