APR-246 potently inhibits tumour growth and overcomes chemoresistance in preclinical models of oesophageal adenocarcinoma.

OBJECTIVES: p53 is a critical tumour suppressor and is mutated in 70% of oesophageal adenocarcinomas (OACs), resulting in chemoresistance and poor survival. APR-246 is a first-in-class reactivator of mutant p53 and is currently in clinical trials. In this study, we characterised the activity of APR-246 and its effect on p53 signalling in a large panel of cell line xenograft (CLX) and patient-derived xenograft (PDX) models of OAC. DESIGN: In vitro response to APR-246 was assessed using clonogenic survival, cell cycle and apoptosis assays. Ectopic expression, gene knockdown and CRISPR/Cas9-mediated knockout studies of mutant p53 were performed to investigate p53-dependent drug effects. p53 signalling was examined using quantitative RT-PCR and western blot. Synergistic interactions between APR-246 and conventional chemotherapies were evaluated in vitro and in vivo using CLX and PDX models. RESULTS: APR-246 upregulated p53 target genes, inhibited clonogenic survival and induced cell cycle arrest as well as apoptosis in OAC cells harbouring p53 mutations. Sensitivity to APR-246 correlated with cellular levels of mutant p53 protein. Ectopic expression of mutant p53 sensitised p53-null cells to APR-246, while p53 gene knockdown and knockout diminished drug activity. Importantly, APR-246 synergistically enhanced the inhibitory effects of cisplatin and 5-fluorouracil through p53 accumulation. Finally, APR-246 demonstrated potent antitumour activity in CLX and PDX models, and restored chemosensitivity to a cisplatin/5-fluorouracil-resistant xenograft model. CONCLUSIONS: APR-246 has significant antitumour activity in OAC. Given that APR-246 is safe at therapeutic levels our study strongly suggests that APR-246 can be translated into improving the clinical outcomes for OAC patients.

Selective targeting BMP2 and 4 in SMAD4 negative esophageal adenocarcinoma inhibits tumor growth and aggressiveness in preclinical models.

PURPOSE: Abnormalities within the Sonic Hedgehog (SHH), Bone Morphogenetic Protein (BMP) and SMAD4 signalling pathways have been associated with the malignant behavior of esophageal adenocarcinoma (EAC). We recently developed two specific llama-derived antibodies (VHHs), C4C4 and C8C8, which target BMP4 and BMP2/4, respectively. Here we aimed to demonstrate the feasibility of the VHHs for the treatment of EAC and to elucidate its underlying mechanism. METHODS: Gene Set Enrichment Analysis (GSEA) was performed on a TCGA dataset, while expression of SHH, BMP2/4 and SMAD4 was validated in a cohort of EAC patients. The effects of the VHHs were tested on the recently established SMAD4(-) ISO76A primary EAC cell line and its counterpart SMAD4(+) ISO76A. In a patient-derived xenograft (PDX) model, the VHHs were evaluated for their ability to selectively target tumor cells and for their effects on tumor growth and survival. RESULTS: High expression of BMP2/4 was detected in all SMAD4 negative EACs. SHH upregulated BMP2/4 expression and induced p38 MAPK signaling in the SMAD4(-) ISO76A cells. Inhibition of BMP2/4 by VHHs decreased the aggressive and chemo-resistant phenotype of the SMAD4(-) ISO76A but not of the SMAD4(+) ISO76A cells. In the PDX model, in vivo imaging indicated that VHHs effectively targeted tumor cells. Both VHHs significantly inhibited tumor growth and acted synergistically with cisplatin. Furthermore, we found that C8C8 significantly improved survival of the mice. CONCLUSIONS: Our data indicate that increased BMP2/4 expression triggers aggressive non-canonical BMP signaling in SMAD4 negative EAC. Inhibiting BMP2/4 decreases malignant behavior and improves survival. Therefore, VHHs directed against BMP2/4 hold promise for the treatment of SMAD4 negative EAC.

Lamina-specific changes in hippocampal GABA(A)/cBZR and mossy fibre sprouting during and following amygdala kindling in the rat.

Alterations in hippocampal GABA(A)/central benzodiazepine receptor (GABA(A)/cBZR) expression and mossy fibre sprouting (MFS) may have aetiological significance in temporal lobe epileptogenesis. Their relationship with each other is also unknown. We utilised [3H]-flumazenil autoradiography to quantify changes in GABA(A)/cBZR density and affinity in all hippocampal laminae, and Timm's staining for MFS at different stages of epileptogenesis in the amygdala kindling rat model (after 24 stimulations, 48 stimulations and two weeks post-kindling). During kindling, receptor density was significantly elevated within the dentate stratum moleculare and granulosum, but decreased within the stratum radiatum of CA3 and CA2. Two weeks post-kindling, receptor density remained upregulated in the dentate stratum moleculare and was also upregulated in CA3 stratum oriens and CA1 stratum moleculare. MFS was significantly increased in the dentate stratum moleculare at two weeks post-kindling, with a strong inverse correlation between MFS and GABA(A)/cBZR density in this region. No changes in GABA(A)/cBZR binding affinity were detected for any hippocampal subregion at any time point. Our results demonstrate that changes in hippocampal GABA(A)/cBZR expression are lamina- and time-specific. Within the dentate gyrus, receptor density is upregulated throughout epileptogenesis, whilst within the hippocampus proper, receptor density is downregulated early in epileptogenesis but upregulated at the chronic phase. A novel association between MFS and GABA(A)/cBZR density has been demonstrated by this study, which could represent an important compensatory or pathological mechanism associated with epileptogenesis.

Medication chart intervention improves inpatient thromboembolism prophylaxis.

BACKGROUND: Inpatient VTE prophylaxis is underused. This study evaluated the effectiveness of the low-cost, multifaceted Australian National Inpatient Medication Chart (NIMC) intervention on improving the quality of VTE prophylaxis and reducing disease. The NIMC intervention incorporated (1) a VTE risk stratification and appropriate prophylaxis guidance tool, (2) a prophylaxis contraindication screening instrument, and (3) a prophylaxis prescription prompt. METHODS: Retrospective analysis of 2,371 consecutive medical and surgical admissions was performed at a regional referral hospital over 1 year both before and after the intervention. Outcomes measured included the frequency of prophylaxis use, timing of prophylaxis initiation, adherence of the prescribed prophylaxis regimen to guidelines, incidence of VTE disease, and prophylaxis-related complications. RESULTS: Following NIMC intervention, prophylaxis use increased from 52.7% to 66.5% in medical patients and from 77.5% to 89.1% in surgical patients (P < .001). This increase was still evident 12 months postintervention. After intervention, prophylaxis initiated on admission increased from 65.0% to 83.6% in medical patients and from 60.7% to 78.0% in surgical patients (P < .01); adherence rates to recommended guidelines increased from 55.6% to 71.0% in medical patients and from 53.6% to 75.6% in surgical patients (P < .01). More VTE risk factors independently triggered prophylaxis usage postintervention. The improved quality of prophylaxis did not significantly reduce VTE incidence (risk ratio, 0.88; 95% CI, 0.48-1.62). The rate of prophylaxis-related complications remained similar before and after intervention. CONCLUSIONS: The multifaceted NIMC intervention resulted in a sustained increase in appropriate and timely VTE prophylaxis in medical and surgical inpatients.