Biology subjects

Hanna, G.

Publications and source records attributed to Hanna, G..

4 recordsLinked to original sources

bioRxiv2026

Dual-inactivation of Regnase-1 and SOCS1 rewires exhausted CD8+ T cell fate to enhance anti-tumor functionality

The solid tumor microenvironment inhibits the functionality of tumor infiltrating T cells recognizing cognate tumor antigen, driving their differentiation towards terminal exhaustion. Interventions are sought to enhance the anti-tumor functionality of tumor-reactive T cells for clinical benefit. The functional genome regulating CD8+ T cell function against solid tumors was mapped by performing genome-wide, focused, and combination in vivo CRISPR/Cas9 screens using OT1 and PMEL TCR transgenic T cells in B16-OVA, MC38-gp100 and EG7-OVA syngeneic tumor models. The ability of the top single hits and combinations, which include Regnase-1 and SOCS1, to enhance CD8+ T cell anti-tumor function was evaluated in the OT1/B16-OVA model with large and established tumors, the disseminated PMEL/B16F10 tumor model, and in a novel murine TIL syngeneic model. The impact of Regnase-1 and SOCS1 single and dual-inactivation on the differentiation of exhausted CD8+ T cell subsets and on long-term persistent memory following tumor clearance was evaluated in OT1 CD8+ T cells in the B16-OVA model. The impact of single and dual-inactivation of Regnase-1 and SOCS1 on the anti-tumor function of experimental human T cell therapeutics was characterized in CRISPR/Cas9-engineered human TIL derived in vitro and in mesothelin-targeting CAR-Ts in vivo. NF-{kappa}B and cytokine signaling were identified as the top pathways regulating CD8+ T cell anti-tumor function, with Regnase-1 and Suppressor of Cytokine Signaling 1 (SOCS1) the top single and combination edits regulating the accumulation of tumor-specific TCR transgenic CD8+ T cells in syngeneic tumor models. Dual-inactivation of Regnase-1 and SOCS1 cooperated through non-redundant mechanisms to strongly expand intermediate (Texint) and effector (Texeff) exhausted CD8+ T cells within lymphoid tissues and tumor, with CD8+ T cells rewired to display an enhanced effector state and suppressed expression of TOX. Dual-edited persistent T effector memory cells (Tem) were formed following tumor clearance. Lastly, Regnase-1 and SOCS1 inactivation enhanced human Tumor Infiltrating Lymphocyte (TIL) and chimeric antigen receptor T cells (CAR-T) therapy functionality. Collectively, this study systematically mapped pathways regulating CD8+ T cell anti-tumor functionality, with Regnase-1 and SOCS1 dual-inactivation found to maximize anti-tumor function through non-redundant mechanisms.

immunology↗

bioRxiv2025

Missense3D-PTMdb an interactive web resource to explore and visualize genetic variants and post-translational modifications sites (PTMs) using AlphaFold 3D models

Only a fraction of the >11 million missense variants identified in the human population has a known damaging or tolerated clinical impact. Post-translational modifications (PTMs), such as phosphorylation, glycosylation and ubiquitination, are key regulators of protein function and structure, and are critical for protein localisation, stability and interactions with other molecules. The ability of a protein to undergo PTMs, is subject to a correctly folded protein structure, and the recognition and binding of enzymes to specific amino acid motifs in close proximity to residues that undergo PTMs [PTM residue]. AlphaFold models provide an unprecedented opportunity to perform sequence-structure mapping of variants, which are in close linear or spatial proximity to a PTM site and should have their impact on protein function experimentally investigated. We present Missense3D-PTMdb, a "one-stop-shop" interactive web tool that provides a user-friendly sequence-structure mapping of 20,235 human proteins to 11,544,303 naturally occurring human missense variants, 203,775 PTM sites and their neighbours in sequence and 3D structure space using AlphaFold generated 3D models of the human proteome. Additionally, the sequence-structure mapping tool allows visualization and exploration of any human variant not currently stored in the database. Missense3D-PTMDb is freely available at https://missense3d.bc.ic.ac.uk/ptmdb.

genetics↗

bioRxiv2023

A new web resource to predict the impact of missense variants at protein interfaces using 3D structural data: Missense3D-PPI

In 2019, we released Missense3D which identifies stereochemical features that are disrupted by a missense variant, such as introducing a buried charge. Missense3D analyses the effect of a missense variant on a single structure and thus may fail to identify as damaging surface variants disrupting a protein interface i.e., a protein-protein interaction (PPI) site. Here we present Missense3D-PPI designed to predict missense variants at PPI interfaces. Our development dataset comprised of 1,279 missense variants (pathogenic n=733, benign n=546) in 434 proteins and 545 experimental structures of PPI complexes. Benchmarking of Missense3D-PPI was performed after dividing the dataset in training (320 benign and 320 pathogenic variants) and testing (226 benign and 413 pathogenic). Structural features affecting PPI, such as disruption of interchain bonds and introduction of unbalanced charged interface residues, were analysed to assess the impact of the variant at PPI. Missense3D-PPIs performance was superior to that of Missense3D: sensitivity 42% versus 8% and accuracy 58% versus 40%, p=4.23x10-16 However, the specificity of Missense3D-PPI was slightly lower compared to Missense3D (84% versus 98%). On our dataset, Missense3D-PPIs accuracy was superior to BeAtMuSiC (p=2.3x10-5), mCSM-PPI2 (p=3.2x10-12) and MutaBind2 (p=0.003). Missense3D-PPI represents a valuable tool for predicting the structural effect of missense variants on biological protein networks and is available at the Missense3D web portal (http://missense3d.bc.ic.ac.uk/missense3d/indexppi.html).

bioinformatics↗

bioRxiv2020

Structure, function and variants analysis of the androgen-regulated TMPRSS2, a drug target candidate for COVID-19 infection

SARS-CoV-2 is a novel virus causing mainly respiratory, but also gastrointestinal symptoms. Elucidating the molecular processes underlying SARS-CoV-2 infection, and how the genetic background of an individual is responsible for the variability in clinical presentation and severity of COVID-19 is essential in understanding this disease. Cell infection by the SARS-CoV-2 virus requires binding of its Spike (S) protein to the ACE2 cell surface protein and priming of the S by the serine protease TMPRSS2. One may expect that genetic variants leading to a defective TMPRSS2 protein can affect SARS-CoV-2 ability to infect cells. We used a range of bioinformatics methods to estimate the prevalence and pathogenicity of TMPRSS2 genetic variants in the human population, and assess whether TMPRSS2 and ACE2 are co-expressed in the intestine, similarly to what is observed in lungs. We generated a 3D structural model of the TMPRSS2 extracellular domain using the prediction server Phyre and studied 378 naturally-occurring TMPRSS2 variants reported in the GnomAD database. One common variant, p.V160M (rs12329760), is predicted damaging by both SIFT and PolyPhen2 and has a MAF of 0.25. Valine 160 is a highly conserved residue within the SRCS domain. The SRCS is found in proteins involved in host defence, such as CD5 and CD6, but its role in TMPRSS2 remains unknown. 84 rare variants (53 missense and 31 leading to a prematurely truncated protein, cumulative minor allele frequency (MAF) 7.34x10-4) cause structural destabilization and possibly protein misfolding, and are also predicted damaging by SIFT and PolyPhen2 prediction tools. Moreover, we extracted gene expression data from the human protein atlas and showed that both ACE2 and TMPRSS2 are expressed in the small intestine, duodenum and colon, as well as the kidneys and gallbladder. The implications of our study are that: i. TMPRSS2 variants, in particular p.V160M with a MAF of 0.25, should be investigated as a possible marker of disease severity and prognosis in COVID-19 and ii. in vitro validation of the co-expression of TMPRSS2 and ACE2 in gastro-intestinal is warranted.

bioinformatics↗