ABSTRACT KDM5B, a member of the KDM5 family of histone demethylases, plays a critical role in transcriptional repression by demethylating H3K4me2/3. It regulates key biological processes such as development, stem cell maintenance, and oncogenesis, and its aberrant expression is implicated in various cancers. Accordingly, KDM5B is considered a promising therapeutic target in cancer drug discovery. In this study, we performed a screening to identify novel inhibitors of KDM5B. Through this screening, we identified a common core scaffold associated with KDM5B inhibitory activity, and subsequent optimization of the side‐chain structures led to the identification of a series of compounds with IC50 values ranging from several nanomolar to several micromolar. Evaluation of growth inhibitory activity using the JFCR39 human cancer cell line panel revealed that the final lead compounds, JB‐157 and JB‐161, exhibited GI50 values in the low micromolar range. In a xenograft model, where the human colorectal cancer cell line HT‐29 was implanted into NOD/SCID mice, administration of these compounds resulted in antitumour effects, with JB‐161 showing particularly pronounced tumor suppression in a subset of treated animals. Furthermore, combination treatment with in vivo CAR‐T cell therapy and JB‐161 in C57BL/6J mice was associated with alterations in chromatin accessibility in tumor and immune cell populations. Taken together, these results suggest that JB‐161 is a candidate KDM5B inhibitor associated with chromatin accessibility changes and antitumour activity.

Fusion genes and mutations of PI3K-related genes in sarcoma cell lines.

Simultaneous inhibition of PI3Kα with PI3Kβ and/or PI3Kδ results in synergistic antitumor effects and apoptosis induction in TRS cells. A–D, Fluorescence time-lapse imaging analysis in Aska-SS (A and B) and SYO-1 cells (C and D) stained with SiR-DNA and SYTOX Green followed by treatment with alpelisib, TGX-221, and idelalisib alone or in combination for 48 hours. Drugs were added at 1 µmol/L for Aska-SS cells and at 8 μmol/L for SYO-1 cells. Representative images of Aska-SS (A) and SYO-1 cells (C) at 48 hours are presented. The growth rate of live cells (top left), the fragmentation index (top right), and the proportion of dead cells to total cells (bottom) in Aska-SS (B) and SYO-1 cells (D) were calculated by quantitative image analysis. Data are presented as the mean ± SD (n = 5). E, Representative flow cytometry plots of Aska-SS cells stained with Annexin V–FITC/PI after treatment with alpelisib, TGX-221, and idelalisib alone or in combination for 48 hours. F, Summary of Annexin V–FITC-positive cells (%) in E. Data are presented as the mean ± SD (n = 3). Statistical analysis was performed by one-way ANOVA with Dunnett post hoc test. n.s., not significant; *, P < 0.05; **, P < 0.01.

Shimojilide (1), a new cyclic lipopeptide, was isolated from a marine cyanobacterium Okeania sp. Its structure was elucidated by NMR and chemical derivatization, revealing a peptide-polyketide hybrid with a skipped diene and β-branched methyl group. Shimojilide (1) exhibited potent and selective antiproliferative activity with a unique JFCR39 fingerprint that poorly correlates with those of known anticancer agents, suggesting a distinct mechanism of action. Shimojilide (1) represents a promising lead for anticancer drug discovery.

Abstract Current precision oncology—molecular targeted therapies and immunotherapies—relies on genomic or expressed biomarkers, yet most cancer patients remain ineligible for these treatments. Here, we establish enzyme activity as an actionable and orthogonal axis for precision cancer medicine. Strategic activity-based screening of mouse organs and human clinical specimens with a panel of enzyme-reactive fluorescence probes identified β-galactosidase 1 (GLB1) and β-hexosaminidases (HEX) as broadly elevated tumor-selective biomarkers. Leveraging these activities, we developed 7-ethyl-10-hydroxycamptothecin (SN38)-based GLB1-and HEX-reactive prodrugs. These prodrugs exhibited dramatically reduced systemic toxicities and improved therapeutic windows, compared to a clinically used SN38-based prodrug, irinotecan (CPT-11). Both prodrugs demonstrated activity-dependent therapeutic efficacy, affording a dramatic reduction of tumor volumes across multiple in vivo models, including a subcutaneous patient-derived xenograft (PDX) of lung squamous cell carcinoma that lacked genetic alterations targeted by current precision medicine. Furthermore, this strategy is broadly applicable across various cytotoxic payloads, establishing a generalizable platform for small-molecule precision medicines. Our results define an enzyme-targeting paradigm for precision oncology, in which fluorescence probes serve as companion diagnostic tools to guide development and selection of appropriately targeted prodrugs, which are expected to provide safer and more efficacious treatment options for cancer patients with elevated enzyme activities.

Simultaneous inhibition of PI3Kα with PI3Kβ/δ induces significant apoptosis associated with suppression of Akt/mTOR signaling

Growth inhibition of TRS cell lines by PI3K inhibitors

Among class I PI3K isoforms, PI3Kα is the dominant isoform in TRSs. A, The expression of PIK3CA, PIK3CB, PIK3CD, PIK3CG, and PTEN in the indicated sarcoma cell lines, a gastric cancer cell line, and diffuse large B-cell lymphoma cell lines determined by RNA-seq. B, Immunoblots presenting the expression of p110α, p110β, p110δ, p110γ, and PTEN in the indicated cell lines. Tubulin was used as a loading control. C, Concentration–response curves of alpelisib, TGX-221, idelalisib, and ZSTK474 in the indicated TRS cell lines. D, Dot plots presenting differences in the GI50 values of alpelisib, TGX-221, idelalisib, and ZSTK474 in the indicated TRS cell lines. GI50 value was calculated using the growth curves presented in C and Supplementary Fig. S5. Statistical analysis was performed by one-way repeated-measures ANOVA with Bonferroni post hoc test. *, P < 0.05. E, Immunoblots of the indicated proteins in Aska-SS and SYO-1 cells treated with alpelisib, TGX-221, idelalisib, and ZSTK474 for 48 hours. Tubulin was used as a loading control. F, Immunoblots of the indicated proteins in Aska-SS cells transfected with siRNAs specific for PIK3CA, PIK3CB, and PIK3CD. Tubulin was used as a loading control.

Simultaneous inhibition of PI3Kα with PI3Kβ/δ induces synergistic antitumor effects and apoptosis in SJCRH30 but not MKN1 cells

Expression of PI3K pathway genes in sarcoma cell lines and clinical samples

Simultaneous inhibition of PI3Kα with PI3Kβ/δ enhances suppression of cell growth without inducing apoptosis in non-sarcoma cancer cell lines

Simultaneous inhibition of PI3Kα with PI3Kβ and/or PI3Kδ enhances growth inhibition but does not induce apoptosis in diverse nonsarcoma cancer cell lines. A, The list of cell lines used in this study and their mutation status for PIK3CA, PTEN, and KRAS. Cell lines shown in red are classified as the PIK3CA-mutated group (PTEN-wild and KRAS-wild), and those in blue are classified as the PTEN-deficient group (PIK3CA-wild and KRAS-wild). B, Box plots showing growth rate (%) of nonsarcoma cells treated with indicated drugs at 8 μmol/L for 48 hours, comparing between PIK3CA-mutated, PTEN-deficient, and other cell lines. Statistical analysis was performed by one-way ANOVA with Dunnett post hoc test. n.s., not significant; ***, P < 0.001. C and D, Box plots showing growth rate (%) of nonsarcoma cells (C) and TRS cells (D) at 48 hours, comparing the indicated drug treatments. Statistical analysis was performed by one-way ANOVA with Dunnett post hoc test. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. E and F, Dot plots showing SYTOX positivity (%) of nonsarcoma cells (E) and TRS cells (F) at 48 hours, comparing the indicated drug treatments.

Phosphoproteomic analysis reveals that ZSTK474, a pan-PI3K inhibitor, more potently inhibits Akt/mTOR signaling than the PI3Kα-specific inhibitor alpelisib. A, MA plots of phosphopeptides in DMSO-treated Aska-SS cells vs. alpelisib-treated Aska-SS cells (top) or DMSO-treated Aska-SS cells vs. ZSTK474-treated Aska-SS cells (bottom). Drugs were added at 1 μmol/L for 1 hour. The dotted lines indicate the boundary of the twofold change. Phosphopeptides in Akt/mTOR signaling are highlighted by red plots. Among them, representative phosphopeptides in Akt/mTOR signaling are indicated. B, GO analysis of phosphoproteins with greater than twofold changes in abundance between DMSO and alpelisib treatment (top) or between DMSO and ZSTK474 treatment (bottom). C, KSEA revealing activated or inactivated kinases in DMSO-treated Aska-SS cells vs. alpelisib-treated Aska-SS cells (top) or DMSO-treated Aska-SS cells vs. ZSTK474-treated Aska-SS cells (bottom). Positive kinase Z-scores indicate that the kinase is activated in Aska-SS cells treated with alpelisib or ZSTK474 compared with the findings in Aska-SS cells treated with DMSO, whereas negative kinase Z-scores indicate that the kinase is inactivated in Aska-SS cells treated with alpelisib or ZSTK474. P values were calculated using the KSEA algorithm. *, P < 0.05. D, MA plot of phosphopeptides with greater than twofold changes in signals between DMSO and ZSTK474 treatment in alpelisib-treated Aska-SS cells vs. ZSTK474-treated Aska-SS cells. The dotted lines indicate the boundary of the twofold change. Phosphopeptides in Akt/mTOR signaling are highlighted in red plots. Among them, representative phosphopeptides in Akt/mTOR signaling are indicated. E, GO analysis of phosphoproteins with greater than twofold changes in abundance between alpelisib and ZSTK474 treatment among phosphoproteins with greater than twofold changes in abundance between DMSO and ZSTK474 treatment. F, KSEA revealing activated or inactivated kinases in alpelisib-treated Aska-SS cells vs. ZSTK474-treated Aska-SS cells. P values were calculated using the KSEA algorithm. *, P < 0.05. G, Immunoblots of the indicated proteins in Aska-SS cells treated with alpelisib or ZSTK474 at 1 μmol/L for 1 hour. Tubulin was used as a loading control.

Simultaneous inhibition of PI3Kα with PI3Kβ and/or PI3Kδ significantly suppresses Akt/mTOR signaling. A and B, Immunoblots of the indicated proteins in Aska-SS and SYO-1 cells treated with alpelisib, TGX-221, and idelalisib alone or in combination at 1 μmol/L for 3 hours (A) or 48 hours (B). Tubulin was used as a loading control. C, Immunoblots of the indicated proteins in Aska-SS cells treated with alpelisib, TGX-221, and idelalisib at 1 μmol/L for 48 hours after transfection with siRNAs specific for PIK3CA, PIK3CB, or PIK3CD. Tubulin was used as a loading control. D, Immunoblots of the indicated proteins in SYO-1 cells with PIK3CB and/or PIK3CD knockout that were treated with alpelisib for 48 hours. Tubulin was used as a loading control.

Simultaneous inhibition of PI3Kα with PI3Kβ/δ suppresses tumor growth with PI3K pathway inhibition and apoptosis induction in the SYO-1 xenograft model

Simultaneous inhibition of PI3Kα with PI3Kβ/δ enhances suppression of cell growth and cell cycle in SJCRH30 but not MKN1 cells

The transcriptional cofactors YAP1 and TAZ regulate target gene expression by binding to the transcription factor TEAD. Due to their roles in cancer initiation, progression, and drug resistance, YAP1 and TAZ are promising targets for cancer therapy. SAMD4A/B are RNA-binding proteins that are broadly expressed across human tissues, but few of their molecular targets and biological functions have been identified. In Drosophila, the SAMD4A/B homolog Smaug participates in early embryonic development by disrupting the stability and translation of maternal mRNA. To discover targets inhibiting the YAP1/TAZ-TEAD oncogenic transcription program, we screened a whole-genome siRNA library and identified siSAMD4B as potently suppressing TEAD activity in human cancer cells. We showed that SAMD4A/B increased TEAD activity by destabilizing and repressing the translation of VGLL4 mRNA, promoting cancer progression in vitro. Conversely, inhibiting either SAMD4A or SAMD4B elevated VGLL4 mRNA, which suppressed TEAD activity and inhibited cancer progression. Notably, transgenic mice expressing liver-specific SAMD4B exhibited accelerated development of intrahepatic cholangiocarcinomas in an Nf2-deficient background. These tumors appeared in the mutants at one week of age and caused death due to hepatic failure by 100 days. Thus, SAMD4A/B may be a promising target for anticancer drugs designed to inhibit TEAD activation.