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Pexidartinib (TURALIO™) is an orally administered small molecule tyrosine kinase inhibitor with selective activity against the colony-stimulating factor 1 (CSF1) receptor, KIT proto-oncogene receptor tyrosine kinase (KIT) and FMS-like tyrosine kinase 3 harboring an internal tandem duplication mutation (FLT3-ITD). In August 2019, the US FDA approved pexidartinib capsules for the treatment of adult patients with symptomatic tenosynovial giant cell tumor (TGCT) associated with severe morbidity or functional limitations and not amenable to improvement with surgery. This approval was based on positive results from the phase III ENLIVEN trial. Pexidartinib is being investigated in various malignancies as monotherapy or combination therapy. This article summarizes the milestones in the development of pexidartinib leading to its first approval for TGCT.

Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists. Era+ breast cancer patients often develop resistance to endocrine therapy. Here, the authors show that FGFR1 amplification is a resistance mechanism to CDK4/6 inhibitor and endocrine therapy and that combined treatment with FGFR, CDK4/6, and anti-estrogens is a potential therapeutic strategy in Era+ breast cancer tumors.

The cyclin-dependent kinase (CDK) 4/6 inhibitors belong to a new class of drugs that interrupt proliferation of malignant cells by inhibiting progression through the cell cycle. Three such inhibitors, palbociclib, ribociclib, and abemaciclib were recently approved for breast cancer treatment in various settings and combination regimens. On the basis of their impressive efficacy, all three CDK4/6 inhibitors now play an important role in the treatment of patients with HR+, HER2− breast cancer; however, their optimal use still needs to be established. The three drugs have many similarities in both pharmacokinetics and pharmacodynamics. However, there are some differences on the basis of which the choice for a particular CDK4/6 inhibitor for an individual patient can be important. In this article, the clinical pharmacokinetic and pharmacodynamic profiles of the three CDK4/6 inhibitors are reviewed and important future directions of the clinical applicability of CDK4/6 inhibitors will be discussed.

Immunotherapy is a powerful treatment for certain cancers. Yet for those patients that do not respond, simultaneous strategies that mobilize the immune system and directly target malignant cells may be more effective. Ruscetti et al. report that combining two clinically approved cancer drugs promoted immune surveillance and killing of KRAS-mutant lung tumors in mice (see the Perspective by Cornen and Vivier). The two small molecules—a mitogen-activated protein kinase inhibitor and a cyclin-dependent kinase 4/6 inhibitor—induced natural killer (NK) cell recruitment and elimination of senescent lung cancer cells, which did not occur when either agent was used alone. Science , this issue p. 1416 ; see also p. 1355 Combined MEK and CDK4/6 inhibition triggers senescence-associated NK cell–mediated tumor regression. Molecularly targeted therapies aim to obstruct cell autonomous programs required for tumor growth. We show that mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase 4/6 inhibitors act in combination to suppress the proliferation of KRAS-mutant lung cancer cells while simultaneously provoking a natural killer (NK) cell surveillance program leading to tumor cell death. The drug combination, but neither agent alone, promotes retinoblastoma (RB) protein-mediated cellular senescence and activation of the immunomodulatory senescence-associated secretory phenotype (SASP). SASP components tumor necrosis factor–α and intercellular adhesion molecule–1 are required for NK cell surveillance of drug-treated tumor cells, which contributes to tumor regressions and prolonged survival in a KRAS-mutant lung cancer mouse model. Therefore, molecularly targeted agents capable of inducing senescence can produce tumor control through non–cell autonomous mechanisms involving NK cell surveillance.

Somatic mutations in the isocitrate dehydrogenase 2 gene ( IDH2 ) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG) 1 – 8 . Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants 9 , 10 . In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2 -mutant AML 11 . Here we describe two patients with IDH2 -mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans , such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis , which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH -mutant malignancies. A new mechanism of acquired clinical resistance in two patients with acute myeloid leukaemia driven by mutant IDH2 is described, in which a second-site mutation on the wild-type allele induces therapeutic resistance to IDH2 inhibitors.

Crystal structures of the human GLP-1 receptor in complex with two negative allosteric modulators reveal a common binding pocket, and, together with mutagenesis and modelling studies, further our understanding of the receptor activation mechanism.Author: Please check the wording of the following statement, which will appear online only. Full-length class B GPCR structures The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) belong to the class B G-protein-coupled receptor family and have opposing physiological roles in glucose homeostasis and insulin release. As such, they are important in regulating metabolism and appetite and offer significant treatment possibilities for type 2 diabetes. However, as yet, no full-length structures of these receptors have been solved. Three papers in this issue of Nature report the structure of GLP-1R. Ray Stevens and colleagues describe the crystal structure of the human GLP-1R transmembrane domain in an inactive state in complex with negative allosteric modulators. Fiona Marshall and colleagues describe the active-state full-length receptor in complex with truncated peptide agonists, which have potent activity in mice on oral administration. Georgios Skiniotis, Brian Kobilka and colleagues describe the cryo-electron microscopy structure of an unmodified GLP-1R in complex with its endogenous peptide ligand, GLP-1, and the heterotrimeric G protein. Finally, in a fourth paper in this week's issue of Nature , Beili Wu and colleagues report the crystal structure of the full-length GCGR in an inactive conformation. Taken together, these studies provide key insights into the activation and signalling mechanisms of class B receptors and provide therapeutic opportunities for targeting this receptor family. The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis 1 . The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner 2 . Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR 3 and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs 4 , 5 , 6 . Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

ObjectiveIn the tumour microenvironment, critical drivers of immune escape include the oncogenic activity of the tumour cell-intrinsic osteopontin (OPN), the expression of programmed death ligand 1 (PD-L1) and the expansion of tumour-associated macrophages (TAMs). We investigated the feasibility of targeting these pathways as a therapeutic option in hepatocellular carcinoma (HCC) mouse models.DesignWe analysed the number of tumour-infiltrating immune cells and the inflammatory immune profiles in chemically induced liver tumour isolated from wild-type and OPNknockout (KO) mice. In vitro cell cocultures were further conducted to investigate the crosstalk between TAMs and HCC cells mediated by OPN, colony stimulating factor-1 (CSF1) and CSF1 receptor (CSF1R). The in vivo efficacy of anti-PD-L1 and CSF1/CSF1R inhibition was evaluated in OPN overexpressing subcutaneous or orthotopic mouse model of HCC.ResultsThe numbers of TAMs, as well as the expression levels of M2 macrophage markers and PD-L1 were significantly decreased, but the levels of cytokines produced by T-helper 1 (Th1) cells were upregulated in tumour tissues from OPN KO mice compared with that from the controls. In addition, we observed a positive association between the OPN and PD-L1 expression, and OPN expression and TAM infiltration in tumour tissues from patients with HCC. We further demonstrated that OPN facilitates chemotactic migration, and alternative activation of macrophages, and promotes the PD-L1 expression in HCC via activation of the CSF1-CSF1R pathway in macrophages. Combining anti-PD-L1 and CSF1R inhibition elicited potent antitumour activity and prolonged survival of OPNhigh tumour-bearing mice. Histological, flow cytometric and ELISA revealed increased CD8+ T cell infiltration, reduced TAMs and enhanced Th1/Th2 cytokine balance in multiple mouse models of HCC.ConclusionsOPN/CSF1/CSF1R axis plays a critical role in the immunosuppressive nature of the HCC microenvironment. Blocking CSF1/CSF1R prevents TAM trafficking and thereby enhances the efficacy of immune checkpoint inhibitors for the treatment of HCC.

Aims/hypothesisSodium–glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i) constitute a novel class of glucose-lowering (type 2) kidney-targeted agents. We recently reported that the SGLT2i empagliflozin (EMPA) reduced cardiac cytosolic Na+ ([Na+]c) and cytosolic Ca2+ ([Ca2+]c) concentrations through inhibition of Na+/H+ exchanger (NHE). Here, we examine (1) whether the SGLT2i dapagliflozin (DAPA) and canagliflozin (CANA) also inhibit NHE and reduce [Na+]c; (2) a structural model for the interaction of SGLT2i to NHE; (3) to what extent SGLT2i affect the haemodynamic and metabolic performance of isolated hearts of healthy mice.MethodsCardiac NHE activity and [Na+]c in mouse cardiomyocytes were measured in the presence of clinically relevant concentrations of EMPA (1 μmol/l), DAPA (1 μmol/l), CANA (3 μmol/l) or vehicle. NHE docking simulation studies were applied to explore potential binding sites for SGTL2i. Constant-flow Langendorff-perfused mouse hearts were subjected to SGLT2i for 30 min, and cardiovascular function, O2 consumption and energetics (phosphocreatine (PCr)/ATP) were determined.ResultsEMPA, DAPA and CANA inhibited NHE activity (measured through low pH recovery after NH4+ pulse: EMPA 6.69 ± 0.09, DAPA 6.77 ± 0.12 and CANA 6.80 ± 0.18 vs vehicle 7.09 ± 0.09; p < 0.001 for all three comparisons) and reduced [Na+]c (in mmol/l: EMPA 10.0 ± 0.5, DAPA 10.7 ± 0.7 and CANA 11.0 ± 0.9 vs vehicle 12.7 ± 0.7; p < 0.001). Docking studies provided high binding affinity of all three SGLT2i with the extracellular Na+-binding site of NHE. EMPA and CANA, but not DAPA, induced coronary vasodilation of the intact heart. PCr/ATP remained unaffected.Conclusions/interpretationEMPA, DAPA and CANA directly inhibit cardiac NHE flux and reduce [Na+]c, possibly by binding with the Na+-binding site of NHE-1. Furthermore, EMPA and CANA affect the healthy heart by inducing vasodilation. The [Na+]c-lowering class effect of SGLT2i is a potential approach to combat elevated [Na+]c that is known to occur in heart failure and diabetes.

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism. Approximately 30% of known drugs target G protein-coupled receptors (GPCRs), but all the published structures of GPCRs to date are from the class A family of GPCRs; here the first X-ray crystal structure of a member of the class B family of GPCRs, the human corticotropin-releasing factor receptor 1, is determined. Two class B human GPCR receptors G-protein-coupled receptors (GPCRs) are membrane proteins that act as sensors for a broad range of extracellular signals, including photons, ions, small organic molecules and even entire proteins. Approximately a third of known drugs target GPCRs. Until now all the published structures of GPCRs have been from class A GPCRs. In this issue of Nature two groups independently report the crystal structures of two receptors of the B family, the second largest of four family divisions based on primary sequence and pharmacology. Hollenstein et al . solved the structure of human corticotropin-releasing factor receptor 1. This GPCR binds to corticotropin-releasing hormone, a potent mediator of endocrine, autonomic, behavioral and immune responses to stress. In all known class A GPCRs, the ligand-binding sites are close to the extracellular boundaries of the receptors; in this GPCR, the antagonist (CP-376395) binds in a hydrophobic pocket located in the cytoplasmic half of the V-shaped receptor. Siu et al . solved the X-ray crystal structure of the human glucagon receptor. This GPCR binds to the glucagon peptide, which triggers the release of glucose from the liver, making it a potential drug target for type 2 diabetes. The structure reveals a larger ligand-binding pocket than that seen in class A GPCRs.

The development of selective ubiquitin-specific protease-7 (USP7) inhibitors GNE-6640 and GNE-6776, which induce tumour cell death and reveal differential kinetics of Lys-48 and Lys-63-linked ubiquitin chain depolymerization by USP7. Interfering inhibitors show toxicity to tumours Deubiquitinating enzymes remove the small modifier protein ubiquitin from target substrates regulating their stability. One such enzyme, USP7, is a potential target for anti-cancer therapy, as its inhibition would result in the degradation of the ubiquitinated oncoprotein MDM2, leading to reactivation of the tumour suppressor protein p53. However, selective inhibitors of USP7 have remained elusive. Here, Ingrid Wertz and team develop two USP7 inhibitors, providing structural insights into the mode of action of these compounds and demonstrating their toxicity towards tumour cells. Elsewhere in this issue, David Komander and colleagues independently report the identification of two small molecules that inhibit USP7 with high affinity and specificity both in vitro and within cells, also demonstrating their ability to inhibit tumour growth. The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation 1 . Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease 2 ; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival 3 . However, developing selective deubiquitinase inhibitors has been challenging 4 and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain 5 , suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.