Antiangiogenic and cytotoxic effects are considered the principal mechanisms of action of sorafenib, a multitarget kinase inhibitor approved for the treatment of hepatocellular carcinoma (HCC). We report that sorafenib also acts through direct immune modulation, indispensable for its antitumor activity. In vivo cell depletion experiments in two orthotopic HCC mouse models as well as in vitro analysis identified macrophages (MΦ) as the key mediators of the antitumoral effect and demonstrate a strong interdependency of MΦ and natural killer (NK) cells for efficient tumor cell killing. Caspase 1 analysis in sorafenib-treated MΦ revealed an induction of pyroptosis. As a result, cytotoxic NK cells become activated when cocultured with sorafenib-treated MΦ, leading to tumor cell death. In addition, sorafenib was found to down-regulate major histocompatibility complex class I expression of tumor cells, which may reduce the tumor responsiveness to immune checkpoint therapies and favor NK-cell response. In vivo cytokine blocking revealed that sorafenib efficacy is abrogated after inhibition of interleukins 1B and 18. Conclusion:We report an immunomodulatory mechanism of sorafenib involving MΦ pyroptosis and unleashing of an NK-cell response that sets it apart from other spectrum kinase inhibitors as a promising immunotherapy combination partner for the treatment of HCC. (Hepatology 2019;70:1280-1297). L iver cancer is the second leading cause of cancer-related deaths worldwide.(1) Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer, with increasing incidence and dismal prognosis. Hepatitis B and C viral infection, alcoholism, as well as nonalcoholic steatohepatitis are predominant risk factors for HCC development. (2) Sorafenib, a broad-spectrum kinase inhibitor, has been approved since 2007 for treating patients with unresectable HCC. (3) This adenosine triphosphate-competitive kinase inhibitor targets B-Raf, C-Raf, mitogen-activated protein (MAP) kinases, vascular endothelial growth factor (VEGF) receptor, and platelet-derived growth factor

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αβ heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in Plasmodium and MIC2 in Toxoplasma. Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.

During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma-membrane spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here we show that different mutations in profilin, not affecting actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow-down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.

Transmission of the malaria parasite Plasmodium to mosquitoes necessitates gamete egress from red blood cells to allow zygote formation and ookinete motility to enable penetration of the midgut epithelium. Both processes are dependent on the secretion of proteins from distinct sets of specialized vesicles. Inhibiting some of these proteins has shown potential for blocking parasite transmission to the mosquito. To identify new transmission blocking vaccine candidates, we defined the microneme content from ookinetes of the rodent model organism Plasmodium berghei using APEX2-mediated rapid proximity-dependent biotinylation. Besides known proteins of ookinete micronemes, this identified over 50 novel candidates and sharpened the list of a previous survey based on subcellular fractionation. Functional analysis of a first candidate uncovered a dual role for this membrane protein in male gametogenesis and ookinete midgut traversal. Mutation of a putative trafficking motif in the C-terminus led to its mis-localization in ookinetes and affected ookinete to oocyst transition but not gamete formation. This suggests the existence of distinct functional and transport requirements for Plasmodium proteins in different parasite stages.SignificanceThe genome of the malaria parasite Plasmodium contains over 5500 genes, of which over 30% have no assigned function. Transmission of Plasmodium spp. to the mosquito contains several essential steps that can be inhibited by antibodies or chemical compounds. Yet few proteins involved in these processes are characterized, thus limiting our capacity to generate transmission interfering tools. Here, we establish a method to rapidly identify proteins in a specific compartment within the parasite that is essential for establishment of an infection within the mosquito, and identify over 50 novel candidate proteins. Functional analysis of the top candidate identifies a protein with two independent essential functions in subsequent steps along the Plasmodium life cycle within the mosquito.Highlightsfirst use of APEX based proximity ligation in Apicomplexaidentification of >50 putative ookinete surface proteinsnovel membrane protein essential for microgamete egress and ookinete migrationputative trafficking motif essential in ookinetes but not gametes