Supplemental Methods and Figure Legends

Abstract Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAFV600E has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAFV600E also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway–driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting. Cancer Immunol Res; 3(6); 602–9. ©2015 AACR.

We present a new method to synthesize hydrogel particles by exploiting the interface formed between two immiscible liquids. Spherical monomer droplets, of varying diameters, could be suspended at the planar interface formed between two immiscible liquids. While suspended, polymerization could proceed, after which the hydrogel particles that were formed could be collected. Using this approach, we were able to synthesize particles containing various monomers/co-monomers including: N-isopropylacrylamide, 2-hydroxyethyl methacrylate, and N-(3-aminopropyl)methacrylamide hydrochloride. The approach also allowed for the facile encapsulation of various inorganic nanoparticles and small molecules, including: Au nanoparticles, Ag nanoparticles, magnetic cobalt (Co) nanoparticles, tris(4-(dimethylamino)phenyl)methylium chloride (crystal violet) and fluorescein isothiocyanate isomer I (FITC). The benefit of this approach is the ability to load polymer particles with a wide variety of moieties without the need to optimize any reaction conditions. So long as the species to be encapsulated in the particle are soluble in water, and minimally soluble in the solvents used to form the interface, they will be incorporated in the polymerized particle.

Abstract 2406 The death receptor Fas has a key role in mediating homeostasis, elimination of defective cells and more recently promotion of cancer. Many effective anti-cancer therapies depend on Fas-mediated apoptosis to eradicate tumor cells and ineffective Fas- apoptotic signaling is a basis for primary as well as acquired resistance to chemotherapy. We hypothesized that Fas is subjected to direct regulation and inhibition of Fas attained by cancer cells and may explain the emergence of chemoresistance. To screen for potential binding modulators of Fas, we analyzed lymphoma cells for Fas binding proteins. We first purified Fas associated proteins by using activating CH-11 antibody bound to intact BJAB cells. After immunoprecipitation, any remaining Fas, considered activation–resistant, was subjected to the second immunoprecipitation with Fas antibody B-10 followed by liquid chromatography and tandem mass spectroscopy. This purification scheme identified high scoring peptides derived from nucleolin, a nuclear protein known to be overexpressed in cancer. Nucleolin is selectively expressed on the surfaces of cancer cells and blood vessels undergoing angiogenesis. In a cell culture system, we confirmed binding of nucleolin to Fas and the presence of nucleolin-Fas complexes on the surface of lymphoma cells by surface biotin labeling. Using deletion mutants of nucleolin, we identified RNA binding domain 4 and glycine/arginine rich region to be required for the binding to Fas. BJAB cells with partially knockdown (KD) nucleolin showed significantly higher rates of apoptosis in response to stimulation with CH-11 and FasL when compared to nontarget KD controls. Importantly, the lower levels of nucleolin in knockdown cells did not affect total and surface Fas expression. Nucleolin present on the cell surface prevented binding of FasL and CH-11 to the receptor and thus provides a mechanism for blocking activation of Fas apoptosis. To examine the role of nucleon in vivo, we transfected mice with nucleolin-expressing plasmids using the hydrodynamic transfection method. The mice overexpressing nucleolin showed significantly higher survival rates than vector control transfected mice (P=.01) after a challenge with a lethal dose of agonistic anti-Fas antibody. We next examined the expression of nucleolin in human lymphomas. Cell lines derived from lymphomas of different histological types consistently expressed nucleolin protein. We found nucleolin expressed on the surface of cells in over 20 primary lymphoma isolates, whereas peripheral blood lymphocytes showed low or undetectable levels. Lymphoma tissue microarray staining showed a correlation between nucleolin and Ki-67 expression. Whether nucleolin expression also correlates with adverse clinical features in lymphoma is currently under evaluation. Taken together, we show here that the known cancer associated protein nucleolin is overexpressed on surface of lymphoma cells where it binds to Fas receptor and blocks Fas signaling and apoptosis. We expect that further analysis of nucleolin properties will reveal how Fas-nucleolin interaction can be targeted to enhance killing of cancer cells leading eventually to cell surface nucleolin targeting therapy. Disclosures: Fayad: Roche: Research Funding.

We show that hydrogel particles could be easily synthesized by adding drops of aqueous solution containing monomer, comonomer, crosslinker, and initiator to the surface of a polytetrafluoroethylene (PTFE or Teflon) disk immersed in 2,2,4-trimethylpentane (TMP). This system yielded conditions that allowed for a spherical drop to be stable at that interface while the polymerization occurred. This approach was used to synthesize particles composed of poly(N-isopropylacrylamide) (pNIPAm) and poly(2-hydroxyethyl methacrylate) (pHEMA). Furthermore, the polymer particles could easily be doped with a variety of nanoparticles and small molecules simply by adding the components to the monomer/crosslinker solution prior to polymerization. This synthetic route is advantageous because it only appears to depend on the ability to form a water drop at the PTFE–TMP interface, thus this approach can be used to synthesize particles with a variety of different functionalities and compositions. This can lead to direct applications in oral drug delivery and tissue engineering.

Abstract Abstract 1327 Objective: Chemotherapies and irradiation depend on an intact Fas signaling system to eradicate cancer cells. Defective Fas signaling is an important cause of acquired resistance to cancer therapy. If we were able to restore Fas apoptosis or sensitize cancer cells to Fas-mediated apoptosis, we could improve the efficacy of many current cancer therapies. To elucidate defects of Fas signaling in cancer cells, we sought to identify potential modulators of Fas selectively expressed in cancers cells and target them to sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectroscopy was used to identify Fas-associated proteins. Co-immunoprecipitation (co-IP) and Western Blot (WB) were used to detect/confirm interactions of PML and PMLRARα with Fas, and components of death-inducing signaling complex (DISC) FADD, c-FLIP, and caspase-8 cleavage in tissues from PML wild-type (WT) and knock-out (KO) mice and acute promyelocytic leukemia (APL) cells. Deletional mutagenesis was used to map protein interacting domains. PML shRNA lentivirus and As2O3 were used to downregulate PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis in response to Fas stimulation. Mice transfected with PMLRARα were monitored for survival after a lethal challenge with agonistic Fas antibody Jo2 and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: The promyelocytic leukemia protein (PML) was identified as a Fas-binding protein by mass spectroscopy analysis. Using co-IP/WB analysis of tissues from PML WT and KO mice, we found PML interaction with Fas and FADD in PML WT MEF cells and liver cells but absent in KO MEF and liver cells; PML-Fas complexes were exclusively present in the membrane/cytoplasmic extracts but not in the nuclear extracts. The B-box domain of PML was found to be required for Fas binding. Knockdown of PML was associated with suppressed rates of Fas-mediated apoptosis compared to non-targeted knockdown cells; PML KO cells reconstituted with cytoplasmic PML were sensitized to Fas apoptosis. Furthermore, we found that liver cells from PML KO mouse showed impaired assembly of the Fas death-inducing signaling complex (DISC) in response to Fas activation when compared to PML WT. PML functions are known to be blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in primary human and transgenic mouse APL cells blocked Fas-mediated apoptosis. Blockage of apoptosis was mediated through PMLRARα -dependent recruitment of c-FLIPL/Sto and exclusion of procaspase-8 from the DISC. PMLRARα effects were also observed in vivo, as expression of PMLRARα protected mice against a lethal effect of agonistic anti-Fas antibody (P<.001). Livers from PMLRARα -transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions: PML binds to Fas and promotes Fas-mediated apoptosis through enhancing Fas DISC formation while binding of PMLRARα to Fas blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex enriched in c-FLIP. Our data suggest that PML plays a critical role in initiation of Fas signaling; in contrast, the dominant-negative mutant PMLRARα is a confirmed cancer specific inhibitor of Fas-mediated apoptosis. Thus, deficiency of PML or expression of PMLRARα can contribute to cancer development and resistance to chemotherapy. The newly discovered PML-Fas and PMLRARα -Fas complexes can be sites for modulation of apoptosis. Thus, by neutralizing the inhibitory effect of Fas-binding proteins such as PMLRARα and/or promoting positive Fas modulators such as PML, we can improve responses to chemotherapy that depend on activation of death receptors for effective elimination of cancer cells. Disclosures: No relevant conflicts of interest to declare.

ABSTRACT For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.

Mitochondrial antiviral signaling protein (MAVS) is an essential component of virus-activated signaling pathways that induce protective IFN responses. Its localization to the outer mitochondrial membrane suggests an important yet unexplained role for mitochondria in innate immunity. Here, we show that hepatitis A virus (HAV), a hepatotropic picornavirus, ablates type 1 IFN responses by targeting the 3ABC precursor of its 3C pro cysteine protease to mitochondria where it colocalizes with and cleaves MAVS, thereby disrupting activation of IRF3 through the MDA5 pathway. The 3ABC cleavage of MAVS requires both the protease activity of 3C pro and a transmembrane domain in 3A that directs 3ABC to mitochondria. Lacking this domain, mature 3C pro protease is incapable of MAVS proteolysis. HAV thus disrupts host signaling by a mechanism that parallels that of the serine NS3/4A protease of hepatitis C virus, but differs in its use of a stable, catalytically active polyprotein processing intermediate. The unique requirement for mitochondrial localization of 3ABC underscores the importance of mitochondria to host control of virus infections within the liver.