Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

The dl1102 mutant prolongs time to progression in combination with docetaxel in PC-3 xenografts <i>in vivo.</i>

<p>A) Animals with PC-3 subcutaneous tumor xenografts were treated with the <i>dl</i>1102 (filled triangle) or <i>dl</i>1104 (filled circle) mutants or mock treated with <i>dl</i>312 (filled square) at 1×10⁹ vp (i.t. injections on day 1, 3, and 5) with and without docetaxel at 10 mg/kg (D10; i.p. administration on day 2 and 8, open squares), and tumor growth was monitored. *p<0.05, treatments compared with mock and single-agent treatments (one-way ANOVA), p<0.05 for <i>dl</i>1102 alone compared to mock, n = 6. B). In a second study animals with PC-3 subcutaneous tumor xenografts were treated as above with the indicated suboptimal doses of mutants at 1×10⁹ vp and docetaxel at 10 mg/kg (D10) or the respective combinations. Median time to tumor progression (tumor volume >500 µl) was determined by Kaplan-Meier survival analysis (8–10 animals per group). *p<0.05, combination-treated compared with docetaxel. C) PC-3 cells infected with the indicated mutants and treated with docetaxel at four constant ratios; 0.5, 2.5, 12.5 and 62.5 ppc/nM drug (indicated by the wedges). CI values were calculated from isobolograms and CI≤0.9 were considered synergistic, averages ±SEM, n = 3, *p<0.05 vs the theoretical additive values (0.9</p

Potent cell killing of prostate cancer cell lines by replicating E1A-deletion mutants in combination with mitoxantrone.

<p>A) The replicating viruses used in the study had intact E1A-region (E1A13S) except for the indicated deletions. The replication-defective mutants were based on the E1A12S construct with the same deletions as in the replicating viruses; AdE1A1102 (Δ26–35), AdE1A1104 (Δ48–60), AdE1A1108 (Δ124–127), in addition to deletion of the CR3-region, responsible for viral transcriptional activity. B) EC₅₀ values for the replicating mutants were determined from dose-response curves and presented as averages ± SD, n = 3. Significantly different values compared to Ad5 are indicated. C) Sensitization of the human PC-3, 22Rv1 and DU145 cells to mitoxantrone by fixed doses of each virus at EC₁₀ and EC₂₅. Data presented as percentages of mitoxantrone EC₅₀ values in each cell line, averages ± SD, n = 3. Statistical analysis by 1-way Anova, *p<0.05 for drug EC₅₀ values that were significantly lower than the corresponding Ad5 values. The <i>dl</i>312 (ΔE1A) non-replicating virus served as negative control. D) Graphic representation of combination indexes (CI) generated from synergy studies with mitoxantrone in combination with each replicating viral mutant at two constant ratios 0.5 and 2.5 viral particles per cell (ppc)/nM drug. Synergistic interactions are represented by CI≤0.9, antagonism by CI≥1.1 and additive effects by 0.9</p

All replication-defective E1A12S mutants sensitise prostate cancer cells to mitoxantrone and docetaxel except the AdE1A1104 virus.

<p>A) Drug dose responses in each cell line were evaluated after infection with AdE1A12S, AdE1A1102, AdE1A1104, and AdE1A1108 mutants with AdGFP as negative control to determine changes in drug EC₅₀ values. All cell lines were infected at doses killing <10% of cells alone; PC-3 cells at 100 ppc (left panel), DU145 cells at 10 ppc (mid panel) and 22Rv1 cells at 2.5 ppc (right panel). Data represent averages ±SD, n = 4–5 independent experiments analysed by t-test comparing EC₅₀ values for each combination to that of drug alone, expressed as percentages, *p<0.05 and °p<0.01. B) EC₅₀ values for mitoxantrone were determined with and without simultaneous infection with viral mutants at 2.5, 10 and 100 ppc for 22Rv1, DU145 and PC-3 respectively, and with (grey bar) and without (black bar) the addition of the pan-caspase inhibitor zVAD-fmk at 25 µM. EC₅₀ values are expressed as percentages of mitoxantrone alone (Ctrl), averages ± SD, n = 3. C) Flow cytometry of cells infected with the AdE1A12S mutants or treated with mitoxantrone (50 nM) alone and in combination and analysed for tetramethylrhodamine uptake (TMRE) as an indicator of mitochondrial depolarisation and apoptosis induction. AdE1A12S, AdE1A1102, AdE1A1104, AdE1A1108, and AdGFP alone (solid arrow; all cell lines) and AdE1A12S, AdE1A1102, AdE1A1104 and AdE1A1108 in combination with mitoxantrone (dashed arrow; 22Rv1 cells). Data expressed as % apoptotic cells; percentages of cells that showed mitochondrial depolarisation, averages ± SD, n = 3. D) Cells infected with each mutant at 100 (PC-3) or 2.5 ppc (22Rv), mock infected and treated with or without mitoxantrone at 50 nM. Changes in cell cycle were analysed by flow cytometry at 24, 48 and 72 h after infection and drug treatment in PC-3 cells or after 48 h for 22Rv1 cells, one representative study (n = 3), *p<0.05 comparing G2/M-phase in combination treated vs mitoxantrone alone, °p<0.05 G2/M-phase for mitoxantrone vs mock treated.</p

EC₅₀ values for mitoxantrone and docetaxel in prostate cancer cells transfected with E1A12S or GFP expressing plasmids.

<p>Data from one representative experiment treated with mitoxantrone for 3 days after transfection with pcDNA plasmids expressing the respective proteins, n = 3.</p

Synergistic cell killing with a replication-defective virus expressing the small AdE1A12S protein, in combination with cytotoxic drugs.

<p>A) Isobolograms generated from EC₅₀ values for combinations of the AdE1A12S mutant with mitoxantrone (Mit) or docetaxel (Doc) at four constant ratios (0.5. 2.5, 12.5 and 62.5 ppc/nM drug) in PC-3 and DU145 cells. The straight lines represent the theoretical values for additive effects and points below the line synergistic cell killing, one representative study (n = 3–4). B) Characterization of replication of the AdE1A12S, AdE1A1102, AdE1A1108 and AdE1A1104 mutants in PC-3, DU145 and 22Rv1 cells. Levels of viral replication determined by the limiting dilution assay (TCID₅₀) for replicating and replication-defective mutants with identical E1A-deletions except for the additional deletion of the CR3-domain in E1A12S. Cells were infected with each mutant at 100 ppc and harvested 72 h later, averages ±SD, n≥3. The non-replicating AdGFP mutant was used as a control in all assays, *p<0.001 for the replicating compared to the corresponding replication-defective mutant (t-test). C) qPCR analysis of cells infected as described for the replication assays and harvested 24, 48 and 72 h later. Total copy number at each time point was normalised to the copy numbers detected 3 h after infection in 10 ng of total DNA, averages ± SEM, n = 2–3. D) Viral replication in normal human primary bronchial epithelial cells (NHBE) determined by TCID₅₀ for Ad5wt, <i>dl</i>1102 and <i>dl</i>1104 mutants infected at 100 ppc, n = 3, *p<0.005.</p

EC₅₀ values (ppc) for the replication-defective AdE1A12S, AdE1A1102, AdE1A1104 and AdE1A1108 viral mutants and wild type virus (Ad5).

<p>Data are averages ± SD, n = 3, p<0.001 for all mutants vs Ad5 (t-test). The non-replicating <i>dl</i>312 and AdGFP control viruses had EC₅₀ values >1×10⁵ ppc.</p

Immunoglobulin, glucocorticoid, or combination therapy for multisystem inflammatory syndrome in children: a propensity-weighted cohort study

Background: Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory condition associated with SARS-CoV-2 infection, has emerged as a serious illness in children worldwide. Immunoglobulin or glucocorticoids, or both, are currently recommended treatments. Methods: The Best Available Treatment Study evaluated immunomodulatory treatments for MIS-C in an international observational cohort. Analysis of the first 614 patients was previously reported. In this propensity-weighted cohort study, clinical and outcome data from children with suspected or proven MIS-C were collected onto a web-based Research Electronic Data Capture database. After excluding neonates and incomplete or duplicate records, inverse probability weighting was used to compare primary treatments with intravenous immunoglobulin, intravenous immunoglobulin plus glucocorticoids, or glucocorticoids alone, using intravenous immunoglobulin as the reference treatment. Primary outcomes were a composite of inotropic or ventilator support from the second day after treatment initiation, or death, and time to improvement on an ordinal clinical severity scale. Secondary outcomes included treatment escalation, clinical deterioration, fever, and coronary artery aneurysm occurrence and resolution. This study is registered with the ISRCTN registry, ISRCTN69546370. Findings: We enrolled 2101 children (aged 0 months to 19 years) with clinically diagnosed MIS-C from 39 countries between June 14, 2020, and April 25, 2022, and, following exclusions, 2009 patients were included for analysis (median age 8·0 years [IQR 4·2–11·4], 1191 [59·3%] male and 818 [40·7%] female, and 825 [41·1%] White). 680 (33·8%) patients received primary treatment with intravenous immunoglobulin, 698 (34·7%) with intravenous immunoglobulin plus glucocorticoids, 487 (24·2%) with glucocorticoids alone; 59 (2·9%) patients received other combinations, including biologicals, and 85 (4·2%) patients received no immunomodulators. There were no significant differences between treatments for primary outcomes for the 1586 patients with complete baseline and outcome data that were considered for primary analysis. Adjusted odds ratios for ventilation, inotropic support, or death were 1·09 (95% CI 0·75–1·58; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids and 0·93 (0·58–1·47; corrected p value=1·00) for glucocorticoids alone, versus intravenous immunoglobulin alone. Adjusted average hazard ratios for time to improvement were 1·04 (95% CI 0·91–1·20; corrected p value=1·00) for intravenous immunoglobulin plus glucocorticoids, and 0·84 (0·70–1·00; corrected p value=0·22) for glucocorticoids alone, versus intravenous immunoglobulin alone. Treatment escalation was less frequent for intravenous immunoglobulin plus glucocorticoids (OR 0·15 [95% CI 0·11–0·20]; p<0·0001) and glucocorticoids alone (0·68 [0·50–0·93]; p=0·014) versus intravenous immunoglobulin alone. Persistent fever (from day 2 onward) was less common with intravenous immunoglobulin plus glucocorticoids compared with either intravenous immunoglobulin alone (OR 0·50 [95% CI 0·38–0·67]; p<0·0001) or glucocorticoids alone (0·63 [0·45–0·88]; p=0·0058). Coronary artery aneurysm occurrence and resolution did not differ significantly between treatment groups. Interpretation: Recovery rates, including occurrence and resolution of coronary artery aneurysms, were similar for primary treatment with intravenous immunoglobulin when compared to glucocorticoids or intravenous immunoglobulin plus glucocorticoids. Initial treatment with glucocorticoids appears to be a safe alternative to immunoglobulin or combined therapy, and might be advantageous in view of the cost and limited availability of intravenous immunoglobulin in many countries. Funding: Imperial College London, the European Union's Horizon 2020, Wellcome Trust, the Medical Research Foundation, UK National Institute for Health and Care Research, and National Institutes of Health