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KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.

ABSTRACT: Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway.

SUBMITTER: Krall EB

PROVIDER: S-EPMC5305212 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.

Krall Elsa B EB Wang Belinda B Munoz Diana M DM Ilic Nina N Raghavan Srivatsan S Niederst Matthew J MJ Yu Kristine K Ruddy David A DA Aguirre Andrew J AJ Kim Jong Wook JW Redig Amanda J AJ Gainor Justin F JF Williams Juliet A JA Asara John M JM Doench John G JG Janne Pasi A PA Shaw Alice T AT McDonald Iii Robert E RE Engelman Jeffrey A JA Stegmeier Frank F Schlabach Michael R MR Hahn William C WC

eLife 20170201

Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, an ...[more]

PMID: 28145866

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Project description:Aberrant fibroblast growth factor receptor (FGFR) activation/expression is a common feature in lung cancer (LC). In this study, we evaluated the antitumor activity of and the mechanisms underlying acquired resistance to two potent selective FGFR inhibitors, AZD4547 and BAY116387, in LC cell lines. The antitumor activity of AZD4547 and BAY1163877 was screened in 24 LC cell lines, including 5 with FGFR1 amplification. Two cell lines containing FGFR1 amplifications, H1581 and DMS114, were sensitive to FGFR inhibitors (IC50<250 nm). Clones of FGFR1-amplified H1581 cells resistant to AZD4547 or BAY116387 (H1581AR and H1581BR cells, respectively) were established. Receptor tyrosine kinase (RTK) array and immunoblotting analyses showed strong overexpression and activation of Met in H1581AR/BR cells, compared with that in the parental cells. Gene set enrichment analysis against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that cytokine-cytokine receptor interaction pathways were significantly enriched in H1581AR/BR cells, with Met contributing significantly to the core enrichment. Genomic DNA quantitative PCR and fluorescent in situ hybridization analyses showed MET amplification in H1581AR, but not in H1581BR, cells. Met amplification drives acquired resistance to AZD4547 in H1581AR cells by activating ErbB3. Combination treatment with FGFR inhibitors and an anaplastic lymphoma kinase (ALK)/Met inhibitor, crizotinib, or Met-specific short interfering RNA (siRNA) synergistically inhibited cell proliferation in both H1581AR and H1581BR cells. Conversely, ectopic expression of Met in H1581 cells conferred resistance to AZD4547 and BAY1163877. Acquired resistance to FGFR inhibitors not only altered cellular morphology, but also promoted migration and invasion of resistant clones, in part by inducing epithelial-to-mesenchymal transition. Taken together, our data suggest that Met activation is sufficient to bypass dependency on FGFR signaling. Concurrent inhibition of the Met and FGFR pathways may have synergistic clinical benefits when targeting FGFR-dependent LC.

Project description:BackgroundHER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype.MethodsWe generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets.ResultsTreatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts.ConclusionOur results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Dataset Information

KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.

Publications

KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.

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