Splicing neoantigen discovery with SNAF reveals shared targets for cancer immunotherapy.

Immunotherapy has emerged as a crucial strategy to combat cancer by "reprogramming" a patient's own immune system. Although immunotherapy is typically reserved for patients with a high mutational burden, neoantigens produced from posttranscriptional regulation may provide an untapped reservoir of common immunogenic targets for new targeted therapies. To comprehensively define tumor-specific and likely immunogenic neoantigens from patient RNA-Seq, we developed Splicing Neo Antigen Finder (SNAF), an easy-to-use and open-source computational workflow to predict splicing-derived immunogenic MHC-bound peptides (T cell antigen) and unannotated transmembrane proteins with altered extracellular epitopes (B cell antigen). This workflow uses a highly accurate deep learning strategy for immunogenicity prediction (DeepImmuno) in conjunction with new algorithms to rank the tumor specificity of neoantigens (BayesTS) and to predict regulators of mis-splicing (RNA-SPRINT). T cell antigens from SNAF were frequently evidenced as HLA-presented peptides from mass spectrometry (MS) and predict response to immunotherapy in melanoma. Splicing neoantigen burden was attributed to coordinated splicing factor dysregulation. Shared splicing neoantigens were found in up to 90% of patients with melanoma, correlated to overall survival in multiple cancer cohorts, induced T cell reactivity, and were characterized by distinct cells of origin and amino acid preferences. In addition to T cell neoantigens, our B cell focused pipeline (SNAF-B) identified a new class of tumor-specific extracellular neoepitopes, which we termed ExNeoEpitopes. ExNeoEpitope full-length mRNA predictions were tumor specific and were validated using long-read isoform sequencing and in vitro transmembrane localization assays. Therefore, our systematic identification of splicing neoantigens revealed potential shared targets for therapy in heterogeneous cancers.

Characteristics of Immune Memory and Effector Activity to Cancer-Expressed MHC Class I Phosphopeptides Differ in Healthy Donors and Ovarian Cancer Patients.

Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.

Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

BACKGROUND: Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy. METHODOLOGY/PRINCIPAL FINDINGS: This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150-300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway. CONCLUSIONS/SIGNIFICANCE: The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical environments encountered by cancer cells upon metastasis to distant sites.

RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration.

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.