Xenografted Tumors Share Comparable Fraction Unbound and Can Be Surrogated by Mouse Lung Tissue.

Free (unbound) drug concentration at the site of action is the key determinant of biological activity since only unbound drugs can exert pharmacological and toxicological effects. Unbound drug concentration in tumors for solid cancers is needed to understand/explain/predict pharmacokinetics (PK), pharmacodynamics (PD), and efficacy relations. Fraction unbound (fu) in tumors is usually determined across several xenografted tumors derived from various cell lines in the drug discovery stage, which is time-consuming and a resource burden. In this study, we determined the fu values for a set of diverse compounds (comprising acid, base, neutral, zwitterion, and covalent drugs) across five different xenografted tumors and five commercially available mouse tissues to explore the correlation of fu between tumors and the possibility of surrogate tissue(s) for tumor fu (fu,tumor) determination. The cross-tumor comparison showed fu,tumor values across tumors are largely comparable, and systematic tissue vs. tumor comparison demonstrated only lung tissue had comparable fu to all five tumors (fu values within 2-fold change for >80% compounds in both comparisons). These results indicated mouse lung tissue can be used as a surrogate matrix for fu,tumor assay. This study will increase efficiency in fu,tumor assessment and reduce animal use (adapting the 3Rs principle: replace, reduce, and refine) in drug discovery Significance Statement The free drug concept is a well-accepted principle in drug discovery research. Currently, fu,tumor is determined in several tumors derived from different cell lines to estimate free drug concentrations of a compound. The results from this study indicated fu,tumor across xenografted tumors are comparable and fu,tumor can be estimated using a surrogate tissue, mouse lung. The results will increase efficiency in fu,tumor assessment and reduce animal use in drug discovery.

Rilotumumab Resistance Acquired by Intracrine Hepatocyte Growth Factor Signaling.

Drug resistance is a long-standing impediment to effective systemic cancer therapy and acquired drug resistance is a growing problem for molecularly-targeted therapeutics that otherwise have shown unprecedented successes in disease control. The hepatocyte growth factor (HGF)/Met receptor pathway signaling is frequently involved in cancer and has been a subject of targeted drug development for nearly 30 years. To anticipate and study specific resistance mechanisms associated with targeting this pathway, we engineered resistance to the HGF-neutralizing antibody rilotumumab in glioblastoma cells harboring autocrine HGF/Met signaling, a frequent abnormality of this brain cancer in humans. We found that rilotumumab resistance was acquired through an unusual mechanism comprising dramatic HGF overproduction and misfolding, endoplasmic reticulum (ER) stress-response signaling and redirected vesicular trafficking that effectively sequestered rilotumumab and misfolded HGF from native HGF and activated Met. Amplification of MET and HGF genes, with evidence of rapidly acquired intron-less, reverse-transcribed copies in DNA, was also observed. These changes enabled persistent Met pathway activation and improved cell survival under stress conditions. Point mutations in the HGF pathway or other complementary or downstream growth regulatory cascades that are frequently associated with targeted drug resistance in other prevalent cancer types were not observed. Although resistant cells were significantly more malignant, they retained sensitivity to Met kinase inhibition and acquired sensitivity to inhibition of ER stress signaling and cholesterol biosynthesis. Defining this mechanism reveals details of a rapidly acquired yet highly-orchestrated multisystem route of resistance to a selective molecularly-targeted agent and suggests strategies for early detection and effective intervention.

Dual Inhibition of Angiopoietin-TIE2 and MET Alters the Tumor Microenvironment and Prolongs Survival in a Metastatic Model of Renal Cell Carcinoma.

Receptor tyrosine kinase inhibitors have shown clinical benefit in clear cell renal cell carcinoma (ccRCC), but novel therapeutic strategies are needed. The angiopoietin/Tie2 and MET pathways have been implicated in tumor angiogenesis, metastases, and macrophage infiltration. In our study, we used trebananib, an angiopoietin 1/2 inhibitor, and a novel small-molecule MET kinase inhibitor in patient-derived xenograft (PDX) models of ccRCC. Our goal was to assess the ability of these compounds to alter the status of tumor-infiltrating macrophages, inhibit tumor growth and metastases, and prolong survival. Seven-week-old SCID mice were implanted subcutaneously or orthotopically with human ccRCC models. One month postimplantation, mice were treated with angiopoietin 1/2 inhibitor trebananib (AMG 386), MET kinase inhibitor, or combination. In our metastatic ccRCC PDX model, RP-R-02LM, trebananib alone, and in combination with a MET kinase inhibitor, significantly reduced lung metastases and M2 macrophage infiltration (P = 0.0075 and P = 0.0205, respectively). Survival studies revealed that treatment of the orthotopically implanted RP-R-02LM tumors yielded a significant increase in survival in both trebananib and combination groups. In addition, resection of the subcutaneously implanted primary tumor allowed for a significant survival advantage to the combination group compared with vehicle and both single-agent groups. Our results show that the combination of trebananib with a MET kinase inhibitor significantly inhibits the spread of metastases, reduces infiltrating M2-type macrophages, and prolongs survival in our highly metastatic ccRCC PDX model, suggesting a potential use for this combination therapy in treating patients with ccRCC.

Discovery of a Covalent Inhibitor of KRASG12C (AMG 510) for the Treatment of Solid Tumors.

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Discovery of ( R)-8-(6-Methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4- b]pyrrol-2-yl)-3-(1-methylcyclopropyl)-2-((1-methylcyclopropyl)amino)quinazolin-4(3 H)-one, a Potent and Selective Pim-1/2 Kinase Inhibitor for Hematological Malignancies.

Pim kinases are a family of constitutively active serine/threonine kinases that are partially redundant and regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with improved potency, solubility, and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16 h postdose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100 mg/kg.

Selective MET Kinase Inhibition in MET-Dependent Glioma Models Alters Gene Expression and Induces Tumor Plasticity.

The receptor tyrosine kinase (RTK) MET represents a promising tumor target in a subset of glioblastomas. Most RTK inhibitors available in the clinic today, including those inhibiting MET, affect multiple targets simultaneously. Previously, it was demonstrated that treatment with cabozantinib (MET/VEGFR2/RET inhibitor) prolonged survival of mice carrying orthotopic patient-derived xenografts (PDX) of the MET-addicted glioblastoma model E98, yet did not prevent development of recurrent and cabozantinib-resistant tumors. To exclude VEGFR2 inhibition-inflicted blood-brain barrier normalization and diminished tumor distribution of the drug, we have now investigated the effects of the novel MET-selective inhibitor Compound A in the orthotopic E98 xenograft model. In vitro, Compound A proved a highly potent inhibitor of proliferation of MET-addicted cell lines. In line with its target selectivity, Compound A did not restore the leaky blood-brain barrier and was more effective than cabozantinib in inhibiting MET phosphorylation in vivo Compound A treatment significantly prolonged survival of mice carrying E98 tumor xenografts, but did not prevent eventual progression. Contrasting in vitro results, the Compound A-treated xenografts displayed high levels of AKT phosphorylation despite the absence of phosphorylated MET. Profiling by RNA sequencing showed that in vivo transcriptomes differed significantly from those in control xenografts.Implications: Collectively, these findings demonstrate the plasticity of paracrine growth factor receptor signaling in vivo and urge for prudency with in vitro drug-testing strategies to validate monotherapies. Mol Cancer Res; 15(11); 1587-97. ©2017 AACR.

Discovery and Optimization of Quinazolinone-pyrrolopyrrolones as Potent and Orally Bioavailable Pan-Pim Kinase Inhibitors.

The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.

Preclinical Evaluation of AMG 337, a Highly Selective Small Molecule MET Inhibitor, in Hepatocellular Carcinoma.

Aberrant hepatocyte growth factor (HGF)/MET signaling has been implicated in hepatocarcinogenesis, suggesting that MET may serve as an attractive therapeutic target in hepatocellular carcinoma. We sought to investigate the in vitro and in vivo antitumor activity of AMG 337, a potent and highly selective small molecule MET kinase inhibitor, in preclinical models of hepatocellular carcinoma. The antiproliferative activity of AMG 337 was evaluated across a panel of hepatocellular carcinoma cell lines in a viability assay. Daily oral administration was used to evaluate the in vivo antitumor activity of AMG 337 in two patient-derived xenograft (PDX) models of hepatocellular carcinoma (LI0612 and LI1078). AMG 337 exerted potent antiproliferative activity against 2 of 40 hepatocellular carcinoma cell lines, namely, MHCC97H (IC50, 0.015 µmol/L) and HCCLM3 (IC50, 0.025 µmol/L). Both sensitive cell lines showed MET amplification (MET/CEN-7 >2.0) assessed by FISH, and high MET expression (3+ IHC) assessed by IHC. AMG 337 potently inhibited p-MET in all cell lines with detectable levels of total MET. However, the dose-dependent inhibition of downstream effectors of HGF/MET signaling, including p-GAB1, p-AKT, and p-ERK, was limited to those cell lines sensitive to AMG 337 in a viability assay (MHCC97H and HCCLM3). AMG 337 significantly inhibited tumor growth at all doses tested in the MET-amplified and MET-high-expressing hepatocellular carcinoma PDX model LI0612 and had no effect on tumor growth in the non-MET-amplified and MET-low-expressing hepatocellular carcinoma PDX model LI1078. AMG 337 represents a promising and novel therapeutic strategy for targeting hepatocellular carcinomas with a dependence on HGF/MET signaling. Mol Cancer Ther; 15(6); 1227-37. ©2016 AACR.

In Vitro and In Vivo Activity of AMG 337, a Potent and Selective MET Kinase Inhibitor, in MET-Dependent Cancer Models.

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568-79. ©2016 AACR.

Discovery and Optimization of Macrocyclic Quinoxaline-pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors.

The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.

Discovery of (R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET with High Unbound Target Coverage and Robust In Vivo Antitumor Activity.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Discovery of potent and selective 8-fluorotriazolopyridine c-Met inhibitors.

The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

Structure guided design of a series of sphingosine kinase (SphK) inhibitors.

Sphingosine-1-phosphate (S1P) signaling plays a vital role in mitogenesis, cell migration and angiogenesis. Sphingosine kinases (SphKs) catalyze a key step in sphingomyelin metabolism that leads to the production of S1P. There are two isoforms of SphK and observations made with SphK deficient mice show the two isoforms can compensate for each other's loss. Thus, inhibition of both isoforms is likely required to block SphK dependent angiogenesis. A structure based approach was used to design and synthesize a series of SphK inhibitors resulting in the identification of the first potent inhibitors of both isoforms of human SphK. Additionally, to our knowledge, this series of inhibitors contains the only sufficiently potent inhibitors of murine SphK1 with suitable physico-chemical properties to pharmacologically interrogate the role of SphK1 in rodent models and to reproduce the phenotype of SphK1 (-/-) mice.

Sphingosine kinase activity is not required for tumor cell viability.

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.

Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model.

INTRODUCTION: Dysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using (18)F-FDG and (18)F-FLT PET. METHODS: U-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 µg) were administered, and mice were evaluated with (18)F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 µg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using (18)F-FDG and (18)F-FLT PET. RESULTS: In the dose response study, rilotumumab at doses of 300 and 500 µg was similarly effective against tumor growth. Treatment with 300 and 500 µg rilotumumab inhibited (18)F-FDG accumulation with significant decreases of -37% and -40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 µg rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation with a maximum %ID/g of -41% and -64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed. CONCLUSIONS: Rilotumumab inhibited (18)F-FDG and (18)F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate (18)F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.

Discovery and optimization of a potent and selective triazolopyridinone series of c-Met inhibitors.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

Structure-based design of novel class II c-Met inhibitors: 1. Identification of pyrazolone-based derivatives.

Deregulation of c-Met receptor tyrosine kinase activity leads to tumorigenesis and metastasis in animal models. More importantly, the identification of activating mutations in c-Met, as well as MET gene amplification in human cancers, points to c-Met as an important target for cancer therapy. We have previously described two classes of c-Met kinase inhibitors (class I and class II) that differ in their binding modes and selectivity profiles. The class II inhibitors tend to have activities on multiple kinases. Knowledge of the binding mode of these molecules in the c-Met protein led to the design and evaluation of several new class II c-Met inhibitors that utilize various 5-membered cyclic carboxamides to conformationally restrain key pharmacophoric groups within the molecule. These investigations resulted in the identification of a potent and novel class of pyrazolone c-Met inhibitors with good in vivo activity.

Context-dependent role of angiopoietin-1 inhibition in the suppression of angiogenesis and tumor growth: implications for AMG 386, an angiopoietin-1/2-neutralizing peptibody.

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings.