Identification of Antigenic Targets.

The ideal cancer target antigen (Ag) is expressed at high copy numbers on neoplastic cells, absent on normal tissues, and contributes to the survival of cancer cells. Despite significant investments in the identification of cell surface Ags, there is a paucity of targets that meet such ideal cancer target criteria. Recent clinical trials in patients with cancer treated with immune checkpoint inhibitors (ICIs) indicate that cluster of differentiation (CD)8+ T cells, by means of their T cell receptors (TCRs) recognizing intracellular targets presented as peptides in the context of human leukocyte antigen (peptide-human leukocyte antigen complex; pHLA) molecules on tumor cells, can mediate deep and long-lasting antitumor responses in patients with solid tumors. Therefore, pHLA-target Ags may represent the long sought-after, ideal targets for solid tumor targeting by high-potency oncology compounds.

Functional significance of U2AF1 S34F mutations in lung adenocarcinomas.

The functional role of U2AF1 mutations in lung adenocarcinomas (LUADs) remains incompletely understood. Here, we report a significant co-occurrence of U2AF1 S34F mutations with ROS1 translocations in LUADs. To characterize this interaction, we profiled effects of S34F on the transcriptome-wide distribution of RNA binding and alternative splicing in cells harboring the ROS1 translocation. Compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3' splice junctions. The presence of S34F caused a shift in cross-linking at 3' splice sites, which was significantly associated with alternative splicing of skipped exons. U2AF1 S34F induced expression of genes involved in the epithelial-mesenchymal transition (EMT) and increased tumor cell invasion. Finally, S34F increased splicing of the long over the short SLC34A2-ROS1 isoform, which was also associated with enhanced invasiveness. Taken together, our results suggest a mechanistic interaction between mutant U2AF1 and ROS1 in LUAD.

Intracellular targets as source for cleaner targets for the treatment of solid tumors.

High-potency oncology compounds such as antibody- drug conjugates, T cell redirecting, and CAR-T cell therapies have provided transformational responses in patients with liquid tumors. However, they delivered only limited benefit to solid tumor patients due to the frequent onset of dose limiting toxicities in normal tissues. Such on-target, off-tumor toxicities are caused by recognition of targets present at low-levels on normal tissues. The apparent imbalance between the rapid development of high-potency therapeutic modalities and the slow progress in identification of cleaner targets is illustrated by the fact that most high-potency compounds currently developed in the clinic target cell surface antigens identified over 20 years ago. Therefore, identification of novel, truly tumor-specific targets is critical for the future success of high-potency oncology compounds in solid tumors. One of the most promising approaches to overcome the limitations of targeting cell surface antigens are intracellular targets. The renewed interest in this class of targets is due to the success of immune checkpoint inhibitors, which mediate their anti-tumor responses by activation of cytotoxic T cells recognizing peptide fragments of intracellular targets presented by human leukocyte antigens (HLAs) on the surface of tumor cells. Importantly, many intracellular targets belong to the class of tumor specific antigens (TSAs), which lack presentation on normal tissues. In this report we review the main classes of tumor specific antigens, including viral, neoantigens and shared self-antigens as well as tumor associated antigens (TAAs) and their relevance for therapeutic targeting of solid tumors by high-potency therapeutic modalities.

Integrating Tumor and Stromal Gene Expression Signatures With Clinical Indices for Survival Stratification of Early-Stage Non-Small Cell Lung Cancer.

BACKGROUND: Accurate survival stratification in early-stage non-small cell lung cancer (NSCLC) could inform the use of adjuvant therapy. We developed a clinically implementable mortality risk score incorporating distinct tumor microenvironmental gene expression signatures and clinical variables. METHODS: Gene expression profiles from 1106 nonsquamous NSCLCs were used for generation and internal validation of a nine-gene molecular prognostic index (MPI). A quantitative polymerase chain reaction (qPCR) assay was developed and validated on an independent cohort of formalin-fixed paraffin-embedded (FFPE) tissues (n = 98). A prognostic score using clinical variables was generated using Surveillance, Epidemiology, and End Results data and combined with the MPI. All statistical tests for survival were two-sided. RESULTS: The MPI stratified stage I patients into prognostic categories in three microarray and one FFPE qPCR validation cohorts (HR = 2.99, 95% CI = 1.55 to 5.76, P < .001 in stage IA patients of the largest microarray validation cohort; HR = 3.95, 95% CI = 1.24 to 12.64, P = .01 in stage IA of the qPCR cohort). Prognostic genes were expressed in distinct tumor cell subpopulations, and genes implicated in proliferation and stem cells portended poor outcomes, while genes involved in normal lung differentiation and immune infiltration were associated with superior survival. Integrating the MPI with clinical variables conferred greatest prognostic power (HR = 3.43, 95% CI = 2.18 to 5.39, P < .001 in stage I patients of the largest microarray cohort; HR = 3.99, 95% CI = 1.67 to 9.56, P < .001 in stage I patients of the qPCR cohort). Finally, the MPI was prognostic irrespective of somatic alterations in EGFR, KRAS, TP53, and ALK. CONCLUSION: The MPI incorporates genes expressed in the tumor and its microenvironment and can be implemented clinically using qPCR assays on FFPE tissues. A composite model integrating the MPI with clinical variables provides the most accurate risk stratification.

FACTERA: a practical method for the discovery of genomic rearrangements at breakpoint resolution.

UNLABELLED: For practical and robust de novo identification of genomic fusions and breakpoints from targeted paired-end DNA sequencing data, we developed Fusion And Chromosomal Translocation Enumeration and Recovery Algorithm (FACTERA). Our method has minimal external dependencies, works directly on a preexisting Binary Alignment/Map file and produces easily interpretable output. We demonstrate FACTERA's ability to rapidly identify breakpoint-resolution fusion events with high sensitivity and specificity in patients with non-small cell lung cancer, including novel rearrangements. We anticipate that FACTERA will be broadly applicable to the discovery and analysis of clinically relevant fusions from both targeted and genome-wide sequencing datasets. AVAILABILITY AND IMPLEMENTATION: http://factera.stanford.edu.

Carbachol-mediated endocytosis of NHE3 involves a clathrin-independent mechanism requiring lipid rafts and Cdc42.

BACKGROUND: In intestinal epithelial cells, acute regulation of the brush border Na(+)/H(+) exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca(2+) ([Ca(2+)]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. METHODS: The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches. RESULTS: Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K(+) depletion) supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MßCD (to disrupt lipid rafts) as well as in Cdc42 knockdown cells but was unaffected by CME blockers. CONCLUSION: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42.

PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.

Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

NHERF3 (PDZK1) contributes to basal and calcium inhibition of NHE3 activity in Caco-2BBe cells.

Elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca(2+) ionophore, 4-bromo-A23187 (0.5 mum): 1) inhibited NHE3 V(max) activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca(2+)](i) by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca(2+)](i) inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca(2+)](i); 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca(2+).