Dabrafenib plus trametinib in unselected advanced BRAF V600-mut melanoma: a non-interventional, multicenter, prospective trial.

OBJECTIVE: The efficacy of combined BRAF and MEK inhibition for BRAF V600-mutant melanoma in a broad patient population, including subgroups excluded from phase 3 trials, remains unanswered. This noninterventional study (DATUM-NIS) assessed the real-world efficacy, safety and tolerability of dabrafenib plus trametinib in Austrian patients with unresectable/metastatic melanoma. METHODS: This multicenter, open-label, non-interventional, post-approval, observational study investigated the effectiveness of dabrafenib plus trametinib prescribed in day-to-day clinical practice to patients ( N  = 79) with BRAF V600-mutant unresectable/metastatic melanoma with M1c disease (American Joint Committee on Cancer staging manual version 7), ECOG > 1, and elevated serum lactate dehydrogenase (LDH). The primary endpoint was 6-, 12- and 18-month progression-free survival (PFS) rates. Secondary endpoints were median PFS, disease control rate and overall survival (OS). RESULTS: The 6-, 12- and 18-month PFS rates were 76%, 30.6% and 16.2%, respectively. Subgroup analysis showed a significant PFS benefit in the absence of lung metastasis. The median PFS and OS were 9.1 (95% CI, 7.1-10.3) months and 17.9 (95% CI, 12.7-27.8) months, respectively. The 12- and 24-month OS rates were 62.7% and 26.8%, respectively. Subgroup analyses showed significant OS benefits in the absence of bone or lung metastasis and the presence of other metastases (excluding bone, lung, brain, liver and lymph nodes). Furthermore, S100 and Eastern Cooperative Oncology Group performance status (ECOG PS) showed a significant impact on survival. No new safety signals were observed. CONCLUSION: Despite an unselected population of melanoma patients with higher M1c disease, ECOG PS > 1 and elevated LDH, this real-world study demonstrated comparable efficacy and safety with the pivotal phase 3 clinical trials for dabrafenib-trametinib.

Real-World Evidence Data on Metastatic Renal-Cell Carcinoma Treatment in Austria: The RELACS Study.

BACKGROUND: Treatment decisions in routine clinical practice are based on reports of clinical trials, which represent highly selected populations. Limited studies reported real-world evidences representing routine clinical practices in patients with renal-cell carcinoma (RCC) in Europe. The aim of this retrospective, noninterventional chart review was to collect data on the treatment landscape for patients with advanced/metastatic RCC in routine clinical practice in a broader patient population in Austria. PATIENTS AND METHODS: Patients with advanced/metastatic RCC receiving systemic treatment between June 2010 and June 2016 across 12 centers in Austria were included. Parameters were entered into an electronic case report form from the participating sites via the application Hermesoft electronic data capture system. Progression-free survival (PFS) and overall survival (OS) were the 2 primary end points. RESULTS: The median PFS and OS were 12 months and 44 months, respectively (first-line PFS was 14 months for pazopanib and 13 months for sunitinib; first-line OS was 44 months for pazopanib and 48 months for sunitinib). Factors influencing the OS were sex, with female patients at a significantly higher risk than male patients (hazard ratio = 1.719), Eastern Cooperative Oncology Group performance status > 0 increased the risk twice (hazard ratio = 2.048), and number of metastases > 3 before the first line doubled the risk compared to metastases (hazard ratio = 2.064). CONCLUSION: OS in this retrospective chart review was considerably longer than the previous reports in real-world patients, underlining the benefit of current RCC treatment options in routine clinical practice.

The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection.

Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections.

T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.

TNF Counterbalances the Emergence of M2 Tumor Macrophages.

Cancer can involve non-resolving, persistent inflammation where varying numbers of tumor-associated macrophages (TAMs) infiltrate and adopt different activation states between anti-tumor M1 and pro-tumor M2 phenotypes. Here, we resolve a cascade causing differential macrophage phenotypes in the tumor microenvironment. Reduction in TNF mRNA production or loss of type I TNF receptor signaling resulted in a striking pattern of enhanced M2 mRNA expression. M2 gene expression was driven in part by IL-13 from eosinophils co-recruited with inflammatory monocytes, a pathway that was suppressed by TNF. Our data define regulatory nodes within the tumor microenvironment that balance M1 and M2 populations. Our results show macrophage polarization in cancer is dynamic and dependent on the balance between TNF and IL-13, thus providing a strategy for manipulating TAMs.

Tristetraprolin Limits Inflammatory Cytokine Production in Tumor-Associated Macrophages in an mRNA Decay-Independent Manner.

Tristetraprolin (TTP) is an inducible zinc finger AU-rich RNA-binding protein essential for enforcing degradation of mRNAs encoding inflammatory chemokines and cytokines. Most studies on TTP center on the connection between mRNA half-life and inflammatory output, because loss of TTP amplifies inflammation by increasing the stability of AU-rich mRNAs. Here, we focused on how TTP controls cytokine and chemokine production in the nonresolving inflammation of cancer using tissue-specific approaches. In contrast with model in vitro macrophage systems, we found constitutive TTP expression in late-stage tumor-associated macrophages (TAM). However, TTP's effects on AU-rich mRNA stability were negligible and limited by constitutive p38α MAPK activity, which was the main driver of proinflammatory cytokine production in TAMs at the posttranscriptional level. Instead, elimination of TTP caused excessive protein production of inflammatory mediators, suggesting TTP-dependent translational suppression of AU-rich mRNAs. Manipulation of the p38α-TTP axis in macrophages has significant effects on the growth of tumors and therefore represents a means to manipulate inflammation in the tumor microenvironment.

Macrophages and cancer: from mechanisms to therapeutic implications.

Infiltration by immune cells is a hallmark of most forms of malignancy. In this context, tumor-associated macrophages (TAMs) represent key regulators of the complex interplay between the immune system and cancer. We discuss evidence indicating that in many settings TAMs fuel, rather than limit, tumor progression, and negatively impact on responses to therapy. We discuss how the unique functional properties of TAMs are shaped by tumor-derived signals, placing TAM development in the context of the broader understanding of the cellular and molecular mechanisms controlling macrophage origin, differentiation, and maintenance in tissues. Finally, we provide examples of how a molecular understanding of the relationships between TAMs and the tumor microenvironment may lead to improved cancer therapies.

Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation.

For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.

Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.

Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs.

AREsite: a database for the comprehensive investigation of AU-rich elements.

AREsite is an online resource for the detailed investigation of AU-rich elements (ARE) in vertebrate mRNA 3'-untranslated regions (UTRs). AREs are one of the most prominent cis-acting regulatory elements found in 3'-UTRs of mRNAs. Various ARE-binding proteins that possess RNA stabilizing or destabilizing functions are recruited by sequence-specific motifs. Recent findings suggest an essential role of the structural mRNA context in which these sequence motifs are embedded. AREsite is the first database that allows to quantify the structuredness of ARE motif sites in terms of opening energies and accessibility probabilities. Moreover, we also provide a detailed phylogenetic analysis of ARE motifs and incorporate information about experimentally validated targets of the ARE-binding proteins TTP, HuR and Auf1. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite.

Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10.

IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls proinflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is TNF-alpha, a potent proinflammatory mediator that is the target for multiple anti-TNF-alpha clinical strategies in Crohn's disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. In this study, we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNF-alpha and IL-1alpha in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3' untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38 MAPK activity. The reduction of p38 MAPK activity releases TTP from the p38 MAPK-mediated inhibition, thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.

Regulation of matrilysin expression in endothelium by fibroblast growth factor-2.

Matrilysin (MMP7) is a secreted matrix metalloproteinase, which contributes to angiogenesis by breaking down basement membranes. We show that the angiogenic factor FGF-2 induces MMP7 expression in human endothelial cells. The promoter contains a Lef/Tcf consensus sequence, but using wildtype or Lef/Tcf-mutated promoter constructs, FGF-2-induced MMP7 reporter activity is independent from Lef/Tcf sites. Instead, we show that overexpression of a dominant negative Stat3 mutant reduces FGF-2-mediated MMP7 promoter activity. However, Stat3 does not bind to the MMP7 promoter, but activates MMP7 gene expression indirectly via AP-1. This is confirmed by MMP7 promoter constructs with mutated AP-1 sites which did not respond to FGF-2 and by siRNAs against Stat1 and Stat3, which repressed FGF-2-induced MMP7 protein expression. In conclusion, we show that FGF-2-induced MMP7 expression in endothelium depends on AP-1 and FGF-2 signaling to AP-1 involves a Stat1/3-dependent pathway.