Citations

Abstract Background Caesarean sections (CSs) constitute a large proportion of fetal deliveries, yet no consensus exists on optimal surgical method. Objectives To investigate the safety and efficacy of the Pfannenstiel‐Kerr method (PKM) in comparison to Misgav‐Ladach method (MLM) and Modified Misgav‐Ladach method (MMLM) for CS. Search Strategy Cochrane, Embase, PubMed, CiNAHL, Scopus, and Web of Science were searched from database inception to March 2023. Selection Criteria Prospective studies comparing MLM with PKM or MMLM with PKM. Data Collection and Analysis Three authors independently conducted data extraction and assessed quality. Pooled means and proportions were analyzed using inverse variance and Mantel‐Haenszel methods. Main Results Twenty‐seven studies with 15 251 patients, met the selection criteria. Both MLM (mean difference [MD]: −12.09 min, 95% confidence interval [CI]: −14.40, −9.77, Tau 2 = 16.79, I 2 = 97%), (MD: −125.98 mL, 95% CI: −172.13, −79.83, Tau 2 = 3458.59, I 2 = 87%) and MMLM (MD: −11.38 min, 95% CI: −14.72, −8.04, Tau 2 = 30.56, I 2 = 100%), (MD: −62.61 mL, 95% CI: −103.13, −22.09, Tau 2 = 1451.35, I 2 = 92%) demonstrated shorter operative time and decreased blood loss, respectively, compared to PKM. Similarly, both MLM (MD: −1.70 min, 95% CI: −2.26, −1.14, Tau 2 = 0.51, I 2 = 97%), (MD: −0.77 d, 95% CI: −1.35, −0.19, Tau 2 = 0.60, I 2 = 98%) and MMLM (MD: −2.80 min, 95% CI: −3.89, −1.70, Tau 2 = 1.74, I 2 = 100%), (MD: −0.58 d, 95% CI: −1.00, −0.17, Tau 2 = 0.29, I 2 = 100%) demonstrated shorter fetal extraction time and length of hospital stay, respectively, compared to PKM. Conclusions MLM and MMLM should be considered safe and effective alternative approaches to CSs.

Abstract OBJECTIVE We sought to define the mechanisms by which purines regulate DNA repair and therapy response. METHODS Phosphoproteomics was used to identify GTP-dependent (de)phosphorylation events after radiation (RT) and antibodies generated against novel sites. Animal models of glioblastoma (GBM) and normal tissues were used to assess DNA repair and treatment responses in vivo. RESULTS Pharmacogenomic inhibition of GTP (but not ATP) synthesis sensitized GBM cells to RT by inhibiting the activity of non-homologous end joining, but not homologous recombination. We found a GTP-dependent RT-induced dephosphorylation event on Abl interactor 1 (Abi-1) serine 323 (S323) using phosphoproteomics. We generated a new antibody for p-Abi-1 (S323), validated its specificity, and confirmed that RT causes a GTP-dependent dephosphorylation of Abi-1 (S323). Knockout of Abi-1 slowed RT-induced double-strand break (DSB) repair, and this was rescued by re-expression of dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D). Abi-1 canonically binds to G protein Rac1. Expression of constitutively active Rac1 promoted but dominant negative Rac1 blocked the dephosphorylation of Abi-1 (S323) and DSB repair. Knock-down or inhibition of protein phosphatase 5 reversed the GTP- and Rac1-mediated dephosphorylation of Abi-1 and DSB repair. In GBM PDX samples, p-Abi-1 (S323) levels negatively correlated with Rac1 activity and predicted favorable efficacy of genotoxic treatments. In orthotopic GBM mouse models, inhibiting Rac1 enhanced RT responses and suppressed Abi-1 (S323) dephosphorylation. Abi-1 knockout enhanced efficacy of genotoxic treatments and could be rescued by Abi-1 S323A (but not Abi-1 S323D) re-expression. This regulation is generalizable beyond brain cancer, as GTP supplementation promoted DNA repair and p-Abi1 (S323) dephosphorylation in non-malignant cells and protected mice from RT-mediated gastrointestinal injury and bleomycin-induced pulmonary fibrosis. CONCLUSION The GTP-Rac1-PP5-Abi-1 signaling axis links metabolism and DNA repair. Disrupting this pathway can overcome GBM resistance to genotoxic therapy while augmenting it can mitigate genotoxic injury of normal tissues.

Abstract Glioblastoma (GBM) is aggressive form of brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Immunocompetent mice harboring intracranial gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. Using parabiosis, we determined that T cell lymphopenia can be transferred from glioma-bearing to healthy mice via circulation This led to the identification of a high molecular weight immunosuppressive factor in sera of glioma-bearing mice that inhibits T cell proliferation ex vivo. Proteomics analysis identified 119 proteins uniquely upregulated in the sera of glioma-bearing mice compared to controls. These proteins are associated with pathways involved in DNA response elements, cell death, and DNA-histone complexes which imply DNA release. Additional analysis revealed serum isolated from glioma-bearing mice harbor significantly higher concentrations of double stranded cell free DNA (cfDNA) compared to controls. Reducing DNA content using DNase partially reverses the proliferation defects observed in T cells when exposed to serum of glioma-bearing mice. This is while, addition of cfDNA purified from serum of glioma-bearing mice, but not genomic DNA, potently inhibited T cell function. These results were AIM2 independent implicating a novel pathway of cfDNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reduce circulating cfDNA is therefore a therapeutic approach that could improve outcomes in GBM patients.

Abstract BACKGROUND New therapies are needed for meningioma since treatment options are limited once patients have exhausted surgery and radiation. FAK, CDK4/6 and RAF/MEK represent potential targets because of synthetic lethality with NF2, loss of CDKN2A, and activation of MAPK signaling pathway in meningioma, respectively. The objective of this study was to evaluate the efficacy of targeting FAK, CDK4/6 and RAF/MEK as monotherapies and combination therapies in traditional and patient-derived cell lines and xenografts. METHODS In vitro, cell viability and proliferation assays were used to test abemaciclib (CDK4/6 inhibitor), defactinib (FAK inhibitor) and VS-6766 (RAF/MEK inhibitor) as monotherapy and in combination. To better recapitulate the meningioma microenvironment, targeted agents were evaluated with Matrigel, an artificial extracellular matrix. The efficacy of VS-4718 (in vivo formulation of defactinib) and abemaciclib were evaluated in a subcutaneous IOMM-lee meningioma model (having CDKN2A loss) in nude mice. Furthermore, we are establishing cell cultures and patient-derived cell and xenograft (PDX) models using freshly-resected tissue from patients with recurrent or high-grade (WHO grade 2-3) meningiomas. RESULTS In vitro evaluation identified the combination of defactinib with abemaciclib resulted in a synergistic decrease in cell viability relative to each monotherapy alone, while the combination of defactinib+VS-6766 was not beneficial. The presence of Matrigel enhanced the sensitivity of all three agents. In vivo, we observed improved survival in mice treated with abemaciclib vs control (45 days vs 26 days; p=0.03), as well as combination of VS-4718 + abemaciclib vs control (44 days vs 26 days; p=0.04, Gehan-Breslow-Wilcoxon test). We did not observe survival extension with VS-4718 monotherapy. Lastly, we successfully established an orthotopic meningioma PDX using a WHO Grade 3 tumor for evaluation of novel therapies in meningioma. CONCLUSION Abemaciclib showed promising efficacy in preclinical meningioma xenografts with CDKN2A loss. Studies to evaluate these therapies in other orthotopic models are underway.

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

Abstract Background The co-stimulatory CD40–CD40 ligand axis is central in atherogenesis. Upon activation, CD40 recruits tumor necrosis factor receptor-associated factors (TRAFs) to induce downstream signaling. Murine studies with mutated CD40-TRAF binding sites in macrophages have shown that the CD40-TRAF6, rather than CD40-TRAF2/3/5, axis is crucial for atherosclerosis. We thus synthesized the small molecule inhibitor TRAF-STOP 6877002, that selectively blocks CD40-TRAF6 signaling. 6877002 successfully reduced the onset and development of atherosclerosis by reducing monocyte recruitment and macrophage activation in ApoE-/- mice without having immunosuppressive side effects. Purpose To assess whether 6877002 is a suitable candidate to pass the translational pipeline towards a clinical application. Methods Elaborate in vitro and in vivo assays evaluating the pharmacological properties and potential toxicity of 6877002. Results Together with Cyprotex, we performed in vitro assays examining the ADME profile of 6877002. In an intestinal permeability assay, 6877002 had low to moderate resorption with no significant efflux, suggesting it is not a substrate for the efflux transporters Pgp/BCRP or MRP2. A plasma protein binding assay showed very low unbound 6877002 fractions, indicating that it is highly bound to plasma proteins. The potential to inhibit cytochrome P450 enzymes was assessed using human liver microsomes in the presence of enzyme-specific probe substrates. While 6877002 inhibited CYP1A2 (IC50=4.01 µM), no potential for inhibiting other CYP isoforms was detected, suggesting low drug-drug interaction potential. To screen for putative cardiovascular risks, hERG ion channel inhibition was assessed and no IC50 could be determined. Hepatic clearance was rapid in mice, rats and dogs, while minipig and human hepatocytes had lower intrinsic clearance. Together with Aptuit, we developed suitable formulations for oral and i.v. administration. In vivo pharmacokinetics studies in rats showed low bioavailability (8.43%) and rapid clearance of orally administered 6877002 compared to i.v. injected 6877002, likely – and in line with our in vitro data – due to fast first-past effect in mouse hepatocytes and uptake by macrophages. To explain the high efficacy of 6877002 despite its rapid metabolism, we assessed whether 6877002 has active metabolites that bind the CD40-TRAF6 pocket and performed an LC-MS/MS-based metabolite profiling. We detected 17 metabolites. Further MD simulations confirmed that Metabolite (M)4, the most abundant metabolite, binds the CD40-TRAF6 pocket, reflected by the highest binding mode (-19.6; defined as Binding Free Energy derived from 40-50 ns of MD run as compared to -16.5 for 6877002). Similar to 6877002, M4 reduced atherosclerosis by 54% in Western diet-fed ApoE-/- mice, indicative of M4 being an active metabolite. Conclusion CD40-TRAF6 inhibitors are an attractive strategy to combat atherosclerosis with high translational potential.

Abstract Background Atherosclerosis is a chronic inflammatory disease, characterized by the formation of luminal plaques in arteries. Atheroprogression is accompanied by strong immune activation, during which T cells play a pivotal role. Genes driving T-cell activation are regulated by the polycomb repressive complex 2(PRC2), whose major component is the Enhancer of Zeste Homolog 2(EZH2), responsible for the methylation of Histone 3 Lysine 27(H3K27me3) epigenetic mark. Purpose We hypothesize that EZH2 mediates T-cell activation and modulates T-helper (Th) cell polarization, thus atherogenesis. Methods To study the effect of EZH2 in atherosclerosis, we backcrossed mice with Ezh2 flanked by loxP-sites sensitive to Cre-mediated inactivation in T cells to apolipoprotein E (Apoe-/-)mice and fed a high cholesterol diet to have hyperlipidemic Ezh2fl/flCd4CreApoe-/-mice and controls. Atherosclerotic plaque progression and immune cell phenotyping were assessed by histology, flow cytometry (FC), gene and protein expression analysis and single cell (sc)RNA-sequencing (Seq) as well as by functional in vitro assays. Results T cell-specific EZH2 deficiency resulted in a significant reduction in H3K27me3 in T-cells. Female Ezh2fl/flCd4Cre mice developed 34% less atherosclerotic plaques in the aortic root compared to wildtype (WT) littermates. FC analysis of spleen and lymph nodes of Ezh2fl/flCd4Cre mice revealed a shift from naive to effector CD4+ T cells, and the majority were Th2 cells (p<0.0001). Accordingly, plasma levels of lL4 and aortic IL4 transcripts were increased in Ezh2fl/flCd4Cre mice(p=0.05 and p=0.0079, respectively). Functional analysis revealed that splenic CD4+ T cells from Ezh2fl/flCd4Cre mice showed decreased migratory capacity towards the chemokines CXCL10, CCL17, CCL22, CCL19. To unravel mechanisms driving the type 2 immune response, bulk RNA-Seq of CD4+ T cells and scRNA-Seq of splenic CD3+ T cells from Ezh2fl/flCd4Cre and WT mice revealed that CD4+ T cell EZH2 deficiency caused upregulation of promyelocytic leukemia zinc finger (Plzf), a transcription factor of NKT cells of the NKT2 subtype, characterized by high IL4 production. Via FC we confirmed the increase in splenic NKT cells in Ezh2fl/flCd4Cre mice. Likewise, Plzf aortic mRNA levels were also significantly increased(p=0.002). To exploit the translational potential of T cell-specific EZH2 targeting, we treated Jurkat T cells with EZH2 inhibitors, the FDA-approved drug Tazemetostat and GSK126. Corroborating our in vivo data, EZH2 inhibitor-treated cells showed decreased H3K27me3 and 70% reduced migration towards the chemokine CXCL12. Conclusions Our study demonstrates that CD4+ T-cell specific EZH2 deficiency impairs T cell-migration and skews the immune response towards a type 2 response, resulting in protection against atherosclerosis. We aim at investigating whether the epigenetic mark H3K27me3 in T cells may be a promising therapeutic target for future cardiovascular immunotherapies.

PURPOSE Hepatocellular carcinoma (HCC), the fourth most common cancer in Africa, has a dismal overall survival of only 3 months like in sub-Saharan Africa. This is affected by the low gross domestic product and human development index, absence of coherent guidelines, and other factors. METHODS An open forum for HCC-experienced health care workers from Africa and the rest of the world was held in October 2021. Participants completed a survey to help assess the real-life access to screening, diagnoses, and treatment in the North and Southern Africa (NS), East and West Africa (EW), Central Africa (C), and the rest of the world. RESULTS Of 461 participants from all relevant subspecialties, 372 were from Africa. Most African participants provided hepatitis B vaccination and treatment for hepatitis B and C. More than half of the participants use serum alpha-fetoprotein and ultrasound for surveillance. Only 20% reported using image-guided diagnostic liver biopsy. The Barcelona Clinic Liver Cancer is the most used staging system (52%). Liver transplant is available for only 28% of NS and 3% EW. C reported a significantly lower availability of resection. Availability of local therapy ranged from 94% in NS to 62% in C. Sorafenib is the most commonly used systemic therapy (66%). Only 12.9% reported access to other medications including immune checkpoint inhibitors. Besides 42% access to regorafenib in NS, second-line treatments were not provided. CONCLUSION Similarities and differences in the care for patients with HCC in Africa are reported. This reconfirms the major gaps in access and availability especially in C and marginally less so in EW. This is a call for concerted multidisciplinary efforts to achieve and sustain a reduction in incidence and mortality from HCC in Africa.

Novel targeted therapies (small molecule inhibitors, antibody–drug conjugates, and CD19-directed therapies) have changed the treatment landscape of relapsed/refractory B-cell lymphomas. Bruton’s tyrosine kinase (BTK) inhibitors continue to evolve in the management of mantle cell lymphoma (MCL), in both the relapsed/refractory and the frontline setting. Anti-CD19 CAR T-cell therapies are now effective and approved treatment options for relapsed/refractory follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and MCL. Bispecific T-cell engagers represent a novel immunotherapeutic approach for relapsed FL and DLBCL after multiple lines of therapies, including prior CAR T-cell therapy. These NCCN Guideline Insights highlight the significant updates to the NCCN Guidelines for B-Cell Lymphomas for the treatment of FL, DLBCL, and MCL.