Capmatinib is an effective treatment for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy.

BACKGROUND: Pediatric-type diffuse high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGG and are associated with devastating morbidity and mortality. METHODS: To identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion IHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy. RESULTS: Capmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib in both models. The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib extended survival and induced long-term progression-free survival when combined with radiotherapy in two complementary mouse models. Capmatinib treatment increased radiation-induced DNA double-strand breaks and delayed their repair. CONCLUSIONS: We comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies, and insights into molecular mechanism underlying increased efficacy. Taken together, we demonstrate the groundbreaking efficacy of capmatinib and radiation as a highly promising concept for future clinical trials.

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

A high-throughput quality control method for assessing the serial dilution performance of dose-response plates with acoustic ejection mass spectrometry.

This study aimed to develop a streamlined method for evaluating the dilution ratio of drug dose-response plates created by automated liquid handlers in the early stages of drug discovery. The quantitative techniques commonly used for this purpose have restrictions due to their limited linear dynamic range and inaccuracies in assessing serial dilution performance. To address this challenge, we describe a method based on acoustic ejection mass spectrometry (AEMS). The method involves using standard compounds and an internal standard to evaluate each dilution point in quality control (QC) plates. The samples are transferred to a chromatography-free tandem mass spectrometry system through an acoustic source, enabling the analysis of one sample per three seconds from a microtiter plate. This approach provides precise, accurate, label-free, and rapid data acquisition to support high-throughput screening efforts.

Fragment-Based NMR Screening of the BPTF PHD Finger Methyl Lysine Reader Leads to the First Small-Molecule Inhibitors.

Methyl lysine readers, specifically PHD fingers, are emerging epigenetic targets in human diseases. For example, several PHD finger fusions are implicated in clinical cases of acute myeloid leukemia, highlighting the potential for PHD inhibitors in disease regulation. However, limited chemical matter targeting PHD fingers exists. Here we report the first fragment-based screen against the BPTF PHD to identify several of the first reported BPTF PHD-targeting small-molecule ligands. We used ligand-observed NMR to first screen a fragment library, followed by biophysical validation to prioritize two scaffolds, pyrrolidine- and pyridazine-containing fragments. Structural predictions show that these respective scaffolds may engage two distinct subpockets on the protein. The demonstrated ligandability of the BPTF PHD supports the future development of methyl lysine reader chemical probes to study their oncogenic functions.

The net electrostatic potential and hydration of ABCG2 affect substrate transport.

ABCG2 is a medically important ATP-binding cassette transporter with crucial roles in the absorption and distribution of chemically-diverse toxins and drugs, reducing the cellular accumulation of chemotherapeutic drugs to facilitate multidrug resistance in cancer. ABCG2's capacity to transport both hydrophilic and hydrophobic compounds is not well understood. Here we assess the molecular basis for substrate discrimination by the binding pocket. Substitution of a phylogenetically-conserved polar residue, N436, to alanine in the binding pocket of human ABCG2 permits only hydrophobic substrate transport, revealing the unique role of N436 as a discriminator. Molecular dynamics simulations show that this alanine substitution alters the electrostatic potential of the binding pocket favoring hydration of the transport pore. This change affects the contact with substrates and inhibitors, abrogating hydrophilic compound transport while retaining the transport of hydrophobic compounds. The N436 residue is also required for optimal transport inhibition of ABCG2, as many inhibitors are functionally impaired by this ABCG2 mutation. Overall, these findings have biomedical implications, broadly extending our understanding of substrate and inhibitor interactions.

ATM inhibition enhances the efficacy of radiation across distinct molecular subgroups of pediatric high-grade glioma.

BACKGROUND: Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. METHODS: We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wild-type and TP53 mutant orthotopic xenografts. RESULTS: AZD1390 significantly potentiated radiation across molecular subgroups of pHGG by increasing mutagenic nonhomologous end joining and augmenting genomic instability. In contrast to previous reports, ATM inhibition significantly improved the efficacy of radiation in both TP53 wild-type and TP53 mutant isogenic cell lines and distinct orthotopic xenograft models. Furthermore, we identified a novel mechanism of resistance to AZD1390 + radiation that was marked by an attenuated ATM pathway response which dampened sensitivity to ATM inhibition and induced synthetic lethality with ATR inhibition. CONCLUSIONS: Our study supports the clinical evaluation of AZD1390 in combination with radiation in pediatric patients with HGG.

Proteasome inhibition targets the KMT2A transcriptional complex in acute lymphoblastic leukemia.

Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.

Combination of Ribociclib with BET-Bromodomain and PI3K/mTOR Inhibitors for Medulloblastoma Treatment In Vitro and In Vivo.

Despite improvement in the treatment of medulloblastoma over the last years, numerous patients with MYC- and MYCN-driven tumors still fail current therapies. Medulloblastomas have an intact retinoblastoma protein RB, suggesting that CDK4/6 inhibition might represent a therapeutic strategy for which drug combination remains understudied. We conducted high-throughput drug combination screens in a Group3 (G3) medulloblastoma line using the CDK4/6 inhibitor (CDK4/6i) ribociclib at IC20, referred to as an anchor, and 87 oncology drugs approved by FDA or in clinical trials. Bromodomain and extra terminal (BET) and PI3K/mTOR inhibitors potentiated ribociclib inhibition of proliferation in an established cell line and freshly dissociated tumor cells from intracranial xenografts of G3 and Sonic hedgehog (SHH) medulloblastomas in vitro. A reverse combination screen using the BET inhibitor JQ1 as anchor, revealed CDK4/6i as the most potentiating drugs. In vivo, ribociclib showed single-agent activity in medulloblastoma models whereas JQ1 failed to show efficacy due to high clearance and insufficient free brain concentration. Despite in vitro synergy, combination of ribociclib with the PI3K/mTOR inhibitor paxalisib did not significantly improve the survival of G3 and SHH medulloblastoma-bearing mice compared with ribociclib alone. Molecular analysis of ribociclib and paxalisib-treated tumors revealed that E2F targets and PI3K/AKT/MTORC1 signaling genes were depleted, as expected. Importantly, in one untreated G3MB model HD-MB03, the PI3K/AKT/MTORC1 gene set was enriched in vitro compared with in vivo suggesting that the pathway displayed increased activity in vitro. Our data illustrate the difficulty in translating in vitro findings in vivo. See related article in Mol Cancer Ther (2022) 21(8):1306-1317.

Allosteric regulation of DNA binding and target residence time drive the cytotoxicity of phthalazinone-based PARP-1 inhibitors.

Allosteric coupling between the DNA binding site to the NAD+-binding pocket drives PARP-1 activation. This allosteric communication occurs in the reverse direction such that NAD+ mimetics can enhance PARP-1's affinity for DNA, referred to as type I inhibition. The cellular effects of type I inhibition are unknown, largely because of the lack of potent, membrane-permeable type I inhibitors. Here we identify the phthalazinone inhibitor AZ0108 as a type I inhibitor. Unlike the structurally related inhibitor olaparib, AZ0108 induces replication stress in tumorigenic cells. Synthesis of analogs of AZ0108 revealed features of AZ0108 that are required for type I inhibition. One analog, Pip6, showed similar type I inhibition of PARP-1 but was ∼90-fold more cytotoxic than AZ0108. Washout experiments suggest that the enhanced cytotoxicity of Pip6 compared with AZ0108 is due to prolonged target residence time on PARP-1. Pip6 represents a new class of PARP-1 inhibitors that may have unique anticancer properties.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Data vignettes for the application of response surface models in drug combination analysis.

This data set contains the data used in Twarog et al. (2021) to examine the robustness and utility of response surface models in drug combination analysis. It includes simulated experimental data for the evaluation of traditional index methods, as well as a processed library of interaction metrics evaluated on the Merck OncoPolyPharmacology Screen (O'Neil et al., 2016), the scripts used to implement those metrics on all tested combinations in that screen, and scripts to evaluate the performance of those metrics in comparison with real-world mechanistic classifications. Finally, the data set includes data from several published and unpublished drug combination experiments, and scripts which allow the analyses of those experiments to be replicated and applied to new data.

SLFN11 is Widely Expressed in Pediatric Sarcoma and Induces Variable Sensitization to Replicative Stress Caused By DNA-Damaging Agents.

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of sensitivity in vitro and in vivo Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a patient with recurrent Ewing sarcoma who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.

Patient-derived models recapitulate heterogeneity of molecular signatures and drug response in pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.

Small Molecule Sequestration of the Intrinsically Disordered Protein, p27Kip1, Within Soluble Oligomers.

Proteins that exhibit intrinsically disordered regions (IDRs) are prevalent in the human proteome and perform diverse biological functions, including signaling and regulation. Due to these important roles, misregulation of intrinsically disordered proteins (IDPs) is associated with myriad human diseases, including neurodegeneration and cancer. The inherent flexibility of IDPs limits the applicability of the traditional structure-based drug design paradigm; therefore, IDPs have long been considered "undruggable". Using NMR spectroscopy and other methods, we previously discovered small, drug-like molecules that bind specifically, albeit weakly, to dynamic clusters of aromatic residues within p27Kip1 (p27), an archetypal disordered protein involved in cell cycle regulation. Here, using synthetic chemistry, NMR spectroscopy and other biophysical methods, we discovered elaborated analogs of our previously reported molecules with 30-fold increased affinity for p27 (apparent Kd = 57 ± 19 µM). Strikingly, using analytical ultracentrifugation methods, we showed that the highest affinity compounds caused p27 to form soluble, disordered oligomers. Based on these observations, we propose that sequestration within soluble oligomers may represent a general strategy for therapeutically targeting disease-associated IDPs in the future.

Using response surface models to analyze drug combinations.

Quantitative evaluation of how drugs combine to elicit a biological response is crucial for drug development. Evaluations of drug combinations are often performed using index-based methods, which are known to be biased and unstable. We examine how these methods can produce misleadingly structured patterns of bias, leading to erroneous judgments of synergy or antagonism. By contrast, response surface models are less prone to these defects and can be applied to a wide range of data that have appeared in recent literature, including the measurement of combination therapeutic windows and the analysis of discrete experimental measures, three-way drug combinations, and atypical response behaviors.

Model-based evaluation of image-guided fractionated whole-brain radiation therapy in pediatric diffuse intrinsic pontine glioma xenografts.

Radiation therapy (RT) is currently the standard treatment for diffuse intrinsic pontine glioma (DIPG), the most common cause of death in children with brain cancer. A pharmacodynamic model was developed to describe the radiation-induced tumor shrinkage and overall survival in mice bearing DIPG. CD1-nude mice were implanted in the brain cortex with luciferase-labeled patient-derived orthotopic xenografts of DIPG (SJDIPGx7 H3F3AWT / K27 M and SJDIPGx37 H3F3AK27M / K27M ). Mice were treated with image-guided whole-brain RT at 1 or 2 Gy/fraction 5-days-on 2-days-off for a cumulative dose of 20 or 54 Gy. Tumor progression was monitored with bioluminescent imaging (BLI). A mathematical model describing BLI and overall survival was developed with data from mice receiving 2 Gy/fraction and validated using data from mice receiving 1 Gy/fraction. BLI data were adequately fitted with a logistic tumor growth function and a signal distribution model with linear radiation-induced killing effect. A higher tumor growth rate in SJDIPGx37 versus SJDIPGx7 xenografts and a killing effect decreasing with higher tumor baseline (p < 0.0001) were identified. Cumulative radiation dose was suggested to inhibit the tumor growth rate according to a Hill function. Survival distribution was best described with a Weibull hazard function in which the hazard baseline was a continuous function of tumor BLI. Significant differences were further identified between DIPG cell lines and untreated versus treated mice. The model was adequately validated with mice receiving 1 Gy/fraction and will be useful in guiding future preclinical trials incorporating radiation and to support systemic combination therapies with RT.

MAIP: a web service for predicting blood-stage malaria inhibitors.

Malaria is a disease affecting hundreds of millions of people across the world, mainly in developing countries and especially in sub-Saharan Africa. It is the cause of hundreds of thousands of deaths each year and there is an ever-present need to identify and develop effective new therapies to tackle the disease and overcome increasing drug resistance. Here, we extend a previous study in which a number of partners collaborated to develop a consensus in silico model that can be used to identify novel molecules that may have antimalarial properties. The performance of machine learning methods generally improves with the number of data points available for training. One practical challenge in building large training sets is that the data are often proprietary and cannot be straightforwardly integrated. Here, this was addressed by sharing QSAR models, each built on a private data set. We describe the development of an open-source software platform for creating such models, a comprehensive evaluation of methods to create a single consensus model and a web platform called MAIP available at https://www.ebi.ac.uk/chembl/maip/ . MAIP is freely available for the wider community to make large-scale predictions of potential malaria inhibiting compounds. This project also highlights some of the practical challenges in reproducing published computational methods and the opportunities that open-source software can offer to the community.

Evaluating and evolving a screening library in academia: the St Jude approach.

The quality of lead compounds is a key factor for determining the success of chemical probe and drug discovery programs. Given that high-throughput screening (HTS) continues to be a dominant lead generation paradigm, access to high-quality screening libraries is crucial for such efforts in both industry and academia. Here, we discuss the strategy implemented a decade ago to build from scratch one of the largest compound collections in academia, containing ∼575 000 carefully annotated small molecules, and a recent multidisciplinary effort designed to further enhance the collection to meet our research demands for the next decade.