Biology subjects

Shah, K. M.

Publications and source records attributed to Shah, K. M..

10 recordsLinked to original sources

bioRxiv2025

An AGO2 adaptor expands the functional and evolutionary reach of microRNA targeting

Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1+/+, LIMD1+/-, and LIMD1-/- human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2-miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C2H2-zinc-finger genes. Even within canonical seed repertoires of individual AGO2-miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo, inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/680889v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@e0f4f5org.highwire.dtl.DTLVardef@f23fd9org.highwire.dtl.DTLVardef@3de3f2org.highwire.dtl.DTLVardef@1404af2_HPS_FORMAT_FIGEXP M_FIG LIMD1 defines the scope of miRNA-mediated targeting and repression O_LIAGO2-chimeric eCLIP in CRISPR-edited human small airway epithelial cells (hSAECs) shows LIMD1 is required for productive AGO2-miRNA engagement transcriptome-wide. C_LIO_LILIMD1 deficiency reduces the AGO2-miRNA:targetome. Each AGO2-miRNA binds fewer targets, with lower occupancy per site and per transcript and fewer global silencing events. C_LIO_LILIMD1 dependence is strongest for GC-poor seed-sites, less conserved miRNAs and sites, and thermodynamically stronger duplexes. C_LIO_LIDosage-dependent effects of LIMD1 deficiency are broadly observed. C_LIO_LITarget-mRNA decay and translational repression are reduced in LIMD1-deficient hSAECs, increasing protein output. C_LIO_LIIn vivo, LIMD1 LOH-associated deficiency is prevalent and typically clonal in NSCLC. LIMD1 expression inversely correlates with oncogene levels in normal lung and adenocarcinoma, and target dysregulation predicts poor survival. C_LI C_FIG

molecular biology↗

bioRxiv2025

Coenzyme A homeostasis regulates hypoxic signalling via UBFD1in triple-negative breast cancer

Hypoxia-inducible factors (HIFs) orchestrate cellular responses to oxygen deprivation and are frequently activated in triple-negative breast cancer (TNBC), driving metastasis and poor prognosis1,2. However, the full mechanisms of how HIFs are activated are not completely understood. It has been long noted that certain tumours have decreased levels of coenzyme A compared to surrounding tissues3,4, but the functional consequences of this remain unknown. Here, we identify Coenzyme A Synthase (CoAsy) as a novel regulator of HIF signalling. We demonstrate that CoAsy loss stabilises HIF-1 and HIF-2 independently of the canonical oxygen sensing pathway. Proximity-labelling proteomics revealed that CoAsy deficiency disrupts the association between HIF-1 and the proteasome through UBFD1, a novel CoA-binding protein that scaffolds HIF for degradation. UBFD1s CoA-dependent interaction with HIF- is mediated by its PH domain, which undergoes CoAlation, an understudied post-translational modification. COASY loss of heterozygosity occurs in [~]1/3 of breast cancer patients and correlates with increased HIF activity, lack of hormone receptor expression and poor patient outcome. In vivo studies confirm that restoring CoAsy expression in tumours suppresses metastasis to the lung. These findings uncover a critical metabolic checkpoint regulating hypoxic signalling and identify CoAsy as a potential biomarker and therapeutic target in aggressive breast cancers.

molecular biology↗

bioRxiv2025

LIMD1 Loss Confers a GPX4-Dependent Cell State in Lung Cancer

LIMD1 (LIM domains containing 1) is a bona-fide tumour suppressor gene frequently lost during the early stages of non-small cell lung cancer (NSCLC) development, resulting in a worse outcome for LIMD1-deficient patients. LIMD1 deficiency is present in approximately 50% of NSCLC cases, representing at least 21,000 patients in the United Kingdom and 1.2 million worldwide. During cancer progression, cells can accumulate genetic changes that render them more dependent on certain genes for their survival. By performing CRISPR-Cas9 dropout screens, we identified GPX4 as a novel vulnerability in LIMD1 deficient LUAD cells. By targeting GPX4 using RNA interference (RNAi) and pharmacological intervention in our isogenic LUAD lines along with a non-transformed lung cell line, we validated GPX4 as a novel dependency in LIMD1 deficient cells. GPX4 is a key defence mechanism against ferroptosis, an iron-dependent form of regulated cell death. This state of increased ferroptosis susceptibility upon LIMD1 loss is due to increased basal reactive oxygen species levels and lipid peroxidation. Importantly, targeting GPX4 with ferroptosis inducing agents in NSCLC patients with LIMD1 loss may represent a novel therapeutic strategy.

cancer biology↗

bioRxiv2025

LIMD1 Loss as an Early Driver of PD-L1 Upregulation and Immune Evasion in Lung Cancer

LIMD1 is a tumour suppressor gene frequently lost in non-small cell lung cancer (NSCLC), but its role in cancer-immune cell interactions remains unexplored. Here, we demonstrate that LIMD1 loss results in upregulation of the key immune checkpoint protein PD-L1. Using multi-region sequencing from the TRACERx dataset, we identify that LIMD1 loss is clonal in over 80% of squamous cell carcinoma (LUSC) and 40% of lung adenocarcinoma (LUAD) cases, correlating with increased PD-L1 expression. LIMD1 deficiency results in upregulation of basal and IFN{gamma}-induced PD-L1 expression in NSCLC cells and, consistent with its early loss during oncogenesis, in primary human small airway epithelial cells. Mechanistically, we demonstrate that LIMD1 interacts with the E3 ubiquitin ligase ARIH1 to mediate efficient PD-L1 ubiquitination and degradation, a process that is significantly impaired in LIMD1-deficient cells, resulting in increased PD-L1 stability. As a consequence, LIMD1-deficient tumour cells suppressed CD8+ T cell activation in vitro, and blockade of PD-L1 reversed this suppression. Clinically, we show that LIMD1 loss is associated with enhanced response to immune checkpoint inhibitors (ICIs) in NSCLC patient cohorts, revealing a novel cancer cell-intrinsic correlation of ICI efficacy. Our results uncover a tumour suppressor-mediated mechanism of PD-L1 expression and pave the way for stratified immunotherapy approaches in LIMD1-/- NSCLC.

cancer biology↗

bioRxiv2023

A C-terminal HaloTag Impairs AGO2 Function

A full understanding of RNA silencing requires appropriate molecular biology tools to explore the roles of Argonaute 2 (AGO2) and the RNA-induced silencing complex (RISC). Approaches relying on affinity tagging and antibodies have important limitations that can lead to artificial results. Both the N- and C-terminal domains of AGO2 have been shown to be important for correct activity and yet the consequences of appending tags to either terminus have not been fully investigated. N-terminal tags are frequently used to study AGO2 biology. Recently, an N-terminal HaloTag-Ago2 fusion was reported and examined in mice. While the versatile HaloTag provided new opportunities to study RISC biology, the tagged construct showed certain activity changes compared to unmodified AGO2. CRISPaint, a new CRISPR-Cas9 technique, permits the creation of endogenous C-terminal tag fusions. We used CRISPaint to generate the first reported recombinant AGO2 construct with a C-terminal tag: an endogenous C-terminal HaloTag fusion to AGO2 (AGO2HALO) in human (A549) cells. We found that the AGO2HALO fusion protein has a reduced capacity to interac with the key protein binding partner TNRC6A and that the C-terminal HaloTag does not affect cell viability. However, the AGO2HALO fusion significantly impairs RNA cleavage and RNA silencing activity compared to control cells and reduces nuclear localisation of the fusion protein. Using plasmid constructs and transient transfection, we compared AGO2 tagged with EGFP at the N- or C-terminus in siRNA and miRNA reporter gene assays, and cellular localisation. N-terminally tagged AGO2 functioned and localised similarly to WT untagged AGO2, whereas C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing and exhibited poor nuclear and P-body localisation. We conclude that the fusion of a C-terminal HaloTag to AGO2 is not appropriate for studying AGO2 and RISC. Our results assert the importance of comprehensively validating recombinant tagging strategies to ensure that any experimental results generated do not arise from, or are not obscured by, critical functional defects.

molecular biology↗

bioRxiv2022

Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types

Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains following parabiosis. For each cell type, we catalogued alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions, and senescence status. Our analyses identified gene signatures demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest novel strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors.

neuroscience↗

bioRxiv2022

P2RX7 inhibition reduces breast cancer induced osteolytic lesions - implications for bone metastasis

Breast cancer metastasis to bone is a major contributor to morbidity and mortality in patients and remains an unmet clinical need. Purinergic signalling via the P2X7 receptor (P2RX7) in the primary tumour microenvironment is associated with progression of several cancers. It has also now become evident that intra-tumoural hypoxia facilitates cancer metastasis and reduces patient survival. In this study, we present data suggesting that hypoxia regulates the expression of P2RX7 in the primary tumour microenvironment; and importantly, inhibition with a selective antagonist (10mg/kg A740003) increased cancer cell death via apoptosis in a E0771/C57BL-6J syngeneic murine model. Furthermore, micro-computed tomography demonstrated reduced number of osteolytic lesions and lesion area following P2RX7 inhibition in absence of overt metastases by decreasing osteoclast numbers. We also demonstrate that activation of P2RX7 plays a role in the secretion of extracellular vesicles (EVs) from breast cancer cells. Mass-spectrometric analyses showed a distinct protein signature for EVs derived from hypoxic compared with normoxic cancer cells which elicit specific responses in bone cells that are associated with pre-metastatic niche formation. Thus, inhibiting P2RX7 provides a novel opportunity to preferentially target the hypoxic breast cancer cells preventing tumour progression and subsequent metastasis to bone

cancer biology↗

bioRxiv2021

The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties

Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread, meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for development of novel therapeutic strategies. In this study, we have produced in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (herein termed " P2RX7B"), in OS progression and metastasis. Our data shows that P2RX7B expression confers a survival advantage in TE85+P2RX7B and MNNG-HOS+ P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, suggesting a probable metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS+P2RX7B tumours exhibited ectopic bone formation that was abrogated with A740003 treatment. An increased metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of the tumour to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. In conclusion, our data indicates for the first time a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a potential therapeutic target in human OS. Novelty and ImpactWe provide evidence for the pro-tumorigenic role of the B isoform of the P2RX7 purinergic receptor in osteosarcoma (OS). In addition to increasing proliferation, P2RX7B increases the cancerous properties of OS cells, reducing adhesion and increasing migration and invasion. In vivo, P2RX7B does not affect primary tumour growth, but does lead to an increased propensity to metastasize. RNA-seq revealed a new axis of oncogenic genes inhibited by the P2RX7 antagonist and this data could potentially lead to new targets for OS treatment.

cancer biology↗

bioRxiv2021

Targeted therapy for LIMD1-deficient non-small cell lung cancer subtypes.

An early event in lung oncogenesis is loss of the tumour suppressor gene LIMD1 (LIM domains containing 1); this encodes a scaffold protein, which suppresses tumourigenesis via a number of different mechanisms. Approximately 45% of non-small cell lung cancers (NSCLC) are deficient in LIMD11, yet this subtype of NSCLC has been overlooked in preclinical and clinical investigations. Defining therapeutic targets in these LIMD1 loss-of-function patients is difficult due to a lack of druggable targets, thus alternative approaches are required. To this end, we performed the first drug repurposing screen to identify compounds that confer synthetic lethality with LIMD1 loss in NSCLC cells. PF-477736 was shown to selectively target LIMD1 deficient cells in vitro through inhibition of multiple kinases, inducing cell death via apoptosis. Furthermore, PF-477736 was effective in treating LIMD1-/- tumors in subcutaneous xenograft models, with no significant effect in LIMD1+/+ cells. We have identified a novel drug tool with significant preclinical characterization that serves as an excellent candidate to explore and define LIMD1-deficient cancers as a new therapeutic subgroup of critical unmet need. Significance StatementHere we provide the first proof-of-concept data validating the scope for development of a targeted therapy against the non-small cell lung cancers (NSCLC) subtypes deficient in expression of the LIMD1 tumor suppressor gene. Approximately 45% of NSCLC are deficient in LIMD11 representing at least 1.2 million lung cancer patients worldwide; yet this subtype has been ignored in preclinical and clinical investigations with no targeted therapies available. This seminal study applied synthetic lethality drug screening to target the loss/reduction of LIMD1 in lung cancer and normal cell lines, identifying and validating the multi-kinase inhibitor PF-477736 as a selectively cytotoxic compound towards LIMD1 deficient cells. This study provides rationale for further investigation into targeting LIMD1 loss in lung cancer, thereby addressing a critical unmet need for therapeutic approached to targeting LIMD1-deficent cancer subtypes.

cancer biology↗

bioRxiv2019

Decoding the genomic basis of osteoarthritis

Osteoarthritis causes pain and functional disability for a quarter of a billion people worldwide, with no disease-stratifying tools nor modifying therapy. Here, we use primary cartilage and synovium from osteoarthritis patients to construct a molecular quantitative trait locus map of gene expression and protein abundance. By integrating data across omics levels, we identify likely effector genes for osteoarthritis-associated genetic signals. We detect pronounced molecular differences between macroscopically intact and highly degenerated cartilage. We identify molecularly-defined patient subgroups that correlate with clinical characteristics, stratifying patients on the basis of their molecular profile. We construct and validate a 7-gene classifier that reproducibly distinguishes between these disease subtypes, and identify potentially actionable compounds for disease modification and drug repurposing.

genomics↗