PDF file - 237K, Figure S5. Combination treatment consisting of trastuzumab+LJM716 or Cetuximab+LJM716 is well tolerated.

PDF file - 181K, Figure S8. PIK3CA mutation is associated with reduced activity contributed by Trastuzumab (T) in Trastuzumab + LJM716 (L) combinations.

PDF file - 272K, Figure S4. The NRG1/ HER3 binding affinity is unaffected by LJM716.

PDF file - 1205K, Figure S7. Treatment of HER2 amplified cell lines with LJM716/ trastuzumab combinations is synergistic in a subset of cell lines.

PDF file - 239K, Figure S3. LJM716 is specific for HER3 and binds a conformational epitope that includes key residues in D2.

PDF file - 459K, Figure S1. HER2, HER3 and members of the PI3K signaling pathway are activated in HER2 amplified cell lines.

PDF file - 268K, Figure S2. LJM716 inhibits HER3 mediated PI3K signaling.

PDF file - 416K, Figure S6. Trastuzumab/LJM716 combination inhibits 3D spheroid growth of breast cancer cell lines more effectively than trastuzumab/pertuzumab combination.

BTLA is a co-inhibitory immune checkpoint molecule sharing sequence and structural homology with PD-1 and CTLA4. Ligation of HVEM with BTLA induces recruitment of SHP1 and SHP2, and triggers inhibition of T cell proliferation and cytokine production. In melanoma, HVEM is described to have a broader expression than PD-L1 and constituted a negative prognostic marker; in PD-L1 negative NSCLC, the expression of HVEM has been shown to contribute to immune escape. Therefore BTLA-HVEM axis could play an important role in the immune escape, thus BTLA blockade in combination with PD-1/PD-L1 blockade could represent an effective therapeutic option. Using our proprietary microfluidic-based single cell platform CelliGO™, we identified a series of anti-human BTLA antibodies that were characterized for their binding affinity, cross-reactivity, selectivity and functional activity. Amongst them, HFB200603 was identified as a single-digit nanomolar binder to human and cynomolgus BTLA, capable of reversing HVEM-mediated immune suppression in a BTLA-HVEM reporter system and in a primary CD4+ T cell proliferation assay. HFB200603 showed synergistic effect with anti-PD-1 to enhance IFN-γ production in an MLR assay, and demonstrated favorable developability and pharmacokinetic profiles. Profiling of tumor infiltrating lymphocytes demonstrated that BTLA+PD-1+ T cells are present in melanoma, NSCLC, and HCC. Blockade of the BTLA-HVEM interaction with HFB200603 alone or in combination with anti-PD-1 led to increases in IFN-γ, CXCL9, IP-10 and other proinflammatory cytokines in primary dissociated tumor cultures. Based on its favorable pharmacological activity and excellent developability, HFB200603 is currently being developed as a potential novel immunotherapy coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Juying Li, Qian Zhang, Bingqing Shen, Manan Shah, Mingjie Chen, Sharon Li, Ling Dong, Francisco Adrian, Liang Schweizer. HFB200603, a novel anti-BTLA monoclonal antibody that provides therapeutic potential for immune escape and synergizes with anti-PD-1 treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4248.

Abstract PURPOSE: Several observational studies and multinutrient interventional trials suggest that selenium compounds have potential chemopreventive effects on esophageal and gastric carcinogenesis, presumably via their influence on selenoproteins. We aimed to investigate associations between genetic variants in selenoprotein genes and risk of esophageal squamous cell carcinoma (ESCC), gastric carcinoma (GC) and GC subsets: cardia (GCA) and non-cardia (GNCA) using data from a genome-wide association study in a Han Chinese population in which ESCC and GC are predominant cancers. METHODS: 1944 ESCC cases, 1758 GC cases (1126 GCA and 632 GNCA) and 2111 controls from the Shanxi Upper GI Cancer Genetics project and the Linxian Nutritional Intervential Trials were genotyped for 243 SNPs in 24 selenoprotein genes. Logistic regression models were applied to evaluate SNP-level associations. Pathway- and gene-level associations were determined using the resampling-based adaptive rank-truncated product approach. RESULTS: Selenoproteins as a whole pathway were not significantly associated with ESCC (p=0.398) or GC (p=0.608), but five significant (p<0.05) gene-level associations were identified, including GPX2 (p=0.008) and GPX5 (p=0.006) with ESCC, DIO2 (p=0.026) with GC, and SEPP1 (p=0.007) and SEPW1 (p=0.004) with GCA. CONCLUSION: Our results show evidence for an association between specific selenoprotein genes and the risk of ESCC and GC, warranting further studies to validate these associations and investigate underlying mechanisms. Citation Format: Sharon Li, Paula L. Hyland, Neal D. Freedman, Nan Hu, Hua Su, Lemin Wang, Chaoyu Wang, Ti Ding, Yuan Wang, Jin-Hu Fan, You-Lin Qiao, Xiaoqin Xiong, Kai Yu, Alisa M. Goldstein, Sanford M. Dawsey, Philip R. Taylor, Christian C. Abnet, Shih-Wen Lin. Genetic variants in selenoprotein genes and risk of esophageal squamous cell carcinoma and gastric cancer in a Chinese population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2206. doi:10.1158/1538-7445.AM2014-2206

The mechanisms that underlie metazoan DNA replication initiation, especially the connection between transcription and replication origin activation, are not well understood. To probe the role of transcription in origin activation, we exploited a specific replication origin in Drosophila melanogaster follicle cells, ori62, which coincides with the yellow-g2 transcription unit and exhibits transcription-dependent origin firing. Within a 10-kb genomic fragment that contains ori62 and is sufficient for amplification, RNA-sequencing analysis revealed that all detected RNAs mapped solely to the yellow-g2 gene. To determine whether transcription is required in cis for ori62 firing, we generated a set of tagged yellow-g2 transgenes in which we could prevent local transcription across ori62 by deletions in the yellow-g2 promoter. Surprisingly, inhibition of yellow-g2 transcription by promoter deletions did not affect ori62 firing. Our results reveal that transcription in cis is not required for ori62 firing, raising the possibility that a trans-acting factor is required specifically for the activation of ori62. This finding illustrates that a diversity of mechanisms can be used in the regulation of metazoan DNA replication initiation.

206 words Text (incl. abstract) but excluding references, acknowledgements and supplementary information: 5212 words Figures: 5 (all full color) Research. on January 22, 2018. © 2013 American Association for Cancer cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/0008-5472.CAN-13-1198

We are utilizing the Drosophila follicle cell gene amplification system to better understand the mechanisms of DNA replication origin activation in metazoans. During gene amplification, specific origins of replication undergo repeated rounds of activation at six distinct sites in the follicle cell genome. These Drosophila Amplicons in Follicle Cells (DAFCs) utilize the same replication machinery as normal DNA replication, including the Origin Recognition Complex (ORC), DUP/Cdt1, and the MCM2–7 helicase. However, the mechanisms controlling the activation of amplification origins remain poorly understood. We identified an origin at DAFC‐62D whose activation is dependent upon transcription. We will present experiments utilizing transgenic DAFC‐62D transposon insertions to test whether transcription is required locally for origin activation. Additionally, we are investigating the effects of local chromatin on origin activation. We have identified cis‐acting position effects acting over at least 5 kb that repress ORC binding and amplification at DAFC‐22B.

The trans -translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans -translation as a target for antibiotic development, a high-throughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans -translation tagging reaction with an IC 50 = 0.9 µM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans -translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.

Abstract HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven 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 with similar activity 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. Cancer Res; 73(19); 6024–35. ©2013 AACR.

Abstract HER3 (ErbB3) is a member of the ErbB family of receptor tyrosine kinases (RTK). Inappropriate HER2/ HER3 dimerization as a result of HER2 or neuregulin (NRG) over-expression in cancer results in HER3 mediated activation of PI3K signaling. Consequently, HER3 is a mediator of oncogenic transformation. Although, ligand blocking HER3 antibodies inhibit growth of neuregulin driven xenograft models they are ineffective in models of HER2 amplified cancer as HER2 mediated activation of HER3 occurs in a ligand- independent manner. LJM716 is a high affinity HER3- targeted antibody selected from a Human Combinatorial Antibody Library (HuCAL) specifically for its ability to neutralize multiple modes of HER3 activation. LJM716 is a potent inhibitor of HER3/ AKT phosphorylation and proliferation in a range of HER2 amplified and NRG expressing cell lines in vitro. LJM716 induced tumor regression in Fadu (NRG expressing, HNSCC) tumor xenografts and significant tumor growth inhibition (>80%) in a variety of xenograft models including BT474 (HER2 amplified breast). Furthermore, the combination of LJM716 with trastuzumab, cetuximab or PI3K- targeted agents was synergistic in a panel of in vitro cell lines while the in vivo combination of LJM716 with trastuzumab or erlotinib was efficacious in BT474 and L3.3 (pancreatic) tumor xenografts respectively. To further understand the mechanism by which LJM716 inhibits multiple modes of HER3 activation we solved the crystal structure of LJM716 bound to the HER3 extra-cellular domain. This data revealed that LJM716 binds to a novel conformational epitope contained within domains 2 and 4 and appears to trap HER3 in the inactive conformation. Interestingly, LJM716 does not block NRG binding to HER3 nor does it affect the binding affinity of the HER3/ NRG interaction. LJM716 therefore possesses a novel mechanism of action; it prevents the structural rearrangements required for HER3 activation induced by either HER2 or NRG. Thus LJM716 is the first HER3 antibody to display efficacy in both HER2 and ligand driven xenograft models. Based on preclinical data, combining LJM716 with either trastuzumab, cetuximab or PI3K- targeted agents may lead to greater and more sustained clinical efficacy in ErbB driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2733. doi:1538-7445.AM2012-2733

Preferential induction of apoptosis in cancer cells has been the objective of therapeutic strategies targeting apoptotic pathways. To this end, multiple therapeutic agonists of Death Receptors 4 and 5 (DR4, DR5), have been developed and are under clinical evaluation. Although these agonists, including antibodies and soluble ligand TRAIL, demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation for the discrepant pre-clinical and clinical results is that DR5 may play a more prominent role in in vitro model systems as opposed to cancers in humans. Alternatively, these results might indicate that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients. In particular, naturally dimeric antibody agonists require secondary cross-linking via Fcα receptors expressed on immune cells present in the tumor microenvironment to achieve optimal clustering of DR5 into a ternary active state. Because immune cell content in the tumor can be heterogeneous, reliance on this secondary mechanism for activity may limit the potency of these antibodies. To overcome this limitation, a novel nanobody approach was taken to eliminate the need for cross-linking and improve receptor activation with the goal of generating a significantly more potent DR5 agonist. Nanobodies are a class of therapeutic proteins based on single, high affinity heavy chain domain (VHH) antibodies that naturally occur in camelid species, and these VHH domains can be linked to form multivalent structures (di-, tri-, tetra-, etc). This approach led to the development of a tetrameric DR5 targeted agonist, TAS266, with significantly greater avidity for DR5 binding. TAS266 activates downstream caspases with more rapid kinetics and is up to 1000-fold more potent in cell death assays when compared to a cross-linked DR5 antibody or TRAIL. In vivo, TAS266 elicits single dose tumor regressions in multiple tumor xenograft models and sustained tumor regressions after treatment cessation. TAS266 showed superior anti-tumor activity compared to a DR5 agonist antibody and TRAIL, including the ability to induce tumor regression in a patient-derived primary pancreatic tumor model that is insensitive to the agonist antibody. Thus, TAS266 has the potential for superior clinical activity in settings insensitive to the conventional therapeutic approaches to DR5. First-in-man trials are expected to begin in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3853. doi:1538-7445.AM2012-3853

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.

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B , does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.

Material Supplemental http://genesdev.cshlp.org/content/suppl/2011/06/30/25.13.1384.DC1.html References http://genesdev.cshlp.org/content/25/13/1384.full.html#ref-list-1 This article cites 44 articles, 22 of which can be accessed free at: Open Access Open Access option. Genes & Development Freely available online through the Service Email Alerting click here. right corner of the article or Receive free email alerts when new articles cite this article sign up in the box at the top