Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.

LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth. Using this assay, along with genome-wide CRISPR screens and validation with orthogonal methods, we discovered that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase, which promotes the internalization of wild-type EGFR. Our findings reveal a new mechanism of regulation of EGFR, which may have implications for the treatment of LKB1 -mutant LUAD.

MEK inhibitor resistance in lung adenocarcinoma is associated with addiction to sustained ERK suppression.

MEK inhibitors (MEKi) have limited efficacy in KRAS mutant lung adenocarcinoma (LUAD) patients, and this is attributed to both intrinsic and adaptive mechanisms of drug resistance. While many studies have focused on the former, there remains a dearth of data regarding acquired resistance to MEKi in LUAD. We established trametinib-resistant KRAS mutant LUAD cells through dose escalation and performed targeted MSK-IMPACT sequencing to identify drivers of MEKi resistance. Comparing resistant cells to their sensitive counterparts revealed alteration of genes associated with trametinib response. We describe a state of "drug addiction" in resistant cases where cells are dependent on continuous culture in trametinib for survival. We show that dependence on ERK2 suppression underlies this phenomenon and that trametinib removal hyperactivates ERK, resulting in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant-specific activity with AMG 510 rescues the lethality associated with trametinib withdrawal. Furthermore, we show that increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study determines the drug-addicted phenotype in lung cancer is associated with KRAS amplification and demonstrates that toxic acquired genetic changes can develop de novo in the background of MAPK suppression with MEK inhibitors. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a "drug holiday" to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.

CCL5 production in lung cancer cells leads to an altered immune microenvironment and promotes tumor development.

Current immunotherapies for lung cancer are only effective in a subset of patients. Identifying tumor-derived factors that facilitate immunosuppression offers the opportunity to develop novel strategies to supplement and improve current therapeutics. We sought to determine whether expression of driver oncogenes in lung cancer cells affects cytokine secretion, alters the local immune environment, and influences lung tumor progression. We demonstrate that oncogenic EGFR and KRAS mutations, which are early events in lung tumourigenesis, can drive cytokine and chemokine production by cancer cells. One of the most prominent changes was in CCL5, which was rapidly induced by KRASG12V or EGFRL858R expression, through MAPK activation. Immunocompetent mice implanted with syngeneic KRAS-mutant lung cancer cells deficient in CCL5 have decreased regulatory T cells (Tregs), evidence of T cell exhaustion, and reduced lung tumor burden, indicating tumor-cell CCL5 production contributes to an immune suppressive environment in the lungs. Furthermore, high CCL5 expression correlates with poor prognosis, immunosuppressive regulatory T cells, and alteration to CD8 effector function in lung adenocarcinoma patients. Our data support targeting CCL5 or CCL5 receptors on immune suppressive cells to prevent formation of an immune suppressive tumor microenvironment that promotes lung cancer progression and immunotherapy insensitivity.

Co-occurrence and mutual exclusivity: what cross-cancer mutation patterns can tell us.

Cancer is the dysregulated proliferation of cells caused by acquired mutations in key driver genes. The most frequently mutated driver genes promote tumorigenesis in various organisms, cell types, and genetic backgrounds. However, recent cancer genomics studies also point to the existence of context-dependent driver gene functions, where specific mutations occur predominately or even exclusively in certain tumor types or genetic backgrounds. Here, we review examples of co-occurring and mutually exclusive driver gene mutation patterns across cancer genomes and discuss their underlying biology. While co-occurring driver genes typically activate collaborating oncogenic pathways, we identify two distinct biological categories of incompatibilities among the mutually exclusive driver genes depending on whether the mutated drivers trigger the same or divergent tumorigenic pathways. Finally, we discuss possible therapeutic avenues emerging from the study of incompatible driver gene mutations.

Drug Sensitivity and Allele Specificity of First-Line Osimertinib Resistance EGFR Mutations.

Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFRL858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance in vivo. SIGNIFICANCE: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/2017/F1.large.jpg.

Generation of pulmonary neuroendocrine cells and SCLC-like tumors from human embryonic stem cells.

Cancer models based on cells derived from human embryonic stem cells (hESCs) may reveal why certain constellations of genetic changes drive carcinogenesis in specialized lineages. Here we demonstrate that inhibition of NOTCH signaling induces up to 10% of lung progenitor cells to form pulmonary neuroendocrine cells (PNECs), putative precursors to small cell lung cancers (SCLCs), and we can increase PNECs by reducing levels of retinoblastoma (RB) proteins with inhibitory RNA. Reducing levels of TP53 protein or expressing mutant KRAS or EGFR genes did not induce or expand PNECs, but tumors resembling early-stage SCLC grew in immunodeficient mice after subcutaneous injection of PNEC-containing cultures in which expression of both RB and TP53 was blocked. Single-cell RNA profiles of PNECs are heterogeneous; when RB levels are reduced, the profiles resemble those from early-stage SCLC; and when both RB and TP53 levels are reduced, the transcriptome is enriched with cell cycle-specific RNAs. Our findings suggest that genetic manipulation of hESC-derived pulmonary cells will enable studies of this recalcitrant cancer.

Integrative Genomic Analyses Identifies GGA2 as a Cooperative Driver of EGFR-Mediated Lung Tumorigenesis.

INTRODUCTION: Targeted therapies for lung adenocarcinoma (LUAD) have improved patient outcomes; however, drug resistance remains a major problem. One strategy to achieve durable response is to develop combination-based therapies that target both mutated oncogenes and key modifiers of oncogene-driven tumorigenesis. This is based on the premise that mutated oncogenes, although necessary, are not sufficient for malignant transformation. We aimed to uncover genetic alterations that cooperate with mutant EGFR during LUAD development. METHODS: We performed integrative genomic analyses, combining copy number, gene expression and mutational information for over 500 LUAD tumors. Co-immunoprecipitation and Western blot analysis were performed in LUAD cell lines to confirm candidate interactions while RNA interference and gene overexpression were used for in vitro and in vivo functional assessment. RESULTS: We identified frequent amplifications/deletions of chromosomal regions affecting the activity of genes specifically in the context of EGFR mutation, including amplification of the mutant EGFR allele and deletion of dual specificity phosphatase 4 (DUSP4), which have both previously been reported. In addition, we identified the novel amplification of a segment of chromosome arm 16p in mutant-EGFR tumors corresponding to increased expression of Golgi Associated, Gamma Adaptin Ear Containing, ARF Binding Protein 2 (GGA2), which functions in protein trafficking and sorting. We found that GGA2 interacts with EGFR, increases EGFR protein levels and modifies EGFR degradation after ligand stimulation. Furthermore, we show that overexpression of GGA2 enhances EGFR mediated transformation while GGA2 knockdown reduces the colony and tumor forming ability of EGFR mutant LUAD. CONCLUSIONS: These data suggest that overexpression of GGA2 in LUAD tumors results in the accumulation of EGFR protein and increased EGFR signaling, which helps drive tumor progression. Thus, GGA2 plays a cooperative role with EGFR during LUAD development and is a potential therapeutic target for combination-based strategies in LUAD.

Hyperactivation of ERK by multiple mechanisms is toxic to RTK-RAS mutation-driven lung adenocarcinoma cells.

Synthetic lethality results when mutant KRAS and EGFR proteins are co-expressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for mutual exclusivity of KRAS and EGFR mutations. We have now defined the biochemical events responsible for the toxic effects by combining pharmacological and genetic approaches and to show that signaling through extracellular signal-regulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes in the RAS pathway must restrain the activity of ERK1/2 to avoid toxicities and enable tumor growth. A dual specificity phosphatase, DUSP6, that negatively regulates phosphorylation of (P)-ERK is up-regulated in EGFR- or KRAS-mutant LUAD, potentially protecting cells with mutations in the RAS signaling pathway, a proposal supported by experiments with DUSP6-specific siRNA and an inhibitory drug. Targeting DUSP6 or other negative regulators might offer a treatment strategy for certain cancers by inducing the toxic effects of RAS-mediated signaling.

How Cancer Genomics Drives Cancer Biology: Does Synthetic Lethality Explain Mutually Exclusive Oncogenic Mutations?

Large-scale analyses of cancer genomes are revealing patterns of mutations that suggest biologically significant ideas about many aspects of cancer, including carcinogenesis, classification, and preventive and therapeutic strategies. Among those patterns is "mutual exclusivity," a phenomenon observed when two or more mutations that are commonly observed in samples of a type of cancer are not found combined in individual tumors. We have been studying a striking example of mutual exclusivity: the absence of coexisting mutations in the KRAS and EGFR proto-oncogenes in human lung adenocarcinomas, despite the high individual frequencies of such mutations in this common type of cancer. Multiple lines of evidence suggest that toxic effects of the joint expression of KRAS and EGFR mutant oncogenes, rather than loss of any selective advantages conferred by a second oncogene that operates through the same signaling pathway, are responsible for the observed mutational pattern. We discuss the potential for understanding the physiological basis of such toxicity, for exploiting it therapeutically, and for extending the studies to other examples of mutual exclusivity.

Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma.

Human lung adenocarcinomas (LUAD) contain mutations in EGFR in ∼15% of cases and in KRAS in ∼30%, yet no individual adenocarcinoma appears to carry activating mutations in both genes, a finding we have confirmed by re-analysis of data from over 600 LUAD. Here we provide evidence that co-occurrence of mutations in these two genes is deleterious. In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene. We also show that forced expression of a second oncogene in human cancer cell lines with an endogenous mutated oncogene is deleterious. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype. Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

Intrinsic sensor of oncogenic transformation induces a signal for innate immunosurveillance.

Multiple cell-autonomous mechanisms exist in complex metazoans to resist oncogenic transformation, including a variety of tumor- suppressor pathways that control cell proliferation and apoptosis. In vertebrates, additional mechanisms of tumor resistance could potentially rely on cancer cell elimination by specialized cytotoxic leukocytes, such as natural killer (NK) cells. Such mechanisms would require that cancer cells be reliably distinguished from normal cells. The ligands for NKG2D, an activating NK cell receptor, are expressed on many tumor cell lines and at least some primary human tumors. However, it is unknown whether their expression is induced as a direct result of oncogenic transformation in vivo. We provide evidence that NKG2D ligands are induced on spontaneously arising tumors in a murine model of lymphomagenesis and that c-Myc is involved in their regulation. Expression of NKG2D ligands is induced at an early, distinct stage of tumorigenesis upon acquisition of genetic lesions unique to cancer cells, potentially defining a critical step in carcinogenesis. This finding suggests that the regulation of NKG2D ligands depends on a mechanism for intrinsic sensing of oncogenic transformation.