Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein.

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAFV600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAFV600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC50 values in the 40-88 nM range. Selected compounds inhibited BRAFV600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.

Transcriptome-wide gene expression outlier analysis pinpoints therapeutic vulnerabilities in colorectal cancer.

Multiple strategies are continuously being explored to expand the drug target repertoire in solid tumors. We devised a novel computational workflow for transcriptome-wide gene expression outlier analysis that allows the systematic identification of both overexpression and underexpression events in cancer cells. Here, it was applied to expression values obtained through RNA sequencing in 226 colorectal cancer (CRC) cell lines that were also characterized by whole-exome sequencing and microarray-based DNA methylation profiling. We found cell models displaying an abnormally high or low expression level for 3533 and 965 genes, respectively. Gene expression abnormalities that have been previously associated with clinically relevant features of CRC cell lines were confirmed. Moreover, by integrating multi-omics data, we identified both genetic and epigenetic alternations underlying outlier expression values. Importantly, our atlas of CRC gene expression outliers can guide the discovery of novel drug targets and biomarkers. As a proof of concept, we found that CRC cell lines lacking expression of the MTAP gene are sensitive to treatment with a PRMT5-MTA inhibitor (MRTX1719). Finally, other tumor types may also benefit from this approach.

Modulating Tumor-Associated Macrophage Polarization by Synthetic and Natural PPARγ Ligands as a Potential Target in Breast Cancer.

Activation of peroxisome proliferator-activated receptor gamma (PPARγ) elicits anti-proliferative effects on different tumor cells, including those derived from breast cancer. PPARγ is also expressed in several cells of the breast tumor microenvironment, among which tumor associated macrophages (TAMs) play a pivotal role in tumor progression and metastasis. We explored the ability of synthetic and natural PPARγ ligands to modulate TAM polarization. The ligands included rosiglitazone (BRL-49653), and two docosahexaenoic acid (DHA) conjugates, N-docosahexaenoyl ethanolamine (DHEA) and N-docosahexaenoyl serotonin (DHA-5-HT). Human THP-1 monocytic cells were differentiated into M0, M1 and M2 macrophages that were characterized by qRT-PCR, ELISA and western blotting. A TAM-like phenotypic state was generated by adding two different breast cancer cell conditioned media (BCC-CM) to the cultures. Macrophages exposed to BCC-CM concomitantly exhibited M1 and M2 phenotypes. Interestingly, rosiglitazone, DHEA and DHA-5-HT attenuated cytokine secretion by TAMs, and this effect was reversed by the PPARγ antagonist GW9662. Given the key role played by PPARγ in the crosstalk between cancer cells and TAMs in tumor progression, its activation via endogenous or synthetic ligands may lead to novel strategies that target both epithelial neoplastic cells and the tumor microenvironment.

N-Eicosapentaenoyl Dopamine, A Conjugate of Dopamine and Eicosapentaenoic Acid (EPA), Exerts Anti-inflammatory Properties in Mouse and Human Macrophages.

A large body of evidence suggests that dietary n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), contribute to a reduced inflammatory tone thereby lowering the risk for several chronic and degenerative diseases. Different mechanisms have been proposed to explain these anti-inflammatory effects, including those involving endocannabinoids and endocannabinoid-like molecules. In this context, fatty acid amides (FAAs), conjugates of fatty acids with amines or amino acids, are an emerging class of compounds. Dopamine conjugates of DHA (N-docosahexaenoyl dopamine, DHDA) and EPA (N-eicosapentaenoyl dopamine, EPDA) have previously been shown to induce autophagy, apoptosis, and cell death in different tumor lines. Additionally, DHDA has displayed anti-inflammatory properties in vitro. Here, we tested the immune-modulatory properties of EPDA in mouse RAW 264.7 and human THP-1 macrophages stimulated with lipopolysaccharide (LPS). EPDA suppressed the production of monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in both cell lines, and nitric oxide (NO), and macrophage-inflammatory protein-3α (MIP3A) in RAW 264.7 macrophages. At a transcriptional level, EPDA attenuated cyclooxygenase-2 (COX-2) expression in both cell lines and that of MCP-1, IL-6, and interleukin-1ß (IL-1ß) in THP-1 macrophages. Although further research is needed to reveal whether EPDA is an endogenous metabolite, our data suggest that this EPA-derived conjugate possesses interesting immune-modulating properties.

Ligand-activated PPARγ downregulates CXCR4 gene expression through a novel identified PPAR response element and inhibits breast cancer progression.

Stromal Derived Factor-1α (SDF-1α) and its cognate receptor CXCR4 play a key role in mediating breast cancer cell invasion and metastasis. Therefore, drugs able to inhibit CXCR4 activation may add critical tools to reduce tumor progression, especially in the most aggressive form of the breast cancer disease. Peroxisome Proliferator-Activated Receptor (PPAR) γ, a member of the nuclear receptor superfamily, has been found to downregulate CXCR4 gene expression in different cancer cells, however the molecular mechanism underlying this effect is not fully understood. Here, we identified a novel PPARγ-mediated mechanism that negatively regulates CXCR4 expression in both epithelial and stromal breast cancer cells. We found that ligand-activated PPARγ downregulated CXCR4 transcriptional activity through the recruitment of the silencing mediator of retinoid and thyroid hormone receptor (SMRT) corepressor onto a newly identified PPAR response element (PPRE) within the CXCR4 promoter in breast cancer cell lines. As a consequence, the PPARγ agonist rosiglitazone (BRL) significantly inhibited cell migration and invasion and this effect was PPARγ-mediated, since it was reversed in the presence of the PPARγ antagonist GW9662. According to the ability of cancer-associated fibroblasts (CAFs), the most abundant component of breast cancer stroma, to secrete high levels of SDF-1α, BRL reduced migratory promoting activities induced by conditioned media (CM) derived from CAFs and affected CXCR4 downstream signaling pathways activated by CAF-CM. In addition, CAFs exposed to BRL showed a decreased expression of CXCR4, a reduced motility and invasion along with a phenotype characterized by an altered morphology. Collectively, our findings provide novel insights into the role of PPARγ in inhibiting breast cancer progression and further highlight the utility of PPARγ ligands for future therapies aimed at targeting both cancer and surrounding stromal cells in breast cancer patients.