Yu-Wang Liu

Transforming growth factor-β receptor type 1 (TGFβRI) kinase activity but not p38 activation is required for TGFβRI-induced myofibroblast differentiation and profibrotic gene expression

Transforming growth factor-β (TGFβ) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFβ also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFβRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFβRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFβ-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFβ-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.

Intratumoural heterogeneity generated by Notch signalling promotes small-cell lung cancer

The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10–50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.

Pharmacological Properties of SD-282 – An α-Isoform Selective Inhibitor for p38 MAP Kinase

The effects of small-molecule p38 inhibitors in numerous models of different disease states have been published, including those of SD-282, an indole-5-carboxamide inhibitor. The aim of the present study was to evaluate the pharmacological activity of SD-282 on cytokine production in vitro as well as in 2 in vivo models of inflammation in order to illuminate the role of this particular inhibitor in diverse disease states. The results presented here provide further characterization of SD-282 and provide a context in which to interpret the activity of this p38 inhibitor in models of arthritis, pain, myocardial injury, sepsis and asthma; all of which have an inflammatory component. SD-282 represents a valuable tool to elucidate the role of p38 MAP kinase in multiple models of inflammation.

Abstract 4652: Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors

Background: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers, the leading cause of cancer-related deaths. Notch signaling has been shown to play an important role in lung cancer initiation and progression. Delta-like ligand 4 (DLL4) activates the Notch pathway and is important for cancer stem cell (CSC) survival. Demcizumab (OMP-21M18) is a humanized IgG2 anti-DLL4 antibody currently being tested in a Phase 2 trial in combination with pemetrexed and carboplatin for first-line treatment of patients with NSCLC. Previously, OMP-21M18 in combination with its mouse anti-DLL4 surrogate has been shown to inhibit tumor growth, decrease cancer stem cell frequency, and cause dysfunctional sprouting of new vessels resulting in an anti-angiogenic effect in patient-derived tumor xenograft (PDX) models in breast, colon, ovarian, and pancreatic cancers. Here we show results from NSCLC PDX models. Methods and Results: Anti-DLL4 treatment was tested in a series of NSCLC PDX models. Because DLL4 inhibition has been shown to have effects on the tumor as well as the vasculature, the combination of OMP-21M18 (targeting human DLL4) and 21R30 (antibody targeting mouse DLL4) treatment in the PDX models was used to model demcizumab treatment in humans. Treatment with anti-DLL4 in combination with chemotherapy inhibited tumor growth in a series of NSCLC PDX models. Additionally, a tumorigenicity assay showed a decrease in the frequency of tumor-initiating cells following treatment with anti-DLL4 and chemotherapy. Gene expression analysis of tumor samples provided insights into the mechanism of action. Conclusions: Anti-DLL4 treatment in a panel of NSCLC PDX tumor models in vivo showed inhibition of tumor growth and a decrease in the frequency of tumor-initiating cells. Mechanism of action and gene expression analysis of these models treated with anti-DLL4 will be presented. These findings provide additional evidence supporting demcizumab as an effective treatment for NSCLC patients. Citation Format: Alayne Brunner, Fiore Cattaruzza, Wan-Ching Yen, Pete Yeung, Marcus Fischer, Belinda Cancilla, Gilbert O’Young, Raymond Tam, Yu-Wang Liu, Austin Gurney, John Lewicki, Tim Hoey, Min Wang, Ann M. Kapoun. Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4652.

Abstract A030: Biomarker study of vantictumab plus paclitaxel in HER2- breast cancer patients

Introduction: We have developed a monoclonal antibody, vantictumab, that blocks canonical Wnt/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, and 8). This antibody inhibits the growth of several tumor types, including breast. Vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care (SOC) agents (Gurney et al., 2012). To confirm the mechanism of action and to potentially target breast cancer patients most likely to respond to vantictumab, we undertook a biomarker study. Methods: We previously identified a 6-gene Wnt pathway-related signature, FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, based on microarray gene expression data from 8 BC patient-derived xenograft (PDX) models with established in vivo response to vantictumab plus SOC. This signature successfully predicted the response of 8 additional and independent PDX breast tumors. We further developed a qPCR Research Use Only (RUO) assay for the 6 genes for use on FFPE human breast tumor samples. This assay was evaluated in the phase 1b study of vantictumab in combination with paclitaxel in locally recurrent or metastatic HER2- breast cancer (NCT01973309) and the signature was refined using a Lasso model with overall survival as the outcome. A repeated 10 fold cross-validation was used to evaluate the performance of the gene signature. The association of the signature with progression-free survival (PFS) and overall survival (OS) was examined (n=40 patients). Furthermore, pharmacodynamic (PD) biomarker analyses were performed on tumor biopsies and hair follicles by comparing gene expression data from post-treatment time points versus baseline data (Affymetrix U133 plus 2 Microarrays). Results: A potential predictive 6-gene Wnt pathway biomarker was identified based on preclinical data and the biomarker was evaluated and refined in a phase 1b study of vantictumab in combination with paclitaxel in HER2- breast cancer. In the phase 1b study, AUC = 75% with repeated 10 fold cross-validation measuring the performance of the gene signature. Based on this analysis, two genes, RHOU and DKK1, were dropped from the preclinical gene signature, which was consistent with the feature ranking in the preclinical qPCR data. The refined 4-gene signature was significantly associated with both PFS and OS at a 50% percentile cut-off. In addition, analysis of PD biomarkers demonstrated that Wnt pathway target genes including AXIN2, LEF1, and CTNNB1 were downregulated while differentiation markers, e.g., KRT19 and Wnt pathway inhibitors, e.g., SFRP1, DKK3 were upregulated by vantictumab plus paclitaxel. Conclusions: We developed a 4-gene signature as a potential predictive biomarker for the response to vantictumab plus paclitaxel in HER2- breast cancer. PD biomarker analysis in tumors and hair follicles confirmed the mechanism of action of vantictumab in patient samples. Preliminary efficacy of vantictumab plus paclitaxel in the phase 1b study was encouraging, particularly in breast cancer patients positive for the 4-gene signature. Updated biomarker and PK/PD data from the phase 1b trial (NCT01973309) will also be presented. Citation Format: Chun Zhang, William R. Henner, Min Wang, Fiore Cattaruzza, Pete Yeung, Gilbert O9Young, Yuwang Liu, Gretchen Argast, Lu Xu, Shailaja Uttamsingh, John Lewicki, Ann M. Kapoun. Biomarker study of vantictumab plus paclitaxel in HER2- breast cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A030.

Abstract 2612: Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2017-2612

Abstract 599: Pharmacodynamic biomarkers for anti-TIGIT treatment and prevalence of TIGIT expression in multiple solid tumor types

TIGIT (T cell immunoreceptor with Ig ITIM domain) is a co-inhibitory receptor and its signaling axis inhibits T cell and Natural Killer (NK) cell activity in the healthy immune system. In tumors TIGIT is highly expressed on a subset of dysfunctional T and NK cells and of highly suppressive regulatory T cells (Treg). Loss of TIGIT signaling enhances NK cell activity, CD4+ T cell priming and CD8+ T cell effector functions, suggesting a role in anti-tumor immunity. We have developed an anti-TIGIT blocking antibody that shows potent antitumor efficacy in in multiple syngeneic mouse models, including CT26WT colon, B16F10 melanoma and 4T1 breast cancer. Dose-dependent (12.5-0.1 mg/Kg) single agent efficacy was demonstrated in established tumors, and pharmacodynamic (PD) biomarkers in blood and in tumors were identified. Consistent with TIGIT’s role as a co-inhibitory receptor, anti-TIGIT promoted a dose-dependent increase in activation of CD8+ and CD4+ tumor infiltrating leukocytes (TIL) and NK cells, as shown by increased staining of IFN-γ and CD69 by flow cytometry, when compared to controls. Anti-TIGIT also increased CD4 and CD8 T cell frequency in the tumor, measured by immunohistochemistry (IHC). Furthermore, anti-TIGIT caused an increase in splenic NK cell cytotoxicity, which correlated with dose and efficacy. To identify gene expression biomarkers in tumor and in blood, we used microarray analysis, and found similar immune gene changes between the two tissues. As expected, anti-TIGIT increased the expression of genes associated with CD8+ T cells, CD4+ T cells, and NK cells. Markers indicative of cytotoxic activity and Th1 response were also induced by anti-TIGIT. The microarray data were validated by qRT-PCR and results were consistent with flow cytometry and cytotoxicity results, and underlie the mechanism of action of anti-TIGIT. We have developed an IHC assay to evaluate TIGIT expression patterns in tumor and the associated stroma and TILs. Using this assay, we profiled 17 tumor types to evaluate patterns of TIGIT expression. Expression was high on immune cells in the stroma and on TILs in 8 tumor types, while it was generally low on tumor cells. TIGIT expression was also low on tumor cells in a panel of 27 PDX models. Consistent with the IHC results, analysis of 33 tumor types in the TCGA by RNA-Seq showed a good correlation of the expression levels of TIGIT and T cell markers, suggesting that TIGIT is mostly expressed on immune cells in tumors. In conclusion, we have identified PD biomarkers for anti-TIGIT in tumors and in surrogate tissues in syngeneic mouse models. Analysis of the biomarkers demonstrates activation of T cells and NK cells upon inhibition of TIGIT signaling. These biomarkers can be used in the clinic to demonstrate target engagement. In addition, we have profiled the expression of TIGIT in multiple solid tumor types and characterized prevalence of TIGIT-positive cells. Citation Format: Fiore Cattaruzza, Pete Yeung, Min Wang, Alayne Brunner, Erwan Le Scolan, Jennifer Cain, Gretechen Argast, Gilbert O9Young, YuWang Liu, Belinda Cancilla, Austin Gurney, Tim Hoey, John Lewicki, Ann Kapoun. Pharmacodynamic biomarkers for anti-TIGIT treatment and prevalence of TIGIT expression in multiple solid tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 599. doi:10.1158/1538-7445.AM2017-599

Abstract 404: Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors

R-Spondin (RSPO) proteins bind to LGR receptors and potentiate Wnt/β-catenin signaling. We have identified a therapeutic anti-RSPO3 antibody targeting the RSPO-LGR pathway. In preclinical studies, RSPO3 gene expression has shown correlation with anti-RSPO3 antibody efficacy in multiple solid tumor types. A qPCR-based RSPO3 assay has been developed as a predictive biomarker for response to the anti-RSPO3 antibody. In addition, RSPO gene fusions may play a role in the activation of Wnt signaling. A gene fusion detection workflow consisting of a RSPO3 CLIA assay, a RSPO3 RUO assay and next generation sequencing (NGS) has also been developed. We designed 6 qPCR-based assays for the RSPO3 CLIA assay development and 2 assays for the RUO assay. These assays were designed to span exon-exon junctions or target microarray probe set sequences. Amplification sensitivity and specificity were assessed for assay selection. The analytic performance of the candidate RSPO3 CLIA assay and quality control measures were established in a validation study. The validation study included: 1) performance specifications of the RSPO3 assay including analytical sensitivity, linearity, and precision, 2) determination of a reportable range, 3) establishment of a cut-off for the RSPO3 CLIA assay for patient selection, and 4) establishment of quality control procedures. 104 human cancer tissues and 24 independent patient-derived tumor xenografts (PDX) were used in these studies. To evaluate the fusion detection workflow, the RUO assay was performed on samples that tested above the CLIA assay cut-off. The delta Ct difference between the CLIA and RUO assays was calculated to identify potential fusions. The limit of quantification was established for the RSPO3 CLIA assay. The 95% reference interval was estimated to be (-2.44, 16.02) with 90% confidence interval for the lower bound (-3.45, -2.12) and upper bound (15.26, 16.57). The delta Ct cut-off for the RSPO3 CLIA assay was set based on sensitivity, specificity and prevalence. No statistically significant difference in the total variance across the tested samples was observed. A549 and OV56 were identified to be cell line controls with established acceptable delta Ct limits. Using NGS, RSPO3 fusions were identified in 6 PDX tumors with delta Ct RUO - delta Ct CLIA>7, including a novel fusion. This cut-off was further refined with NGS of 9 clinical samples. Prevalence of the RSPO3 expression and fusions will be presented. A qPCR based RSPO3 assay was developed and CLIA-validated for use as a potential predictive biomarker for response to anti-RSPO3 therapy. This RSPO3 CLIA assay, together with the fusion detection workflow, will be evaluated in a Phase 1a/b dose escalation study of anti-RSPO3 (OMP-131R10) in advanced solid tumors and in combination with FOLFIRI in metastatic colorectal cancer (NCT02482441). Citation Format: Chun Zhang, Yuwang Liu, Min Wang, Gilbert OYoung, Joy Kavanagh, Cheryl McFarlane, Fiore Cattaruzza, Pete Yeung, Jennifer Cain, Wan-Ching Yen, Marcus Fischer, Belinda Cancilla, Edwina Dobbin, Michelle McCarthy, Austin Gurney, Leonardo Faoro, John Lewicki, Tim Hoey, Ann M. Kapoun. Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 404.

Abstract 910: NOTCH3 expression is predictive of efficacy in pancreas tumor models treated with OMP-59R5, a monoclonal antibody targeting the NOTCH2 and NOTCH3 receptors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The Notch signaling pathway regulates key functions during embryonic development, stem cell maintenance and differentiation in adult tissues, and is implicated in many human cancers. OMP-59R5 is a ligand-blocking antibody targeting both the NOTCH2 and NOTCH3 receptors. We have developed a series of primary human xenograft models from patients with pancreatic cancer and used these models to examine efficacy response to OMP-59R5. We found that anti-NOTCH2/3, either as a single agent or in combination with chemotherapeutic agents, was efficacious in pancreatic tumor models. Expression of NOTCH3 mRNA by next-generation sequencing in ten baseline pancreatic tumors correlates with response to OMP-59R5, where growth of tumors with moderate to high expression of NOTCH3 was significantly reduced compared to tumors with low expression. We developed a Research-Use-Only (RUO) qPCR assay for measuring NOTCH3 mRNA expression using Formalin-Fixed, Paraffin-Embedded (FFPE) samples. This assay shows consistent NOTCH3 expression data with the next-generation sequencing data in the ten pancreatic xenograft tumors. Expression levels of NOTCH3 were also examined in ∼120 human metastatic pancreatic specimens to determine the reportable range of the assay and to identify association with clinical factors. This analysis showed that NOTCH3 gene expression maintained the same distribution across different specimen types, such as biopsy, surgical biopsy and surgical resection, etc. Samples with clinically relevant sites of recurrence also showed a similar range in NOTCH3 gene expression. Moreover, we developed an immunohistochemistry (IHC) assay for NOTCH3 protein expression. The correlation between the IHC assay and the qPCR assay was examined in both the metastatic pancreatic human specimens and the primary human pancreatic xenograft models. A significant correlation was found between the gene and protein levels, suggesting that both NOTCH3 gene expression and protein expression may predict the response to OMP-59R5 in pancreatic cancer. We are evaluating NOTCH3 levels and patient response in ALPINE, a Ph1b/2 Anti-NOTCH2/3 trial in first-line advanced pancreatic cancer patients. Citation Format: Belinda Cancilla, Wan-Ching Yen, Chun Zhang, Marcus M. Fischer, May Ji, Tracy Tang, Yu-Wang Liu, Raymond S. Tam, Min Wang, Austin Gurney, Timothy Hoey, John Lewicki, Ann M. Kapoun. NOTCH3 expression is predictive of efficacy in pancreas tumor models treated with OMP-59R5, a monoclonal antibody targeting the NOTCH2 and NOTCH3 receptors. [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 910. doi:10.1158/1538-7445.AM2014-910

Abstract A29: Discovery and preclinical to clinical evaluation of biomarkers for Anti-DLL4, a monoclonal antibody targeting cancer stem cells

Blockade of DLL4 signaling reduces tumor growth in preclinical human xenograft models by disrupting productive angiogenesis and reducing proliferation of tumor cells. Importantly in these models, blocking DLL4 also reduces cancer stem cell (CSC) frequency in multiple solid tumor types. Currently, there exists an important need to identify and evaluate pharmacodynamic biomarkers that indicate the biological activity of agents, such as anti-DLL4, that are in clinical studies. Towards this aim, we used numerous human xenograft models derived from primary human tumors to identify and characterize biomarkers for anti-DLL4. Here, we present data from these studies and describe anti-DLL4 biomarkers in tumors, in hair follicles, and in blood. We show that several vascular-related genes are significantly upregulated by anti-DLL4 in tumors including, Egfl7, Apln, Cldn5, and Notch4. We also demonstrate significant downregulation of CSC gene signatures and modulation of differentiation genesets. A subset of the identified markers were validated at the protein level and also tested in a human skin graft model, where the human tumor is engrafted in a human microenvironment/vasculature. Finally, a DLL4-Notch gene signature was identified that showed a significant association with breast cancer survival using publicly available cancer gene sets. In surrogate tissues, we identified anti-DLL4 biomarkers in blood and in hair follicles. These markers from surrogate tissues were translated into a phase I trial of Anti-DLL4 antibody (OMP-21M18), and an interim analysis of this data demonstrates that OMP-21M18 affects the Notch pathway in patient samples. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A29.