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Invasive fungal disease is an emerging and serious public health threat globally. The expanding population of susceptible individuals, together with the rapid emergence of multidrug-resistant fungi pathogens, call for the development of novel therapeutic strategies beyond the limited repertoire of licensed antifungal drugs. Card9 is a critical signaling molecule involved in antifungal defense; we have previously identified Dok3 to be a key negative regulator of Card9 activity in neutrophils. In this study, we identified two synthetic peptides derived from the coiled-coil domain of Card9, which can specifically block Dok3–Card9 binding. We showed that these peptides are cell-permeable, non-toxic, and can enhance antifungal cytokine production and the phagocytosis of human neutrophils upon fungal infection. Collectively, these data provide a proof of concept that disrupting the Dok3–Card9 interaction can boost the antifungal effector functions of neutrophils; they further suggest the potential utility of these peptide inhibitors as an immune-based therapeutic to fight fungal infection.

The N-terminal transactivation domain (TAD) of p53 is a disordered region with multiple phosphorylation sites. Phosphorylation at Thr18 is crucial for the release of p53 from its negative regulator, MDM2. In stressed cells, CK1δ is responsible for phosphorylating Thr18, but requires Ser15 to be phosphorylated. To understand the mechanistic underpinnings of this sequential phosphorylation, molecular modeling and molecular dynamics simulation studies of these phosphorylation events were carried out. Our models suggest that a positively charged region on CK1δ near the adenosine triphosphate (ATP) binding pocket, which is conserved across species, sequesters the negatively charged pSer15, thereby constraining the positioning of the rest of the peptide, such that the side chain of Thr18 is positioned close to the γ-phosphate of ATP. Furthermore, our studies show that the phosphorylated p53 TAD1 (p53pSer15) peptide binds more strongly to CK1δ than does p53. p53 adopts a helical structure when bound to CK1δ, which is lost upon phosphorylation at Ser15, thus gaining higher flexibility and ability to morph into the binding site. We propose that upon phosphorylation at Ser15 the p53 TAD1 peptide binds to CK1δ through an electrostatically driven induced fit mechanism resulting in a flanking fuzzy complex.

On-target resistance to next-generation TRK inhibitors in TRK fusion–positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations.Significance:In TRK fusion–positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation.This article is highlighted in the In This Issue feature, p. 1

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Stapled α-helical peptides can bind to and modulate historically intractable targets while addressing the traditional liabilities associated with peptide therapeutics. However, their pipeline advancement has been impeded by the challenges of identifying peptides with sufficient cellular uptake to engage the target protein while lacking off-target toxicities. Here, we advance the field to arrive at a workflow for identifying advanced stapled peptide lead molecules with on-target in vivo activity with no off-target cell proliferation effects. Specifically, we generated a >350-member library based on ATSP-7041, a stapled peptide Mdm2(X) antagonist with validated on-target cellular effects but with significant off-target activity. Key insights from library analysis include 1) a clear correlation between lipophilicity and permeability, 2) removal of positive charge to avoid off-target toxicities, 3) judicious placement of anionic residues to enhance peptide solubility/behavior, 4) optimization of C-terminal length and helicity to enhance cell activity, 5) optimization of staple type/number to avoid polypharmacology. Incorporation of one or more of these attributes led to molecules with improved in vitro and in vivo activities (up to a >292x improved cell proliferation EC50). A subset of peptides were devoid of off-target cell proliferation effects in cell lines lacking wild-type p53 protein (up to a >3800x on-target index). This latter improvement contrasted with clinical Mdm2 antagonistic molecules. Application of these ‘design rules’ to a distinct Mdm2(X) peptide series resulted in rapid improvement in cellular activity (>150x) and removal of off-target toxicities. Overall, the detailed workflow outlined here should help researchers identify stapled α-helical peptides for therapeutic impact.

Macrocyclic peptides have the potential to address intracellular protein–protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure–activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight – the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry – an approach that has yet to succeed in the clinic despite a long history of attempts – carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers – some of the most treatment-resistant human malignancies., Targeting undruggable intracellular proteins with peptides: novel on-target macrocyclic peptide inhibitors of KRAS with broad inhibition of proliferation of multiple KRAS-dependent cancer cell lines.

Mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1/2) represent a central class of enzymes that are activated by extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein (MAP) kinases. MNK1 and MNK2 coordinate cellular signaling, control production of inflammatory chemokines, and regulate cell proliferation and survival. MNK1/2 are referred to as serine/threonine kinases as they phosphorylate serine or threonine residues on their substrates. Upon activation, MNK1/2 phosphorylate eukaryotic translation initiation factor 4E (eIF4E) at Ser209, which in turn initiates ribosome assembly and protein translation. Deleterious overexpression of MNK1/2 and/or eIF4E have been reported in several diseases including cancers, neurological disorders, autism, and inflammation. Recently, there have been intense efforts toward the development of potent and selective inhibitors of MNK1/2 in both academia and industry. Herein, we review the current understanding of the structural and biological aspects of MNK1/2 and provide an update of pharmacological inhibitors of MNK1/2 including candidates in clinical trials.

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies. This study has been supported by the Susan G. Komen Foundation (CCR15330331) and by the Catalan Agency AGAUR (2017 SGR 540) [to V.S.]. V.S. received funds from the Instituto de Salud Carlos III: grants PI13/01714, CP14/00228, MV15/00041, CPII19/00033 and PI20/00892. M.P. received a Juan de la Cierva Grant from the Ministerio de Economía y Competitividad (FJCI-2015-25412), L.Mo. a grant from FI-AGAUR (2019 FI_B 01199), F.B-M. a grant from the Fundación Científica Asociación Española Contra el Cáncer (AECC_Postdoctoral17-1062) and M.S-G, a Marie Slodowska-Curie Innovative Training Networks PhD fellowship (H2020-MSCA-ITN-2015_675392). This work was supported by Breast Cancer Research Foundation (BCRF-19-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181) and Asociación Española Contra el Cáncer (to J.A.). R.B.C. laboratory is supported by Breast Cancer Now (grant numbers: MAN-Q1 and MAN-Q2), NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20007) and EdiREX Horizon 2020 grant No.731105. The xenograft program in the C.C. laboratory is supported by Cancer Research UK and also received funding from an EU H2020 Network of Excellence (EuroCAN). This work has been supported by NIH grants P30 CA008748 and RO1CA190642-01, the CDMRP grant BC171535P1, and the Breast Cancer Research Foundation [to M.S.]. A.P. received funds from Instituto de Salud Carlos III—PI16/00904 and PI19/01846, Breast Cancer Now—2018NOVPCC1294, Breast Cancer Research Foundation-AACR Career Development Awards for Translational Breast Cancer Research 19-20-26-PRAT, Fundació La Marató TV3 201935-30, the European Union’s Horizon 2020 research and innovation program H2020-SC1-BHC-2018-2020. IRB Barcelona is a recipient of a Severo Ochoa Centre of Excellence Award from the Spanish Ministry of Economy and Competitiveness (MINECO; Government of Spain) and is supported by CERCA (Generalitat de Catalunya). C. S. Verma reports grants from MSD International and grants from Ipsen outside the submitted work.

The death domain associated protein 6 (DAXX) is frequently upregulated in a number of common cancers where its suppression has been linked to reduced tumour progression. As a master regulator protein, with >70 reported protein interaction partners, the role of DAXX in its oncogenecity remains unclear. We designed and developed a set of novel stapled/stitched peptides that target a surface on the N-terminal helical bundle domain of DAXX which is the anchor-point for binding to multiple interaction partners (including Rassf1C, P53, Mdm2 and ATRX) and also for the auto regulation of the DAXX N-terminal SUMO interaction motif (SIM). We demonstrate that these peptides bind to and inhibit DAXX with an affinity higher than those reported for the known interaction partners and release the auto-inhibited SIM for interaction with SUMO-1. NanoBret assays show that the peptides enter cells and that their intracellular concentrations remain at nanomolar levels even after 24 hours, without causing membrane perturbation. Together our data suggest that these peptides are both tools for probing the molecular interactions of DAXX and potential precursors to the development of therapeutics.

Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure‐guided semi‐rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated., Two engineered halogenase enzymes were developed by switching substrate specificity of the parental enzyme from tryptophan to indole compounds. They can halogenate several industrially important indole derivatives more efficiently than the parental enzyme; this includes enzymatic chlorination/bromination of novel substrates that were not reported previously. The engineered enzymes could be useful in regioselective synthesis of drug intermediates.

Previous study has shown that Hibiscus sulfite oxidase (SO) interacts with Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) and triggers sulfur enhanced defense (SED). In this study, we show the interaction of Arabidopsis SO (AtSO) and Turnip crinkle virus (TCV) CP in Arabidopsis thaliana plants. We identified the binding sites of TCV CP (W274) and AtSO (D223) using bioinformatics and confirmed it experimentally. Mutation of binding site W274 to A274 in TCV CP resulted in failure of TCV infection. TCV accumulation in SO over-expression (SO_OE) plants was lower than that in wild-type (WT) and SO knock-out (SO_KO) plants at 7 dpi but reached a level similar to that of WT and SO_KO plants at 10 dpi. AtSO competed with Argonaute 1 (AGO1) for TCV CP binding in vitro. AtSO may serve as an anti-viral factor through sequestering TCV CP for binding with AGO1 and confers virus resistance.

Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically., Codon 158 gain-of-function mutant p53 (158-mutp53) promotes tumourigenesis in lung cancer. Here, the authors show that 158-mutp53 render cancers sensitive to cisplatin and p53 acetylation agents through a mechanism where acetylated mutant p53 upregulates TRAIP and inhibits NF-ĸB signaling.

c-MET receptors are activated in cancers through genomic events like tyrosine kinase domain mutations, juxtamembrane splicing mutation and amplified copy numbers, which can be inhibited by c-MET small molecule inhibitors. Here, we discover that the most common polymorphism known to affect MET gene (N375S), involving the semaphorin domain, confers exquisite binding affinity for HER2 and enables METN375S to interact with HER2 in a ligand-independent fashion. The resultant METN375S/HER2 dimer transduces potent proliferative, pro-invasive and pro-metastatic cues through the HER2 signaling axis to drive aggressive squamous cell carcinomas of the head and neck (HNSCC) and lung (LUSC), and is associated with poor prognosis. Accordingly, HER2 blockers, but not c-MET inhibitors, are paradoxically effective at restraining in vivo and in vitro models expressing METN375S. These results establish METN375S as a biologically distinct and clinically actionable molecular subset of SCCs that are uniquely amenable to HER2 blocking therapies., The MET receptor is frequently activated in cancer. Here, the authors show that in head and neck and lung squamous carcinoma, a polymorphic MET variant enhances binding to HER2, resulting in activation of HER2 signalling and progression of the cancers.

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein–protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable via small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from D-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-D α-helical stapled and stitched peptides. As a template, we used a recently reported all D-linear peptide that is a potent inhibitor of the p53–Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-D-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all D-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

Solute-solvent interactions are critical for biomolecular stability and recognition. Explicit solvent molecular dynamics (MD) simulations are routinely used to probe such interactions. However, detailed analyses and interpretation of the hydration patterns seen in MD simulations can be both complex and time-consuming. A variety of approaches/tools to compute and interrogate hydration properties in structural ensembles of proteins, nucleic acids, or in general any molecule are available and are complemented here with a new and free software package ("JAL"). Central to "JAL" is an intuitive atom centric approach of computing hydration properties. In addition to the standard metrics commonly used to understand hydration, "JAL" introduces two nonstandard utilities: a program to rapidly compute buried waters in an MD trajectory and a new method to compute multiwater bridges around a solute. We demonstrate the utility of the package by probing the hydration characteristics of the tumor suppressor protein p53 and the translation initiation factor eif4E. "JAL" is hosted online and can be accessed for free at http://mspc.bii.a-star.edu.sg/minhn/jal.html .

S100B(ββ) is a member of the S100B protein family and is distributed in a cell-specific manner. Its levels are elevated in several cancers such as malignant melanoma and correlate directly with poor prognosis in patients. S100B(ββ) directly interacts with the tumor suppressor p53, inhibiting tetramerization and protein kinase C-dependent phosphorylation, consequently decreasing p53 DNA binding and transcriptional activity, and preventing apoptosis. Thus, S100B(ββ) is being pursued as a target for therapeutic inhibition. However, development of small molecule inhibitors targeting p53-interactions has met with limited success. In this work, we present a set of designed stapled peptide inhibitors of S100B(ββ), guided by the structure of the C-terminal domain of p53 complexed with S100B(ββ). We further modified a tightly binding stapled peptide with imaging agents and propose these as potential diagnostic agents to detect S100B(ββ) as a biomarker. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) EDB (Economic Devt. Board, S’pore) Published version

Purpose: MET fusions are oncogenic drivers of multiple cancers including non-small cell lung cancer (NSCLC). While MET fusion-positive tumors can be sensitive to MET tyrosine kinase inhibitors (TKI), to date no mechanisms of TKI resistance have been identified or characterized for MET fusion-positive disease. Experimental Design: Serial targeted genomic sequencing (MSK-IMPACT) was performed in clinical samples from a MET fusion-positive NSCLC treated with the type Ia MET TKI crizotinib. Structural modeling and in vitro kinase assays were used to characterize crizotinib resistance and to test the activity of MET inhibitors with alternative binding modes. Results: A novel MET G1090A resistance mutation was identified in a patient with a CD47-MET-rearranged NSCLC after crizotinib progression. In silico structural modeling and simulations indicated that the 2,6-dichloro-3-fluorophenyl moiety present on crizotinib directly binds to the side chain of the Y1230 residue in the activation loop of the MET wild type (WT) kinase. In the MET G1090A mutant kinase, the position of the Y1230 residue is predicted to be flipped roughly 180°. As a result, the interaction between crizotinib and Y1230 is compromised; this in turn is predicted to reduce crizotinib’s binding affinity for the mutant kinase. Similar results were obtained when MET WT and G1090A mutant kinases were modeled in complex with selective type Ib MET TKI (i.e., capmatinib, savolitinib and tepotinib). Conversely, we found that type II agents (i.e., cabozantinib and foretinib) were not predicted to interact with Y1230. Therefore, their binding affinity to the mutant MET kinase was predicted to be retained. In vitro kinase assays performed using titrations of crizotinib and capmatinib (representative type I agents) and of cabozantinib (representative type II agent) showed that all three TKI were active against the MET WT kinase (calculated IC50 of 2.5nM, 3.89nM and 1.59nM, respectively). However, the calculated IC50 for crizotinib and capmatinib were 6.04- and 48.4-fold higher than the calculated IC50 for cabozantinib against the MET G1090A mutant kinase. Consistent with the prediction of type II inhibitor activity, the patient was transitioned to cabozantinib. A marked clinical, radiographic, and metabolic response was observed. Conclusions: In MET fusion-positive cancers, the novel MET G1090A resistance mutation drives resistance to type I MET kinase inhibition. Drug binding mode switching to type II MET inhibition overcomes preclinical and clinical resistance, highlighting the need for further rational type II inhibitor development. Citation Format: Yonina R. Murciano-Goroff, Srinivasaraghavan Kannan, Jason C. Chang, Ryma Benayed, Ilya Blokhin, Natasha Rekhtman, Ann Elizabeth Sisk, Jaime Gibson, Lia Judka, Lauren Kaplanis, Madeline Merrill, Erica Sgroe, Chandra S. Verma, Alexander Drilon, Emiliano Cocco. Switching inhibitor class overcomes crizotinib resistance in a MET fusion-positive NSCLC with a novel acquired MET G1090A mutation [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 5239.

Recent research on the SARS-CoV-2 pandemic has exploded around the furin-cleavable polybasic insert PRRAR↓S, found within the spike protein. The insert and the receptor-binding domain, (RBD), are vital clues in the Sherlock Holmes-like investigation into the origin of the virus and in its zoonotic crossover. Based on comparative analysis of the whole genome and the sequence features of the insert and the RBD domain, the bat and the pangolin have been proposed as very likely intermediary hosts. In this study, using the various databases, in-house developed tools, sequence comparisons, structure-guided docking, and molecular dynamics simulations, we cautiously present a fresh, theoretical perspective on the SARS-CoV-2 virus activation and its intermediary host. They are a) the SARS-CoV-2 has not yet acquired a fully optimal furin binding site or this seemingly less optimal sequence, PRRARS, has been selected for survival; b) in structural models of furin complexed with peptides, PRRAR↓S binds less well and with distinct differences as compared to the all basic RRKRR↓S; c) these differences may be exploited for the design of virus-specific inhibitors; d) the novel polybasic insert of SARS-CoV-2 may be promiscuous enough to be cleaved by multiple enzymes of the human airway epithelium and tissues which may explain its unexpected broad tropism; e) the RBD domain of the feline coronavirus spike protein carries residues that are responsible for high-affinity binding of the SARS-CoV-2 to the ACE 2 receptor; f) en route zoonotic transfer, the virus may have passed through the domestic cat whose very human-like ACE2 receptor and furin may have played some role in optimizing the traits required for zoonotic transfer., Highlights • Polybasic insert of the SARS-CoV-2 spike protein is rare among several hundred proteins with a motif ‘RRARS’. • SARS CoV-2 shares furin-like site and RBD interface residues including hotspot sites, with some of the lethal form of Feline coronavirus spike protein and those from the healthy cats. • Polybasic sequence PRRARS binds less well to furin in structural models. • SARS-CoV-2 may have passed through the domestic cat during zoonotic transfer.

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.

Glioblastoma (GBM) is a uniformly lethal disease driven by glioma stem cells (GSCs). Here, we use a chemical biology approach to unveil previously unknown GBM dependencies. By studying sulconazole (SN) with anti-GSC properties, we find that SN disrupts biotin distribution to the carboxylases and histones. Transcriptomic and metabolomic analyses of SN-treated GSCs reveal metabolic alterations that are characteristic of biotin-deficient cells, including intracellular cholesterol depletion, impairment of oxidative phosphorylation, and energetic crisis. Furthermore, SN treatment reduces histone biotinylation, histone acetylation, and expression of superenhancer-associated GSC critical genes, which are also observed when biotin distribution is genetically disrupted by holocarboxylase synthetase (HLCS) depletion. HLCS silencing impaired GSC tumorigenicity in an orthotopic xenograft brain tumor model. In GBM, high HLCS expression robustly indicates a poor prognosis. Thus, the dependency of GBM on biotin distribution suggests that the rational cotargeting of biotin-dependent metabolism and epigenetic pathways may be explored for GSC eradication. National Research Foundation (NRF) National University of Singapore (NUS), Temasek Laboratories Published version This work was supported by the National Research Foundation Fellowship NRF-NRFF2017-01 (D.S.T.O.), National University of Singapore (NUS) start-up grant (D.S.T.O. and M.J.K.), NUS President’s Assistant Professorship (D.S.T.O.), and NUS Research Scholarships (M.J.Y.A., B.W.L.L., Y.H.C., and R.T.M.). S.K. and C.S.V. thank A*STAR and NSCC for support.

On-target resistance to next-generation TRK inhibitors in TRK fusion–positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. Significance: In TRK fusion–positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation. This article is highlighted in the In This Issue feature, p. 1

S100B(&beta, &beta, ) proteins are a family of multifunctional proteins that are present in several tissues and regulate a wide variety of cellular processes. Their altered expression levels have been associated with several human diseases, such as cancer, inflammatory disorders and neurodegenerative conditions, and hence are of interest as a therapeutic target and a biomarker. Small molecule inhibitors of S100B(&beta, ) have achieved limited success. Guided by the wealth of available experimental structures of S100B(&beta, ) in complex with diverse peptides from various protein interacting partners, we combine comparative structural analysis and molecular dynamics simulations to design a series of peptides and their analogues (stapled) as S100B(&beta, ) binders. The stapled peptides were subject to in silico mutagenesis experiments, resulting in optimized analogues that are predicted to bind to S100B(&beta, ) with high affinity, and were also modified with imaging agents to serve as diagnostic tools. These stapled peptides can serve as theranostics, which can be used to not only diagnose the levels of S100B(&beta, ) but also to disrupt the interactions of S100B(&beta, ) with partner proteins which drive disease progression, thus serving as novel therapeutics.