Roche (Estonia)

Hepatocellular carcinomas (HCCs) constitute one of the few cancer indications for which mortality rates continue to rise. While Notch signaling dictates a key progenitor lineage choice during development, its role in HCC has remained controversial. Using therapeutic antibodies targeting Notch ligands and receptors to screen over 40 patient-derived xenograft models, we here identify progenitor-like HCCs that crucially depend on a tumor-intrinsic JAG1-NOTCH2 signal. Inhibiting this signal induces tumor regressions by triggering progenitor-to-hepatocyte differentiation, the same cell fate-switch that Notch controls during development. Transcriptomic analysis places the responsive tumors within the well-characterized progenitor subclass, a poor prognostic group of highly proliferative tumors, providing a diagnostic method to enrich for Notch-dependent HCCs. Furthermore, single-cell RNA sequencing uncovers a heterogeneous population of tumor cells and reveals how Notch inhibition shifts cells from a mixed cholangiocyte-hepatocyte lineage to one resembling mature hepatocytes. Analyzing the underlying transcriptional programs brings molecular detail to this process by showing that Notch inhibition de-represses expression of CEBPA, which enables the activity of HNF4α, a hepatocyte lineage factor that is otherwise quiescent. We thus describe a compelling and targetable dependency in a poor-prognosis class of HCCs.

Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly in the context of therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates and are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments. Our approach is based on the fast and scaled accessibility of chemically modified shortmer monophosphates as substrates for the T3 DNA ligase. This method has shown high tolerance to chemical modifications, flexibility and overall efficiency, thereby granting access to an ultimately broad range of modified oligonucleotides of different lengths (20 →160 nucleotides). We have applied this method to the synthesis of clinically relevant antisense drugs and highly modified ultramers. Furthermore, the designed chemoenzymatic approach has great potential in numerous applications in oligonucleotide therapeutics, bioorganic chemistry, pharmacology, and chemical biology.

Abstract The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, the neuroprotective and anti-inflammatory effects of the ECS have been studied, and inhibiting the degradation of the endocannabinoid 2-arachydonoylglycerol (2-AG) is emerging as a promising strategy to counteract brain damage in MS. In this study, a systemic and preventive in vivo treatment with MAGLi 432, the reversible inhibitor of monoacylglycerol lipase (MAGLi), was performed in experimental autoimmune encephalomyelitis (EAE) mice. Clinical, biochemical, electrophysiological and immunofluorescence analyses were carried out to evaluate the impact of the drug on motor disability, neuroinflammation and synaptic damage. MAGLi 432 induced a less severe EAE disease, accompanied by an increase of 2-AG and a reduction of acid arachidonic (AA) and prostaglandins (PGs) brain levels. MAGLi 432 reversed the striatal synaptic hyperexcitability promoted by EAE neuroinflammatory insult. Furthermore, MAGLi 432 reduced both astrogliosis and microgliosis in EAE, as microglia/macrophages density was reduced, in parallel with a less reactive morphology. By flow cytometry analysis, we also observed less infiltrating CD45 + and CD3 + cells in the brain of MAGLi 432-treated EAE mice. In conclusion, in vivo MAGL inhibition promotes a significant amelioration of EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects, including an enhanced 2-AG tone and reduced PGs levels, gliosis, and lymphocyte infiltration. Overall, these data provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and reveal the therapeutic potential of MAGLi-based drugs in MS inflammatory and neurodegenerative brain damage.

Abstract Background : Intraneuronal accumulation of a-synuclein (αSyn) is key in Parkinson’s disease (PD) pathogenesis. The pathogenic process is suggested to begin in the enteric nervous system decades before diagnosis of PD and then propagate into the brain. The triggers for these events are unclear but, in some patients, colitis might play a critical role. Methods : We administered lipopolysaccharide (LPS) or dextran sulfate sodium (DSS) to assess the effect of different types of experimental colitis on αSyn accumulation in the gut of αSyn transgenic and wild type mice and quantified local gene expression by RT-PCR and level of αSyn accumulation by immunofluorescence imaging. Immune modulation during the DSS colitis paradigm in the αSyn transgenic mice included genetic ablation of Cx3cr1 or treatment with recombinant IL-10. To determine long-term effects of experimental colitis, we induced DSS colitis in young αSyn transgenic mice and aged them under normal conditions up to nine or 21 months before analyzing their brains by immunohistochemistry. In vivo experiments were performed in randomized cohorts. Blinded experimenters performed image analysis and statistical analysis depended on data type (i.e., Student’s t-test, ANOVA, mixed-effects model). Results : We demonstrate that mild sustained or one strong insult of experimental DSS colitis triggers αSyn accumulation in the submucosal plexus of wild type and αSyn transgenic mice, while short-term mild DSS experimental colitis or inflammation induced by LPS does not have such an effect. Lack of macrophage-related Cx3cr1-signalling during DSS colitis increases accumulation of αSyn in the colonic submucosal plexus of αSyn transgenic mice while systemic treatment with immune-dampening IL-10 ameliorates this phenomenon. Additionally, DSS colitis-induced αSyn accumulation in young αSyn transgenic mice persists for months and is exacerbated by lack of Cx3cr1-signaling. Remarkably, experimental colitis at three months of age exacerbates the accumulation of aggregated phospho-Serine 129 αSyn in the midbrain (including the substantia nigra), in 21- but not 9-month-old αSyn transgenic mice. This increase in midbrain αSyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons. Conclusions : Our data suggest that specific types of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of PD.

Abstract Background The HER2 extracellular domain shed in blood (HER2 ECD ) is reported to rise and fall in parallel with HER2+ breast cancer behavior. In this study, we evaluated the clinical relevance of plasma HER2 ECD values in patients with metastatic breast cancer treated in the SAKK22/99 trial comparing trastuzumab monotherapy followed by trastuzumab-chemotherapy combination at progression versus upfront combination therapy. Methods Quantitative assessment of plasma HER2 ECD was performed in 133 patients at baseline; after 2-24 hours; at 3 weeks; at first response evaluation (8-9 weeks); and at tumor progression. Associations with tumor characteristics, disease course and trial treatment were evaluated. Results Baseline HER2 ECD levels were stable within 24 hours after the first trastuzumab injection. These plasma values correlated positively with the HER2 gene ratio (r s =0.39, P <0.001) and HER2 protein expression levels (r s =0.36, P <0.001) but not with ER/PR status of the primary tumor. HER2 ECD baseline levels were positively associated with the presence of visceral disease ( P =0.05) and poor patients’ outcome (Cox-regression: P =0.009). Patients with high baseline levels ( > 35ng/ml) had the worst overall survival ( P =0.03) if treated with upfront combination therapy. Conversely, patients with low HER2 ECD baseline values (<15ng/ml) had longer time to progression on combined trastuzumab-chemotherapy when first treated with trastuzumab monotherapy ( P =0.02). Monitoring HER2 ECD levels during the course of the trial revealed significant time ( P =0.001) and time-treatment arm interactions ( P =0.0007). Under upfront trastuzumab alone, the HER2 ECD levels remained stable until just before disease progression. In patients responding to combination treatment HER2 ECD levels decreased to > 20%. Conclusions Plasma HER2 ECD levels in patients with metastatic breast cancer reflect HER2 disease status. This robust biomarker might help identifying patients without visceral disease profiting from a sequential treatment’s modality. Monitoring HER2 ECD levels during trastuzumab monotherapy could help defining the optimal time to introduce chemotherapy.

Abstract Background The HER2 extracellular domain shed in blood (HER2 ECD ) is reported to rise and fall in parallel with HER2+ breast cancer behavior. In this study, we evaluated the clinical relevance of plasma HER2 ECD values in patients with metastatic breast cancer treated in the SAKK22/99 trial comparing trastuzumab monotherapy followed by trastuzumab-chemotherapy combination at progression versus upfront combination therapy. Methods Quantitative assessment of plasma HER2 ECD was performed in 133 patients at baseline; after 2-24 hours; at 3 weeks; at first response evaluation (8-9 weeks); and at tumor progression. Associations with tumor characteristics, disease course and trial treatment were evaluated. Results Baseline HER2 ECD levels were stable within 24 hours after the first trastuzumab injection. These plasma values correlated positively with the HER2 gene ratio (r s =0.39, P <0.001) and HER2 protein expression levels (r s =0.36, P <0.001) but not with ER/PR status of the primary tumor. HER2 ECD baseline levels were positively associated with the presence of visceral disease ( P =0.05) and poor patients’ outcome (Cox-regression: P =0.009). Patients with high baseline levels ( > 35ng/ml) had the worst overall survival ( P =0.03) if treated with upfront combination therapy. Conversely, patients with low HER2 ECD baseline values (<15ng/ml) had longer time to progression on combined trastuzumab-chemotherapy when first treated with trastuzumab monotherapy ( P =0.02). Monitoring HER2 ECD levels during the course of the trial revealed significant time ( P =0.001) and time-treatment arm interactions ( P =0.0007). Under upfront trastuzumab alone, the HER2 ECD levels remained stable until just before disease progression. In patients responding to combination treatment HER2 ECD levels decreased to > 20%. Conclusions Plasma HER2 ECD levels in patients with metastatic breast cancer reflect HER2 disease status. This robust biomarker might help identifying patients without visceral disease profiting from a sequential treatment’s modality. Monitoring HER2 ECD levels during trastuzumab monotherapy could help defining the optimal time to introduce chemotherapy.