The STAT3 inhibitor pyrimethamine displays anti-cancer and immune stimulatory effects in murine models of breast cancer.

The transcription factor signal activator and transducer or transcription (STAT3), which regulates genes controlling proliferation, survival, and invasion, is activated inappropriately in many human cancers, including breast cancer. Activation of STAT3 can lead to both malignant cellular behavior and suppression of immune cell function in the tumor microenvironment. Through a chemical-biology screen, pyrimethamine (PYR), an FDA approved anti-microbial drug, was identified as an inhibitor of STAT3 function at concentrations known to be achieved safely in humans. We report that PYR shows therapeutic activity in two independent mouse models of breast cancer, with both direct tumor inhibitory and immune stimulatory effects. PYR-inhibited STAT3 activity in TUBO and TM40D-MB metastatic breast cancer cells in vitro and inhibited tumor cell proliferation and invasion into Matrigel basement membrane matrix. In tumor-transplanted mice, PYR had both direct and indirect tumor inhibitory effects. Tumor-bearing mice treated with PYR showed reduced STAT3 activation in tumor cells, attenuated tumor growth, and reduced tumor-associated inflammation. In addition, expression of Lamp1 by tumor infiltrating CD8+ T cells was elevated, indicating enhanced release of cytotoxic granules. These findings suggest that PYR may have beneficial effects in the treatment of breast cancer.

Gene expression-based discovery of atovaquone as a STAT3 inhibitor and anticancer agent.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated inappropriately in a wide range of hematological and solid cancers, but clinically available therapies targeting STAT3 are lacking. Using a computational strategy to identify compounds opposing the gene expression signature of STAT3, we discovered atovaquone (Mepron), an antimicrobial approved by the US Food and Drug Administration, to be a potent STAT3 inhibitor. We show that, at drug concentrations routinely achieved clinically in human plasma, atovaquone inhibits STAT3 phosphorylation, the expression of STAT3 target genes, and the viability of STAT3-dependent hematological cancer cells. These effects were also observed with atovaquone treatment of primary blasts isolated from patients with acute myelogenous leukemia or acute lymphocytic leukemia. Atovaquone is not a kinase inhibitor but instead rapidly and specifically downregulates cell-surface expression of glycoprotein 130, which is required for STAT3 activation in multiple contexts. The administration of oral atovaquone to mice inhibited tumor growth and prolonged survival in a murine model of multiple myeloma. Finally, in patients with acute myelogenous leukemia treated with hematopoietic stem cell transplantation, extended use of atovaquone for Pneumocystis prophylaxis was associated with improved relapse-free survival. These findings establish atovaquone as a novel, clinically accessible STAT3 inhibitor with evidence of anticancer efficacy in both animal models and humans.

Mutant KRAS Downregulates the Receptor for Leukemia Inhibitory Factor (LIF) to Enhance a Signature of Glycolysis in Pancreatic Cancer and Lung Cancer.

Pancreatic cancer is characterized by aberrant activity of oncogenic KRAS, which is mutated in 90% of pancreatic adenocarcinomas. Because KRAS itself is a challenging therapeutic target, we focused on understanding key signaling pathways driven by KRAS as a way to reveal dependencies that are amenable to therapeutic intervention. Analyses in primary human pancreatic cancers and model systems revealed that the receptor for the cytokine leukemia inhibitory factor (LIF) is downregulated by mutant KRAS. Furthermore, downregulation of the LIF receptor (LIFR) is necessary for KRAS-mediated neoplastic transformation. We found LIFR exerts inhibitory effects on KRAS-mediated transformation by inhibiting expression of the glucose transporter GLUT1, a key mediator of the enhanced glycolysis found in KRAS-driven malignancies. Decreased LIFR expression leads to increased GLUT1 as well as increases in glycolysis and mitochondrial respiration. The repression of GLUT1 by LIFR is mediated by the transcription factor STAT3, indicating a tumor-suppressive role for STAT3 within cancer cells with mutated KRAS. Finally, reflecting a clinically important tumor-suppressive role of LIFR, decreased LIFR expression correlates with shorter survival in pancreatic cancer patients with mutated KRAS. Similar findings were found in non-small cell lung cancers driven by mutated KRAS, suggesting that silencing LIFR is a generalized mechanism of KRAS-mediated cellular transformation. These results indicate that the LIFR/STAT3 pathway may mediate either tumor-promoting or tumor-suppressive signaling pathways depending on the genetic background of tumor cells, and may play diverse roles within other cells in the tumor microenvironment. IMPLICATIONS: Mutant KRAS drives downregulation of the receptor for LIF, thereby allowing an increase in expression of the glucose transporter GLUT1 and increases in glycolysis and mitochondrial respiration.

IL-6 Mediates Cross-Talk between Tumor Cells and Activated Fibroblasts in the Tumor Microenvironment.

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR.

Coordination of the third step of protein splicing in two cyanobacterial inteins.

The third step of protein splicing is cyclization of Asn coupled to peptide bond cleavage. In two related cyanobacterial inteins, this step is facilitated by Asn or Gln. For a Synechococcus sp. PCC7002 intein, the isolated third step of protein splicing is more efficient with its native Asn than with substitution to Gln. For a Trichodesmium erythraeum intein, its native Gln facilitates the third step as efficiently as with Asn. Despite these differences, the yield of splicing is not affected, suggesting that the third step is influenced by mechanism-linked conformational changes. A conserved catalytic His and the penultimate residue also play roles in promoting side-chain cyclization.