We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.
Polo-like kinase 3 (Plk3) is an important mediator of the cellular responses to genotoxic stresses. In this study, we examined the physiologic function of Plk3 by generating Plk3-deficient mice. Plk3 À/À mice displayed an increase in weight and developed tumors in various organs at advanced age. Many tumors in Plk3 À/À mice were large in size, exhibiting enhanced angiogenesis. Plk3 À/À mouse embryonic fibroblasts were hypersensitive to the induction of hypoxiainducible factor-1A (HIF-1A) under hypoxic conditions or by nickel and cobalt ion treatments. Ectopic expression of the Plk3-kinase domain (Plk3-KD), but not its Polo-box domain or a Plk3-KD mutant, suppressed the nuclear accumulation of HIF-1A induced by nickel or cobalt ions. Moreover, hypoxiainduced HIF-1A expression was tightly associated with a significant down-regulation of Plk3 expression in HeLa cells. Given the importance of HIF-1A in mediating the activation of the ''survival machinery'' in cancer cells, these studies strongly suggest that enhanced tumorigenesis in Plk3-null mice is at least partially mediated by a deregulated HIF-1 pathway.
Objective The recurrence rate of anti-SSA/Ro associated congenital heart block (CHB) is 17%. Reversal of 3rd degree block has never been achieved. Based on potential reduction of maternal autoantibody titers as well as fetal inflammatory responses, IVIG was evaluated as a preventative therapy for CHB. Methods A multicenter open-label study based on Simon’s 2-stage optimal design was initiated. Enrollment criteria included: maternal anti-SSA/Ro antibody, a previous child with CHB/rash, = 20 mg prednisone, < 12 weeks pregnant. IVIG (400mg/kg) was given every 3 weeks from 12 to 24 weeks of gestation. The primary outcome was the development of 2nd or 3rd degree CHB. Results Twenty mothers completed the IVIG protocol before reaching the pre-determined stopping rule of three cases of advanced CHB. CHB was detected at 19, 20 and 25 weeks; none followed an abnormal PR interval. One of these mothers had two previous children with CHB. One child without CHB developed a transient rash consistent with neonatal lupus. Sixteen children had no manifestations of neonatal lupus at birth. No significant changes in maternal antibody titers to SSA/Ro, SSB/La, or Ro52 were detected over the course of therapy or at delivery. There were no safety issues. Conclusions IVIG at doses consistent with replacement does not prevent the recurrence of CHB or reduce maternal antibody titers. Having established safety with this protocol and feasibility of patient enrollment, subsequent preventative studies may be considered, perhaps to include higher doses of IVIG.
Activation of TLR by ssRNA after FcγR-mediated phagocytosis of immune complexes (IC) may be relevant in autoimmune-associated congenital heart block (CHB) where the obligate factor is a maternal anti-SSA/Ro Ab and the fetal factors, protein/RNA on an apoptotic cardiocyte and infiltrating macrophages. This study addressed the hypothesis that Ro60-associated ssRNAs link macrophage activation to fibrosis via TLR engagement. Both macrophage transfection with noncoding ssRNA that bind Ro60 and an IC generated by incubation of Ro60-ssRNA with an IgG fraction from a CHB mother or affinity purified anti-Ro60 significantly increased TNF-α secretion, an effect not observed using control RNAs or normal IgG. Dependence on TLR was supported by the significant inhibition of TNF-α release by IRS661 and chloroquine. The requirement for FcγRIIIa-mediated delivery was provided by inhibition with an anti-CD16a Ab. Fibrosis markers were noticeably increased in fetal cardiac fibroblasts after incubation with supernatants generated from macrophages transfected with ssRNA or incubated with the IC. Supernatants generated from macrophages with ssRNA in the presence of IRS661 or chloroquine did not cause fibrosis. In a CHB heart, but not a healthy heart, TLR7 immunostaining was localized to a region near the atrioventricular groove at a site enriched in mononuclear cells and fibrosis. These data support a novel injury model in CHB, whereby endogenous ligand, Ro60-associated ssRNA, forges a nexus between TLR ligation and fibrosis instigated by binding of anti-Ro Abs to the target protein likely accessible via apoptosis.
Gastric cancers are the most frequent gastric malignancy and usually arise in the sequence of Helicobacter pylori-associated chronic gastritis. CpG methylation is a central mechanism of epigenetic gene regulation affecting cancer-related genes, and occurs early in gastric carcinogenesis. DNA samples from non-metaplastic gastric mucosa with variable levels of gastritis (non-metaplastic mucosa), intestinal metaplasia, or gastric cancer were screened with methylation arrays for CpG methylation of cancer-related genes and 30 gene targets were further characterized by high-definition bisulfite next-generation sequencing. In addition, data from The Cancer Genome Atlas were analyzed for correlation of methylation with gene expression. Overall, 13 genes had significantly increased CpG methylation in gastric cancer vs non-metaplastic mucosa (BRINP1, CDH11, CHFR, EPHA5, EPHA7, FGF2, FLI1, GALR1, HS3ST2, PDGFRA, SEZ6L, SGCE, and SNRPN). Further, most of these genes had corresponding reduced expression levels in gastric cancer compared with intestinal metaplasia, including novel hypermethylated genes in gastric cancer (FLI1, GALR1, SGCE, and SNRPN), suggesting that they may regulate neoplastic transformation from non-malignant intestinal metaplasia to cancer. Our data suggest a tumor-suppressor role for FLI1 in gastric cancer, consistent with recently reported data in breast cancer. For the genes with strongest methylation/expression correlation, namely FLI1, the expression was lowest in microsatellite-unstable tumors compared with other gastric cancer molecular subtypes. Importantly, reduced expression of hypermethylated BRINP1 and SGCE was significantly associated with favorable survival in gastric cancer. In summary, we report novel methylation gene targets that may have functional roles in discrete stages of gastric carcinogenesis and may serve as biomarkers for diagnosis and prognosis of gastric cancer.
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