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PDF - 60K, Linear correlation observed between tumor growth inhibition (% T/C) or tumor regression and the fraction of time over the in vivo S473P-Akt IC80 in different cancer cell line-derived tumor xenografts implanted in nude mice (represented as dots) and nude rats (represented as triangles) following NVP-BYL719 treatment (R2=0.77, p

Complete Data for Genetic Analysis of ctDNA and Tumor Tissue for All Samples.

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials. Mol Cancer Ther; 13(5); 1117–29. ©2014 AACR.

Activity of FGF401 in biochemical kinase assays

Details on materials and methods

Supplementary Figure S1. Confirmation of secondary point mutation in FGFR2-ZMYM4 allele. Supplementary Figure S2. Homology between FGFR2 and FGFR3 proteins. Supplementary Figure S3. FGFR2-OPTN fusion detected in all autopsy lesions. Supplementary Figure S4. FoundationOne analysis of autopsy lesions. Supplementary Figure S5. PTEN loss of heterozygosity (LOH) identified in Patient #2. Supplementary Figure S6. Mutational analysis of original resection specimen from Patient #2.

PDF - 129K, Legends for Supplementary Figures 1 through 4 and Supplementary Tables 1 through 7. Supplementary Table 1. Determination of NVP-BYL719 in vitro effects on human protein kinases. Supplementary Table 2. Activity profile of NVP-BYL719 in the Invitrogen Kinase panel. Supplementary Table 3. Activity profile of NVP-BYL719 in the Ambit Kinase panel. Supplementary Table 4. NVP-BYL719 Kd (nmol/L) determination in the Ambit Kinase panel for the hits found to be inhibited >90%. Supplementary Table 5. NVP-BYL719 anti-tumor effects in diverse cancer cell line-derived xenograft models. Supplementary Table 6. CCLE cell lines responsive to NVP-BYL719. Supplementary Table 7 Anti-tumor effect of NVP-BYL719 in patient-derived xenograft models carrying PIK3CA genetic alterations.

PK and PK/PD of FGF401 in mice

PDF - 71K, Rat1-myr-p110alpha (blue), beta (green) or delta (red) cells were treated with increasing concentrations of NVP-BYL719 for 30 minutes. Levels of S473P-Akt in cell extracts were quantified by Reverse Phase Protein Array as described in (18) and plotted as percentage of untreated control cells. The graph illustrates n=3 independent experiments. IC50s (plus/minus) SD and IC80s (plus/minus) SD of n=3 independent experiments are reported..

PDF - 92K, NVP-BYL719 does not inhibit mTOR and PIKKs involved in DNA damage-repair processes. A. TSC1 -/- MEFs cells were grown in a 96-well format and treated for 1 h with increased concentrations of RAD001 or NVP-BYL719 (from 0.5 nmol/L to 10 ?mol/L in 1 third dilution steps) and immediately fixed. S235/236P-RPS6 levels were measured and IC50 determined with the Excel module XLfit. Background (no primary Ab incubated); BL, Baseline. B: TSC1 -/- MEFs cells were treated with increasing concentrations of NVP-BYL719 as indicated or RAD001 at 500 nmol/L or an equivalent DMSO concentration for 30 minutes. Levels of S235/236P-RPS6 and total RPS6 in protein- normalized lysates were detected by Western blot analyzis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. C: 24 h post seeding, A549 cells were treated at the same time with Actinomycin D (Act D) at a concentration of 5 ?mol/L (an agent used to induce DNA damage), and with increasing concentrations of NVP-BYL719 as indicated or with the vehicle control (DMSO) for 1 h. Levels of S15P-p53 and tubulin in protein-normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. D: 24 h post seeding, U2OS cells were pre-treated for 1 h with increased concentrations of NVP-BYL719 or KU55933 a specific small molecular mass inhibitor of ATM (Supplementary reference 1) at a concentration of 10 ?mol/L or with the vehicle control (DMSO). The cells were then irradiated with 15 Gy and re-incubated at 37 degrees C for 1 h and then lysed. Levels of S1981P-ATM in protein- normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and it is the third leading cause of cancer-related deaths worldwide. Recently, aberrant signaling through the FGF19/FGFR4 axis has been implicated in HCC. Here, we describe the development of FGF401, a highly potent and selective, first in class, reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4. FGF401 is exquisitely selective for FGFR4 versus the other FGFR paralogues FGFR1, FGFR2, FGFR3, and all other kinases in the kinome. FGF401 has excellent drug-like properties showing a robust pharmacokinetic/pharmacodynamics/efficacy relationship, driven by a fraction of time above the phospho-FGFR4 IC90 value. FGF401 has remarkable antitumor activity in mice bearing HCC tumor xenografts and patient-derived xenograft models that are positive for FGF19, FGFR4, and KLB. FGF401 is the first FGFR4 inhibitor to enter clinical trials, and a phase I/II study is currently ongoing in HCC and other solid malignancies.

PDF - 37K, NVP-BYL719 was tested at 10 different concentrations (from 0.5 nmol/L to 10 μmol/L in 1 third dilution steps) against CLK2 (A) and LRRK2 (B). SelectScreenTM Kinase Profiling Service uses XLfit from IDBS to determine the IC50 values (IC50 CLK2=621 nmol/L; IC50 LRRK2=3'220 nmol/L). The dose response curve is curve fit to model number 205 (sigmoidal dose-response model).

Median of the shRNAs and RSA values. File containing median counts and log fold changes of each gene from Supplementary Data File S1. According significance values of each gene as activator or sensitizer compared to DMSO control (see Methods) are included.

Purpose:The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection.Experimental Design:The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line– or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models.Results:Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations.Conclusions:Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

Supplementary Fig from Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

Supplementary text and figures Supplementary Figure 1 A, visualization of the "inflection point" (or EC50) and "Amax" parameters that are shown in Fig. 2A. B, HGF mRNA expression (by RNA-seq) vs HGF protein in culture supernatant for 76 cancer cell lines. Cell lines with high HGF mRNA expression generally led to increased protein levels in supernatant. U87-MG and IM95 are labeled, because in vivo efficacy data are presented for those models. U87-MG shows a remarkably high level of HGF protein secretion despite relatively low HGF mRNA. C, labeled cell lines scored as hits with at least 2 out of 4 MET inhibitors, each screened twice across a subset of the CCLE. Hits were defined as Amax {less than or equal to} â^'25% for all compounds, and inflection point {less than or equal to} 100 nmol/L for capmatinib, {less than or equal to} 1 ï�­mol/L for crizotinib and JNJ-38877605, and {less than or equal to} 500 nmol/L for PF-4217903. The upper panel shows MET vs HGF mRNA expression, while the lower panel shows MET expression vs MET copy number for the same cell lines. Three regions that among themselves contain all hits (amplified, overexpression, autocrine) were defined such that the hit with the lowest value for the respective expression or copy number value sets the cut-off. Number of hits compared to total number of cell lines in the respective regions are indicated in brackets. Colors as in Figure 2. D, Time course of MET phosphorylation after a 5 minute pulse treatment with 100 ng/mL recombinant HGF in 2 cell lines. NCI-H596 cells bear a MET exon14 skipping mutation while A549 cells do not. Supplementary Figure 2 A, MET dependency (x axis) vs MET gene copy number (y axis), downloaded from https://depmap.org. MET gene dependency was estimated by applying the DEMETER2 model to a combination of 3 large-scale RNAi screens (Broad Achilles, Novartis DRIVE, Marcotte et al.) (7). B, MET dependency (x axis) vs HGF mRNA expression by RNA-seq (y axis), downloaded from https://depmap.org. MET gene dependency was determined from pooled CRISPR screening data (Avana 1.0 library, Broad Institute) applying the CERES algorithm (8,9). Supplementary Figure 3 A, potency comparison of several clinical MET inhibitors in cellular proliferation assays with EBC-1 cells. Representative dose-response curves are shown on the left, numerical inflection point values (mean {plus minus} standard deviation, n = 3) are displayed on the right. B, MET mRNA expression measured by Affymetrix human genome U133 Plus 2.0 arrays (x axis) or RNA-seq (y axis) in 67 lung cancer PDX models (Supplementary Table 3). Identifiers of the 3 models with highest expression are indicated. C, same models as in B, but displaying MET mRNA by RNA-seq (x axis) vs MET copy number by Affymetrix SNP 6.0 array (y axis). D, data as in Fig. 3B but showing individual tumor volumes under treatment with capmatinib. E, mouse body weights corresponding to the experiment shown in Fig. 3B. F, data as in Fig. 3C but phospho- MET and total MET values from multi-spot ELISA shown separately. G, repeat of capmatinib efficacy study with lung PDX tumors bearing a MET exon 14 skipping mutation (model LU5381) as in Fig. 3D, but longer treatment duration. An average regression of ~60% was observed on day 12. A vehicle control was not repeated in this study. H, antitumor efficacy of capmatinib (dosed as indicated) against xenografts of the gastric cancer cell line IM95, which expresses HGF. Supplementary Figure 4 A, Loewe excess for data shown in Fig. 4C (upper panel), and % inhibition as well as Loewe excess for a combination matrix of capmatinib and the selective BCL2 inhibitor venetoclax (lower panel), in the cell line NCI-H1993. Percent dead cells were quantified by dual imaging with propidium iodide (PI) and Hoechst 33342. B, Percent dead cells by PI/Hoechst (upper panel) and Loewe excess (lower panel) for EBC-1 cells treated as in Fig. 4C and Supplementary Fig. 4A. C, Loewe excess for data shown in Fig. 4D, cell lines as indicated. Here, data are based on a CellTiter-Glo readout with quantification of seeded cells at time of compound addition ("growth inhibition" calculation). Loewe excess also refers to the "growth inhibition" data. D, treatment of EBC-1 cells with the indicated dose matrix of capmatinib and erlotinib. Experimental setup and analysis as in Fig. 4D (docetaxel combination). Supplementary Figure 5 A, HCC827 or HCC827 GR lung cancer cells were exposed to gefitinib, capmatinib, or combinations in a fixed ratio for 72 hours before measuring cell viability using a resazurin assay. The x axis label corresponds to gefitinib concentrations, while capmatinib was used at 10-fold lower concentrations due to its higher potency. In combination, gefitinib and capmatinib were mixed at a ratio of 10:1. B, HCC827 or NCI-H3255 cells were treated with a dilution series of gefitinib in the presence or absence of 50 ng/mL recombinant HGF. Cell viability was measured after 96 hours (HCC827) or 72 hours (NCIH3255) using a resazurin assay. The initial amount of viable cells was quantified at the time of compound addition (dashed line), and cell growth on the y axis is expressed as a multiple of this value. C, lysates of the lung cancer PDX model X-1787, either treated with vehicle or crizotinib, were incubated on a phospho-RTK array. Phospho-MET is clearly detectable in vehicle-treated lysate, suggesting that the high MET mRNA expression in this model leads to MET protein expression and activation. D, anti-tumor efficacy of crizotinib against X-1787 PDX tumors, characterized by presence of EML4-ALK and high MET expression. N = 4 per arm. E, RKO cells (BRAF V600E-mutant colorectal cancer cells secreting HGF) were exposed to capmatinib and fixed ratios of dabrafenib and trametinib in a dose matrix as indicated. After 3 days, viable cells were quantified by CellTiter-Glo. Inhibition is quantified in 2 different ways, without or with regard of the initial amount of viable cells at the time of compound addition (left and middle panel, respectively). Calculations are explained in detail in Materials and Methods. The right panel shows the Loewe excess for "% inhibition."

Supplementary Material from Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

Activation of p53 by inhibitors of the p53–MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53–MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo. Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2–p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure–response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53–MDM2 inhibitors.Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257–67. ©2018 AACR.

Supplementary material and methods include the description for the bioanalytical method for HDM201 detection in plasma and tumor, the human and mouse gene expression analysis in vitro and in vivo, the live-cell quantification of cleaved-caspase activation, the western blot analysis, the immunohistochemistry, the splinkerette PCR for the amplification of transposon integration sites and the tumor sequencing, mapping of insertion sequences to the mouse genome and identification of common integration site, additional information on the shRNA screen and the tumor models and supplementary references. Supplementary figures include: • Fig S1: the SJSA-1 inhibition growth curves when treated with HDM201 at different doses and for different times and the data for MOLM-3. • Fig S2: the cumulative percentage of cleaved-caspase-3/7 positive cells over the time, the GI50 of HDM201, CGM097 or nutlin-3a on SJSA-1 cells and the cellular apoptosis, as judged by AUC of cleaved-caspase-3/7 positive cells, induced by these compounds. • Fig S3: the PK profile in plasma and tumor of HDM201 in SJSA-1 tumors-bearing rat after p.o. and i.v. treatment, the Bcl-xl mRNA levels in tumors after HDM201 treatment, representative images of SJSA-1 tumors stained with p53 and cleaved-caspase 3 antibodies after HDM201 treatment and the individual data for the efficacy experiment in SJSA-1 tumor-bearing rats. • Fig S4: the PD of HDM201 in PB tumor bearing nude mice after single dose administration. Supplementary tables include: • Table S1: Biochemical profile of HDM201. • Table S2: List of cell lines tested for their sensitivity to HDM201 (n=291) • Table S3: Contingency table indicating association between sensitivity to HDM201 and TP53 wild-type status. • Table S4: List of cell lines tested for their sensitivity to both MDM2 knock-down by shRNA and HDM201 (n=261) • Table S5: Contingency table indicating association between sensitivity to HDM201 and sensitivity to MDM2 shRNA. • Table S6: List of significant rescuer and sensitizer genes following both HDM201 treatment types • Table S7: Pharmacokinetic parameters for HDM201 after p.o. and i.v. dosing in rat. • Table S8: Summary of primary PK parameters for HDM201 daily regimen after single dose (Day 1) in patients. • Table S9: Summary of primary PK parameters for HDM201 daily regimen on Day 14 in patients. • Table S10: Summary of primary PK parameters for HDM201 q3w regimen after single dose in patients.

The recent discovery of an acquired activating point mutation in JAK2, substituting valine at amino acid position 617 for phenylalanine, has greatly improved our understanding of the molecular mechanism underlying chronic myeloproliferative neoplasms. Strikingly, the JAK2V617F mutation is found in nearly all patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia and primary myelofibrosis. Thus, JAK2 represents a promising target for the treatment of myeloproliferative neoplasms and considerable efforts are ongoing to discover and develop inhibitors of the kinase. Here, we report potent inhibition of JAK2V617F and JAK2 wild-type enzymes by a novel substituted quinoxaline, NVP-BSK805, which acts in an ATP-competitive manner. Within the JAK family, NVP-BSK805 displays more than 20-fold selectivity towards JAK2 in vitro, as well as excellent selectivity in broader kinase profiling. The compound blunts constitutive STAT5 phosphorylation in JAK2V617F-bearing cells, with concomitant suppression of cell proliferation and induction of apoptosis. In vivo, NVP-BSK805 exhibited good oral bioavailability and a long half-life. The inhibitor was efficacious in suppressing leukemic cell spreading and splenomegaly in a Ba/F3 JAK2V617F cell-driven mouse mechanistic model. Furthermore, NVP-BSK805 potently suppressed recombinant human erythropoietin-induced polycythemia and extramedullary erythropoiesis in mice and rats. Mol Cancer Ther; 9(7); 1945–55. ©2010 AACR.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials. [Mol Cancer Ther 2008;7(7):1–13 [Mol Cancer Ther 2008;7(7):1851–13]

Supplementary Table 1. IC50 values for capmatinib obtained in BaF3 TRP-MET cells and mutant variants as indicated after 3-day proliferation assays with a resazurin readout. MET kinase domain mutations were either generated by site-directed mutagenesis or obtain by random mutagenesis and selection with another selective MET inhibitor (5). Supplementary Table 2. Numerical data that are graphically represented in Figure 2. Supplementary Table 3. Combined data from CCLE screens with 4 MET inhibitors (capmatinib, crizotinib, JNJ- 38877605, and PF-4217903). Related to Supplementary Fig. 1C. Supplementary Table 4. MET genomics data and additional model information for a collection of lung cancer PDX models downloaded from https://compendium.criver.com/. The Excel table is filtered down to those 66 models with complete data for MET mRNA (both Affymetrix and RNA-seq), MET mutations, and MET gene copy number, but other lung cancer models with partial data are also contained within the table. The 3 models that were used for capmatinib in vivo efficacy studies are highlighted in yellow.

Supplementary Data from Potent and Selective Inhibition of Polycythemia by the Quinoxaline JAK2 Inhibitor NVP-BSK805

Supplementary Figure 1 from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

Supplementary Figures 1-2, Tables 1-4, Methods from A Drug Resistance Screen Using a Selective MET Inhibitor Reveals a Spectrum of Mutations That Partially Overlap with Activating Mutations Found in Cancer Patients

Supplementary Figure 3 from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

Supplementary Figure 4 from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

Supplementary Figure Legends 1-4, Table Legend from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

Supplementary Table 1 from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

Supplementary Figure 2 from The Vascular Endothelial Growth Factor Receptor Inhibitor PTK787/ZK222584 Inhibits Aromatase

Disruption of the YAP-TEAD protein-protein interaction is an attractive therapeutic strategy for oncology to suppress tumour progression and cancer metastasis. YAP binds to TEAD at a large flat binding interface (~3500 2) devoid of a well-defined druggable pocket, so it has been difficult to design low molecular weight compounds to abrogate this protein-protein interaction directly. Recently, work by Furet and coworkers (ChemMedChem 2022, DOI: 10.1002/cmdc.202200303) reported the discovery of the first class of small molecules able to efficiently disrupt the transcriptional activity of TEAD by binding to a specific interaction site of the YAP-TEAD binding interface. Using high-throughput in silico docking and structure-based drug design, they identified a virtual screening hit from a hot-spot derived from their previously rationally designed peptidic inhibitor. Given advances in rapid high-throughput screening and rational approaches to peptidic ligand discovery for challenging targets, we analyzed the pharmacophore features involved in transferring from their peptidic to small molecule inhibitor that could enable small molecule discovery for such targets. Here, we show that pharmacophore analysis augmented by solvation analysis of molecular dynamics trajectories can guide the designs while binding free energy calculations provide greater insight into the binding conformation and energetics accompanying the association event. The computed binding free energy estimates agree with experimental findings and offer useful insight into structural determinants that influence ligand binding to the TEAD interaction surface, even for such a shallow binding site. In general, our results provide rationale for the structure-based design efforts in compound optimization that led to a significant gain in potency among the first class of potent small molecule YAP:TEAD protein-protein inhibitors.

Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor

Inhibition of the YAP-TEAD protein-protein interaction is an attractive therapeutic concept under intense investigation with the objective to treat cancers associated with a dysregulation of the Hippo pathway. However, owing to the very extended surface of interaction of the two proteins, the identification of small drug-like molecules able to efficiently prevent YAP from binding to TEAD by direct competition has been elusive so far. We disclose here the discovery of the first class of small molecules potently inhibiting the YAP-TEAD interaction by binding at one of the main interaction sites of YAP at the surface of TEAD. These inhibitors, providing a path forward to pharmacological intervention in the Hippo pathway, evolved from a weakly active virtual screening hit advanced to high potency by structure-based design.

The YAP-TEAD protein-protein interaction (PPI) is a critical event known to mediate YAP oncogenic functions downstream of the Hippo pathway. Current advanced pharmacological agents which aim at inhibiting YAP-TEAD oncogenic function do so by engaging into the lipid pocket of TEAD. Thereby the consequences of a direct pharmacological disruption of the interface of YAP and TEADs remain unexplored. Here we report the identification of IAG933, the first molecule able to potently and directly disrupt the YAP/TAZ-TEADs PPI with suitable properties to enter in clinical trial. The path to drug discovery was established by structure-based optimization of a truncated natural YAP peptide allowing the pharmacophore mapping of TEAD coil binding site. Based on in silico screening, validated hit was optimized using structure- and property-based lead optimization yielding IAG933, whose chemical structure will be for the first time disclosed here. Biochemical and cellular assays demonstrate that IAG933 specifically abrogates the interaction between YAP/TAZ coactivators and all four TEAD isoforms, thus selectively inhibiting TEAD-driven transcriptional activity and inducing anti-cancer effects. At the epigenome level, YAP eviction from chromatin was observed upon treatment with IAG933, while leaving TEADs genomic occupancy unaffected. Concomitantly, engagement of co-repressor VGLL4 translated to a decrease in enhancer activity with rapid and progressive changes in transcription of Hippo target genes. In preclinical experiments, IAG933 linear pharmacokinetics was consistent with dose proportional TEAD transcriptional inhibition and anti-tumor efficacy in xenograft and primary-tumor derived malignant pleural mesothelioma models. Daily treatment with IAG933 elicited complete tumor regression in the MSTO-211H xenograft model at well-tolerated doses. In line with the current clinical strategy for IAG933, robust anti-tumor efficacy in cancer models bearing NF2 loss of function or expressing TAZ-fusions was observed. Moreover, we provide evidence for combination benefits of IAG933 with several MAPK/KRAS inhibitors, both in vitro and in vivo, in non-Hippo altered models including lung, pancreatic and colorectal cancer. Overall, our results provide a rationale of progressing IAG933 as a monotherapy in patients with Hippo-mutated cancers, and as a combination partner in MAPK-dependent cancers, with the potential to treat patient populations of high unmet medical need. Citation Format: Tobias Schmelzle, Emilie Chapeau, Daniel Bauer, Patrick Chene, Jason Faris, Cesar Fernandez, Pascal Furet, Giorgio Galli, Jiachang Gong, Stephanie Harlfinger, Francesco Hofmann, Eloisa Jimenez Nunez, Joerg Kallen, Thanos Mourikis, Laurent Sansregret, Paulo Santos, Clemens Scheufler, Holger Sellner, Markus Voegtle, Markus Wartmann, Peter Wessels, Frederic Zecri, Nicolas Soldermann. IAG933, a selective and orally efficacious YAP1/WWTR1(TAZ)-panTEAD protein-protein interaction inhibitor with pre-clinical activity in monotherapy and combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB319.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

FGF19 signaling through the FGFR4/β-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.

The Hippo pathway is a key signaling pathway in the control of organ size and development. The most distal elements of this pathway, the TEAD transcription factors, are regulated by several proteins, such as YAP (Yes‐associated protein), TAZ (transcriptional co‐activator with PDZ‐binding motif) and VGLL1‐4 (Vestigial‐like members 1–4). In this article, combining structural data and motif searches in protein databases, we identify two new TEAD interactors: FAM181A and FAM181B. Our structural data show that they bind to TEAD via an Ω‐loop as YAP/TAZ do, but only FAM181B possesses the LxxLF motif (x any amino acid) found in YAP/TAZ. The affinity of different FAM181A/B fragments for TEAD is in the low micromolar range and full‐length FAM181A/B proteins interact with TEAD in cells. These findings, together with a recent report showing that FAM181A/B proteins have a role in nervous system development, suggest a potential new involvement of the TEAD transcription factors in the development of this tissue.

The YAP-TEAD protein-protein interaction is a potential therapeutic target to treat cancers in which the Hippo signaling pathway is deregulated. However, the extremely large surface of interaction between the two proteins presents a formidable challenge for a small molecule interaction disrupter approach. We have accomplished progress towards showing the feasibility of this approach by the identification of a 15-mer peptide able to potently (nanomolar range) disrupt the YAP-TEAD interaction by targeting only one of the two important sites of interaction. This peptide, incorporating non-natural amino acids selected by structure-based design, is derived from the Ω-loop sequence 85-99 of YAP.

Purpose: The selective MET inhibitor capmatinib is being investigated in multiple clinical trials, both as a single agent and in combination. Here, we describe the preclinical data of capmatinib, which supported the clinical biomarker strategy for rational patient selection. Experimental Design: The selectivity and cellular activity of capmatinib were assessed in large cellular screening panels. Antitumor efficacy was quantified in a large set of cell line– or patient-derived xenograft models, testing single-agent or combination treatment depending on the genomic profile of the respective models. Results: Capmatinib was found to be highly selective for MET over other kinases. It was active against cancer models that are characterized by MET amplification, marked MET overexpression, MET exon 14 skipping mutations, or MET activation via expression of the ligand hepatocyte growth factor (HGF). In cancer models where MET is the dominant oncogenic driver, anticancer activity could be further enhanced by combination treatments, for example, by the addition of apoptosis-inducing BH3 mimetics. The combinations of capmatinib and other kinase inhibitors resulted in enhanced anticancer activity against models where MET activation co-occurred with other oncogenic drivers, for example EGFR activating mutations. Conclusions: Activity of capmatinib in preclinical models is associated with a small number of plausible genomic features. The low fraction of cancer models that respond to capmatinib as a single agent suggests that the implementation of patient selection strategies based on these biomarkers is critical for clinical development. Capmatinib is also a rational combination partner for other kinase inhibitors to combat MET-driven resistance.

Radiation sensitivity varies greatly between tissues. The transcription factor p53 mediates the response to radiation; however, the abundance of p53 protein does not correlate well with the extent of radiosensitivity across tissues. Given recent studies showing that the temporal dynamics of p53 influence the fate of cultured cells in response to irradiation, we set out to determine the dynamic behavior of p53 and its impact on radiation sensitivity in vivo. We find that radiosensitive tissues show prolonged p53 signaling after radiation, while more resistant tissues show transient p53 activation. Sustaining p53 using a small molecule (NMI801) that inhibits Mdm2, a negative regulator of p53, reduced viability in cell culture and suppressed tumor growth. Our work proposes a mechanism for the control of radiation sensitivity and suggests tools to alter the dynamics of p53 to enhance tumor clearance. Similar approaches can be used to enhance killing of cancer cells or reduce toxicity in normal tissues following genotoxic therapies., p53 mediates the response to irradiation, however, tissues with similar levels of p53 have different radiation sensitivities. Here, the authors show that the in vivo p53 dynamics varies in these tissues after radiation, and the use of Mdm2 inhibitor to sustain p53 activity enhances radiosensitivity.

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

Activation of p53 by inhibitors of the p53–MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53–MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo. Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2–p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure–response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53–MDM2 inhibitors. Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257–67. ©2018 AACR.