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

Animal cell shape changes are controlled by the actomyosin cortex, a peripheral actin network tethered to the plasma membrane by membrane-to-cortex attachment (MCA) proteins. Previous studies have focused on how myosin motors or actin turnover can generate the local deformations required for morphogenesis. However, how the cell controls local actin nucleation remains poorly understood. By combining molecular engineering with biophysical approaches and in situ characterization of cortical actin network architecture, we show that membrane-to-cortex tethering determines the distance between the plasma membrane and the actomyosin cortex at the nanoscale of single actin nucleators. In turn, the size of this gap dictates actin filament production and the mechanical properties of the cell surface. Specifically, it tunes formin activity, controlling actin bundling and cortical tension. Our study defines the membrane-to-cortex distance as a nanogate that cells can open or close by MCA proteins to control the activity of key molecules at the cell surface.

Cell-cell and cell-substrate adhesion is critical for many functions in life. In eukaryotes, I-domains mediate functions as divergent as tissue traversal by malaria-causing Plasmodium parasites as well as cell adhesion and migration by human leucocytes. The Idomain containing protein TRAP is important for Plasmodium sporozoite motility and invasion. Here we show that the I-domain of TRAP is required to mediate adhesional properties which can be partially preserved when the native I-domain is replaced by Idomains from human integrins or from an apicomplexan parasite that does not infect insects.By putting in vivo data and structural features in perspective we conclude that polyspecificity and positive charge around the ligand binding site of the I-domain are important for TRAP function. Our data suggest a highly preserved functionality of I-domains across eukaryotic evolution that is used by apicomplexan parasites to invade a broad range of tissues in a variety of hosts. the domain (Figure 1A compared to 1B and Figure 1D compared to 1E). The distance pistoned is equivalent to two turns of an α-helix. Uniquely in the apicomplexan I-domains, a segment N-terminal to the I-domain is disulfide linked to the last helical turn of the α7-helix in its closed conformation (Figure 1A-C). As this segment pistons out of contact with the remainder of the I-domain in the open conformation, the last two turns of the α7-helix with its cysteine unwind, the N-terminal segment with its cysteine moves in a similar direction, and these segments reshape to form a β-ribbon (Figure 1A). Because of close structural homology between human integrin αI-and apicomplexan adhesin I-domains in the regions shown in green in Figure 1A-E, we were able to engineer exchanges between them in this study. P. berghei parasites expressing TRAP without an I-domain show the I-domain is essential for motility and invasion. Parasites expressing TRAP with I-domains from other organisms including humans can have nearly intact motility and invasion. However, a poly-specific, cationic integrin αI-domain functions much better than a more specific, anionic integrin αIdomain. Together with altering the charge of the native TRAP adhesin, our results suggest that cationic surface charge around the MIDAS motif can enable polyspecificity and permit TRAP to function as an adhesin with diverse ligands in both vertebrate and arthropod hosts.

Introduction Immunotherapy is a promising treatment strategy for hepatocellular carcinoma (HCC). A phase III trial for advanced HCC shows favorable results for nivolumab compared to sorafenib. In order to evaluate the outcome of different therapeutic strategies, we compared two HCC mouse models. The orthotopic transplanted HCC fragment Hep-55.1c model and the inducible HCC mouse model (iAST) were extensively characterized ex vivo and in vivo in response to immunotherapies, and were compared to the clinical situation of HCC patients. Experimental Procedures Fragments of mouse Hep-55.1c tumors were implanted in the left lateral liver lobe of C57/Bl6 mice and tumor growth kinetic was monitored by µ-CT. HCC tumor growth in iAST mice was induced via intravenous injection of the adenovirus expressing Cre recombinase. iAST mice express a loxP flanked stop cassette and the SV40 large T-antigen under control of a hepatocyte-specific albumin promoter. Both tumor models were characterized with respect to immune infiltration, cytokine release, somatic mutational load and histopathological characteristics. Mice were treated using different immunotherapeutic agents in monotherapy or in combination such as anti-PD-1, anti-CTLA-4 or the TLR7/8 agonist R848. Human HCC samples were analyzed for mutational load by sequencing, while tumor architecture and immune infiltrate were analyzed by histology. Results Hep-55.1c tumors showed high stroma content accompanied by a low and disorganized vasculature whereas multinodular iAST tumors were highly vascularized lacking stroma content. Furthermore, a high mutational load and a strong immune cell infiltrate including cytotoxic T cells, NK cells and myeloid cells were found in Hep-55.1c tumors. The iAST tumors were characterized by a relatively low immune infiltrate and a small number of mutations. Comparison of these baseline results with data obtained from immune-histopathological analysis and sequencing of human HCC samples confirmed this differentiated picture in the clinics. Following treatment, iAST mice showed no response to immune checkpoint monotherapies and only a marginal number of reactive T cells was found within the tumor. In contrast, tyrosine kinase inhibitor sorafenib led to tumor growth inhibition in iAST model. Treatment of mice bearing Hep-55.1c tumors with immunotherapy e.g. anti-PD-1 showed good response. Combination of anti-PD-1 with R848 increased therapeutic efficacy compared to monotherapies. Conclusions In this study, we have established two orthotopic HCC mouse models that reflect the diverse clinical situation of human HCC. Our findings demonstrate that the composition of the tumor microenvironment has a tremendous influence on the outcome of therapeutic strategies for HCC. Hence, thorough characterization of tumor patients is indispensable in order to predict response to immunotherapy. Citation Format: Carina Hage, Sabine Hoves, Mailin Ashoff, Leanne Strauss, Mario Perro, Frank Herting, Fabian Kiessling, Thomas Pöschinger. Identifying characteristics of orthotopic HCC mouse models to predict response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4084.