Jie Li

Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974

Significance Targeting the Wnt pathway in cancer is an attractive therapeutic approach. However, success has been limited because of the lack of effective therapeutic agents and the lack of biomarkers to define the patient population that would benefit from such a therapy. Herein, we report the discovery of LGK974, a drug that targets Porcupine, a Wnt-specific acyltransferase. We show that LGK974 potently inhibits Wnt signaling, has strong efficacy in rodent tumor models, and is well-tolerated. We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide a strategy and tools for targeting Wnt-driven cancer. Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2. LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV–Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wnt-driven cancers through the inhibition of PORCN.

A genomic screen identifies TYRO3 as a MITF regulator in melanoma

Malignant melanoma is the most aggressive form of cutaneous carcinoma, accounting for 75% of all deaths caused by skin cancers. Microphthalmia-associated transcription factor (MITF) is a master gene regulating melanocyte development and functions as a “lineage addiction” oncogene in malignant melanoma. We have identified the receptor protein tyrosine kinase TYRO3 as an upstream regulator of MITF expression by a genome-wide gain-of-function cDNA screen and show that TYRO3 induces MITF-M expression in a SOX10-dependent manner in melanoma cells. Expression of TYRO3 is significantly elevated in human primary melanoma tissue samples and melanoma cell lines and correlates with MITF-M mRNA levels. TYRO3 overexpression bypasses BRAF(V600E)-induced senescence in primary melanocytes, inducing transformation of non-tumorigenic cell lines. Furthermore, TYRO3 knockdown represses cellular proliferation and colony formation in melanoma cells, and sensitizes them to chemotherapeutic agent-induced apoptosis; TYRO3 knockdown in melanoma cells also inhibits tumorigenesis in vivo. Taken together, these data indicate that TYRO3 may serve as a target for the development of therapeutic agents for melanoma.

EGF816 Exerts Anticancer Effects in Non–Small Cell Lung Cancer by Irreversibly and Selectively Targeting Primary and Acquired Activating Mutations in the EGF Receptor

Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M.

Regenerative phenotype in mice with a point mutation in transforming growth factor β type I receptor (TGFBR1)

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N-ethyl-N-nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-β, TGFBR1. Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-β target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.

Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T790M) EGFR Mutants for the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers.

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

Discovery of Pyridinyl Acetamide Derivatives as Potent, Selective, and Orally Bioavailable Porcupine Inhibitors

Blockade of aberrant Wnt signaling is an attractive therapeutic approach in multiple cancers. We developed and performed a cellular high-throughput screen for inhibitors of Wnt secretion and pathway activation. A lead structure (GNF-1331) was identified from the screen. Further studies identified the molecular target of GNF-1331 as Porcupine, a membrane bound O-acyl transferase. Structure-activity relationship studies led to the discovery of a novel series of potent and selective Porcupine inhibitors. Compound 19, GNF-6231, demonstrated excellent pathway inhibition and induced robust antitumor efficacy in a mouse MMTV-WNT1 xenograft tumor model.

Abstract B232: Activity of a potent and selective phase I ALK inhibitor LDK378 in naive and crizotinib-resistant preclinical tumor models.

A c-MET/ALK kinase inhibitor crizotinib has demonstrated significant activity in lung cancer patients with EML4-ALK in clinical studies. However relapse (or acquired resistance) has also been reported. We have developed crizotinib resistant tumor models and used the models to evaluate the ALK inhibitor LDK378. LDK378 is a potent and selective ALK inhibitor that does not cross react with c-MET. In a mouse xenograft tumor model derived from the EML4-ALK+ lung cancer cell line NCI-H2228, LDK378 caused complete tumor regression when dosed orally at 25 mg/kg/day. After tumor bearing animals had been treated with LDK378 at 50 mg/kg/day for 14 days, remission was maintained for more than 4 months. In several NCI-H2228 tumor models that were induced to become resistant to crizotinib, LDK378 demonstrated efficacy at 50 mg/kg/day. Based on 4-wk GLP toxicology studies the drug exposure associated with this dose is predicted to be below the exposure at the MTD in humans. ALK resistance mutations reported in crizotinib relapsed patients were also found in the crizotinib resistant NCI-H2228 tumor models. The results from these preclinical studies suggest that LDK378 may be active in crizotinib-relapsed patients. A phase I clinical study of LDK378 has recently begun in both crizotinib-relapsed and crizotinib-naive patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B232.

Abstract 1445: STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity

Stimulator of Interferon Genes (STING) is a critical signaling sensor of the innate immune system. STING binds cyclic dinucleotides (CDN) produced by an intracellular enzyme in response to presence of intracellular DNA, including tumor-derived DNA. STING-mediated production of host type I interferon within the tumor microenvironment (TME) leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. A novel synthetic CDN derivative (ADU-S100), with superior STING-activating and anti-tumor properties, was developed for clinical translation. ADU-S100 has enhanced cellular uptake properties and stability, as compared to bacterial- and mammalian-derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING alleles, including a homozygous haplotype for the most refractory human allele (R232H), indicate that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through intratumoral (IT) administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provides long-lived immunologic memory. Mechanistic studies demonstrate that STING-mediated anti-tumor immunity is due in part to an acute pro-inflammatory cytokine response as well as a tumor-specific CD8+ T cell response. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, for example anti-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. Citation Format: Laura Hix Glickman, David B. Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, Kelsey Sivick Gauthier, Weiwen Deng, Anthony L. Desbien, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffrey M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Sarah M. McWhirter. STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity. [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 1445.

Abstract B020: STING activation in the tumor microenvironment using a synthetic human STING-activating cyclic dinucleotide induces potent antitumor immunity

Stimulator of Interferon Genes (STING) is a critical component of the cytosolic DNA sensing pathway of the innate immune system. STING is activated by cyclic dinucleotides (CDNs), a product of the intracellular enzyme, cyclic GMP-AMP synthase (cGAS), in response to presence of cytosolic DNA, including tumor-derived DNA. Production of type I interferon within the tumor microenvironment (TME), mediated by the STING pathway, leads to the priming and activation of systemic tumor antigen-specific CD8 + T-cell immunity and tumor regression. Therapeutic activation of STING through intratumoral (IT) administration of CDNs results in anti-tumor efficacy and long-lived survival in several mouse syngeneic tumor models. Rational design of synthetic CDN derivatives has shown that certain modifications alter STING binding, increase cellular potency, enhance maturation of human dendritic cells to promote in vitro T cell expansion, and are able to broadly activate all human STING haplotypes. Mechanistic studies in mouse tumor models demonstrate that CDNs mediate anti-tumor immunity by inducing an acute innate immune response, leading to collapse of the injected tumor, and promoting a tumor-specific CD8 + T cell response that protects against tumor re-challenge. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, informing future clinical development. The ability to elicit innate and adaptive anti-tumor immunity via activation of STING in the TME demonstrates that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. The design of an ongoing Phase 1 first-in-human clinical study to evaluate the safety, tolerability and possible antitumor activity of ADU-S100 in subjects with cutaneously accessible tumors and lymphomas will also be presented. Citation Format: Sarah M. McWhirter, Laura Hix Glickman, Tony Desbien, Kelsey Sivick Gauthier, David Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, George Katibah, Ed Lemmens, Leticia Corrales, Meredith Leong, Chudi Ndubaku, Justin Leong, Leonard Sung, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Jr.. STING activation in the tumor microenvironment using a synthetic human STING-activating cyclic dinucleotide induces potent antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B020.

Abstract SY39-02: Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity

Recent reports have provided the mechanistic insight of how innate immune activation promotes priming of anti-tumor immunity and inform the development of clinical approaches to facilitate this process. Spontaneous T cell infiltration of melanoma lesions in humans is correlated with a type I interferon (IFN) transcriptional profile in the tumor microenvironment (TME) and infiltration of lymphocytes, indicative of ongoing innate immune recognition within the tumor. Substantial evidence indicates that tumor infiltrating lymphocytes (TILs), including activated CD8+ T cells, is predictive of a positive clinical outcome in response to several immunotherapy strategies. Similarly, in mice bearing melanoma, there is a correlation between expression of IFN-β by tumor-resident dendritic cells (DCs), and spontaneous priming of tumor-specific immunity. Induction of IFN-β expression and co-regulated IFN-responsive genes and pro-inflammatory chemokines is dependent upon activation of the STING (Stimulator of Interferon Genes) pathway, mediated through sensing of tumor dsDNA in TME-resident CD8α+ DCs by cyclic GMP-AMP (cGAMP) synthase (cGAS), which in turn synthesizes cGAMP. The cyclic dinucleotide (CDN) cGAMP produced by cGAS is the natural STING agonist ligand. Thus, the cGAS-STING signaling axis has emerged as a central node for sensing damage in the host. We hypothesized that direct activation of the STING pathway in the TME by intratumoral (IT) injection of specific CDNs would be an effective therapeutic strategy to promote broad tumor-initiated T cell priming against an individual9s tumor antigen repertoire. There are five variant human STING alleles that exist at varying frequencies. While the natural STING ligand cGAMP activates signaling in all variants, other natural CDNs, including those produced by bacteria, have structural differences and are unable to activate particular STING variants, such as the REF (R232H) allele, informing the development of compounds that activate all human STING alleles. We sought to develop synthetic CDN compounds with increased activity in human cells as well as the ability to engage all known polymorphic human STING molecules. Using human 293T cell lines engineered to express the various STING proteins, we screened a large panel of CDN derivatives that varied in purine nucleotide base, structure of the phosphate bridge linkage, and substitution of the non-bridging oxygen atoms at the phosphate bridge with sulfur atoms. ADU-S100 is composed of two adenosine monophosphate (AMP) analogues cyclized via a 2’-5’ (non-canonical) and a 3’-5’ (canonical) phosphodiester bond, and was selected for clinical translation based on properties of enhanced cellular uptake, human STING activation, stability and anti-tumor efficacy, as compared to bacterial and mammalian derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING haplotypes, including donors with a homozygous haplotype for the refractory human REF allele, indicates that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through IT administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. A bell-shaped ADU-S100 dose response curve (which varied based on tumor model) delineated regression of injected tumor, induction of tumor-specific CD8+ T cell immunity, and regression of distal non-injected tumors (abscopal effect), and/or protection against autologous tumor challenge. At low dose levels, regression of the treated tumor was suboptimal. At optimal doses, regression of the treated and distal untreated tumors, or protection against tumor re-challenge was observed, and correlated with induction of a robust tumor Ag-specific CD8+ T cell response. At higher dose levels there was a loss of protection against tumor re-challenge which correlated with increases in acute systemic cytokines and reduction in CD8+ T cell responses even though growth of the treated tumor was inhibited. These results suggest that the mechanism of ADU-S100-induced tumor regression is due to both an acute pro-inflammatory cytokine response and also tumor-specific CD8+ T cell immunity. The local anti-tumor effect without systemic immunity is consistent with well-established data in which excessive innate immune stimulation and induction of pro-inflammatory cytokines such as TNF-α are known to inhibit both priming of CD8+ T cell immunity and establishment of a stable and self-renewing memory CD8+ T cell population. In addition, anti-tumor efficacy was enhanced by combination with immune checkpoint inhibitors, for example α-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. A Phase 1 clinical study to evaluate the safety and tolerability and possible anti-tumor effects in subjects with cutaneously accessible non UV-induced and UV-induced malignancies or lymphomas given repeated IT doses of ADU-S100 is planned. Citation Format: Laura Hix Glickman, Leticia Corrales, David B. Kanne, Shailaja Kasibhatla, Jie Li, Anne Marie Culazzo Pferdekamper, Kelsey Sivick Gauthier, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Weiwen Deng, Anthony L. Desbien, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Sarah M. McWhirter, Thomas F. Gajewski, Thomas W. Dubensky. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. [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 SY39-02.