Darlene Barnard

Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association

The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Specifically, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491–508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCα phosphorylation sites failed to significantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no effect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of different stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a significant mechanism of Raf-1 kinase regulation in COS-7 cells.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1s role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe. Mol Cancer Ther; 14(9); 2004–13. ©2015 AACR.

Cyclo-oxygenase-2 expression in primary cancers of the lung and bladder compared to normal adjacent tissue.

Multiple observations in the laboratory and the clinical setting have linked expression of the enzyme cyclo-oxygenase-2 (COX-2) to carcinogenesis. The frequency and amount of COX-2 and Bcl-2 expression in primary lung and bladder cancer sites were detennined by immunoblot analysis of cell lysates prepared from frozen human tumor tissue and matched normal adjacent tissue. COX-2 protein was expressed statistically more frequently and at a higher level in primary adenocarcinomas and squamous cell carcinomas of the lung as well as transitional cell carcinomas of the bladder than in normal adjacent tissue. No correlation was observed between COX-2 and Bcl-2 expression in either the lung or bladder cancer specimens. Immunohistochemistry was also employed to localize COX-2 expression. In addition to expression in the malignant tissues, COX-2 was occasionally localized to the normal bronchial and transitional cell epithelia of the normal adjacent tissue. Detection of COX-2 in histologically normal appearing adjacent tissue suggests that COX-2 may be involved in early cellular changes leading to the development of lung and bladder cancer.

Abstract A108: LY2606368, a second generation Chk1 inhibitor, inhibits growth of ovarian carcinoma xenografts either as monotherapy or in combination with standard-of-care agents.

Chk1 is a key component of the cellular response to DNA damage resulting from exposure to various genotoxic agents. When damaged DNA is present, Chk1 maintains genomic integrity by stalling cell cycle progression thereby providing time for the appropriate repair to occur. Inhibition of Chk1 kinase activity leads to a loss in the regulation of this damage response and results in abrogation of cell cycle arrest and ultimately to cell death as cells with damaged DNA proceed inappropriately into mitosis. Thus first generation small molecule inhibitors of Chk1 kinase were developed to act as companion compounds that would be used together with established standard -of-care (SoC) cancer treatments that target DNA to enhance the effectiveness of such treatments. As a second generation inhibitor, LY2606368 distinguishes itself from other molecules in this class in that it has been shown to inhibit tumor growth when used either as monotherapy or in combination with SoC agents. The single agent activity of LY2606368 is likely related to the ability of this molecule to potently inhibit Chk1 thereby effectively interfering with its function to support normal DNA replication via regulation of origin firing and/or aspects of chromosomal dynamics during mitosis which are thought to be dependent upon Chk1 kinase activity. The studies presented here have evaluated the potential of LY2606368 to inhibit ovarian tumor growth both as monotherapy and in combination with cisplatin or paclitaxel. Treatment of tumor-bearing animals on a BID X 3 schedule with LY2606368 alone resulted in profound growth inhibition such that greater than 80% inhibition was observed at doses of 12mg/kg in both subcutaneous and orthotopic models. Moreover, the studies in the orthotopic SKOV3 model demonstrated that treatment with LY2606368 not only robustly inhibited growth of the primary tumor but also significantly reduced the incidence of metastases and accumulation of ascites fluid. Subsequent studies in SKOV3 subcutaneous xenografts revealed that significant growth inhibition could be achieved with various combination strategies which include agents that are commonly used in the treatment of ovarian cancer. In particular, the extent of growth inhibition that could be achieved with cisplatin or paclitaxel alone was augmented by combining these agents with LY2606368. Overall these animal studies indicate the potential therapeutic benefit in ovarian cancer that could be achieved with LY2606368 either as monotherapy or in combination with SoC agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A108.

Abstract B248: Characterization and preclinical development of LCI‐1, a selective and potent Chk1 inhibitor in phase 1 clinical trials

Interference of DNA damage checkpoints has been demonstrated to be a highly effective means of increasing the cytotoxicity of a wide number of anti‐cancer therapies. Ionizing radiation, DNA cross‐linkers, topoisomerase inhibitors and anti‐metabolites all cause severe cellular DNA damage and activation of multiple DNA damage checkpoints. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until the DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA proceed into premature mitosis followed by rapid apoptotic death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 leads to abrogation of the DNA damage checkpoints and has been shown to enhance the preclinical activity of many standard of care chemotherapeutic agents. LCI‐1 is a novel small molecule inhibitor of Chk1 (IC50 = 7 nM). In cell‐based experiments LCI‐1 inhibited doxorubicin‐dependent autophosphorylation of Chk1 (IC50 = 52 nM). Treatment of cells with LCI‐1 alone caused a phenotype identical to that reported for cells depleted of Chk1, demonstrating the mechanism‐dependent activity of the compound. Treated with 100 nM doxorubicin, HeLa cells arrested at the G2M checkpoint. When treated 24 hours later with LCI‐ 1, the arrested cells traversed the G2M checkpoint, allowing cells to proceed into mitosis with unresolved replicated chromosomes. Consistent with abrogation of the Chk1‐dependent G2M checkpoint, HT‐29 cells (mutant p53) treated with LCI‐1 were more sensitive to killing by gemcitabine then were LCI‐1 treated HCT116 cells (wild‐type p53). In vivo, LCI‐1 effectively inhibited Chk1 autophosphorylation, as well as released the S and G2M block induced by gemcitabine treatment. When used preclinically in combination with DNA damaging agents, LCI‐1 increased DNA damage and cell death over gemcitabine alone. These results indicate that LCI‐1 may prove to be an effective potentiator of DNA damaging therapies in the clinic. LCI‐1 is currently undergoing clinical testing in combination with gemcitabine and pemetrexed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B248.

Abstract A49: Circulating tumor cell (CTC) assay development for detection of H2AX as a clinical pharmacodynamic (PD) marker for Chk1 kinase inhibitors.

Circulating tumor cells are prognostic of survival in metastatic breast, colon, and prostate cancers. Additionally, CTCs are of interest because they may be representative of the phenotype/genotype of the primary and metastatic tumors, and a useful tool (e.g. patient stratification) for drug development. CTCs, as “liquid biopsies” are potentially useful as a pharmacodynamic marker allowing easy repeat sampling before and after drug treatment. We describe the development of a CTC assay measuring H2AX induction, a marker of DNA damage. The CTC assay was developed using the FDA cleared Veridex/CellSearch™ instrument and reagents for enumeration of CTCs from blood. CellSearch defines CTCs as EpCam+/DAPI+/CK 8,18,19+/CD45−. For assay development, cells from tumor cell lines representing major solid tumor types were chosen, cultured, treated with inhibitors of checkpoint kinase 1 (Chk1, LY2603618, LY2606368) or with various standard-of-care (SOC) chemotherapeutics. These treated cells were then spiked into whole blood drawn into CellSave™ (preservative+EDTA). LY2603618 and LY2606368 are inhibitors of Chk1 kinase currently in clinical development. LY2603618 is meant to be used in combination with a DNA damaging agent, while LY2606368 has potent single agent activity. Inhibition of Chk1 in cells with DNA damage allows the cells to progress into mitosis without DNA repair, leading to cell death. In tumor xenograft mouse models, treatment reduced tumor volume with elevation of γH2AX in tumors and anagen hair follicles at drug exposure levels similar to concentrations used for the CTC γH2AX development assay. For initial assay development, mouse whole blood was used and results reproduced subsequently with human whole blood from healthy subjects. The spiked tumor cells in blood samples were recovered using the CellSearch Mouse/Rat CTC kit (for mouse blood) or human CXC kit (for human blood), supplemented with the R-PE conjugated anti-γH2AX antibody. γH2AX in the recovered tumor cells was detected in the open/fourth channel on the CellSearch instrument. Results were confirmed by flow analysis. Without drug treatment, only about 0.2–2% of all cultured tumor cells were γH2AX positive. The magnitude of the induction of γH2AX in the cells after treatment was dose and cell-line dependent. Significant induction of H2AX (>20% cells) was observed after 24 hour treatment with only a few SOC agents (cisplatin, etoposide) in sensitive tumor cell lines; the majority induced moderate to low levels (usually Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A49.