Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Discovery of indazole-pyridinone derivatives as a novel class of potent and selective MNK1/2 kinase inhibitors that protecting against endotoxin-induced septic shock.

The mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNKs 1/2) and their downstream target eIF4E, play a role in oncogenic transformation, progression and metastasis. These results provided rationale for development of first MNKs inhibitors, currently in clinical trials for cancer treatment. Inhibitors of the MNKs/eIF4E pathway are also proposed as treatment strategy for inflammatory conditions. Here we present results of optimization of indazole-pyridinone derived MNK1/2 inhibitors among which compounds 24 and 26, selective and metabolically stable derivatives. Both compounds decreased levels of eIF4E Ser206 phosphorylation (pSer209-eIF4E) in MOLM16 cell line. When administered in mice compounds 24 and 26 significantly improved survival rates of animals in the endotoxin lethal dose challenge model, with concomitant reduction of proinflammatory cytokine levels - TNFα and IL-6 in serum. Identified MNK1/2 inhibitors represent a novel class of immunomodulatory compounds with a potential for the treatment of inflammatory diseases including sepsis.

Limited Practical Utility of Liquid Biopsy in the Treated Patients with Advanced Breast Cancer.

Recently, liquid biopsy has emerged as a tool to monitor oncologic disease progression and the effects of treatment. In this study we aimed to determine the clinical utility of liquid biopsy relative to conventional oncological post-treatment surveillance. Plasma cell-free (cf) DNA was collected from six healthy women and 37 patients with breast cancer (18 and 19 with stage III and IV tumors, respectively). CfDNA was assessed using the Oncomine Pan-Cancer Cell-Free Assay. In cfDNA samples from patients with BC, 1112 variants were identified, with only a few recurrent or hotspot mutations within specific regions of cancer genes. Of 65 potentially pathogenic variants detected in tumors, only 19 were also discovered in at least one blood sample. The allele frequencies of detected variants (VAFs) were <1% in cfDNA from all controls and patients with stage III BC, and 24/85 (28.2%) variants had VAFs > 1% in only 8 of 25 (32%) patients with stage IV BC. Copy number variations (CNVs) spanning CDK4, MET, FGFR1, FGFR2, ERBB2, MYC, and CCND3 were found in 1 of 12 (8%) and 8 of 25 (32%) patients with stage III and IV tumors, respectively. In healthy controls and patients without BC progression after treatment, VAFs were <1%, while in patients with metastatic disease and/or more advanced genomic alterations, VAFs > 1% and/or CNV were detected in approximately 30%. Therefore, most patients with stage IV BC could not be distinguished from those with stage III disease following therapy, based on liquid biopsy results.

Mutation Profiling of Premalignant Colorectal Neoplasia.

Accumulation of allelic variants in genes that regulate cellular proliferation, differentiation, and apoptosis may result in expansion of the aberrant intestinal epithelium, generating adenomas. Herein, we compared the mutation profiles of conventional colorectal adenomas (CNADs) across stages of progression towards early carcinoma. DNA was isolated from 17 invasive adenocarcinomas (ACs) and 58 large CNADs, including 19 with low-grade dysplasia (LGD), 21 with LGD adjacent to areas of high-grade dysplasia and/or carcinoma (LGD-H), and 28 with high-grade dysplasia (HGD). Ion AmpliSeq Comprehensive Cancer Panel libraries were prepared and sequenced on the Ion Proton. We identified 956 unique allelic variants; of these, 499 were considered nonsynonymous variants. Eleven genes (APC, KRAS, SYNE1, NOTCH4, BLNK, FBXW7, GNAS, KMT2D, TAF1L, TCF7L2, and TP53) were mutated in at least 15% of all samples. Out of frequently mutated genes, TP53 and BCL2 had a consistent trend in mutation prevalence towards malignancy, while two other genes (HNF1A and FBXW7) exhibited the opposite trend. HGD adenomas had significantly higher mutation rates than LGD adenomas, while LGD-H adenomas exhibited mutation frequencies similar to those of LGD adenomas. A significant increase in copy number variant frequency was observed from LGD through HGD to malignant samples. The profiling of advanced CNADs demonstrated variations in mutation patterns among colorectal premalignancies. Only limited numbers of genes were repeatedly mutated while the majority were altered in single cases. Most genetic alterations in adenomas can be considered early contributors to colorectal carcinogenesis.

Imatinib Inhibits the Renewal and Tumorigenicity of CT-26 Colon Cancer Cells after Cytoreductive Treatment with Doxorubicin.

Conventional anti-cancer drugs preferentially eliminate differentiated cancer cells but those cells that are spared (i.e. cancer stem cells: CSC), initiate recurrence. We tested whether drugs that target receptor tyrosine kinases (RTKs) involved in developmental signaling cascades and activated in CSC, could be used to silence and/or to eliminate colorectal cancer cells refractory to conventional treatment with cytoreductive drugs. A sequential treatment model was thereby developed with doxorubicin (DOX) and imatinib. CT-26 mouse colon carcinoma cells were pre-treated with DOX to select DOX-refractory cells with CSC properties, which were then subsequently treated with RTK inhibitor imatinib, where their regrowth was found to be inhibited. Under both normoxic and hypoxic conditions, imatinib potently inhibited clonogenicity of DOX-refractory CT-26 cells. Treatment with DOX did not eliminate tumorigenic CT-26 cells, since CT-26 cells pre-exposed to DOX in vitro, when inoculated subcutaneously, induced tumors in 90 % of mice, as opposed to a 100 % rate in the case of chemonaive CT-26 cells. In mice inoculated with chemonaive CT-26 cells, tumor formation was not prevented by imatinib. However, imatinib prevented tumor formation in 50 % of mice inoculated with CT-26 cells pre-exposed to DOX in vitro, with the remaining 50 % mice showing delayed tumor formation. These results suggest that the sequential use of the drug imatinib, as a drug targeting cancer cells expressing stem cell features after conventional cytoreductive treatment, is a promising future strategy for preventing tumor recurrence.

Chronic mild stress facilitates melanoma tumor growth in mouse lines selected for high and low stress-induced analgesia.

Both chronic stress conditions and hyperergic reaction to environmental stress are known to enhance cancer susceptibility. We described two mouse lines that displayed high (HA) and low (LA) swim stress-induced analgesia (SSIA) to investigate the relationship between inherited differences in sensitivity to stress and proneness to an increased growth rate of subcutaneously inoculated melanoma. These lines display several genetic and physiological differences, among which distinct sensitivity to mutagens and susceptibility to cancer are especially noticeable. High analgesic mice display high proneness both to stress and a rapid local spread of B16F0 melanoma. However, stress-resistant LA mice do not develop melanoma tumors after inoculation, or if so, tumors regress spontaneously. We found that the chronic mild stress (CMS) procedure leads to enhanced interlinear differences in melanoma susceptibility. Tumors developed faster in stress conditions in both lines. However, LA mice still displayed a tendency for spontaneous regression, and 50% of LA mice did not develop a tumor, even under stressed conditions. Moreover, we showed that chronic stress, but not tumor progression, induces depressive behavior, which may be an important clue in cancer therapy. Our results clearly indicate how the interaction between genetic susceptibility to stress and environmental stress determine the risk and progression of melanoma. To our knowledge, HA/LA mouse lines are the first animal models of distinct melanoma progression mediated by inherited differences in stress reactivity.

Soluble TNF-α receptor I encoded on plasmid vector and its application in experimental gene therapy of radiation-induced lung fibrosis.

Post-radiation inflammatory reaction leads to an irreversible pulmonary fibrosis which may cause lethal respiratory insufficiency. Pathological inflammatory and fibrotic changes might be attenuated by inhibiting tumour necrosis factor (TNF)-α activity using TNF-α soluble receptors. Thus, an experimental antifibrotic gene therapy with the plasmid vector encoding a mouse soluble receptor I for TNF-α (psTNFR-I) was assessed. Soluble TNFR-I encoding gene was cloned into pcDNA3.1 plasmid. The ability of psTNFR-I expressing vector to transfect cells, and its biological activity in vitro and in vivo were examined by PCR, RT-PCR, MTT assay and ELISA. The C57Bl/6J mice received single intramuscular injection of psTNFR-I, conjugated with polyetylenimine (PEI) 25 kDa, equally divided to both hind legs, 3 days before irradiation (20 Gy, Co60), and either a single injection or ten injections once a week after irradiation. The data proved the effectiveness of psTNFR-I product to neutralise TNF-α activity in vitro. The in vivo plasmid incorporation and maintenance was confirmed. Measurements of plasma soluble TNFR-I levels showed that the in vivo gene transfer was effective. PEI was found to enhance transfection efficiency in vivo. The psTNFR-I/PEI complexes caused no toxicity in the transfected mice. C57Bl/6J mice that received prolonged psTNFR-I/PEI injections developed lethal fibrotic syndrome and died 8 weeks later than the mice treated with a double plasmid injection and the control mice treated with a control plasmid. Sequential administration of soluble TNFR-I by a nonviral, intramuscular gene transduction in the early and late post-radiation inflammatory phase prolonged survival of irradiated mice and attenuated the symptoms of lung fibrosis. The psTNFR-I gene transduction may provide a safe and simple method to partially neutralise TNF-α activity and prevent radiation-induced lung injury.

Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development.

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts' outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions. To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney. We conclude that somatic cells (FFs) can proliferate in early embryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.

Opposite effects of modifiers of the ApcMin mutation in intestine and mammary gland.

Patterns of tumor susceptibility in different organs are widely divergent in mouse strains: one strain may be highly susceptible to tumors in one organ but resistant in another organ, whereas another strain may exhibit the opposite pattern (P. Demant, Semin. Cancer Biol., 3: 159-166, 1992). Therefore, susceptibility to tumors in different organs is assumed to be controlled by different sets of genes. On the other hand, many oncogenes and tumor suppressor genes are mutated in tumors from different organs, indicating that similar tumorigenic pathways operate in various tissues. To obtain insight into the interactions of susceptibility genes with one of such pathways, we compared tumorigenesis in intestine and mammary gland in recombinant congenic strains (RCSs) carrying the Apc(Min) mutation, affecting the Wnt pathway. The presence of Apc(Min) increased considerably the incidence of intestinal and mammary tumors. The individual RCSs differed in the number and latency of Apc(Min)-induced intestinal and mammary tumors and histological type of the latter. Unexpectedly, the strain distribution of susceptibility to the intestinal and mammary tumors in the Apc(Min)-bearing mice was opposite in the RCSs; the strains most susceptible for intestinal tumors were most resistant to mammary tumors and vice versa. This suggests that a set of genes controls the impact of the Apc(Min) mutation in both organs but with opposite effects. Elucidation of the basis of the observed strain differences in organ-specific Wnt pathway-mediated tumorigenesis will help to understand the interactions between germ-line encoded allelic differences in susceptibility genes and the spectrum of somatic mutations in tumor cells.