B-cell receptor (BCR) signalling is critical for the survival of B-cell lymphomas and is a therapeutic target of drugs such as Ibrutinib. However, the role of T-cell receptor (TCR) signalling in the survival of T/Natural Killer (NK) lymphomas is not clear. ZAP-70 (zeta associated protein-70) is a cytoplasmic tyrosine kinase with a critical role in T-cell receptor (TCR) signalling. It has also been shown to play a role in normal NK cell signalling and activation. High ZAP-70 expression has been detected by immunohistochemistry in peripheral T cell lymphoma (PTCL) and NK cell lymphomas (NKTCL). We therefore, studied the role of TCR pathways in mediating the proliferation and survival of these malignancies through ZAP-70 signalling. ZAP-70 protein was highly expressed in T cell lymphoma cell lines (JURKAT and KARPAS-299) and NKTCL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1 and SNK-6), but not in multiple B-cell lymphoma cell lines. siRNA depletion of ZAP-70 suppressed the phosphorylation of ZAP-70 substrates, SLP76, LAT and p38MAPK, but did not affect cell viability or induce apoptosis in these cell lines. Similarly, while stable overexpression of ZAP-70 mediates increased phosphorylation of target substrates in the TCR pathway, it does not promote increased survival or growth of NKTCL cell lines. The epidermal growth factor receptor (EGFR) inhibitor Gefitinib, which has off-target activity against ZAP-70, also did not show any differential cell kill between ZAP-70 overexpressing (OE) or knockdown (KD) cell lines. Whole transcriptome RNA sequencing highlighted that there was very minimal differential gene expression in three different T/NK cell lines induced by ZAP-70 KD. Importantly, ZAP-70 KD did not significantly enrich for any downstream TCR related genes and pathways. Altogether, this suggests that high expression and constitutive signalling of ZAP-70 in T/NK lymphoma is not critical for cell survival or downstream TCR-mediated signalling and gene expression. ZAP-70 therefore may not be a suitable therapeutic target in T/NK cell malignancies.
Abstract Tumor-specific neoantigens have emerged as a promising source for cancer immunotherapy. These tumor-specific neoantigens could arise from somatic mutations, aberrant splicing and RNA editing. Since intronic polyadenylation has similar potential as mutations to generate tumor-specific transcripts and peptides, it may serve as another neoantigen source, which has not been explored. We developed a novel computational pipeline for identification of tumor-specific transcripts and their translated neoantigens derived from intronic polyadenylation. Applying it to RNA-seq data from 5,654 tumor samples of various cancers and 11,000 + normal samples of different tissues, we observed widespread tumor-specific intronic polyadenylated transcripts and their corresponding neoantigens. In addition, we also discovered complementary effects of neoantigens derived from different sources, identified neoantigens arising from recurrent intronic polyadenylated transcripts, and validated their immunogenicity. Here, we have demonstrated that we were able to identify and predict neoantigens from intronic polyadenylated transcripts using RNA sequencing data, hence, allowing us to explore such neoantigens as potential candidates for cancer immunotherapy.
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