First-in-human phase 1 study of margetuximab (MGAH22), an Fc-modified chimeric monoclonal antibody, in patients with HER2-positive advanced solid tumors.

Background: Margetuximab is an anti-HER2 antibody that binds with elevated affinity to both the lower and higher affinity forms of CD16A, an Fc-receptor important for antibody dependent cell-mediated cytotoxicity (ADCC) against tumor cells. A Phase 1 study was initiated to evaluate the toxicity profile, maximum tolerated dose (MTD), pharmacokinetics, and antitumor activity of margetuximab in patients with HER2-overexpressing carcinomas. Patients and methods: Patients with HER2-positive breast or gastric cancer, or other carcinomas that overexpress HER2, for whom no standard therapy was available, were treated with margetuximab by intravenous infusion at doses of 0.1-6.0 mg/kg for 3 of every 4 weeks (Regimen A) or once every 3 weeks (10-18 mg/kg) (Regimen B). Results: Sixty-six patients received margetuximab (34 patients for Regimen A and 32 patients for Regimen B). The MTD was not reached for either regimen. Treatment was well-tolerated, with mostly Grade 1 and 2 toxicities consisting of constitutional symptoms such as pyrexia, nausea, anemia, diarrhea, and fatigue. Among 60 response-evaluable patients, confirmed partial responses and stable disease were observed in 7 (12%) and 30 (50%) patients, respectively; 26 (70%) of these patients had received prior HER2-targeted therapy. Tumor reductions were observed in over half (18/23, 78%) of response-evaluable patients with breast cancer including durable (>30 weeks) responders. Ex vivo analyses of patient peripheral blood mononuclear cell samples confirmed the ability of margetuximab to support enhanced ADCC compared with trastuzumab. Conclusions: Margetuximab was well-tolerated and has promising single-agent activity. Further development efforts of margetuximab as single agent and in combination with other therapeutic agents are ongoing. Trial Registration ID: NCT01148849.

Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.

Smad proteins transduce signals from TGF-beta receptors and regulate transcription of target genes either directly or in combination with other sequence-specific transcription factors. AP-1 sites and their cognate transcription factors also play important roles in the gene regulatory activities of TGF-beta. In this report, we have investigated the functional interactions of the Smad and AP-1 transcription factors. We demonstrate that Smad and AP-1 complexes specifically bind to their cognate cis-elements and do not interact with each other on-DNA, whereas off-DNA interactions occur between Smad3 and both c-Jun and JunB. Using both artificial constructs specific for either the Smad or AP-1 signaling pathways or natural promoters known to be TGF-beta-responsive, we have determined that Jun family members downregulate Smad3-mediated gene transactivation whereas AP-1-dependent promoters are synergistically activated by Smad3 and Jun proteins. We propose a model where the presence of Smad- and/or AP-1-specific cis-elements within TGF-beta-responsive genes allows dynamic modulation of gene expression, in contrast to the existing model where interactions between Smad and AP-1 proteins are merely an on/off mechanism to regulate TGF-beta/Smad targets.

Transforming growth factor-beta receptor-associated protein 1 is a Smad4 chaperone.

Members of the transforming growth factor-beta (TGF-beta) superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-beta receptors and suggested to play a role in TGF-beta signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-beta and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-beta signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-beta signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases.

Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

Targeted mutagenesis of Smad1 reveals an essential role in chorioallantoic fusion.

The Smad family of intracellular signaling intermediates transduce signals downstream from the transforming growth factor beta (TGF-beta) family of receptor serine threonine kinases. The original member of this family, Smad1, has been shown to mediate signals from receptors for the bone morphogenetic proteins (BMPs), a large group of ligands in the TGF-beta superfamily that mediate important developmental events. We have targeted the Smad1 gene in mice and created mutants null at this locus. Smad1 mutant mice die at approximately 9.5 days postcoitum due to defects in allantois formation. In Smad1 mutant mice, the allantois fails to fuse to the chorion, resulting in a lack of placenta and failure to establish a definitive embryonic circulation. Although vasculogenesis is initiated in the mutant allantois, the vessels formed are disorganized, and VCAM-1 protein, a marker for distal allantois development, is not expressed. Smad1 null fibroblasts are still able to respond to BMP2, however, suggesting that the defect observed in the developing extraembryonic tissue is caused by a very specific loss of transcriptional activity regulated by Smad1. Our data further demonstrate that although highly similar structurally, Smad proteins are not functionally homologous.

A novel smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction.

Members of the transforming growth factor-beta superfamily play critical roles in controlling cell growth and differentiation. Effects of TGF-beta family ligands are mediated by Smad proteins. To understand the mechanism of Smad function, we sought to identify novel interactors of Smads by use of a yeast two-hybrid system. A 396-amino acid nuclear protein termed SNIP1 was cloned and shown to harbor a nuclear localization signal (NLS) and a Forkhead-associated (FHA) domain. The carboxyl terminus of SNIP1 interacts with Smad1 and Smad2 in yeast two-hybrid as well as in mammalian overexpression systems. However, the amino terminus of SNIP1 harbors binding sites for both Smad4 and the coactivator CBP/p300. Interaction between endogenous levels of SNIP1 and Smad4 or CBP/p300 is detected in NMuMg cells as well as in vitro. Overexpression of full-length SNIP1 or its amino terminus is sufficient to inhibit multiple gene responses to TGF-beta and CBP/p300, as well as the formation of a Smad4/p300 complex. Studies in Xenopus laevis further suggest that SNIP1 plays a role in regulating dorsomedial mesoderm formation by the TGF-beta family member nodal. Thus, SNIP1 is a nuclear inhibitor of CBP/p300 and its level of expression in specific cell types has important physiological consequences by setting a threshold for TGF-beta-induced transcriptional activation involving CBP/p300.

The MSG1 non-DNA-binding transactivator binds to the p300/CBP coactivators, enhancing their functional link to the Smad transcription factors.

The MSG1 nuclear protein has a strong transcriptional activating activity but does not bind directly to DNA. When cotransfected, MSG1 enhances transcription mediated by the Smad transcription factors in mammalian cells in a manner dependent on ligand-induced Smad hetero-oligomerization. However, the mechanism of this MSG1 effect has been unknown. We now show that MSG1 directly binds to the p300/cAMP-response element-binding protein-binding protein (CBP) transcriptional coactivators, which in turn bind to the Smads, and enhances Smad-mediated transcription in a manner dependent on p300/CBP. The C-terminal transactivating domain of MSG1 is required for binding to p300/CBP and enhancement of Smad-mediated transcription; the viral VP16 transactivating domain could not substitute for it. In the N-terminal region of MSG1, we identified a domain that is necessary and sufficient to direct the specific interaction of MSG1 with Smads. We also found that the Hsc70 heat-shock cognate protein also forms complex with MSG1 in vivo, suppressing both binding of MSG1 to p300/CBP and enhancement of Smad-mediated transcription by MSG1. These results indicate that MSG1 interacts with both the DNA-binding Smad proteins and the p300/CBP coactivators through its N- and C-terminal regions, respectively, and enhances the functional link between Smads and p300/CBP.

Selective loss of the transforming growth factor-beta apoptotic signaling pathway in mutant NRP-154 rat prostatic epithelial cells.

Retroviral insertional mutagenesis was used to select mutant NRP-154 rat prostate carcinoma cells resistant to transforming growth factor (TGF)-beta-induced cell death. Similar to the parental cells, a mutant clone, M-NRP1, expressed TGF-beta receptors and was still responsive to induction both of direct target genes by TGF-beta and of apoptosis by staurosporine or okadaic acid. In contrast, indicators of cell growth, strongly suppressed by TGF-beta in the parental cells, were unaffected in M-NRP1 cells. M-NRP1 cells overexpress the antiapoptotic protein, Bcl-xL, and show dysregulated expression and localization of a protein related to a novel human septin, ARTS (designation of apoptotic response to TGF-beta signals), cloned by homology to an exonic sequence flanked by the viral long terminal repeats in M-NRP1 cells and shown to make cells competent to undergo apoptosis in response to TGF-beta. We propose that ARTS might operate within the same apoptotic pathway as Bcl-xL and that M-NRP1 cells could serve as a useful model for characterization of this pathway.

The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

A novel mitochondrial septin-like protein, ARTS, mediates apoptosis dependent on its P-loop motif.

Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-beta signalling pathway). ARTS is expressed in many cells and acts to enhance cell death induced by TGF-beta or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.

Functional analysis of human Smad1: role of the amino-terminal domain.

The signals originating from transforming growth factor beta/activin/bone morphogenetic proteins (BMPs) are transduced by a set of evolutionarily conserved family of Smad proteins which, upon activation, directly translocate to the nucleus where they may activate transcription. Smad proteins of different species contain conserved amino- (N) and carboxy- (C) terminal domains separated by a proline-rich linker. Human, Drosophila, and Xenopus Smad1 all have been shown to mediate the biological effects of BMP-4 in Xenopus embryos. We have investigated the functional domains of human Smad1 (hSmad1) using the Xenopus embryo system. Dorsal injection of hSmad1 RNA into the 4-cell-stage embryos results in embryonic ventralization. Since the C-terminus of Smads has been shown to mediate the transcriptional activity, whereas this activity is masked by the presence of the N-terminus, we tested the effect of a hSmad1 construct lacking the C-terminal domain [hSmad1(N)] in the Xenopus embryo system. Surprisingly, we found that hSmad1(N) not only synergizes with hSmad1 in embryonic ventralization, but induces ventralization by itself. Ectopic expression of a dominant negative BMP receptor (DN-BR) as well as neural inducers noggin and chordin induce neurogenesis in the animal cap, which is inhibited by co-expression of either hSmad1 or hSmad1(N). Ventral expression of DN-BR induces formation of a second body axis at tailbud stage, which is also prevented by hSmad1 and hSmad1(N). It has recently been reported that calmodulin interacts with the N-terminal domain of Smad proteins. We demonstrate that the ventralizing activity of hSmad1 and hSmad1(N) is markedly inhibited by calmodulin. Thus, calmodulin acts as a Smad1 inhibitor. A model is proposed to accomodate these findings.

Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.

SMAD3 is one of the intracellular mediators that transduces signals from transforming growth factor-beta (TGF-beta) and activin receptors. We show that SMAD3 mutant mice generated by gene targeting die between 1 and 8 months due to a primary defect in immune function. Symptomatic mice exhibit thymic involution, enlarged lymph nodes, and formation of bacterial abscesses adjacent to mucosal surfaces. Mutant T cells exhibit an activated phenotype in vivo, and are not inhibited by TGF-beta1 in vitro. Mutant neutrophils are also impaired in their chemotactic response toward TGF-beta. Chronic intestinal inflammation is infrequently associated with colonic adenocarcinoma in mice older than 6 months of age. These data suggest that SMAD3 has an important role in TGF-beta-mediated regulation of T cell activation and mucosal immunity, and that the loss of these functions is responsible for chronic infection and the lethality of Smad3-null mice.

SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.

The human type VII collagen gene (COL7A1) recently has been identified as an immediate-early response gene for transforming growth factor beta (TGF-beta)/SMAD signaling pathway. In this study, by using MDA-MB-468 SMAD4-/- breast carcinoma cells, we demonstrate that expression of SMAD4 is an absolute requirement for SMAD-mediated promoter activity. We also demonstrate that the SMAD binding sequence (SBS) representing the TGF-beta response element in the region -496/-444 of the COL7A1 promoter functions as an enhancer in the context of a heterologous promoter. Electrophoretic mobility-shift assays with nuclear extracts from COS-1 cells transfected with expression vectors for SMADs 1-5 indicate that SMAD3 forms a complex with a migration similar to that of the endogenous TGF-beta-specific complex observed in fibroblast extracts. Electrophoretic mobility-shift assays using recombinant glutathione S-transferase-SMAD fusion proteins indicate that both SMAD4 and C-terminally truncated SMAD3, but not SMAD2, can bind the COL7A1 SBS. Coexpression of SMAD3 and SMAD4 in COS-1 cells leads to the formation of two complexes: a DNA/protein complex containing SMAD3 alone and another slower-migrating complex containing both SMAD3 and SMAD4, the latter complex not being detected in fibroblasts. Maximal transactivation of COL7A1 SBS-driven promoters in either MDA-MB-468 carcinoma cells or fibroblasts requires concomitant overexpression of SMAD3 and SMAD4. These data may represent the first identification of a functional homomeric SMAD3 complex regulating a human gene.

Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator.

Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor beta signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.

Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases.

SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.

Smad-dependent transcriptional activation of human type VII collagen gene (COL7A1) promoter by transforming growth factor-beta.

We have previously shown that transforming growth factor-beta (TGF-beta) increases type VII collagen gene (COL7A1) expression in human dermal fibroblasts in culture (Mauviel, A., Lapière, J.-C., Halcin, C., Evans, C. H., and Uitto, J. (1994) J. Biol. Chem. 269, 25-28). To gain insight into the molecular mechanisms underlying the up-regulation of COL7A1 by this growth factor, we performed transient cell transfections with a series of 5'-deletion promoter/chloramphenicol acetyltransferase reporter gene constructs. We identified a 68-base pair region between nucleotides -524 and -456, relative to the transcription start site, as critical for TGF-beta response. Using electrophoresis mobility shift assays (EMSAs) with an oligonucleotide spanning the region from -524 to -444, we discovered that a TGF-beta-specific protein-DNA complex was formed as early as 11 min after TGF-beta stimulation and persisted for 1 h after addition of the growth factor. Deletion analysis of the TGF-betaresponsive region of the COL7A1 promoter by EMSA identified segment -496/-444 as the minimal fragment capable of binding the TGF-beta-induced complex. Furthermore, two distinct segments, -496/-490 and -453/-444, appeared to be necessary for TGF-beta-induced DNA binding activity, suggesting a bipartite element. Supershift experiments with a pan-Smad antibody unambiguously identified the TGF-beta-induced complex as containing a Smad member. This is the first direct identification of binding of endogenous Smad proteins to regulatory sequences of a human gene.

Characterization of functional domains within Smad4/DPC4.

Smad proteins are a family of highly conserved, intracellular proteins that signal cellular responses downstream of transforming growth factor-beta (TGF-beta) family serine/threonine kinase receptors. One of these molecules, Smad4, originally identified as the candidate tumor suppressor gene dpc-4, reconstitutes TGF-beta- and activin-dependent transcriptional responses in Smad4 null cell lines and interacts in a ligand-dependent manner with other Smad family members in both TGF-beta, activin, and bone morphogenetic protein-2/-4 pathways. Here, we used an assay based on the restoration of ligand-dependent transcriptional responses in a Smad4 null cell line to characterize functional domain structures within Smad4. We showed that restoration of TGF-beta-induced transcriptional responses by Smad4 was inhibited by co-transfection with a kinase dead TGF-beta type II receptor and that constitutive activation was blocked with TGF-beta neutralizing antibodies, confirming the essential role of Smad4 in TGF-beta signaling. Using a series of Smad4 mutation, deletion, and Smad1/Smad4 chimera constructs we identified a 47-amino acid deletion within the middle-linker region of Smad4 that is essential for the mediation of signaling responses. In addition, we showed that the NH2-terminal domain of Smad4 augments ligand-dependent activation associated with the middle-linker region, indicating that there is a distinct ligand-response domain within the N terminus of this molecule.

The immunophilin FKBP12 functions as a common inhibitor of the TGF beta family type I receptors.

The immunophilin FKBP12 is an evolutionarily conserved abundant protein; however, its physiological roles remain poorly defined. Here we report that FKBP12 is a common cytoplasmic interactor of TGF beta family type I receptors. FKBP12 binds to ligand-free TGF beta type I receptor, from which it is released upon a ligand-induced, type II receptor mediated phosphorylation of the type I receptor. Blocking FKBP12/type I receptor interaction with FK506 nonfunctional derivatives enhances the ligand activity, indicating that FKBP12 binding is inhibitory to the signaling pathways of the TGF beta family ligands. Overexpression of a myristylated FKBP12 in Mv1Lu cell specifically inhibits two separate pathways activated by TGF beta, and two point mutations on FKBP12 (G89P, I90K) abolish the inhibitory activity of FKBP12, suggesting that FKBP12 may dock a cytoplasmic protein to the type I receptors to inhibit TGF beta family mediated signaling.

Serine phosphorylation, chromosomal localization, and transforming growth factor-beta signal transduction by human bsp-1.

The transforming growth factor-beta (TGF-beta) superfamily regulates a multitude of cellular and developmental events. TGF-beta family ligands signal through transmembrane serine/threonine kinase receptors whose downstream effectors are largely unknown. Using genetic data from the fruit fly, we have identified a downstream effector of TGF-beta-induced signaling. TGF-beta signaling protein-1 (BSP-1) is rapidly phosphorylated in response to TGF-beta. Localization of bsp-1 to chromosome 4q28 suggests a role in carcinogenesis. These data suggest that BSP-1 is the prototype of a new class of signaling molecules.