Peter McNamara

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.

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.

Discovery of trifluoromethyl(pyrimidin-2-yl)azetidine-2-carboxamides as potent, orally bioavailable TGR5 (GPBAR1) agonists: structure-activity relationships, lead optimization, and chronic in vivo efficacy.

Activation of the G-protein coupled receptor (GPCR) Takeda G-protein receptor 5 (TGR5), also known as G-protein bile acid receptor 1 (GPBAR1), has been shown to play a key role in pathways associated with diabetes, metabolic syndrome, and autoimmune disease. Nipecotamide 5 was identified as an attractive starting point after a high-throughput screen (HTS) for receptor agonists. A comprehensive hit-to-lead effort culminated in the discovery of 45h as a potent, selective, and bioavailable TGR5 agonist to test in preclinical metabolic disease models. In genetically obese mice (ob/ob), 45h was as effective as a dipeptidyl peptidase-4 (DPP-4) inhibitor at reducing peak glucose levels in an acute oral glucose tolerance test (OGTT), but this effect was lost upon chronic dosing.

Discovery of Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH)

The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto a trisubstituted isoxazole scaffold. Herein, we report the discovery of 1 (tropifexor, LJN452), a novel and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models by measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC.

Discovery of structurally novel, potent and orally efficacious GPR119 agonists.

Screening hit 5 was identified in a biochemical screen for GPR119 agonists. Compound 5 was structurally novel, displayed modest biochemical activity and no oral exposure, but was structurally distinct from typical GPR119 agonist scaffolds. Systematic optimization led to compound 36 with significantly improved in vitro activity and oral exposure, to elevate GLP1 acutely in an in vivo mouse model at a dose of 10mg/kg.

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.

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.

Novel tricyclic pyrazolopyrimidines as potent and selective GPR119 agonists

Systematic SAR optimization of the GPR119 agonist lead 1, derived from an internal HTS campaign, led to compound 29. Compound 29 displays significantly improved in vitro activity and oral exposure, leading to GLP1 elevation in acutely dosed mice and reduced glucose excursion in an OGTT study in rats at doses ⩾10 mg/kg.

Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells

CRISPR–Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells—an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR–Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.Guide Swap challenges the hypothesis that Cas9–sgRNA binding is irreversible. The authors find that instead, nontargeting sgRNAs are swapped for targeting sgRNAs in the Cas9 complex. The method allows genome-scale functional screens in primary cells

Abstract IA35: Discovery of porcupine inhibitors targeting Wnt signaling in cancer

Wnt signaling is tightly controlled during cellular proliferation, differentiation and embryonic morphogenesis. Aberrant activation of this pathway plays a critical role in a variety of cancers. Blockade of Wnt signaling is therefore an attractive therapeutic approach for anticancer therapy. In this presentation, we will discuss our approach to search for inhibitors of Wnt ligand secretion. We developed and performed a cellular high-throughput screen using a co-culture system. Lead structure (GNF-1331) was identified and further target elucidation revealed Porcupine, a membrane bound O-acyl transferase, as its molecular target. Further structure-activity relationship studies led to the discovery of WNT974, a potent and specific Porcupine inhibitor. Treatment of WNT974 leads to tumor regression in a Wnt dependent MMTV-Wnt1 mouse model at well tolerated doses. WNT974 is currently in Phase 1 clinical trials. Citation Format: Shifeng Pan, Jun Liu, Dai Cheng, Dong Han, Guobao Zhang, Mindy Hsieh, Nicholas Ng, Chun Li, Shailaja Kasibhatla, Peter McNamara, H. Martin Seidel, Jennifer Harris. Discovery of porcupine inhibitors targeting Wnt signaling in cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA35.