Collin M. Blakely

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Hormone-Induced Protection against Mammary Tumorigenesis Is Conserved in Multiple Rat Strains and Identifies a Core Gene Expression Signature Induced by Pregnancy

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-beta signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer.

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

Regulation of Interferon-Induced Protein Kinase PKR: Modulation of P58IPK Inhibitory Function by a Novel Protein, P52rIPK

ABSTRACT The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58IPK, a cellular PKR inhibitor. P58IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2α subunit (eIF-2α) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58IPK-interacting protein. This protein, designated P52rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52rIPK and P58IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58IPKrepressed PKR-mediated eIF-2α phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52rIPK into these strains resulted in restoration of both PKR activity and eIF-2α phosphorylation, concomitant with growth suppression due to inhibition of P58IPK function. Furthermore, P52rIPKinhibited P58IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58IPKis inhibited through a direct interaction with P52rIPKwhich, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.

Developmental stage determines the effects of MYC in the mammary epithelium

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.

Inactivation of Capicua drives cancer metastasis

Metastasis is the leading cause of death in people with lung cancer, yet the molecular effectors underlying tumor dissemination remain poorly defined. Through the development of an in vivo spontaneous lung cancer metastasis model, we show that the developmentally regulated transcriptional repressor Capicua (CIC) suppresses invasion and metastasis. Inactivation of CIC relieves repression of its effector ETV4, driving ETV4-mediated upregulation of MMP24, which is necessary and sufficient for metastasis. Loss of CIC, or an increase in levels of its effectors ETV4 and MMP24, is a biomarker of tumor progression and worse outcomes in people with lung and/or gastric cancer. Our findings reveal CIC as a conserved metastasis suppressor, highlighting new anti-metastatic strategies that could potentially improve patient outcomes.

Tissue specific expression of PKR protein kinase in aging B6D2F1 mice

A decline in the rate of protein synthesis is a common biochemical change observed with aging in a wide variety of cells and organisms. The double stranded RNA-dependent protein kinase PKR has been shown to phosphorylate eukaryotic initiation factor 2 alpha (eIF-2alpha), a well-characterized factor for down-regulating protein synthesis, in response to environmental stress conditions. Therefore, we were interested in evaluating the role of PKR in the aging process. Tissues from 2- and 20-month-old B6D2F1 male mice were evaluated by Western blot analysis. PKR was detected in all tissues of aging mice confirming its ubiquitous nature. Tissues examined from young mice showed little evidence of PKR expression, suggesting an age-associated up-regulation. P58(IPK), a cellular inhibitor of PKR, was expressed in tissues from both age groups but to a greater extent in tissues of aging mice suggesting an up-regulation to control PKR activity. Hyperphosphorylated eIF-2alpha was increased in selected tissues from older mice compared with tissues from younger mice indicating a possible correlation between PKR expression and kinase function. The data suggest that translational activity is slowing down in a tissue specific manner during the aging process in mice, possibly as the result of increased levels of PKR, and could be a factor in the reduction of the rate of protein synthesis during senescence seen in specific tissues of many organisms.

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/β-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.

Resiliency of Lung Cancers to EGFR Inhibitor Treatment Unveiled, Offering Opportunities to Divide and Conquer EGFR Inhibitor Resistance

The clinical success of EGF receptor (EGFR) inhibitors in patients with lung cancer is limited by the inevitable development of treatment resistance. Two reports in this issue of Cancer Discovery uncover additional mechanisms by which EGFR-mutant lung cancers escape from EGFR kinase inhibitor treatment. These findings pave the way for clinical testing of new rational therapeutic strategies to prevent or overcome resistance to EGFR kinase inhibitors in the clinic.

Emerging antiangiogenic therapies for non-small-cell lung cancer

Lung cancer remains the leading cause of cancer-related deaths. Antiangiogenic therapy has increasingly been studied for advanced non-small-cell lung cancer (NSCLC). Bevacizumab is the only approved antiangiogenic agent for NSCLC and has shown progression-free survival benefits in large Phase III studies and an overall survival benefit in the Phase III E4599 trial in advanced nonsquamous NSCLC. New antiangiogenic treatment strategies are being evaluated that target multiple receptors within a family (VEGF receptor [VEGFR]-1, VEGFR-2) or multiple angiogenic pathways (targets VEGFR and PDGF receptor pathways), and agents that inhibit alternative mediators of angiogenesis (integrins and established vasculature). As data become available from ongoing studies, it will be important to determine how these new antiangiogenic agents will best fit into the current NSCLC treatment paradigm.