Significance Overcoming resistance to targeted kinase inhibitors is a major clinical challenge in oncology. Development of crizotinib resistance through the emergence of a secondary ROS1 mutation, ...ROS1 ᴳ²⁰³²ᴿ, was observed in patients with ROS1 fusion-positive lung cancer. In addition, a novel ROS1 fusion recently has been identified in glioblastoma. A new agent with robust activity against the ROS1 ᴳ²⁰³²ᴿ mutation and with CNS activity is needed to address these unmet medical needs. Here we report the identification of PF-06463922, a ROS1/anaplastic lymphoma kinase (ALK) inhibitor, with exquisite potency against ROS1 fusion kinases, capable of inhibiting the ROS1 ᴳ²⁰³²ᴿ mutation and FIG-ROS1–driven glioblastoma tumor growth in preclinical models. PF-06463922 demonstrated excellent therapeutic potential against ROS1 fusion-driven cancers, and it currently is undergoing phase I/II clinical trial investigation.
Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1 ᴳ²⁰³²ᴿ mutation and the ROS1 ᴳ²⁰²⁶ᴹ gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1 ᴳ²⁰³²ᴿ mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.
A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic
lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of ...anaplastic large-cell lymphoma (ALCL). PF-2341066 was
recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK
fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC 50 value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically
relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was
associated with G 1 -S–phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC 50 values, ∼30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide
transferase–mediated nick-end labeling and Annexin V staining (IC 50 values, 25–50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299
ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d
dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition
of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators,
including phospholipase C-γ, signal transducers and activators of transcription 3, extracellular signal-regulated kinases,
and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation
and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of
patients with ALK-positive ALCL. Mol Cancer Ther 2007;6(12):3314–22
The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of several human cancers and are attractive therapeutic targets. PF-2341066 ...was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. PF-2341066 was selective for c-Met (and anaplastic lymphoma kinase) compared with a panel of >120 diverse tyrosine and serine-threonine kinases. PF-2341066 potently inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, or invasion of human tumor cells in vitro (IC(50) values, 5-20 nmol/L). In addition, PF-2341066 potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties. PF-2341066 showed efficacy at well-tolerated doses, including marked cytoreductive antitumor activity, in several tumor models that expressed activated c-Met. The antitumor efficacy of PF-2341066 was dose dependent and showed a strong correlation to inhibition of c-Met phosphorylation in vivo. Near-maximal inhibition of c-Met activity for the full dosing interval was necessary to maximize the efficacy of PF-2341066. Additional mechanism-of-action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), induction of apoptosis (caspase-3), and reduction of microvessel density (CD31). These results indicated that the antitumor activity of PF-2341066 may be mediated by direct effects on tumor cell growth or survival as well as antiangiogenic mechanisms. Collectively, these results show the therapeutic potential of targeting c-Met with selective small-molecule inhibitors for the treatment of human cancers.
The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely ...understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.
•NPM-ALK induces a metabolic shift toward biomass production.•NPM-ALK phosphorylates Y105-PKM2 to regulate metabolism and tumorigenesis.
ObjectiveTo evaluate safety and mechanism of action of mezagitamab (TAK-079), an anti-CD38 monoclonal antibody, in patients with moderate to severe systemic lupus erythematosus (SLE).MethodsA phase ...1b double-blind, placebo-controlled, multicentre study was conducted in patients with SLE receiving standard background therapy. Eligible patients were adults who met the 2012 SLICC or ACR criteria for diagnosis, had a baseline SLE Disease Activity Index 2000 (SLEDAI-2K) score of ≥6 and were positive for anti-double-stranded DNA antibodies and/or anti-extractable nuclear antigens antibodies. Patients received 45 mg, 90 mg or 135 mg of mezagitamab or placebo every 3 weeks over 12 weeks. Primary endpoints were safety and tolerability. Secondary endpoints included pharmacokinetics and pharmacodynamics. Exploratory assessments included disease activity scales, deep immune profiling and interferon pathway analysis.Results22 patients received at least one dose of either mezagitamab or placebo. In patients exposed to mezagitamab (n=17), drug was well tolerated. Adverse event (AEs) were balanced across treatment groups, with no treatment emergent AEs exceeding grade 2. Responder analyses for Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI) and SLEDAI-2K did not reveal any observable differences across treatment groups. However, there was a trend for more profound skin responses among patients with higher CLASI scores (>10) at baseline. Pharmacodynamic analysis showed median CD38 receptor occupancy up to 88.4% on CD38+ natural killer cells with concurrent depletion of these cells up to 90% in the 135 mg group. Mean reductions in IgG and autoantibodies were less than 20% in all dose groups. Cytometry by time of flight and type 1 interferon gene analysis revealed unique fingerprints that are indicative of a broad immune landscape shift following CD38 targeting.ConclusionsMezagitamab had a favourable safety profile in patients with moderate to severe SLE and elicited a pharmacodynamic effect consistent with CD38+ cell depletion. These findings reveal novel insights into the drug’s mechanism of action and support the continued investigation of mezagitamab in autoimmune diseases.
Although anti-tumor activities of type I interferons (IFNs) have been recognized for decades, the molecular mechanisms contributing to clinical response remain poorly understood. The complex ...functions of these pleiotropic cytokines include stimulation of innate and adaptive immune responses against tumors as well as direct inhibition of tumor cells. In high-grade, Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer, nadofaragene firadenovec, a non-replicating adenovirus administered locally to express the IFNα2b transgene, embodies a novel approach to deploy the therapeutic activity of type I IFNs while minimizing systemic toxicities. Deciphering which functions of type I IFN are required for clinical activity will bolster efforts to maximize the efficacy of nadofaragene firadenovec and other type I IFN-based therapies, and inform strategies to address resistance. As such, we characterized the phenotypic and molecular response of human bladder cancer cell lines to IFNα delivered in multiple contexts, including adenoviral delivery. We found that constitutive activation of the type I IFN signaling pathway is a biomarker for resistance to both transcriptional response and direct cytotoxic effects of IFNα. We present several genes that discriminate between sensitive and resistant tumor cells, suggesting they should be explored for utility as biomarkers in future clinical trials of type I IFN-based anti-tumor therapies.
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Jørn Jakobsen and colleagues characterize molecular biomarkers of the anti-tumor effects of IFNα, including a non-replicating adenovirus expressing transgene IFNα2b, on bladder cancer cell lines. Their results will assist design and analysis of future clinical trials to optimize treatment approach to provide maximal benefit to patients with bladder cancer.
PKM2 (pyruvate kinase M2), a critical regulator of glycolysis, is phosphorylated by numerous growth factor receptors and oncogenic tyrosine kinases including NPM-ALK which is expressed in a subset of ...aggressive T-cell non-Hodgkin lymphomas known as anaplastic large cell lymphoma, ALK-positive. Our previous work demonstrated that phosphorylation of Y105-PKM2 by NPM-ALK regulates a major metabolic shift to promote lymphomagenesis. In addition to its role in metabolism, recent studies have shown that PKM2 promotes oncogenesis by phosphorylating nuclear STAT3 (signal transducer and activator of transcription 3) and regulating transcription of genes involved in cell survival and proliferation. We hypothesized that identification of novel PKM2 interactors could provide additional insights into its expanding functional role in cancer. To this end, immunocomplexes of FLAG-tagged PKM2 were isolated from NPM-ALK-positive ALCL (anaplastic large cell lymphoma) cells and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) which led to the identification of polypyrimidine tract-binding protein (PTBP1) as a novel interactor of PKM2. The interaction between PTBP1 and PKM2 was restricted to the nucleus and was dependent on NPM-ALK mediated Y105 phosphorylation of PKM2. Stable shRNA-mediated silencing of PTBP1 resulted in a marked decrease in pY105-PKM2 and pY705-STAT3 which led to decreased ALCL cell proliferation and colony formation. Overall, our data demonstrate that PTBP1 interacts with PKM2 and promotes ALCL oncogenesis by facilitating PKM2-dependent activation of STAT3 within the nucleus.
The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-κB in lymphocytes, thereby representing a cornerstone ...of the adaptive immune response. Although CARMA1 is restricted to cells of the immune system, the analogous CARMA3 protein has a much wider expression pattern. Emerging evidence suggests that CARMA3 can substitute for CARMA1 in non-immune cells to assemble a CARMA3-Bcl10-MALT1 signalosome and mediate G protein-coupled receptor activation of NF-κB. Here we show that one G protein-coupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-κB in endothelial and vascular smooth muscle cells, thereby inducing pro-inflammatory signals within the vasculature, a key factor in atherogenesis. Further, we demonstrate that Bcl10-deficient mice are protected from developing angiotensin-dependent atherosclerosis and aortic aneurysms. By uncovering a novel vascular role for the CBM signalosome, these findings illustrate that CBM-dependent signaling has functions outside the realm of adaptive immunity and impacts pathobiology more broadly than previously known.
1 Diabetes Biology Department, 2 Biochemical Pharmacology and 3 Structural Biology, Pfizer Incorporated, San Diego, California; and 4 Division of Cardiovascular, Metabolic and Endocrine Diseases, ...Pfizer Incorporated, Groton, Conneticut
Submitted 17 March 2008
; accepted in final form 25 August 2008
c-Jun NH 2 -terminal kinase (JNK) plays an important role in insulin resistance; however, identification of pharmacologically potent and selective small molecule JNK inhibitors has been limited. Compound A has a cell IC 50 of 102 nM and is at least 100-fold selective against related kinases and 27-fold selective against glycogen synthase kinase-3β and cyclin-dependent kinase-2. In C57BL/6 mice, compound A reduced LPS-mediated increases in both plasma cytokine levels and phosphorylated c-Jun in adipose tissue. Treatment of mice fed a high-fat diet with compound A for 3 wk resulted in a 13.1 ± 1% decrease in body weight and a 9.3 ± 1.5% decrease in body fat, compared with a 6.6 ± 2.1% increase in body weight and a 6.7 ± 2.1% increase in body fat in vehicle-treated mice. Mice pair fed to those that received compound A exhibited a body weight decrease of 7 ± 1% and a decrease in body fat of 1.6 ± 1.3%, suggesting that reductions in food intake could not account solely for the reductions in adiposity observed. Compound A dosed at 30 mg/kg for 13 days in high-fat fed mice resulted in a significant decrease in phosphorylated c-Jun in adipose tissue accompanied by a decrease in weight and reductions in glucose and triglycerides and increases in insulin sensitivity to levels comparable with those in lean control mice. The ability of compound A to reduce the insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) von Ser307 and partially reverse the free fatty acid inhibition of glucose uptake in 3T3L1 adipocytes, suggests that enhancement of insulin signaling in addition to weight loss may contribute to the effects of compound A on insulin sensitization in vivo. Pharmacological inhibition of JNK using compound A may therefore offer an effective therapy for type 2 diabetes mediated at least in part via weight reduction.
Jun NH 2 -terminal kinase; insulin sensitizer; weight loss; diet-induced obese mice
Address for reprint requests and other correspondence: C. P. Briscoe, Johnson and Johnson, 3210 Merryfield Row, San Diego, CA 92121 (e-mail: Cbriscoe{at}prdus.jnj.com )
Abstract
Background:
Over 70% of patients newly diagnosed with bladder cancer will present with non-muscle invasive disease (NMIBC). The standard of care treatment to prevent recurrence and ...progression is tumor resection followed by Bacillus Calmette-Guerin (BCG). While this treatment regimen is effective for many patients, a significant fraction become resistant over time. Adenoviral-delivered IFNα gene therapy is emerging as a safe and efficacious treatment for BCG-refractory NMIBC. IFNα is a type 1 interferon that mediates complex tumor-immune interactions, and also has direct cytotoxic effect on tumors. While the mechanisms of IFNα have been extensively studied, it is unclear which of these mechanisms are most relevant for driving therapeutic response. It is also poorly understood why some tumors are sensitive to the direct cytotoxic effects of IFNα and others are resistant.
Methods:
Here, we investigate the molecular mechanisms of tumor-intrinsic sensitivity and resistance to IFNα. We characterized response of a panel of bladder cancer cell lines to treatment with IFNα, and performed concomitant gene expression profiling to identify molecular features and pathways associated with response and sensitivity. These features were then compared across responsive and non-responsive cancer cell line models from other tissues of origin including skin, pancreas, liver and lung.
Results/Conclusions:
Several bladder cancer models exhibited a dose-dependent cytotoxic response to IFNα treatments, while others were resistant to these effects. Genomic and mRNA features associated with IFNα sensitivity/resistance were identified. Pan-cancer cell line analysis identified conserved and putative tissue-specific differences in IFNα downstream signaling genes in resistant vs sensitive cell lines. These features are being evaluated and will inform future investigation of the mechanism of IFNα anti-tumor activity in patients.
Citation Format: Robin Osterhout, Jennifer Green, Scott McDonnell, Amy Klova, Adeela Kamal. Molecular characterization of response to interferon alpha in sensitive and resistant bladder cancer models abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5857.
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