The signaling network downstream of the ErbB family of receptors has been extensively targeted by cancer therapeutics; however, understanding the relative importance of the different components of the ErbB network is nontrivial. To explore the optimal way to therapeutically inhibit combinatorial, ligand-induced activation of the ErbB-phosphatidylinositol 3-kinase (PI3K) axis, we built a computational model of the ErbB signaling network that describes the most effective ErbB ligands, as well as known and previously unidentified ErbB inhibitors. Sensitivity analysis identified ErbB3 as the key node in response to ligands that can bind either ErbB3 or EGFR (epidermal growth factor receptor). We describe MM-121, a human monoclonal antibody that halts the growth of tumor xenografts in mice and, consistent with model-simulated inhibitor data, potently inhibits ErbB3 phosphorylation in a manner distinct from that of other ErbB-targeted therapies. MM-121, a previously unidentified anticancer therapeutic designed using a systems approach, promises to benefit patients with combinatorial, ligand-induced activation of the ErbB signaling network that are not effectively treated by current therapies targeting overexpressed or mutated oncogenes.

The muscle LIM protein (MLP) is a muscle-specific LIM-only factor that exhibits a dual subcellular localization, being present in both the nucleus and in the cytoplasm. Overexpression of MLP in C2C12 myoblasts enhances skeletal myogenesis, whereas inhibition of MLP activity blocks terminal differentiation. Thus, MLP functions as a positive developmental regulator, although the mechanism through which MLP promotes terminal differentiation events remains unknown. While examining the distinct roles associated with the nuclear and cytoplasmic forms of MLP, we found that nuclear MLP functions through a physical interaction with the muscle basic helix-loop-helix (bHLH) transcription factors MyoD, MRF4, and myogenin. This interaction is highly specific since MLP does not associate with nonmuscle bHLH proteins E12 or E47 or with the myocyte enhancer factor-2 (MEF2) protein, which acts cooperatively with the myogenic bHLH proteins to promote myogenesis. The first LIM motif in MLP and the highly conserved bHLH region of MyoD are responsible for mediating the association between these muscle-specific factors. MLP also interacts with MyoD-E47 heterodimers, leading to an increase in the DNA-binding activity associated with this active bHLH complex. Although MLP lacks a functional transcription activation domain, we propose that it serves as a cofactor for the myogenic bHLH proteins by increasing their interaction with specific DNA regulatory elements. Thus, the functional complex of MLP-MyoD-E protein reveals a novel mechanism for both initiating and maintaining the myogenic program and suggests a global strategy for how LIM-only proteins may control a variety of developmental pathways.Skeletal myogenesis involves a series of well-orchestrated developmental events in which embryonic stem cells become committed to the myogenic cell lineage, producing muscle precursor cells known as myoblasts (reviewed in reference 49). Myoblasts subsequently withdraw from the cell cycle, fuse into multinucleate syncytia, and synthesize a large number of muscle-specific genes. The onset and progression of the entire myogenic program are controlled through a complex set of signaling pathways that are mediated by environmental cues (reviewed in references 34, 40, and 43). Many of these control points directly affect transcriptional events, often impinging on several muscle-specific transcription factors.Of the many known transcription factor families that regulate myogenesis, the MyoD family stands out as a key regulator. This family of proteins, which includes MyoD, myogenin, Myf-5, and MRF4, shares the remarkable property of inducing myogenic terminal differentiation when expressed in a variety of nonmuscle cells (reviewed in reference 40). The muscle regulatory factors (MRFs) are proteins containing a basic helix-loop-helix (bHLH) motif, with the basic region responsible for DNA binding and the HLH domain mediating proteinprotein interactions. The MRFs heterodimerize with widely expressed bHLH E proteins, such as E12 and E47, and activate mus...

Abstract. Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-1 and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis.These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-1 and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo. p RIMARY skeletal muscle cells and many skeletal muscle cell lines are repressed from terminal differentiation by FGFs. Nine members of the FGF family have been identified (FGF-1 through FGF-9). The hallmarks of this family include (a) high affinity for heparin or heparan sulfate; (b) two invariant conserved cysteine residues; and (c) an overall homology of 30% (Baird and Klagsbrun, 1991). While exogenously added FGFs repress differentiation of cultured skeletal muscle cells (Gospodarowicz et al., 1975;Linkhart et al., 1981;Olwin and Hauschka, 1986), the role of endogenously expressed FGFs, particularly those lacking signal peptide sequences, are not clear. FGF-2, FGF-4, FGF-5, FGF-6, and FGF-8 mRNA appear to be expressed in skeletal muscle cells as they have been localized to the myotomal muscle region of the somites and in the developing limb muscle masses (for review see Olwin et al., 1994 pressed in proliferating or in differentiated muscle cells, we examined FGF expression patterns in mouse myoblasts. Moreover, localization of FGF mRNAs cannot identify functional roles for these proteins in the regulation of skeletal muscle development.Of the three members of the FGF family (FGF-1, FGF-2, and FGF-9) that lack a classical secretory signal sequence, FGF-1 and FGF-2 are implicated in regulating skeletal...

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF ␤ receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF ␤ receptor activated the PDGF ␤ receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF ␤ receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF ␤ receptor-and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF ␤ receptor. Activation of PDGF ␤ receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation. Intracellular signaling cascades initiated by growth factor binding to cell surface tyrosine kinase receptors transmit signals that control cell proliferation, differentiation, cell migration, and cell fate. We are examining the fibroblast growth factor (FGF)-mediated signaling pathways that control proliferation and differentiation of skeletal muscle cells. The effects of FGF on both skeletal muscle cell proliferation and differentiation have been well documented in studies using cell lines (40) and primary cell cultures (1,46,53).A satellite cell line (MM14) derived from an adult mouse muscle exhibits an absolute dependence on exogenously supplied FGFs (12, 34). Removal of FGF from the culture medium results in terminal differentiation, which initiates with an irreversible withdrawal from the cell cycle, is followed by expression of muscle-specific genes and culminates with fusion into multinucleated myotubes. The biological activity of FGFs on these cells requires an interaction with heparan sulfate proteoglycans (HSPGs) (43,48). HSPGs have been proposed to form a high-affinity FGF-binding and signaling complex with the FGF receptor tyrosine ki...

Ligand-stimulated activation of FGF receptors (FGFRs) in skeletal muscle cells represses terminal myogenic differentiation. Skeletal muscle cell lines and subsets of primary cells are dependent on FGFs to repress myogenesis and maintain growth. To understand the intracellular events that transduce these signals, MM14 skeletal muscle cells were transfected with expression vectors encoding chimeric receptors. The chimeras are comprised of the PDGF β receptor (PDGFβR) extracellular domain, the FGFR-1 intracellular domain, and either the PDGFβR or FGFR-1 transmembrane domain. The chimeric receptors were autophosphorylated upon PDGF-BB stimulation and are capable of stimulating mitogen-activated protein kinase activity. Activation of the tyrosine kinase domain of either chimera repressed myogenesis, suggesting intracellular responses regulating skeletal muscle differentiation are transduced by activation of the FGFR-1 tyrosine kinase. Unexpectedly, we found that activation of either chimeric receptor failed to stimulate cellular proliferation. Thus, it appears that regulation of skeletal muscle differentiation by FGFs requires only activation of the FGFR tyrosine kinase. In contrast, stimulation of proliferation may require additional, as yet unidentified, signals involving the receptor ectodomain, the FGF ligand, and heparan sulfate either alone, or in combination.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.

Oncogenic Ras (H-Ras G12V) inhibits skeletal myogenesis through multiple signaling pathways. Previously, we demonstrated that the major downstream e ectors of Ras (i.e., MEK/MAPK, RalGDS and Rac/Rho) play a minor, if any, role in the di erentiation-defective phenotype of Ras myoblasts. Recently, NFkB, another Ras signaling target, has been shown to inhibit myogenesis presumably by stimulating cyclin D1 accumulation and cell cycle progression. In this study, we address the involvement of NFkB activation in the Ras-induced inhibition of myogenesis. Using H-Ras G12V and three G12V e ector-loop variants, we detect high levels of NFkB transcriptional activity in C3H10T1/2-MyoD cells treated with di erentiation medium. Myogenesis is blocked by all Ras proteins tested, yet only in the case of H-Ras G12V are cyclin D1 levels increased and cell cycle progression maintained. Expression of IkBa SR, an inhibitor of NFkB, does not reverse the di erentiation-defective phenotype of Ras expressing cultures, but does induce di erentiation in cultures treated with tumor necrosis factor (TNFa) or in cultures expressing the RelA/p65 subunit of NFkB. These data con®rm that NFkB is a target of Ras and suggest that the cellular actions of NFkB require additional signals that are discriminated by the Ras e ector-loop variants. Results with IkBa SR convincingly demonstrate that H-Ras G12V does not rely on NFkB activity to block myogenesis, an observation that continues to implicate another unidenti®ed signaling pathway(s) in the inhibition of skeletal myogenesis by Ras. Oncogene (2001) 20, 1276 ± 1286