Blunt, M. D. | bioRxiv Science

bioRxiv2022

Network analysis reveals a major role for 14q32 cluster miRNAs in determining transcriptional differences between IGHV-mutated and unmutated CLL

Tumour cells from patients with chronic lymphocytic leukaemia (CLL) can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear to what extent these differences reflect the tumour cells innate pre/post germinal centre origin or their BCR signalling competence, we applied RNA sequencing, small RNA sequencing and DNA methylation array analysis to 38 CLL cases categorised into three groups by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. A significant proportion of the IGHV-associated miRNAs derived from chr14q32 clusters (14/38 (37%)), where all miRNAs were co-expressed with the MEG3 lncRNA, as part of the DLK1-DIO3 genomic imprinted region, a locus of known importance in the pathogenesis of other human tumours. Integrative in silico analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was predicted to be regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

cancer biology↗

bioRxiv2020

A DNA vaccine strategy to activate natural killer cells for therapeutic targeting of XPO1-positive tumors

Natural killer (NK) cells are key components of the immune response to viral infections and cancer. Their functions are controlled by activating and inhibitory killer-cell immunoglobulin-like receptors (KIR) which have MHC class I ligands. KIR2DS2 is an activating KIR, that binds conserved viral peptides in the context of HLA-C and has been associated with protective responses to both cancer and viral infections. We sought to investigate whether NK cells can be specifically activated in a peptide:MHC dependent manner to generate functional immune responses as a potential immunotherapeutic strategy. We developed a peptide-based KIR targeting DNA vaccine. Immunizing KIR-Tg mice with the vaccine construct generated in vivo peptide-specific activation of KIR2DS2-positive NK cells leading to canonical and cross-reactive peptide specific immune responses in vitro, and also in vivo inhibition of tumor growth. Using immunopeptidomics we identified that the nuclear export protein XPO1, which has been associated with a poor prognosis in many different human cancers, furnishes an HLA-C restricted cancer-associated peptide ligand for KIR2DS2-positive NK cells. We thus define a novel strategy to activate KIR in a peptide-specific manner and identify a rationale for its use in cancer immunotherapy. Significance statementNatural killer (NK) cells are known to have important roles in determining the outcomes of viral infections and cancer. The killer cell immunoglobulin-like receptors (KIR), and in particular the activating receptor KIR2DS2, have been associated with the outcome of a number of different human cancers. Specific activation of NK cells through KIR2DS2 is challenging because it shares high (>98%) sequence homology with related inhibitory KIR. We have used a peptide:MHC targeting strategy to activate NK cells through KIR2DS2 and identified a novel cancer-associated ligand for this receptor. The work provides a proof-of-concept for targeting NK cells through activating KIR as a cancer immunotherapy strategy.

immunology↗