Peripheral blood metabolite concentrations vary with food intake and time of day, risking confounding effects in metabolomics studies with non-standardised sampling conditions or incomplete metadata. Such effects are often overlooked during study design, limiting the clinical translation of biomarkers and wasting resources for researchers, funders and clinicians. In our random sample of 100 human metabolomics studies, 56% did not control for food intake, and 59% did not explicitly control for sampling time. To provide a study design resource, we analysed a liquid-chromatography-mass-spectrometry-targeted dataset from controlled laboratory studies of 24 young, healthy participants (12 male, 12 female) sampled every 2 h for 34 h, with fixed-macronutrient meals provided at set times. Acute postprandial responses were quantified by effect size using pre- and post-meal windows, while daily rhythmicity was assessed using a mixed-effects cosinor model. Analyses were sex-stratified, and metabolites were classified as meal-responsive, time-of-day-responsive, both, or neither. Amino acids and their derivatives showed strong postprandial increases, whereas lipid classes showed minimal changes. Rhythmicity varied across metabolites, enabling the identification of features sensitive to meal timing and/or time of day. These results aim to provide a comprehensive dictionary of metabolite effect sizes for study design and metadata collection to support reproducibility and the clinical translation of potential biomarkers.

Experimental and predicted combinations. A, Clustered heatmap of Bliss synergy scores was experimentally measured for six cell lines treated with 21 two-drug combinations. B, Histogram representing the EPS rankings of nodes of targets of drugs in the top 25% highest Bliss synergy scores, that is, “most synergistic” (left), or the EPS rankings of nodes of targets of drugs in the 25% lowest Bliss synergy scores, that is, “least synergistic” (right). There is a significant bias toward higher EPS rankings for the most synergistic drug targets, with a significant Mann–Whitney U test P value of 0.0038875, indicating a biased distribution of rankings. C, Simulation of the Mann–Whitney U test P values obtained from 10,000-fold random permutations of EPS ranking, demonstrating the robustness of this P value.

Prediction of drug sensitivity using phosphoproteomic analysis. A, Classification of cell line–drug single-agent sensitivities into four quartiles, with Q1 = most sensitive and Q4 = least sensitive. B, Feature importance of phosphoproteins based on elastic net analysis shown. Features are described previously as significant if the weight is greater than +0.1 or lesser than −0.1. C, Performance of predictions of sensitivity quartile based on phosphoproteomic changes using elastic net analysis. D, Performance of prediction of sensitivity quartile based on three clinically relevant mutations (EGFR, PIK3CA, and KRAS) using elastic net analysis.

The lack of curative therapies for acute myeloid leukaemia (AML) remains an ongoing challenge despite recent advances in the understanding of the molecular basis of the disease. Here we identify the WNK1-OXSR1/STK39 pathway as a previously uncharacterised dependency in AML. We show that genetic depletion and pharmacological inhibition of WNK1 or its downstream phosphorylation targets OXSR1 and STK39 strongly reduce cell proliferation and induce apoptosis in leukaemia cells in vitro and in vivo. Furthermore, we show that the WNK1-OXSR1/STK39 pathway controls mTORC1 signalling via regulating amino acid uptake through a mechanism involving the phosphorylation of amino acid transporters, such as SLC38A2. Our findings underscore an important role of the WNK1-OXSR1/STK39 pathway in regulating amino acid uptake and driving AML progression.

Supplementary Figures S1-S3

Dynamic changes in phosphoproteins and EPS. Exemplar of results in a cell line HCC827. A and B, Network diagrams showing phosphoproteomic changes and drug targets with color gradient blue (−1.7) and red (+1.7). Nodes that are drug targets but where phosphorylation has not been measured are denoted in gray, that is, HSP90, PI3K, and BRAF. A, Phosphoproteomic changes related to exposure to the AKT inhibitor capivasertib. B, Phosphoproteomic changes related to exposure to the MEK inhibitor trametinib. C and D, EPS calculated for nodes that are tractable on CanSAR. C, EPS scores upon exposure to the AKT inhibition capivasertib. D, EPS scores upon exposure the MEK inhibitor trametinib.

Abstract Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease requiring additional therapeutic strategies. MCL1, an anti-apoptotic BCL2 family member, promotes cancer-cell survival, but its role in mCRPC remains poorly understood. Here, we characterise MCL1 in multiple mCRPC biopsy cohorts and patient-derived models, assessing responses to MCL1 inhibition. MCL1 copy number gain (14%-34%) correlates with increased MCL1 expression and worse outcomes. MCL1 inhibition exhibits anti-tumour effects in MCL1-gained mCRPC models. Co-inhibition of MCL1 and AKT induces cancer-specific cell death in PTEN-loss/PI3K-activated models in vitro and in vivo, modulating BAD-BCLXL and BIM-MCL1 interactions, with durable anti-tumour activity in models with AKT inhibitor acquired resistance. Finally, CDK9-mediated MCL1 downregulation combined with AKT inhibition recapitulates these findings, providing further opportunities for clinical translation. These data support early phase clinical trials targeting MCL1, both as monotherapy for MCL1-gained mCRPC, and in combination with AKT inhibition for PTEN-loss/PI3K-activated mCRPC.

Comparison phosphoprotein changes in patient samples and cell lines. A, 3D plot showing that for the first three principal components of the phosphoproteomic data, patient samples (blue diamonds) show comparable distribution with cell line data (yellow circles), indicating that changes in phosphorylation in cell line panels could potentially reflect changes within clinical samples. B, Probability density functions of cell line and patient data, showing a strong overlap in distribution and peak values between the two sample types, despite a Welch Two Sample t test, indicating that the two groups have different means (P = 0.006804). Here, x-axis plots the value of dynamic phosphoprotein changes, and the y-axis (density) is proportional to frequency.

Abstract Using innovative phenotypic screening, targeting the Heat Shock Factor 1 (HSF1) pathway, followed by multiparameter medicinal chemistry optimization, we discovered NXP800, an orally active, potent inhibitor of cell proliferation. Evaluation in a mini-panel of human cancer cell lines and tumor xenografts revealed high sensitivity in ARIDIA-deficient human ovarian cancer models, confirmed in the large Sanger panel and isogenic systems. By RNAseq we identified overlapping gene expression changes in human cancer cell lines exposed to NXP800, including expected changes in HSF1-regulated genes plus alterations in ATF4-regulated gene expression associated with activation of the integrated stress response (ISR). This did not indicate a global stress response to NXP800 as we saw no activation of the unfolded protein response. Consistent with activation of the ISR, NXP800 induced phosphorylation of EIF2A and increased expression of downstream ISR markers/effectors ATF4, CHAC1 and CHOP both in human ovarian cells in vitro and corresponding tumor xenograft models in vivo. Using an siRNA approach, we found that blocking the induction of ATF4 reduced the response of sensitive, ARID1A mutant SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. Phosphorylation of EIF2A is tightly regulated by four stress-controlled kinases, GCN2, HRI, PKR and PERK. Using either systematic siRNA knockdown or inhibition by two small-molecule tool compounds from different chemotypes, we discovered that GCN2 alone was required for ISR activation by NXP800. Also, inactivation of GCN2 markedly reduced the antiproliferative activity of NXP800. Global phospho-proteome analysis demonstrated defined changes in response to NXP800 which were reversed on co-treatment with a GCN2 inhibitor. Furthermore, ISR induction inhibited HSF1 activation, confirming the mechanistic link between ISR activation and inhibition of HSF1-mediated transcription. In summary, we discovered the mechanistically novel drug NXP800 which acts on cancer cells to stimulate GCN2 and thereby activate the ISR pathway, leading to inhibition of cap-dependent protein translation. NXP800 shows highly promising activity in human ovarian cancer, including tumor regression of ARID1A-deficient ovarian cancer xenografts. Studies are currently underway to determine precisely how NXP800 stimulates GCN2 activity and the role of ARID1A deficiency. With Nuvectis Pharma, the Phase 1a dose escalation study is completing and the multicentre Phase 1b expansion cohort study in platinum-resistant ARID1A-mutated ovarian cancer is now initiated (NCT05226507) in collaboration with the GOG Foundation and the European Network of Gynecological Oncological Trial Group (ENGOT). FDA has issued a Fast Track designation to NXP800 in this setting. Citation Format: Marissa V. Powers, Swee Y. Swap, Robert te Poele, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Suzenne Eccles, Florence Raynaud, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by NXP800, an orally available, clinical-stage, investigational agent in ARID1A-mutated, platinum resistant ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-002.

Bile acids are trans-genomic molecules arising from the concerted metabolism of the human host and the intestinal microbiota and are important for digestion, energy homeostasis and metabolic regulation. While diurnal variation has been demonstrated in the enterohepatic circulation and the gut microbiota, existing human data are poorly resolved, and the influence of the host circadian system has not been determined. Using entrained laboratory protocols, we demonstrate robust daily rhythms in the circulating bile acid pool in healthy male participants. We identify temporal relationships between bile acids and plasma lipids and show that these relationships are lost following sleep deprivation. We also highlight that bile acid rhythmicity is predominantly lost when environmental timing cues are held constant. Here we show that the environment is a stronger determinant of these temporal dynamics than the intrinsic circadian system of the host. This has significance for the intimate relationship between circadian timing and metabolism. Bile acids are important for digestion, energy homeostasis and metabolic regulation. Here the authors show daily rhythms in the circulating bile acid pool which are lost when environmental timing cues are held constant indicating that environment is a stronger determinant of these dynamics than the circadian system.

Background We proposed to quantify reduction of functional DNA damage response (DDR) mechanisms caused by the combination of CHK1 and WEE1 inhibitors. Methods Survival of cells and tumor growth in-vitro and in-vivo caused by the combination of the CHK1 inhibitor SRA737 and the WEE1 inhibitor adavosertib was studied in OVCAR3 and MDA-MB 436 cells. Functional DNA damage was quantified using in vitro cell free DNA assays. Results The combination of SRA737 and adavosertib caused significant reduction of survival of cells and DNA damage in-vitro and growth inhibition in-vivo. Studies using functional DDR assays found significant changes in the functional capacity of OVCAR3 but not MDA-MB 436 cells to repair DNA damage using multiple mechanisms including intra strand cross link repair, nucleotide excision repair, homologous recombination and non-homologous end joining. This study, for the first time provides a mechanistic insight into differences in the reduction in functional capacity of cells to repair DNA when exposed to CHK1 and WEE1 inhibitors. Conclusion The combination of the CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib causes growth inhibition in-vitro and in-vivo, but differential functional inhibition of DDR in the models studied.

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6441.

Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are lethal brain tumors lacking targeted therapies. They originate from interneuronal precursors; however, leveraging this origin for therapeutic insights remains unexplored. Here, we delineate a cellular hierarchy along the interneuron lineage development continuum, revealing that DHG-H3G34 mirror spatial patterns of progenitor streams surrounding interneuron nests, as seen during human brain development. Integrating these findings with genome-wide CRISPR-Cas9 screens identifies genes upregulated in interneuron lineage progenitors as major dependencies. Among these, CDK6 emerges as a targetable vulnerability: DHG-H3G34 tumor cells show enhanced sensitivity to CDK4/6 inhibitors and a CDK6-specific degrader, promoting a shift toward more mature interneuron-like states, reducing tumor growth, and prolonging xenograft survival. Notably, a patient with progressive DHG-H3G34 treated with a CDK4/6 inhibitor achieved 17 months of stable disease. This study underscores interneuronal progenitor-like states, organized in characteristic niches, as a distinct vulnerability in DHG-H3G34, highlighting CDK6 as a promising clinically actionable target.

Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on N-terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays. Our results show that a central amide linkage, a secondary amine, and (R)-configuration at the 4-position of the core are three key factors for inhibitory potency. Several potent analogues with low- or sub-μM IC50 against purified HHAT also inhibit Sonic Hedgehog (SHH) palmitoylation in cells and suppress the SHH signaling pathway. This work identifies IMP-1575 as the most potent cell-active chemical probe for HHAT function, alongside an inactive control enantiomer, providing tool compounds for validation of HHAT as a target in cellular assays.

XLS file - 703K, Gene expression data for CCT128930: Genes that showed at least a 1.5 Fold Change in at least one treated sample compared to vehicle control and were differentially expressed between the treatment groups (Welch ANOVA FDR5%)

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