Edwina C. Lerner

Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes

Ras-induced malignant transformation requires Ras farnesylation, a lipid posttranslational modification catalyzed by farnesyltransferase (FTase). Inhibitors of this enzyme have been shown to block Ras-dependent transformation, but the mechanism by which this occurs remains largely unknown. We have designed FTI-276, a peptide mimetic of the COOH-terminal Cys-Val-Ile-Met of K-Ras4B that inhibited potently FTase in vitro (IC = 500 pM) and was highly selective for FTase over geranylgeranyltransferase I (GGTase I) (IC = 50 nM). FTI-277, the methyl ester derivative of FTI-276, was extremely potent (IC = 100 nM) at inhibiting H-Ras, but not the geranylgeranylated Rap1A processing in whole cells. Treatment of H-Ras oncogene-transformed NIH 3T3 cells with FTI-277 blocked recruitment to the plasma membrane and subsequent activation of the serine/threonine kinase c-Raf-1 in cells transformed by farnesylated Ras (H-RasF), but not geranylgeranylated, Ras (H-RasGG). FTI-277 induced accumulation of cytoplasmic non-farnesylated H-Ras that was able to bind Raf and form cytoplasmic Ras/Raf complexes in which Raf kinase was not activated. Furthermore, FTI-277 blocked constitutive activation of mitogen-activated protein kinase (MAPK) in H-RasF, but not H-RasGG, or Raf-transformed cells. FTI-277 also inhibited oncogenic K-Ras4B processing and constitutive activation of MAPK, but the concentrations required were 100-fold higher than those needed for H-Ras inhibition. The results demonstrate that FTI-277 blocks Ras oncogenic signaling by accumulating inactive Ras/Raf complexes in the cytoplasm, hence preventing constitutive activation of the MAPK cascade.

Disruption of oncogenic K-Ras4B processing and signaling by a potent geranylgeranyltransferase I inhibitor

Prenylation of the carboxyl-terminal CAAX (C, cysteine; A, aliphatic acid; and X, any amino acid) of Ras is required for its biological activity. We have designed a CAAX peptidomimetic, GGTI-287, which is 10 times more potent toward inhibiting geranylgeranyltransferase I (GGTase I) in vitro (IC = 5 nM) than our previously reported farnesyltransferase inhibitor, FTI-276. In whole cells, the methyl ester derivative of GGTI-287, GGTI-286, was 25-fold more potent (IC = 2 μM) than the corresponding methyl ester of FTI-276, FTI-277, toward inhibiting the processing of the geranylgeranylated protein Rap1A. Furthermore, GGTI-286 is highly selective for geranylgeranylation over farnesylation since it inhibited the processing of farnesylated H-Ras only at much higher concentrations (IC > 30 μM). While the processing of H-Ras was very sensitive to inhibition by FTI-277 (IC = 100 nM), that of K-Ras4B was highly resistant (IC = 10 μM). In contrast, we found the processing of K-Ras4B to be much more sensitive to GGTI-286 (IC = 2 μM). Furthermore, oncogenic K-Ras4B stimulation of mitogen-activated protein (MAP) kinase was inhibited potently by GGTI-286 (IC = 1 μM) but weakly by FTI-277 (IC = 30 μM). Significant inhibition of oncogenic K-Ras4B stimulation of MAP kinase by GGTI-286 occurred at concentrations (1-3 μM) that did not inhibit oncogenic H-Ras stimulation of MAP kinase. The data presented in this study provide the first demonstration of selective disruption of oncogenic K-Ras4B processing and signaling by a CAAX peptidomimetic. The higher sensitivity of K-Ras4B toward a GGTase I inhibitor has a tremendous impact on future research directions targeting Ras in anticancer therapy.

HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer

Purpose: We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling. Experimental Design: Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice. Results: c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa. Conclusions: These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.

Control of myeloid differentiation and survival by Stats.

Hematopoiesis involves a complex array of growth factors that regulate the survival and proliferation of immature progenitors, influence differentiation commitment, and modulate end-stage cell functions. This mini-review is focused on the role of Stat activation in the development of myeloid cells in response to hematopoietic cytokines. Much of the evidence implicating Stats in these cellular processes comes from studies of mutant cytokine receptors selectively uncoupled from Stat activation, dominant-inhibitory Stat mutants, and mice with targeted disruptions of Stat genes. Together these approaches provide strong evidence that Stat activation, particularly of Stat3 and Stat5, plays an important role in myeloid differentiation and survival.

SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo

Src family protein-tyrosine kinase activity is suppressed by two intramolecular interactions. These involve binding of the SH2 domain to the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the SH2-kinase linker. Here we show that SH3-dependent activation of the Src family member Hck by HIV-1 Nef binding or by SH2-kinase linker mutation does not affect tail tyrosine phosphorylation in fibroblasts. Surprisingly, replacement of the wild type Hck tail with a high-affinity SH2 domain-binding sequence did not affect Hck activation or downstream signaling by these SH3-dependent mechanisms, suggesting that activation through SH3 occurs without SH2–tail dissociation. These results identify SH3–linker interaction as an independent mode of Hck kinase regulation in vivo and suggest that different mechanisms of Src kinase activation may generate distinct output signals because of differences in SH2 or SH3 domain accessibility.

Differential function of STAT5 isoforms in head and neck cancer growth control.

Up-regulation of the epidermal growth factor receptor (EGFR) is critical for the loss of growth control in a variety of human cancers, including squamous cell carcinoma of the head and neck (SCCHN). Stimulation of EGFR results in activation of mitogenic signaling pathways including Signal Transducers and Activators of Transcription (STATs). Stat5 activation has been primarily demonstrated in hematopoietic malignancies. Gene disruption studies suggest potentially distinct functions of the Stat5 isoforms, Stat5a and Stat5b, which are encoded by two genes closely linked on human chromosome 17. To determine the function of Stat5 in SCCHN growth control, we studied the expression and constitutive activation of Stat5a and Stat5b in normal and transformed human squamous epithelial cells. Increased constitutive activation of Stat5 was detected in transformed compared with normal squamous cells. Blockade of TGF-α or EGFR, abrogated Stat5 activation. Targeting of Stat5b using antisense oligonucleotides inhibited SCCHN growth. In addition, SCCHN cells stably transfected with dominant negative mutant Stat5b failed to proliferate in vitro. In contrast, targeting of Stat5a using either antisense or dominant negative strategies had no effect on cell growth. These results suggest that TGF-α/EGFR-mediated autocrine growth of transformed epithelial cells is dependent on activation of Stat5b but not Stat5a.

Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds.

Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent antiviral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling noncatalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry.

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.

Activation of the SRC-family kinase HCK without SH3-linker release

Src family protein-tyrosine kinases are regulated by intramolecular binding of the SH2 domain to the C-terminal tail and association of the SH3 domain with the SH2 kinase-linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To address this question, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. Surface plasmon resonance analysis demonstrated tight intramolecular binding of the modified HAL sequence to SH3. Hck-HAL was then combined with a tail tyrosine mutation (Y501F) and expressed in Rat-2 fibroblasts. Surprisingly, Hck-HAL-Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3-linker release is not required for SH2-based kinase activation. In Saccharomyces cerevisiae, which lacks the negative regulatory tail kinase Csk, wild-type Hck was more strongly activated in the presence of an SH3-binding protein (human immunodeficiency virus-1 Nef), indicating persistence of native SH3-linker interaction in an active Hck conformation. Taken together, these data support the existence of multiple active conformations of Src family kinases that may generate unique downstream signals.

Lyn regulates the cell death response to ultraviolet radiation through c-Jun N terminal Kinase-dependent Fas ligand activation

The Src-related tyrosine kinase, Lyn, plays an important role in mediating the cell cycle arrest and cell death response to genotoxic agents such as ionizing radiation. In this report we provide evidence to show that the catalytic function of Lyn is required for ultraviolet radiation (UV)- and methyl methanesulfonate (MMS)- but not for cisplatin (CDDP)- or ionizing radiation (IR)-induced cell death. Consequently, fibroblasts deficient in Lyn function were protected against cell death induction by UV and MMS, but showed normal cell death to IR and CDDP treatment. In Lyn(-/-) cells, UV-induced activation of stress-responsive kinases, Erk1/2 and p38, was normal; however, JNK activation was diminished. In addition, FasL induction by UV was also diminished in these cells. Reintroduction of wild-type Lyn restored JNK activation, FasL induction, and sensitivity to UV and MMS. A role for FasL in the cell death induction by Lyn-JNK signaling is indicated by the inhibition of cell death response by FasL neutralizing antibody. Together, the results support the presence of the Lyn-JNK signaling pathway that mediates the cell death response to UV and MMS treatment through FasL induction.