Abnormal DNA hypermethylation mediated by DNA methyltransferases (DNMT) is a nearly universal hallmark of human cancers. However, while DNA methyltransferase inhibitors (DNMTi) such as decitabine and azacitidine are effective in treating myelodysplatic syndrome/leukemia, they have had limited utility for the majority of other cancers. Through a chemical library screen, we identify that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, and multiple of its analogs, significantly augment the epigenetic and anti-cancer effects of decitabine in vitro and in vivo. These effects are attributable to inhibition of DCTPP1-mediated cleavage of 5-aza-deoxycytidine triphosphate, the convergent activated metabolite of nucleoside DNMTi, leading to enhanced drug incorporation into genomic DNA, increased DNMT degradation, enhanced DNA demethylation and associated transcriptional reprogramming. We show that high DCTPP1 expression mediates cell-intrinsic resistance to nucleoside DNMTi, and that triptolide and its analogs could overcome this resistance. These findings nominate combining DNMTi with triptolide or its analogs as a rational cancer therapeutic strategy. Abnormal DNA hypermethylation is common in cancer, but DNA methyltransferase inhibitors show limited activity in many tumors. Here, the authors employ a chemical library screen to identify triptolide as an active agent that enhances these drugs by blocking DCTPP1, increasing drug incorporation into DNA, promoting demethylation and epigenetic reprogramming, and improving anti-cancer effects.

The 26S proteasome is an essential regulator of protein homeostasis and a clinically validated therapeutic target in multiple myeloma (MM). Rapaprotin, a novel macrocycle identified from a rapamycin-inspired rapafucin library, disrupts 26S proteasome function by inducing disassembly of the 19S regulatory particle in the 26S proteasome, leading to apoptosis in MM cells. Its bioactivation requires prolyl endopeptidase (PREP)-mediated cleavage to generate Rapaprotin-L, a negatively charged, linear metabolite with potent proteasome-disassembly activity. Using the PRISM cancer cell line profiling platform, we identified high P-glycoprotein (P-gp/ABCB1) expression as a major determinant of Rapaprotin resistance in solid tumor cell lines. Efflux assays confirmed Rapaprotin-L, but not its parent Rapaprotin, as a high-efficiency P-gp substrate. Co-treatment with the third-generation P-gp inhibitor tariquidar restored the intracellular accumulation of Rapaprotin-L, reinstating proteasome inhibition and consequent apoptosis in Rapaprotin-resistant colorectal cancer cell lines. Strong synergy between Rapaprotin and tariquidar was observed in a 3D spheroid model. These results establish P-gp as a key mediator of resistance to Rapaprotin and identify a rare example of a negatively charged Rapaprotin-L as a P-gp substrate. Together, these findings expand the potential therapeutic scope of Rapaprotin beyond hematologic malignancies to a broader range of solid tumors.

Inspired by the anticancer marine natural product superstolide A, we previously designed and synthesized a truncated analogue, designated ZJ-101, that retains the potent anticancer activity of the original natural product. In this study, using ZJ-101 as a lead compound, we conducted molecular design, convergent synthesis, and structure-activity optimization, resulting in the discovery of new analogues exhibiting anticancer activity approximately 30-80-fold more potent than ZJ-101, with IC50 values predominantly in the single-digit nanomolar range.

Lipoproteins are essential for lipid transport in all bilaterians. A single Apolipoprotein B (ApoB) molecule is the inseparable structural scaffold of each ApoB-containing lipoprotein (B-lps), which are responsible for transporting lipids to peripheral tissues. The cellular mechanisms that regulate ApoB and B-lp production, secretion, transport, and degradation remain to be fully defined. In humans, elevated levels of vascular B-lps play a causative role in cardiovascular disease. Previously, we have detailed that human B-lp biology is remarkably conserved in the zebrafish using an in vivo chemiluminescent reporter of ApoB (LipoGlo) that does not disrupt ApoB function. Thus, the LipoGlo model is an ideal system for identifying novel mechanisms of ApoB modulation and, due to the ability of zebrafish to generate many progeny, is particularly amenable to large-scale phenotypic drug screening. Here, we report a screen of roughly 3000 compounds that identified 49 unique ApoB-lowering hits. Nineteen hits passed orthogonal screening criteria, and seven were subjected to extensive phenotyping. A licorice root component, enoxolone, significantly lowered B-lps only in animals that express a functional allele of the nuclear hormone receptor Hepatocyte Nuclear Factor 4⍺ (HNF4⍺). Consistent with this result, inhibitors of HNF4⍺ also reduce B-lp levels. These data demonstrate that mechanism(s) of action can be rapidly determined from a whole-animal zebrafish phenotypic screen. Given the well-documented role of HNF4⍺ in human B-lp biology, these data validate the LipoGlo screening platform for identifying small-molecule modulators of B-lps that play a critical role in a leading cause of worldwide mortality.

BackgroundThe Food and Drug Administration has approved the Serine/threonine-protein kinase B-raf (BRAF) inhibitor and Mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor combo as the first-line treatment for individuals with metastatic melanoma, although the majority of these patients exhibit primary or secondary drug resistance in the clinic. Dihydrotanshinone I (DHT) is a lipophilic compound extracted from the root of Salvia miltiorrhiza that has been linked to multiple antitumor activities. In this study, we investigated the effect of dihydrotanshinone I on the MAPK pathway inhibitor resistance of BRAF mutant malignant melanoma.MethodAfter treating A375, A375R, and A2058 cells with DHT or a combination of DHT and BRAF/MEK inhibitors, WB and Real-Time RT-qPCR were used to confirm the activation of the MAPK and STAT3/SOX2 pathways. CCK-8 was used to assess cell viability, while flow cytometry was used to identify apoptosis. In addition, mice were inoculated with A375 cells to establish a model of tumour formation, and various drug groups and treatment models were utilized. The diameter and weight of tumours in each group were then measured, and IHC and HE staining were used to assess the expression of two pathways and cytotoxicity, respectively.ResultsThis study found that DHT directly interacts with STAT3 protein and it can stop the feedback activation of the STAT3/SOX2 pathway caused by the use of MAPK pathway inhibitors. In addition, the combination of DHT and BRAF/MEK inhibitors can inhibit the proliferation and growth of BRAF mutant melanoma cells and primary and secondary drug-resistant cells. Finally, we proved that the combined therapy of DHT and BRAF/MEK inhibitors is reliable and effective at animal and cell levels.ConclusionIn BRAF mutant melanoma cells, DHT suppresses the STAT3/SOX2 signaling pathway. Combining DHT, BRAF inhibitors, and MEK inhibitors can help treat treatment-resistant BRAF mutant melanoma cells. Experimental results both in vitro and in vivo have shown that the combination of DHT and an inhibitor of the MAPK pathway is safer and more successful than using an inhibitor of the MAPK pathway alone when treating BRAF mutant melanoma.

Rapaglutin A (RgA) is a potent pan-class I glucose transporter (GLUT) inhibitor identified from the rapafucin library. However, its development is hindered by plasma instability due to the rapid hydrolysis of lactone moieties by carboxylesterases, especially in rodents. To improve its stability, we designed and synthesized RgA analogues in which the lactones were substituted with more stable lactams. We found that substitution of either lactone-enhanced plasma stability, while dual lactam replacement produced the greatest improvement in mouse plasma, which has a high esterase activity. Importantly, the lactam analogues retained most of the inhibitory activity of the parent RgA against GLUT in DLD1 cells. This approach offers a promising strategy to enhance the plasma stability of RgA without significantly compromising its activity, with potential applicability across the rapafucin class of macrocycles.

Signet ring cell carcinoma (SRCC) poses a considerable challenge in terms of treatment, given its refractory nature and poor outcomes. Unlike other cancers, SRCC exhibits significant MDM2 copy number gains, with elevated MDM2 expression linked to poor prognosis. MDM2 inhibition induces a morphological transition in SRCC cells by suppressing E-cadherin degradation, which may render these cells vulnerable to a second drug. Using a high-throughput drug screen, our study demonstrated that the combination of MDM2 inhibitors with G2/M checkpoint inhibitors, including WEE1 or CHK1 inhibitors, can elicit a synergistic antitumor response in SRCC cells by inducing DNA damage. Furthermore, pharmacological inhibition of MDM2, WEE1, or CHK1 significantly impeded tumor growth in in vivo mouse models and organoids of SRCC. Collectively, our findings indicate that MDM2 inhibition-induced morphological changes may enhance the efficacy of G2/M checkpoint inhibitors, presenting a promising combined treatment for SRCC.

Natural products have a long history of providing probes into protein biosynthesis, with many of these compounds serving as therapeutics. The marine natural product girolline has been described as an inhibitor of protein synthesis. Its precise mechanism of action, however, has remained unknown. The data we present here suggests that girolline is a sequence-selective modulator of translation factor eIF5A. Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling where translational progress is impeded, including on AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in ribosome-associated quality control and in maintaining the efficiency of translational progress. Girolline helped to deepen our understanding of the interplay between protein production and quality control in a physiological setting and offers a potent chemical tool to selectively modulate gene expression.

Nanoparticle-based small interfering RNA (siRNA) therapeutics have revealed potential applications in the treatment of osteosarcoma. Nevertheless, charge-related toxicity and nuclease clearance severely limit the applicability of siRNAs in osteosarcoma therapeutic field. Herein, a GSH-responsive diblock copolymer siRNAdisulfide-poly(2-(diisopropylamino)ethyl methacrylate) (siRNA-SS-PDPA) is designed and synthesized, of which the siRNA micelles are generated via self-assembly strategy. Specifically, the derived siRNA micelles not only possess the features of effective cellular internalization, endosomal escape, prolonged blood circulation time, stable and efficient release, and no charge toxicity, but also effectively knock down the EPHA2 gene that mediates the insensitivity of tumor cells to imatinib (IMA). Resultantly, EPHA2 is capable of being knocked down by siRNA micelles, which enhances the sensitivity of MG63 cell line to imatinib, synergistically inhibiting the PI3K-AKT pathway and inducing apoptosis in combination with IMA. Simultaneously, the siRNA micelles demonstrate favorable biosafety in both cell lines and animal experiments, which significantly inhibit the in-situ growth of MG63 xenograft osteosarcoma by synergizing with IMA and markedly improve the survival rate. Conclusively, such cation-free siRNA micelles provide a novel drug-encoded delivery platform for GSH-triggered synergistic treatment of osteosarcoma with RNAi and chemotherapeutic agents.

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

The 26S proteasome is an essential regulator of protein homeostasis and a clinically validated therapeutic target in multiple myeloma (MM). Rapaprotin, a novel macrocycle identified from a rapamycin-inspired rapafucin library, disrupts 26S proteasome function by inducing disassembly of the 19S regulatory particle in the 26S proteasome, leading to apoptosis in MM cells. Its bioactivation requires prolyl endopeptidase (PREP)-mediated cleavage to generate Rapaprotin-L, a negatively charged, linear metabolite with potent proteasome-disassembly activity. Using the PRISM cancer cell line profiling platform, we identified high P-glycoprotein (P-gp/ABCB1) expression as a major determinant of Rapaprotin resistance in solid tumor cell lines. Efflux assays confirmed Rapaprotin-L, but not its parent Rapaprotin, as a high-affinity P-gp substrate. Co-treatment with the third-generation P-gp inhibitor tariquidar restored the intracellular accumulation of Rapaprotin-L, reinstating proteasome inhibition and consequent apoptosis of Rapaprotin-resistant colorectal cancer cell lines. Strong synergy between Rapaprotin and tariquidar was observed in a 3D spheroid model. These results establish P-gp as a key mediator of resistance to Rapaprotin and identify a rare example of a negatively charged Rapaprotin-L as a P-gp substrate. Together, these findings expand the potential therapeutic scope of Rapaprotin beyond hematologic malignancies to a broader range of solid tumors.

The 19S regulatory particle (RP) associates with the 20S core particle (CP) to form the 26S proteasome, an evolutionarily conserved holoenzyme that plays key roles in both physiological and pathological processes. Proteasome inhibitors that target the catalytic subunits within the 20S have proven to be valuable research tools and therapeutics for various cancers. Herein we report the discovery of rapaprotin, a 26S proteasome assembly inhibitor from our natural product-inspired hybrid macrocycle rapafucin library. Rapaprotin induces apoptosis in both myeloma and leukemia cell lines. Genome-wide CRISPR-Cas9 screen identified a cytosolic enzyme, prolyl endopeptidase (PREP) that is required for the pro-apoptotic activity of rapaprotin. Further mechanistic studies revealed that rapaprotin acts as a molecular transformer, changing from an inactive cyclic form into an active linear form, rapaprotin-L, upon PREP cleavage, to block 26S proteasome activity. Time-resolved cryogenic electron microscopy (cryo-EM) revealed that rapaprotin-L induces dissociation of the 19S RP from the 26S holoenzyme, which was verified in cells. Furthermore, rapaprotin exhibits a marked synergistic effect with FDA-approved proteasome inhibitors and resensitizes drug-resistant multiple myeloma cells from patients to bortezomib. Taken together, these results suggest that rapaprotin is a new chemical tool to probe the dynamics of the 26S proteasome assembly and a promising anticancer drug lead.

Natural products rapamycin and FK506 are macrocyclic compounds with therapeutic benefits whose unique scaffold inspired the generation and exploration of hybrid macrocycle rapafucins. From this library, a potent inhibitor of the facilitative glucose transporter (GLUT), rapaglutin A (RgA), was previously identified. RgA is a pan-GLUT inhibitor of Class I isoforms GLUT1, GLUT3, and GLUT4. Herein, we report the discovery of rapaglutin E (RgE). Unlike RgA, RgE is highly specific for GLUT1. Further characterization revealed that RgE and RgA likely bound to distinct sites on GLUT1 despite their shared FKBP-binding domain, suggesting that the distinct effector domains of RgE and RgA play key roles in the recognition of GLUTs.

Objective To determine the association between concurrent statin use with immune checkpoint inhibitors (ICIs) and lung cancer-specific and overall mortality in patients with non-small cell lung cancer (NSCLC). Materials and Methods SEER-Medicare was used to conduct a retrospective study of Medicare beneficiaries ≥65 years of age diagnosed with NSCLC between 2007-2017 treated with an ICI. Patients were followed from date of first ICI claim until death, 1 month from last ICI claim, or 12/31/2018, whichever came first. Associations for time-updated statin use and lung cancer-specific mortality, and overall mortality were estimated using Cox regression models adjusted for demographic, pathological, and treatment-related factors as well as a propensity score for statin use. Results Among 1,401 patients, concurrent statin use with any ICI was associated with 41% lower risk of lung-cancer specific mortality compared to patients receiving ICI not using a statin (HR=0.59; 95% CI=0.35-0.99). Statin use was associated with a similarly lower risk of overall mortality (HR=0.62; 95% CI=0.41 to 0.94). Consistent inverse associations were observed when restricting to PD-1 inhibitors and by statin type. Limited anti-PD-L1 treatment prevented analysis in this subgroup. Conclusion Concurrent statin use with ICIs was associated with lower risk of lung cancer-specific and overall mortality in a population-based sample of older patients with NSCLC. Future work is needed to confirm these findings in prospective studies and randomized trials, including evaluating concurrent statin use with frontline ICIs, deciphering the underlying mechanism of this purported synergy, and determining the optimal statin-ICI combination that maximize clinical benefit. Microabstract Resistance to anti-PD(L)1 immune checkpoint inhibitors (ICIs) in advanced non-small cell lung cancer (NSCLC) is common, and activation of the YAP-TAZ signaling pathway has been implicated in resistance. We used the SEER-Medicare database to analyze the association between statin use (potent YAP inhibitor) and both lung cancer-specific and overall mortality in patients with advanced NSCLC receiving ICIs. Concurrent statin plus ICI was associated with significantly lower lung cancer-specific and overall survival.

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

As an executor of pyroptosis, gasdermin D (GSDMD) plays a critical role in inflammatory diseases and cancer. Thus, GSDMD is currently being widely explored as a drug target. Existing inhibitors targeting GSDMD, such as necrosulfonamide, disulfiram, and fumarate, primarily prevent pyroptosis by modifying human/mouse C191/C192 in the N-terminal fragment of GSDMD. However, cysteine modification can prevent the function of important proteins or enzymes, thereby leading to adverse reactions. Here, we chose an alternative key intervention site for GSDMD activation, which is located at the oligomerization interface I of its pore-forming structure. Through high-throughput virtual and experimental screening and in combination with efficacy and pharmacological validation, we have identified two safe, specific “repurposed drugs” that potently suppress GSDMD-mediated pyroptosis. Moreover, the candidates exhibited synergistic therapeutic effects of “1 + 1>2” in murine sepsis and tumorigenesis models. These recently identified GSDMD inhibitors hold great promise for clinical translation in the development of anti-inflammatory and anti-cancer immunotherapies.

SUMMARY While nucleoside DNA methyltransferase inhibitors (DNMTi) such as decitabine and azacitidine are effective in treating myelodysplatic syndrome (MDS)/leukemia, they have had limited utility for the majority of other cancers. Through a chemical library screen, we identified that triptolide, a diterpenoid epoxide from Tripterygium wilfordii , or analogs significantly augmented the epigenetic and anti-cancer effects of decitabine in vitro and in vivo . These effects were attributable to inhibition of DCTPP1-mediated cleavage of 5-aza-deoxycytidine triphosphate, the convergent activated metabolite of nucleoside DNMTi, leading to enhanced drug incorporation into genomic DNA, increased DNMT degradation, enhanced global DNA demethylation and associated transcriptional reprogramming. We show that high DCTPP1 expression was associated with cell-intrinsic resistance to nucleoside DNMTi, and that triptolide and its analogs could overcome this resistance. SIGNIFICANCE We screened a library of existing drugs to identify those capable of enhancing the anti-cancer effects of the nucleoside DNMTi decitabine. The combination of triptolide and decitabine synergistically inhibited cancer cell growth and survival in vitro , and was highly effective in inhibiting xenograft growth in vivo . Biochemical, genetic and structural biology studies with triptolide and its analogs revealed that this synergy was due to their inhibition of DCTPP1-mediated pyrophosphate cleavage from 5-aza-deoxycytidine triphosphate, the active metabolite of DNMTi. The genomic incorporation and efficacy of decitabine in cancer cell lines were significantly correlated with DCTPP1 expression more so than those of other nucleoside metabolizing genes. Triptolide and its analogs comprise rational adjuncts to nucleoside DNMTi ripe for further pre-clinical/clinical translation. HIGHLIGHTS Triptolide synergistically sensitizes cancer cells to DNMTi in vitro . Triptolide and decitabine combination shows favorable efficacy and safety in vivo . Synergy of triptolide and decitabine is mediated through inhibition of DCTPP1. High DCTPP1 expression confers cell intrinsic resistance to DNMTi.

This study investigates the molecular mechanism of HMGA2-mediated regulation of IGFBP2 expression in the PI3K/ AKT/VEGFA signaling pathway, which is involved in angiogenesis and LUAD metastasis. Target genes with prognostic implications for LUAD patients were selected using bioinformatics, and previously published literature was referenced to predict the molecular regulatory mechanisms. A549 cells were used for in vitro validation. Cell proliferation and viability were assessed using CCK-8 and EdU assays, while cell migration ability was evaluated using Transwell and wound healing assays. Changes in angiogenesis were examined using an angiogenesis assay. The targeted binding of HMGA2 with the IGFBP2 promoter was confirmed through dual luciferase reporter gene experiments and ChIP assays. In vivo validation was performed using a xenograft mouse model, and changes in angiogenesis and tumor metastasis were observed using western blot, immunofluorescence, and H&E staining. Bioinformatics analysis revealed that HMGA2 was one of the AAGs that differed between normal individuals and LUAD patients and could serve as a critical mRNA for predicting LUAD prognosis. Results from in vitro experiments demonstrated that the expression of the HMGA2 gene was significantly upregulated in LUAD cell lines. Through mediating the expression of IGFBP2, the HMGA2 gene activated the PI3K/AKT/VEGFA signaling pathway, promoting the proliferation, migration, and angiogenesis of A549 cells. In vivo , animal experiments further confirmed that HMGA2 facilitated angiogenesis and the development and metastasis of LUAD through mediating IGFBP2 expression and activating the PI3K/AKT/VEGFA signaling pathway. HMGA2 promotes angiogenesis and healthy growth and metastasis of LUAD by activating the PI3K/AKT/VEGFA signaling pathway by mediating IGFBP2 expression.

ZJ-101, a structurally simplified analog of marine natural product superstolide A, was previously designed and synthesized in our laboratory. In the present study four new analogs of ZJ-101 were designed and synthesized to investigate the structure-activity relationship of the acetamide moiety of the molecule. The biological evaluation showed that the amide moiety is important for the molecule's anticancer activity. Replacing the amide with other functional groups such as a sulfonamide group, a carbamate group, and a urea group resulted in the decrease in anticancer activity.

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.