A guide to ERK dynamics, part 1: mechanisms and models.

Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.

A guide to ERK dynamics, part 2: downstream decoding.

Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer.

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Deciphering the History of ERK Activity from Fixed-Cell Immunofluorescence Measurements.

The Ras/ERK pathway drives cell proliferation and other oncogenic behaviors, and quantifying its activity in situ is of high interest in cancer diagnosis and therapy. Pathway activation is often assayed by measuring phosphorylated ERK. However, this form of measurement overlooks dynamic aspects of signaling that can only be observed over time. In this study, we combine a live, single-cell ERK biosensor approach with multiplexed immunofluorescence staining of downstream target proteins to ask how well immunostaining captures the dynamic history of ERK activity. Combining linear regression, machine learning, and differential equation models, we develop an interpretive framework for immunostains, in which Fra-1 and pRb levels imply long term activation of ERK signaling, while Egr-1 and c-Myc indicate recent activation. We show that this framework can distinguish different classes of ERK dynamics within a heterogeneous population, providing a tool for annotating ERK dynamics within fixed tissues.

Spatiotemporal Clusters of ERK Activity Coordinate Cytokine-induced Inflammatory Responses in Human Airway Epithelial Cells.

RATIONALE: Spatially coordinated ERK signaling events ("SPREADs") transmit radially from a central point to adjacent cells via secreted ligands for EGFR and other receptors. SPREADs maintain homeostasis in non-pulmonary epithelia, but it is unknown whether they play a role in the airway epithelium or are dysregulated in inflammatory disease. OBJECTIVES: (1) To characterize spatiotemporal ERK activity in response to pro-inflammatory ligands, and (2) to assess pharmacological and metabolic regulation of cytokine-mediated SPREADs. METHODS: SPREADs were measured by live-cell ERK biosensors in human bronchial epithelial cell lines (HBE1 and 16HBE) and primary human bronchial epithelial (pHBE) cells, in both submerged and biphasic Air-Liquid Interface (ALI) culture conditions (i.e., differentiated cells). Cells were exposed to pro-inflammatory cytokines relevant to asthma and chronic obstructive pulmonary disease (COPD), and to pharmacological treatments (gefitinib, tocilizumab, hydrocortisone) and metabolic modulators (insulin, 2-deoxyglucose) to probe the airway epithelial mechanisms of SPREADs. Phospho-STAT3 immunofluorescence was used to measure localized inflammatory responses to IL-6. RESULTS: Pro-inflammatory cytokines significantly increased the frequency of SPREADs. Notably, differentiated pHBE cells display increased SPREAD frequency that coincides with airway epithelial barrier breakdown. SPREADs correlate with IL-6 peptide secretion and localized pSTAT3. Hydrocortisone, inhibitors of receptor signaling, and suppression of metabolic function decreased SPREAD occurrence. CONCLUSIONS: Pro-inflammatory cytokines modulate SPREADs in human airway epithelial cells via both secreted EGFR and IL6R ligands. SPREADs correlate with changes in epithelial barrier permeability, implying a role for spatiotemporal ERK signaling in barrier homeostasis and dysfunction during inflammation. The involvement of SPREADs in airway inflammation suggests a novel signaling mechanism that could be exploited clinically to supplement corticosteroid treatment for asthma and COPD.

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors.

Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling-dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.

FOXF1 Defines the Core-Regulatory Circuitry in Gastrointestinal Stromal Tumor.

The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behavior and shapes treatment responses in distinct cancer types. Here, we uncover that in gastrointestinal stromal tumor (GIST), the forkhead family member FOXF1 directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal lineage specification and GIST tumorigenesis. Further, FOXF1 colocalizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST lineage-specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance, and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behavior and treatment response.Significance: We uncover that FOXF1 defines the core-regulatory circuitry in GIST through both direct transcriptional regulation and pioneer factor function. The unique and simultaneous control of signaling and transcriptional circuitry by FOXF1 sets up an enforced transcriptional addiction to FOXF1 in GIST, which can be exploited diagnostically and therapeutically. Cancer Discov; 8(2); 234-51. ©2017 AACR.See related commentary by Lee and Duensing, p. 146This article is highlighted in the In This Issue feature, p. 127.

ETV1-Positive Cells Give Rise to BRAFV600E -Mutant Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumor (GIST) is the most common subtype of sarcoma. Despite clinical advances in the treatment of KIT/PDGFRA-mutant GIST, similar progress against KIT/PDGFRA wild-type GIST, including mutant BRAF-driven tumors, has been limited by a lack of model systems. ETV1 is a master regulator in the intestinal cells of Cajal (ICC), thought to be the cells of origin of GIST. Here, we present a model in which the ETV1 promoter is used to specifically and inducibly drive Cre recombinase in ICC as a strategy to study GIST pathogenesis. Using a conditional allele for BrafV600E , a mutation observed in clinical cases of GIST, we observed that BrafV600E activation was sufficient to drive ICC hyperplasia but not GIST tumorigenesis. In contrast, combining BrafV600E activation with Trp53 loss was sufficient to drive both ICC hyperplasia and formation of multifocal GIST-like tumors in the mouse gastrointestinal tract with 100% penetrance. This mouse model of sporadic GIST model was amenable to therapeutic intervention, and it recapitulated clinical responses to RAF inhibition seen in human GIST. Our work offers a useful in vivo model of human sporadic forms of BRAF-mutant GIST to help unravel its pathogenesis and therapeutic response to novel experimental agents. Cancer Res; 77(14); 3758-65. ©2017 AACR.

Aberrant Activation of a Gastrointestinal Transcriptional Circuit in Prostate Cancer Mediates Castration Resistance.

Prostate cancer exhibits a lineage-specific dependence on androgen signaling. Castration resistance involves reactivation of androgen signaling or activation of alternative lineage programs to bypass androgen requirement. We describe an aberrant gastrointestinal-lineage transcriptome expressed in ∼5% of primary prostate cancer that is characterized by abbreviated response to androgen-deprivation therapy and in ∼30% of castration-resistant prostate cancer. This program is governed by a transcriptional circuit consisting of HNF4G and HNF1A. Cistrome and chromatin analyses revealed that HNF4G is a pioneer factor that generates and maintains enhancer landscape at gastrointestinal-lineage genes, independent of androgen-receptor signaling. In HNF4G/HNF1A-double-negative prostate cancer, exogenous expression of HNF4G at physiologic levels recapitulates the gastrointestinal transcriptome, chromatin landscape, and leads to relative castration resistance.

A Tmprss2-CreERT2 Knock-In Mouse Model for Cancer Genetic Studies on Prostate and Colon.

Fusion between TMPRSS2 and ERG, placing ERG under the control of the TMPRSS2 promoter, is the most frequent genetic alteration in prostate cancer, present in 40-50% of cases. The fusion event is an early, if not initiating, event in prostate cancer, implicating the TMPRSS2-positive prostate epithelial cell as the cancer cell of origin in fusion-positive prostate cancer. To introduce genetic alterations into Tmprss2-positive cells in mice in a temporal-specific manner, we generated a Tmprss2-CreERT2 knock-in mouse. We found robust tamoxifen-dependent Cre activation in the prostate luminal cells but not basal epithelial cells, as well as epithelial cells of the bladder and gastrointestinal (GI) tract. The knock-in allele on the Tmprss2 locus does not noticeably impact prostate, bladder, or gastrointestinal function. Deletion of Pten in Tmprss2-positive cells of adult mice generated neoplasia only in the prostate, while deletion of Apc in these cells generated neoplasia only in the GI tract. These results suggest that this new Tmprss2-CreERT2 mouse model will be a useful resource for genetic studies on prostate and colon.

Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape.

Several types of pediatric cancers reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here we report that the H3 lysine 36-to-methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified.

Combined inhibition of MAP kinase and KIT signaling synergistically destabilizes ETV1 and suppresses GIST tumor growth.

UNLABELLED: Gastrointestinal stromal tumor (GIST), originating from the interstitial cells of Cajal (ICC), is characterized by frequent activating mutations of the KIT receptor tyrosine kinase. Despite the clinical success of imatinib, which targets KIT, most patients with advanced GIST develop resistance and eventually die of the disease. The ETS family transcription factor ETV1 is a master regulator of the ICC lineage. Using mouse models of Kit activation and Etv1 ablation, we demonstrate that ETV1 is required for GIST initiation and proliferation in vivo, validating it as a therapeutic target. We further uncover a positive feedback circuit where MAP kinase activation downstream of KIT stabilizes the ETV1 protein, and ETV1 positively regulates KIT expression. Combined targeting of ETV1 stability by imatinib and MEK162 resulted in increased growth suppression in vitro and complete tumor regression in vivo. The combination strategy to target ETV1 may provide an effective therapeutic strategy in GIST clinical management. SIGNIFICANCE: ETV1 is a lineage-specific oncogenic transcription factor required for the growth and survival of GIST. We describe a novel strategy of targeting ETV1 protein stability by the combination of MEK and KIT inhibitors that synergistically suppress tumor growth. This strategy has the potential to change first-line therapy in GIST clinical management.