MAPK/ERK Activation in Macrophages Promotes Leishmania Internalization and Pathogenesis.

The obligate intracellular parasite Leishmania binds several receptors to trigger uptake by phagocytic cells, ultimately resulting in visceral or cutaneous leishmaniasis. A series of signaling pathways in host cells, which are critical for establishment and persistence of infection, are activated during Leishmania internalization. Thus, preventing Leishmania uptake by phagocytes could be a novel therapeutic strategy for leishmaniasis. However, the host cellular machinery mediating promastigote and amastigote uptake is not well understood. Here, using small molecule inhibitors of Mitogen-activated protein/Extracellular signal regulated kinases (MAPK/ERK), we demonstrate that ERK1/2 mediates Leishmania amazonensis uptake and (to a lesser extent) phagocytosis of beads by macrophages. We find that inhibiting host MEK1/2 or ERK1/2 leads to inefficient amastigote uptake. Moreover, using inhibitors and primary macrophages lacking spleen tyrosine kinase (SYK) or Abl family kinases, we show that SYK and Abl family kinases mediate Raf, MEK, and ERK1/2 activity and are necessary for uptake. Finally, we demonstrate that trametinib, a MEK1/2 inhibitor used to treat cancer, reduces disease severity and parasite burden in Leishmania-infected mice, even if it is started after lesions develop. Our results show that maximal Leishmania infection requires MAPK/ERK and highlight potential for MAPK/ERK-mediated signaling pathways to be novel therapeutic targets for leishmaniasis.

Src- and Abl-family kinases activate spleen tyrosine kinase to maximize phagocytosis and Leishmania infection.

Leishmania spp. are obligate intracellular parasites that must be internalized by phagocytic cells to evade immune responses and cause disease. The uptake of both Leishmania promastigotes (insect-stage parasites) and amastigotes (proliferative-stage parasites in humans and mice) by phagocytes is thought to be mainly host cell driven, not parasite driven. Our previous work indicates that host Src- and Abl-family kinases facilitate Leishmania entry into macrophages and pathogenesis in murine cutaneous leishmaniasis. Here, we demonstrate that host spleen tyrosine kinase (SYK) is required for efficient uptake of Leishmania promastigotes and amastigotes. A Src-family kinase-Abl-family kinase-SYK signaling cascade induces Leishmania amastigote internalization. Finally, lesion size and parasite burden during Leishmania infection is significantly decreased in mice lacking SYK in monocytes or by treatment with the SYK inhibitor entospletinib. In summary, SYK is required for maximal Leishmania uptake by macrophages and disease in mice. Our results suggest potential for treating leishmaniasis using host cell-directed agents.

Tumoral CD105 promotes immunosuppression, metastasis, and angiogenesis in renal cell carcinoma.

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4+ and CD8+ T cells. Finally, while CD105 expression by endothelial cells is a well-established contributor to tumor angiogenesis, we also find that tumor cell-expressed CD105 significantly contributes to tumor angiogenesis in RCC.

Modulation of protein S and growth arrest specific 6 protein signaling inhibits pancreatic cancer cell survival and proliferation.

Thrombotic complications and hypercoagulopathies are commonly associated with the progression of pancreatic ductal adenocarcinoma (PDAC). Although the mechanistic link between the two phenomena is uncertain, there is evidently an increase in procoagulant proteins and a decrease in anticoagulants in PDAC patients. For example, the anticoagulant protein S (PS) is decreased during the progression of PDAC, a condition that possibly contributes to the hypercoagulopathies. PS is also an important signaling molecule that binds a family of tyrosine kinase receptors known as TAM (Tyro3, Axl and Mer) receptors; TAM receptors are often upregulated in different cancers. Growth Arrest Specific 6 or GAS6 protein, a homolog of PS, is also a TAM receptor family ligand. The downstream signaling pathways triggered by this ligand­receptor interaction perform diverse functions, such as cell survival, proliferation, efferocytosis, and apoptosis. Targeting the TAM receptors to treat cancer has had limited success; side effects are a significant obstacle due to the widespread numerous functions of TAM receptors. In the present study, it was revealed that PS­TAM interaction was pro­apoptotic, whereas GAS6­mediated TAM signaling promoted proliferation and survival in select PDAC cell lines. Furthermore, by regulating the balance between these two signaling pathways (by overexpressing PS or knocking down GAS6), the proliferative potential of the cells was decreased. Both long­term and short­term effects of natural PS overexpression were comparable to the treatment of the cells with the drug UNC2025, which inhibits the Mer­receptor. The present study lays the foundation for investigation of PS as a therapeutic agent to control cancer progression and to concurrently arrest thrombotic events.

Padua FIXa resistance to Protein S and a potential therapy for hyperactive FIXa.

INTRODUCTION: Abnormalities in the levels and functions of proteins that maintain hemostasis can cause thrombosis. Factor IX (FIX) R338L, i.e., Factor IX Padua, is a hyperactive clotting factor that promotes thrombosis. The R338L mutation increases the clotting rate by 8-fold despite increasing the Factor IXa enzymatic activity by only 2-fold. Protein S (PS) is a natural anticoagulant that directly inhibits FIXa. Because individuals affected by the R338L mutation have normal concentrations of PS, we speculated that the Padua hypercoagulation phenotype is due to decreased inhibition of FIXa R338L by PS. METHODS: We measured the ability of PS to inhibit FIX R338L, and we assessed the ability of PS to mitigate the prothrombotic effect FIX R338L. RESULTS: Plasma clotting assays demonstrated that 3-fold more PS was required to inhibit FIXa R338L compared with inhibition of wild type FIXa. Thrombin generation assays with Padua patient plasma recapitulated this biochemical consequence of the R338L mutation. Importantly, the less efficient inhibition of FIXa R338L was reversed by increasing PS concentration. Binding and co-immunoprecipitation studies revealed that the decrease in the inhibition of FIXa R338L by PS was caused by a 3- to 4-fold reduction in FIXa R338L affinity for PS. CONCLUSION: In summary, the resistance of FIXa R338L to inhibition by PS likely contributes to the unexpectedly high clotting rate in Padua individuals. Moreover, PS-mediated reversal of the pathological properties of FIXa R338L suggests that PS administration may be a novel and effective means to mitigate thrombophilia caused by any source of elevated FIXa activity.

miR-17 deregulates a core RUNX1-miRNA mechanism of CBF acute myeloid leukemia.

BACKGROUND: Core Binding Factor acute myeloid leukemia (CBF-AML) with t(8;21) RUNX1-MTG8 or inv(16) CBFB-MYH11 fusion proteins often show upregulation of wild type or mutated KIT receptor. However, also non-CBF-AML frequently displays upregulated KIT expression. In the first part of this study we show that KIT expression can be also upregulated by miR-17, a regulator of RUNX1, the gene encoding a CBF subunit. Interestingly, both CBF leukemia fusion proteins and miR-17, which targets RUNX1-3'UTR, negatively affect a common core RUNX1-miRNA mechanism that forces myeloid cells into an undifferentiated, KIT-induced, proliferating state. In the second part of this study we took advantage of the conservation of the core RUNX1-miRNA mechanism in mouse and human, to mechanistically demonstrate in a mouse myeloid cell model that increased KIT-induced proliferation is per se a mechanism sufficient to delay myeloid differentiation. METHODS: Human (U937) or mouse (32D) myeloid clonal lines were used, respectively, to test: 1) the effect of RUNX1-MTG8 and CBFB-MYH11 fusion proteins, or upregulation of miR-17, on KIT-induced proliferation and myeloid differentiation, and 2) the effect of upregulation of KIT-induced proliferation per se on myeloid cell differentiation. RESULTS: In the first part of this study we found that stable miR-17 upregulation affects, like the CBF-AML fusion proteins (RUNX1-MTG8 or CBFB-MYH11), a core RUNX1-miRNA mechanism leading to KIT-induced proliferation of differentiation-arrested U937 myeloid cells. In the second part of the study we harnessed the conservation of this core mechanism in human and mouse to demonstrate that the extent of KIT upregulation in 32D mouse myeloid cells with wild type RUNX1 can per se delay G-CSF-induced differentiation. The integrated information gathered from the two myeloid cell models shows that RUNX1 regulates myeloid differentiation not only by direct transcriptional regulation of coding and non-coding myeloid differentiation functions (e.g. miR-223), but also by modulating KIT-induced proliferation via non-coding miRNAs (e.g. miR-221). CONCLUSIONS: The novelty of this study is dual. On the one hand, miRNAs (e.g. miR-17) can mimic the effects of CBF-AML fusion proteins by affecting a core RUNX1-miRNA mechanism of KIT-induced proliferation of undifferentiated myeloid cells. On the other hand, the extent of KIT-induced proliferation itself can modulate myeloid differentiation of cells with wild type RUNX1 function.