Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1-and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1-and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wntdriven cancer.antibody therapeutics | cancer T he Wnt/β-catenin pathway regulates diverse biological processes during development and tissue homeostasis by modulating the protein stability of β-catenin (1-3). In the absence of extracellular Wnt proteins, cytoplasmic β-catenin is associated with the β-catenin destruction complex and degraded by ubiquitinmediated proteolysis. Wnt signals are transduced by two distinct receptors, the serpentine receptor Frizzled (Frz) and the singlespan transmembrane proteins LRP5 or LRP6. Wnt proteins promote the assembly of the Frz-LRP5/6 signaling complex and induce phosphorylation of LRP5 or LRP6. Phosphorylated LRP5 or LRP6 inactivates the β-catenin degradation complex, allowing stabilized β-catenin to enter the nucleus, bind to the TCF transcription factors, and act as a transcriptional coactivator.The extracellular domain of LRP5 or LRP6 contains four YWTD-type β-propeller domains each followed by an EGF-like domain and an LDLR domain. Each propeller contains six YWTD motifs that form a six-bladed β-propeller structure (4). Biochemical studies suggest that Wnt proteins physically interact with both Frz and LRP6 and induce the formation of an Frz-

Polyploid or polytene cells, which have more than 2C DNA content, are widespread throughout nature and present in most differentiated Drosophila tissues. These cells also can display differential replication, that is, genomic regions of increased or decreased DNA copy number relative to overall genomic ploidy. How frequently differential replication is used as a developmental strategy remains unclear. Here, we use genome-wide array-based comparative genomic hybridization (aCGH) to profile differential DNA replication in isolated and purified larval fat body and midgut tissues of Drosophila, and we compare them with recent aCGH profiles of the larval salivary gland. We identify sites of euchromatic underreplication that are common to all three tissues and others that are tissue specific. We demonstrate that both common and tissuespecific underreplicated sites are dependent on the Suppressor of Underreplication protein, SUUR. mRNA-seq profiling shows that whereas underreplicated regions are generally transcriptionally silent in the larval midgut and salivary gland, transcriptional silencing and underreplication have been uncoupled in the larval fat body. In addition to revealing the prevalence of differential replication, our results show that transcriptional silencing and underreplication can be mechanistically uncoupled.

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HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of PI3K signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-drive tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells and it displayed single agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations and was similarly active in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope within domains 2 and 4 that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that in combination with HER2 or EGFR-targeted agents may leverage their clinical efficacy in ErbB-driven cancers.