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The ability to effectively target mutated KRAS has remained elusive despite decades of research. The recent identification of KRASG12C inhibitors has provided an effective treatment option for patients harboring this particular mutation and has also provided insight toward targeting other KRAS mutants, including KRASG12D. MRTX1133 was identified via a structure-based drug design (SBDD) strategy as a potent, selective, and non-covalent KRASG12D inhibitor directed at the switch II binding pocket. MRTX1133 demonstrated a high-affinity interaction with KRASG12D with KD or IC50 values each determined at ~0.2 pM or 1000-fold selectivity for inhibition of ERK1/2 phosphorylation in KRASG12D-mutant cell lines compared with KRASWT cell lines. Dose-dependent inhibition of KRAS-mediated signal transduction was also observed in multiple KRASG12D-mutant tumor models in vivo. MRTX1133 demonstrated marked tumor regression (>30%) in a subset of KRASG12D-mutant cell line- and patient-derived xenograft (PDX) models, including 8 out of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models evaluated. Pharmacological studies and CRISPR-based screens demonstrated co-targeting KRASG12D in concert with putative feedback or bypass pathways including EGFR and PI3Kα led to enhanced anti-tumor activity relative to targeting each individual protein. Together, these data indicate the feasibility of utilizing SBDD approaches to selectively target alternative KRAS mutant variants with non-covalent, high-affinity small molecules targeting either the active or inactive state of KRAS. In addition, these data illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors, including PDAC, on KRAS for tumor cell growth and survival.

Recent progress in targeting KRAS

The fibroblast growth factor receptor (FGFR) family of protein tyrosine kinases plays a role in many physiological processes including angiogenesis and wound healing. FGFR mutations, fusions, rearrangements, and amplifications have been linked to the pathogenesis of multiple tumor types. For example, approximately 10-15% of patients with intrahepatic cholangiocarcinoma have FGFR2 fusions, and amplification of FGFR2 is prevalent in gastric cancer and implicated in tumor growth. Approved FGFR inhibitors produce responses in patients that harbor FGFR genetic alterations but show reduced activity against key mutations (e.g., V565, N550, etc.). These pan-FGFR inhibitors also frequently show dose-limiting toxicities including hyperphosphatemia which can be linked to the inhibition of FGFR1. To address these issues, a novel series of FGFR inhibitors that are potent and selective reversible inhibitors of FGFR2 with selectivity over FGFR1 have been identified. This series also maintains activity against clinically relevant mutations in the FGFR2 protein. Inhibition of cellular FGFR phosphorylation was measured using engineered cell lines to demonstrate that the inhibitors target both wild-type FGFR2 and activating FGFR2 mutations while sparing FGFR1. In addition, CDX tumor models showed in vivo target engagement and FGFR isoform selectivity. Herein, the in vitro and in vivo characterization of a representative selective reversible FGFR2 inhibitor is described. Citation Format: John Fischer, Karyn Bouhana, Mark J. Chicarelli, Josh Dahlke, Brad Fell, Jennifer Fulton, Anna Guarnieri, Ravi Jalluri, Amber Johnson, Brent Mclean, Max Mejia, Rob Rieger, John Robinson, Mareli Rodriguez, Francis Sullivan, Yang Wang, Shannon Winski, Yeyun Zhou. Pre-clinical characterization of a novel series of FGFR2 selective inhibitors with potency against clinically relevant mutations [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 167.

KIT is a well characterized oncogenic driver, mutated in up to 80% of gastro-intestinal stromal tumors (GIST). Front-line imatinib is effective in many patients but durable responses in the setting of imatinib failure are rarely achieved due to complex secondary mutations that arise in the KIT ATP-binding pocket (exons 13/14) or the activation (A)loop (exons 17/18). Second-line sunitinib is active against exon 13/14 mutations but not A-loop mutations and identifying inhibitors that target the spectrum of possible mutations without incurring off-target toxicities related to broad-spectrum kinase inhibition has been challenging. Bezuclastinib (CGT9486) is a novel type I tyrosine kinase inhibitor that was designed as a potent and selective inhibitor of KIT A-loop mutations to address this unmet medical need. Herein, we present nonclinical data on the differentiated selectivity profile of bezuclastinib compared with other KIT inhibitors. We show that bezuclastinib is a low nanomolar inhibitor of KIT A-loop mutants which uniquely avoids targeting closely related kinases with known liabilities (e.g., PDGFRA, PDGFRB, CSF1R, and KDR) versus other approved inhibitors. In vivo pharmacokinetic/pharmacodynamic (PKPD) relationships have been evaluated utilizing either HMC1.2 human mast cell leukemia tumors for KIT phosphorylation or a Ba/F3-KIT-D816V splenomegaly model. In these models, plasma exposure associated with 50% reduction in splenomegaly or KIT phosphorylation was observed at 3-10 mg/kg/day corresponding to exposures 1.5-2.2 µg∙hr/mL. This correlated well with plasma exposure required to cause significant tumor growth inhibition in a GIST patient-derived xenograft model harboring an A-loop mutation (~1.3 µg∙hr/mL). Maximal efficacy in the PKPD models and tumor regression in GIST disease models was observed in exposure ranges of 19-36 or 11-44 µg∙hr/mL, respectively, which corresponds to clinically reached exposures in a recent GIST combination study of bezuclastinib with sunitinib. Importantly, in nonclinical studies, we also show that bezuclastinib has minimal brain penetration, a preferred feature of an anti-KIT molecule due to CNS-related adverse events observed in this targeted class. We present a tissue distribution study which demonstrates a brain:plasma ratio Citation Format: Anna Guarnieri, Karyn Bouhana, LouAnn Cable, Rob Rieger, Leyla Haygood, John Robinson, Shannon Winski. Bezuclastinib is a differentiated KIT inhibitor that exhibits unique selectivity to KIT A-loop mutations, minimal brain penetration, and favorable pharmacokinetic properties in preclinical models [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 147.

Capping off an era marred by drug development failures and punctuated by waning interest and presumed intractability toward direct targeting of KRAS, new technologies and strategies are aiding in the target's resurgence. As previously reported, the tetrahydropyridopyrimidines were identified as irreversible covalent inhibitors of KRAS

[Image: see text] KRAS is the most frequently mutated driver oncogene in human cancer, and KRAS mutations are commonly associated with poor prognosis and resistance to standard treatment. The ability to effectively target and block the function of mutated KRAS has remained elusive despite decades of research. Recent findings have demonstrated that directly targeting KRAS-G12C with electrophilic small molecules that covalently modify the mutated codon 12 cysteine is feasible. We have discovered a series of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRAS-G12C with in vivo activity. The PK/PD and efficacy of compound 13 will be highlighted.

The molecular pathogenesis of Systemic Mastocytosis (SM) is driven by mutations in the KIT gene, with 95% of patients having a mutation in exon 17, D816V, leading to constant proliferation of mast cells (Garcia-Montero et al, 2006; Jara-Acevedo et al, 2015; Vaes et al, 2017). Targeted therapeutics have revealed clinical activity in these patients, but toxicities such as cognitive effects, intracranial hemorrhage, hypertension, and edema may limit dosing and availability of these therapies. While the exact cause of these effects is difficult to determine, numerous closely related kinases, such as wild type PDGFRα, PDGFRβ, KIT, VEGFR2 (KDR), and CSF1R (FMS), are considered to be anti-targets, with previous evidence of their inhibition linked to observed clinical toxicities (Liu & Kurzrock, 2015; Giles et al., 2009; Jayson et al., 2005). Bezuclastinib (CGT9486) was designed to selectively inhibit KIT D816V versus these other closely related kinase anti-targets. Additionally, we demonstrate that bezuclastinib has minimal brain penetration, together with no observed CNS-related toxicities in nonclinical studies. Herein, we present results from cell-based kinase profiling assays, which demonstrate that bezuclastinib has a significant and unique selectivity to KIT D816V relative to the aforementioned kinases when tested head-to-head against other clinically relevant compounds in SM. Additionally, a similar selectivity profile was observed for a broader panel of kinases, ion channels, transporters, and enzymes, which will be presented here, including drug concentrations and target engagement achieved with recent in vivo studies. Importantly, we also show that bezuclastinib has minimal brain penetration, a preferred feature of an anti-Kit molecule due to CNS-related adverse events observed in these indications. In a tissue distribution study performed in rats, bezuclastinib shows a brain:plasma ratio Disclosures Guarnieri: Cogent Biosciences: Current Employment. Cable: Cogent Biosciences: Current Employment. Bouhana: Cogent Biosciences: Current Employment. Sullivan: Cogent Biosciences: Current Employment. Ball: Cogent Biosciences: Current Employment. Sachs: Cogent Biosciences: Current Employment. Winski: Cogent Biosciences: Current Employment. Robinson: Cogent Biosciences: Current Employment.

The TAM family of receptor tyrosine kinases (MERTK, AXL, and TYRO3) are expressed on DCs and macrophages, and they serve a critical role in efferocytosis, by which apoptotic bodies are cleared by engulfment. Their activation drives phagocytic activity concomitant to promoting an immuno-suppressive, tolerogenic state in DCs and the M2 polarization of macrophages. This dual signaling mechanism allows for the clearance of cells undergoing non-pathologic apoptotic turnover without inappropriately breaking self-tolerance and driving auto-immunity. Unlike most immuno-therapy targets, the KOs of the TAM kinases, PD-1, and CTLA-4 share common auto-immune disease phenotypes such as splenomegaly, immune infiltration of the liver, arthritis, and lupus. Furthermore, viruses have evolved to promote PD-1/CTLA-4 signaling to suppress the anti-viral immune response, and they similarly target the TAM receptors via apoptotic mimicry to reduce the inflammatory response. This confluence of immune phenotypes provided the biological rationale for the discovery of PF-5807/ARRY-067, a potent and selective inhibitor of MERTK/AXL as a DC-activating immuno-oncology therapeutic. Based on the IC50 for inhibition of phospho-MERTK/AXL in cell-based assays, our mouse PK and PK/PD studies determined that 50 mg/kg PF-5807 provided IC90 plasma coverage for 12 hours. This PK profile supported a twice-daily dosing schedule for maintaining continuous suppression of MERTK and AXL, which was used for mechanistic and efficacy studies in mouse models. To investigate DC and T cell responses to PF-5807, mice were adoptively-transferred with OT-I T cells and challenged with ovalbumin immunization; flow cytometry analysis indicated that PF-5807 treatment increased the fraction of activated, Class II MHC-positive DCs, as well as cross-primed IFNγ+ TNFα+ OT-I T cells. Also, we found that cytotoxic T lymphocytes were necessary for tumor control in the E0771 mouse syngeneic tumor model, as depletion of CD8+ T cells abrogated all activity by PF-5807 in inhibiting tumor growth. This anti-tumor activity was further demonstrated in the MC-38 and EMT6 tumor models, where single agent PF-5807 effected 57-88% TGI and a 13-38% cure rate, while combination with PD-1 blockade drove 99-100% TGI and a 75-100% cure rate. Finally, these complete responders rejected tumor re-challenge without further treatment, resulting in extended survival and durable cures that corroborate the development of immunologic memory. Therefore, we find that PF-5807 activates DCs to cross-prime CD8+ T cells, which are necessary for tumor control and are likely responsible for the establishment of tumor-specific memory T cells. PF-5807 is currently in an ongoing Phase I clinical trial in patients with advanced or metastatic solid tumors, with the intent to explore combination with anti-PD-1/anti-PD-L1 antibodies. Citation Format: Jim Wong, Jacqueline Harrison, Karyn Bouhana, Nhan Nguyen, Namir Shaabani, Vincent F. Vartabedian, LouAnn Cable, Robert Rieger, Lisa Pieti Opie, Shelley Allen, Adam Cook, Joshua Dahlke, Ronald Hinklin, David Chantry, Shannon Winski, John Teijaro. The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1735.

Although approved BRAF inhibitors have transformed the treatment of patients with certain BRAF V600 mutant cancers, their long-term efficacy is thought to be limited by poor brain penetration. As a result, disease progression in the brain is a significant cause of morbidity and mortality. PF-07284890 (ARRY-461) is an orally bioavailable, brain penetrant, potent, small molecule inhibitor targeting BRAF V600 mutant tumors and is in clinical development in patients with BRAF V600 metastatic melanoma with progression to the brain. In biochemical in vitro studies, PF-07284890 inhibits BRAF and CRAF with IC50's of 5.8 and 4.1 nM, respectively. Additionally, PF-07284890 inhibits the clinically relevant kinase domain BRAF V600 mutants (V600E and V600K) (IC50 = 24-25 nM). In cell-based systems, PF-07284890 potently inhibits phosphorylation of ERK, a downstream marker of BRAF inhibition, and potently inhibits proliferation of BRAF V600E/K mutant melanoma cell lines (IC50 18-38 nM). PF-07284890 was designed to distribute to the brain and, as such, in vitro experiments indicate that PF-07284890 has high cellular membrane permeability and is not a substrate for human P glycoprotein (P-gp). After oral administration of PF-07284890 to mice and rats, PF-07284890 distributes to the brain where the free fraction adjusted exposure in the brain was approximately proportional to the free fraction adjusted exposure in plasma (i.e., Cbrain,u/Cplasma,u approaches 1.0). In vivo, PF-07284890 inhibits phosphorylation of ERK in A375 BRAF V600E tumors, achieving maximal target inhibitions at a dose of 10 mg/kg. PF-07284890 has been evaluated for its ability to control BRAF V600E cell line and patient-derived melanoma xenograft tumor growth in nude mice when implanted both subcutaneously and intracranially. Dose-related tumor growth inhibition was demonstrated at dose levels ranging from 1 to 30 mg/kg, BID in both subcutaneous and intracranial xenograft models. In all models, regardless of tumor location, maximal efficacy was observed in dose ranges of 10-30 mg/kg BID. In the intracranial A375-luc BRAF V600E melanoma xenograft model, significant and durable tumor regressions were seen at doses of 10 and 30 mg/kg/day which translated to a profound survival benefit in animals receiving PF-07284890 (median survival > 55 days post-implantation) compared to control animals (median survival = 15 days). GLP safety studies demonstrated a good safety profile for PF-07284890. PF-07284890 is fully brain penetrant, with the potential to address this key unmet medical need and thereby defines a new class of brain penetrant, potent and selective BRAF inhibitors. Citation Format: Karyn Bouhana, Deborah Anderson, Walter DeWolf, Suzy Brown, Lance Williams, Li Ren, David Moreno, Ross Wallace, Jay Brad Fell, Dylan Hartley, Patrice Lee. Nonclinical development of PF-07284890 (ARRY-461), a potent, brain-penetrant, small molecule inhibitor of BRAF V600-mutation-driven tumors in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1473.

The ability to effectively target mutated KRAS has remained elusive despite decades of research. By solving a highly informative set of ligand-complexed co-crystal structures coupled with iterative structure-based drug design, substituted tetrahydropyridopyrimidines were identified as selective, covalent inhibitors of mutant KRAS G12C. Key molecular interactions with the protein were optimized, with the potency of lead compounds evaluated by (a) mass spectrometric quantification of modified KRAS protein with and without treatment of test compounds, and (b) measurement of phospho-ERK in a whole-cell assay using H358 cells after incubation with test compounds for 3 hours. These efforts identified MRTX1257 as a potent and selective inhibitor of mutant KRAS G12C activity. This lead compound was utilized as a research tool to aid in a deeper understanding of therapeutic susceptibility and KRAS dependence. MRTX1257 makes a covalent bond with the codon 12 cysteine and binds in the “Switch-2” pocket of KRAS, stabilizing the protein in the inactive, GDP-bound state. MRTX1257 contains a cyanomethyl group that displaces a water found near Gly10 in co-crystal structures of less potent analogs and contains an 8-methylnaphthyl group that fills a hydrophobic pocket, resulting in enhanced potency compared with unsubstituted naphthyl analogs. MRTX1257 demonstrated rapid, irreversible modification of GDP-bound recombinant KRAS G12C and suppressed ERK phosphorylation with an IC50 = 1 nM in the H358 cell line. In proteomics studies designed to assess global protein modification, MRTX1257 was shown to be highly selective for the targeted Cys12 of KRAS G12C versus other surface-exposed cysteine residues in NCI-H358 cells. Finally, at a 30mg/kg PO dose, MRTX1257 exhibited 31% bioavailability in mouse, demonstrated near-complete inhibition of KRAS signaling in tumor tissue, and complete durable tumor regression in MIA PaCa-2 xenografts. The discovery of the tetrahydropyridopyrimidine series, the structure-based optimization to MRTX1257 and its preclinical potency, selectivity, ADME and efficacy profile will be presented. Citation Format: Matthew A. Marx, Brian R. Baer, Joshua Ballard, James F. Blake, Karyn Bouhana, David M. Briere, Laurence E. Burgess, Michael R. Burkhard, Harrah Chiang, Mark J. Chicarelli, James G. Christensen, John P. Fischer, Jill Hallin, Macedonia J. Mejia, Peter Olson, Pavel Savechenkov, Niranjan Sudhakar, Tony P. Tang, Guy P. Vigers, Jay B. Fell. Structure-based drug discovery of MRTX1257, a selective, covalent KRAS G12C inhibitor with oral activity in animal models of cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B30.

Background Alterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations. Patients and methods Potent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly. Results LOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein −/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved. Conclusions These results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.

Background: Inflammation is an essential component of arthritis pain. Nerve growth factor (NGF) plays a key role in acute and chronic pain states especially those associated with inflammation. NGF acts through tropomyosin-receptor-kinase A (TrkA). NGF blockade has reduced arthritis pain in clinical trials. We explored the mechanisms within the joint which may contribute to the analgesic effects of NGF by selectively inhibiting TrkA in carrageenan-induced or collagen-induced joint pain behaviour. The goal of the current study was to elucidate whether inflammation is central to the efficacy for NGF blockade.Methods: Rats were injected in their left knees with 2 % carrageenan or saline. Collagen-induced arthritis (CIA) was induced by intradermal injections of a mixture of bovine type II collagen (0.2 mg) and incomplete Freund’s adjuvant (0.2 mg). Oral doses (30 mg/kg) of AR786 or vehicle control were given twice daily after arthritis induction. Ibuprofen-treated (35 mg/kg, orally, once daily) rats with CIA were used as positive analgesic controls. Pain behaviour was measured as hind-limb weight-bearing asymmetry and hind-paw withdrawal thresholds to von Frey hair stimulation (carrageenan synovitis), or withdrawal to joint compression using a Randall Selitto device (CIA). Inflammation was measured as increased knee joint diameter and by histopathological analysis.Results: Intra-articular injections of carrageenan or induction of CIA was each associated with pain behaviour and synovial inflammation. Systemic administration of the TrkA inhibitor AR786 reduced carrageenan-induced or CIA-induced pain behaviour to control values, and inhibited joint swelling and histological evidence of synovial inflammation and joint damage.Conclusions: By using two models of varying inflammation we demonstrate for the first time that selective inhibition of TrkA may reduce carrageenan-induced or CIA-induced pain behaviour in rats, in part through potentially inhibiting synovial inflammation, although direct effects on sensory nerves are also likely. Our observations suggest that inflammatory arthritis causes pain and the presence of inflammation is fundamental to the beneficial effects (reduction in pain and pathology) of NGF blockade. Further research should determine whether TrkA inhibition may ameliorate human inflammatory arthritis.

Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5′ untranslated region, the coding region, and the 3′ untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.

Background: Activating mutations and oncogenic fusions of the RET receptor tyrosine kinase have been identified in multiple tumor types, including thyroid, lung, breast and colon carcinoma. Furthermore, tyrosine kinase inhibitors (TKIs) with anti-RET activity have produced clinical responses in patients whose tumors harbor RET alterations. However, currently available RET inhibitors were initially developed to target kinases other than RET and are only moderately potent against RET, inhibit multiple kinases other than RET or poorly inhibit secondary resistance mutations (e.g. gatekeeper mutations) common to other TKIs. We have discovered novel, potent and selective RET inhibitors. The resulting compounds exhibit nanomolar potency against wild type RET and select RET mutants, including the KIF5B-RET fusion and V804M gatekeeper mutation, in both enzyme and cellular assays, with minimal activity against highly related kinases. AR025 is representative of this series; the activity of AR025 and related analogs in relevant in vitro and in vivo models will be presented here. Methods: In vitro and in vivo evaluations, including enzyme and cell-based assays, pharmacokinetic (PK)/pharmacodynamics (PD) correlations, drug metabolism characterization, and non-clinical safety evaluation, were conducted using standard methods. Tumor growth inhibition and PD studies were carried out using subcutaneous allografts of NIH-3T3 cells expressing KIF5B-RET in nude mice. Results: AR025 demonstrated nanomolar potency against both wild type and mutant RET proteins in enzyme and cellular assays. AR025 had minimal activity against an enzyme panel of >200 diverse kinases and demonstrated >50x cellular selectivity against VEGFR, with similar selectivity against other related kinases. AR025 possessed low intravenous clearance and high oral exposure in mice, rats and dogs. Finally, a single oral dose of 30mg/kg produced more than 90% inhibition of phospho-RET in NIH-3T3-KIF5B-RET mouse allografts, while twice-per-day continuous dosing resulted in greater than 90% tumor growth inhibition. Notably, AR025 was minimally toxic at doses up to 100mg/kg per day. Conclusions: We have identified a series of potent and selective RET inhibitors with high oral bioavailability and favorable PK properties in animals. One of these, AR025, demonstrated potent inhibition of RET in enzyme and cellular assays, with minimal activity against highly related kinases. In an NIH3T3-KIF5B-RET allograft model, AR025 effectively inhibited phospho-RET and caused dramatic tumor growth inhibition without significant toxicity. The identification of potent and selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of currently available inhibitors with anti-RET activity. Citation Format: Barbara J. Brandhuber, Nisha Nanda, Julia Haas, Karyn Bouhana, Lance Williams, Shannon Winski, Michael Burkard, Brian Tuch, Kevin Ebata, Jennifer Low, Francis Sullivan, Lauren Hanson, Tony Morales, Guy Vigers, Jessica Gaffney, Ross D. Wallace, James Blake, Yutong Jiang, S. Michael Rothenberg, Steven Andrews. Identification and characterization of highly potent and selective RET kinase inhibitors for the treatment of RET-driven cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B192.

High-grade serous ovarian carcinoma (HGSOC) has the poorest prognosis amongst all gynecological cancers with a median survival of approximately 5 years. While most patients (pts) initially respond to therapy, few achieve long-term remission or cure; thus, new treatment strategies are needed. Paclitaxel (Pac), an agent commonly used in the treatment of HGSOC, induces the Ras/Raf/MEK/ERK pathway and resistance may be partially mediated through activation of MEK/ERK, suggesting a rationale for combining a MEK inhibitor with Pac. Binimetinib (MEK162) is an oral, potent, selective, allosteric small-molecule MEK1/2 inhibitor. The combination of binimetinib and Pac was studied in a panel of in vivo ovarian carcinoma xenograft models derived from either cell lines (A2780, SK-OV-3) or primary patient tumor resections pathologically confirmed to be HGSOC (OVC38B1, OVC604, OVC629, OVC104) and propagated solely in mice (pt-derived xenograft [PDX]). Three of these PDX models were from newly diagnosed pts, while 1 (OVC38B1) was from a pt who had relapsed following a prior response to a platinum/taxane regimen. All models were sequenced for cancer-specific genes by next-generation sequencing; no mutations were found in Ras, Raf, MEK or ERK. To evaluate single-agent and combination activity, tumor-bearing mice received 25 mg/kg Pac IP (days 1,5,9) and/or 30 mg/kg binimetinib PO (days 2-15). Single-agent binimetinib was modestly effective in 3/6 models (tumor growth inhibition [TGI] ≥ 50%), which is consistent with the lack of MAPK pathway mutations in these models. Single-agent Pac was highly effective in 3/6 models (TGI ≥ 90%) including OVC38B1 and 2 PDX models from newly diagnosed pts; the remaining models were taxane-resistant. This is consistent with expected high rates of response in the setting of newly diagnosed pts and/or prior responders. Binimetinib plus Pac had superior efficacy to either agent alone in 4/6 models including all taxane-resistant models. In 2 PDX models from newly diagnosed pts, combination activity was not improved, but Pac was highly effective as a single agent (72-82% regression) leaving little room for enhancement. Encouragingly, the combination induced tumor regressions in 2 taxane-resistant models in which no single-agent regressions were observed. In conclusion, in vivo drug sensitivity studies with binimetinib and Pac recapitulate the response expected in HGSOC based on stage, genetic background and clinical history. In models of taxane-resistant disease, combination with binimetinib had improved activity over taxane alone warranting further study of this approach in HGSOC. Citation Format: Shannon L. Winski, Karyn Bouhana, Susan Rhodes, LouAnn Cable, Deborah Anderson, Lance Williams, Brian Tunquest, Tiffany Logan, Guy Vigers, Patrice Lee. Activity of the MEK inhibitor Binimetinib (MEK162) in combination with paclitaxel in patient-derived xenograft models of high-grade serous ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 666. doi:10.1158/1538-7445.AM2015-666

A nuclease resistant ribozyme targeting the 5′ untranslated region (5′ UTR) of hepatitis C virus (HCV) at site 195 has been identified. To investigate the therapeutic utility of this ribozyme, we evaluated the pharmacokinetics and tissue distribution with two labeled forms of this ribozyme. [32P]-labeled ribozyme was administered as a single subcutaneous (SC) or intravenous (IV) bolus at a dose of 10 mg/kg or 30 mg/kg in C57Bl/6 mice. Regardless of route of administration, peak liver concentrations achieved were greater than the concentration necessary to inhibit HCV-IRES-luciferase expression in cell culture. The ribozyme was well absorbed after SC administration (89%) and had an elimination half-life of 23 minutes. To show intracellular localization of the ribozyme in target tissue, a tetramethyl rhodamine (TMR)–labeled ribozyme was administered as a single SC or IV bolus at a dose of 30 mg/kg in C57Bl/6 mice. Mice treated SC or IV with TMR–labeled ribozyme had positive fluorescence in the liver from 15 minutes to 48 hours after dosing. Definite positive fluorescence was still present at 72 hours in the mice dosed via the IV route. At early time points (15 and 30 minutes postinjection), nuclear and possibly cytoplasmic fluorescence was present in the hepatocytes, and sinusoidal fluorescence was intense. At the later time points, fluorescence became more punctate. Abundant staining was often present in Kupffer cells. This study confirms the retention of ribozyme in liver cells and supports the potential of an anti-HCV ribozyme as a therapeutic agent for treatment of chronic hepatitis C. (Hepatology 2000;32:640-646.)

Vascular endothelial growth factor (VEGF) is a growth factor that contributes to the angiogenesis of developing tumors. To interfere with the action of VEGF, a nuclease-stabilized ribozyme, ANGIOZYME, has been developed against VEGF receptor subtype Flt-1 mRNA. To determine which routes of administration would be useful for systemic delivery of this ribozyme, a dose of 30 mg/kg [32P]ANGIOZYME was administered as an i.v., i.p., or s.c. bolus. Concentrations of ANGIOZYME in plasma, femur, kidney, liver, and lung were examined. ANGIOZYME was well absorbed after i.p. (90%) or s.c. administration (77%), with peak plasma concentrations occurring 30 minutes after dosing. Total body clearance after a single dose of 30 mg/kg ANGIOZYME was 20 ml/min/kg, and the elimination half-life was 33 minutes. The apparent volume of distribution at steady-state ranged from 0.5 to 1.3 L/kg. ANGIOZYME was detected in the four tissues examined through the 3 hour sampling period after i.v. or i.p. administration. After s.c. administration, ANGIOZYME was detected in femur, kidney, and lung but not in the liver. The highest concentrations of ANGIOZYME were found in kidney and femur with all three routes. Because of the rapid and extensive absorption after extravascular injections, either i.p. or s.c. administration could be considered for use in pharmacodynamic studies examining the effects of ANGIOZYME or other ribozymes with similar chemical modifications.

Historically, human tumor cell line cultures and xenografts have served as standard models for identifying and optimizing anticancer drugs. Despite wide use, cell line models are poor predictors of clinical activity of drugs, particularly cytotoxics. Primary human tumor explant models have significant potential to inform clinical direction. These models have not undergone genetic drift or selection for growth in culture. Furthermore, low-passage explants retain the morphology and aspects of the tumor microenvironment reflecting the primary cancer in patients. These models further represent patients who have been treated with current chemotherapeutics. ARRY-520 is a novel kinesin spindle protein inhibitor which has been investigated in clinical studies in both solid and hematological cancers. In cell line xenografts, ARRY-520 has significant activity in solid tumors and even greater activity in hematological cell lines both in vitro and in vivo. In clinical studies, while ARRY-520 has demonstrated significant single-agent activity in multiple myeloma, activity in solid tumors has been modest. Thus, while preclinical data in hematological models has correlated with clinical activity in myeloma, the impressive solid tumor data has not translated to clinical activity in solid tumors. To probe the disparity of preclinical and clinical activity in solid tumors, we have investigated the ability of preclinical models to inform clinical success by retrospective analysis of the activity of ARRY-520 in solid tumor cell line and explant models, as compared to clinical data of this molecule in these indications. ARRY-520 shows potent activity in human tumor cell lines both in vitro, (IC50 0.5-14nM) and in vivo (50% tumor regression in 4/5 cell line xenografts CRC and PaCa including 100% regression in 2 models). By contrast, in 6 primary CRC and PaCa explants, ARRY-520 showed minimal activity (transient stable disease in 1/6 models and progressive disease in 5/6 models). Phase 1 solid tumor data on ARRY-520 was reported at ASCO 2010. In this study, 30 patients with solid tumors, treated at or above the MTD, were evaluable for response with best response of SD>12 weeks in 2 patients (7%). Within this study, there were 4 CRC and 3 PaCa patients, all of whom experienced progressive disease within 12 weeks of initiation. Further investigation of ARRY-520 in solid tumor explants is ongoing. Analysis of responsive and non-responsive models will be used to identify solid tumor types for further clinical development. In summary, in this study, human solid tumor explant xenografts more accurately predicted the actual clinical results from a Phase 1 solid tumor trial. These data suggest that solid tumor explant models may have significant utility in informing clinical decisions. 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 1782. doi:1538-7445.AM2012-1782

The Trk family of genes, which includes trkA/NTRK1, trkB/NTRK2 and trkC/NTRK3, encode the tyrosine kinase receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activity of trk family members is associated with human cancer. Oncogenic translocations involving trkC kinase domain have been identified in AML, salivary gland carcinoma, adult secretory breast cancer, congenital fibrosarcoma, and pediatric nephroma. Oncogenic trkA translocations have been reported in papillary thyroid and colorectal cancers. We have identified orally bioavailable, potent and selective ATP-competitive inhibitors of the trk family of receptor tyrosine kinases and are developing these for the treatment of Trk-driven cancers. AR523 is a pan-Trk inhibitor which demonstrates similar activity against TrkA, TrkB and TrkC receptors in a cell based assay (IC50 ∼10 nM). In a screen at 1 µM against a panel of 230 kinases, AR523 inhibited only three additional kinases more than 50% (TNK2, Bmx and Txk). In cell culture AR523 was found to exhibit selective anti-proliferative activity toward the cell line KM12, which harbors the oncogenic TPM-TrkA translocation, while exhibiting no activity toward HT29, a cell line with no trk gene rearrangements. Studies in mice engrafted with KM12 tumors revealed significant anti-tumor and pharmacodynamic activity of AR523. Administration of a single oral dose of AR523 reduced tyrosine phosphorylation of TrkA by nearly 80% at twelve hours after dosing. Parallel analysis of Akt and Erk revealed reduced phosphorylation of these downstream effectors. Administration of 100 mg AR523 daily for two weeks to mice with KM12 xenografts produced mean tumor growth inhibition of 86% and a mean of 42% tumor regression. AR523 exhibits dose dependent inhibition of tumor growth at 10 and 30 mg/kg in KM12 xenografts with 54 and 72% TGI respectively. AR523 was well-tolerated, causing no weight loss or deaths compared with vehicle control. Increasingly Trk mutations have been shown to be activating and the use of Trk inhibitors may provide a new therapeutic strategy for targeted treatment. 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 1798. doi:1538-7445.AM2012-1798

Macrophages are believed to play a crucial role in wound healing by synthesizing and secreting numerous cytokines. Some of these cytokines, such as transforming growth factor-beta and tumor necrosis factor-alpha, promote fibrosis and repair. We have shown that macrophages are recruited to sterile fetal wounds and have the potential to regulate repair by synthesizing transforming growth factor-beta(1), transforming growth factor-beta(2), and tumor necrosis factor-alpha. Transforming growth factor-beta was present in fetal lamb wounds in higher amounts than in adult sheep wounds. Furthermore, the concentrations and ratios of the transforming growth factor-beta isoforms in wounds that healed without scarring were different from those in wounds that scarred; transforming growth factor-beta(2) was highest in fetal wounds that did not scar and lowest in adult wounds. These data suggest that concentrations of transforming growth factor-beta isoforms rather than total transforming growth factor-beta concentration may be important in the regulation of fibrosis in prenatal and postnatal wound healing.

Activation of redundant receptor tyrosine kinases (RTKs) in tumors can confer resistance to selective RTK inhibitors. In particular, activation of multiple ErbB-family kinases is associated with diminished activity of selective EGFR or ErbB2 inhibitors. ErbB-family ligands are commonly expressed in cancers and can activate multiple redundant ErbB dimers. We hypothesize that ligand activation of multiple ErbB-family kinases in tumors can confer resistance to selective ErbB inhibitors, but not pan-ErbB inhibitors, suggesting a setting where pan-ErbB inhibitors may be advantageous over selective inhibitors. One such candidate currently in Phase 1/2 clinical trials is ARRY-334543, an oral, reversible pan-ErbB inhibitor with potent in vivo activity in both EGFR- and ErbB2-dependent human tumor xenografts. We tested this hypothesis in vitro with N87 gastric carcinoma cells that express high levels of constitutively active ErbB2 and low levels of EGFR. In the absence of EGF, ErbB2 homodimers are predominant and cells are highly sensitive to ErbB2 inhibition. ARRY-334543 and the ErbB2-selective inhibitor, ARRY-380, strongly inhibited N87 proliferation (IC50 = 60 nM and 5 nM, respectively) while the EGFR-selective inhibitor erlotinib was active only at high concentrations (IC50 = 860 nM). No benefit was observed by combining ARRY-380 and erlotinib. EGF-stimulation of N87 cells caused the formation of EGFR homodimers and EGFR/ErbB2 heterodimers which significantly alters cellular responses to selective ErbB inhibitors. The cellular IC50 for ARRY-380 increased 57-fold while erlotinib activity was relatively unchanged, suggesting a loss of dependence on ErbB2 signaling alone. The dual inhibition of EGFR and ErbB2, either by ARRY-334543 or the combination of ARRY-380 and erlotinib, largely maintained potency with only a modest increase in IC50 (9- to 10-fold). These data suggest that activation of EGFR can significantly diminish the activity of selective ErbB inhibitors, but not pan-ErbB inhibitors. Dual-responsiveness was also observed in vivo where both EGFR and ErbB2 homodimers were detected in N87 xenografts and dual EGFR/ErbB2 treatment resulted in superior tumor growth inhibition (TGI). ARRY-334543 was highly active with 90% TGI (100 mg/kg BID). Treatment with the combination of ARRY-380 and erlotinib (50 mg/kg QD each, MTD when given in combination) had similar activity with 82% TGI. Treatment with either erlotinib (100 mg/kg QD) or ARRY-380 (50 mg/kg QD) alone was less efficacious. These results suggest that the pan-ErbB inhibitor, ARRY-334543, can be differentiated from selective ErbB inhibitors by targeting tumors that signal through multiple ErbB-family receptors. A wide range of tumor types has been shown to activate multiple ErbB-family members and clinical evaluation of this hypothesis is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3610.

ARRY-380 is an orally active, potent small molecule tyrosine kinase inhibitor targeting ErbB2. The compound is a reversible, ATP-competitive inhibitor with nanomolar potency against ErbB2 in both in vitro and in cell-based assays. This compound has very good in vivo and in vitro PK/ADME properties and has shown excellent activity in numerous mouse tumor models including breast (BT-474, MDA-MB-453), ovarian (SKOV-3) and gastric (N87) carcinoma models. Here we demonstrate excellent single agent activity and combinability with trastuzumab, docetaxel or bevacizumab in breast and ovarian carcinoma models. For the BT-474 studies, female SCID beige mice were implanted with tumor fragments. For the SKOV-3 tumor studies, female nude mice were inoculated with cells subcutaneously in the flank. Animals received: doses of ARRY-380 ranging up to 200 mg/kg/d, PO; and/or trastuzumab at 20 mg/kg, IP, Q3D or QW; and/or docetaxel at 10 mg/kg, IV, Q3D; and/or bevacizumab at 10 mg/kg, IP, Q4D x3. Tumor size was measured at regular intervals and subsets of animals were monitored for up to 90 days to determine tumor-free survival. In the BT-474 model, ARRY-380 demonstrated significant dose-related tumor growth inhibition (TGI; 50% at 50 mg/kg/d and 96% at 100 mg/kg/d) with numerous partial regressions (>50% reduction from baseline size) at the higher dose level in 9/12 animals. One complete response was observed at the higher dose. Trastuzumab alone provided a 45% TGI with no regressions. ARRY-380 (50 mg/kg/d) in combination with trastuzumab showed a 98% TGI with complete regressions in 9/12 animals and two partial regressions. At dose of 100 mg/kg/d of ARRY-380 in combination with trastuzumab, there was 100% TGI and all animals had complete responses. Docetaxel as a single agent produced a 55% TGI with no regressions. In combination with ARRY-380 (50 mg/kg/d), there was an 81% TGI and five partial regressions. In the SKOV-3 model, ARRY-380 demonstrated significant dose-related tumor growth inhibition (TGI; 39% at 50 mg/kg, BID and 96% at 100 mg/kg, BID) with partial regressions (>50% reduction from baseline size) at the higher dose level in all animals. Bevacizumab alone provided a 55% TGI with no regressions. ARRY-380 (50 mg/kg, BID) in combination with bevacizumab showed 80% TGI with partial responses in 7/8 animals and one stable disease. From this work we have demonstrated superb single agent activity for ARRY-380 in the BT-474 human breast carcinoma xenograft model and the SKOV-3 human ovarian carcinoma model. In addition, ARRY-380 has shown additive activity and tolerability with trastuzumab, docetaxel and bevacizumab. ARRY-380 has entered Phase I clinical trials in patients with advanced cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5104.