Discovery of Pyrido[2,3-d]pyrimidin-7-one Derivatives as Highly Potent and Efficacious Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Cancer Treatment.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an extracellular enzyme responsible for hydrolyzing cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), the endogenous agonist for the stimulator of interferon genes (STING) pathway. Inhibition of ENPP1 can trigger STING and promote antitumor immunity, offering an attractive therapeutic target for cancer immunotherapy. Despite progress in the discovery of ENPP1 inhibitors, the diversity in chemical structures and the efficacy of the agents are far from desirable, emphasizing the demand for novel inhibitors. Herein, we describe the design, synthesis, and biological evaluation of a series of ENPP1 inhibitors based on the pyrido[2,3-d]pyrimidin-7-one scaffold. Optimization efforts led to compound 31 with significant potency in both ENPP1 inhibition and STING pathway stimulation in vitro. Notably, 31 demonstrated in vivo efficacy in a syngeneic 4T1 mouse triple negative breast cancer model. These findings provide a promising lead compound with a novel scaffold for further drug development in cancer immunotherapy.

Second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP): the cell autonomous and non-autonomous roles in cancer progression.

Cytosolic double-stranded DNA (dsDNA) is frequently accumulated in cancer cells due to chromosomal instability or exogenous stimulation. Cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor, which is activated upon binding to dsDNA to synthesize the crucial second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) that in turn triggers stimulator of interferon genes (STING) signaling. The canonical role of cGAS-cGAMP-STING pathway is essential for innate immunity and viral defense. Recent emerging evidence indicates that 2'3'-cGAMP plays an important role in cancer progression via cell autonomous and non-autonomous mechanisms. Beyond its role as an intracellular messenger to activate STING signaling in tumor cells, 2'3'-cGAMP also serves as an immunotransmitter produced by cancer cells to modulate the functions of non-tumor cells especially immune cells in the tumor microenvironment by activating STING signaling. In this review, we summarize the synthesis, transmission, and degradation of 2'3'-cGAMP as well as the dual functions of 2'3'-cGAMP in a STING-dependent manner. Additionally, we discuss the potential therapeutic strategies that harness the cGAMP-mediated antitumor response for cancer therapy.

Protocol for PPP1R15A-inhibited mouse model establishment with subcutaneous B16F1 tumor and single-cell analysis.

Here, we present a protocol for exploring the effects of PPP1R15A inhibitor, Sephin1, on antitumor immunity of B16F1 subcutaneous tumor in mice. We describe steps for constructing single-cell transcriptome and TCR libraries, sequencing, and using sequencing data for the integration of expression and TCR data. We then detail procedures for gene differentiation, regulon and cell-cell communication analysis, and validation of single-cell analysis results. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Discovery, SAR Study of GST Inhibitors from a Novel Quinazolin-4(1H)-one Focused DNA-Encoded Library.

The DNA-encoded library (DEL) is a powerful hit-generation tool in drug discovery. This study describes a new DEL with a privileged scaffold quinazolin-4(3H)-one developed by a robust DNA-compatible multicomponent reaction and a series of novel glutathione S-transferase (GST) inhibitors that were identified through affinity-mediated DEL selection. A novel inhibitor 16 was subsequently verified with an inhibitory potency value of 1.55 ± 0.02 µM against SjGST and 2.02 ± 0.20 µM against hGSTM2. Further optimization was carried out via various structure-activity relationship studies. And especially, the co-crystal structure of the compound 16 with the SjGST was unveiled, which clearly demonstrated its binding mode was quite different from the known GSH-like compounds. This new type of probe is likely to play a different role compared with the GSH, which may provide new opportunities to discover more potent GST inhibitors.

Covalent inhibitor targets KRasG12C: A new paradigm for drugging the undruggable and challenges ahead.

KRAS is one of the most commonly mutated oncogenes in cancers and therapeutics directly targeting the KRas have been challenging. Among the different known mutants, KRasG12C has been proved to be successfully targeted recently. Several covalent inhibitors selectively targeting KRasG12C have shown promising efficacy against cancers harboring KRASG12C mutation in clinical trials and AMG510 (sotorasib) has been approved for the treatment of KRASG12C-mutated locally advanced or metastatic non-small cell lung cancer. However, the overall responsive rate of KRasG12C inhibitors was around 50% in patients with non-small cell lung cancer and the efficacy in patients with colorectal cancer or appendiceal cancer appears to be less desirable. It is of great importance to discover biomarkers to distinguish patients who are likely benefitted. Moreover, adaptive resistance would occur inevitably with the persistent administration like other molecularly targeted therapies. Several combinatorial regimens have been studied in an effort to potentiate the efficacy of KRasG12C inhibitors in preclinical settings. This review summarized the recent progress of covalent KRasG12C inhibitors with a focus on identifying biomarkers to predict or monitor the efficacy and proposing rational drug combinations based on elucidation of the mechanisms of drug resistance.

Intact regulation of G1/S transition renders esophageal squamous cell carcinoma sensitive to PI3Kα inhibitors.

Phosphatidylinositol 3-kinase alpha (PI3Kα) inhibitors are currently evaluated for the therapy of esophageal squamous cell carcinoma (ESCC). It is of great importance to identify potential biomarkers to predict or monitor the efficacy of PI3Kα inhibitors in an aim to improve the clinical responsive rate in ESCC. Here, ESCC PDXs with CCND1 amplification were found to be more sensitive to CYH33, a novel PI3Kα-selective inhibitor currently in clinical trials for the treatment of advanced solid tumors including ESCC. Elevated level of cyclin D1, p21 and Rb was found in CYH33-sensitive ESCC cells compared to those in resistant cells. CYH33 significantly arrested sensitive cells but not resistant cells at G1 phase, which was associated with accumulation of p21 and suppression of Rb phosphorylation by CDK4/6 and CDK2. Hypo-phosphorylation of Rb attenuated the transcriptional activation of SKP2 by E2F1, which in turn hindered SKP2-mediated degradation of p21 and reinforced accumulation of p21. Moreover, CDK4/6 inhibitors sensitized resistant ESCC cells and PDXs to CYH33. These findings provided mechanistic rationale to evaluate PI3Kα inhibitors in ESCC patients harboring amplified CCND1 and the combined regimen with CDK4/6 inhibitors in ESCC with proficient Rb.

Discovery of 7H-Pyrrolo[2,3-d]pyrimidine Derivatives as potent hematopoietic progenitor kinase 1 (HPK1) inhibitors.

Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cells and is a negative regulator of T cell receptor (TCR) signaling. Recent studies have demonstrated that HPK1 is a promising therapeutic target for cancer immunotherapy. However, despite significant progress in the development of HPK1 inhibitors, none of them has been approved for cancer therapy. Development of HPK1 inhibitors with a structurally distinct scaffold is still needed. Herein, we describe the design and synthesis of a series of HPK1 inhibitors with a 7H-pyrrolo[2,3-d]pyrimidine scaffold, exemplified by 31. Compound 31 showed potent inhibitory activity against HPK1 with an IC50 value of 3.5 nM and favorable selectivity within a panel of kinases. It also potently inhibited the phosphorylation level of SLP76, a substrate of HPK1, and enhanced the IL-2 secretion in Jurkat cells (human T cell leukemia). Our findings provide new clues for further optimization and development to generate HPK1 inhibitors for cancer immunotherapy.

Xanthine dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation.

Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown. Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis. Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells. Similar results were obtained in XDH-knockout cells. Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells. Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH. Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo. In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity.

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, T cell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific T cells was inhibited by Sephin1. A special TCR + macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

Targeting PI3Kα overcomes resistance to KRasG12C inhibitors mediated by activation of EGFR and/or IGF1R.

Although several KRasG12C inhibitors have displayed promising efficacy in clinical settings, acquired resistance developed rapidly and circumvented the activity of KRasG12C inhibitors. To explore the mechanism rendering acquired resistance to KRasG12C inhibitors, we established a series of KRASG12C-mutant cells with acquired resistance to AMG510. We found that differential activation of receptor tyrosine kinases (RTKs) especially EGFR or IGF1R rendered resistance to AMG510 in different cellular contexts by maintaining the activation of MAPK and PI3K signaling. Simultaneous inhibition of EGFR and IGF1R restored sensitivity to AMG510 in resistant cells. PI3K integrates signals from multiple RTKs and the level of phosphorylated AKT was revealed to negatively correlate with the anti-proliferative activity of AMG510 in KRASG12C-mutant cells. Concurrently treatment of a novel PI3Kα inhibitor CYH33 with AMG510 exhibited a synergistic effect against parental and resistant KRASG12C-mutant cells in vitro and in vivo, which was accompanied with concomitant inhibition of AKT and MAPK signaling. Taken together, these findings revealed the potential mechanism rendering acquired resistance to KRasG12C inhibitors and provided a mechanistic rationale to combine PI3Kα inhibitors with KRasG12C inhibitors for therapy of KRASG12C-mutant cancers in future clinical trials.

Advances in the Pharmacological Activities and Effects of Perilla Ketone and Isoegomaketone.

As components of a traditional Chinese herbal medicine with many physiological activities, perilla ketone and isoegomaketone isolated from perilla essential oil are important active components of Perilla frutescens. Recent studies have shown that these two compounds have promising antitumor, antifungal, antirheumatoid arthritis, antiobesity, anti-inflammatory, healing-promoting, and other activities and can be used to combat toxicity from immunotherapy. Therefore, the multitude of pharmacological activities and effects demonstrate the broad research potential of perilla ketone and isoegomaketone. However, no reviews have been published related to the pharmacological activities or effects of perilla ketone and isoegomaketone. The purpose of this review is as follows: (1) outline the recent advances made in understanding the pharmacological activities of perilla ketone and isoegomaketone; (2) summarize their effects; and (3) discuss future research perspectives.

Comparative genomic analysis of esophageal squamous cell carcinoma and adenocarcinoma: New opportunities towards molecularly targeted therapy.

Esophageal cancer is one of the most lethal cancers worldwide because of its rapid progression and poor prognosis. Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) are two major subtypes of esophageal cancer. ESCC predominantly affects African and Asian populations, which is closely related to chronic smoking and alcohol consumption. EAC typically arises in Barrett's esophagus with a predilection for Western countries. While surgical operation and chemoradiotherapy have been applied to combat this deadly cancer, molecularly targeted therapy is still at the early stages. With the development of large-scale next-generation sequencing, various genomic alterations in ESCC and EAC have been revealed and their potential roles in the initiation and progression of esophageal cancer have been studied. Potential therapeutic targets have been identified and novel approaches have been developed to combat esophageal cancer. In this review, we comprehensively analyze the genomic alterations in EAC and ESCC and summarize the potential role of the genetic alterations in the development of esophageal cancer. Progresses in the therapeutics based on the different tissue types and molecular signatures have also been reviewed and discussed.

Genome-wide gain-of-function screening identifies EZH2 mediating resistance to PI3Kα inhibitors in oesophageal squamous cell carcinoma.

Phosphoinositide-3 kinase alpha (PI3Kα) has been confirmed to be a potential therapeutic target for esophageal squamous cell carcinoma (ESCC), while the potency of PI3Kα inhibitors is often attenuated by concurrent oncogenic signalling pathways. We performed genome-wide gain-of-function screening with a CRISPR-SAM library and identified enhancer of zeste homolog 2 (EZH2) rendering ESCC cells resistant to the PI3Kα inhibitor CYH33. Enhanced expression of EZH2 frequently occurs in ESCC and is related to poor prognosis. Overexpression of full-length EZH2 but not methyltransferase-deficient EZH2 conferred resistance to CYH33, while downregulating EZH2 expression restored sensitivity. EZH2 expression was negatively related to the activity of CYH33 against the proliferation of ESCC cell lines and patient-derived cells. Transcriptomic analysis revealed that EZH2 abrogated CYH33-mediated cell cycle regulation. EZH2 epigenetically suppressed the transcription of CDKN1A, promoting RB phosphorylation and cell cycle progression. Concurrently targeting EZH2 significantly potentiated CYH33 to inhibit the growth of ESCC cells and patient-derived xenografts accompanied by enhanced cell cycle arrest. Taken together, our study demonstrated that an EZH2-p21-RB axis remodeled cell cycle regulation and rendered resistance to PI3Kα inhibitors in ESCC. Simultaneously targeting PI3Kα and EZH2 may provide an effective strategy for ESCC therapy with high expression of EZH2.

SAF-248, a novel PI3Kδ-selective inhibitor, potently suppresses the growth of diffuse large B-cell lymphoma.

PI3Kδ is expressed predominately in leukocytes and overexpressed in B-cell-related malignances. PI3Kδ has been validated as a promising target for cancer therapy, and specific PI3Kδ inhibitors were approved for clinical practice. However, the substantial toxicity and relatively low efficacy as a monotherapy in diffuse large B-cell lymphoma (DLBCL) limit their clinical use. In this study, we described a novel PI3Kδ inhibitor SAF-248, which exhibited high selectivity for PI3Kδ (IC50 = 30.6 nM) over other PI3K isoforms at both molecular and cellular levels, while sparing most of the other human protein kinases in the kinome profiling. SAF-248 exhibited superior antiproliferative activity against 27 human lymphoma and leukemia cell lines compared with the approved PI3Kδ inhibitor idelalisib. In particular, SAF-248 potently inhibited the proliferation of a panel of seven DLBCL cell lines (with GI50 values < 1 µM in 5 DLBCL cell lines). We demonstrated that SAF-248 concentration-dependently blocked PI3K signaling followed by inducing G1 phase arrest and apoptosis in DLBCL KARPAS-422, Pfeiffer and TMD8 cells. Its activity against the DLBCL cells was negatively correlated to the protein level of PI3Kα. Oral administration of SAF-248 dose-dependently inhibited the growth of xenografts derived from Pfeiffer and TMD8 cells. Activation of mTORC1, MYC and JAK/STAT signaling was observed upon prolonged treatment and co-targeting these pathways would potentiate the activity of SAF-248. Taken together, SAF-248 is a promising selective PI3Kδ inhibitor for the treatment of DLBCL and rational drug combination would further improve its efficacy.

Repressing MYC by targeting BET synergizes with selective inhibition of PI3Kα against B cell lymphoma.

Phosphatidylinositol 3-kinase (PI3K) δ-specific inhibitors have been approved for the therapy of certain types of B cell lymphoma (BCL). However, their clinical use is limited by the substantial toxicity and lack of efficacy in other types of BCL. Emerging evidence indicates that PI3Kα plays important roles in the progression of B cell lymphoma. In this study, we revealed that PI3Kα was important for the PI3K signaling and proliferation in BCL cells. A novel clinical PI3Kα-selective inhibitor CYH33 possessed superior activity against BCL compared to the marketed PI3Kα-selective inhibitor Alpelisib and PI3Kδ-selective inhibitor Idelalisib. Though CYH33 was able to inhibit PI3K/AKT signaling in tested BCL cells, differential activity against proliferation was observed. Transcriptome profiling revealed that CYH33 down-regulated "MYC-targets" gene set in sensitive but not resistant cells. CYH33 inhibited c-MYC transcription in sensitive cells, which was attributed to a decrease in acetylated H3 bound to the promoter and super-enhancer region of c-MYC. Accordingly, CYH33 treatment resulted in phosphorylation and proteasomal degradation of the histone acetyltransferase p300. An unbiased screening with drugs approved or in clinical trials for the therapy of BCL identified that the clinical BET (Bromodomain and Extra Terminal domain) inhibitor OTX015 significantly potentiated the activity of CYH33 against BCL in vitro and in vivo, which was associated with enhanced inhibition on c-MYC expression and induction of cell cycle arrest and apoptosis. Our findings provide the rationale of combined CYH33 with BET inhibitors for the therapy of B cell lymphoma.

Identification and optimization of biphenyl derivatives as novel tubulin inhibitors targeting colchicine-binding site overcoming multidrug resistance.

Microtubule targeting agents (MTAs) are among the most successful chemotherapeutic drugs, but their efficacy is often limited by the development of multidrug resistance (MDR). Therefore, the development of novel MTAs with the ability to overcome MDR is urgently needed. In this contribution, through modification of the unsymmetric biaryl compounds, we discovered a novel compound dxy-1-175 with potent anti-proliferative activity against cancer cells. Mechanistic study revealed that dxy-1-175 inhibited tubulin polymerization by interacting with the colchicine-binding site of tubulin, which caused cell cycle arrest at G2/M phase. Based on the predicted binding model of dxy-1-175 with tubulin, a series of new 4-benzoylbiphenyl analogues were designed and synthesized. Among them, the hydrochloride compound 12e with improved solubility and good stability in human liver microsome, exhibited the most potent anti-proliferative activity with IC50 value in the low nanomolar range, and markedly inhibited the growth of breast cancer 4T1 xenograft in vivo. Notably, 12e effectively overcame P-gp-mediated MDR and our preliminary data suggested that 12e may not be a substrate of P-glycoprotein (P-gp). Taken together, our study reveals a novel MTA 12e targeting the colchicine-binding site with potent anticancer activity and the ability to circumvent MDR.

The double faced role of xanthine oxidoreductase in cancer.

Xanthine oxidoreductase (XOR) is a critical, rate-limiting enzyme that controls the last two steps of purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. It also produces reactive oxygen species (ROS) during the catalytic process. The enzyme is generally recognized as a drug target for the therapy of gout and hyperuricemia. The catalytic products uric acid and ROS act as antioxidants or oxidants, respectively, and are involved in pro/anti-inflammatory actions, which are associated with various disease manifestations, including metabolic syndrome, ischemia reperfusion injury, cardiovascular disorders, and cancer. Recently, extensive efforts have been devoted to understanding the paradoxical roles of XOR in tumor promotion. Here, we summarize the expression of XOR in different types of cancer and decipher the dual roles of XOR in cancer by its enzymatic or nonenzymatic activity to provide an updated understanding of the mechanistic function of XOR in cancer. We also discuss the potential to modulate XOR in cancer therapy.

Assessment of FSDAF Accuracy on Cotton Yield Estimation Using Different MODIS Products and Landsat Based on the Mixed Degree Index with Different Surroundings.

Research on fusion modeling of high spatial and temporal resolution images typically uses MODIS products at 500 m and 250 m resolution with Landsat images at 30 m, but the effect on results of the date of reference images and the 'mixed pixels' nature of moderate-resolution imaging spectroradiometer (MODIS) images are not often considered. In this study, we evaluated those effects using the flexible spatiotemporal data fusion model (FSDAF) to generate fusion images with both high spatial resolution and frequent coverage over three cotton field plots in the San Joaquin Valley of California, USA. Landsat images of different dates (day-of-year (DOY) 174, 206, and 254, representing early, middle, and end stages of the growing season, respectively) were used as reference images in fusion with two MODIS products (MOD09GA and MOD13Q1) to produce new time-series fusion images with improved temporal sampling over that provided by Landsat alone. The impact on the accuracy of yield estimation of the different Landsat reference dates, as well as the degree of mixing of the two MODIS products, were evaluated. A mixed degree index (MDI) was constructed to evaluate the accuracy and time-series fusion results of the different cotton plots, after which the different yield estimation models were compared. The results show the following: (1) there is a strong correlation (above 0.6) between cotton yield and both the Normalized Difference Vegetation Index (NDVI) from Landsat (NDVIL30) and NDVI from the fusion of Landsat with MOD13Q1 (NDVIF250). (2) Use of a mid-season Landsat image as reference for the fusion of MODIS imagery provides a better yield estimation, 14.73% and 17.26% higher than reference images from early or late in the season, respectively. (3) The accuracy of the yield estimation model of the three plots is different and relates to the MDI of the plots and the types of surrounding crops. These results can be used as a reference for data fusion for vegetation monitoring using remote sensing at the field scale.

PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8+T cells and promoting fatty acid metabolism.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) is frequently hyperactivated in cancer and plays important roles in both malignant and immune cells. The effect of PI3Kα inhibitors on the tumor microenvironment (TME) remains largely unknown. Here, we investigated the modulation of the TME by a clinical PI3Kα-specific inhibitor CYH33. METHODS: The activity of CYH33 against a panel of murine tumors in the immune-competent context or athymic mice was detected. Single-cell RNA sequencing and multi-parameter flow cytometry were performed to determine the immune profiling of TME. The effect of CYH33 on immune cells was conducted with primary murine cells. RESULTS: CYH33 exhibited more potent antitumor activity in immune-competent context. CYH33 enhanced the infiltration and activation of CD8+T and CD4+T cells, while attenuating M2-like macrophages and regulatory CD4+T cells. Increase in memory T cells was confirmed by the induction of long-term immune memory on CYH33 treatment. Mechanistically, CYH33 relieved the suppressed expansion of CD8+T cells via preferential polarization of the macrophages to the M1 phenotype. CYH33 promoted fatty acid (FA) metabolism in the TME, while FA enhanced the activity of CD8+T cells in vitro. The combination of CYH33 with the FA synthase (FASN) inhibitor C75 synergistically inhibited tumor growth with enhanced host immunity. CONCLUSIONS: CYH33 induces immune activation and synergizes with FASN inhibitor to further promote the antitumor immunity, which gains novel insights into how PI3K inhibitors exert their activity by modulating TME and provides a rationale for the concurrent targeting of PI3K and FASN in breast cancer treatment.