Spatially-resolved transcriptomics reveal macrophage heterogeneity and prognostic significance in diffuse large B-cell lymphoma.

Macrophages are abundant immune cells in the microenvironment of diffuse large B-cell lymphoma (DLBCL). Macrophage estimation by immunohistochemistry shows varying prognostic significance across studies in DLBCL, and does not provide a comprehensive analysis of macrophage subtypes. Here, using digital spatial profiling with whole transcriptome analysis of CD68+ cells, we characterize macrophages in distinct spatial niches of reactive lymphoid tissues (RLTs) and DLBCL. We reveal transcriptomic differences between macrophages within RLTs (light zone /dark zone, germinal center/ interfollicular), and between disease states (RLTs/ DLBCL), which we then use to generate six spatially-derived macrophage signatures (MacroSigs). We proceed to interrogate these MacroSigs in macrophage and DLBCL single-cell RNA-sequencing datasets, and in gene-expression data from multiple DLBCL cohorts. We show that specific MacroSigs are associated with cell-of-origin subtypes and overall survival in DLBCL. This study provides a spatially-resolved whole-transcriptome atlas of macrophages in reactive and malignant lymphoid tissues, showing biological and clinical significance.

Discovery of 4-aminophenylacetamide derivatives as intestine-specific farnesoid X receptor antagonists for the potential treatment of nonalcoholic steatohepatitis.

Farnesoid X receptor (FXR) plays a key role in bile acid homeostasis, inflammation, fibrosis, lipid and glucose metabolism and is emerging as a promising therapeutic target for nonalcoholic steatohepatitis (NASH). Emerging evidence suggested that intestine-specific FXR antagonists exhibited remarkable metabolic improvements and slowed NASH progression. In this study, we discovered several potent FXR antagonists using a multistage ligand- and structure-based virtual screening approach. Notably, compound V023-9340, which possesses a 4-aminophenylacetamide scaffold, emerged as the most potent FXR antagonist with an IC50 value of 4.27 µM. In vivo, V023-9340 demonstrated selective accumulation in the intestine, substantially ameliorating high-fat diet (HFD)-induced NASH in mice by mitigating hepatic steatosis and inflammation. Mechanistic studies revealed that V023-9340 strongly inhibited intestinal FXR while concurrently feedback-activated hepatic FXR. Further structure-activity relationship optimization employing V023-9340 has resulted in the synthesis of a more efficacious compound V02-8 with an IC50 value of 0.89 µM, which exhibited a 4.8-fold increase in FXR antagonistic activity compared to V023-9340. In summary, 4-aminophenylacetamide derivative V023-9340 represented a novel intestine-specific FXR antagonist and showed improved effects against HFD-induced NASH in mice, which may serve as a promising lead in discovering potential therapeutic drugs for NASH treatment.

Patterns of Oncogene Coexpression at Single-Cell Resolution Influence Survival in Lymphoma.

Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6- (M+2+6-) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6- percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6- unfavorable biology. Similar analyses evaluating oncogenic combinations at single-cell resolution in other cancers may facilitate an understanding of cancer evolution and therapy resistance. SIGNIFICANCE: Using single-cell-resolved multiplexed imaging, we show that selected subpopulations of cells expressing specific combinations of oncogenes influence clinical outcomes in lymphoma. We describe a probabilistic metric for the estimation of cellular oncogenic coexpression from IHC or bulk transcriptomes, with possible implications for prognostication and therapeutic target discovery in cancer. This article is highlighted in the In This Issue feature, p. 1027.

Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma.

BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition.

Excessive genomic instability coupled with abnormalities in DNA repair pathways induces high levels of 'replication stress' when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention towards targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1, and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective ATR kinase inhibitor elimusertib (BAY 1895344) has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identification of novel inhibitors of S-adenosyl-L-homocysteine hydrolase via structure-based virtual screening and molecular dynamics simulations.

S-adenosyl-L-homocysteine hydrolase (SAHase) is an important regulator in the methylation reactions in many organisms and thus is crucial for numerous cellular functions. In recent years, SAHase has become one of the popular targets for drug design, and SAHase inhibitors have exhibited potent antiviral activity. In this study, we established the complex-based pharmacophore models based on the known crystal complex of SAHase (PDB ID: 1A7A) to screen the drug-likeness compounds of ChEMBL database. Then, three molecular docking programs were used to validate the reliability of compounds, involving Libdock, CDOCKER, and AutoDock Vina programs. The four promising hit compounds (CHEMBL420751, CHEMBL346387, CHEMBL1569958, and CHEMBL4206648) were performed molecular dynamics simulations and MM-PBSA calculations to evaluate their stability and binding-free energy in the binding site of SAHase. After screening and analyzing, the hit compounds CHEMBL420751 and CHEMBL346387 were suggested to further research to obtain novel potential SAHase inhibitors. A series of computer-aided drug design methods, including pharmacophore, molecular docking, molecular dynamics simulation and MM-PBSA calculations, were employed in this study to identity novel inhibitors of S-adenosyl-L-homocysteine hydrolase (SAHase). Some compounds from virtual screening could form various interactions with key residues of SAHase. Among them, compounds CHEMBL346387 and CHEMBL420751 exhibited potent binding affinity from molecular docking and MM-PBSA, and maintained good stability at the binding site during molecular dynamics simulations as well. All these results indicated that the selected compounds might have the potential to be novel SAHase inhibitors.

Synthesis and biological evaluation of novel pentanediamide derivatives as S-adenosyl-l-homocysteine hydrolase inhibitors.

A series of novel pentanediamide derivatives were designed, synthesized and evaluated as S-adenosyl-l-homocysteine hydrolase (SAHase) inhibitors in this study. Some compounds showed good inhibitory activity against SAHase. The optimal compound 7i showed good inhibitory activity against SAHase with IC50 value of 3.58 ± 0.19 µM, cytotoxicity with IC50 values ranging from 13.16 ± 1.44 to 21.23 ± 0.73 µM against four tumor cell lines (MCF-7, A549, MGC-803, Hela) and very weak cytotoxicity (IC50 = 84.22 ± 1.89 µM) on normal LO2 cells. In addition, compound 7i showed potency against respiratory syncytial virus with EC50 value of 27.4 µM and selectivity index of 6.84. Further molecular simulation study suggested that compound 7i had good ADMET properties, and strongly binds to the active site of SAHase. In summary, compound 7i could serve as a new lead compound for further screening novel non-adenosine SAHase inhibitors.

PLK1 inhibition selectively induces apoptosis in ARID1A deficient cells through uncoupling of oxygen consumption from ATP production.

Inhibitors of the mitotic kinase PLK1 yield objective responses in a subset of refractory cancers. However, PLK1 overexpression in cancer does not correlate with drug sensitivity, and the clinical development of PLK1 inhibitors has been hampered by the lack of patient selection marker. Using a high-throughput chemical screen, we discovered that cells deficient for the tumor suppressor ARID1A are highly sensitive to PLK1 inhibition. Interestingly this sensitivity was unrelated to canonical functions of PLK1 in mediating G2/M cell cycle transition. Instead, a whole-genome CRISPR screen revealed PLK1 inhibitor sensitivity in ARID1A deficient cells to be dependent on the mitochondrial translation machinery. We find that ARID1A knock-out (KO) cells have an unusual mitochondrial phenotype with aberrant biogenesis, increased oxygen consumption/expression of oxidative phosphorylation genes, but without increased ATP production. Using expansion microscopy and biochemical fractionation, we see that a subset of PLK1 localizes to the mitochondria in interphase cells. Inhibition of PLK1 in ARID1A KO cells further uncouples oxygen consumption from ATP production, with subsequent membrane depolarization and apoptosis. Knockdown of specific subunits of the mitochondrial ribosome reverses PLK1-inhibitor induced apoptosis in ARID1A deficient cells, confirming specificity of the phenotype. Together, these findings highlight a novel interphase role for PLK1 in maintaining mitochondrial fitness under metabolic stress, and a strategy for therapeutic use of PLK1 inhibitors. To translate these findings, we describe a quantitative microscopy assay for assessment of ARID1A protein loss, which could offer a novel patient selection strategy for the clinical development of PLK1 inhibitors in cancer.

Early enteral feeding versus traditional feeding in neonatal congenital gastrointestinal malformation undergoing intestinal anastomosis: A randomized multicenter controlled trial of an enhanced recovery after surgery (ERAS) component.

PURPOSE: the aim of this clinical trial was to evaluate the safety and efficacy of early enteral feeding (EEN) following intestinal anastomosis in neonates with congenital gastrointestinal malformation. METHODS: a multicenter, prospective, randomized controlled trial (registered under chictr.org.cn Identifier no.ChiCTR-INR-17014179) was conducted between 2018 and 2019. Four centers in China analyzed 156 newborns of congenital gastrointestinal malformation undergoing intestinal anastomosis to EEN group (n = 78) or control (C) group (n = 78). The primary outcomes of this study were length of postoperative stay (LOPS) and time to full feeds. Secondary outcomes included morbidity of complications, parenteral nutrition (PN) duration, feeding intolerance, 30 day mortality rate and 30 day readmission rate. RESULTS: the mean time to full feeds and LOPS in the EEN group were 15.0 (9.8-22.8) days and 17.6 (12.0-29.8) days, while that were 18.0 (12.0-24.0) days and 20.0 (15.0-30.3) days in C groups respectively. There was no significant difference between two groups(P >0.05). No significant intergroup difference was found with respect to postoperative morbidity, PN duration or feeding intolerance(P >0.05). CONCLUSIONS: early enteral feeding following intestinal anastomosis in neonates with congenital gastrointestinal malformation is safe. Post-operative outcomes demonstrated a trend toward improvement. LEVEL OF EVIDENCE: Level Ⅰ.

PRDM15 is a key regulator of metabolism critical to sustain B-cell lymphomagenesis.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.

Discovery of novel N-aryl pyrrothine derivatives as bacterial RNA polymerase inhibitors.

Bacterial RNA polymerase (RNAP) is a validated drug target for broad-spectrum antibiotics, and its "switch region" is considered as the promising binding site for novel antibiotics. Based on the core scaffold of dithiolopyrrolone, a series of N-aryl pyrrothine derivatives was designed, synthesized, and evaluated for their antibacterial activity. Compounds generally displayed more active against Gram-positive bacteria, but less against Gram-negative bacteria. Among them, compound 6e exhibited moderate antibacterial activity against clinical isolates of rifampin-resistant Staphylococcus aureus with minimum inhibition concentration value of 1-2 µg/ml and inhibited Escherichia coli RNAP with IC50 value of 12.0 ± 0.9 µM. In addition, compound 6e showed certain degree of cytotoxicity against HepG2 and LO2 cells. Furthermore, molecular docking studies suggested that compound 6e might interact with the switch region of bacterial RNAP in a similar conformation to myxopyronin A. Together, the N-aryl pyrrothine scaffold is a promising lead for discovery of antibacterial drugs acting against bacterial RNAP.

Design, synthesis and biological evaluation of novel hybrids of N-aryl pyrrothine-base α-pyrone as bacterial RNA polymerase inhibitors.

Antibiotic resistance in bacteria has been an emerging public health problem, thus discovery of novel and effective antibiotics is urgent. A series of novel hybrids of N-aryl pyrrothine-base α-pyrone hybrids was designed, synthesized and evaluated as bacterial RNA polymerase (RNAP) inhibitors. Among them, compound 13c exhibited potent antibacterial activity against antibiotic-resistant S. aureus with the minimum inhibitory concentration (MIC) in the range of 1-4 µg/mL. Moreover, compound 13c exhibited strong inhibitory activity against E.coli RNAP with IC50 value of 16.06 µM, and cytotoxicity in HepG2 cells with IC50 value of 7.04 µM. The molecular docking study further suggested that compound 13c binds to the switch region of bacterial RNAP. In summary, compound 13c is a novel bacterial RNAP inhibitor, and a promising lead compound for further optimization.

Modulating Macrophage Phenotype by Sustained MicroRNA Delivery Improves Host-Implant Integration.

Biomedical implant failure due to the host's response remains a challenging problem. In particular, the formation of the fibrous capsule is a common barrier for the normal function of implants. Currently, there is mounting evidence indicating that the polarization state of macrophages plays an important role in effecting the foreign body reaction (FBR). This opens up a potential avenue for improving host-implant integration. Here, electrospun poly(caprolactone-co-ethyl ethylene phosphate) nanofiber scaffolds are utilized to deliver microRNAs (miRs) to induce macrophage polarization and modulate FBR. Specifically, C57BL/6 mice that are treated with M2-inducing miRs, Let-7c and miR-124, display relatively thinner fibrous capsule formation around the scaffolds at both Week 2 and 4, as compared to treatment with M1-inducing miR, Anti-Let-7c. Histological analysis shows that the density of blood vessels in the scaffolds are the highest in miR-124 treatment group, followed by Anti-Let-7c and Let-7c treatment groups. Based on immunohistochemical quantifications, these miR-encapsulated nanofiber scaffolds are useful for localized and sustained delivery of functional miRs and are able to modulate macrophage polarization during the first 2 weeks of implantation to result in significant alteration in host-implant integration at longer time points.

Comparison of outcomes following three surgical techniques for patients with severe jejunoileal atresia.

BACKGROUND: Severe jejunoileal atresia is associated with prolonged parenteral nutrition, higher mortality and secondary surgery. However, the ideal surgical management of this condition remains controversial. This study aimed to compare the outcomes of patients with severe jejunoileal atresia treated by three different procedures. METHODS: From January 2007 to December 2016, 105 neonates with severe jejunoileal atresia were retrospectively reviewed. Of these, 42 patients (40.0%) underwent the Bishop-Koop procedure (BK group), 49 (46.7%) underwent primary anastomosis (PA group) and 14 (13.3%) underwent Mikulicz double-barreled ileostomy (DB group). Demographics, treatment and outcomes including mortality, morbidity and nutrition status were reviewed and were compared among the three groups. RESULTS: The total mortality rate was 6.7%, showing no statistical difference among the three groups (P = 0.164). The BK group had the lowest post-operative complication rate (33.3% vs 65.3% for the PA group and 71.4% for the DB group, P = 0.003) and re-operation rate (4.8% vs 38.8% for the PA group and 14.3% for the DB group, P < 0.001). Compared with the BK group, the PA group showed a positive correlation with the complication rate and re-operation rate, with an odds ratio of 4.15 [95% confidence interval (CI): 1.57, 10.96] and 12.78 (95% CI: 2.58, 63.29), respectively. The DB group showed a positive correlation with the complication rate when compared with the BK group, with an odds ratio of 7.73 (95% CI: 1.67, 35.72). The weight-for-age Z-score at stoma closure was -1.22 (95% CI: -1.91, -0.54) in the BK group and -2.84 (95% CI: -4.28, -1.40) in the DB group (P = 0.039). CONCLUSIONS: The Bishop-Koop procedure for severe jejunoileal atresia had a low complication rate and re-operation rate, and the nutrition status at stoma closure was superior to double-barreled enterostomy. The Bishop-Koop procedure seems to be an appropriate choice for severe jejunoileal atresia.

Photoconductive Micro/Nanoscale Interfaces of a Semiconducting Polymer for Wireless Stimulation of Neuron-Like Cells.

We report multiscale structured fibers and patterned films based on a semiconducting polymer, poly(3-hexylthiophene) (P3HT), as photoconductive biointerfaces to promote neuronal stimulation upon light irradiation. The micro/nanoscale structures of P3HT used for neuronal interfacing and stimulation include nanofibers with an average diameter of 100 nm, microfibers with an average diameter of about 1 µm, and lithographically patterned stripes with width of 3, 25, and 50 µm, respectively. The photoconductive effect of P3HT upon light irradiation provides electrical stimulation for neuronal differentiation and directed growth. Our results demonstrate that neurons on P3HT nanofibers showed a significantly higher total number of branches, while neurons grown on P3HT microfibers had longer and thinner neurites. Such a combination strategy of topographical and photoconductive stimulation can be applied to further enhance neuronal differentiation and directed growth. These photoconductive polymeric micro/nanostructures demonstrated their great potential for neural engineering and development of novel neural regenerative devices.

Sub-10 nm Theranostic Unimolecular Micelles with High Tumor-Specific Accumulation, Retention, and Inhibitory Effect.

Theranostic agents that integrate far-red/near-infrared fluorescence and anticancer drugs are useful for biomedical applications such as imaging-guided therapy of cancers. However, the clinical translation of previously reported theranostic agents is still limited by factors such as weak fluorescence of the imaging probe, premature and off-target release of fluorophores and drugs during blood circulation, the long-term retention in the reticuloendothelial system, and side effects of toxicity. Here, we report a new type of ultrasmall theranostic unimolecular micelles with an average diameter below 10 nm, and dual functionalities of bright fluorescence in the spectral window of 600-800 nm toward noninvasive in vivo bioimaging and covalently bound anticancer drugs for specific cancer treatment. Each unimolecular micelle is formed by an amphiphilic bottlebrush copolymer containing a fluorescent conjugated backbone of poly(fluorene-alt-(4,7-bis(hexylthien)-2,1,3-benzothiadiazole)), from which hydrophobic disulfide-linked camptothecin as an anticancer drug and hydrophilic oligo(ethylene glycol) are grafted. These ultrasmall unimolecular micelles exhibit remarkably high efficiency of accumulation and retention in tumor tissues with a tumor inhibitory rate of 50%, but little distribution in other healthy organs and tissues. Such a feature of enhanced tumor targeting and reduced toxic side effects against healthy cells and tissues is promising for future clinical translation of imaging-guided cancer therapy.

Is the Bishop-Koop procedure useful in severe jejunoileal atresia?

PURPOSE: The aim of this study was to report our experience using the Bishop-Koop procedure for the treatment of various surgical problems of jejunoileal atresia including luminal discrepancy, complex meconium peritonitis, type IIIb and type IV atresia which we defined as severe jejunoileal atresia. METHODS: This retrospective study was performed on the patients with severe jejunoileal atresia who underwent Bishop-Koop procedure at a tertiary center in China over a five year of period. The mortality, complication rate, nutrition status and the risk factors for postoperative adverse outcomes were explored. RESULTS: A total of 41 neonates underwent the Bishop-Koop procedure. The median duration of the hospital stay and total parenteral nutrition and the point at which oral feeding was initiated postoperatively were 24 days (95% CI =18.99-29.01), 13 days (95% CI = 9.03-16.97) and 11 days (95% CI = 10.17-11.83) respectively. The mortality rate was 7.32% (3/41). The complication rate was 41.4% (17/41) including anastomotic leak, intestinal obstruction, high output stoma and cholestasis. The weight for age Z-score at stoma closure was restored to normal levels (-0.86, 95% confidence interval (CI) = -1.44, -0.28). The main factor associated with adverse outcomes in severe jejunoileal atresia was premature delivery (odds ratio (OR) = 4.44, 95% CI = 1.06-18.67). CONCLUSIONS: Bishop-Koop procedure appears to be a technically efficient method for severe jejunoileal atresia, although larger studies are needed to compare Bishop-Koop procedure and other operation techniques. TYPE OF STUDY: Therapeutic. LEVEL OF EVIDENCE: Level IV.